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An extensive gastroenteritis outbreak after drinking-water contamination by sewage effluent, Finland
|
SUMMARY
An inappropriate cross-connection between sewage- and drinking-water pipelines contaminated tap water in a Finnish town, resulting in an extensive waterborne gastroenteritis outbreak in this developed country. According to a database and a line-list, altogether 1222 subjects sought medical care as a result of this exposure. Seven pathogens were found in patient samples of those who sought treatment. To establish the true disease burden from this exposure, we undertook a population-based questionnaire investigation with a control population, infrequently used to study waterborne outbreaks. The study covered three areas, contaminated and uncontaminated parts of the town and a control town. An estimated 8453 residents fell ill during the outbreak, the excess number of illnesses being 6501. Attack rates were 53% [95% confidence interval (CI) 49.5–56.4] in the contaminated area, 15.6% (95% CI 13.1–18.5) in the uncontaminated area and 6.5% (95% CI 4.8–8.8) in the control population. Using a control population allowed us to differentiate baseline morbidity from the observed morbidity caused by the water contamination, thus enabling a more accurate estimate of the disease burden of this outbreak.
Key words: Gastroenteritis, outbreak, waterborne infections.
INTRODUCTION
Safe water is essential to life. Globally, the lack of clean water is estimated to cause about 4% of total mortality [1], predominantly in the developing world. However, waterborne outbreaks also remain a source of concern in developed countries. In the USA, 20 million people are estimated to contract a waterborne infection annually [2]. Over 4 00 000 people fell ill with cryptosporidiosis in Milwaukee in 1993 as a consequence of inadequate water filtration [3]. In Canada, the Walkerton outbreak resulted in seven fatalities attributable to Escherichia coli O157:H7 infection [4]. Waterborne outbreaks have been reported from many European countries including England and Wales [5], Norway [6], Sweden [7–10], Italy, Spain and Switzerland [11].
Finland is a large country (3 38 424 km2) with a small population (5.4 million). The number of waterworks is high (almost 2000) and most serve small communities. The majority (95%) use groundwater. However, almost half of the population are served by surface-water plants of the largest cities. Groundwater is usually not disinfected for use, while surface water is filtered and disinfected before distribution to consumers [12]. Although drinking water is generally considered safe in Finland, a number of waterborne outbreaks have occurred. Twenty-four outbreaks affecting 7700 inhabitants were spontaneously reported during the period 1980–1992 [13]; since 1997 reporting of outbreaks has been mandatory, and 14 outbreaks affecting 7300 people were registered during the first 2 years [12]. The most widespread outbreak to date affected 5500 people [14], and a number of other substantial waterborne outbreaks have been reported [15–17]. The most common microbes have been norovirus [18] and Campylobacter [19].
We report the investigation of a serious waterborne gastroenteritis outbreak that occurred in a Finnish town in autumn 2007.
Setting
Nokia (population 30 016) is a town located in southern Finland. Over 90% of the population are served by the municipal drinking-water supply. The average daily water consumption in Nokia is 4250 m3 (year 2006). The waterworks uses groundwater, which is treated with sodium hypochlorite for disinfection and pH adjustment. Sewage is processed in one municipal waste water plant, which covers 86% of the population. The town has a municipal health centre for primary care and a hospital for primary in-patient care and limited secondary care. Secondary and tertiary care are provided by Tampere University Hospital 20 km distant from Nokia. The Centre for Laboratory Medicine provides the municipalities with clinical laboratory services. Environmental health surveillance and monitoring of water quality is organized in collaboration with five neighbouring municipalities.
The outbreak
On Wednesday, 28 November 2007, maintenance work was conducted at the waste water plant in Nokia. The same day, technical problems were encountered in the municipal drinking-water system, and during the days following, customers complained of a bad smell and taste in their tap water. These were first misinterpreted as being caused by harmless deposits loosened from the inner walls of the pipeline and discharged during the maintenance work on 28 November. From 30 November, environmental health and municipal waterworks officials received reports of bouts of diarrhoea and vomiting among residents, and during that same day an increase in the number of patients with gastroenteritis was observed at the health centre. Extensive water and pipeline sampling was initiated and a boil-water notice issued, and water chlorine concentration was increased. The water contamination and its source became evident the same day. A valve in the waste-water plant connecting the drinking-water line and a waste-water effluent line had been opened during the maintenance work and inadvertently left open for 2 days. As a consequence, the drinking-water network became contaminated with sewage effluent. According to flow data, an estimated 450 m3 of waste water was mixed with the drinking water and distributed to customers, heavily contaminating household water and pipelines in southern parts of the town. The population in this area numbered over 9500.
Distribution of clean water was organized by municipal officials, the military and volunteers, and the health centre focused on treating outbreak patients, cutting back on other activities. The University Hospital prepared to open a temporary ward, which was in fact eventually not needed. Schools and daycare centres were closed from 6 to 9 December owing to teachers’ and children’s sick leave and the need to reduce the possible horizontal spread of gastrointestinal viruses. After 12 days, the boil-water notice was withdrawn in the uncontaminated area. In the contaminated area, adenoviruses and noroviruses were repeatedly detected in water-supply samples despite chlorination and pipeline flushing. Finally, after extensive decontamination procedures including shock chlorination, precautionary restrictions were discontinued in the contaminated area on 18 February 2008, almost 3 months after the incident.
We conducted a comprehensive investigation to determine the magnitude and consequences of the waterborne outbreak including describing the clinical aspects, reviewing microbiological findings and estimating the disease burden using a population-based survey.
METHODS
Descriptive epidemiology
Patients visiting the municipal health centre between 28 November and 31 December 2007 because of acute gastroenteritis were registered on a line-list, including name, age, gender, address and personal identity number. The timing and range of symptoms were recorded. Prospective line-listing was initiated 6 days after the beginning of the contamination visits prior to that being added retrospectively from patient charts. The information collected from the patient register included the number of primary-care visits to the centre because of gastroenteritis as defined by the international classification of primary care (ICPC-1, WHO, Geneva) codes D01, D09, D10, D11, D25, D70, D73 or D96. The information on reported symptoms was collected from the line-list. Daily admissions to Tampere University Hospital were counted by emergency-department personnel.
Analytical epidemiology
The town was divided into two study areas, contaminated (population 9538) and uncontaminated (population 20 478). The division was based on microbiological data from distribution system samples and technical modelling of water-flow directions within the network. Another municipality (population 27 259) 35 km distant from Nokia was chosen as a control population. One thousand persons for each study group were randomly selected from the population register. Groups were matched by age and gender and all age groups were included. Only one participant per household was included. The survey was conducted using postal questionnaires, the form being posted 8 weeks after the contamination had occurred. A repeat questionnaire was sent out 3 weeks later to non-responders.
Participants were asked about the timing and nature of gastrointestinal symptoms. A case was defined as a person suffering acute diarrhoea (o3 loose stools/day) or vomiting between 28 November 2007 and 20 January 2008. The occurrence of gastroenteritis during a 10-month period prior to the outbreak was also enquired about to ensure the comparability of the study groups.
Microbiological investigation
Faecal specimens for bacterial pathogens were not investigated from all the patients or from representative samples from consecutive cases. Instead, bacterial samples were available from cases with more severe symptoms. Specimens for detection of norovirus were collected from five randomly selected patients within the first week of the outbreak. Other viral pathogens were studied in samples from children admitted to the emergency department of the University Hospital. In order to detect enteric parasites specimens were taken from ten consecutive patients during the second week of the outbreak. Subsequently, when cases with giardiasis were detected, parasites were investigated in all symptomatic patients. Specimens were taken at the health centre or in the University Hospital.
Stool specimens were cultured for the presence of Campylobacter, Salmonella, Yersinia and Shigella spp. Norovirus was investigated using electron microscopy and PCR; rotavirus antigens were detected by enzyme immunological assay (EIA). Protozoan pathogens were investigated using microscopy of formalin-fixed samples. Subsequently, because no other protozoan pathogens were identified, only antigen detection of Giardia was used instead of microscopy. If a patient reported bloody stools, Shiga toxin producing Escherichia coli (STEC) was examined for by culture and toxin detection (EIA). Only samples taken between 28 November and 31 December 2007 were included. For Giardia the time-period for inclusion was extended to 31 May 2008 (6 months) because of the longer incubation time and gradual case detection.
The methods used in water and pipeline analysis will be described in detail in a forthcoming paper. Briefly, samples from the drinking-water distribution system were analysed by cultivation for the presence of Campylobacter, Salmonella and Clostridium spp. PCR was utilized to analyse enteric viruses as previously described by Maunula and colleagues [20]. The presence of Giardia cysts and Cryptosporidium oocysts was detected using concentration, immunomagnetic separation and epifluorescence microscopy as described in detail by Rimhanen-Finne and colleagues [21].
The study areas were compared using x2 or Kruskal– Wallis rank sum tests whenever appropriate. Attack rates were calculated as percentage of cases in a sample population. Total numbers of cases were calculated by extrapolating the age- and sex-adjusted attack rates to the total population of each area. To assess baseline rates of gastroenteritis, the attack rates from the control population were used to estimate the number of cases in the contaminated population that would have occurred in the absence of the water contamination. Estimations were made adjusting for age and sex. Confidence intervals for the total numbers were obtained using bootstrap methods [21]. All statistical analyses were performed using R version 2.9 (or later versions) [22].
RESULTS
Healthcare visits
According to the health centre database, 1222 visits due to gastrointestinal symptoms were made between 28 November and 31 December 2007. During the busiest week, the number of patients presenting with gastrointestinal complaints was 53-fold higher than expected compared to the weekly median number of cases in a 7-month period preceding the outbreak. Children aged <10 years constituted the largest group (Figs 1, 2).
Line-list data were available on 1024 patients. Only one visit per person was included. The most common symptoms reported were diarrhoea (n=813, 79%), vomiting (n=601, 59%), fever (n=337, 33%) and abdominal pain (n=291, 28%). Altogether 204 visits were made to emergency rooms in Tampere University Hospital, 145 (71%) of them involving children. To our knowledge, the outbreak did not lead directly to fatalities.
Questionnaire study
A total of 2154 participants returned the questionnaire. Thirty-one were excluded as being inadequately completed or because the respondent could not be identified; 2123 forms were finally accepted for the analysis (response rate 71%). The response rate was highest in the contaminated area and lowest in the control population. The background characteristics of the study groups showed no significant demographic differences between the study populations (Table 1). Altogether, 17% of the responders reported having gastroenteritis during the 10 months prior to the outbreak. Most subjects fell ill within 1 week of the contamination of the water supply (Fig. 3).
The most common symptoms were diarrhoea, vomiting, nausea and abdominal pain (Fig. 4). Overall, 579 respondents met the case definition, of whom 540 (93%) were residents of Nokia. Extrapolating the result for the whole population, an estimated 8453 (95% CI 7568–9363) residents fell ill with gastroenteritis in the whole of Nokia (Table 2). The excess number of cases was 6500 (95% CI 5613–7370) in Nokia, compared to the control population. The gastroenteritis attack rate was eightfold higher in the polluted area (53.0%) and 2.4- fold higher in the unpolluted area (15.6%) compared to the control municipality (6.5%). The median duration of gastroenteritis was 3 days for cases in the polluted area, 2 days in the unpolluted area and 2 days in the control population (P<0.001). The probability of falling ill increased along with the quantity of tap water consumed in the contaminated area, but not in the uncontaminated area or in the control population.
Microbiological findings
Seven enteropathogens were detected in faecal specimens (Table 3). The most frequently encountered pathogen was Campylobacter sp., with C. jejuni representing most of them, followed by Giardia. Five random samples investigated for norovirus were collected on 2 December 2007 and all were positive. Other findings included non typhoidal salmonellas, Clostridium difficile and rotavirus. Four stool cultures grew Shigella boydii. In ten persons, two different bacterial pathogens were found in faecal specimens. Except for Shigella boydii, the same pathogens also were detected in water-distribution system samples. Ninety-eight percent (158/162) of Campylobacter- and Salmonella-positive cultures occurred within the first 3 weeks of the outbreak.
DISCUSSION
This paper describes the largest reported waterborne outbreak in Finland to date. In the town of Nokia, over 8000 residents (28% of the population) were estimated to have fallen ill with gastroenteritis, 6500 of them as a consequence of water contamination. Over half of the population in the contaminated area suffered from gastroenteritis.
Although faecal contamination is a known cause of waterborne epidemics [8, 23–26], the chain of events in Nokia was exceptional. In most circumstances, faecal contamination occurs when small amounts of sewage are mixed with clean water as a result of an obstructed or broken waste-water line or drainage from surface sources, e.g. after heavy rainfall. However, in the present case, sewage had direct entry to the distribution line through an inappropriate crossconnection. As a result, the magnitude of contamination was substantial. The purpose and origin of the inappropriate valve was not ascertained in the investigation. It was built some decades ago, and was not prohibited during that time. After the outbreak, the national authorities required waterworks across the country to rule out the presence of similar cross-connection constructions. Contacts with healthcare are often used to quantify the burden of illness in outbreak investigations. However, experience from previous outbreaks indicates that a large proportion of affected subjects have a selflimiting disease and do not contact healthcare providers [17]. Thus, if only health centre visitors’ data are used, the burden of disease would be underestimated. In this study, a population-based survey was employed to avoid this problem. Since gastroenteritis is a common disease, a survey without proper controls might overestimate the magnitude of an outbreak if assessment of background morbidity is not done [27]. To obviate this challenge, we used a control population chosen from the same region but on the opposite side of the Tampere city area. Daily contacts between these communities are likely to be minimal, so that baseline morbidity could be estimated from the control population, lending accuracy to conclusions regarding the morbidity from the water system contamination.
The gastroenteritis attack rate was high in the contaminated area, where over half of the population fell ill. This probably reflects the high concentration of pathogens in the drinking water as well as their infectivity, especially that of viruses [20]. An even higher attack rate has been reported during a large waterborne norovirus outbreak in Finland [14]. In that case, however, the epidemic lasted longer and was investigated using an internet survey, making selection bias more likely. There was also excess morbidity among residents in the uncontaminated area of the town. This can be explained by exposure to polluted water upon visiting the affected area. Another likely explanation is person-to-person transmission of certain pathogens, especially viruses. The incidence in the uncontaminated area increased later and less sharply than in the contaminated area, which would support the hypothesis of secondary spread. Furthermore, what is referred to here as the affected area is an estimate, which was grounded on a thorough judgement of available network and microbiological data. However, it may not be completely precise.
The duration of illness was longer in those living in the affected area. Bacterial infections were possibly more common in residents in the contaminated area, while in the uncontaminated area secondary spread of viral infections may have been more common. Another reason may be the quantity of exposure. Those living in the affected area were probably more extensively exposed to contaminated water than others, who perhaps ingested contaminated water only occasionally. Furthermore, those most intensively exposed may have been infected with several pathogens, either simultaneously or consecutively. The frequency of previous episodes of gastroenteritis during the 10 months preceding the outbreak was 17% in the whole study population without differences between study groups. This is in good agreement with the yearly incidence of gastroenteritis (19.4/100 person-years) in a British cohort [28]. The survey was conducted as soon as possible after the outbreak. Since the outbreak took place shortly before the Christmas and New Year holiday season, it still took over 2 months before the survey could be launched. This conceivably resulted in some degree of recall bias. In addition, this outbreak gained wide coverage in the public media, which may have caused over-reporting of symptoms. The response rate of those who fell ill during the outbreak may have been higher, which might lead to overestimation of the disease burden. Nevertheless, the overall response rates in all study areas were relatively good, making significant bias unlikely. The spectrum of pathogens in both water and patient samples was wide, consistent with the mechanism of contamination [24, 25]. This distinguishes the outbreak from most Finnish waterborne epidemics, where usually only Campylobacter and/or norovirus are seen. Although samples were not systematically studied in all symptomatic patients, almost 200 Campylobacter-associated cases were confirmed in this outbreak, which is about 200 times more than the mean monthly number (n=1) for Campylobacter cases during the 24 pre-outbreak months in Nokia (National Infectious Disease Register).
Only a few samples were collected to detect norovirus in the early days of the epidemic, but all (5/5) were positive. Despite the small number of confirmed cases, we regard norovirus as a major pathogen in this outbreak. A number of faecal specimens collected for other purposes were later analysed for enteric viruses, and almost a third of them contained norovirus [20]. Astroviruses, adenoviruses, rotaviruses and enteroviruses were also detected. In another study connected to this outbreak and covering children attending the University Hospital, 40% (20/50) of samples yielded norovirus. In the same study, a variety of other viral pathogens were also detected, and a notable proportion of patients yielded mixed viral findings in their stools [29]. In addition to Campylobacter and norovirus, Giardia was regarded as a major pathogen. Although Giardia has been isolated from natural sources in Finland [30], domestic infections are uncommon. Drinking-water contamination was considered to be the obvious source of this outbreak, and this study provides strong evidence to support the assumption. Most subjects fell ill shortly after the valve was opened and microbes found in stools were in accord with findings in pipeline samples.
Finally, the incidence of the cases was highest in the contaminated area. Regardless of the severity of the event, the clinical consequences were milder than feared. Most of those afflicted in Nokia suffered self-limiting gastroenteritis and no permanent damage to their health is anticipated. To our knowledge, there were no obvious fatal outcomes. However, Contributory influences in the case of deaths mainly for other reasons cannot be excluded. If waterborne outbreaks cause fatalities, these are most often caused by STEC O157:H7. Although O157:H7 was present in Finland during the 1990s, it is nowadays uncommon [31] and was not detected in this outbreak. Hepatitis A virus (HAV) is another severe infection associated with waterborne exposure. HAV is highly uncommon in the Finnish population, but sporadic cases do occur. HAV was not found in water samples during this outbreak, and no hepatitis cases were detected. Clostridium difficile was identified, but the hypervirulent ribotype O27 was absent (S. Kotila et al., unpublished observation). This study demonstrates the value of a populationbased controlled survey in estimating disease burden in outbreaks when illnesses are mild or moderate. In this particular case, the survey showed that the actual number of those affected was almost seven times higher than the number of people seeking medical care. Furthermore, the use of a control population affords greater accuracy when assessing excess morbidity connected to an outbreak.
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How to prevent this?
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401
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An extensive gastroenteritis outbreak after drinking-water contamination by sewage effluent, Finland
|
SUMMARY
An inappropriate cross-connection between sewage- and drinking-water pipelines contaminated tap water in a Finnish town, resulting in an extensive waterborne gastroenteritis outbreak in this developed country. According to a database and a line-list, altogether 1222 subjects sought medical care as a result of this exposure. Seven pathogens were found in patient samples of those who sought treatment. To establish the true disease burden from this exposure, we undertook a population-based questionnaire investigation with a control population, infrequently used to study waterborne outbreaks. The study covered three areas, contaminated and uncontaminated parts of the town and a control town. An estimated 8453 residents fell ill during the outbreak, the excess number of illnesses being 6501. Attack rates were 53% [95% confidence interval (CI) 49.5–56.4] in the contaminated area, 15.6% (95% CI 13.1–18.5) in the uncontaminated area and 6.5% (95% CI 4.8–8.8) in the control population. Using a control population allowed us to differentiate baseline morbidity from the observed morbidity caused by the water contamination, thus enabling a more accurate estimate of the disease burden of this outbreak.
Key words: Gastroenteritis, outbreak, waterborne infections.
INTRODUCTION
Safe water is essential to life. Globally, the lack of clean water is estimated to cause about 4% of total mortality [1], predominantly in the developing world. However, waterborne outbreaks also remain a source of concern in developed countries. In the USA, 20 million people are estimated to contract a waterborne infection annually [2]. Over 4 00 000 people fell ill with cryptosporidiosis in Milwaukee in 1993 as a consequence of inadequate water filtration [3]. In Canada, the Walkerton outbreak resulted in seven fatalities attributable to Escherichia coli O157:H7 infection [4]. Waterborne outbreaks have been reported from many European countries including England and Wales [5], Norway [6], Sweden [7–10], Italy, Spain and Switzerland [11].
Finland is a large country (3 38 424 km2) with a small population (5.4 million). The number of waterworks is high (almost 2000) and most serve small communities. The majority (95%) use groundwater. However, almost half of the population are served by surface-water plants of the largest cities. Groundwater is usually not disinfected for use, while surface water is filtered and disinfected before distribution to consumers [12]. Although drinking water is generally considered safe in Finland, a number of waterborne outbreaks have occurred. Twenty-four outbreaks affecting 7700 inhabitants were spontaneously reported during the period 1980–1992 [13]; since 1997 reporting of outbreaks has been mandatory, and 14 outbreaks affecting 7300 people were registered during the first 2 years [12]. The most widespread outbreak to date affected 5500 people [14], and a number of other substantial waterborne outbreaks have been reported [15–17]. The most common microbes have been norovirus [18] and Campylobacter [19].
We report the investigation of a serious waterborne gastroenteritis outbreak that occurred in a Finnish town in autumn 2007.
Setting
Nokia (population 30 016) is a town located in southern Finland. Over 90% of the population are served by the municipal drinking-water supply. The average daily water consumption in Nokia is 4250 m3 (year 2006). The waterworks uses groundwater, which is treated with sodium hypochlorite for disinfection and pH adjustment. Sewage is processed in one municipal waste water plant, which covers 86% of the population. The town has a municipal health centre for primary care and a hospital for primary in-patient care and limited secondary care. Secondary and tertiary care are provided by Tampere University Hospital 20 km distant from Nokia. The Centre for Laboratory Medicine provides the municipalities with clinical laboratory services. Environmental health surveillance and monitoring of water quality is organized in collaboration with five neighbouring municipalities.
The outbreak
On Wednesday, 28 November 2007, maintenance work was conducted at the waste water plant in Nokia. The same day, technical problems were encountered in the municipal drinking-water system, and during the days following, customers complained of a bad smell and taste in their tap water. These were first misinterpreted as being caused by harmless deposits loosened from the inner walls of the pipeline and discharged during the maintenance work on 28 November. From 30 November, environmental health and municipal waterworks officials received reports of bouts of diarrhoea and vomiting among residents, and during that same day an increase in the number of patients with gastroenteritis was observed at the health centre. Extensive water and pipeline sampling was initiated and a boil-water notice issued, and water chlorine concentration was increased. The water contamination and its source became evident the same day. A valve in the waste-water plant connecting the drinking-water line and a waste-water effluent line had been opened during the maintenance work and inadvertently left open for 2 days. As a consequence, the drinking-water network became contaminated with sewage effluent. According to flow data, an estimated 450 m3 of waste water was mixed with the drinking water and distributed to customers, heavily contaminating household water and pipelines in southern parts of the town. The population in this area numbered over 9500.
Distribution of clean water was organized by municipal officials, the military and volunteers, and the health centre focused on treating outbreak patients, cutting back on other activities. The University Hospital prepared to open a temporary ward, which was in fact eventually not needed. Schools and daycare centres were closed from 6 to 9 December owing to teachers’ and children’s sick leave and the need to reduce the possible horizontal spread of gastrointestinal viruses. After 12 days, the boil-water notice was withdrawn in the uncontaminated area. In the contaminated area, adenoviruses and noroviruses were repeatedly detected in water-supply samples despite chlorination and pipeline flushing. Finally, after extensive decontamination procedures including shock chlorination, precautionary restrictions were discontinued in the contaminated area on 18 February 2008, almost 3 months after the incident.
We conducted a comprehensive investigation to determine the magnitude and consequences of the waterborne outbreak including describing the clinical aspects, reviewing microbiological findings and estimating the disease burden using a population-based survey.
METHODS
Descriptive epidemiology
Patients visiting the municipal health centre between 28 November and 31 December 2007 because of acute gastroenteritis were registered on a line-list, including name, age, gender, address and personal identity number. The timing and range of symptoms were recorded. Prospective line-listing was initiated 6 days after the beginning of the contamination visits prior to that being added retrospectively from patient charts. The information collected from the patient register included the number of primary-care visits to the centre because of gastroenteritis as defined by the international classification of primary care (ICPC-1, WHO, Geneva) codes D01, D09, D10, D11, D25, D70, D73 or D96. The information on reported symptoms was collected from the line-list. Daily admissions to Tampere University Hospital were counted by emergency-department personnel.
Analytical epidemiology
The town was divided into two study areas, contaminated (population 9538) and uncontaminated (population 20 478). The division was based on microbiological data from distribution system samples and technical modelling of water-flow directions within the network. Another municipality (population 27 259) 35 km distant from Nokia was chosen as a control population. One thousand persons for each study group were randomly selected from the population register. Groups were matched by age and gender and all age groups were included. Only one participant per household was included. The survey was conducted using postal questionnaires, the form being posted 8 weeks after the contamination had occurred. A repeat questionnaire was sent out 3 weeks later to non-responders.
Participants were asked about the timing and nature of gastrointestinal symptoms. A case was defined as a person suffering acute diarrhoea (o3 loose stools/day) or vomiting between 28 November 2007 and 20 January 2008. The occurrence of gastroenteritis during a 10-month period prior to the outbreak was also enquired about to ensure the comparability of the study groups.
Microbiological investigation
Faecal specimens for bacterial pathogens were not investigated from all the patients or from representative samples from consecutive cases. Instead, bacterial samples were available from cases with more severe symptoms. Specimens for detection of norovirus were collected from five randomly selected patients within the first week of the outbreak. Other viral pathogens were studied in samples from children admitted to the emergency department of the University Hospital. In order to detect enteric parasites specimens were taken from ten consecutive patients during the second week of the outbreak. Subsequently, when cases with giardiasis were detected, parasites were investigated in all symptomatic patients. Specimens were taken at the health centre or in the University Hospital.
Stool specimens were cultured for the presence of Campylobacter, Salmonella, Yersinia and Shigella spp. Norovirus was investigated using electron microscopy and PCR; rotavirus antigens were detected by enzyme immunological assay (EIA). Protozoan pathogens were investigated using microscopy of formalin-fixed samples. Subsequently, because no other protozoan pathogens were identified, only antigen detection of Giardia was used instead of microscopy. If a patient reported bloody stools, Shiga toxin producing Escherichia coli (STEC) was examined for by culture and toxin detection (EIA). Only samples taken between 28 November and 31 December 2007 were included. For Giardia the time-period for inclusion was extended to 31 May 2008 (6 months) because of the longer incubation time and gradual case detection.
The methods used in water and pipeline analysis will be described in detail in a forthcoming paper. Briefly, samples from the drinking-water distribution system were analysed by cultivation for the presence of Campylobacter, Salmonella and Clostridium spp. PCR was utilized to analyse enteric viruses as previously described by Maunula and colleagues [20]. The presence of Giardia cysts and Cryptosporidium oocysts was detected using concentration, immunomagnetic separation and epifluorescence microscopy as described in detail by Rimhanen-Finne and colleagues [21].
The study areas were compared using x2 or Kruskal– Wallis rank sum tests whenever appropriate. Attack rates were calculated as percentage of cases in a sample population. Total numbers of cases were calculated by extrapolating the age- and sex-adjusted attack rates to the total population of each area. To assess baseline rates of gastroenteritis, the attack rates from the control population were used to estimate the number of cases in the contaminated population that would have occurred in the absence of the water contamination. Estimations were made adjusting for age and sex. Confidence intervals for the total numbers were obtained using bootstrap methods [21]. All statistical analyses were performed using R version 2.9 (or later versions) [22].
RESULTS
Healthcare visits
According to the health centre database, 1222 visits due to gastrointestinal symptoms were made between 28 November and 31 December 2007. During the busiest week, the number of patients presenting with gastrointestinal complaints was 53-fold higher than expected compared to the weekly median number of cases in a 7-month period preceding the outbreak. Children aged <10 years constituted the largest group (Figs 1, 2).
Line-list data were available on 1024 patients. Only one visit per person was included. The most common symptoms reported were diarrhoea (n=813, 79%), vomiting (n=601, 59%), fever (n=337, 33%) and abdominal pain (n=291, 28%). Altogether 204 visits were made to emergency rooms in Tampere University Hospital, 145 (71%) of them involving children. To our knowledge, the outbreak did not lead directly to fatalities.
Questionnaire study
A total of 2154 participants returned the questionnaire. Thirty-one were excluded as being inadequately completed or because the respondent could not be identified; 2123 forms were finally accepted for the analysis (response rate 71%). The response rate was highest in the contaminated area and lowest in the control population. The background characteristics of the study groups showed no significant demographic differences between the study populations (Table 1). Altogether, 17% of the responders reported having gastroenteritis during the 10 months prior to the outbreak. Most subjects fell ill within 1 week of the contamination of the water supply (Fig. 3).
The most common symptoms were diarrhoea, vomiting, nausea and abdominal pain (Fig. 4). Overall, 579 respondents met the case definition, of whom 540 (93%) were residents of Nokia. Extrapolating the result for the whole population, an estimated 8453 (95% CI 7568–9363) residents fell ill with gastroenteritis in the whole of Nokia (Table 2). The excess number of cases was 6500 (95% CI 5613–7370) in Nokia, compared to the control population. The gastroenteritis attack rate was eightfold higher in the polluted area (53.0%) and 2.4- fold higher in the unpolluted area (15.6%) compared to the control municipality (6.5%). The median duration of gastroenteritis was 3 days for cases in the polluted area, 2 days in the unpolluted area and 2 days in the control population (P<0.001). The probability of falling ill increased along with the quantity of tap water consumed in the contaminated area, but not in the uncontaminated area or in the control population.
Microbiological findings
Seven enteropathogens were detected in faecal specimens (Table 3). The most frequently encountered pathogen was Campylobacter sp., with C. jejuni representing most of them, followed by Giardia. Five random samples investigated for norovirus were collected on 2 December 2007 and all were positive. Other findings included non typhoidal salmonellas, Clostridium difficile and rotavirus. Four stool cultures grew Shigella boydii. In ten persons, two different bacterial pathogens were found in faecal specimens. Except for Shigella boydii, the same pathogens also were detected in water-distribution system samples. Ninety-eight percent (158/162) of Campylobacter- and Salmonella-positive cultures occurred within the first 3 weeks of the outbreak.
DISCUSSION
This paper describes the largest reported waterborne outbreak in Finland to date. In the town of Nokia, over 8000 residents (28% of the population) were estimated to have fallen ill with gastroenteritis, 6500 of them as a consequence of water contamination. Over half of the population in the contaminated area suffered from gastroenteritis.
Although faecal contamination is a known cause of waterborne epidemics [8, 23–26], the chain of events in Nokia was exceptional. In most circumstances, faecal contamination occurs when small amounts of sewage are mixed with clean water as a result of an obstructed or broken waste-water line or drainage from surface sources, e.g. after heavy rainfall. However, in the present case, sewage had direct entry to the distribution line through an inappropriate crossconnection. As a result, the magnitude of contamination was substantial. The purpose and origin of the inappropriate valve was not ascertained in the investigation. It was built some decades ago, and was not prohibited during that time. After the outbreak, the national authorities required waterworks across the country to rule out the presence of similar cross-connection constructions. Contacts with healthcare are often used to quantify the burden of illness in outbreak investigations. However, experience from previous outbreaks indicates that a large proportion of affected subjects have a selflimiting disease and do not contact healthcare providers [17]. Thus, if only health centre visitors’ data are used, the burden of disease would be underestimated. In this study, a population-based survey was employed to avoid this problem. Since gastroenteritis is a common disease, a survey without proper controls might overestimate the magnitude of an outbreak if assessment of background morbidity is not done [27]. To obviate this challenge, we used a control population chosen from the same region but on the opposite side of the Tampere city area. Daily contacts between these communities are likely to be minimal, so that baseline morbidity could be estimated from the control population, lending accuracy to conclusions regarding the morbidity from the water system contamination.
The gastroenteritis attack rate was high in the contaminated area, where over half of the population fell ill. This probably reflects the high concentration of pathogens in the drinking water as well as their infectivity, especially that of viruses [20]. An even higher attack rate has been reported during a large waterborne norovirus outbreak in Finland [14]. In that case, however, the epidemic lasted longer and was investigated using an internet survey, making selection bias more likely. There was also excess morbidity among residents in the uncontaminated area of the town. This can be explained by exposure to polluted water upon visiting the affected area. Another likely explanation is person-to-person transmission of certain pathogens, especially viruses. The incidence in the uncontaminated area increased later and less sharply than in the contaminated area, which would support the hypothesis of secondary spread. Furthermore, what is referred to here as the affected area is an estimate, which was grounded on a thorough judgement of available network and microbiological data. However, it may not be completely precise.
The duration of illness was longer in those living in the affected area. Bacterial infections were possibly more common in residents in the contaminated area, while in the uncontaminated area secondary spread of viral infections may have been more common. Another reason may be the quantity of exposure. Those living in the affected area were probably more extensively exposed to contaminated water than others, who perhaps ingested contaminated water only occasionally. Furthermore, those most intensively exposed may have been infected with several pathogens, either simultaneously or consecutively. The frequency of previous episodes of gastroenteritis during the 10 months preceding the outbreak was 17% in the whole study population without differences between study groups. This is in good agreement with the yearly incidence of gastroenteritis (19.4/100 person-years) in a British cohort [28]. The survey was conducted as soon as possible after the outbreak. Since the outbreak took place shortly before the Christmas and New Year holiday season, it still took over 2 months before the survey could be launched. This conceivably resulted in some degree of recall bias. In addition, this outbreak gained wide coverage in the public media, which may have caused over-reporting of symptoms. The response rate of those who fell ill during the outbreak may have been higher, which might lead to overestimation of the disease burden. Nevertheless, the overall response rates in all study areas were relatively good, making significant bias unlikely. The spectrum of pathogens in both water and patient samples was wide, consistent with the mechanism of contamination [24, 25]. This distinguishes the outbreak from most Finnish waterborne epidemics, where usually only Campylobacter and/or norovirus are seen. Although samples were not systematically studied in all symptomatic patients, almost 200 Campylobacter-associated cases were confirmed in this outbreak, which is about 200 times more than the mean monthly number (n=1) for Campylobacter cases during the 24 pre-outbreak months in Nokia (National Infectious Disease Register).
Only a few samples were collected to detect norovirus in the early days of the epidemic, but all (5/5) were positive. Despite the small number of confirmed cases, we regard norovirus as a major pathogen in this outbreak. A number of faecal specimens collected for other purposes were later analysed for enteric viruses, and almost a third of them contained norovirus [20]. Astroviruses, adenoviruses, rotaviruses and enteroviruses were also detected. In another study connected to this outbreak and covering children attending the University Hospital, 40% (20/50) of samples yielded norovirus. In the same study, a variety of other viral pathogens were also detected, and a notable proportion of patients yielded mixed viral findings in their stools [29]. In addition to Campylobacter and norovirus, Giardia was regarded as a major pathogen. Although Giardia has been isolated from natural sources in Finland [30], domestic infections are uncommon. Drinking-water contamination was considered to be the obvious source of this outbreak, and this study provides strong evidence to support the assumption. Most subjects fell ill shortly after the valve was opened and microbes found in stools were in accord with findings in pipeline samples.
Finally, the incidence of the cases was highest in the contaminated area. Regardless of the severity of the event, the clinical consequences were milder than feared. Most of those afflicted in Nokia suffered self-limiting gastroenteritis and no permanent damage to their health is anticipated. To our knowledge, there were no obvious fatal outcomes. However, Contributory influences in the case of deaths mainly for other reasons cannot be excluded. If waterborne outbreaks cause fatalities, these are most often caused by STEC O157:H7. Although O157:H7 was present in Finland during the 1990s, it is nowadays uncommon [31] and was not detected in this outbreak. Hepatitis A virus (HAV) is another severe infection associated with waterborne exposure. HAV is highly uncommon in the Finnish population, but sporadic cases do occur. HAV was not found in water samples during this outbreak, and no hepatitis cases were detected. Clostridium difficile was identified, but the hypervirulent ribotype O27 was absent (S. Kotila et al., unpublished observation). This study demonstrates the value of a populationbased controlled survey in estimating disease burden in outbreaks when illnesses are mild or moderate. In this particular case, the survey showed that the actual number of those affected was almost seven times higher than the number of people seeking medical care. Furthermore, the use of a control population affords greater accuracy when assessing excess morbidity connected to an outbreak.
|
What were the investigation steps?
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{
"answer_start": [],
"text": []
}
|
402
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An extensive gastroenteritis outbreak after drinking-water contamination by sewage effluent, Finland
|
SUMMARY
An inappropriate cross-connection between sewage- and drinking-water pipelines contaminated tap water in a Finnish town, resulting in an extensive waterborne gastroenteritis outbreak in this developed country. According to a database and a line-list, altogether 1222 subjects sought medical care as a result of this exposure. Seven pathogens were found in patient samples of those who sought treatment. To establish the true disease burden from this exposure, we undertook a population-based questionnaire investigation with a control population, infrequently used to study waterborne outbreaks. The study covered three areas, contaminated and uncontaminated parts of the town and a control town. An estimated 8453 residents fell ill during the outbreak, the excess number of illnesses being 6501. Attack rates were 53% [95% confidence interval (CI) 49.5–56.4] in the contaminated area, 15.6% (95% CI 13.1–18.5) in the uncontaminated area and 6.5% (95% CI 4.8–8.8) in the control population. Using a control population allowed us to differentiate baseline morbidity from the observed morbidity caused by the water contamination, thus enabling a more accurate estimate of the disease burden of this outbreak.
Key words: Gastroenteritis, outbreak, waterborne infections.
INTRODUCTION
Safe water is essential to life. Globally, the lack of clean water is estimated to cause about 4% of total mortality [1], predominantly in the developing world. However, waterborne outbreaks also remain a source of concern in developed countries. In the USA, 20 million people are estimated to contract a waterborne infection annually [2]. Over 4 00 000 people fell ill with cryptosporidiosis in Milwaukee in 1993 as a consequence of inadequate water filtration [3]. In Canada, the Walkerton outbreak resulted in seven fatalities attributable to Escherichia coli O157:H7 infection [4]. Waterborne outbreaks have been reported from many European countries including England and Wales [5], Norway [6], Sweden [7–10], Italy, Spain and Switzerland [11].
Finland is a large country (3 38 424 km2) with a small population (5.4 million). The number of waterworks is high (almost 2000) and most serve small communities. The majority (95%) use groundwater. However, almost half of the population are served by surface-water plants of the largest cities. Groundwater is usually not disinfected for use, while surface water is filtered and disinfected before distribution to consumers [12]. Although drinking water is generally considered safe in Finland, a number of waterborne outbreaks have occurred. Twenty-four outbreaks affecting 7700 inhabitants were spontaneously reported during the period 1980–1992 [13]; since 1997 reporting of outbreaks has been mandatory, and 14 outbreaks affecting 7300 people were registered during the first 2 years [12]. The most widespread outbreak to date affected 5500 people [14], and a number of other substantial waterborne outbreaks have been reported [15–17]. The most common microbes have been norovirus [18] and Campylobacter [19].
We report the investigation of a serious waterborne gastroenteritis outbreak that occurred in a Finnish town in autumn 2007.
Setting
Nokia (population 30 016) is a town located in southern Finland. Over 90% of the population are served by the municipal drinking-water supply. The average daily water consumption in Nokia is 4250 m3 (year 2006). The waterworks uses groundwater, which is treated with sodium hypochlorite for disinfection and pH adjustment. Sewage is processed in one municipal waste water plant, which covers 86% of the population. The town has a municipal health centre for primary care and a hospital for primary in-patient care and limited secondary care. Secondary and tertiary care are provided by Tampere University Hospital 20 km distant from Nokia. The Centre for Laboratory Medicine provides the municipalities with clinical laboratory services. Environmental health surveillance and monitoring of water quality is organized in collaboration with five neighbouring municipalities.
The outbreak
On Wednesday, 28 November 2007, maintenance work was conducted at the waste water plant in Nokia. The same day, technical problems were encountered in the municipal drinking-water system, and during the days following, customers complained of a bad smell and taste in their tap water. These were first misinterpreted as being caused by harmless deposits loosened from the inner walls of the pipeline and discharged during the maintenance work on 28 November. From 30 November, environmental health and municipal waterworks officials received reports of bouts of diarrhoea and vomiting among residents, and during that same day an increase in the number of patients with gastroenteritis was observed at the health centre. Extensive water and pipeline sampling was initiated and a boil-water notice issued, and water chlorine concentration was increased. The water contamination and its source became evident the same day. A valve in the waste-water plant connecting the drinking-water line and a waste-water effluent line had been opened during the maintenance work and inadvertently left open for 2 days. As a consequence, the drinking-water network became contaminated with sewage effluent. According to flow data, an estimated 450 m3 of waste water was mixed with the drinking water and distributed to customers, heavily contaminating household water and pipelines in southern parts of the town. The population in this area numbered over 9500.
Distribution of clean water was organized by municipal officials, the military and volunteers, and the health centre focused on treating outbreak patients, cutting back on other activities. The University Hospital prepared to open a temporary ward, which was in fact eventually not needed. Schools and daycare centres were closed from 6 to 9 December owing to teachers’ and children’s sick leave and the need to reduce the possible horizontal spread of gastrointestinal viruses. After 12 days, the boil-water notice was withdrawn in the uncontaminated area. In the contaminated area, adenoviruses and noroviruses were repeatedly detected in water-supply samples despite chlorination and pipeline flushing. Finally, after extensive decontamination procedures including shock chlorination, precautionary restrictions were discontinued in the contaminated area on 18 February 2008, almost 3 months after the incident.
We conducted a comprehensive investigation to determine the magnitude and consequences of the waterborne outbreak including describing the clinical aspects, reviewing microbiological findings and estimating the disease burden using a population-based survey.
METHODS
Descriptive epidemiology
Patients visiting the municipal health centre between 28 November and 31 December 2007 because of acute gastroenteritis were registered on a line-list, including name, age, gender, address and personal identity number. The timing and range of symptoms were recorded. Prospective line-listing was initiated 6 days after the beginning of the contamination visits prior to that being added retrospectively from patient charts. The information collected from the patient register included the number of primary-care visits to the centre because of gastroenteritis as defined by the international classification of primary care (ICPC-1, WHO, Geneva) codes D01, D09, D10, D11, D25, D70, D73 or D96. The information on reported symptoms was collected from the line-list. Daily admissions to Tampere University Hospital were counted by emergency-department personnel.
Analytical epidemiology
The town was divided into two study areas, contaminated (population 9538) and uncontaminated (population 20 478). The division was based on microbiological data from distribution system samples and technical modelling of water-flow directions within the network. Another municipality (population 27 259) 35 km distant from Nokia was chosen as a control population. One thousand persons for each study group were randomly selected from the population register. Groups were matched by age and gender and all age groups were included. Only one participant per household was included. The survey was conducted using postal questionnaires, the form being posted 8 weeks after the contamination had occurred. A repeat questionnaire was sent out 3 weeks later to non-responders.
Participants were asked about the timing and nature of gastrointestinal symptoms. A case was defined as a person suffering acute diarrhoea (o3 loose stools/day) or vomiting between 28 November 2007 and 20 January 2008. The occurrence of gastroenteritis during a 10-month period prior to the outbreak was also enquired about to ensure the comparability of the study groups.
Microbiological investigation
Faecal specimens for bacterial pathogens were not investigated from all the patients or from representative samples from consecutive cases. Instead, bacterial samples were available from cases with more severe symptoms. Specimens for detection of norovirus were collected from five randomly selected patients within the first week of the outbreak. Other viral pathogens were studied in samples from children admitted to the emergency department of the University Hospital. In order to detect enteric parasites specimens were taken from ten consecutive patients during the second week of the outbreak. Subsequently, when cases with giardiasis were detected, parasites were investigated in all symptomatic patients. Specimens were taken at the health centre or in the University Hospital.
Stool specimens were cultured for the presence of Campylobacter, Salmonella, Yersinia and Shigella spp. Norovirus was investigated using electron microscopy and PCR; rotavirus antigens were detected by enzyme immunological assay (EIA). Protozoan pathogens were investigated using microscopy of formalin-fixed samples. Subsequently, because no other protozoan pathogens were identified, only antigen detection of Giardia was used instead of microscopy. If a patient reported bloody stools, Shiga toxin producing Escherichia coli (STEC) was examined for by culture and toxin detection (EIA). Only samples taken between 28 November and 31 December 2007 were included. For Giardia the time-period for inclusion was extended to 31 May 2008 (6 months) because of the longer incubation time and gradual case detection.
The methods used in water and pipeline analysis will be described in detail in a forthcoming paper. Briefly, samples from the drinking-water distribution system were analysed by cultivation for the presence of Campylobacter, Salmonella and Clostridium spp. PCR was utilized to analyse enteric viruses as previously described by Maunula and colleagues [20]. The presence of Giardia cysts and Cryptosporidium oocysts was detected using concentration, immunomagnetic separation and epifluorescence microscopy as described in detail by Rimhanen-Finne and colleagues [21].
The study areas were compared using x2 or Kruskal– Wallis rank sum tests whenever appropriate. Attack rates were calculated as percentage of cases in a sample population. Total numbers of cases were calculated by extrapolating the age- and sex-adjusted attack rates to the total population of each area. To assess baseline rates of gastroenteritis, the attack rates from the control population were used to estimate the number of cases in the contaminated population that would have occurred in the absence of the water contamination. Estimations were made adjusting for age and sex. Confidence intervals for the total numbers were obtained using bootstrap methods [21]. All statistical analyses were performed using R version 2.9 (or later versions) [22].
RESULTS
Healthcare visits
According to the health centre database, 1222 visits due to gastrointestinal symptoms were made between 28 November and 31 December 2007. During the busiest week, the number of patients presenting with gastrointestinal complaints was 53-fold higher than expected compared to the weekly median number of cases in a 7-month period preceding the outbreak. Children aged <10 years constituted the largest group (Figs 1, 2).
Line-list data were available on 1024 patients. Only one visit per person was included. The most common symptoms reported were diarrhoea (n=813, 79%), vomiting (n=601, 59%), fever (n=337, 33%) and abdominal pain (n=291, 28%). Altogether 204 visits were made to emergency rooms in Tampere University Hospital, 145 (71%) of them involving children. To our knowledge, the outbreak did not lead directly to fatalities.
Questionnaire study
A total of 2154 participants returned the questionnaire. Thirty-one were excluded as being inadequately completed or because the respondent could not be identified; 2123 forms were finally accepted for the analysis (response rate 71%). The response rate was highest in the contaminated area and lowest in the control population. The background characteristics of the study groups showed no significant demographic differences between the study populations (Table 1). Altogether, 17% of the responders reported having gastroenteritis during the 10 months prior to the outbreak. Most subjects fell ill within 1 week of the contamination of the water supply (Fig. 3).
The most common symptoms were diarrhoea, vomiting, nausea and abdominal pain (Fig. 4). Overall, 579 respondents met the case definition, of whom 540 (93%) were residents of Nokia. Extrapolating the result for the whole population, an estimated 8453 (95% CI 7568–9363) residents fell ill with gastroenteritis in the whole of Nokia (Table 2). The excess number of cases was 6500 (95% CI 5613–7370) in Nokia, compared to the control population. The gastroenteritis attack rate was eightfold higher in the polluted area (53.0%) and 2.4- fold higher in the unpolluted area (15.6%) compared to the control municipality (6.5%). The median duration of gastroenteritis was 3 days for cases in the polluted area, 2 days in the unpolluted area and 2 days in the control population (P<0.001). The probability of falling ill increased along with the quantity of tap water consumed in the contaminated area, but not in the uncontaminated area or in the control population.
Microbiological findings
Seven enteropathogens were detected in faecal specimens (Table 3). The most frequently encountered pathogen was Campylobacter sp., with C. jejuni representing most of them, followed by Giardia. Five random samples investigated for norovirus were collected on 2 December 2007 and all were positive. Other findings included non typhoidal salmonellas, Clostridium difficile and rotavirus. Four stool cultures grew Shigella boydii. In ten persons, two different bacterial pathogens were found in faecal specimens. Except for Shigella boydii, the same pathogens also were detected in water-distribution system samples. Ninety-eight percent (158/162) of Campylobacter- and Salmonella-positive cultures occurred within the first 3 weeks of the outbreak.
DISCUSSION
This paper describes the largest reported waterborne outbreak in Finland to date. In the town of Nokia, over 8000 residents (28% of the population) were estimated to have fallen ill with gastroenteritis, 6500 of them as a consequence of water contamination. Over half of the population in the contaminated area suffered from gastroenteritis.
Although faecal contamination is a known cause of waterborne epidemics [8, 23–26], the chain of events in Nokia was exceptional. In most circumstances, faecal contamination occurs when small amounts of sewage are mixed with clean water as a result of an obstructed or broken waste-water line or drainage from surface sources, e.g. after heavy rainfall. However, in the present case, sewage had direct entry to the distribution line through an inappropriate crossconnection. As a result, the magnitude of contamination was substantial. The purpose and origin of the inappropriate valve was not ascertained in the investigation. It was built some decades ago, and was not prohibited during that time. After the outbreak, the national authorities required waterworks across the country to rule out the presence of similar cross-connection constructions. Contacts with healthcare are often used to quantify the burden of illness in outbreak investigations. However, experience from previous outbreaks indicates that a large proportion of affected subjects have a selflimiting disease and do not contact healthcare providers [17]. Thus, if only health centre visitors’ data are used, the burden of disease would be underestimated. In this study, a population-based survey was employed to avoid this problem. Since gastroenteritis is a common disease, a survey without proper controls might overestimate the magnitude of an outbreak if assessment of background morbidity is not done [27]. To obviate this challenge, we used a control population chosen from the same region but on the opposite side of the Tampere city area. Daily contacts between these communities are likely to be minimal, so that baseline morbidity could be estimated from the control population, lending accuracy to conclusions regarding the morbidity from the water system contamination.
The gastroenteritis attack rate was high in the contaminated area, where over half of the population fell ill. This probably reflects the high concentration of pathogens in the drinking water as well as their infectivity, especially that of viruses [20]. An even higher attack rate has been reported during a large waterborne norovirus outbreak in Finland [14]. In that case, however, the epidemic lasted longer and was investigated using an internet survey, making selection bias more likely. There was also excess morbidity among residents in the uncontaminated area of the town. This can be explained by exposure to polluted water upon visiting the affected area. Another likely explanation is person-to-person transmission of certain pathogens, especially viruses. The incidence in the uncontaminated area increased later and less sharply than in the contaminated area, which would support the hypothesis of secondary spread. Furthermore, what is referred to here as the affected area is an estimate, which was grounded on a thorough judgement of available network and microbiological data. However, it may not be completely precise.
The duration of illness was longer in those living in the affected area. Bacterial infections were possibly more common in residents in the contaminated area, while in the uncontaminated area secondary spread of viral infections may have been more common. Another reason may be the quantity of exposure. Those living in the affected area were probably more extensively exposed to contaminated water than others, who perhaps ingested contaminated water only occasionally. Furthermore, those most intensively exposed may have been infected with several pathogens, either simultaneously or consecutively. The frequency of previous episodes of gastroenteritis during the 10 months preceding the outbreak was 17% in the whole study population without differences between study groups. This is in good agreement with the yearly incidence of gastroenteritis (19.4/100 person-years) in a British cohort [28]. The survey was conducted as soon as possible after the outbreak. Since the outbreak took place shortly before the Christmas and New Year holiday season, it still took over 2 months before the survey could be launched. This conceivably resulted in some degree of recall bias. In addition, this outbreak gained wide coverage in the public media, which may have caused over-reporting of symptoms. The response rate of those who fell ill during the outbreak may have been higher, which might lead to overestimation of the disease burden. Nevertheless, the overall response rates in all study areas were relatively good, making significant bias unlikely. The spectrum of pathogens in both water and patient samples was wide, consistent with the mechanism of contamination [24, 25]. This distinguishes the outbreak from most Finnish waterborne epidemics, where usually only Campylobacter and/or norovirus are seen. Although samples were not systematically studied in all symptomatic patients, almost 200 Campylobacter-associated cases were confirmed in this outbreak, which is about 200 times more than the mean monthly number (n=1) for Campylobacter cases during the 24 pre-outbreak months in Nokia (National Infectious Disease Register).
Only a few samples were collected to detect norovirus in the early days of the epidemic, but all (5/5) were positive. Despite the small number of confirmed cases, we regard norovirus as a major pathogen in this outbreak. A number of faecal specimens collected for other purposes were later analysed for enteric viruses, and almost a third of them contained norovirus [20]. Astroviruses, adenoviruses, rotaviruses and enteroviruses were also detected. In another study connected to this outbreak and covering children attending the University Hospital, 40% (20/50) of samples yielded norovirus. In the same study, a variety of other viral pathogens were also detected, and a notable proportion of patients yielded mixed viral findings in their stools [29]. In addition to Campylobacter and norovirus, Giardia was regarded as a major pathogen. Although Giardia has been isolated from natural sources in Finland [30], domestic infections are uncommon. Drinking-water contamination was considered to be the obvious source of this outbreak, and this study provides strong evidence to support the assumption. Most subjects fell ill shortly after the valve was opened and microbes found in stools were in accord with findings in pipeline samples.
Finally, the incidence of the cases was highest in the contaminated area. Regardless of the severity of the event, the clinical consequences were milder than feared. Most of those afflicted in Nokia suffered self-limiting gastroenteritis and no permanent damage to their health is anticipated. To our knowledge, there were no obvious fatal outcomes. However, Contributory influences in the case of deaths mainly for other reasons cannot be excluded. If waterborne outbreaks cause fatalities, these are most often caused by STEC O157:H7. Although O157:H7 was present in Finland during the 1990s, it is nowadays uncommon [31] and was not detected in this outbreak. Hepatitis A virus (HAV) is another severe infection associated with waterborne exposure. HAV is highly uncommon in the Finnish population, but sporadic cases do occur. HAV was not found in water samples during this outbreak, and no hepatitis cases were detected. Clostridium difficile was identified, but the hypervirulent ribotype O27 was absent (S. Kotila et al., unpublished observation). This study demonstrates the value of a populationbased controlled survey in estimating disease burden in outbreaks when illnesses are mild or moderate. In this particular case, the survey showed that the actual number of those affected was almost seven times higher than the number of people seeking medical care. Furthermore, the use of a control population affords greater accuracy when assessing excess morbidity connected to an outbreak.
|
What did the investigation find?
|
{
"answer_start": [],
"text": []
}
|
403
|
An extensive gastroenteritis outbreak after drinking-water contamination by sewage effluent, Finland
|
SUMMARY
An inappropriate cross-connection between sewage- and drinking-water pipelines contaminated tap water in a Finnish town, resulting in an extensive waterborne gastroenteritis outbreak in this developed country. According to a database and a line-list, altogether 1222 subjects sought medical care as a result of this exposure. Seven pathogens were found in patient samples of those who sought treatment. To establish the true disease burden from this exposure, we undertook a population-based questionnaire investigation with a control population, infrequently used to study waterborne outbreaks. The study covered three areas, contaminated and uncontaminated parts of the town and a control town. An estimated 8453 residents fell ill during the outbreak, the excess number of illnesses being 6501. Attack rates were 53% [95% confidence interval (CI) 49.5–56.4] in the contaminated area, 15.6% (95% CI 13.1–18.5) in the uncontaminated area and 6.5% (95% CI 4.8–8.8) in the control population. Using a control population allowed us to differentiate baseline morbidity from the observed morbidity caused by the water contamination, thus enabling a more accurate estimate of the disease burden of this outbreak.
Key words: Gastroenteritis, outbreak, waterborne infections.
INTRODUCTION
Safe water is essential to life. Globally, the lack of clean water is estimated to cause about 4% of total mortality [1], predominantly in the developing world. However, waterborne outbreaks also remain a source of concern in developed countries. In the USA, 20 million people are estimated to contract a waterborne infection annually [2]. Over 4 00 000 people fell ill with cryptosporidiosis in Milwaukee in 1993 as a consequence of inadequate water filtration [3]. In Canada, the Walkerton outbreak resulted in seven fatalities attributable to Escherichia coli O157:H7 infection [4]. Waterborne outbreaks have been reported from many European countries including England and Wales [5], Norway [6], Sweden [7–10], Italy, Spain and Switzerland [11].
Finland is a large country (3 38 424 km2) with a small population (5.4 million). The number of waterworks is high (almost 2000) and most serve small communities. The majority (95%) use groundwater. However, almost half of the population are served by surface-water plants of the largest cities. Groundwater is usually not disinfected for use, while surface water is filtered and disinfected before distribution to consumers [12]. Although drinking water is generally considered safe in Finland, a number of waterborne outbreaks have occurred. Twenty-four outbreaks affecting 7700 inhabitants were spontaneously reported during the period 1980–1992 [13]; since 1997 reporting of outbreaks has been mandatory, and 14 outbreaks affecting 7300 people were registered during the first 2 years [12]. The most widespread outbreak to date affected 5500 people [14], and a number of other substantial waterborne outbreaks have been reported [15–17]. The most common microbes have been norovirus [18] and Campylobacter [19].
We report the investigation of a serious waterborne gastroenteritis outbreak that occurred in a Finnish town in autumn 2007.
Setting
Nokia (population 30 016) is a town located in southern Finland. Over 90% of the population are served by the municipal drinking-water supply. The average daily water consumption in Nokia is 4250 m3 (year 2006). The waterworks uses groundwater, which is treated with sodium hypochlorite for disinfection and pH adjustment. Sewage is processed in one municipal waste water plant, which covers 86% of the population. The town has a municipal health centre for primary care and a hospital for primary in-patient care and limited secondary care. Secondary and tertiary care are provided by Tampere University Hospital 20 km distant from Nokia. The Centre for Laboratory Medicine provides the municipalities with clinical laboratory services. Environmental health surveillance and monitoring of water quality is organized in collaboration with five neighbouring municipalities.
The outbreak
On Wednesday, 28 November 2007, maintenance work was conducted at the waste water plant in Nokia. The same day, technical problems were encountered in the municipal drinking-water system, and during the days following, customers complained of a bad smell and taste in their tap water. These were first misinterpreted as being caused by harmless deposits loosened from the inner walls of the pipeline and discharged during the maintenance work on 28 November. From 30 November, environmental health and municipal waterworks officials received reports of bouts of diarrhoea and vomiting among residents, and during that same day an increase in the number of patients with gastroenteritis was observed at the health centre. Extensive water and pipeline sampling was initiated and a boil-water notice issued, and water chlorine concentration was increased. The water contamination and its source became evident the same day. A valve in the waste-water plant connecting the drinking-water line and a waste-water effluent line had been opened during the maintenance work and inadvertently left open for 2 days. As a consequence, the drinking-water network became contaminated with sewage effluent. According to flow data, an estimated 450 m3 of waste water was mixed with the drinking water and distributed to customers, heavily contaminating household water and pipelines in southern parts of the town. The population in this area numbered over 9500.
Distribution of clean water was organized by municipal officials, the military and volunteers, and the health centre focused on treating outbreak patients, cutting back on other activities. The University Hospital prepared to open a temporary ward, which was in fact eventually not needed. Schools and daycare centres were closed from 6 to 9 December owing to teachers’ and children’s sick leave and the need to reduce the possible horizontal spread of gastrointestinal viruses. After 12 days, the boil-water notice was withdrawn in the uncontaminated area. In the contaminated area, adenoviruses and noroviruses were repeatedly detected in water-supply samples despite chlorination and pipeline flushing. Finally, after extensive decontamination procedures including shock chlorination, precautionary restrictions were discontinued in the contaminated area on 18 February 2008, almost 3 months after the incident.
We conducted a comprehensive investigation to determine the magnitude and consequences of the waterborne outbreak including describing the clinical aspects, reviewing microbiological findings and estimating the disease burden using a population-based survey.
METHODS
Descriptive epidemiology
Patients visiting the municipal health centre between 28 November and 31 December 2007 because of acute gastroenteritis were registered on a line-list, including name, age, gender, address and personal identity number. The timing and range of symptoms were recorded. Prospective line-listing was initiated 6 days after the beginning of the contamination visits prior to that being added retrospectively from patient charts. The information collected from the patient register included the number of primary-care visits to the centre because of gastroenteritis as defined by the international classification of primary care (ICPC-1, WHO, Geneva) codes D01, D09, D10, D11, D25, D70, D73 or D96. The information on reported symptoms was collected from the line-list. Daily admissions to Tampere University Hospital were counted by emergency-department personnel.
Analytical epidemiology
The town was divided into two study areas, contaminated (population 9538) and uncontaminated (population 20 478). The division was based on microbiological data from distribution system samples and technical modelling of water-flow directions within the network. Another municipality (population 27 259) 35 km distant from Nokia was chosen as a control population. One thousand persons for each study group were randomly selected from the population register. Groups were matched by age and gender and all age groups were included. Only one participant per household was included. The survey was conducted using postal questionnaires, the form being posted 8 weeks after the contamination had occurred. A repeat questionnaire was sent out 3 weeks later to non-responders.
Participants were asked about the timing and nature of gastrointestinal symptoms. A case was defined as a person suffering acute diarrhoea (o3 loose stools/day) or vomiting between 28 November 2007 and 20 January 2008. The occurrence of gastroenteritis during a 10-month period prior to the outbreak was also enquired about to ensure the comparability of the study groups.
Microbiological investigation
Faecal specimens for bacterial pathogens were not investigated from all the patients or from representative samples from consecutive cases. Instead, bacterial samples were available from cases with more severe symptoms. Specimens for detection of norovirus were collected from five randomly selected patients within the first week of the outbreak. Other viral pathogens were studied in samples from children admitted to the emergency department of the University Hospital. In order to detect enteric parasites specimens were taken from ten consecutive patients during the second week of the outbreak. Subsequently, when cases with giardiasis were detected, parasites were investigated in all symptomatic patients. Specimens were taken at the health centre or in the University Hospital.
Stool specimens were cultured for the presence of Campylobacter, Salmonella, Yersinia and Shigella spp. Norovirus was investigated using electron microscopy and PCR; rotavirus antigens were detected by enzyme immunological assay (EIA). Protozoan pathogens were investigated using microscopy of formalin-fixed samples. Subsequently, because no other protozoan pathogens were identified, only antigen detection of Giardia was used instead of microscopy. If a patient reported bloody stools, Shiga toxin producing Escherichia coli (STEC) was examined for by culture and toxin detection (EIA). Only samples taken between 28 November and 31 December 2007 were included. For Giardia the time-period for inclusion was extended to 31 May 2008 (6 months) because of the longer incubation time and gradual case detection.
The methods used in water and pipeline analysis will be described in detail in a forthcoming paper. Briefly, samples from the drinking-water distribution system were analysed by cultivation for the presence of Campylobacter, Salmonella and Clostridium spp. PCR was utilized to analyse enteric viruses as previously described by Maunula and colleagues [20]. The presence of Giardia cysts and Cryptosporidium oocysts was detected using concentration, immunomagnetic separation and epifluorescence microscopy as described in detail by Rimhanen-Finne and colleagues [21].
The study areas were compared using x2 or Kruskal– Wallis rank sum tests whenever appropriate. Attack rates were calculated as percentage of cases in a sample population. Total numbers of cases were calculated by extrapolating the age- and sex-adjusted attack rates to the total population of each area. To assess baseline rates of gastroenteritis, the attack rates from the control population were used to estimate the number of cases in the contaminated population that would have occurred in the absence of the water contamination. Estimations were made adjusting for age and sex. Confidence intervals for the total numbers were obtained using bootstrap methods [21]. All statistical analyses were performed using R version 2.9 (or later versions) [22].
RESULTS
Healthcare visits
According to the health centre database, 1222 visits due to gastrointestinal symptoms were made between 28 November and 31 December 2007. During the busiest week, the number of patients presenting with gastrointestinal complaints was 53-fold higher than expected compared to the weekly median number of cases in a 7-month period preceding the outbreak. Children aged <10 years constituted the largest group (Figs 1, 2).
Line-list data were available on 1024 patients. Only one visit per person was included. The most common symptoms reported were diarrhoea (n=813, 79%), vomiting (n=601, 59%), fever (n=337, 33%) and abdominal pain (n=291, 28%). Altogether 204 visits were made to emergency rooms in Tampere University Hospital, 145 (71%) of them involving children. To our knowledge, the outbreak did not lead directly to fatalities.
Questionnaire study
A total of 2154 participants returned the questionnaire. Thirty-one were excluded as being inadequately completed or because the respondent could not be identified; 2123 forms were finally accepted for the analysis (response rate 71%). The response rate was highest in the contaminated area and lowest in the control population. The background characteristics of the study groups showed no significant demographic differences between the study populations (Table 1). Altogether, 17% of the responders reported having gastroenteritis during the 10 months prior to the outbreak. Most subjects fell ill within 1 week of the contamination of the water supply (Fig. 3).
The most common symptoms were diarrhoea, vomiting, nausea and abdominal pain (Fig. 4). Overall, 579 respondents met the case definition, of whom 540 (93%) were residents of Nokia. Extrapolating the result for the whole population, an estimated 8453 (95% CI 7568–9363) residents fell ill with gastroenteritis in the whole of Nokia (Table 2). The excess number of cases was 6500 (95% CI 5613–7370) in Nokia, compared to the control population. The gastroenteritis attack rate was eightfold higher in the polluted area (53.0%) and 2.4- fold higher in the unpolluted area (15.6%) compared to the control municipality (6.5%). The median duration of gastroenteritis was 3 days for cases in the polluted area, 2 days in the unpolluted area and 2 days in the control population (P<0.001). The probability of falling ill increased along with the quantity of tap water consumed in the contaminated area, but not in the uncontaminated area or in the control population.
Microbiological findings
Seven enteropathogens were detected in faecal specimens (Table 3). The most frequently encountered pathogen was Campylobacter sp., with C. jejuni representing most of them, followed by Giardia. Five random samples investigated for norovirus were collected on 2 December 2007 and all were positive. Other findings included non typhoidal salmonellas, Clostridium difficile and rotavirus. Four stool cultures grew Shigella boydii. In ten persons, two different bacterial pathogens were found in faecal specimens. Except for Shigella boydii, the same pathogens also were detected in water-distribution system samples. Ninety-eight percent (158/162) of Campylobacter- and Salmonella-positive cultures occurred within the first 3 weeks of the outbreak.
DISCUSSION
This paper describes the largest reported waterborne outbreak in Finland to date. In the town of Nokia, over 8000 residents (28% of the population) were estimated to have fallen ill with gastroenteritis, 6500 of them as a consequence of water contamination. Over half of the population in the contaminated area suffered from gastroenteritis.
Although faecal contamination is a known cause of waterborne epidemics [8, 23–26], the chain of events in Nokia was exceptional. In most circumstances, faecal contamination occurs when small amounts of sewage are mixed with clean water as a result of an obstructed or broken waste-water line or drainage from surface sources, e.g. after heavy rainfall. However, in the present case, sewage had direct entry to the distribution line through an inappropriate crossconnection. As a result, the magnitude of contamination was substantial. The purpose and origin of the inappropriate valve was not ascertained in the investigation. It was built some decades ago, and was not prohibited during that time. After the outbreak, the national authorities required waterworks across the country to rule out the presence of similar cross-connection constructions. Contacts with healthcare are often used to quantify the burden of illness in outbreak investigations. However, experience from previous outbreaks indicates that a large proportion of affected subjects have a selflimiting disease and do not contact healthcare providers [17]. Thus, if only health centre visitors’ data are used, the burden of disease would be underestimated. In this study, a population-based survey was employed to avoid this problem. Since gastroenteritis is a common disease, a survey without proper controls might overestimate the magnitude of an outbreak if assessment of background morbidity is not done [27]. To obviate this challenge, we used a control population chosen from the same region but on the opposite side of the Tampere city area. Daily contacts between these communities are likely to be minimal, so that baseline morbidity could be estimated from the control population, lending accuracy to conclusions regarding the morbidity from the water system contamination.
The gastroenteritis attack rate was high in the contaminated area, where over half of the population fell ill. This probably reflects the high concentration of pathogens in the drinking water as well as their infectivity, especially that of viruses [20]. An even higher attack rate has been reported during a large waterborne norovirus outbreak in Finland [14]. In that case, however, the epidemic lasted longer and was investigated using an internet survey, making selection bias more likely. There was also excess morbidity among residents in the uncontaminated area of the town. This can be explained by exposure to polluted water upon visiting the affected area. Another likely explanation is person-to-person transmission of certain pathogens, especially viruses. The incidence in the uncontaminated area increased later and less sharply than in the contaminated area, which would support the hypothesis of secondary spread. Furthermore, what is referred to here as the affected area is an estimate, which was grounded on a thorough judgement of available network and microbiological data. However, it may not be completely precise.
The duration of illness was longer in those living in the affected area. Bacterial infections were possibly more common in residents in the contaminated area, while in the uncontaminated area secondary spread of viral infections may have been more common. Another reason may be the quantity of exposure. Those living in the affected area were probably more extensively exposed to contaminated water than others, who perhaps ingested contaminated water only occasionally. Furthermore, those most intensively exposed may have been infected with several pathogens, either simultaneously or consecutively. The frequency of previous episodes of gastroenteritis during the 10 months preceding the outbreak was 17% in the whole study population without differences between study groups. This is in good agreement with the yearly incidence of gastroenteritis (19.4/100 person-years) in a British cohort [28]. The survey was conducted as soon as possible after the outbreak. Since the outbreak took place shortly before the Christmas and New Year holiday season, it still took over 2 months before the survey could be launched. This conceivably resulted in some degree of recall bias. In addition, this outbreak gained wide coverage in the public media, which may have caused over-reporting of symptoms. The response rate of those who fell ill during the outbreak may have been higher, which might lead to overestimation of the disease burden. Nevertheless, the overall response rates in all study areas were relatively good, making significant bias unlikely. The spectrum of pathogens in both water and patient samples was wide, consistent with the mechanism of contamination [24, 25]. This distinguishes the outbreak from most Finnish waterborne epidemics, where usually only Campylobacter and/or norovirus are seen. Although samples were not systematically studied in all symptomatic patients, almost 200 Campylobacter-associated cases were confirmed in this outbreak, which is about 200 times more than the mean monthly number (n=1) for Campylobacter cases during the 24 pre-outbreak months in Nokia (National Infectious Disease Register).
Only a few samples were collected to detect norovirus in the early days of the epidemic, but all (5/5) were positive. Despite the small number of confirmed cases, we regard norovirus as a major pathogen in this outbreak. A number of faecal specimens collected for other purposes were later analysed for enteric viruses, and almost a third of them contained norovirus [20]. Astroviruses, adenoviruses, rotaviruses and enteroviruses were also detected. In another study connected to this outbreak and covering children attending the University Hospital, 40% (20/50) of samples yielded norovirus. In the same study, a variety of other viral pathogens were also detected, and a notable proportion of patients yielded mixed viral findings in their stools [29]. In addition to Campylobacter and norovirus, Giardia was regarded as a major pathogen. Although Giardia has been isolated from natural sources in Finland [30], domestic infections are uncommon. Drinking-water contamination was considered to be the obvious source of this outbreak, and this study provides strong evidence to support the assumption. Most subjects fell ill shortly after the valve was opened and microbes found in stools were in accord with findings in pipeline samples.
Finally, the incidence of the cases was highest in the contaminated area. Regardless of the severity of the event, the clinical consequences were milder than feared. Most of those afflicted in Nokia suffered self-limiting gastroenteritis and no permanent damage to their health is anticipated. To our knowledge, there were no obvious fatal outcomes. However, Contributory influences in the case of deaths mainly for other reasons cannot be excluded. If waterborne outbreaks cause fatalities, these are most often caused by STEC O157:H7. Although O157:H7 was present in Finland during the 1990s, it is nowadays uncommon [31] and was not detected in this outbreak. Hepatitis A virus (HAV) is another severe infection associated with waterborne exposure. HAV is highly uncommon in the Finnish population, but sporadic cases do occur. HAV was not found in water samples during this outbreak, and no hepatitis cases were detected. Clostridium difficile was identified, but the hypervirulent ribotype O27 was absent (S. Kotila et al., unpublished observation). This study demonstrates the value of a populationbased controlled survey in estimating disease burden in outbreaks when illnesses are mild or moderate. In this particular case, the survey showed that the actual number of those affected was almost seven times higher than the number of people seeking medical care. Furthermore, the use of a control population affords greater accuracy when assessing excess morbidity connected to an outbreak.
|
How was the infrastructure affected?
|
{
"answer_start": [
16339
],
"text": [
"sewage had direct entry to the distribution line through an inappropriate crossconnection"
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|
404
|
An extensive gastroenteritis outbreak after drinking-water contamination by sewage effluent, Finland
|
SUMMARY
An inappropriate cross-connection between sewage- and drinking-water pipelines contaminated tap water in a Finnish town, resulting in an extensive waterborne gastroenteritis outbreak in this developed country. According to a database and a line-list, altogether 1222 subjects sought medical care as a result of this exposure. Seven pathogens were found in patient samples of those who sought treatment. To establish the true disease burden from this exposure, we undertook a population-based questionnaire investigation with a control population, infrequently used to study waterborne outbreaks. The study covered three areas, contaminated and uncontaminated parts of the town and a control town. An estimated 8453 residents fell ill during the outbreak, the excess number of illnesses being 6501. Attack rates were 53% [95% confidence interval (CI) 49.5–56.4] in the contaminated area, 15.6% (95% CI 13.1–18.5) in the uncontaminated area and 6.5% (95% CI 4.8–8.8) in the control population. Using a control population allowed us to differentiate baseline morbidity from the observed morbidity caused by the water contamination, thus enabling a more accurate estimate of the disease burden of this outbreak.
Key words: Gastroenteritis, outbreak, waterborne infections.
INTRODUCTION
Safe water is essential to life. Globally, the lack of clean water is estimated to cause about 4% of total mortality [1], predominantly in the developing world. However, waterborne outbreaks also remain a source of concern in developed countries. In the USA, 20 million people are estimated to contract a waterborne infection annually [2]. Over 4 00 000 people fell ill with cryptosporidiosis in Milwaukee in 1993 as a consequence of inadequate water filtration [3]. In Canada, the Walkerton outbreak resulted in seven fatalities attributable to Escherichia coli O157:H7 infection [4]. Waterborne outbreaks have been reported from many European countries including England and Wales [5], Norway [6], Sweden [7–10], Italy, Spain and Switzerland [11].
Finland is a large country (3 38 424 km2) with a small population (5.4 million). The number of waterworks is high (almost 2000) and most serve small communities. The majority (95%) use groundwater. However, almost half of the population are served by surface-water plants of the largest cities. Groundwater is usually not disinfected for use, while surface water is filtered and disinfected before distribution to consumers [12]. Although drinking water is generally considered safe in Finland, a number of waterborne outbreaks have occurred. Twenty-four outbreaks affecting 7700 inhabitants were spontaneously reported during the period 1980–1992 [13]; since 1997 reporting of outbreaks has been mandatory, and 14 outbreaks affecting 7300 people were registered during the first 2 years [12]. The most widespread outbreak to date affected 5500 people [14], and a number of other substantial waterborne outbreaks have been reported [15–17]. The most common microbes have been norovirus [18] and Campylobacter [19].
We report the investigation of a serious waterborne gastroenteritis outbreak that occurred in a Finnish town in autumn 2007.
Setting
Nokia (population 30 016) is a town located in southern Finland. Over 90% of the population are served by the municipal drinking-water supply. The average daily water consumption in Nokia is 4250 m3 (year 2006). The waterworks uses groundwater, which is treated with sodium hypochlorite for disinfection and pH adjustment. Sewage is processed in one municipal waste water plant, which covers 86% of the population. The town has a municipal health centre for primary care and a hospital for primary in-patient care and limited secondary care. Secondary and tertiary care are provided by Tampere University Hospital 20 km distant from Nokia. The Centre for Laboratory Medicine provides the municipalities with clinical laboratory services. Environmental health surveillance and monitoring of water quality is organized in collaboration with five neighbouring municipalities.
The outbreak
On Wednesday, 28 November 2007, maintenance work was conducted at the waste water plant in Nokia. The same day, technical problems were encountered in the municipal drinking-water system, and during the days following, customers complained of a bad smell and taste in their tap water. These were first misinterpreted as being caused by harmless deposits loosened from the inner walls of the pipeline and discharged during the maintenance work on 28 November. From 30 November, environmental health and municipal waterworks officials received reports of bouts of diarrhoea and vomiting among residents, and during that same day an increase in the number of patients with gastroenteritis was observed at the health centre. Extensive water and pipeline sampling was initiated and a boil-water notice issued, and water chlorine concentration was increased. The water contamination and its source became evident the same day. A valve in the waste-water plant connecting the drinking-water line and a waste-water effluent line had been opened during the maintenance work and inadvertently left open for 2 days. As a consequence, the drinking-water network became contaminated with sewage effluent. According to flow data, an estimated 450 m3 of waste water was mixed with the drinking water and distributed to customers, heavily contaminating household water and pipelines in southern parts of the town. The population in this area numbered over 9500.
Distribution of clean water was organized by municipal officials, the military and volunteers, and the health centre focused on treating outbreak patients, cutting back on other activities. The University Hospital prepared to open a temporary ward, which was in fact eventually not needed. Schools and daycare centres were closed from 6 to 9 December owing to teachers’ and children’s sick leave and the need to reduce the possible horizontal spread of gastrointestinal viruses. After 12 days, the boil-water notice was withdrawn in the uncontaminated area. In the contaminated area, adenoviruses and noroviruses were repeatedly detected in water-supply samples despite chlorination and pipeline flushing. Finally, after extensive decontamination procedures including shock chlorination, precautionary restrictions were discontinued in the contaminated area on 18 February 2008, almost 3 months after the incident.
We conducted a comprehensive investigation to determine the magnitude and consequences of the waterborne outbreak including describing the clinical aspects, reviewing microbiological findings and estimating the disease burden using a population-based survey.
METHODS
Descriptive epidemiology
Patients visiting the municipal health centre between 28 November and 31 December 2007 because of acute gastroenteritis were registered on a line-list, including name, age, gender, address and personal identity number. The timing and range of symptoms were recorded. Prospective line-listing was initiated 6 days after the beginning of the contamination visits prior to that being added retrospectively from patient charts. The information collected from the patient register included the number of primary-care visits to the centre because of gastroenteritis as defined by the international classification of primary care (ICPC-1, WHO, Geneva) codes D01, D09, D10, D11, D25, D70, D73 or D96. The information on reported symptoms was collected from the line-list. Daily admissions to Tampere University Hospital were counted by emergency-department personnel.
Analytical epidemiology
The town was divided into two study areas, contaminated (population 9538) and uncontaminated (population 20 478). The division was based on microbiological data from distribution system samples and technical modelling of water-flow directions within the network. Another municipality (population 27 259) 35 km distant from Nokia was chosen as a control population. One thousand persons for each study group were randomly selected from the population register. Groups were matched by age and gender and all age groups were included. Only one participant per household was included. The survey was conducted using postal questionnaires, the form being posted 8 weeks after the contamination had occurred. A repeat questionnaire was sent out 3 weeks later to non-responders.
Participants were asked about the timing and nature of gastrointestinal symptoms. A case was defined as a person suffering acute diarrhoea (o3 loose stools/day) or vomiting between 28 November 2007 and 20 January 2008. The occurrence of gastroenteritis during a 10-month period prior to the outbreak was also enquired about to ensure the comparability of the study groups.
Microbiological investigation
Faecal specimens for bacterial pathogens were not investigated from all the patients or from representative samples from consecutive cases. Instead, bacterial samples were available from cases with more severe symptoms. Specimens for detection of norovirus were collected from five randomly selected patients within the first week of the outbreak. Other viral pathogens were studied in samples from children admitted to the emergency department of the University Hospital. In order to detect enteric parasites specimens were taken from ten consecutive patients during the second week of the outbreak. Subsequently, when cases with giardiasis were detected, parasites were investigated in all symptomatic patients. Specimens were taken at the health centre or in the University Hospital.
Stool specimens were cultured for the presence of Campylobacter, Salmonella, Yersinia and Shigella spp. Norovirus was investigated using electron microscopy and PCR; rotavirus antigens were detected by enzyme immunological assay (EIA). Protozoan pathogens were investigated using microscopy of formalin-fixed samples. Subsequently, because no other protozoan pathogens were identified, only antigen detection of Giardia was used instead of microscopy. If a patient reported bloody stools, Shiga toxin producing Escherichia coli (STEC) was examined for by culture and toxin detection (EIA). Only samples taken between 28 November and 31 December 2007 were included. For Giardia the time-period for inclusion was extended to 31 May 2008 (6 months) because of the longer incubation time and gradual case detection.
The methods used in water and pipeline analysis will be described in detail in a forthcoming paper. Briefly, samples from the drinking-water distribution system were analysed by cultivation for the presence of Campylobacter, Salmonella and Clostridium spp. PCR was utilized to analyse enteric viruses as previously described by Maunula and colleagues [20]. The presence of Giardia cysts and Cryptosporidium oocysts was detected using concentration, immunomagnetic separation and epifluorescence microscopy as described in detail by Rimhanen-Finne and colleagues [21].
The study areas were compared using x2 or Kruskal– Wallis rank sum tests whenever appropriate. Attack rates were calculated as percentage of cases in a sample population. Total numbers of cases were calculated by extrapolating the age- and sex-adjusted attack rates to the total population of each area. To assess baseline rates of gastroenteritis, the attack rates from the control population were used to estimate the number of cases in the contaminated population that would have occurred in the absence of the water contamination. Estimations were made adjusting for age and sex. Confidence intervals for the total numbers were obtained using bootstrap methods [21]. All statistical analyses were performed using R version 2.9 (or later versions) [22].
RESULTS
Healthcare visits
According to the health centre database, 1222 visits due to gastrointestinal symptoms were made between 28 November and 31 December 2007. During the busiest week, the number of patients presenting with gastrointestinal complaints was 53-fold higher than expected compared to the weekly median number of cases in a 7-month period preceding the outbreak. Children aged <10 years constituted the largest group (Figs 1, 2).
Line-list data were available on 1024 patients. Only one visit per person was included. The most common symptoms reported were diarrhoea (n=813, 79%), vomiting (n=601, 59%), fever (n=337, 33%) and abdominal pain (n=291, 28%). Altogether 204 visits were made to emergency rooms in Tampere University Hospital, 145 (71%) of them involving children. To our knowledge, the outbreak did not lead directly to fatalities.
Questionnaire study
A total of 2154 participants returned the questionnaire. Thirty-one were excluded as being inadequately completed or because the respondent could not be identified; 2123 forms were finally accepted for the analysis (response rate 71%). The response rate was highest in the contaminated area and lowest in the control population. The background characteristics of the study groups showed no significant demographic differences between the study populations (Table 1). Altogether, 17% of the responders reported having gastroenteritis during the 10 months prior to the outbreak. Most subjects fell ill within 1 week of the contamination of the water supply (Fig. 3).
The most common symptoms were diarrhoea, vomiting, nausea and abdominal pain (Fig. 4). Overall, 579 respondents met the case definition, of whom 540 (93%) were residents of Nokia. Extrapolating the result for the whole population, an estimated 8453 (95% CI 7568–9363) residents fell ill with gastroenteritis in the whole of Nokia (Table 2). The excess number of cases was 6500 (95% CI 5613–7370) in Nokia, compared to the control population. The gastroenteritis attack rate was eightfold higher in the polluted area (53.0%) and 2.4- fold higher in the unpolluted area (15.6%) compared to the control municipality (6.5%). The median duration of gastroenteritis was 3 days for cases in the polluted area, 2 days in the unpolluted area and 2 days in the control population (P<0.001). The probability of falling ill increased along with the quantity of tap water consumed in the contaminated area, but not in the uncontaminated area or in the control population.
Microbiological findings
Seven enteropathogens were detected in faecal specimens (Table 3). The most frequently encountered pathogen was Campylobacter sp., with C. jejuni representing most of them, followed by Giardia. Five random samples investigated for norovirus were collected on 2 December 2007 and all were positive. Other findings included non typhoidal salmonellas, Clostridium difficile and rotavirus. Four stool cultures grew Shigella boydii. In ten persons, two different bacterial pathogens were found in faecal specimens. Except for Shigella boydii, the same pathogens also were detected in water-distribution system samples. Ninety-eight percent (158/162) of Campylobacter- and Salmonella-positive cultures occurred within the first 3 weeks of the outbreak.
DISCUSSION
This paper describes the largest reported waterborne outbreak in Finland to date. In the town of Nokia, over 8000 residents (28% of the population) were estimated to have fallen ill with gastroenteritis, 6500 of them as a consequence of water contamination. Over half of the population in the contaminated area suffered from gastroenteritis.
Although faecal contamination is a known cause of waterborne epidemics [8, 23–26], the chain of events in Nokia was exceptional. In most circumstances, faecal contamination occurs when small amounts of sewage are mixed with clean water as a result of an obstructed or broken waste-water line or drainage from surface sources, e.g. after heavy rainfall. However, in the present case, sewage had direct entry to the distribution line through an inappropriate crossconnection. As a result, the magnitude of contamination was substantial. The purpose and origin of the inappropriate valve was not ascertained in the investigation. It was built some decades ago, and was not prohibited during that time. After the outbreak, the national authorities required waterworks across the country to rule out the presence of similar cross-connection constructions. Contacts with healthcare are often used to quantify the burden of illness in outbreak investigations. However, experience from previous outbreaks indicates that a large proportion of affected subjects have a selflimiting disease and do not contact healthcare providers [17]. Thus, if only health centre visitors’ data are used, the burden of disease would be underestimated. In this study, a population-based survey was employed to avoid this problem. Since gastroenteritis is a common disease, a survey without proper controls might overestimate the magnitude of an outbreak if assessment of background morbidity is not done [27]. To obviate this challenge, we used a control population chosen from the same region but on the opposite side of the Tampere city area. Daily contacts between these communities are likely to be minimal, so that baseline morbidity could be estimated from the control population, lending accuracy to conclusions regarding the morbidity from the water system contamination.
The gastroenteritis attack rate was high in the contaminated area, where over half of the population fell ill. This probably reflects the high concentration of pathogens in the drinking water as well as their infectivity, especially that of viruses [20]. An even higher attack rate has been reported during a large waterborne norovirus outbreak in Finland [14]. In that case, however, the epidemic lasted longer and was investigated using an internet survey, making selection bias more likely. There was also excess morbidity among residents in the uncontaminated area of the town. This can be explained by exposure to polluted water upon visiting the affected area. Another likely explanation is person-to-person transmission of certain pathogens, especially viruses. The incidence in the uncontaminated area increased later and less sharply than in the contaminated area, which would support the hypothesis of secondary spread. Furthermore, what is referred to here as the affected area is an estimate, which was grounded on a thorough judgement of available network and microbiological data. However, it may not be completely precise.
The duration of illness was longer in those living in the affected area. Bacterial infections were possibly more common in residents in the contaminated area, while in the uncontaminated area secondary spread of viral infections may have been more common. Another reason may be the quantity of exposure. Those living in the affected area were probably more extensively exposed to contaminated water than others, who perhaps ingested contaminated water only occasionally. Furthermore, those most intensively exposed may have been infected with several pathogens, either simultaneously or consecutively. The frequency of previous episodes of gastroenteritis during the 10 months preceding the outbreak was 17% in the whole study population without differences between study groups. This is in good agreement with the yearly incidence of gastroenteritis (19.4/100 person-years) in a British cohort [28]. The survey was conducted as soon as possible after the outbreak. Since the outbreak took place shortly before the Christmas and New Year holiday season, it still took over 2 months before the survey could be launched. This conceivably resulted in some degree of recall bias. In addition, this outbreak gained wide coverage in the public media, which may have caused over-reporting of symptoms. The response rate of those who fell ill during the outbreak may have been higher, which might lead to overestimation of the disease burden. Nevertheless, the overall response rates in all study areas were relatively good, making significant bias unlikely. The spectrum of pathogens in both water and patient samples was wide, consistent with the mechanism of contamination [24, 25]. This distinguishes the outbreak from most Finnish waterborne epidemics, where usually only Campylobacter and/or norovirus are seen. Although samples were not systematically studied in all symptomatic patients, almost 200 Campylobacter-associated cases were confirmed in this outbreak, which is about 200 times more than the mean monthly number (n=1) for Campylobacter cases during the 24 pre-outbreak months in Nokia (National Infectious Disease Register).
Only a few samples were collected to detect norovirus in the early days of the epidemic, but all (5/5) were positive. Despite the small number of confirmed cases, we regard norovirus as a major pathogen in this outbreak. A number of faecal specimens collected for other purposes were later analysed for enteric viruses, and almost a third of them contained norovirus [20]. Astroviruses, adenoviruses, rotaviruses and enteroviruses were also detected. In another study connected to this outbreak and covering children attending the University Hospital, 40% (20/50) of samples yielded norovirus. In the same study, a variety of other viral pathogens were also detected, and a notable proportion of patients yielded mixed viral findings in their stools [29]. In addition to Campylobacter and norovirus, Giardia was regarded as a major pathogen. Although Giardia has been isolated from natural sources in Finland [30], domestic infections are uncommon. Drinking-water contamination was considered to be the obvious source of this outbreak, and this study provides strong evidence to support the assumption. Most subjects fell ill shortly after the valve was opened and microbes found in stools were in accord with findings in pipeline samples.
Finally, the incidence of the cases was highest in the contaminated area. Regardless of the severity of the event, the clinical consequences were milder than feared. Most of those afflicted in Nokia suffered self-limiting gastroenteritis and no permanent damage to their health is anticipated. To our knowledge, there were no obvious fatal outcomes. However, Contributory influences in the case of deaths mainly for other reasons cannot be excluded. If waterborne outbreaks cause fatalities, these are most often caused by STEC O157:H7. Although O157:H7 was present in Finland during the 1990s, it is nowadays uncommon [31] and was not detected in this outbreak. Hepatitis A virus (HAV) is another severe infection associated with waterborne exposure. HAV is highly uncommon in the Finnish population, but sporadic cases do occur. HAV was not found in water samples during this outbreak, and no hepatitis cases were detected. Clostridium difficile was identified, but the hypervirulent ribotype O27 was absent (S. Kotila et al., unpublished observation). This study demonstrates the value of a populationbased controlled survey in estimating disease burden in outbreaks when illnesses are mild or moderate. In this particular case, the survey showed that the actual number of those affected was almost seven times higher than the number of people seeking medical care. Furthermore, the use of a control population affords greater accuracy when assessing excess morbidity connected to an outbreak.
|
What were the infrastructure complaints?
|
{
"answer_start": [
16339
],
"text": [
"sewage had direct entry to the distribution line through an inappropriate crossconnection"
]
}
|
405
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
What happened?
|
{
"answer_start": [
13
],
"text": [
"outbreak of viral gastroenteritis"
]
}
|
406
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
What was the event?
|
{
"answer_start": [
13
],
"text": [
"outbreak of viral gastroenteritis linked to municipal drinking water"
]
}
|
407
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
When did this happen?
|
{
"answer_start": [
113
],
"text": [
"June 2009"
]
}
|
408
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
When did this event start?
|
{
"answer_start": [
627
],
"text": [
"9 June 2009"
]
}
|
409
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
What is the date of this event?
|
{
"answer_start": [
627
],
"text": [
"9 June 2009"
]
}
|
410
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
How long was the event?
|
{
"answer_start": [
129
],
"text": [
"one month"
]
}
|
411
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
How long did the event last?
|
{
"answer_start": [
129
],
"text": [
"one month"
]
}
|
412
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
In which street did this happen?
|
{
"answer_start": [],
"text": []
}
|
413
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
In which city did this happen?
|
{
"answer_start": [
688
],
"text": [
"San Felice del Benaco"
]
}
|
414
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
In which region did this happen?
|
{
"answer_start": [
761
],
"text": [
"Lombardy"
]
}
|
415
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
In which region did this happen?
|
{
"answer_start": [
95
],
"text": [
"northern Italy"
]
}
|
416
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
In which country did this happen?
|
{
"answer_start": [
104
],
"text": [
"Italy"
]
}
|
417
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
Where did this happen?
|
{
"answer_start": [
761
],
"text": [
"Lombardy region, north Italy"
]
}
|
418
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
Where did this happen?
|
{
"answer_start": [
82
],
"text": [
"in a town in northern Italy"
]
}
|
419
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
What caused the event?
|
{
"answer_start": [
7382
],
"text": [
"contaminated municipal water supply"
]
}
|
420
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
What caused the contaminated municipal water supply?
|
{
"answer_start": [],
"text": []
}
|
421
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
What was the cause of the event?
|
{
"answer_start": [
7382
],
"text": [
"contaminated municipal water supply"
]
}
|
422
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
What source started the event?
|
{
"answer_start": [
57
],
"text": [
"municipal drinking water"
]
}
|
423
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
How was the event first detected?
|
{
"answer_start": [
640
],
"text": [
"a general practitioner from the municipality of San Felice del Benaco notified to the local health authority"
]
}
|
424
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
How was the event first detected?
|
{
"answer_start": [
7604
],
"text": [
"cluster of gastroenteritis in a hotel"
]
}
|
425
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
How many people were ill?
|
{
"answer_start": [
153
],
"text": [
"299"
]
}
|
426
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
How many people were hospitalized?
|
{
"answer_start": [
4279
],
"text": [
"Four"
]
}
|
427
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
How many people were dead?
|
{
"answer_start": [
4341
],
"text": [
"no fatality"
]
}
|
428
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
Which contaminants or viruses or bacteria were found?
|
{
"answer_start": [
241
],
"text": [
"norovirus, rotavirus, enterovirus or astrovirus"
]
}
|
429
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
Which were the symptoms?
|
{
"answer_start": [
868
],
"text": [
"vomiting, diarrhoea and fever"
]
}
|
430
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
What did the patients have?
|
{
"answer_start": [
868
],
"text": [
"vomiting, diarrhoea and fever"
]
}
|
431
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
What were the first steps?
|
{
"answer_start": [
6200
],
"text": [
"restricted the use of municipal water"
]
}
|
432
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
What did they do to control the problem?
|
{
"answer_start": [
6200
],
"text": [
"restricted the use of municipal water"
]
}
|
433
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
What did they do to control the problem?
|
{
"answer_start": [
6327
],
"text": [
"provided alternative water supplies to the population via water tankers"
]
}
|
434
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
What did they do to control the problem?
|
{
"answer_start": [
6430
],
"text": [
"door-to-door information campaign and distributed leaflets"
]
}
|
435
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
What did they do to control the problem?
|
{
"answer_start": [
6906
],
"text": [
"Regular water sampling and testing"
]
}
|
436
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
What did the local authorities advise?
|
{
"answer_start": [
6261
],
"text": [
"not to use municipal water for drinking and cooking"
]
}
|
437
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
What were the control measures?
|
{
"answer_start": [
6577
],
"text": [
"disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid"
]
}
|
438
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
What type of samples were examined?
|
{
"answer_start": [
3032
],
"text": [
"water samples from the lake"
]
}
|
439
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
What did they test for in the samples?
|
{
"answer_start": [
241
],
"text": [
"norovirus, rotavirus, enterovirus or astrovirus"
]
}
|
440
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
What is the concentration of the pathogens?
|
{
"answer_start": [],
"text": []
}
|
441
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
What steps were taken to restore the problem?
|
{
"answer_start": [
6577
],
"text": [
"disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid"
]
}
|
442
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
What was done to fix the problem?
|
{
"answer_start": [
6577
],
"text": [
"disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid"
]
}
|
443
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
What could have been done to prevent the event?
|
{
"answer_start": [],
"text": []
}
|
444
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
How to prevent this?
|
{
"answer_start": [
9206
],
"text": [
"well-maintained and monitored water supplies"
]
}
|
445
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
What were the investigation steps?
|
{
"answer_start": [
6906
],
"text": [
"Regular water sampling and testing"
]
}
|
446
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
What did the investigation find?
|
{
"answer_start": [
5765
],
"text": [
"1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l"
]
}
|
447
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
How was the infrastructure affected?
|
{
"answer_start": [],
"text": []
}
|
448
|
An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009
|
We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.
Introduction
On 9 June 2009, a general practitioner from the municipality of San Felice del Benaco notified to the local health authority of Brescia (Lombardy region, north Italy) 21 cases of gastroenteritis among guests of a hotel. Patients presented with vomiting, diarrhoea and fever. In the following days, there were also reports of cases among local residents. Located near the lake of Garda, San Felice del Benaco has 3,360 residents but is very touristic during the summer months. We investigated the outbreak in order to identify the source of infection and implement appropriate control measures.
Methods
We defined a probable outbreak case as a person who fell sick with vomiting or diarrhoea after 7 June 2009 and who stayed prior to disease onset in San Felice del Benaco. A confirmed outbreak case was defined as a person who fulfilled the criteria of a probable case and whose stool sample was laboratory-confirmed for at least
one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Probable cases who tested negative for the presence of virus in the stools were still considered as probable cases.
Active case finding was performed as follows: a public hotline was set up where people could call the health authority for information regarding the disease and report symptoms, date of onset and basic demographic data. In parallel, the outbreak investigation team collected daily information on case-patients presenting at the emergency unit of the local hospital and collected stool samples when possible.
The local and regional health authorities initiated an environmental investigation at the hotel on 9 June 2009, taking food samples from the kitchen, interviewing and collecting stool samples for microbiological testing from 20 probable cases (both guests and hotel staff). When it was clear that the outbreak was spreading to the larger community (apart from three campsites with their own private water supply, where no cases were reported), the environmental investigation was extended and included collection of water samples from the municipal water supply. Municipal water comes from the nearby lake. Before being distributed to the town as drinking water, it is treated with chlorine dioxide and hypochlorite and passes through sand filters. The investigators collected a total of 94 water samples from the lake at the location where the water is pumped, from filters and from public fountains. Samples were sent to the Lombardy and Emilia Romagna Experimental Zooprophylactic Institute (IZSLER) to test for the presence of bacterial pathogens (Salmonella sp., Shigella sp., Campylobacter sp., E. coli O157, Yersinia enterocolitica, Aeromonas sp., Clostridium perfringens toxins), parasites (Cryptosporidium sp.) and viral pathogens (norovirus, rotavirus, enterovirus, astrovirus). Virological methods included negative staining electron microscopy, type A rotavirus ELISA and PCR methods for norovirus, rotavirus, enterovirus and astrovirus.
Results
A total of 299 persons fulfilled the outbreak case definition, including 269 probable and 30 confirmed cases. The epidemic curve in Figure 1 shows the probable and confirmed outbreak cases by date of onset. The outbreak occurred between 8 June and 4 July 2009 and peaked on the 15 and 16 June with 47 outbreak cases per day. The attack rate for the town of San Felice del Benaco was 8.9% (299/3,360). Age group-specific attack rates ranged from 7% (50/713) in persons aged 65 years and older to 14% (34/242) in the age group 15-24 years (Figure 2). Four cases were hospitalised, all of them children. There was no fatality. Stool samples obtained from 36 probable cases were examined at the laboratory. Of these, 17 (47.2%) tested positive for norovirus, 19 (52.8%) for rotavirus, 12 (33.3%) for
enterovirus and 4 (11.1%) for astrovirus. Eight cases had both norovirus and rotavirus in the stools and two cases tested positive for norovirus, rotavirus and enterovirus. The laboratory did not find any virus in six cases (but we still included them among probable outbreak cases because of compatible symptoms). Salmonella
sp., Clostridium perfringens and Campylobacter sp. were found in samples from two, one and one cases, respectively.
The mean age of confirmed cases of rotavirus was 29 years (range: 0-71) compared to the mean age of 39 years (range: 0-88) for cases of norovirus and 39 years (9-88) for cases of enterovirus. The age distribution of confirmed cases is shown in Figure 3. Food samples from the hotel tested negative for the presence of pathogens. On 16 June 2009, preliminary environmental investigation results showed abnormally high levels of Clostridium perfringens (4 UFC/100 ml) and Aeromonas hydrophyla (16 UFC/100 ml) in water samples from two public fountains. Tests on 44 water samples from from the municipal water system (water from fountains and filers) showed the presence of norovirus and enterovirus. Examination of the municipal water network revealed that: 1) the water company had undertaken work on the collection reservoir which might have limited the effect of chlorination; 2) two filters were 10 years old (cleaned weekly but not disinfected); 3) the chlorine concentration in the water before it passed through the filters was 0.4 mg/l; in filtered water it was only 0.08 mg/l.
Control measures
On 17 June 2009, a special ordinance from the municipality restricted the use of municipal water (inhabitants were told not to use municipal water for drinking and cooking purposes) and provided alternative water supplies to the population via water tankers. Local authorities organised a door-to-door information campaign and distributed leaflets in order to reach as many people as possible. On 19 June 2009, the municipality started disinfecting the water system with chlorine dioxide (0.2 mg/l) and sand filters with peracetic acid. When the presence of norovirus in water and stools of cases was confirmed, the residual concentration of chlorine dioxide in terminal points of the network was increased to 3.4 mg/l for three consecutive days from 23 June 2009. Regular water sampling and testing was performed to monitor the efficiency of control measures. The ordinance on drinking water was maintained until all water quality tests complied with safety norms. Water samples collected after the first treatment with chlorine dioxide and peracetic acid all tested negative for the presence of norovirus.
Conclusion and discussion
An outbreak of viral gastroenteritis has been microbiologically linked to a contaminated municipal water supply in a small town of Lombardy. Timely control measures and good compliance of the population following the information campaign prevented a much higher attack rate. The alert came from a cluster of gastroenteritis in a hotel. The direction of the hotel promptly informed a general practitioner who notified the cluster to the public health authorities. An increase of gastroenteritis in the general population was noticed one day after the initial alert. The hotel is located along the lake, near the water reservoir, which could explain why the guests and its staff were among the first to be affected (see the first peak on the epidemic curve on 9 July 2009). Although the number of residents in San Felice Del Benaco is 3,360, it is important to note that in the summer season many tourists stay in the town and the total population is multiplied by three. Therefore, the attack rates reported above (based on the resident population) are probably overestimates even though the surveillance system did not capture all cases. All age groups were affected. This is consistent with an exposure that is equally distributed across all ages. The relatively high mean age of confirmed rotavirus cases (29 years) is also consistent with an exposure that is not limited to young children. The municipal water is taken from the lake at a place where the water is stagnant. So far, water samples from the lake tested negative for the presence of norovirus, rotavirus, enterovirus or astrovirus. However, we cannot exclude contamination of the lake due to over-capacity of the sewage system and/or illegal wastage.*In Italy, municipal water systems have been identified as the source of water-borne infections in several norovirus outbreaks (1, 2). It reminds us of the public health importance of well-maintained and monitored water supplies in our towns and cities (3).
|
What were the infrastructure complaints?
|
{
"answer_start": [
5890
],
"text": [
"two filters were 10 years old"
]
}
|
449
|
Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
|
Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
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Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
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Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
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Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
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Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
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When did this happen?
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452
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Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
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Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
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When did this event start?
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453
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Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
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Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
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What is the date of this event?
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475
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"2019"
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454
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Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
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Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
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Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
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Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
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Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
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Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
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Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
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Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
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Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
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Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
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In which region did this happen?
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Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
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Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
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In which country did this happen?
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{
"answer_start": [
459
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"text": [
"Portugal"
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|
460
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Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
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Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
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Where did this happen?
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461
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Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
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Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
|
What caused the event?
|
{
"answer_start": [
194
],
"text": [
"consumption of contaminated water"
]
}
|
462
|
Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
|
Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
|
What was the cause of the event?
|
{
"answer_start": [
194
],
"text": [
"consumption of contaminated water"
]
}
|
463
|
Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
|
Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
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Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
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Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
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Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
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Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
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How many people were ill?
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Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
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Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
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How many people were hospitalized?
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467
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Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
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Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
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How many people were dead?
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468
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Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
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Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
|
Which contaminants or viruses or bacteria were found?
|
{
"answer_start": [
10
],
"text": [
"Hepatitis E virus"
]
}
|
469
|
Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
|
Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
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Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
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Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
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What did the patients have?
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Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
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Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
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What were the first steps?
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495
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"text": [
"detection and eventual quantification of enteric viruses in samples from surface and drinking water"
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472
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Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
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Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
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What did they do to control the problem?
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Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
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Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
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What did the local authorities advise?
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"text": []
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Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
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Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
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What were the control measures?
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Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
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Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
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What type of samples were examined?
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Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
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Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
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What did they test for in the samples?
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Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
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Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
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What is the concentration of the pathogens?
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478
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Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
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Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
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What steps were taken to restore the problem?
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479
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Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
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Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
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Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
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Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
|
What could have been done to prevent the event?
|
{
"answer_start": [
1177
],
"text": [
"effective virological control of water"
]
}
|
481
|
Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
|
Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
|
How to prevent this?
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{
"answer_start": [
1177
],
"text": [
"effective virological control of water"
]
}
|
482
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Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
|
Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
|
What were the investigation steps?
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483
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Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
|
Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
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What did the investigation find?
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484
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Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
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Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
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How was the infrastructure affected?
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485
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Assessment of the Presence of Hepatitis E Virus in Surface Water and Drinking Water in Portugal
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Abstract: Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.
Keywords: drinking water; enteric viruses; Hepatitis E virus; RT-qPCR; surface water; Vero E6 cell line; viral infectivity; water quality; water treatment
1. Introduction
Diseases transmitted by water and food have high socioeconomic impacts on public health all around the world, especially at a time when climate change has caused alterations in the occurrence, distribution and seasonal variation of several pathogens, increasing the risk of exposure [1–3]. Viruses are among the most worrying groups of pathogens. Enteric viruses, which include, among others, enteroviruses, noroviruses, rotaviruses, Hepatitis A virus and Hepatitis E virus (HEV) are excreted in large quantities in human feces and transmitted mainly by the fecal–oral route [3–6]. They do not have a lipid envelope and possess a robust protein capsid, which is responsible for their high resistance to environmental stress such as heat, extreme pH, desiccation, and organic solvents [2]. Typical treatments used to inactivate/remove bacterial pathogens or enveloped viruses (for example, Influenza) in food and water, such as filtration and chemical oxidants, may not be effective against this group of viruses. In Korea, infectious enteric viruses such as adenovirus and enterovirus were detected in tap water, despite treatments [7]. Further, their stability also makes them quite resistant to the most common sanitation treatments and allows their maintenance in wastewater for long periods [2,8,9]. Recently, in Canada, several infectious enteric viruses, such as rotavirus, have been detected in surface water in the country’s largest rivers, where many wastewater sources are discharged [10].
According to the World Health Organization (WHO), enteric viruses have moderate to high significance in human health [4] and the existence of 20 million infections worldwide was estimated for HEV alone, with 3.3 million symptomatic cases of acute hepatitis E. In 2015, HEV caused approximately 44,000 deaths worldwide, representing 3.3% of viral hepatitis mortality [11]. In Europe, 68,000 HEV infections had recently been estimated in France, 100,000 in the United Kingdom and 300,000 in Germany, per year. The number of infections has been increasing dramatically, and now hepatitis E is considered an emerging disease in Europe and Asia [2,5].
HEV is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepeviridae family [12,13]. HEV is transmitted mainly by the consumption of contaminated water or contaminated undercooked or raw food such as vegetables, meat (pork, mutton, rabbit, poultry) and dairy products [2,5,14,15]. It is important to note that contaminated water is not only a cause of direct transmission but is also related to indirect transmission due to its use in agriculture practices, namely in the irrigation of vegetables, where the virus may accumulate and be delivered as infectious particles [16–20]. Although less frequent, HEV can also be transmitted by transfusions with contaminated blood and by vertical (mother-to-child) transmission [14,21,22]. HEV was responsible for both sporadic and epidemic outbreaks in several countries [23,24], and has been detected in natural waters from Colombia, Italy, and Sweden [9,25,26], in drinking water in Sweden and India [9,27], and in wastewater in Italy, Sweden, Pakistan, Colombia, Portugal and Spain [14,25,28–31].
After entering the human body, HEV is associated with clinical manifestations such as jaundice, vomiting, loss of appetite, fatigue, fever, darkened urine, hepatalgia and hepatomegaly. Although less common, this virus may also be associated with neurological complications such as Guillain–Barré syndrome, neuralgic amyotrophy, inflammatory polyradiculopathy, ataxia/encephalitis, and peripheral neuropathy. The mortality rate associated with HEV is approximately 2% in the general population, although in pregnant women the rate increases to 20% [21,32,33]. HEV infectious dose is not known [3]. A vaccine to prevent HEV infection was developed in China but is not yet available in most countries [11]. Antibiotics are ineffective and only a small number of antivirals, such as ribavirin have been indicated to treat infected individuals [34,35]. For all these reasons, there is a growing need for an effective surveillance system for the detection and quantification of this pathogen, mainly in water sources delivered to large populations, in order to reduce its transmission to humans and prevent potential epidemic situations with remarkable human and economic impacts (Figure 1) [3,5,36].
This study aimed to evaluate the presence of HEV in two sources of surface water and in drinking water sampled at two water treatment plants (WTPs), located at the central region of Portugal and at a point in the water distribution network. Our first approach was a quantitative molecular method (reverse transcription quantitative PCR (RT-qPCR)) applied to RNA extracted from sampled water in order to identify HEV-positive samples. A second approach, applied to HEV-positive samples, consisted of their inoculation in cultured cells (Vero E6 cell line); RNA was extracted from putative viral particles produced in these cells and HEV replication was detected/confirmed by RT-qPCR.
2. Materials and Methods
2.1. Study Sites
This study was carried out in two water matrices (surface water and drinking water) localized at the central region of Portugal. The sampling points of surface water were in a river and in a dam reservoir. Two sampling points of drinking water were located at the end of the treatment process in two water treatment plants (WTPs) and another one, at one point in the water distribution network. WTP_R treats surface water from the river and WTP_D treats surface water from the dam reservoir. In WTP_R, surface water goes through the following treatment processes: pre-oxidation with ozone, pH adjustment, activated carbon adsorption, coagulation/flocculation, sedimentation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP is composed of two independent treatment lines, each one with the capacity to produce 120,000 m3/day. In WTP_D, surface water undergoes the following treatment processes: pre-oxidation with chlorine, remineralization and correction of aggressiveness, coagulation, filtration with sand filters, pH correction and disinfection with chlorine. This WTP has two independent treatment lines with the capacity to produce 500,000 m3/day (line 1) and 125,000 m3/day (line 2). These two WTPs provide water for more than three million inhabitants [37].
2.2. Water Collection and Primary Concentration
The sampled water analyzed was collected and processed (concentrated) in 2019, between January and December. Sampling sites, dates and volumes of sampled water are indicated in Table 1. Large starting volumes of water (Table 1) were concentrated using NanoceramR PAC-AG electropositive filters (Argonite; Sanford, FL, USA) set up in housing chambers [38–40]. The housings with the filters immersed in water (500 mL) were transported refrigerated to the laboratory, as soon as possible and processed within 72 h.
2.3. Elution and Secondary Concentration The procedure was carried out according to EPA Method 1615 (EPA/600/R-10/181) with some modifications [41]. Before eluting the filter, 10 µL of Mengo virus solution (process control virus) with 105 copies/µL (bioMérieux; Marcy-l’Etoile, France) was added to the water inside the housing with the filter [42,43]; then, this water was passed through the filter to be discarded. Following this procedure, the NanoceramR PAC-AG filters (Argonite; Sanford, FL, USA) were eluted with 1 L of 3% beef extract (BD Bioscience; Franklin Lakes, NJ, USA). The resulting eluted solution was subjected to an organic flocculation process with pH adjustment to 3.5, followed by centrifugation at 2500× g, 4 ◦C for 15 min The pellet was resuspended in sodium phosphate pH 7.0–7.5 and, after adjusting the pH to 9.0, a new centrifugation was performed at 5000× g, 4 ◦C for 10 min. The resulting supernatant was transferred to a new tube and the pH adjusted to 7.0–7.5. Samples were filtered through Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA) with a pore size of 0.22 µm and the resulting volume (35–40 mL) was divided into three parts: 20 mL for RNA extraction and further evaluation by RT-qPCR, 10 mL for inoculation into cell cultures and 5 to 10 mL for storage. The samples were kept at −70 ◦C until use.
2.4. Tertiary Concentration and Nucleic Acid Extraction
Samples reserved for RT-qPCR analysis (20 mL) were thawed and applied to VivaspinR concentrators (Sartorius; Goettingen, Germany) that were centrifuged at 8000× g and 4 ◦C for several hours including, in the final, two wash steps with 1 mL of 1 M sodium phosphate (pH 7.0–7.5), until the sample volume was less than 1 mL. The final concentrate was transferred from the VivaspinR concentrator to a 1.5 mL microtube. All final concentrates were subjected to RNA extraction and purification with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany) [44,45] according to the manufacturer’s instructions.
2.5. Detection and Quantification of Viral Genomes
RT-qPCR amplifications were performed in a StepOnePlus thermocycler (Applied Biosystems; Foster City, CA, USA), in reaction mixtures of 25 µL containing 5 µL of extracted RNA. Each template RNA was assayed in duplicate and negative controls without nucleic acid as well as positive controls were introduced in each run. HEV and Mengo virus were assayed with a CeeramTools Hepatitis E kit (bioMérieux; Marcy-l’Etoile, France) and a Mengo virus Extraction Control kit (bioMérieux; Marcy-l’Etoile, France), respectively. HEV amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Mengo virus amplification conditions were reverse transcription at 45 ◦C for 10 min, enzyme activation at 95 ◦C for 10 min, followed by 45 cycles of amplification with denaturation at 95 ◦C for 15 s and data collection at 60 ◦C for 45 s. Quantification of HEV was estimated by standard curves, with five points constructed with serial dilutions (1:10) of control HEV RNA (CeeramTools Hepatitis E Standard kit; bioMérieux; Marcy-l’Etoile, France). Mengo virus quantification was performed with a four-point quantitation curve (0.1%, 1%, 10%, and 100%), prepared with RNA extracted from 10 µL of Mengo virus solution (105 copies) with the QIAamp viral RNA Mini kit (Qiagen; Hilden, Germany). Only the results that met the quality criteria established by the mentioned kits were considered. Values given were the average of the results obtained in two independent RT-qPCR reactions. Initial results, expressed in genomic copies per five microliters of RNA (gc/5µL) in the reaction mixture, were converted in number of genomic copies per liter (gc/L) of sampled water, based on the data presented in Table 1. For Mengo virus, the results were expressed in percentage (%) of genomic copies (according to the quantitation curve).
2.6. Infectivity Assays
Concentrated water samples reserved for assays of infectivity were thawed and maintained at 4 ◦C, until inoculation into Vero E6 cultures (Vero C 1008, ATCC CRL–1586) [46,47]. Cells were grown at 37 ◦C in 25 cm2 flasks (T25) in the following culture medium (culture medium, FBS10)
CO2-Independent Medium (Gibco, Thermo Fisher Scientific; Waltham, MA, USA) with additional 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific; Waltham, MA, USA), and 0.5 mg/mL gentamicin (Gibco, Thermo Fisher Scientific; Waltham, MA, USA). Cells were routinely sub-cultured at confluency, by action of TrypLE Express cell dissociation reagent (Thermo Fisher Scientific; Waltham, MA, USA). Cell inoculations with water samples were carried out in identical culture conditions, except that the concentration of FBS was 2% instead. The original culture medium was discarded from each T25 with a sub-confluent cell culture, prior to its inoculation with 1 mL of water sample diluted 1:2 in culture medium, FBS2. After 3 h of incubation with gentle agitation, 4 mL of FBS2 was added and incubations occurred until a cytopathic effect (CPE) was observed or for a maximum period of 15 days. Cultures were observed daily and when longer incubations were needed, there was a replacement of the culture medium every 5 days, keeping the previous medium refrigerated. Finally, cells (in suspension or manually detached) and culture medium were frozen/thawed (-70 ◦C/37 ◦C) twice and centrifuged at 3000× g for 5 min. Supernatants with the first-passage (P1) intra and extracellular putative viral particles (pVPs) were used in a new round of inoculations, performed as described above, in order to produce pVPs from a second passage (P2). However, P2 pVPs differ from P1 pVPs because at the end of the incubation period, the culture medium with cells was directly subjected to centrifugation to recover the supernatant, mostly consisting of extracellular pVPs. The P2 pVPs were subjected to a 6 h centrifugation at 17,000× g and 4 ◦C and the pellets were suspended in 140 µL of phosphate-buffered saline (PBS) in order to be processed for RNA extraction and purification, as indicated in 2.4. RT-qPCR targeting HEV RNA was performed as described in 2.5.
2.7. Statistical Analyses
All statistical analyses were conducted using Microsoft Excel 2017 (Microsoft Inc., Redmond, WS, USA) and IBM SPSS Statistics (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was applied to assess the normality of the data within each data set; a p-value greater than 0.05 was indicative of a normal distribution. Once the normality was confirmed, the Student’s t-test could be used to compare the data. Differences were considered significant for a p-value less than 0.05.
3. Results
3.1. RT-qPCR HEV RNA Detection and Quantification in Water Samples
3.1.1. HEV RNA in Surface Water Samples
From the 27 concentrated samples from surface water, HEV RNA was detected and quantified in 21 (77.8%) (Figures 2 and 3).
In the river, in February, March, August, September, and October, two sampling campaigns were carried out (in the first and second half of each month) and, of the 17 concentrated samples, 15 (88.2%) were positive for HEV RNA (Figure 2a). HEV RNA was detected in 11 of the 12 months of campaign, in concentrations fluctuating between 0 and 7383.2 gc/L, with a maximum in April (7383.2 gc/L) and a very sharp decrease from August to December (Figure 2a).
In the 10 concentrated surface water samples from the dam reservoir, HEV RNA was detected in six (60%) (Figure 2b). HEV RNA was detected and quantified in concentrations ranging between 0 and 10,9687.5 gc/L (the highest concentration, which was in April), with a concentration lower than 2411.9 gc/L in the remaining months (Figure 2b).
3.1.2. HEV RNA in Drinking Water Samples
From the 36 samples of concentrated drinking water, HEV RNA was detected and quantified in 24 (66.7%) (Figures 2 and 3).
In WTP_R, two sampling campaigns were carried out in February, March, August, September, and October (in the first and second half of each month). HEV RNA was detected in 11 out of 17 concentrated samples (64.7%) (Figure 2a), presenting its highest concentration (2,379.3 gc/L) in April, which then declined sharply to 0. It was not detected in January, March (first and second half), August and October (second half in both months), and December.
In the 10 concentrated drinking water samples from WTP_D, HEV RNA was detected in five (50%), corresponding to five months (Figure 2b) and was not detected in January, March, May, November, and December. From 75.151 gc/L detected in February and a maximum concentration of 5617.1 gc/L reached in April, HEV RNA concentrations significantly decreased, presenting values between 58.725 gc/L to no detection, from May to December.
In the concentrated samples of drinking water from the sampling point in the distribution network, HEV RNA was detected in eight out of nine samples (88.9%) (eight months) (Figure 3), in concentrations fluctuating between 5645 and 8926.6 gc/L. The highest concentration occurred in April and then decreased until December. It was not detected in February.
3.2. Evaluation of the Efficacy of Water Treatment Plants (WTPs) in HEV RNA Elimination
3.2.1. River vs WTP_R
In January and the first half of March, HEV RNA was not detected either in the surface water of the river or in its treated drinking water from WTP_R. Along the remaining months (15 sampling dates), HEV RNA was detected in the river, always in higher concentrations than in the WTP_R (9.8–100% reduction, as shown in Table 2). The lowest reduction values were obtained in February (9.8 and 37%, in the first and in the second half, respectively), while the highest values (100%) were obtained in the second half of March, August, October, and December; values higher than 87.6% were obtained in the first half of these months. High reduction values (>90%) were also obtained in four other situations (June, July, and in the first and second half of September), while intermediate values were obtained in the remaining comparisons (77.9 and 67.8%, in May and April, respectively). There were statistically significant differences in the concentrations of HEV RNA between samples from the river and from WPT_R, i.e., between non-treated and treated water samples (Student’s t-test, p < 0.05).
3.2.2. Dam Reservoir vs WTP_D In January, March, and December, HEV RNA was not detected either in surface water from the dam reservoir or in its treated drinking water from WTP_D, and in February and June, there was no reduction with the treatment. However, a reduction of 73.3% was observed in September and a reduction of over 94.9% in April, May, October, and November (Table 2). There were no statistically significant differences in the concentrations of HEV RNA between samples from the dam reservoir and from WTP_D, i.e., between non-treated and treated water samples (Student’s t-test, p > 0.05).
3.3. Recovery of the Process Control Virus (Mengo Virus) in the Water Samples Subjected to the Survey
Mengo virus recovery was evaluated by RT-qPCR in the 59 water samples subjected to this survey
25 from surface water and 34 from drinking water. The percentages of recovery were low (0.1–5.0%) in most samples (63%), and it was not even detected in 25%. Recoveries higher than 5% were found in 12% of the samples (Figure 4a). The results, shown in Figure 4b, evidence much lower recoveries in surface water samples than in drinking water samples. There were statistically significant differences in the recovery of Mengo virus between surface water and drinking water (Student’s t-test, p < 0.05).
3.4. Potential Infectivity of the Water Samples
3.4.1. Effect on Vero E6 Cultures and Production of Putative Viral Particles
Thirty-four concentrated samples from water sampled in 2019, between January and August and covering all sampling sites, were considered for infectivity assays in Vero E6 cultures. However, samples previously identified as negative for HEV RNA were not selected, except when the related sample (collected on the same date in the associated matrix) was positive. This was the case of the four HEV RNA negative samples, whose infectivity results are shown in Tables 3 and 4.
Most cultures (19 in 32) did not develop CPEs during the incubation period (15 days). Nevertheless, putative viral particles (pVPs) from this first passage were collected and used to infect new cultures. From these, 17 developed CPEs within 2–6 days post-inoculation; pVPs from this second passage, with or without CPEs, were collected and subjected to RNA extraction.
3.4.2. RT-qPCR Evaluation of Putative Infectious HEV Produced in Vero E6 Cultures
RNA extracted from pVPs produced as referred to above was subjected to RT-qPCR evaluation. From the samples evaluated, 18 were related samples from the river and WTP_R (eight from each) (Table 3), eight from the dam reservoir and WTP_D (four from each) (Table 4), and six were from the sampling point in the distribution network (Table 5). HEV infectivity was confirmed in samples from all matrixes (globally 25%): 3/13 (23.0%) from surface water were positive (two from the river and one from the dam reservoir: 22.2% and 25.0%, respectively) as well as 5/18 (27.7%) from drinking water (three from WTP_R, one from WTP_D and one from the sampling point in the distribution network
33.3%, 25.0% and 16.6%, respectively) (Tables 3–5).
It was also possible to determine that 1) most positive samples for HEV infectivity had also tested positive for HEV RNA (exceptions were WTP_R from August and dam reservoir from June) and 2) positive samples for HEV infectivity were frequently found in related samples, i.e., in river/WTP_R and dam reservoir/WTP_D sampled on the same date (one exception was found in river/WTP_R from May, where only WTP_R was positive for infectious HEV) (Tables 3–5). Moreover, a relationship was not evidenced between the number of RNA copies detected in a water sample and its potential infectivity because, from the 11 samples presenting more than 1000 gc/L, only one (river from June) evidenced infectivity; values of gc/L between 0 and 428 had been found in all the others able to produce infectious HEV in Vero E6 cells.
4. Discussion
Although a preliminary work, this study followed a complex and complete approach in order to assess the presence of HEV, starting from high volumes of water and combining, in the same procedure, the possibility to detect viral RNA by RT-qPCR as well as evaluate infectivity. Other methods are limited to a maximum starting volume of five liters of water, as described in ISO 15216-1:2017 [42,43], but EPA Method 1615 of the United States Environmental Protection Agency [41], which supported this experimental protocol, is based on the collection of much larger volumes, making the results more reliable [40,48]. The use of cell cultures also overcame the limitation of evaluations based only on RT-qPCR. In fact, RT-qPCR has been increasingly used to detect enteric viruses in water and food samples, with high specificity/sensitivity and the possibility of obtaining results in less than four hours [3,48,49]. However, this methodology does not allow assessing the infectivity associated with the viral genomes detected in the reaction [3,25,40]. Beyond the confirmation of viral genomes, it is crucial in the evaluation of risks to public health in order to determine whether they correspond to viral particles with the ability to infect human cells as well [48–51]. Despite being expensive and time consuming, relying on cell cultures, it is the most used standard method for assessing the infectivity of viral particles, by observing cytopathic effects (CPEs) [3,6,52].
This one-year survey evaluated the presence of HEV in concentrated samples from two bodies of water (a river and a dam reservoir) and from the drinking water sampled on their water treatment plants (WTP_R and WTP_D, respectively) at the end of the treatment process. A mammal cell line (Vero E6) derived from African green monkey (Cercopithecus aethiops) kidney was used for the first time in order to assay the potential infectivity of water samples where HEV RNA had been detected by RT-qPCR. The rationale for the utilization of this cell line was its capability to replicate many different viruses [46,53], also taking into account that HEV has a large host range [12,15]. This approach effectively resulted in the detection of infectious HEV in several samples, by induction of CPEs in cultured cells with subsequent confirmation of HEV replication through RT-qPCR to RNA extracted from extracellular viral particles. Our results agree with a recent study [54] demonstrating that a wild-derived HEV strain replicated in Vero cells, the cell line from which Vero E6 was derived (46).
HEV was detected in concentrated samples from the two bodies of water, and in an infectious state in some of these samples. Comparing the two bodies of water, the river showed a greater number of positive samples for HEV RNA (88,2%) than the dam reservoir (60%). On the other hand, in both, the maximum concentration (>100,000 gc/L, in the dam reservoir) occurred in April (Spring in Portugal) and decreased until December. The maximum concentrations in April can be explained by the pattern of precipitation in Portugal, as described for Colombia [25]. In April 2019, rainfall was high, the fifth rainiest April since 2000 [55] and, associated with more precipitation, a greater runoff of possible contaminants from the surrounding areas may have disturbed these water bodies [56]. Many of the areas surrounding the sampling sites are places of agriculture and animal production [57], where manure from animal production activities is still used as fertilizer. It is well known that pigs are reservoirs of HEV, which is excreted in feces, and might thus be the possible origin of water contamination [2,58,59]. Concentrated surface water samples also presented infectious HEV in higher percentages (33.3%) in samples from the river than in samples from the dam reservoir (25%). Although the peak HEV RNA concentration was found in April, those samples did not produce infectious HEV in Vero E6 cells. Nevertheless, infectious HEV was detected in both bodies of water in June, when HEV RNA concentrations were high in the river (>1000 gc/L) and zero in the dam reservoir, evidencing inexistence of a direct association between the number of detected RNA copies and potential infectivity.
In drinking water, HEV RNA was also detected, although in a smaller number of concentrated water samples (64.7% in WTP_R, and 50.0% in WTP_D) than was observed in the bodies of water, as also found in Switzerland [9]. After analyzing the concentrated samples of drinking water from the two WTPs and from a point in the distribution network, there was an identical pattern: the peak HEV RNA concentration was detected in April, and subsequently decreased until the end of the year. These results are in complete accordance with what was previously described for the bodies of water that feed these WTPs and the point in the distribution network. Likewise, the highest HEV RNA concentrations were found in two related water matrixes: the dam reservoir and WTP_D. Out of the 18 concentrated drinking water samples selected for evaluation of HEV infectivity, 27.7% were positive. Drinking water from WTP_R presented the highest number of infectious samples (three), followed by WTP_D and the water from the point in the distribution network, both with only one infectious sample. Infectious HEV was detected in samples collected between May and August, after the peak of HEV RNA copies, as also observed in the bodies of water. No clear relationship was found between infectivity and number of HEV RNA copies detected per liter of sampled water.
Even though most of the results did not evidence contradictory aspects, a few should be discussed. One unexpected result was the infectivity of two samples (one from the dam reservoir and the other from the WTP_R) originally identified as negative for HEV RNA. This may be explained by eventual mishaps during the original RNA extraction procedures and emphasizes the relevance of evaluating results achieved by independent approaches. The detection of an infectious drinking water sample (WTP_R) was also unexpected, although the sampled water from its source (river) did not show infectivity. This may be explained by the large differences in the water volumes subjected to sampling
1300 L in the WTP_R and 152 L in the river. It should be noted that the positive result of infectivity in WTP_R means that HEV was also present in the river to such an extent that infectivity remained after treatment.
The results showing the presence of infectious HEV in concentrated samples of drinking water evidence the need to further investigate eventual threats to human health. It is worth noting that Vero E6 cultures were inoculated with 0.5 mL of concentrated (40×, in average) drinking water samples, equivalent to approximately 17.5 L of the sampled water (1400 L, in average). Actually, while healthy individuals drink approximately two liters of water each day [60], this study, as referred above, was conducted with an average volume of 1400 L.
High values of genomic copies per liter found in several samples of natural water may also be attributed to the large volumes sampled. These large volumes were considered necessary to recover enough viruses to allow a reliable quantification based on the low levels of enteric viruses often reported for natural waters.
Low recovery percentages of the process control virus (Mengo virus) used for validation of the experimental procedure [61,62] were often found, as also occurred in Kyria da Silva et al. [63] and Teixeira et al. [43]. Recoveries were even lower in the untreated water, which may be related to the great number of particles and other interfering materials usually present [64]. These results suggest that Mengo virus was not a good recovery control despite the relevance that may be attributed to its presence at the end of the process for the validation of the eventual negative results of the viruses under detection.
Regarding the efficacy of the WTPs in reducing HEV, based only on genome copies, this was observed in both situations, but only with statistically significant differences in WTP_R. Differences in the water volumes sampled in the two types of matrix, as discussed above and evidenced in Table 1, may explain less marked differences. Differences in eliminating viruses may also be attributed to difficulties related to the manipulation of the sample during the various stages of filtration and concentration, including the characteristics of each water matrix. Moreover, the values obtained by RT-qPCR cannot be interpreted as absolute, since there are many steps in the experimental procedures that may lead to RNA degradation [48]. In any case, these results may indicate that the treatment at WTP_R, including pre-oxidation with ozone, adsorption with coal and disinfection with chlorine, may be more effective in the elimination of this virus than WTP_D, which uses neither ozone nor adsorption with coal [25,65,66]. Ozone may be crucial in the control of viruses in water, as found in other studies [67,68], concerning the elimination of enteric viruses and bacteriophages. However, more studies will be needed to confirm this statement.
Finally, climate change will certainly increase the frequency of pathogens in water systems worldwide, whether due to the occurrence of floods, sewage contamination or the scarcity of safe drinking water sources [3]. In this context and considering the results obtained in this study, monitoring the presence of HEV and other viruses in water supply and distribution systems is advisable. A similar approach should be conducted, increasing the sampling effort (number of samples and geographical regions during the entire year to allow the detection of possible seasonality patterns) and implementing the application of the quantitative microbial risk assessment (QMRA) [69].
5. Conclusions
HEV has is gaining an increasing presence in developed societies. As in many other countries in Europe and Asia, this virus also circulates in Portuguese waters. The results of this survey highlight the need for systematic monitoring of the presence of HEV and other emerging enteric viruses in surface and treated waters. It is also recommended to carry out studies targeting water treatment methods to better understand the influence of the various stages of the elimination/inactivation of these viruses in WTPs.
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What were the infrastructure complaints?
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486
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Compliance with boil water advice following a water contamination incident in the Netherlands in 2007
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In May 2007, Escherichia coli was detected in tap water supplied by a company in North Holland. The company issued advice through mass media to boil tap water before consumption; this advice was lifted six days later. A cross-sectional study was implemented to investigate compliance among residents in this area. Based on postcode, a total of 300 households, chosen randomly from a database of a private company performing internet-based surveys for different marketing purposes, were sent a self-administered questionnaire for this study. The questionnaire contained questions on demographic information, source of information regarding the advice, response to it and personal opinions on the company’s reaction and the advice. Ninety-nine (66%) households of the affected area and 90 (60%) households from non-affected areas served by the same company replied to the survey. All respondents knew about the advice. 81.8% of the respondents in the affected area and 5.6% of the non-affected areas reported complying with the advisory. Most respondents from the affected area still used unboiled water to brush teeth, wash salads and fruits. There was no difference in compliance between men and women. Using the mass media was proved to be efficient to inform the public and could be used in the future in similar settings. However, more detailed wording of boiling advices should be considered in the future.
Introduction
Consumption of drinking water may cause waterborne disease which can be prevented by protection of the source water, efî¬cient treatment processes and reliable distribution systems. The European Union Drinking Water Directive [1] demands monitoring of tap water for different parameters, such as Escherichia coli, to indicate possible faecal contamination from humans and animals.
System failure or human error may cause an increase in the level of pathogens in the water posing a risk of waterborne disease. For example, in 2001, a large outbreak of gastroenteritis occurred due to accidental introduction of partially treated water to the drinking water supply system in the Netherlands, resulting in 921 households being exposed to contaminated water [2].
In the event that faecal contamination is detected the drinking water company may issue an advice to boil tap water before using it for domestic purposes. On 15 May 2007, E. coli was detected in samples collected the day before of the î¬nished tap water delivered by a company in the province Noord-Holland (North-Holland) in the Netherlands. For preventive reasons, on the same day the company issued an advice for consumers to boil tap water for two minutes before consumption but that this was not necessary for taking a shower or washing. This information was broadcasted through mass-media including the national and regional television channel, radio and newspapers. In addition, a public website used during emergency situations (www.crisis.nl) and a toll-free telephone number were made available for the public to provide information to households in the affected area.
The boil water advice had an impact on approximately 180,000 households in the affected area comprising 13 municipalities. The advice was lifted a week later, on 22 May 2007, as risk for public health was no longer present. In September 2007, the water company published a press release informing that the cause of the water contamination was due to run-off of rainwater contaminated with faeces of breeding gulls on the roof that had seeped into one of the six storage rooms [3].
Elevated levels of microorganisms in drinking water may represent a public health risk. For this reason, we investigated compliance with boil water advice issued by the private water company following the 2007 incident.
Methods
A cross-sectional study was implemented to investigate factors that may have affected water consumption habits of the residents in the area supplied by the water company. For this purpose, on the company’s behalf, a self-administered questionnaire was sent to 300 households in June 2007. Households were selected on the basis of their residence postcodes; half in the area where the advice was valid and half in areas served by the same company but where the advice did not apply. These participants were derived from a database of a private company that conducts online consumer surveys for marketing purposes.
The questionnaire contained questions on demographic information, level of urbanisation, source and time of receiving the information regarding the advice, initial and secondary response to the advice and personal opinions on the company’s response and the advice itself. The data were sent back to the drinking water company and the National Institute for Public Health and the Environment, where they were analysed. The statistical analysis was done with STATA v10.
Results
Ninety-nine households (66%) from the area affected by water contamination and 90 households (60%) from control areas supplied with water by the same company replied to the survey. Women more often than men responded to the questionnaire in both the affected and the non-affected areas (57.7% of all responders). The respondents represented 189 households with a total population of 505 people, 176 (34.9%) of whom were below the age of 18 years. There was no statistically signiî¬cant difference in the number of children per household between the affected and the non-affected areas (p=0.112). Descriptive results for the two different areas are presented in Table 1.
All 189 respondents (100%) in both areas answered that they had been informed about the advice. Ninety-î¬ve (50.3%) of them said they had first heard about it through the television. Other sources were radio (24.3%), friends, relatives or neighbours (22.8%), newspapers (19.6%) and the internet (7.4%).
Persons living in the affected area were more frequently disappointed (14.1%) about the choice of the company to use mass media for the advice than people residing in the non-affected area (2.2%). In the affected area, seven (9.3%) of the respondents had î¬rst reacted with fear to the information on the possible contamination of water, 34 (45.3%) responded with self-control and 34 (45.3%) with the intention to take measures. The corresponding percentages for the non affected area were 15.7%, 72.9% and 11.4%. About half (48.5%) of the respondents from the affected area said they had looked for more information when they had heard about the advice, while the corresponding proportion of respondents from the non-affected area was only 8.9% (p<0.001). The most common source of active search for more information was the website of the water supply company.
Eighty-one (81.8%) of all respondents in the affected area said they had complied with the advice. This was done by buying bottled water (43.4% of all respondents in affected area) or boiling tap water for two minutes before consuming it (70.7%). None of the respondents in the area stopped consuming tap water completely. Five (5.6%) of the respondents in the non-affected area were buying bottled water and three of them (3.3%) were boiling tap water during the advice. These numbers were considerably lower than the corresponding ones in the affected area, but showed that compliance exceeded beyond the affected area.
Even though it had not been advised to boil water for activities such as washing and showering, 26 (26.3%) of the respondents in the affected area stated that they had not been aware of that.
Concerning the image of the drinking water company, 177 respondents (93.7%) thought that the company had done well informing the consumers about the water contamination and its response to it. This prevailing opinion was not different between respondents from the affected area and those from the non affected area.
The respondents’ compliance with the advice was independent of sex, age and the presence of children in the household. However, the respondents were 138.6 times more likely to follow the advice if a second person in the household was following it as well (p<0.001).
Reasons for non-compliance with the advice are given in Table 2.Some of the respondents replied that they had been using boiled water for uses other than drinking, too. These results are shown in Table 3.The majority of the respondents stated that their image of the company had not changed after the incident and the six-day advice (78.8% in the affected area and 88.9% in the non-affected area).
Factors affecting compliance
The type of mass media from which people in the affected area found out about the advice played no signiî¬cant role in the subsequent compliance of the respondents. The highest compliance rates occurred among those in the affected area who heard about the advice from the internet (90%) or from friends (89.5%). Respondents informed by more than one source were more likely to have complied with the advice (90.9% against 79.2%) but this difference was not statistically signiî¬cant. The source of information did not depend on the age (p=0.6532). Compliance with the advice did not differ between households with children and those without children (p=0.536).
Respondents who undertook active search for more information may have been more likely to follow the advice than those who did not proceed to further active search for more information (89.4% vs. 74.5%, p=0.058).Since all respondents knew about the advice, it was not possible to estimate unwitting compliance rates.
Conclusions
Since excess of standard levels of certain microorganisms such as E. coli indicate faecal contamination and the possible presence of pathogens in tap water, the time between the water sampling, water analysis and the boil water notice is essential. During this period, consumers may be exposed to tap water of unacceptable quality. The choice of mass media for broadcasting the advice is therefore believed to be an effective measure to prevent panic and to protect public health.
From this study, it can be concluded that participating consumers not only thought that they had been informed about the advice in a timely manner, but that also the response of the company to ensure the advice would reach the public had been satisfactory as well as the choice of communication channels. Thus, the incident did not lead to customers’ dissatisfaction or a degradation of the company’s image.
The sample in our study derived from a database of people who subscribed to be included in different research surveys. This could raise questions regarding the representativeness of the study population. We agree that there is a need for similar studies with samples deriving randomly from the whole population and not from potentially biased data sources. For example, 100% of the participants stated that they had been informed about the boiling water advice; however, subscribers to online databases for marketing purposes may be more likely to regularly follow the news than the general population.
In the Netherlands, boil water notices are not harmonised but are determined by the drinking water company itself. This results in different advice with respect to, for instance, boiling time. Internationally recognised guidelines, such as the World Health Organization (WHO) Guidelines for Drinking Water Quality [4], could be taken into consideration in case of similar “crises†in the future. According to data from the water company involved in our study, about thirty boil water advices are issued per year in their responsibility area (ca. 700,000 households); involving on average 100 households per time. So, the chance to receive a boil water advice is small but existing.
The inclusion of recommendations including use of water for brushing teeth, washing fruits and vegetables may also prove helpful in future advice, since it is not only consumption of water through drinking that may pose a risk to the consumer. Bathing and showering may also need to be addressed separately, as a possible link between this kind of exposure to contaminated water and itching has been described elsewhere [2]. Also, although this conclusion does not directly follow from our results, vulnerable groups should be targeted separately in the advice; elderly people and children may easily miss information disseminated through the means of mass media [5,6].
Few studies have been published on boil water notices and their results seldom reach the public. Further research would also be useful to incorporate î¬ndings from compliance studies to model health effects of drinking contaminated water during similar events.
|
What happened?
|
{
"answer_start": [
13
],
"text": [
"Escherichia coli was detected in tap water"
]
}
|
487
|
Compliance with boil water advice following a water contamination incident in the Netherlands in 2007
|
In May 2007, Escherichia coli was detected in tap water supplied by a company in North Holland. The company issued advice through mass media to boil tap water before consumption; this advice was lifted six days later. A cross-sectional study was implemented to investigate compliance among residents in this area. Based on postcode, a total of 300 households, chosen randomly from a database of a private company performing internet-based surveys for different marketing purposes, were sent a self-administered questionnaire for this study. The questionnaire contained questions on demographic information, source of information regarding the advice, response to it and personal opinions on the company’s reaction and the advice. Ninety-nine (66%) households of the affected area and 90 (60%) households from non-affected areas served by the same company replied to the survey. All respondents knew about the advice. 81.8% of the respondents in the affected area and 5.6% of the non-affected areas reported complying with the advisory. Most respondents from the affected area still used unboiled water to brush teeth, wash salads and fruits. There was no difference in compliance between men and women. Using the mass media was proved to be efficient to inform the public and could be used in the future in similar settings. However, more detailed wording of boiling advices should be considered in the future.
Introduction
Consumption of drinking water may cause waterborne disease which can be prevented by protection of the source water, efî¬cient treatment processes and reliable distribution systems. The European Union Drinking Water Directive [1] demands monitoring of tap water for different parameters, such as Escherichia coli, to indicate possible faecal contamination from humans and animals.
System failure or human error may cause an increase in the level of pathogens in the water posing a risk of waterborne disease. For example, in 2001, a large outbreak of gastroenteritis occurred due to accidental introduction of partially treated water to the drinking water supply system in the Netherlands, resulting in 921 households being exposed to contaminated water [2].
In the event that faecal contamination is detected the drinking water company may issue an advice to boil tap water before using it for domestic purposes. On 15 May 2007, E. coli was detected in samples collected the day before of the î¬nished tap water delivered by a company in the province Noord-Holland (North-Holland) in the Netherlands. For preventive reasons, on the same day the company issued an advice for consumers to boil tap water for two minutes before consumption but that this was not necessary for taking a shower or washing. This information was broadcasted through mass-media including the national and regional television channel, radio and newspapers. In addition, a public website used during emergency situations (www.crisis.nl) and a toll-free telephone number were made available for the public to provide information to households in the affected area.
The boil water advice had an impact on approximately 180,000 households in the affected area comprising 13 municipalities. The advice was lifted a week later, on 22 May 2007, as risk for public health was no longer present. In September 2007, the water company published a press release informing that the cause of the water contamination was due to run-off of rainwater contaminated with faeces of breeding gulls on the roof that had seeped into one of the six storage rooms [3].
Elevated levels of microorganisms in drinking water may represent a public health risk. For this reason, we investigated compliance with boil water advice issued by the private water company following the 2007 incident.
Methods
A cross-sectional study was implemented to investigate factors that may have affected water consumption habits of the residents in the area supplied by the water company. For this purpose, on the company’s behalf, a self-administered questionnaire was sent to 300 households in June 2007. Households were selected on the basis of their residence postcodes; half in the area where the advice was valid and half in areas served by the same company but where the advice did not apply. These participants were derived from a database of a private company that conducts online consumer surveys for marketing purposes.
The questionnaire contained questions on demographic information, level of urbanisation, source and time of receiving the information regarding the advice, initial and secondary response to the advice and personal opinions on the company’s response and the advice itself. The data were sent back to the drinking water company and the National Institute for Public Health and the Environment, where they were analysed. The statistical analysis was done with STATA v10.
Results
Ninety-nine households (66%) from the area affected by water contamination and 90 households (60%) from control areas supplied with water by the same company replied to the survey. Women more often than men responded to the questionnaire in both the affected and the non-affected areas (57.7% of all responders). The respondents represented 189 households with a total population of 505 people, 176 (34.9%) of whom were below the age of 18 years. There was no statistically signiî¬cant difference in the number of children per household between the affected and the non-affected areas (p=0.112). Descriptive results for the two different areas are presented in Table 1.
All 189 respondents (100%) in both areas answered that they had been informed about the advice. Ninety-î¬ve (50.3%) of them said they had first heard about it through the television. Other sources were radio (24.3%), friends, relatives or neighbours (22.8%), newspapers (19.6%) and the internet (7.4%).
Persons living in the affected area were more frequently disappointed (14.1%) about the choice of the company to use mass media for the advice than people residing in the non-affected area (2.2%). In the affected area, seven (9.3%) of the respondents had î¬rst reacted with fear to the information on the possible contamination of water, 34 (45.3%) responded with self-control and 34 (45.3%) with the intention to take measures. The corresponding percentages for the non affected area were 15.7%, 72.9% and 11.4%. About half (48.5%) of the respondents from the affected area said they had looked for more information when they had heard about the advice, while the corresponding proportion of respondents from the non-affected area was only 8.9% (p<0.001). The most common source of active search for more information was the website of the water supply company.
Eighty-one (81.8%) of all respondents in the affected area said they had complied with the advice. This was done by buying bottled water (43.4% of all respondents in affected area) or boiling tap water for two minutes before consuming it (70.7%). None of the respondents in the area stopped consuming tap water completely. Five (5.6%) of the respondents in the non-affected area were buying bottled water and three of them (3.3%) were boiling tap water during the advice. These numbers were considerably lower than the corresponding ones in the affected area, but showed that compliance exceeded beyond the affected area.
Even though it had not been advised to boil water for activities such as washing and showering, 26 (26.3%) of the respondents in the affected area stated that they had not been aware of that.
Concerning the image of the drinking water company, 177 respondents (93.7%) thought that the company had done well informing the consumers about the water contamination and its response to it. This prevailing opinion was not different between respondents from the affected area and those from the non affected area.
The respondents’ compliance with the advice was independent of sex, age and the presence of children in the household. However, the respondents were 138.6 times more likely to follow the advice if a second person in the household was following it as well (p<0.001).
Reasons for non-compliance with the advice are given in Table 2.Some of the respondents replied that they had been using boiled water for uses other than drinking, too. These results are shown in Table 3.The majority of the respondents stated that their image of the company had not changed after the incident and the six-day advice (78.8% in the affected area and 88.9% in the non-affected area).
Factors affecting compliance
The type of mass media from which people in the affected area found out about the advice played no signiî¬cant role in the subsequent compliance of the respondents. The highest compliance rates occurred among those in the affected area who heard about the advice from the internet (90%) or from friends (89.5%). Respondents informed by more than one source were more likely to have complied with the advice (90.9% against 79.2%) but this difference was not statistically signiî¬cant. The source of information did not depend on the age (p=0.6532). Compliance with the advice did not differ between households with children and those without children (p=0.536).
Respondents who undertook active search for more information may have been more likely to follow the advice than those who did not proceed to further active search for more information (89.4% vs. 74.5%, p=0.058).Since all respondents knew about the advice, it was not possible to estimate unwitting compliance rates.
Conclusions
Since excess of standard levels of certain microorganisms such as E. coli indicate faecal contamination and the possible presence of pathogens in tap water, the time between the water sampling, water analysis and the boil water notice is essential. During this period, consumers may be exposed to tap water of unacceptable quality. The choice of mass media for broadcasting the advice is therefore believed to be an effective measure to prevent panic and to protect public health.
From this study, it can be concluded that participating consumers not only thought that they had been informed about the advice in a timely manner, but that also the response of the company to ensure the advice would reach the public had been satisfactory as well as the choice of communication channels. Thus, the incident did not lead to customers’ dissatisfaction or a degradation of the company’s image.
The sample in our study derived from a database of people who subscribed to be included in different research surveys. This could raise questions regarding the representativeness of the study population. We agree that there is a need for similar studies with samples deriving randomly from the whole population and not from potentially biased data sources. For example, 100% of the participants stated that they had been informed about the boiling water advice; however, subscribers to online databases for marketing purposes may be more likely to regularly follow the news than the general population.
In the Netherlands, boil water notices are not harmonised but are determined by the drinking water company itself. This results in different advice with respect to, for instance, boiling time. Internationally recognised guidelines, such as the World Health Organization (WHO) Guidelines for Drinking Water Quality [4], could be taken into consideration in case of similar “crises†in the future. According to data from the water company involved in our study, about thirty boil water advices are issued per year in their responsibility area (ca. 700,000 households); involving on average 100 households per time. So, the chance to receive a boil water advice is small but existing.
The inclusion of recommendations including use of water for brushing teeth, washing fruits and vegetables may also prove helpful in future advice, since it is not only consumption of water through drinking that may pose a risk to the consumer. Bathing and showering may also need to be addressed separately, as a possible link between this kind of exposure to contaminated water and itching has been described elsewhere [2]. Also, although this conclusion does not directly follow from our results, vulnerable groups should be targeted separately in the advice; elderly people and children may easily miss information disseminated through the means of mass media [5,6].
Few studies have been published on boil water notices and their results seldom reach the public. Further research would also be useful to incorporate î¬ndings from compliance studies to model health effects of drinking contaminated water during similar events.
|
What was the event?
|
{
"answer_start": [
2345
],
"text": [
"faecal contamination is detected the drinking water"
]
}
|
488
|
Compliance with boil water advice following a water contamination incident in the Netherlands in 2007
|
In May 2007, Escherichia coli was detected in tap water supplied by a company in North Holland. The company issued advice through mass media to boil tap water before consumption; this advice was lifted six days later. A cross-sectional study was implemented to investigate compliance among residents in this area. Based on postcode, a total of 300 households, chosen randomly from a database of a private company performing internet-based surveys for different marketing purposes, were sent a self-administered questionnaire for this study. The questionnaire contained questions on demographic information, source of information regarding the advice, response to it and personal opinions on the company’s reaction and the advice. Ninety-nine (66%) households of the affected area and 90 (60%) households from non-affected areas served by the same company replied to the survey. All respondents knew about the advice. 81.8% of the respondents in the affected area and 5.6% of the non-affected areas reported complying with the advisory. Most respondents from the affected area still used unboiled water to brush teeth, wash salads and fruits. There was no difference in compliance between men and women. Using the mass media was proved to be efficient to inform the public and could be used in the future in similar settings. However, more detailed wording of boiling advices should be considered in the future.
Introduction
Consumption of drinking water may cause waterborne disease which can be prevented by protection of the source water, efî¬cient treatment processes and reliable distribution systems. The European Union Drinking Water Directive [1] demands monitoring of tap water for different parameters, such as Escherichia coli, to indicate possible faecal contamination from humans and animals.
System failure or human error may cause an increase in the level of pathogens in the water posing a risk of waterborne disease. For example, in 2001, a large outbreak of gastroenteritis occurred due to accidental introduction of partially treated water to the drinking water supply system in the Netherlands, resulting in 921 households being exposed to contaminated water [2].
In the event that faecal contamination is detected the drinking water company may issue an advice to boil tap water before using it for domestic purposes. On 15 May 2007, E. coli was detected in samples collected the day before of the î¬nished tap water delivered by a company in the province Noord-Holland (North-Holland) in the Netherlands. For preventive reasons, on the same day the company issued an advice for consumers to boil tap water for two minutes before consumption but that this was not necessary for taking a shower or washing. This information was broadcasted through mass-media including the national and regional television channel, radio and newspapers. In addition, a public website used during emergency situations (www.crisis.nl) and a toll-free telephone number were made available for the public to provide information to households in the affected area.
The boil water advice had an impact on approximately 180,000 households in the affected area comprising 13 municipalities. The advice was lifted a week later, on 22 May 2007, as risk for public health was no longer present. In September 2007, the water company published a press release informing that the cause of the water contamination was due to run-off of rainwater contaminated with faeces of breeding gulls on the roof that had seeped into one of the six storage rooms [3].
Elevated levels of microorganisms in drinking water may represent a public health risk. For this reason, we investigated compliance with boil water advice issued by the private water company following the 2007 incident.
Methods
A cross-sectional study was implemented to investigate factors that may have affected water consumption habits of the residents in the area supplied by the water company. For this purpose, on the company’s behalf, a self-administered questionnaire was sent to 300 households in June 2007. Households were selected on the basis of their residence postcodes; half in the area where the advice was valid and half in areas served by the same company but where the advice did not apply. These participants were derived from a database of a private company that conducts online consumer surveys for marketing purposes.
The questionnaire contained questions on demographic information, level of urbanisation, source and time of receiving the information regarding the advice, initial and secondary response to the advice and personal opinions on the company’s response and the advice itself. The data were sent back to the drinking water company and the National Institute for Public Health and the Environment, where they were analysed. The statistical analysis was done with STATA v10.
Results
Ninety-nine households (66%) from the area affected by water contamination and 90 households (60%) from control areas supplied with water by the same company replied to the survey. Women more often than men responded to the questionnaire in both the affected and the non-affected areas (57.7% of all responders). The respondents represented 189 households with a total population of 505 people, 176 (34.9%) of whom were below the age of 18 years. There was no statistically signiî¬cant difference in the number of children per household between the affected and the non-affected areas (p=0.112). Descriptive results for the two different areas are presented in Table 1.
All 189 respondents (100%) in both areas answered that they had been informed about the advice. Ninety-î¬ve (50.3%) of them said they had first heard about it through the television. Other sources were radio (24.3%), friends, relatives or neighbours (22.8%), newspapers (19.6%) and the internet (7.4%).
Persons living in the affected area were more frequently disappointed (14.1%) about the choice of the company to use mass media for the advice than people residing in the non-affected area (2.2%). In the affected area, seven (9.3%) of the respondents had î¬rst reacted with fear to the information on the possible contamination of water, 34 (45.3%) responded with self-control and 34 (45.3%) with the intention to take measures. The corresponding percentages for the non affected area were 15.7%, 72.9% and 11.4%. About half (48.5%) of the respondents from the affected area said they had looked for more information when they had heard about the advice, while the corresponding proportion of respondents from the non-affected area was only 8.9% (p<0.001). The most common source of active search for more information was the website of the water supply company.
Eighty-one (81.8%) of all respondents in the affected area said they had complied with the advice. This was done by buying bottled water (43.4% of all respondents in affected area) or boiling tap water for two minutes before consuming it (70.7%). None of the respondents in the area stopped consuming tap water completely. Five (5.6%) of the respondents in the non-affected area were buying bottled water and three of them (3.3%) were boiling tap water during the advice. These numbers were considerably lower than the corresponding ones in the affected area, but showed that compliance exceeded beyond the affected area.
Even though it had not been advised to boil water for activities such as washing and showering, 26 (26.3%) of the respondents in the affected area stated that they had not been aware of that.
Concerning the image of the drinking water company, 177 respondents (93.7%) thought that the company had done well informing the consumers about the water contamination and its response to it. This prevailing opinion was not different between respondents from the affected area and those from the non affected area.
The respondents’ compliance with the advice was independent of sex, age and the presence of children in the household. However, the respondents were 138.6 times more likely to follow the advice if a second person in the household was following it as well (p<0.001).
Reasons for non-compliance with the advice are given in Table 2.Some of the respondents replied that they had been using boiled water for uses other than drinking, too. These results are shown in Table 3.The majority of the respondents stated that their image of the company had not changed after the incident and the six-day advice (78.8% in the affected area and 88.9% in the non-affected area).
Factors affecting compliance
The type of mass media from which people in the affected area found out about the advice played no signiî¬cant role in the subsequent compliance of the respondents. The highest compliance rates occurred among those in the affected area who heard about the advice from the internet (90%) or from friends (89.5%). Respondents informed by more than one source were more likely to have complied with the advice (90.9% against 79.2%) but this difference was not statistically signiî¬cant. The source of information did not depend on the age (p=0.6532). Compliance with the advice did not differ between households with children and those without children (p=0.536).
Respondents who undertook active search for more information may have been more likely to follow the advice than those who did not proceed to further active search for more information (89.4% vs. 74.5%, p=0.058).Since all respondents knew about the advice, it was not possible to estimate unwitting compliance rates.
Conclusions
Since excess of standard levels of certain microorganisms such as E. coli indicate faecal contamination and the possible presence of pathogens in tap water, the time between the water sampling, water analysis and the boil water notice is essential. During this period, consumers may be exposed to tap water of unacceptable quality. The choice of mass media for broadcasting the advice is therefore believed to be an effective measure to prevent panic and to protect public health.
From this study, it can be concluded that participating consumers not only thought that they had been informed about the advice in a timely manner, but that also the response of the company to ensure the advice would reach the public had been satisfactory as well as the choice of communication channels. Thus, the incident did not lead to customers’ dissatisfaction or a degradation of the company’s image.
The sample in our study derived from a database of people who subscribed to be included in different research surveys. This could raise questions regarding the representativeness of the study population. We agree that there is a need for similar studies with samples deriving randomly from the whole population and not from potentially biased data sources. For example, 100% of the participants stated that they had been informed about the boiling water advice; however, subscribers to online databases for marketing purposes may be more likely to regularly follow the news than the general population.
In the Netherlands, boil water notices are not harmonised but are determined by the drinking water company itself. This results in different advice with respect to, for instance, boiling time. Internationally recognised guidelines, such as the World Health Organization (WHO) Guidelines for Drinking Water Quality [4], could be taken into consideration in case of similar “crises†in the future. According to data from the water company involved in our study, about thirty boil water advices are issued per year in their responsibility area (ca. 700,000 households); involving on average 100 households per time. So, the chance to receive a boil water advice is small but existing.
The inclusion of recommendations including use of water for brushing teeth, washing fruits and vegetables may also prove helpful in future advice, since it is not only consumption of water through drinking that may pose a risk to the consumer. Bathing and showering may also need to be addressed separately, as a possible link between this kind of exposure to contaminated water and itching has been described elsewhere [2]. Also, although this conclusion does not directly follow from our results, vulnerable groups should be targeted separately in the advice; elderly people and children may easily miss information disseminated through the means of mass media [5,6].
Few studies have been published on boil water notices and their results seldom reach the public. Further research would also be useful to incorporate î¬ndings from compliance studies to model health effects of drinking contaminated water during similar events.
|
When did this happen?
|
{
"answer_start": [
3
],
"text": [
"May 2007"
]
}
|
489
|
Compliance with boil water advice following a water contamination incident in the Netherlands in 2007
|
In May 2007, Escherichia coli was detected in tap water supplied by a company in North Holland. The company issued advice through mass media to boil tap water before consumption; this advice was lifted six days later. A cross-sectional study was implemented to investigate compliance among residents in this area. Based on postcode, a total of 300 households, chosen randomly from a database of a private company performing internet-based surveys for different marketing purposes, were sent a self-administered questionnaire for this study. The questionnaire contained questions on demographic information, source of information regarding the advice, response to it and personal opinions on the company’s reaction and the advice. Ninety-nine (66%) households of the affected area and 90 (60%) households from non-affected areas served by the same company replied to the survey. All respondents knew about the advice. 81.8% of the respondents in the affected area and 5.6% of the non-affected areas reported complying with the advisory. Most respondents from the affected area still used unboiled water to brush teeth, wash salads and fruits. There was no difference in compliance between men and women. Using the mass media was proved to be efficient to inform the public and could be used in the future in similar settings. However, more detailed wording of boiling advices should be considered in the future.
Introduction
Consumption of drinking water may cause waterborne disease which can be prevented by protection of the source water, efî¬cient treatment processes and reliable distribution systems. The European Union Drinking Water Directive [1] demands monitoring of tap water for different parameters, such as Escherichia coli, to indicate possible faecal contamination from humans and animals.
System failure or human error may cause an increase in the level of pathogens in the water posing a risk of waterborne disease. For example, in 2001, a large outbreak of gastroenteritis occurred due to accidental introduction of partially treated water to the drinking water supply system in the Netherlands, resulting in 921 households being exposed to contaminated water [2].
In the event that faecal contamination is detected the drinking water company may issue an advice to boil tap water before using it for domestic purposes. On 15 May 2007, E. coli was detected in samples collected the day before of the î¬nished tap water delivered by a company in the province Noord-Holland (North-Holland) in the Netherlands. For preventive reasons, on the same day the company issued an advice for consumers to boil tap water for two minutes before consumption but that this was not necessary for taking a shower or washing. This information was broadcasted through mass-media including the national and regional television channel, radio and newspapers. In addition, a public website used during emergency situations (www.crisis.nl) and a toll-free telephone number were made available for the public to provide information to households in the affected area.
The boil water advice had an impact on approximately 180,000 households in the affected area comprising 13 municipalities. The advice was lifted a week later, on 22 May 2007, as risk for public health was no longer present. In September 2007, the water company published a press release informing that the cause of the water contamination was due to run-off of rainwater contaminated with faeces of breeding gulls on the roof that had seeped into one of the six storage rooms [3].
Elevated levels of microorganisms in drinking water may represent a public health risk. For this reason, we investigated compliance with boil water advice issued by the private water company following the 2007 incident.
Methods
A cross-sectional study was implemented to investigate factors that may have affected water consumption habits of the residents in the area supplied by the water company. For this purpose, on the company’s behalf, a self-administered questionnaire was sent to 300 households in June 2007. Households were selected on the basis of their residence postcodes; half in the area where the advice was valid and half in areas served by the same company but where the advice did not apply. These participants were derived from a database of a private company that conducts online consumer surveys for marketing purposes.
The questionnaire contained questions on demographic information, level of urbanisation, source and time of receiving the information regarding the advice, initial and secondary response to the advice and personal opinions on the company’s response and the advice itself. The data were sent back to the drinking water company and the National Institute for Public Health and the Environment, where they were analysed. The statistical analysis was done with STATA v10.
Results
Ninety-nine households (66%) from the area affected by water contamination and 90 households (60%) from control areas supplied with water by the same company replied to the survey. Women more often than men responded to the questionnaire in both the affected and the non-affected areas (57.7% of all responders). The respondents represented 189 households with a total population of 505 people, 176 (34.9%) of whom were below the age of 18 years. There was no statistically signiî¬cant difference in the number of children per household between the affected and the non-affected areas (p=0.112). Descriptive results for the two different areas are presented in Table 1.
All 189 respondents (100%) in both areas answered that they had been informed about the advice. Ninety-î¬ve (50.3%) of them said they had first heard about it through the television. Other sources were radio (24.3%), friends, relatives or neighbours (22.8%), newspapers (19.6%) and the internet (7.4%).
Persons living in the affected area were more frequently disappointed (14.1%) about the choice of the company to use mass media for the advice than people residing in the non-affected area (2.2%). In the affected area, seven (9.3%) of the respondents had î¬rst reacted with fear to the information on the possible contamination of water, 34 (45.3%) responded with self-control and 34 (45.3%) with the intention to take measures. The corresponding percentages for the non affected area were 15.7%, 72.9% and 11.4%. About half (48.5%) of the respondents from the affected area said they had looked for more information when they had heard about the advice, while the corresponding proportion of respondents from the non-affected area was only 8.9% (p<0.001). The most common source of active search for more information was the website of the water supply company.
Eighty-one (81.8%) of all respondents in the affected area said they had complied with the advice. This was done by buying bottled water (43.4% of all respondents in affected area) or boiling tap water for two minutes before consuming it (70.7%). None of the respondents in the area stopped consuming tap water completely. Five (5.6%) of the respondents in the non-affected area were buying bottled water and three of them (3.3%) were boiling tap water during the advice. These numbers were considerably lower than the corresponding ones in the affected area, but showed that compliance exceeded beyond the affected area.
Even though it had not been advised to boil water for activities such as washing and showering, 26 (26.3%) of the respondents in the affected area stated that they had not been aware of that.
Concerning the image of the drinking water company, 177 respondents (93.7%) thought that the company had done well informing the consumers about the water contamination and its response to it. This prevailing opinion was not different between respondents from the affected area and those from the non affected area.
The respondents’ compliance with the advice was independent of sex, age and the presence of children in the household. However, the respondents were 138.6 times more likely to follow the advice if a second person in the household was following it as well (p<0.001).
Reasons for non-compliance with the advice are given in Table 2.Some of the respondents replied that they had been using boiled water for uses other than drinking, too. These results are shown in Table 3.The majority of the respondents stated that their image of the company had not changed after the incident and the six-day advice (78.8% in the affected area and 88.9% in the non-affected area).
Factors affecting compliance
The type of mass media from which people in the affected area found out about the advice played no signiî¬cant role in the subsequent compliance of the respondents. The highest compliance rates occurred among those in the affected area who heard about the advice from the internet (90%) or from friends (89.5%). Respondents informed by more than one source were more likely to have complied with the advice (90.9% against 79.2%) but this difference was not statistically signiî¬cant. The source of information did not depend on the age (p=0.6532). Compliance with the advice did not differ between households with children and those without children (p=0.536).
Respondents who undertook active search for more information may have been more likely to follow the advice than those who did not proceed to further active search for more information (89.4% vs. 74.5%, p=0.058).Since all respondents knew about the advice, it was not possible to estimate unwitting compliance rates.
Conclusions
Since excess of standard levels of certain microorganisms such as E. coli indicate faecal contamination and the possible presence of pathogens in tap water, the time between the water sampling, water analysis and the boil water notice is essential. During this period, consumers may be exposed to tap water of unacceptable quality. The choice of mass media for broadcasting the advice is therefore believed to be an effective measure to prevent panic and to protect public health.
From this study, it can be concluded that participating consumers not only thought that they had been informed about the advice in a timely manner, but that also the response of the company to ensure the advice would reach the public had been satisfactory as well as the choice of communication channels. Thus, the incident did not lead to customers’ dissatisfaction or a degradation of the company’s image.
The sample in our study derived from a database of people who subscribed to be included in different research surveys. This could raise questions regarding the representativeness of the study population. We agree that there is a need for similar studies with samples deriving randomly from the whole population and not from potentially biased data sources. For example, 100% of the participants stated that they had been informed about the boiling water advice; however, subscribers to online databases for marketing purposes may be more likely to regularly follow the news than the general population.
In the Netherlands, boil water notices are not harmonised but are determined by the drinking water company itself. This results in different advice with respect to, for instance, boiling time. Internationally recognised guidelines, such as the World Health Organization (WHO) Guidelines for Drinking Water Quality [4], could be taken into consideration in case of similar “crises†in the future. According to data from the water company involved in our study, about thirty boil water advices are issued per year in their responsibility area (ca. 700,000 households); involving on average 100 households per time. So, the chance to receive a boil water advice is small but existing.
The inclusion of recommendations including use of water for brushing teeth, washing fruits and vegetables may also prove helpful in future advice, since it is not only consumption of water through drinking that may pose a risk to the consumer. Bathing and showering may also need to be addressed separately, as a possible link between this kind of exposure to contaminated water and itching has been described elsewhere [2]. Also, although this conclusion does not directly follow from our results, vulnerable groups should be targeted separately in the advice; elderly people and children may easily miss information disseminated through the means of mass media [5,6].
Few studies have been published on boil water notices and their results seldom reach the public. Further research would also be useful to incorporate î¬ndings from compliance studies to model health effects of drinking contaminated water during similar events.
|
When did this event start?
|
{
"answer_start": [
2485
],
"text": [
"15 May 2007"
]
}
|
490
|
Compliance with boil water advice following a water contamination incident in the Netherlands in 2007
|
In May 2007, Escherichia coli was detected in tap water supplied by a company in North Holland. The company issued advice through mass media to boil tap water before consumption; this advice was lifted six days later. A cross-sectional study was implemented to investigate compliance among residents in this area. Based on postcode, a total of 300 households, chosen randomly from a database of a private company performing internet-based surveys for different marketing purposes, were sent a self-administered questionnaire for this study. The questionnaire contained questions on demographic information, source of information regarding the advice, response to it and personal opinions on the company’s reaction and the advice. Ninety-nine (66%) households of the affected area and 90 (60%) households from non-affected areas served by the same company replied to the survey. All respondents knew about the advice. 81.8% of the respondents in the affected area and 5.6% of the non-affected areas reported complying with the advisory. Most respondents from the affected area still used unboiled water to brush teeth, wash salads and fruits. There was no difference in compliance between men and women. Using the mass media was proved to be efficient to inform the public and could be used in the future in similar settings. However, more detailed wording of boiling advices should be considered in the future.
Introduction
Consumption of drinking water may cause waterborne disease which can be prevented by protection of the source water, efî¬cient treatment processes and reliable distribution systems. The European Union Drinking Water Directive [1] demands monitoring of tap water for different parameters, such as Escherichia coli, to indicate possible faecal contamination from humans and animals.
System failure or human error may cause an increase in the level of pathogens in the water posing a risk of waterborne disease. For example, in 2001, a large outbreak of gastroenteritis occurred due to accidental introduction of partially treated water to the drinking water supply system in the Netherlands, resulting in 921 households being exposed to contaminated water [2].
In the event that faecal contamination is detected the drinking water company may issue an advice to boil tap water before using it for domestic purposes. On 15 May 2007, E. coli was detected in samples collected the day before of the î¬nished tap water delivered by a company in the province Noord-Holland (North-Holland) in the Netherlands. For preventive reasons, on the same day the company issued an advice for consumers to boil tap water for two minutes before consumption but that this was not necessary for taking a shower or washing. This information was broadcasted through mass-media including the national and regional television channel, radio and newspapers. In addition, a public website used during emergency situations (www.crisis.nl) and a toll-free telephone number were made available for the public to provide information to households in the affected area.
The boil water advice had an impact on approximately 180,000 households in the affected area comprising 13 municipalities. The advice was lifted a week later, on 22 May 2007, as risk for public health was no longer present. In September 2007, the water company published a press release informing that the cause of the water contamination was due to run-off of rainwater contaminated with faeces of breeding gulls on the roof that had seeped into one of the six storage rooms [3].
Elevated levels of microorganisms in drinking water may represent a public health risk. For this reason, we investigated compliance with boil water advice issued by the private water company following the 2007 incident.
Methods
A cross-sectional study was implemented to investigate factors that may have affected water consumption habits of the residents in the area supplied by the water company. For this purpose, on the company’s behalf, a self-administered questionnaire was sent to 300 households in June 2007. Households were selected on the basis of their residence postcodes; half in the area where the advice was valid and half in areas served by the same company but where the advice did not apply. These participants were derived from a database of a private company that conducts online consumer surveys for marketing purposes.
The questionnaire contained questions on demographic information, level of urbanisation, source and time of receiving the information regarding the advice, initial and secondary response to the advice and personal opinions on the company’s response and the advice itself. The data were sent back to the drinking water company and the National Institute for Public Health and the Environment, where they were analysed. The statistical analysis was done with STATA v10.
Results
Ninety-nine households (66%) from the area affected by water contamination and 90 households (60%) from control areas supplied with water by the same company replied to the survey. Women more often than men responded to the questionnaire in both the affected and the non-affected areas (57.7% of all responders). The respondents represented 189 households with a total population of 505 people, 176 (34.9%) of whom were below the age of 18 years. There was no statistically signiî¬cant difference in the number of children per household between the affected and the non-affected areas (p=0.112). Descriptive results for the two different areas are presented in Table 1.
All 189 respondents (100%) in both areas answered that they had been informed about the advice. Ninety-î¬ve (50.3%) of them said they had first heard about it through the television. Other sources were radio (24.3%), friends, relatives or neighbours (22.8%), newspapers (19.6%) and the internet (7.4%).
Persons living in the affected area were more frequently disappointed (14.1%) about the choice of the company to use mass media for the advice than people residing in the non-affected area (2.2%). In the affected area, seven (9.3%) of the respondents had î¬rst reacted with fear to the information on the possible contamination of water, 34 (45.3%) responded with self-control and 34 (45.3%) with the intention to take measures. The corresponding percentages for the non affected area were 15.7%, 72.9% and 11.4%. About half (48.5%) of the respondents from the affected area said they had looked for more information when they had heard about the advice, while the corresponding proportion of respondents from the non-affected area was only 8.9% (p<0.001). The most common source of active search for more information was the website of the water supply company.
Eighty-one (81.8%) of all respondents in the affected area said they had complied with the advice. This was done by buying bottled water (43.4% of all respondents in affected area) or boiling tap water for two minutes before consuming it (70.7%). None of the respondents in the area stopped consuming tap water completely. Five (5.6%) of the respondents in the non-affected area were buying bottled water and three of them (3.3%) were boiling tap water during the advice. These numbers were considerably lower than the corresponding ones in the affected area, but showed that compliance exceeded beyond the affected area.
Even though it had not been advised to boil water for activities such as washing and showering, 26 (26.3%) of the respondents in the affected area stated that they had not been aware of that.
Concerning the image of the drinking water company, 177 respondents (93.7%) thought that the company had done well informing the consumers about the water contamination and its response to it. This prevailing opinion was not different between respondents from the affected area and those from the non affected area.
The respondents’ compliance with the advice was independent of sex, age and the presence of children in the household. However, the respondents were 138.6 times more likely to follow the advice if a second person in the household was following it as well (p<0.001).
Reasons for non-compliance with the advice are given in Table 2.Some of the respondents replied that they had been using boiled water for uses other than drinking, too. These results are shown in Table 3.The majority of the respondents stated that their image of the company had not changed after the incident and the six-day advice (78.8% in the affected area and 88.9% in the non-affected area).
Factors affecting compliance
The type of mass media from which people in the affected area found out about the advice played no signiî¬cant role in the subsequent compliance of the respondents. The highest compliance rates occurred among those in the affected area who heard about the advice from the internet (90%) or from friends (89.5%). Respondents informed by more than one source were more likely to have complied with the advice (90.9% against 79.2%) but this difference was not statistically signiî¬cant. The source of information did not depend on the age (p=0.6532). Compliance with the advice did not differ between households with children and those without children (p=0.536).
Respondents who undertook active search for more information may have been more likely to follow the advice than those who did not proceed to further active search for more information (89.4% vs. 74.5%, p=0.058).Since all respondents knew about the advice, it was not possible to estimate unwitting compliance rates.
Conclusions
Since excess of standard levels of certain microorganisms such as E. coli indicate faecal contamination and the possible presence of pathogens in tap water, the time between the water sampling, water analysis and the boil water notice is essential. During this period, consumers may be exposed to tap water of unacceptable quality. The choice of mass media for broadcasting the advice is therefore believed to be an effective measure to prevent panic and to protect public health.
From this study, it can be concluded that participating consumers not only thought that they had been informed about the advice in a timely manner, but that also the response of the company to ensure the advice would reach the public had been satisfactory as well as the choice of communication channels. Thus, the incident did not lead to customers’ dissatisfaction or a degradation of the company’s image.
The sample in our study derived from a database of people who subscribed to be included in different research surveys. This could raise questions regarding the representativeness of the study population. We agree that there is a need for similar studies with samples deriving randomly from the whole population and not from potentially biased data sources. For example, 100% of the participants stated that they had been informed about the boiling water advice; however, subscribers to online databases for marketing purposes may be more likely to regularly follow the news than the general population.
In the Netherlands, boil water notices are not harmonised but are determined by the drinking water company itself. This results in different advice with respect to, for instance, boiling time. Internationally recognised guidelines, such as the World Health Organization (WHO) Guidelines for Drinking Water Quality [4], could be taken into consideration in case of similar “crises†in the future. According to data from the water company involved in our study, about thirty boil water advices are issued per year in their responsibility area (ca. 700,000 households); involving on average 100 households per time. So, the chance to receive a boil water advice is small but existing.
The inclusion of recommendations including use of water for brushing teeth, washing fruits and vegetables may also prove helpful in future advice, since it is not only consumption of water through drinking that may pose a risk to the consumer. Bathing and showering may also need to be addressed separately, as a possible link between this kind of exposure to contaminated water and itching has been described elsewhere [2]. Also, although this conclusion does not directly follow from our results, vulnerable groups should be targeted separately in the advice; elderly people and children may easily miss information disseminated through the means of mass media [5,6].
Few studies have been published on boil water notices and their results seldom reach the public. Further research would also be useful to incorporate î¬ndings from compliance studies to model health effects of drinking contaminated water during similar events.
|
What is the date of this event?
|
{
"answer_start": [
2485
],
"text": [
"15 May 2007"
]
}
|
491
|
Compliance with boil water advice following a water contamination incident in the Netherlands in 2007
|
In May 2007, Escherichia coli was detected in tap water supplied by a company in North Holland. The company issued advice through mass media to boil tap water before consumption; this advice was lifted six days later. A cross-sectional study was implemented to investigate compliance among residents in this area. Based on postcode, a total of 300 households, chosen randomly from a database of a private company performing internet-based surveys for different marketing purposes, were sent a self-administered questionnaire for this study. The questionnaire contained questions on demographic information, source of information regarding the advice, response to it and personal opinions on the company’s reaction and the advice. Ninety-nine (66%) households of the affected area and 90 (60%) households from non-affected areas served by the same company replied to the survey. All respondents knew about the advice. 81.8% of the respondents in the affected area and 5.6% of the non-affected areas reported complying with the advisory. Most respondents from the affected area still used unboiled water to brush teeth, wash salads and fruits. There was no difference in compliance between men and women. Using the mass media was proved to be efficient to inform the public and could be used in the future in similar settings. However, more detailed wording of boiling advices should be considered in the future.
Introduction
Consumption of drinking water may cause waterborne disease which can be prevented by protection of the source water, efî¬cient treatment processes and reliable distribution systems. The European Union Drinking Water Directive [1] demands monitoring of tap water for different parameters, such as Escherichia coli, to indicate possible faecal contamination from humans and animals.
System failure or human error may cause an increase in the level of pathogens in the water posing a risk of waterborne disease. For example, in 2001, a large outbreak of gastroenteritis occurred due to accidental introduction of partially treated water to the drinking water supply system in the Netherlands, resulting in 921 households being exposed to contaminated water [2].
In the event that faecal contamination is detected the drinking water company may issue an advice to boil tap water before using it for domestic purposes. On 15 May 2007, E. coli was detected in samples collected the day before of the î¬nished tap water delivered by a company in the province Noord-Holland (North-Holland) in the Netherlands. For preventive reasons, on the same day the company issued an advice for consumers to boil tap water for two minutes before consumption but that this was not necessary for taking a shower or washing. This information was broadcasted through mass-media including the national and regional television channel, radio and newspapers. In addition, a public website used during emergency situations (www.crisis.nl) and a toll-free telephone number were made available for the public to provide information to households in the affected area.
The boil water advice had an impact on approximately 180,000 households in the affected area comprising 13 municipalities. The advice was lifted a week later, on 22 May 2007, as risk for public health was no longer present. In September 2007, the water company published a press release informing that the cause of the water contamination was due to run-off of rainwater contaminated with faeces of breeding gulls on the roof that had seeped into one of the six storage rooms [3].
Elevated levels of microorganisms in drinking water may represent a public health risk. For this reason, we investigated compliance with boil water advice issued by the private water company following the 2007 incident.
Methods
A cross-sectional study was implemented to investigate factors that may have affected water consumption habits of the residents in the area supplied by the water company. For this purpose, on the company’s behalf, a self-administered questionnaire was sent to 300 households in June 2007. Households were selected on the basis of their residence postcodes; half in the area where the advice was valid and half in areas served by the same company but where the advice did not apply. These participants were derived from a database of a private company that conducts online consumer surveys for marketing purposes.
The questionnaire contained questions on demographic information, level of urbanisation, source and time of receiving the information regarding the advice, initial and secondary response to the advice and personal opinions on the company’s response and the advice itself. The data were sent back to the drinking water company and the National Institute for Public Health and the Environment, where they were analysed. The statistical analysis was done with STATA v10.
Results
Ninety-nine households (66%) from the area affected by water contamination and 90 households (60%) from control areas supplied with water by the same company replied to the survey. Women more often than men responded to the questionnaire in both the affected and the non-affected areas (57.7% of all responders). The respondents represented 189 households with a total population of 505 people, 176 (34.9%) of whom were below the age of 18 years. There was no statistically signiî¬cant difference in the number of children per household between the affected and the non-affected areas (p=0.112). Descriptive results for the two different areas are presented in Table 1.
All 189 respondents (100%) in both areas answered that they had been informed about the advice. Ninety-î¬ve (50.3%) of them said they had first heard about it through the television. Other sources were radio (24.3%), friends, relatives or neighbours (22.8%), newspapers (19.6%) and the internet (7.4%).
Persons living in the affected area were more frequently disappointed (14.1%) about the choice of the company to use mass media for the advice than people residing in the non-affected area (2.2%). In the affected area, seven (9.3%) of the respondents had î¬rst reacted with fear to the information on the possible contamination of water, 34 (45.3%) responded with self-control and 34 (45.3%) with the intention to take measures. The corresponding percentages for the non affected area were 15.7%, 72.9% and 11.4%. About half (48.5%) of the respondents from the affected area said they had looked for more information when they had heard about the advice, while the corresponding proportion of respondents from the non-affected area was only 8.9% (p<0.001). The most common source of active search for more information was the website of the water supply company.
Eighty-one (81.8%) of all respondents in the affected area said they had complied with the advice. This was done by buying bottled water (43.4% of all respondents in affected area) or boiling tap water for two minutes before consuming it (70.7%). None of the respondents in the area stopped consuming tap water completely. Five (5.6%) of the respondents in the non-affected area were buying bottled water and three of them (3.3%) were boiling tap water during the advice. These numbers were considerably lower than the corresponding ones in the affected area, but showed that compliance exceeded beyond the affected area.
Even though it had not been advised to boil water for activities such as washing and showering, 26 (26.3%) of the respondents in the affected area stated that they had not been aware of that.
Concerning the image of the drinking water company, 177 respondents (93.7%) thought that the company had done well informing the consumers about the water contamination and its response to it. This prevailing opinion was not different between respondents from the affected area and those from the non affected area.
The respondents’ compliance with the advice was independent of sex, age and the presence of children in the household. However, the respondents were 138.6 times more likely to follow the advice if a second person in the household was following it as well (p<0.001).
Reasons for non-compliance with the advice are given in Table 2.Some of the respondents replied that they had been using boiled water for uses other than drinking, too. These results are shown in Table 3.The majority of the respondents stated that their image of the company had not changed after the incident and the six-day advice (78.8% in the affected area and 88.9% in the non-affected area).
Factors affecting compliance
The type of mass media from which people in the affected area found out about the advice played no signiî¬cant role in the subsequent compliance of the respondents. The highest compliance rates occurred among those in the affected area who heard about the advice from the internet (90%) or from friends (89.5%). Respondents informed by more than one source were more likely to have complied with the advice (90.9% against 79.2%) but this difference was not statistically signiî¬cant. The source of information did not depend on the age (p=0.6532). Compliance with the advice did not differ between households with children and those without children (p=0.536).
Respondents who undertook active search for more information may have been more likely to follow the advice than those who did not proceed to further active search for more information (89.4% vs. 74.5%, p=0.058).Since all respondents knew about the advice, it was not possible to estimate unwitting compliance rates.
Conclusions
Since excess of standard levels of certain microorganisms such as E. coli indicate faecal contamination and the possible presence of pathogens in tap water, the time between the water sampling, water analysis and the boil water notice is essential. During this period, consumers may be exposed to tap water of unacceptable quality. The choice of mass media for broadcasting the advice is therefore believed to be an effective measure to prevent panic and to protect public health.
From this study, it can be concluded that participating consumers not only thought that they had been informed about the advice in a timely manner, but that also the response of the company to ensure the advice would reach the public had been satisfactory as well as the choice of communication channels. Thus, the incident did not lead to customers’ dissatisfaction or a degradation of the company’s image.
The sample in our study derived from a database of people who subscribed to be included in different research surveys. This could raise questions regarding the representativeness of the study population. We agree that there is a need for similar studies with samples deriving randomly from the whole population and not from potentially biased data sources. For example, 100% of the participants stated that they had been informed about the boiling water advice; however, subscribers to online databases for marketing purposes may be more likely to regularly follow the news than the general population.
In the Netherlands, boil water notices are not harmonised but are determined by the drinking water company itself. This results in different advice with respect to, for instance, boiling time. Internationally recognised guidelines, such as the World Health Organization (WHO) Guidelines for Drinking Water Quality [4], could be taken into consideration in case of similar “crises†in the future. According to data from the water company involved in our study, about thirty boil water advices are issued per year in their responsibility area (ca. 700,000 households); involving on average 100 households per time. So, the chance to receive a boil water advice is small but existing.
The inclusion of recommendations including use of water for brushing teeth, washing fruits and vegetables may also prove helpful in future advice, since it is not only consumption of water through drinking that may pose a risk to the consumer. Bathing and showering may also need to be addressed separately, as a possible link between this kind of exposure to contaminated water and itching has been described elsewhere [2]. Also, although this conclusion does not directly follow from our results, vulnerable groups should be targeted separately in the advice; elderly people and children may easily miss information disseminated through the means of mass media [5,6].
Few studies have been published on boil water notices and their results seldom reach the public. Further research would also be useful to incorporate î¬ndings from compliance studies to model health effects of drinking contaminated water during similar events.
|
How long was the event?
|
{
"answer_start": [
202
],
"text": [
"six days"
]
}
|
492
|
Compliance with boil water advice following a water contamination incident in the Netherlands in 2007
|
In May 2007, Escherichia coli was detected in tap water supplied by a company in North Holland. The company issued advice through mass media to boil tap water before consumption; this advice was lifted six days later. A cross-sectional study was implemented to investigate compliance among residents in this area. Based on postcode, a total of 300 households, chosen randomly from a database of a private company performing internet-based surveys for different marketing purposes, were sent a self-administered questionnaire for this study. The questionnaire contained questions on demographic information, source of information regarding the advice, response to it and personal opinions on the company’s reaction and the advice. Ninety-nine (66%) households of the affected area and 90 (60%) households from non-affected areas served by the same company replied to the survey. All respondents knew about the advice. 81.8% of the respondents in the affected area and 5.6% of the non-affected areas reported complying with the advisory. Most respondents from the affected area still used unboiled water to brush teeth, wash salads and fruits. There was no difference in compliance between men and women. Using the mass media was proved to be efficient to inform the public and could be used in the future in similar settings. However, more detailed wording of boiling advices should be considered in the future.
Introduction
Consumption of drinking water may cause waterborne disease which can be prevented by protection of the source water, efî¬cient treatment processes and reliable distribution systems. The European Union Drinking Water Directive [1] demands monitoring of tap water for different parameters, such as Escherichia coli, to indicate possible faecal contamination from humans and animals.
System failure or human error may cause an increase in the level of pathogens in the water posing a risk of waterborne disease. For example, in 2001, a large outbreak of gastroenteritis occurred due to accidental introduction of partially treated water to the drinking water supply system in the Netherlands, resulting in 921 households being exposed to contaminated water [2].
In the event that faecal contamination is detected the drinking water company may issue an advice to boil tap water before using it for domestic purposes. On 15 May 2007, E. coli was detected in samples collected the day before of the î¬nished tap water delivered by a company in the province Noord-Holland (North-Holland) in the Netherlands. For preventive reasons, on the same day the company issued an advice for consumers to boil tap water for two minutes before consumption but that this was not necessary for taking a shower or washing. This information was broadcasted through mass-media including the national and regional television channel, radio and newspapers. In addition, a public website used during emergency situations (www.crisis.nl) and a toll-free telephone number were made available for the public to provide information to households in the affected area.
The boil water advice had an impact on approximately 180,000 households in the affected area comprising 13 municipalities. The advice was lifted a week later, on 22 May 2007, as risk for public health was no longer present. In September 2007, the water company published a press release informing that the cause of the water contamination was due to run-off of rainwater contaminated with faeces of breeding gulls on the roof that had seeped into one of the six storage rooms [3].
Elevated levels of microorganisms in drinking water may represent a public health risk. For this reason, we investigated compliance with boil water advice issued by the private water company following the 2007 incident.
Methods
A cross-sectional study was implemented to investigate factors that may have affected water consumption habits of the residents in the area supplied by the water company. For this purpose, on the company’s behalf, a self-administered questionnaire was sent to 300 households in June 2007. Households were selected on the basis of their residence postcodes; half in the area where the advice was valid and half in areas served by the same company but where the advice did not apply. These participants were derived from a database of a private company that conducts online consumer surveys for marketing purposes.
The questionnaire contained questions on demographic information, level of urbanisation, source and time of receiving the information regarding the advice, initial and secondary response to the advice and personal opinions on the company’s response and the advice itself. The data were sent back to the drinking water company and the National Institute for Public Health and the Environment, where they were analysed. The statistical analysis was done with STATA v10.
Results
Ninety-nine households (66%) from the area affected by water contamination and 90 households (60%) from control areas supplied with water by the same company replied to the survey. Women more often than men responded to the questionnaire in both the affected and the non-affected areas (57.7% of all responders). The respondents represented 189 households with a total population of 505 people, 176 (34.9%) of whom were below the age of 18 years. There was no statistically signiî¬cant difference in the number of children per household between the affected and the non-affected areas (p=0.112). Descriptive results for the two different areas are presented in Table 1.
All 189 respondents (100%) in both areas answered that they had been informed about the advice. Ninety-î¬ve (50.3%) of them said they had first heard about it through the television. Other sources were radio (24.3%), friends, relatives or neighbours (22.8%), newspapers (19.6%) and the internet (7.4%).
Persons living in the affected area were more frequently disappointed (14.1%) about the choice of the company to use mass media for the advice than people residing in the non-affected area (2.2%). In the affected area, seven (9.3%) of the respondents had î¬rst reacted with fear to the information on the possible contamination of water, 34 (45.3%) responded with self-control and 34 (45.3%) with the intention to take measures. The corresponding percentages for the non affected area were 15.7%, 72.9% and 11.4%. About half (48.5%) of the respondents from the affected area said they had looked for more information when they had heard about the advice, while the corresponding proportion of respondents from the non-affected area was only 8.9% (p<0.001). The most common source of active search for more information was the website of the water supply company.
Eighty-one (81.8%) of all respondents in the affected area said they had complied with the advice. This was done by buying bottled water (43.4% of all respondents in affected area) or boiling tap water for two minutes before consuming it (70.7%). None of the respondents in the area stopped consuming tap water completely. Five (5.6%) of the respondents in the non-affected area were buying bottled water and three of them (3.3%) were boiling tap water during the advice. These numbers were considerably lower than the corresponding ones in the affected area, but showed that compliance exceeded beyond the affected area.
Even though it had not been advised to boil water for activities such as washing and showering, 26 (26.3%) of the respondents in the affected area stated that they had not been aware of that.
Concerning the image of the drinking water company, 177 respondents (93.7%) thought that the company had done well informing the consumers about the water contamination and its response to it. This prevailing opinion was not different between respondents from the affected area and those from the non affected area.
The respondents’ compliance with the advice was independent of sex, age and the presence of children in the household. However, the respondents were 138.6 times more likely to follow the advice if a second person in the household was following it as well (p<0.001).
Reasons for non-compliance with the advice are given in Table 2.Some of the respondents replied that they had been using boiled water for uses other than drinking, too. These results are shown in Table 3.The majority of the respondents stated that their image of the company had not changed after the incident and the six-day advice (78.8% in the affected area and 88.9% in the non-affected area).
Factors affecting compliance
The type of mass media from which people in the affected area found out about the advice played no signiî¬cant role in the subsequent compliance of the respondents. The highest compliance rates occurred among those in the affected area who heard about the advice from the internet (90%) or from friends (89.5%). Respondents informed by more than one source were more likely to have complied with the advice (90.9% against 79.2%) but this difference was not statistically signiî¬cant. The source of information did not depend on the age (p=0.6532). Compliance with the advice did not differ between households with children and those without children (p=0.536).
Respondents who undertook active search for more information may have been more likely to follow the advice than those who did not proceed to further active search for more information (89.4% vs. 74.5%, p=0.058).Since all respondents knew about the advice, it was not possible to estimate unwitting compliance rates.
Conclusions
Since excess of standard levels of certain microorganisms such as E. coli indicate faecal contamination and the possible presence of pathogens in tap water, the time between the water sampling, water analysis and the boil water notice is essential. During this period, consumers may be exposed to tap water of unacceptable quality. The choice of mass media for broadcasting the advice is therefore believed to be an effective measure to prevent panic and to protect public health.
From this study, it can be concluded that participating consumers not only thought that they had been informed about the advice in a timely manner, but that also the response of the company to ensure the advice would reach the public had been satisfactory as well as the choice of communication channels. Thus, the incident did not lead to customers’ dissatisfaction or a degradation of the company’s image.
The sample in our study derived from a database of people who subscribed to be included in different research surveys. This could raise questions regarding the representativeness of the study population. We agree that there is a need for similar studies with samples deriving randomly from the whole population and not from potentially biased data sources. For example, 100% of the participants stated that they had been informed about the boiling water advice; however, subscribers to online databases for marketing purposes may be more likely to regularly follow the news than the general population.
In the Netherlands, boil water notices are not harmonised but are determined by the drinking water company itself. This results in different advice with respect to, for instance, boiling time. Internationally recognised guidelines, such as the World Health Organization (WHO) Guidelines for Drinking Water Quality [4], could be taken into consideration in case of similar “crises†in the future. According to data from the water company involved in our study, about thirty boil water advices are issued per year in their responsibility area (ca. 700,000 households); involving on average 100 households per time. So, the chance to receive a boil water advice is small but existing.
The inclusion of recommendations including use of water for brushing teeth, washing fruits and vegetables may also prove helpful in future advice, since it is not only consumption of water through drinking that may pose a risk to the consumer. Bathing and showering may also need to be addressed separately, as a possible link between this kind of exposure to contaminated water and itching has been described elsewhere [2]. Also, although this conclusion does not directly follow from our results, vulnerable groups should be targeted separately in the advice; elderly people and children may easily miss information disseminated through the means of mass media [5,6].
Few studies have been published on boil water notices and their results seldom reach the public. Further research would also be useful to incorporate î¬ndings from compliance studies to model health effects of drinking contaminated water during similar events.
|
How long did the event last?
|
{
"answer_start": [
202
],
"text": [
"six days"
]
}
|
493
|
Compliance with boil water advice following a water contamination incident in the Netherlands in 2007
|
In May 2007, Escherichia coli was detected in tap water supplied by a company in North Holland. The company issued advice through mass media to boil tap water before consumption; this advice was lifted six days later. A cross-sectional study was implemented to investigate compliance among residents in this area. Based on postcode, a total of 300 households, chosen randomly from a database of a private company performing internet-based surveys for different marketing purposes, were sent a self-administered questionnaire for this study. The questionnaire contained questions on demographic information, source of information regarding the advice, response to it and personal opinions on the company’s reaction and the advice. Ninety-nine (66%) households of the affected area and 90 (60%) households from non-affected areas served by the same company replied to the survey. All respondents knew about the advice. 81.8% of the respondents in the affected area and 5.6% of the non-affected areas reported complying with the advisory. Most respondents from the affected area still used unboiled water to brush teeth, wash salads and fruits. There was no difference in compliance between men and women. Using the mass media was proved to be efficient to inform the public and could be used in the future in similar settings. However, more detailed wording of boiling advices should be considered in the future.
Introduction
Consumption of drinking water may cause waterborne disease which can be prevented by protection of the source water, efî¬cient treatment processes and reliable distribution systems. The European Union Drinking Water Directive [1] demands monitoring of tap water for different parameters, such as Escherichia coli, to indicate possible faecal contamination from humans and animals.
System failure or human error may cause an increase in the level of pathogens in the water posing a risk of waterborne disease. For example, in 2001, a large outbreak of gastroenteritis occurred due to accidental introduction of partially treated water to the drinking water supply system in the Netherlands, resulting in 921 households being exposed to contaminated water [2].
In the event that faecal contamination is detected the drinking water company may issue an advice to boil tap water before using it for domestic purposes. On 15 May 2007, E. coli was detected in samples collected the day before of the î¬nished tap water delivered by a company in the province Noord-Holland (North-Holland) in the Netherlands. For preventive reasons, on the same day the company issued an advice for consumers to boil tap water for two minutes before consumption but that this was not necessary for taking a shower or washing. This information was broadcasted through mass-media including the national and regional television channel, radio and newspapers. In addition, a public website used during emergency situations (www.crisis.nl) and a toll-free telephone number were made available for the public to provide information to households in the affected area.
The boil water advice had an impact on approximately 180,000 households in the affected area comprising 13 municipalities. The advice was lifted a week later, on 22 May 2007, as risk for public health was no longer present. In September 2007, the water company published a press release informing that the cause of the water contamination was due to run-off of rainwater contaminated with faeces of breeding gulls on the roof that had seeped into one of the six storage rooms [3].
Elevated levels of microorganisms in drinking water may represent a public health risk. For this reason, we investigated compliance with boil water advice issued by the private water company following the 2007 incident.
Methods
A cross-sectional study was implemented to investigate factors that may have affected water consumption habits of the residents in the area supplied by the water company. For this purpose, on the company’s behalf, a self-administered questionnaire was sent to 300 households in June 2007. Households were selected on the basis of their residence postcodes; half in the area where the advice was valid and half in areas served by the same company but where the advice did not apply. These participants were derived from a database of a private company that conducts online consumer surveys for marketing purposes.
The questionnaire contained questions on demographic information, level of urbanisation, source and time of receiving the information regarding the advice, initial and secondary response to the advice and personal opinions on the company’s response and the advice itself. The data were sent back to the drinking water company and the National Institute for Public Health and the Environment, where they were analysed. The statistical analysis was done with STATA v10.
Results
Ninety-nine households (66%) from the area affected by water contamination and 90 households (60%) from control areas supplied with water by the same company replied to the survey. Women more often than men responded to the questionnaire in both the affected and the non-affected areas (57.7% of all responders). The respondents represented 189 households with a total population of 505 people, 176 (34.9%) of whom were below the age of 18 years. There was no statistically signiî¬cant difference in the number of children per household between the affected and the non-affected areas (p=0.112). Descriptive results for the two different areas are presented in Table 1.
All 189 respondents (100%) in both areas answered that they had been informed about the advice. Ninety-î¬ve (50.3%) of them said they had first heard about it through the television. Other sources were radio (24.3%), friends, relatives or neighbours (22.8%), newspapers (19.6%) and the internet (7.4%).
Persons living in the affected area were more frequently disappointed (14.1%) about the choice of the company to use mass media for the advice than people residing in the non-affected area (2.2%). In the affected area, seven (9.3%) of the respondents had î¬rst reacted with fear to the information on the possible contamination of water, 34 (45.3%) responded with self-control and 34 (45.3%) with the intention to take measures. The corresponding percentages for the non affected area were 15.7%, 72.9% and 11.4%. About half (48.5%) of the respondents from the affected area said they had looked for more information when they had heard about the advice, while the corresponding proportion of respondents from the non-affected area was only 8.9% (p<0.001). The most common source of active search for more information was the website of the water supply company.
Eighty-one (81.8%) of all respondents in the affected area said they had complied with the advice. This was done by buying bottled water (43.4% of all respondents in affected area) or boiling tap water for two minutes before consuming it (70.7%). None of the respondents in the area stopped consuming tap water completely. Five (5.6%) of the respondents in the non-affected area were buying bottled water and three of them (3.3%) were boiling tap water during the advice. These numbers were considerably lower than the corresponding ones in the affected area, but showed that compliance exceeded beyond the affected area.
Even though it had not been advised to boil water for activities such as washing and showering, 26 (26.3%) of the respondents in the affected area stated that they had not been aware of that.
Concerning the image of the drinking water company, 177 respondents (93.7%) thought that the company had done well informing the consumers about the water contamination and its response to it. This prevailing opinion was not different between respondents from the affected area and those from the non affected area.
The respondents’ compliance with the advice was independent of sex, age and the presence of children in the household. However, the respondents were 138.6 times more likely to follow the advice if a second person in the household was following it as well (p<0.001).
Reasons for non-compliance with the advice are given in Table 2.Some of the respondents replied that they had been using boiled water for uses other than drinking, too. These results are shown in Table 3.The majority of the respondents stated that their image of the company had not changed after the incident and the six-day advice (78.8% in the affected area and 88.9% in the non-affected area).
Factors affecting compliance
The type of mass media from which people in the affected area found out about the advice played no signiî¬cant role in the subsequent compliance of the respondents. The highest compliance rates occurred among those in the affected area who heard about the advice from the internet (90%) or from friends (89.5%). Respondents informed by more than one source were more likely to have complied with the advice (90.9% against 79.2%) but this difference was not statistically signiî¬cant. The source of information did not depend on the age (p=0.6532). Compliance with the advice did not differ between households with children and those without children (p=0.536).
Respondents who undertook active search for more information may have been more likely to follow the advice than those who did not proceed to further active search for more information (89.4% vs. 74.5%, p=0.058).Since all respondents knew about the advice, it was not possible to estimate unwitting compliance rates.
Conclusions
Since excess of standard levels of certain microorganisms such as E. coli indicate faecal contamination and the possible presence of pathogens in tap water, the time between the water sampling, water analysis and the boil water notice is essential. During this period, consumers may be exposed to tap water of unacceptable quality. The choice of mass media for broadcasting the advice is therefore believed to be an effective measure to prevent panic and to protect public health.
From this study, it can be concluded that participating consumers not only thought that they had been informed about the advice in a timely manner, but that also the response of the company to ensure the advice would reach the public had been satisfactory as well as the choice of communication channels. Thus, the incident did not lead to customers’ dissatisfaction or a degradation of the company’s image.
The sample in our study derived from a database of people who subscribed to be included in different research surveys. This could raise questions regarding the representativeness of the study population. We agree that there is a need for similar studies with samples deriving randomly from the whole population and not from potentially biased data sources. For example, 100% of the participants stated that they had been informed about the boiling water advice; however, subscribers to online databases for marketing purposes may be more likely to regularly follow the news than the general population.
In the Netherlands, boil water notices are not harmonised but are determined by the drinking water company itself. This results in different advice with respect to, for instance, boiling time. Internationally recognised guidelines, such as the World Health Organization (WHO) Guidelines for Drinking Water Quality [4], could be taken into consideration in case of similar “crises†in the future. According to data from the water company involved in our study, about thirty boil water advices are issued per year in their responsibility area (ca. 700,000 households); involving on average 100 households per time. So, the chance to receive a boil water advice is small but existing.
The inclusion of recommendations including use of water for brushing teeth, washing fruits and vegetables may also prove helpful in future advice, since it is not only consumption of water through drinking that may pose a risk to the consumer. Bathing and showering may also need to be addressed separately, as a possible link between this kind of exposure to contaminated water and itching has been described elsewhere [2]. Also, although this conclusion does not directly follow from our results, vulnerable groups should be targeted separately in the advice; elderly people and children may easily miss information disseminated through the means of mass media [5,6].
Few studies have been published on boil water notices and their results seldom reach the public. Further research would also be useful to incorporate î¬ndings from compliance studies to model health effects of drinking contaminated water during similar events.
|
In which street did this happen?
|
{
"answer_start": [],
"text": []
}
|
494
|
Compliance with boil water advice following a water contamination incident in the Netherlands in 2007
|
In May 2007, Escherichia coli was detected in tap water supplied by a company in North Holland. The company issued advice through mass media to boil tap water before consumption; this advice was lifted six days later. A cross-sectional study was implemented to investigate compliance among residents in this area. Based on postcode, a total of 300 households, chosen randomly from a database of a private company performing internet-based surveys for different marketing purposes, were sent a self-administered questionnaire for this study. The questionnaire contained questions on demographic information, source of information regarding the advice, response to it and personal opinions on the company’s reaction and the advice. Ninety-nine (66%) households of the affected area and 90 (60%) households from non-affected areas served by the same company replied to the survey. All respondents knew about the advice. 81.8% of the respondents in the affected area and 5.6% of the non-affected areas reported complying with the advisory. Most respondents from the affected area still used unboiled water to brush teeth, wash salads and fruits. There was no difference in compliance between men and women. Using the mass media was proved to be efficient to inform the public and could be used in the future in similar settings. However, more detailed wording of boiling advices should be considered in the future.
Introduction
Consumption of drinking water may cause waterborne disease which can be prevented by protection of the source water, efî¬cient treatment processes and reliable distribution systems. The European Union Drinking Water Directive [1] demands monitoring of tap water for different parameters, such as Escherichia coli, to indicate possible faecal contamination from humans and animals.
System failure or human error may cause an increase in the level of pathogens in the water posing a risk of waterborne disease. For example, in 2001, a large outbreak of gastroenteritis occurred due to accidental introduction of partially treated water to the drinking water supply system in the Netherlands, resulting in 921 households being exposed to contaminated water [2].
In the event that faecal contamination is detected the drinking water company may issue an advice to boil tap water before using it for domestic purposes. On 15 May 2007, E. coli was detected in samples collected the day before of the î¬nished tap water delivered by a company in the province Noord-Holland (North-Holland) in the Netherlands. For preventive reasons, on the same day the company issued an advice for consumers to boil tap water for two minutes before consumption but that this was not necessary for taking a shower or washing. This information was broadcasted through mass-media including the national and regional television channel, radio and newspapers. In addition, a public website used during emergency situations (www.crisis.nl) and a toll-free telephone number were made available for the public to provide information to households in the affected area.
The boil water advice had an impact on approximately 180,000 households in the affected area comprising 13 municipalities. The advice was lifted a week later, on 22 May 2007, as risk for public health was no longer present. In September 2007, the water company published a press release informing that the cause of the water contamination was due to run-off of rainwater contaminated with faeces of breeding gulls on the roof that had seeped into one of the six storage rooms [3].
Elevated levels of microorganisms in drinking water may represent a public health risk. For this reason, we investigated compliance with boil water advice issued by the private water company following the 2007 incident.
Methods
A cross-sectional study was implemented to investigate factors that may have affected water consumption habits of the residents in the area supplied by the water company. For this purpose, on the company’s behalf, a self-administered questionnaire was sent to 300 households in June 2007. Households were selected on the basis of their residence postcodes; half in the area where the advice was valid and half in areas served by the same company but where the advice did not apply. These participants were derived from a database of a private company that conducts online consumer surveys for marketing purposes.
The questionnaire contained questions on demographic information, level of urbanisation, source and time of receiving the information regarding the advice, initial and secondary response to the advice and personal opinions on the company’s response and the advice itself. The data were sent back to the drinking water company and the National Institute for Public Health and the Environment, where they were analysed. The statistical analysis was done with STATA v10.
Results
Ninety-nine households (66%) from the area affected by water contamination and 90 households (60%) from control areas supplied with water by the same company replied to the survey. Women more often than men responded to the questionnaire in both the affected and the non-affected areas (57.7% of all responders). The respondents represented 189 households with a total population of 505 people, 176 (34.9%) of whom were below the age of 18 years. There was no statistically signiî¬cant difference in the number of children per household between the affected and the non-affected areas (p=0.112). Descriptive results for the two different areas are presented in Table 1.
All 189 respondents (100%) in both areas answered that they had been informed about the advice. Ninety-î¬ve (50.3%) of them said they had first heard about it through the television. Other sources were radio (24.3%), friends, relatives or neighbours (22.8%), newspapers (19.6%) and the internet (7.4%).
Persons living in the affected area were more frequently disappointed (14.1%) about the choice of the company to use mass media for the advice than people residing in the non-affected area (2.2%). In the affected area, seven (9.3%) of the respondents had î¬rst reacted with fear to the information on the possible contamination of water, 34 (45.3%) responded with self-control and 34 (45.3%) with the intention to take measures. The corresponding percentages for the non affected area were 15.7%, 72.9% and 11.4%. About half (48.5%) of the respondents from the affected area said they had looked for more information when they had heard about the advice, while the corresponding proportion of respondents from the non-affected area was only 8.9% (p<0.001). The most common source of active search for more information was the website of the water supply company.
Eighty-one (81.8%) of all respondents in the affected area said they had complied with the advice. This was done by buying bottled water (43.4% of all respondents in affected area) or boiling tap water for two minutes before consuming it (70.7%). None of the respondents in the area stopped consuming tap water completely. Five (5.6%) of the respondents in the non-affected area were buying bottled water and three of them (3.3%) were boiling tap water during the advice. These numbers were considerably lower than the corresponding ones in the affected area, but showed that compliance exceeded beyond the affected area.
Even though it had not been advised to boil water for activities such as washing and showering, 26 (26.3%) of the respondents in the affected area stated that they had not been aware of that.
Concerning the image of the drinking water company, 177 respondents (93.7%) thought that the company had done well informing the consumers about the water contamination and its response to it. This prevailing opinion was not different between respondents from the affected area and those from the non affected area.
The respondents’ compliance with the advice was independent of sex, age and the presence of children in the household. However, the respondents were 138.6 times more likely to follow the advice if a second person in the household was following it as well (p<0.001).
Reasons for non-compliance with the advice are given in Table 2.Some of the respondents replied that they had been using boiled water for uses other than drinking, too. These results are shown in Table 3.The majority of the respondents stated that their image of the company had not changed after the incident and the six-day advice (78.8% in the affected area and 88.9% in the non-affected area).
Factors affecting compliance
The type of mass media from which people in the affected area found out about the advice played no signiî¬cant role in the subsequent compliance of the respondents. The highest compliance rates occurred among those in the affected area who heard about the advice from the internet (90%) or from friends (89.5%). Respondents informed by more than one source were more likely to have complied with the advice (90.9% against 79.2%) but this difference was not statistically signiî¬cant. The source of information did not depend on the age (p=0.6532). Compliance with the advice did not differ between households with children and those without children (p=0.536).
Respondents who undertook active search for more information may have been more likely to follow the advice than those who did not proceed to further active search for more information (89.4% vs. 74.5%, p=0.058).Since all respondents knew about the advice, it was not possible to estimate unwitting compliance rates.
Conclusions
Since excess of standard levels of certain microorganisms such as E. coli indicate faecal contamination and the possible presence of pathogens in tap water, the time between the water sampling, water analysis and the boil water notice is essential. During this period, consumers may be exposed to tap water of unacceptable quality. The choice of mass media for broadcasting the advice is therefore believed to be an effective measure to prevent panic and to protect public health.
From this study, it can be concluded that participating consumers not only thought that they had been informed about the advice in a timely manner, but that also the response of the company to ensure the advice would reach the public had been satisfactory as well as the choice of communication channels. Thus, the incident did not lead to customers’ dissatisfaction or a degradation of the company’s image.
The sample in our study derived from a database of people who subscribed to be included in different research surveys. This could raise questions regarding the representativeness of the study population. We agree that there is a need for similar studies with samples deriving randomly from the whole population and not from potentially biased data sources. For example, 100% of the participants stated that they had been informed about the boiling water advice; however, subscribers to online databases for marketing purposes may be more likely to regularly follow the news than the general population.
In the Netherlands, boil water notices are not harmonised but are determined by the drinking water company itself. This results in different advice with respect to, for instance, boiling time. Internationally recognised guidelines, such as the World Health Organization (WHO) Guidelines for Drinking Water Quality [4], could be taken into consideration in case of similar “crises†in the future. According to data from the water company involved in our study, about thirty boil water advices are issued per year in their responsibility area (ca. 700,000 households); involving on average 100 households per time. So, the chance to receive a boil water advice is small but existing.
The inclusion of recommendations including use of water for brushing teeth, washing fruits and vegetables may also prove helpful in future advice, since it is not only consumption of water through drinking that may pose a risk to the consumer. Bathing and showering may also need to be addressed separately, as a possible link between this kind of exposure to contaminated water and itching has been described elsewhere [2]. Also, although this conclusion does not directly follow from our results, vulnerable groups should be targeted separately in the advice; elderly people and children may easily miss information disseminated through the means of mass media [5,6].
Few studies have been published on boil water notices and their results seldom reach the public. Further research would also be useful to incorporate î¬ndings from compliance studies to model health effects of drinking contaminated water during similar events.
|
In which city did this happen?
|
{
"answer_start": [],
"text": []
}
|
495
|
Compliance with boil water advice following a water contamination incident in the Netherlands in 2007
|
In May 2007, Escherichia coli was detected in tap water supplied by a company in North Holland. The company issued advice through mass media to boil tap water before consumption; this advice was lifted six days later. A cross-sectional study was implemented to investigate compliance among residents in this area. Based on postcode, a total of 300 households, chosen randomly from a database of a private company performing internet-based surveys for different marketing purposes, were sent a self-administered questionnaire for this study. The questionnaire contained questions on demographic information, source of information regarding the advice, response to it and personal opinions on the company’s reaction and the advice. Ninety-nine (66%) households of the affected area and 90 (60%) households from non-affected areas served by the same company replied to the survey. All respondents knew about the advice. 81.8% of the respondents in the affected area and 5.6% of the non-affected areas reported complying with the advisory. Most respondents from the affected area still used unboiled water to brush teeth, wash salads and fruits. There was no difference in compliance between men and women. Using the mass media was proved to be efficient to inform the public and could be used in the future in similar settings. However, more detailed wording of boiling advices should be considered in the future.
Introduction
Consumption of drinking water may cause waterborne disease which can be prevented by protection of the source water, efî¬cient treatment processes and reliable distribution systems. The European Union Drinking Water Directive [1] demands monitoring of tap water for different parameters, such as Escherichia coli, to indicate possible faecal contamination from humans and animals.
System failure or human error may cause an increase in the level of pathogens in the water posing a risk of waterborne disease. For example, in 2001, a large outbreak of gastroenteritis occurred due to accidental introduction of partially treated water to the drinking water supply system in the Netherlands, resulting in 921 households being exposed to contaminated water [2].
In the event that faecal contamination is detected the drinking water company may issue an advice to boil tap water before using it for domestic purposes. On 15 May 2007, E. coli was detected in samples collected the day before of the î¬nished tap water delivered by a company in the province Noord-Holland (North-Holland) in the Netherlands. For preventive reasons, on the same day the company issued an advice for consumers to boil tap water for two minutes before consumption but that this was not necessary for taking a shower or washing. This information was broadcasted through mass-media including the national and regional television channel, radio and newspapers. In addition, a public website used during emergency situations (www.crisis.nl) and a toll-free telephone number were made available for the public to provide information to households in the affected area.
The boil water advice had an impact on approximately 180,000 households in the affected area comprising 13 municipalities. The advice was lifted a week later, on 22 May 2007, as risk for public health was no longer present. In September 2007, the water company published a press release informing that the cause of the water contamination was due to run-off of rainwater contaminated with faeces of breeding gulls on the roof that had seeped into one of the six storage rooms [3].
Elevated levels of microorganisms in drinking water may represent a public health risk. For this reason, we investigated compliance with boil water advice issued by the private water company following the 2007 incident.
Methods
A cross-sectional study was implemented to investigate factors that may have affected water consumption habits of the residents in the area supplied by the water company. For this purpose, on the company’s behalf, a self-administered questionnaire was sent to 300 households in June 2007. Households were selected on the basis of their residence postcodes; half in the area where the advice was valid and half in areas served by the same company but where the advice did not apply. These participants were derived from a database of a private company that conducts online consumer surveys for marketing purposes.
The questionnaire contained questions on demographic information, level of urbanisation, source and time of receiving the information regarding the advice, initial and secondary response to the advice and personal opinions on the company’s response and the advice itself. The data were sent back to the drinking water company and the National Institute for Public Health and the Environment, where they were analysed. The statistical analysis was done with STATA v10.
Results
Ninety-nine households (66%) from the area affected by water contamination and 90 households (60%) from control areas supplied with water by the same company replied to the survey. Women more often than men responded to the questionnaire in both the affected and the non-affected areas (57.7% of all responders). The respondents represented 189 households with a total population of 505 people, 176 (34.9%) of whom were below the age of 18 years. There was no statistically signiî¬cant difference in the number of children per household between the affected and the non-affected areas (p=0.112). Descriptive results for the two different areas are presented in Table 1.
All 189 respondents (100%) in both areas answered that they had been informed about the advice. Ninety-î¬ve (50.3%) of them said they had first heard about it through the television. Other sources were radio (24.3%), friends, relatives or neighbours (22.8%), newspapers (19.6%) and the internet (7.4%).
Persons living in the affected area were more frequently disappointed (14.1%) about the choice of the company to use mass media for the advice than people residing in the non-affected area (2.2%). In the affected area, seven (9.3%) of the respondents had î¬rst reacted with fear to the information on the possible contamination of water, 34 (45.3%) responded with self-control and 34 (45.3%) with the intention to take measures. The corresponding percentages for the non affected area were 15.7%, 72.9% and 11.4%. About half (48.5%) of the respondents from the affected area said they had looked for more information when they had heard about the advice, while the corresponding proportion of respondents from the non-affected area was only 8.9% (p<0.001). The most common source of active search for more information was the website of the water supply company.
Eighty-one (81.8%) of all respondents in the affected area said they had complied with the advice. This was done by buying bottled water (43.4% of all respondents in affected area) or boiling tap water for two minutes before consuming it (70.7%). None of the respondents in the area stopped consuming tap water completely. Five (5.6%) of the respondents in the non-affected area were buying bottled water and three of them (3.3%) were boiling tap water during the advice. These numbers were considerably lower than the corresponding ones in the affected area, but showed that compliance exceeded beyond the affected area.
Even though it had not been advised to boil water for activities such as washing and showering, 26 (26.3%) of the respondents in the affected area stated that they had not been aware of that.
Concerning the image of the drinking water company, 177 respondents (93.7%) thought that the company had done well informing the consumers about the water contamination and its response to it. This prevailing opinion was not different between respondents from the affected area and those from the non affected area.
The respondents’ compliance with the advice was independent of sex, age and the presence of children in the household. However, the respondents were 138.6 times more likely to follow the advice if a second person in the household was following it as well (p<0.001).
Reasons for non-compliance with the advice are given in Table 2.Some of the respondents replied that they had been using boiled water for uses other than drinking, too. These results are shown in Table 3.The majority of the respondents stated that their image of the company had not changed after the incident and the six-day advice (78.8% in the affected area and 88.9% in the non-affected area).
Factors affecting compliance
The type of mass media from which people in the affected area found out about the advice played no signiî¬cant role in the subsequent compliance of the respondents. The highest compliance rates occurred among those in the affected area who heard about the advice from the internet (90%) or from friends (89.5%). Respondents informed by more than one source were more likely to have complied with the advice (90.9% against 79.2%) but this difference was not statistically signiî¬cant. The source of information did not depend on the age (p=0.6532). Compliance with the advice did not differ between households with children and those without children (p=0.536).
Respondents who undertook active search for more information may have been more likely to follow the advice than those who did not proceed to further active search for more information (89.4% vs. 74.5%, p=0.058).Since all respondents knew about the advice, it was not possible to estimate unwitting compliance rates.
Conclusions
Since excess of standard levels of certain microorganisms such as E. coli indicate faecal contamination and the possible presence of pathogens in tap water, the time between the water sampling, water analysis and the boil water notice is essential. During this period, consumers may be exposed to tap water of unacceptable quality. The choice of mass media for broadcasting the advice is therefore believed to be an effective measure to prevent panic and to protect public health.
From this study, it can be concluded that participating consumers not only thought that they had been informed about the advice in a timely manner, but that also the response of the company to ensure the advice would reach the public had been satisfactory as well as the choice of communication channels. Thus, the incident did not lead to customers’ dissatisfaction or a degradation of the company’s image.
The sample in our study derived from a database of people who subscribed to be included in different research surveys. This could raise questions regarding the representativeness of the study population. We agree that there is a need for similar studies with samples deriving randomly from the whole population and not from potentially biased data sources. For example, 100% of the participants stated that they had been informed about the boiling water advice; however, subscribers to online databases for marketing purposes may be more likely to regularly follow the news than the general population.
In the Netherlands, boil water notices are not harmonised but are determined by the drinking water company itself. This results in different advice with respect to, for instance, boiling time. Internationally recognised guidelines, such as the World Health Organization (WHO) Guidelines for Drinking Water Quality [4], could be taken into consideration in case of similar “crises†in the future. According to data from the water company involved in our study, about thirty boil water advices are issued per year in their responsibility area (ca. 700,000 households); involving on average 100 households per time. So, the chance to receive a boil water advice is small but existing.
The inclusion of recommendations including use of water for brushing teeth, washing fruits and vegetables may also prove helpful in future advice, since it is not only consumption of water through drinking that may pose a risk to the consumer. Bathing and showering may also need to be addressed separately, as a possible link between this kind of exposure to contaminated water and itching has been described elsewhere [2]. Also, although this conclusion does not directly follow from our results, vulnerable groups should be targeted separately in the advice; elderly people and children may easily miss information disseminated through the means of mass media [5,6].
Few studies have been published on boil water notices and their results seldom reach the public. Further research would also be useful to incorporate î¬ndings from compliance studies to model health effects of drinking contaminated water during similar events.
|
In which region did this happen?
|
{
"answer_start": [
2620
],
"text": [
"Noord-Holland"
]
}
|
496
|
Compliance with boil water advice following a water contamination incident in the Netherlands in 2007
|
In May 2007, Escherichia coli was detected in tap water supplied by a company in North Holland. The company issued advice through mass media to boil tap water before consumption; this advice was lifted six days later. A cross-sectional study was implemented to investigate compliance among residents in this area. Based on postcode, a total of 300 households, chosen randomly from a database of a private company performing internet-based surveys for different marketing purposes, were sent a self-administered questionnaire for this study. The questionnaire contained questions on demographic information, source of information regarding the advice, response to it and personal opinions on the company’s reaction and the advice. Ninety-nine (66%) households of the affected area and 90 (60%) households from non-affected areas served by the same company replied to the survey. All respondents knew about the advice. 81.8% of the respondents in the affected area and 5.6% of the non-affected areas reported complying with the advisory. Most respondents from the affected area still used unboiled water to brush teeth, wash salads and fruits. There was no difference in compliance between men and women. Using the mass media was proved to be efficient to inform the public and could be used in the future in similar settings. However, more detailed wording of boiling advices should be considered in the future.
Introduction
Consumption of drinking water may cause waterborne disease which can be prevented by protection of the source water, efî¬cient treatment processes and reliable distribution systems. The European Union Drinking Water Directive [1] demands monitoring of tap water for different parameters, such as Escherichia coli, to indicate possible faecal contamination from humans and animals.
System failure or human error may cause an increase in the level of pathogens in the water posing a risk of waterborne disease. For example, in 2001, a large outbreak of gastroenteritis occurred due to accidental introduction of partially treated water to the drinking water supply system in the Netherlands, resulting in 921 households being exposed to contaminated water [2].
In the event that faecal contamination is detected the drinking water company may issue an advice to boil tap water before using it for domestic purposes. On 15 May 2007, E. coli was detected in samples collected the day before of the î¬nished tap water delivered by a company in the province Noord-Holland (North-Holland) in the Netherlands. For preventive reasons, on the same day the company issued an advice for consumers to boil tap water for two minutes before consumption but that this was not necessary for taking a shower or washing. This information was broadcasted through mass-media including the national and regional television channel, radio and newspapers. In addition, a public website used during emergency situations (www.crisis.nl) and a toll-free telephone number were made available for the public to provide information to households in the affected area.
The boil water advice had an impact on approximately 180,000 households in the affected area comprising 13 municipalities. The advice was lifted a week later, on 22 May 2007, as risk for public health was no longer present. In September 2007, the water company published a press release informing that the cause of the water contamination was due to run-off of rainwater contaminated with faeces of breeding gulls on the roof that had seeped into one of the six storage rooms [3].
Elevated levels of microorganisms in drinking water may represent a public health risk. For this reason, we investigated compliance with boil water advice issued by the private water company following the 2007 incident.
Methods
A cross-sectional study was implemented to investigate factors that may have affected water consumption habits of the residents in the area supplied by the water company. For this purpose, on the company’s behalf, a self-administered questionnaire was sent to 300 households in June 2007. Households were selected on the basis of their residence postcodes; half in the area where the advice was valid and half in areas served by the same company but where the advice did not apply. These participants were derived from a database of a private company that conducts online consumer surveys for marketing purposes.
The questionnaire contained questions on demographic information, level of urbanisation, source and time of receiving the information regarding the advice, initial and secondary response to the advice and personal opinions on the company’s response and the advice itself. The data were sent back to the drinking water company and the National Institute for Public Health and the Environment, where they were analysed. The statistical analysis was done with STATA v10.
Results
Ninety-nine households (66%) from the area affected by water contamination and 90 households (60%) from control areas supplied with water by the same company replied to the survey. Women more often than men responded to the questionnaire in both the affected and the non-affected areas (57.7% of all responders). The respondents represented 189 households with a total population of 505 people, 176 (34.9%) of whom were below the age of 18 years. There was no statistically signiî¬cant difference in the number of children per household between the affected and the non-affected areas (p=0.112). Descriptive results for the two different areas are presented in Table 1.
All 189 respondents (100%) in both areas answered that they had been informed about the advice. Ninety-î¬ve (50.3%) of them said they had first heard about it through the television. Other sources were radio (24.3%), friends, relatives or neighbours (22.8%), newspapers (19.6%) and the internet (7.4%).
Persons living in the affected area were more frequently disappointed (14.1%) about the choice of the company to use mass media for the advice than people residing in the non-affected area (2.2%). In the affected area, seven (9.3%) of the respondents had î¬rst reacted with fear to the information on the possible contamination of water, 34 (45.3%) responded with self-control and 34 (45.3%) with the intention to take measures. The corresponding percentages for the non affected area were 15.7%, 72.9% and 11.4%. About half (48.5%) of the respondents from the affected area said they had looked for more information when they had heard about the advice, while the corresponding proportion of respondents from the non-affected area was only 8.9% (p<0.001). The most common source of active search for more information was the website of the water supply company.
Eighty-one (81.8%) of all respondents in the affected area said they had complied with the advice. This was done by buying bottled water (43.4% of all respondents in affected area) or boiling tap water for two minutes before consuming it (70.7%). None of the respondents in the area stopped consuming tap water completely. Five (5.6%) of the respondents in the non-affected area were buying bottled water and three of them (3.3%) were boiling tap water during the advice. These numbers were considerably lower than the corresponding ones in the affected area, but showed that compliance exceeded beyond the affected area.
Even though it had not been advised to boil water for activities such as washing and showering, 26 (26.3%) of the respondents in the affected area stated that they had not been aware of that.
Concerning the image of the drinking water company, 177 respondents (93.7%) thought that the company had done well informing the consumers about the water contamination and its response to it. This prevailing opinion was not different between respondents from the affected area and those from the non affected area.
The respondents’ compliance with the advice was independent of sex, age and the presence of children in the household. However, the respondents were 138.6 times more likely to follow the advice if a second person in the household was following it as well (p<0.001).
Reasons for non-compliance with the advice are given in Table 2.Some of the respondents replied that they had been using boiled water for uses other than drinking, too. These results are shown in Table 3.The majority of the respondents stated that their image of the company had not changed after the incident and the six-day advice (78.8% in the affected area and 88.9% in the non-affected area).
Factors affecting compliance
The type of mass media from which people in the affected area found out about the advice played no signiî¬cant role in the subsequent compliance of the respondents. The highest compliance rates occurred among those in the affected area who heard about the advice from the internet (90%) or from friends (89.5%). Respondents informed by more than one source were more likely to have complied with the advice (90.9% against 79.2%) but this difference was not statistically signiî¬cant. The source of information did not depend on the age (p=0.6532). Compliance with the advice did not differ between households with children and those without children (p=0.536).
Respondents who undertook active search for more information may have been more likely to follow the advice than those who did not proceed to further active search for more information (89.4% vs. 74.5%, p=0.058).Since all respondents knew about the advice, it was not possible to estimate unwitting compliance rates.
Conclusions
Since excess of standard levels of certain microorganisms such as E. coli indicate faecal contamination and the possible presence of pathogens in tap water, the time between the water sampling, water analysis and the boil water notice is essential. During this period, consumers may be exposed to tap water of unacceptable quality. The choice of mass media for broadcasting the advice is therefore believed to be an effective measure to prevent panic and to protect public health.
From this study, it can be concluded that participating consumers not only thought that they had been informed about the advice in a timely manner, but that also the response of the company to ensure the advice would reach the public had been satisfactory as well as the choice of communication channels. Thus, the incident did not lead to customers’ dissatisfaction or a degradation of the company’s image.
The sample in our study derived from a database of people who subscribed to be included in different research surveys. This could raise questions regarding the representativeness of the study population. We agree that there is a need for similar studies with samples deriving randomly from the whole population and not from potentially biased data sources. For example, 100% of the participants stated that they had been informed about the boiling water advice; however, subscribers to online databases for marketing purposes may be more likely to regularly follow the news than the general population.
In the Netherlands, boil water notices are not harmonised but are determined by the drinking water company itself. This results in different advice with respect to, for instance, boiling time. Internationally recognised guidelines, such as the World Health Organization (WHO) Guidelines for Drinking Water Quality [4], could be taken into consideration in case of similar “crises†in the future. According to data from the water company involved in our study, about thirty boil water advices are issued per year in their responsibility area (ca. 700,000 households); involving on average 100 households per time. So, the chance to receive a boil water advice is small but existing.
The inclusion of recommendations including use of water for brushing teeth, washing fruits and vegetables may also prove helpful in future advice, since it is not only consumption of water through drinking that may pose a risk to the consumer. Bathing and showering may also need to be addressed separately, as a possible link between this kind of exposure to contaminated water and itching has been described elsewhere [2]. Also, although this conclusion does not directly follow from our results, vulnerable groups should be targeted separately in the advice; elderly people and children may easily miss information disseminated through the means of mass media [5,6].
Few studies have been published on boil water notices and their results seldom reach the public. Further research would also be useful to incorporate î¬ndings from compliance studies to model health effects of drinking contaminated water during similar events.
|
In which country did this happen?
|
{
"answer_start": [
2220
],
"text": [
"the Netherlands"
]
}
|
497
|
Compliance with boil water advice following a water contamination incident in the Netherlands in 2007
|
In May 2007, Escherichia coli was detected in tap water supplied by a company in North Holland. The company issued advice through mass media to boil tap water before consumption; this advice was lifted six days later. A cross-sectional study was implemented to investigate compliance among residents in this area. Based on postcode, a total of 300 households, chosen randomly from a database of a private company performing internet-based surveys for different marketing purposes, were sent a self-administered questionnaire for this study. The questionnaire contained questions on demographic information, source of information regarding the advice, response to it and personal opinions on the company’s reaction and the advice. Ninety-nine (66%) households of the affected area and 90 (60%) households from non-affected areas served by the same company replied to the survey. All respondents knew about the advice. 81.8% of the respondents in the affected area and 5.6% of the non-affected areas reported complying with the advisory. Most respondents from the affected area still used unboiled water to brush teeth, wash salads and fruits. There was no difference in compliance between men and women. Using the mass media was proved to be efficient to inform the public and could be used in the future in similar settings. However, more detailed wording of boiling advices should be considered in the future.
Introduction
Consumption of drinking water may cause waterborne disease which can be prevented by protection of the source water, efî¬cient treatment processes and reliable distribution systems. The European Union Drinking Water Directive [1] demands monitoring of tap water for different parameters, such as Escherichia coli, to indicate possible faecal contamination from humans and animals.
System failure or human error may cause an increase in the level of pathogens in the water posing a risk of waterborne disease. For example, in 2001, a large outbreak of gastroenteritis occurred due to accidental introduction of partially treated water to the drinking water supply system in the Netherlands, resulting in 921 households being exposed to contaminated water [2].
In the event that faecal contamination is detected the drinking water company may issue an advice to boil tap water before using it for domestic purposes. On 15 May 2007, E. coli was detected in samples collected the day before of the î¬nished tap water delivered by a company in the province Noord-Holland (North-Holland) in the Netherlands. For preventive reasons, on the same day the company issued an advice for consumers to boil tap water for two minutes before consumption but that this was not necessary for taking a shower or washing. This information was broadcasted through mass-media including the national and regional television channel, radio and newspapers. In addition, a public website used during emergency situations (www.crisis.nl) and a toll-free telephone number were made available for the public to provide information to households in the affected area.
The boil water advice had an impact on approximately 180,000 households in the affected area comprising 13 municipalities. The advice was lifted a week later, on 22 May 2007, as risk for public health was no longer present. In September 2007, the water company published a press release informing that the cause of the water contamination was due to run-off of rainwater contaminated with faeces of breeding gulls on the roof that had seeped into one of the six storage rooms [3].
Elevated levels of microorganisms in drinking water may represent a public health risk. For this reason, we investigated compliance with boil water advice issued by the private water company following the 2007 incident.
Methods
A cross-sectional study was implemented to investigate factors that may have affected water consumption habits of the residents in the area supplied by the water company. For this purpose, on the company’s behalf, a self-administered questionnaire was sent to 300 households in June 2007. Households were selected on the basis of their residence postcodes; half in the area where the advice was valid and half in areas served by the same company but where the advice did not apply. These participants were derived from a database of a private company that conducts online consumer surveys for marketing purposes.
The questionnaire contained questions on demographic information, level of urbanisation, source and time of receiving the information regarding the advice, initial and secondary response to the advice and personal opinions on the company’s response and the advice itself. The data were sent back to the drinking water company and the National Institute for Public Health and the Environment, where they were analysed. The statistical analysis was done with STATA v10.
Results
Ninety-nine households (66%) from the area affected by water contamination and 90 households (60%) from control areas supplied with water by the same company replied to the survey. Women more often than men responded to the questionnaire in both the affected and the non-affected areas (57.7% of all responders). The respondents represented 189 households with a total population of 505 people, 176 (34.9%) of whom were below the age of 18 years. There was no statistically signiî¬cant difference in the number of children per household between the affected and the non-affected areas (p=0.112). Descriptive results for the two different areas are presented in Table 1.
All 189 respondents (100%) in both areas answered that they had been informed about the advice. Ninety-î¬ve (50.3%) of them said they had first heard about it through the television. Other sources were radio (24.3%), friends, relatives or neighbours (22.8%), newspapers (19.6%) and the internet (7.4%).
Persons living in the affected area were more frequently disappointed (14.1%) about the choice of the company to use mass media for the advice than people residing in the non-affected area (2.2%). In the affected area, seven (9.3%) of the respondents had î¬rst reacted with fear to the information on the possible contamination of water, 34 (45.3%) responded with self-control and 34 (45.3%) with the intention to take measures. The corresponding percentages for the non affected area were 15.7%, 72.9% and 11.4%. About half (48.5%) of the respondents from the affected area said they had looked for more information when they had heard about the advice, while the corresponding proportion of respondents from the non-affected area was only 8.9% (p<0.001). The most common source of active search for more information was the website of the water supply company.
Eighty-one (81.8%) of all respondents in the affected area said they had complied with the advice. This was done by buying bottled water (43.4% of all respondents in affected area) or boiling tap water for two minutes before consuming it (70.7%). None of the respondents in the area stopped consuming tap water completely. Five (5.6%) of the respondents in the non-affected area were buying bottled water and three of them (3.3%) were boiling tap water during the advice. These numbers were considerably lower than the corresponding ones in the affected area, but showed that compliance exceeded beyond the affected area.
Even though it had not been advised to boil water for activities such as washing and showering, 26 (26.3%) of the respondents in the affected area stated that they had not been aware of that.
Concerning the image of the drinking water company, 177 respondents (93.7%) thought that the company had done well informing the consumers about the water contamination and its response to it. This prevailing opinion was not different between respondents from the affected area and those from the non affected area.
The respondents’ compliance with the advice was independent of sex, age and the presence of children in the household. However, the respondents were 138.6 times more likely to follow the advice if a second person in the household was following it as well (p<0.001).
Reasons for non-compliance with the advice are given in Table 2.Some of the respondents replied that they had been using boiled water for uses other than drinking, too. These results are shown in Table 3.The majority of the respondents stated that their image of the company had not changed after the incident and the six-day advice (78.8% in the affected area and 88.9% in the non-affected area).
Factors affecting compliance
The type of mass media from which people in the affected area found out about the advice played no signiî¬cant role in the subsequent compliance of the respondents. The highest compliance rates occurred among those in the affected area who heard about the advice from the internet (90%) or from friends (89.5%). Respondents informed by more than one source were more likely to have complied with the advice (90.9% against 79.2%) but this difference was not statistically signiî¬cant. The source of information did not depend on the age (p=0.6532). Compliance with the advice did not differ between households with children and those without children (p=0.536).
Respondents who undertook active search for more information may have been more likely to follow the advice than those who did not proceed to further active search for more information (89.4% vs. 74.5%, p=0.058).Since all respondents knew about the advice, it was not possible to estimate unwitting compliance rates.
Conclusions
Since excess of standard levels of certain microorganisms such as E. coli indicate faecal contamination and the possible presence of pathogens in tap water, the time between the water sampling, water analysis and the boil water notice is essential. During this period, consumers may be exposed to tap water of unacceptable quality. The choice of mass media for broadcasting the advice is therefore believed to be an effective measure to prevent panic and to protect public health.
From this study, it can be concluded that participating consumers not only thought that they had been informed about the advice in a timely manner, but that also the response of the company to ensure the advice would reach the public had been satisfactory as well as the choice of communication channels. Thus, the incident did not lead to customers’ dissatisfaction or a degradation of the company’s image.
The sample in our study derived from a database of people who subscribed to be included in different research surveys. This could raise questions regarding the representativeness of the study population. We agree that there is a need for similar studies with samples deriving randomly from the whole population and not from potentially biased data sources. For example, 100% of the participants stated that they had been informed about the boiling water advice; however, subscribers to online databases for marketing purposes may be more likely to regularly follow the news than the general population.
In the Netherlands, boil water notices are not harmonised but are determined by the drinking water company itself. This results in different advice with respect to, for instance, boiling time. Internationally recognised guidelines, such as the World Health Organization (WHO) Guidelines for Drinking Water Quality [4], could be taken into consideration in case of similar “crises†in the future. According to data from the water company involved in our study, about thirty boil water advices are issued per year in their responsibility area (ca. 700,000 households); involving on average 100 households per time. So, the chance to receive a boil water advice is small but existing.
The inclusion of recommendations including use of water for brushing teeth, washing fruits and vegetables may also prove helpful in future advice, since it is not only consumption of water through drinking that may pose a risk to the consumer. Bathing and showering may also need to be addressed separately, as a possible link between this kind of exposure to contaminated water and itching has been described elsewhere [2]. Also, although this conclusion does not directly follow from our results, vulnerable groups should be targeted separately in the advice; elderly people and children may easily miss information disseminated through the means of mass media [5,6].
Few studies have been published on boil water notices and their results seldom reach the public. Further research would also be useful to incorporate î¬ndings from compliance studies to model health effects of drinking contaminated water during similar events.
|
Where did this happen?
|
{
"answer_start": [
2220
],
"text": [
"the Netherlands"
]
}
|
498
|
Compliance with boil water advice following a water contamination incident in the Netherlands in 2007
|
In May 2007, Escherichia coli was detected in tap water supplied by a company in North Holland. The company issued advice through mass media to boil tap water before consumption; this advice was lifted six days later. A cross-sectional study was implemented to investigate compliance among residents in this area. Based on postcode, a total of 300 households, chosen randomly from a database of a private company performing internet-based surveys for different marketing purposes, were sent a self-administered questionnaire for this study. The questionnaire contained questions on demographic information, source of information regarding the advice, response to it and personal opinions on the company’s reaction and the advice. Ninety-nine (66%) households of the affected area and 90 (60%) households from non-affected areas served by the same company replied to the survey. All respondents knew about the advice. 81.8% of the respondents in the affected area and 5.6% of the non-affected areas reported complying with the advisory. Most respondents from the affected area still used unboiled water to brush teeth, wash salads and fruits. There was no difference in compliance between men and women. Using the mass media was proved to be efficient to inform the public and could be used in the future in similar settings. However, more detailed wording of boiling advices should be considered in the future.
Introduction
Consumption of drinking water may cause waterborne disease which can be prevented by protection of the source water, efî¬cient treatment processes and reliable distribution systems. The European Union Drinking Water Directive [1] demands monitoring of tap water for different parameters, such as Escherichia coli, to indicate possible faecal contamination from humans and animals.
System failure or human error may cause an increase in the level of pathogens in the water posing a risk of waterborne disease. For example, in 2001, a large outbreak of gastroenteritis occurred due to accidental introduction of partially treated water to the drinking water supply system in the Netherlands, resulting in 921 households being exposed to contaminated water [2].
In the event that faecal contamination is detected the drinking water company may issue an advice to boil tap water before using it for domestic purposes. On 15 May 2007, E. coli was detected in samples collected the day before of the î¬nished tap water delivered by a company in the province Noord-Holland (North-Holland) in the Netherlands. For preventive reasons, on the same day the company issued an advice for consumers to boil tap water for two minutes before consumption but that this was not necessary for taking a shower or washing. This information was broadcasted through mass-media including the national and regional television channel, radio and newspapers. In addition, a public website used during emergency situations (www.crisis.nl) and a toll-free telephone number were made available for the public to provide information to households in the affected area.
The boil water advice had an impact on approximately 180,000 households in the affected area comprising 13 municipalities. The advice was lifted a week later, on 22 May 2007, as risk for public health was no longer present. In September 2007, the water company published a press release informing that the cause of the water contamination was due to run-off of rainwater contaminated with faeces of breeding gulls on the roof that had seeped into one of the six storage rooms [3].
Elevated levels of microorganisms in drinking water may represent a public health risk. For this reason, we investigated compliance with boil water advice issued by the private water company following the 2007 incident.
Methods
A cross-sectional study was implemented to investigate factors that may have affected water consumption habits of the residents in the area supplied by the water company. For this purpose, on the company’s behalf, a self-administered questionnaire was sent to 300 households in June 2007. Households were selected on the basis of their residence postcodes; half in the area where the advice was valid and half in areas served by the same company but where the advice did not apply. These participants were derived from a database of a private company that conducts online consumer surveys for marketing purposes.
The questionnaire contained questions on demographic information, level of urbanisation, source and time of receiving the information regarding the advice, initial and secondary response to the advice and personal opinions on the company’s response and the advice itself. The data were sent back to the drinking water company and the National Institute for Public Health and the Environment, where they were analysed. The statistical analysis was done with STATA v10.
Results
Ninety-nine households (66%) from the area affected by water contamination and 90 households (60%) from control areas supplied with water by the same company replied to the survey. Women more often than men responded to the questionnaire in both the affected and the non-affected areas (57.7% of all responders). The respondents represented 189 households with a total population of 505 people, 176 (34.9%) of whom were below the age of 18 years. There was no statistically signiî¬cant difference in the number of children per household between the affected and the non-affected areas (p=0.112). Descriptive results for the two different areas are presented in Table 1.
All 189 respondents (100%) in both areas answered that they had been informed about the advice. Ninety-î¬ve (50.3%) of them said they had first heard about it through the television. Other sources were radio (24.3%), friends, relatives or neighbours (22.8%), newspapers (19.6%) and the internet (7.4%).
Persons living in the affected area were more frequently disappointed (14.1%) about the choice of the company to use mass media for the advice than people residing in the non-affected area (2.2%). In the affected area, seven (9.3%) of the respondents had î¬rst reacted with fear to the information on the possible contamination of water, 34 (45.3%) responded with self-control and 34 (45.3%) with the intention to take measures. The corresponding percentages for the non affected area were 15.7%, 72.9% and 11.4%. About half (48.5%) of the respondents from the affected area said they had looked for more information when they had heard about the advice, while the corresponding proportion of respondents from the non-affected area was only 8.9% (p<0.001). The most common source of active search for more information was the website of the water supply company.
Eighty-one (81.8%) of all respondents in the affected area said they had complied with the advice. This was done by buying bottled water (43.4% of all respondents in affected area) or boiling tap water for two minutes before consuming it (70.7%). None of the respondents in the area stopped consuming tap water completely. Five (5.6%) of the respondents in the non-affected area were buying bottled water and three of them (3.3%) were boiling tap water during the advice. These numbers were considerably lower than the corresponding ones in the affected area, but showed that compliance exceeded beyond the affected area.
Even though it had not been advised to boil water for activities such as washing and showering, 26 (26.3%) of the respondents in the affected area stated that they had not been aware of that.
Concerning the image of the drinking water company, 177 respondents (93.7%) thought that the company had done well informing the consumers about the water contamination and its response to it. This prevailing opinion was not different between respondents from the affected area and those from the non affected area.
The respondents’ compliance with the advice was independent of sex, age and the presence of children in the household. However, the respondents were 138.6 times more likely to follow the advice if a second person in the household was following it as well (p<0.001).
Reasons for non-compliance with the advice are given in Table 2.Some of the respondents replied that they had been using boiled water for uses other than drinking, too. These results are shown in Table 3.The majority of the respondents stated that their image of the company had not changed after the incident and the six-day advice (78.8% in the affected area and 88.9% in the non-affected area).
Factors affecting compliance
The type of mass media from which people in the affected area found out about the advice played no signiî¬cant role in the subsequent compliance of the respondents. The highest compliance rates occurred among those in the affected area who heard about the advice from the internet (90%) or from friends (89.5%). Respondents informed by more than one source were more likely to have complied with the advice (90.9% against 79.2%) but this difference was not statistically signiî¬cant. The source of information did not depend on the age (p=0.6532). Compliance with the advice did not differ between households with children and those without children (p=0.536).
Respondents who undertook active search for more information may have been more likely to follow the advice than those who did not proceed to further active search for more information (89.4% vs. 74.5%, p=0.058).Since all respondents knew about the advice, it was not possible to estimate unwitting compliance rates.
Conclusions
Since excess of standard levels of certain microorganisms such as E. coli indicate faecal contamination and the possible presence of pathogens in tap water, the time between the water sampling, water analysis and the boil water notice is essential. During this period, consumers may be exposed to tap water of unacceptable quality. The choice of mass media for broadcasting the advice is therefore believed to be an effective measure to prevent panic and to protect public health.
From this study, it can be concluded that participating consumers not only thought that they had been informed about the advice in a timely manner, but that also the response of the company to ensure the advice would reach the public had been satisfactory as well as the choice of communication channels. Thus, the incident did not lead to customers’ dissatisfaction or a degradation of the company’s image.
The sample in our study derived from a database of people who subscribed to be included in different research surveys. This could raise questions regarding the representativeness of the study population. We agree that there is a need for similar studies with samples deriving randomly from the whole population and not from potentially biased data sources. For example, 100% of the participants stated that they had been informed about the boiling water advice; however, subscribers to online databases for marketing purposes may be more likely to regularly follow the news than the general population.
In the Netherlands, boil water notices are not harmonised but are determined by the drinking water company itself. This results in different advice with respect to, for instance, boiling time. Internationally recognised guidelines, such as the World Health Organization (WHO) Guidelines for Drinking Water Quality [4], could be taken into consideration in case of similar “crises†in the future. According to data from the water company involved in our study, about thirty boil water advices are issued per year in their responsibility area (ca. 700,000 households); involving on average 100 households per time. So, the chance to receive a boil water advice is small but existing.
The inclusion of recommendations including use of water for brushing teeth, washing fruits and vegetables may also prove helpful in future advice, since it is not only consumption of water through drinking that may pose a risk to the consumer. Bathing and showering may also need to be addressed separately, as a possible link between this kind of exposure to contaminated water and itching has been described elsewhere [2]. Also, although this conclusion does not directly follow from our results, vulnerable groups should be targeted separately in the advice; elderly people and children may easily miss information disseminated through the means of mass media [5,6].
Few studies have been published on boil water notices and their results seldom reach the public. Further research would also be useful to incorporate î¬ndings from compliance studies to model health effects of drinking contaminated water during similar events.
|
What caused the event?
|
{
"answer_start": [],
"text": []
}
|
499
|
Compliance with boil water advice following a water contamination incident in the Netherlands in 2007
|
In May 2007, Escherichia coli was detected in tap water supplied by a company in North Holland. The company issued advice through mass media to boil tap water before consumption; this advice was lifted six days later. A cross-sectional study was implemented to investigate compliance among residents in this area. Based on postcode, a total of 300 households, chosen randomly from a database of a private company performing internet-based surveys for different marketing purposes, were sent a self-administered questionnaire for this study. The questionnaire contained questions on demographic information, source of information regarding the advice, response to it and personal opinions on the company’s reaction and the advice. Ninety-nine (66%) households of the affected area and 90 (60%) households from non-affected areas served by the same company replied to the survey. All respondents knew about the advice. 81.8% of the respondents in the affected area and 5.6% of the non-affected areas reported complying with the advisory. Most respondents from the affected area still used unboiled water to brush teeth, wash salads and fruits. There was no difference in compliance between men and women. Using the mass media was proved to be efficient to inform the public and could be used in the future in similar settings. However, more detailed wording of boiling advices should be considered in the future.
Introduction
Consumption of drinking water may cause waterborne disease which can be prevented by protection of the source water, efî¬cient treatment processes and reliable distribution systems. The European Union Drinking Water Directive [1] demands monitoring of tap water for different parameters, such as Escherichia coli, to indicate possible faecal contamination from humans and animals.
System failure or human error may cause an increase in the level of pathogens in the water posing a risk of waterborne disease. For example, in 2001, a large outbreak of gastroenteritis occurred due to accidental introduction of partially treated water to the drinking water supply system in the Netherlands, resulting in 921 households being exposed to contaminated water [2].
In the event that faecal contamination is detected the drinking water company may issue an advice to boil tap water before using it for domestic purposes. On 15 May 2007, E. coli was detected in samples collected the day before of the î¬nished tap water delivered by a company in the province Noord-Holland (North-Holland) in the Netherlands. For preventive reasons, on the same day the company issued an advice for consumers to boil tap water for two minutes before consumption but that this was not necessary for taking a shower or washing. This information was broadcasted through mass-media including the national and regional television channel, radio and newspapers. In addition, a public website used during emergency situations (www.crisis.nl) and a toll-free telephone number were made available for the public to provide information to households in the affected area.
The boil water advice had an impact on approximately 180,000 households in the affected area comprising 13 municipalities. The advice was lifted a week later, on 22 May 2007, as risk for public health was no longer present. In September 2007, the water company published a press release informing that the cause of the water contamination was due to run-off of rainwater contaminated with faeces of breeding gulls on the roof that had seeped into one of the six storage rooms [3].
Elevated levels of microorganisms in drinking water may represent a public health risk. For this reason, we investigated compliance with boil water advice issued by the private water company following the 2007 incident.
Methods
A cross-sectional study was implemented to investigate factors that may have affected water consumption habits of the residents in the area supplied by the water company. For this purpose, on the company’s behalf, a self-administered questionnaire was sent to 300 households in June 2007. Households were selected on the basis of their residence postcodes; half in the area where the advice was valid and half in areas served by the same company but where the advice did not apply. These participants were derived from a database of a private company that conducts online consumer surveys for marketing purposes.
The questionnaire contained questions on demographic information, level of urbanisation, source and time of receiving the information regarding the advice, initial and secondary response to the advice and personal opinions on the company’s response and the advice itself. The data were sent back to the drinking water company and the National Institute for Public Health and the Environment, where they were analysed. The statistical analysis was done with STATA v10.
Results
Ninety-nine households (66%) from the area affected by water contamination and 90 households (60%) from control areas supplied with water by the same company replied to the survey. Women more often than men responded to the questionnaire in both the affected and the non-affected areas (57.7% of all responders). The respondents represented 189 households with a total population of 505 people, 176 (34.9%) of whom were below the age of 18 years. There was no statistically signiî¬cant difference in the number of children per household between the affected and the non-affected areas (p=0.112). Descriptive results for the two different areas are presented in Table 1.
All 189 respondents (100%) in both areas answered that they had been informed about the advice. Ninety-î¬ve (50.3%) of them said they had first heard about it through the television. Other sources were radio (24.3%), friends, relatives or neighbours (22.8%), newspapers (19.6%) and the internet (7.4%).
Persons living in the affected area were more frequently disappointed (14.1%) about the choice of the company to use mass media for the advice than people residing in the non-affected area (2.2%). In the affected area, seven (9.3%) of the respondents had î¬rst reacted with fear to the information on the possible contamination of water, 34 (45.3%) responded with self-control and 34 (45.3%) with the intention to take measures. The corresponding percentages for the non affected area were 15.7%, 72.9% and 11.4%. About half (48.5%) of the respondents from the affected area said they had looked for more information when they had heard about the advice, while the corresponding proportion of respondents from the non-affected area was only 8.9% (p<0.001). The most common source of active search for more information was the website of the water supply company.
Eighty-one (81.8%) of all respondents in the affected area said they had complied with the advice. This was done by buying bottled water (43.4% of all respondents in affected area) or boiling tap water for two minutes before consuming it (70.7%). None of the respondents in the area stopped consuming tap water completely. Five (5.6%) of the respondents in the non-affected area were buying bottled water and three of them (3.3%) were boiling tap water during the advice. These numbers were considerably lower than the corresponding ones in the affected area, but showed that compliance exceeded beyond the affected area.
Even though it had not been advised to boil water for activities such as washing and showering, 26 (26.3%) of the respondents in the affected area stated that they had not been aware of that.
Concerning the image of the drinking water company, 177 respondents (93.7%) thought that the company had done well informing the consumers about the water contamination and its response to it. This prevailing opinion was not different between respondents from the affected area and those from the non affected area.
The respondents’ compliance with the advice was independent of sex, age and the presence of children in the household. However, the respondents were 138.6 times more likely to follow the advice if a second person in the household was following it as well (p<0.001).
Reasons for non-compliance with the advice are given in Table 2.Some of the respondents replied that they had been using boiled water for uses other than drinking, too. These results are shown in Table 3.The majority of the respondents stated that their image of the company had not changed after the incident and the six-day advice (78.8% in the affected area and 88.9% in the non-affected area).
Factors affecting compliance
The type of mass media from which people in the affected area found out about the advice played no signiî¬cant role in the subsequent compliance of the respondents. The highest compliance rates occurred among those in the affected area who heard about the advice from the internet (90%) or from friends (89.5%). Respondents informed by more than one source were more likely to have complied with the advice (90.9% against 79.2%) but this difference was not statistically signiî¬cant. The source of information did not depend on the age (p=0.6532). Compliance with the advice did not differ between households with children and those without children (p=0.536).
Respondents who undertook active search for more information may have been more likely to follow the advice than those who did not proceed to further active search for more information (89.4% vs. 74.5%, p=0.058).Since all respondents knew about the advice, it was not possible to estimate unwitting compliance rates.
Conclusions
Since excess of standard levels of certain microorganisms such as E. coli indicate faecal contamination and the possible presence of pathogens in tap water, the time between the water sampling, water analysis and the boil water notice is essential. During this period, consumers may be exposed to tap water of unacceptable quality. The choice of mass media for broadcasting the advice is therefore believed to be an effective measure to prevent panic and to protect public health.
From this study, it can be concluded that participating consumers not only thought that they had been informed about the advice in a timely manner, but that also the response of the company to ensure the advice would reach the public had been satisfactory as well as the choice of communication channels. Thus, the incident did not lead to customers’ dissatisfaction or a degradation of the company’s image.
The sample in our study derived from a database of people who subscribed to be included in different research surveys. This could raise questions regarding the representativeness of the study population. We agree that there is a need for similar studies with samples deriving randomly from the whole population and not from potentially biased data sources. For example, 100% of the participants stated that they had been informed about the boiling water advice; however, subscribers to online databases for marketing purposes may be more likely to regularly follow the news than the general population.
In the Netherlands, boil water notices are not harmonised but are determined by the drinking water company itself. This results in different advice with respect to, for instance, boiling time. Internationally recognised guidelines, such as the World Health Organization (WHO) Guidelines for Drinking Water Quality [4], could be taken into consideration in case of similar “crises†in the future. According to data from the water company involved in our study, about thirty boil water advices are issued per year in their responsibility area (ca. 700,000 households); involving on average 100 households per time. So, the chance to receive a boil water advice is small but existing.
The inclusion of recommendations including use of water for brushing teeth, washing fruits and vegetables may also prove helpful in future advice, since it is not only consumption of water through drinking that may pose a risk to the consumer. Bathing and showering may also need to be addressed separately, as a possible link between this kind of exposure to contaminated water and itching has been described elsewhere [2]. Also, although this conclusion does not directly follow from our results, vulnerable groups should be targeted separately in the advice; elderly people and children may easily miss information disseminated through the means of mass media [5,6].
Few studies have been published on boil water notices and their results seldom reach the public. Further research would also be useful to incorporate î¬ndings from compliance studies to model health effects of drinking contaminated water during similar events.
|
What was the cause of the event?
|
{
"answer_start": [
1846
],
"text": [
"faecal contamination"
]
}
|
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