|Burden and molecular epidemiology of Rotavirus causing diarrhea among under-five children: A hospital-based study from Eastern India
Arpit Kumar Shrivastava1, N Samarasimha Reddy2, Sidhartha Giri2, Priyadarshi Soumyaranjan Sahu3, Mirabai Das4, Nirmal Kumar Mohakud5, Rashmi Ranjan Das6
1 Department of Biotechnology, Infection Biology Laboratory, KIIT Deemed to be University, Vellore, Tamil Nadu, India
2 Division of Gastrointestinal Sciences, The Wellcome Trust Research Laboratory, Christian Medical College, Vellore, Tamil Nadu, India
3 Department of Microbiology and Immunology, Medical University of the Americas, Nevis, WI
4 Department of Health, Kalinga Institute of Social Sciences, KISS University, Bhubaneswar, Odisha, India
5 Department of Paediatrics, Kalinga Institute of Medical Sciences, KIIT Deemed to be University, Bhubaneswar, Odisha, India
6 Department of Pediatrics, All India Institute of Medical Sciences, Bhubaneswar, Odisha, India
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|Date of Submission||11-Feb-2019|
|Date of Acceptance||12-Sep-2019|
|Date of Web Publication||26-Nov-2019|
| Abstract|| |
Background: Rotavirus (RVA) causes severe gastroenteritis in under-five children, and there are many diverse strains of the virus that are localized to different parts of the world. Objectives: To study the burden and molecular epidemiology of RVA causing gastroenteritis among children from Eastern India. Materials and Methods: This hospital-based cross-sectional study included children under-five with gastroenteritis. Demographic and clinical parameters were recorded in a predesigned pro forma. Stool samples collected from these children were initially screened for RVA VP6 antigen by enzyme immunoassay (EIA). Each EIA-positive sample was then subjected to RNA extraction, followed by reverse transcription, and heminested multiplex polymerase chain reaction for genotyping of RVA strains. Results: Of 320 included children, RVA was detected in 30.62% (98/320) cases by EIA. The highest incidence for RVA-positive cases (34.61%) was observed among children in the age group of 24–36 months, followed by 0–12 months (33.04%). Of the 97 completely typed samples, single genotype was detected in 85 (87.62%) samples with either G (VP7) or P (VP4) types. However, mixed genotypes were detected in 12 (11.21%) samples. G3P (44.09%) was the most common genotype, followed by G1P (32.65%), G2[P4] (5.10%), G1[P6] (3.06%), and G9[P4] (1.02%). Conclusions: The present study found RVA positivity in 30.62% of children with gastroenteritis, with the highest burden among 24–36 months old. The predominant genotypes were G1, G3, and P. Further large-scale/multicentric studies should be conducted to document the diversity of circulating RVA genotypes in this region for giving inputs for vaccination strategy.
Rotavirus (RVA) is the leading cause of diarrheal death in children under 5 years of age. Virtually, every child around the globe experiences RVA diarrhea by the age of 3–5 years. Majority of the RVA-associated gastroenteritis in developing countries is caused due to Group A RVA; India accounts for an estimated 457,000–884,000 hospitalizations and over 2 million outpatient visits for diarrhea. Group A RVA is a double-stranded RNA virus consisting of 11 segments. Rotaviruses are nonenveloped, icosahedral, triple-layered particles; it contains two outer capsid proteins, VP7 (G genotype) and VP4 (P genotype), which independently elicit serotype-specific neutralizing immune responses that may play an important role in protection against recurrent infections. These VP7 and VP4 encoding genes of RVA are classified into 27 G genotypes (G1–G27) and (37) P genotypes (P(1)–P(37)), respectively.
The World Health Organization (WHO) has recommended the inclusion of RVA vaccination of all infants in the national immunization program. At present, three globally licensed vaccines against RVA gastroenteritis, Rotarix (GlaxoSmithKline Biologicals), RotaTeq (Merck and Co., Inc.), and Rotavac (Bharat Biotech Ltd.), are available in India. Rotavac is an indigenously developed vaccine which is WHO prequalified, which is being introduced in a phased manner into the national immunization program by the Government of India, from 2016.
The enormous diversity of RVA is mainly because of point mutations, genetic reassortment, or introduction of animal viral strains to human beings. With Odisha state being one of the early introductory regions for Rotavac vaccine, it is important to monitor the circulating RVA genotypes to detect changes or the emergence of new strains. Therefore, RVA surveillance is needed to monitor the prevalence and possible changes of the different G and P types circulating in the region.
This study was conducted to estimate the burden of RVA-associated gastroenteritis and identify the circulating RVA strains among children under 5 years of age, immediately after RVA vaccine introduction at a tertiary care teaching hospital in Bhubaneswar, Odisha.
Viral RNA extraction was performed from 20% (W/V) stool suspension (0.2 g stool in 1 ml MEM) using automated method (QIAcube HT, QIAGEN). By reverse transcription, the viral RNA was converted to complementary DNA (cDNA) using random primers (Invitrogen) and 200U/μl of Moloney murine leukemia virus reverse transcriptase enzyme (Superscript II MMLV-RT, Invitrogen). The cDNA was used as a template for genotyping in a heminested multiplex PCR for the detection of VP7 (G type) and VP4 (P type) genes, using published oligonucleotide primers.,,, Primers to identify VP7 genotypes G1, G2, G3, G4, G8, G9, G10, and G12 and VP4 genotypes P(4), P(6), P(8), P(9), P(10), and P(11) were included in the PCR reaction. The genotype identification was based on band sizes of the products. For samples, which were negative by genotyping PCR, a VP6 conventional PCR was performed to rule out RVA positivity. Sanger sequencing was used to further evaluate strains that could not be typed using standard PCR-based methods.
Descriptive analysis was used to report demographic characteristics of the participants. These data were entered into Excel 2003 (Microsoft) and were analyzed to evaluate the overall proportion of various G and P types in RVA-positive specimens. Chi-square test was used for the determination of differences in the proportions of RVA positivity according to gender, age groups, and hospital stay. Student's t-test was used for comparing the means of disease severity by Vesikari score. The level of significance was set at P < 0.05. Statistical analysis was conducted using SPSS software version 21, SPSS Inc., Chicago, Illinois, USA.
Of the 320 diarrheal samples, 98 (30.62%) samples were EIA positive for RVA. There was no missing data as children were hospitalized and discharged as per the criteria. Overall, stool samples from 29.29% of males and 32.68% of females were positive for RVA. Age-wise distribution showed that majority of the RVA-associated gastroenteritis was found in the 24–36 months' age group (34.61%). The prevalence of RVA-associated gastroenteritis was slightly higher in rural areas (32.33%) compared to urban areas (30.34%). Sociodemographic data showed that majority of diarrheal episodes due to RVA were in the population who used shared community water (35.41%), compared to those using covered well (20%) or tap water at home (25.31%). RVA gastroenteritis was also associated with education level of mothers, children of mothers with basic education having higher proportion of diarrheal episodes (36.58%), compared to children whose mothers had university level education (24%), although the difference was not statistically significant [Table 1]. The burden was high during the winter season (November–February) [Figure 1].
The mean duration of hospital stay was longer in RVA diarrhea (3.83 ± 1.77) compared to non-RVA diarrhea (3.43 ± 1.32), and this difference was statistically significant. The mean Vesikari scores of RVA diarrhea group (13.86 ± 1.89) were higher compared to the non-RVA diarrhea group (13.19 ± 1.92), and this difference was also statistically significant. Around 96% (94/98) of the RVA diarrhea were of severe to very severe category, compared to 92% (204/222) of the non-RVA diarrhea [Table 3].
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| Discussion|| |
In the present study, RVA diarrhea was high among children of 24–36 months of age, occurring mostly during winter season. Diversity of RVA genotypes was noted, with G3P(8) and G1P(8) being the most predominant circulating strains in the study area.
In the developing counties of Asia, a high burden of RVA (30%–40%) has been reported among children hospitalized with diarrhea., In this study, 30.62% of cases were positive for RVA, which is similar to the previous study from Eastern India. Earlier reports from India, Japan, Malaysia, and Taiwan showed comparatively lower disease burden (20%–25%), but studies from Thailand, Myanmar, and Vietnam (31.1%–56%) showed a higher burden of RVA-associated gastroenteritis.
In this study, a higher burden (34.61%) of RVA infection was found in the age group of 24–36 months in contrast to other studies where the burden was high within the first 2 years and more so after 7 months of age.,, In Odisha, RVA vaccine was introduced in the routine immunization program in March 2016 (children <1 year being eligible to take vaccine). However, as the study population was not uniformly vaccinated for RVA vaccine, a large portion of 24–36-month-old children were unvaccinated compared to children of <24 months of age, which could be one of the reasons for a higher burden in this age group. The vaccination at an early age can be beneficial to prevent majority of cases of RVA disease. Previous reports have shown that children <4 months of age are initially protected to some extent by maternal antibodies against RVA, which could be one of the reasons for peaking of the disease after 7 months of age. In the present study, RVA positivity was more in <7-month-old children compared to those <7 months old.
RVA infections are predominately observed in winter; however, seasonality may vary from year to year. In the present study, RVA diarrhea was observed throughout the year, but peak in the number of cases was observed between November and February. Similar patterns of seasonal variations have been reported in previous studies.
Studies on RVA epidemiology demonstrate the differences in strain circulation over time., A large number of G-type genotypes (G3, G1, G2, and G9) were observed in the present study unlike the developed countries where one or two genotypes were reported predominant in a season. The most prevalent RVA strains causing childhood diarrhea globally are G1–G4 and G9. In the present study, G3 (44.12%) was the most common strain responsible for severe diarrhea-related hospitalizations, whereas previous studies from India suggested G1 and G2 stains as the most dominant.,, In the present study, the major P-type was P(8) (78.50%), followed by P(4) and P(6); previous studies also suggested similar trend where P(8) or P(6) were the leading strains.,, The most common genotype combinations were found to be G3P(8), followed by G1P(8), G2P[P4], G1P[P6], and G3P[P6]. This pattern is in agreement with findings from other studies.,
[Table 4] shows the list of most common RVA G and P type distribution in different parts of India, compared to the present study.,,, Globally, including India, the most common genotype is G1[P8] unlike the present study where G3[P8] was found commonly. Previous studies from Eastern India conducted during 2005–2008 had found G1[P8] as the dominant genotype, but those conducted during 2011–2013 found G9[P4] as the most common genotype. One study from Odisha conducted during 2003–2005 found G2[P4] and G2[P8] as the most common genotypes, whereas another study from the same region during 2013–2015 found G1[P8] as the most common genotype. This changing pattern of genotype distribution suggests continuous need for RVA disease surveillance and vaccine effectiveness studies for adequate management of RVA diarrhea in this region.
In the present study, RVA gastroenteritis prevalence was 30.66% in under-five children in Bhubaneswar city, with most cases occurring in winter. There was diversity in identified RVA genotypes, with G3P and G1P being more common.
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[Table 1], [Table 2], [Table 3], [Table 4]