Dengue is the predominant mosquito-borne disease in humans, caused by one of the four serotypes of dengue virus (DENV-1 - 4) that belongs to the genus Flavivirus. The virus is transmitted through the bite of an infected female of sympatric Aedes aegypti and Ae. albopictus. Dengue disease is endemic in more than 120 countries with the highest disease burden reported in Asia and Latin America  . Within Asia, Malaysia ranked the third most affected country between the years 1991-2007 . The incidence of dengue in Malaysia showed an escalating trend from 31.6 per 100,000 population in 2000 to 159.7 per 100,000 population in 2010 . Dengue infection affected all ethnicities and gender with an observed predominance in males and Malays  .
In the absence of an effective and safe vaccine against the dengue virus, vector control remains as the primary approach for the prevention and control of dengue. Interruption of dengue transmission through vector control in the Caribbean, America, Cuba, and Singapore has resulted in a substantial reduction of the mosquito population and human cases of dengue     . Unfortunately, these success histories did not sustain and have failed to prevent dengue outbreaks due to several reasons, including inappropriate implementation, lack of sustainability over-time and reduced herd immunity    . These are the compelling reasons for the continuation of research and development on new insecticides and tools for dengue control. At present, there is no consensus regarding the type of vector control approach that can bring the largest impact on dengue transmission for both epidemic situation and sustained management . Hence, there is an urgent need for new or modified approaches such as the residual spraying on the outer walls that can potentially last long enough to control the Aedes population, especially in the outbreak areas. The integration of this modified approach as part of the Integrated Vector Management (IVM) may possess an unexploited potential for an effective dengue control effort if executed correctly.
Indoor residual spraying (IRS) involves the application of long-lasting chemical insecticides on the inner walls, eaves and ceiling of properties where the mosquitoes might encounter the insecticides while resting on these structures . IRS was historically used as the primary tool in the Global Malaria Eradication Program (1955-1969), which has resulted in the total elimination of malaria in 37 out of 143 countries while reducing malaria burden in other countries    . A few studies have shown that the IRS is a promising dengue control tool and has high efficiency in reducing the Aedes populations. The application of IRS using deltamethrin in Peruvian Amazon has appeared to produce >80% mortalities in Ae. aegypti in bioassay test for up to eight weeks post-spraying when applied on wood, unpainted wood and brick, and reduced adult index from 18.5 to 3.1, a month after the intervention . Research in Merida, Mexico showed that treating inner house surfaces with bendiocarb has reduced Ae. aegypti infestation by 60% in three months period , and targeted IRS research in Cairns, Australia demonstrated that IRS along with location-based contact tracing had decreased the likelihood of dengue cases by 86% - 96% in sprayed premises in comparison to unsprayed controls .
Deltamethrin polymer-enhanced suspension concentrate (SC-PE) (K-OthrineÒ Polyzone) is classified as pyrethroid and is one of the WHO recommended insecticides for IRS against malaria vector . The insecticide is a new formulation of existing deltamethrin that has a prolonged residual effect through its property of the proprietary polymer. The polymer layer releases deltamethrin particle over an extended period and protects the deltamethrin particle from rainfall and abrasion. Consequently, less spraying applications are needed that in turn improve the cost-effectiveness of the residual spraying method and minimizing disruption to families. Recent studies in America and Tanzania have shown that deltamethrin SC-PE has superior performance than formerly developed deltamethrin water dispersible granule (WG)   .
Here, we tested deltamethrin SC-PE in the outdoor residual spraying (ORS), a modified approach of an existing IRS method. The insecticide was applied to the outer walls of houses, instead of the indoor wall surfaces. This was done, in part, due to the lack of accessibility to houses and cooperation from the homeowners. ORS targets adult resting population where they may pick up the contact poison at a sufficient amount during resting to cause death. The behavior of Aedes mosquitoes resting on the walls is well established  . The treated walls could also potentially act as barrier walls which prevent the mosquitoes from going inside houses for blood meals. We suggested that ORS is used as part of the national emergency response to a dengue outbreak. We applied deltamethrin SC-PE to the outer walls of terrace houses and apartment units at the application dosage of 30 mg/m2 in the residential areas in Hulu Langat district, Selangor. The objectives of this study were to evaluate the efficacy of ORS using deltamethrin SC-PE and its effect on wild Aedes populations and to assess its residual bio-efficacy on painted cement walls against the pyrethroid-susceptible strains of laboratory-reared Aedes mosquitoes.
2. Materials and Methods
2.1. Study Site and Design
This study was conducted in four residential neighbourhoods in Hulu Langat District, Selangor. The study sites were selected based on epidemiological and ovitrap surveys by the Health District Office that has classified these zones as dengue outbreak hotspots with high vector density of Aedes spp. (≥10% ovitrap index threshold based on MOH guidelines). Two accommodation types were included, i.e. low-rise housing (landed properties with one to three-story design) and high-rise housing (non-landed properties having four stories and above). We chose low-rise housing of terrace houses (Seksyen 2, Bandar Rinching, 2˚55'50.4"N 101˚50'49.0"E and Seksyen 5, Bandar Rinching, 2˚55'50.9"N 101˚51'48.4"E), and high-rise housing of five-story apartments (Pesona Apartment, 2˚58'26.9"N 101˚47'58.1"E and Seraya Apartment, 2˚58'23.5"N 101˚47'55.8"E) as our study sites. A distance of more than 500 m radius separated the unsprayed and treated areas, serving as a buffer zone. The four sites were randomly assigned to control (Seksyen 2 and Pesona Apartment) and treatment (Seksyen 5 and Seraya Apartment) groups.
The study was conducted between January 2014 and June 2015 with 62 weeks of data collection for entomological surveillance. The entomological surveillance was carried out during a four-week of baseline prior to the intervention and following three spray rounds in week 9/2014, week 33/2014 and week 5/2015. A spraying cycle of 4-months was carried out as recommended by the manufacturer for deltamethrin SC-PE (3 months residuality) and we extended another month for entomological surveillance. If there was a dengue incidence in the control and treated areas during the intervention period, the Ministry of Health (MOH) guidelines shall be followed whereby activities under the mosquito control program that includes source reduction, larviciding, and space spraying will be conducted within 200 m radius of the infected house.
2.2. Ethics Statement
This study met with the ethical standard for Human Subject Research by the Medical Research and Ethics Committee (MREC). Study participants were informed on the objectives of the study by oral and information letter. Written informed consent forms were obtained from residents in both control and treated areas. Only around 2% of houses (7 out of 379 units) in the treated low-rise housing did not agree to participate in this study. Public engagement was conducted using banners, posters, and announcement brochures three days before the spraying activities were performed.
2.3. Insecticide Formulation and Application
The insecticide used was polymer-enhanced deltamethrin (K-OthrineÒ Polyzone, Bayer Crop Science, Monheim am Rhein, Germany), which contains 62.5 g/L deltamethrin as its active ingredient and formulated as a suspension concentrate (SC). The insecticide was prepared as recommended by the manufacturer and was sprayed using a calibrated hand compression sprayer (Hudson X-Pert, Chicago, IL, USA) with a flat fan nozzle (Teejet 8002, Spray Systems Co., Bessemer, AL, USA). Some of the external walls were not sheltered walls and were exposed to rainfalls. We decided to use a larger concentration (30 mg/m2) than the WHO-recommended dosage for IRS in malaria control (20 - 25 mg/m2)  to offset the amount of insecticide applied, withstanding the dry and wet season in Malaysia.
All accessible external walls in the house front yards and backyards were sprayed by trained staffs following procedures from WHO . Safety procedures when spraying and handling of the insecticide were also followed . The areas with aquarium or fish ponds, edible plants, surfaces that were near to the electric supply, water filter and water container were avoided. House owners were advised to keep their pets away during the spraying activities until the insecticide has dried completely. The percentage coverage of houses that have been sprayed in both types of accommodation was as described in Table 1. The coverage is defined by the total number of houses sprayed during a spray round divide by the total number of houses that were targeted . The ORS coverage for terrace houses was not 100% due to inaccessibility to the houses since the homeowners were not at home at the time of spraying.
2.4. Ovitrap Surveillance
The number of larvae which translated to population densities of Ae. aegypti and Ae. albopictus was assessed in the control and treated areas via ovitrap surveillance. We performed ovitrapping as described in Lee . Ovitraps (300 ml black plastic containers, diameter 7.0 cm and height 9.0 cm) filled with three-quarter of tap water along with the rough surface of the oviposition paddles (made from hardboard, approximately 10 cm × 2.5 cm × 0.3 cm) facing upwards were placed at the ground level in both semi-indoor and outdoor environments. A total of 60 ovitraps and 40 ovitraps were placed in randomly selected houses in the low-rise
Table 1. The total number of houses and percentage coverage of houses that have been sprayed in the study areas.
and high-rise housing, respectively. The number of ovitraps was equally distributed between the semi-indoor and outdoor environments. Semi-indoor is defined as the area outside of the house units but shielded by the roof, e.g. the porch and hallway. The ovitraps were collected after seven days and immediately transported to the Institute for Medical Research (IMR) laboratory. New ovitraps were placed at the same time when exposed ovitraps were collected. Any disturbances to the exposed ovitraps, e.g. stolen or vandalized, ovitraps were tipped over, invasion by insects were recorded. The recovered ovitrap contents along with the paddle were poured into labelled plastic containers and separated according to the study sites and date of collection. Beef liver powder (Difco Laboratories, MD, USA) was added to the ovitrap contents. Fourth instar larvae were identified and separated according to species by using established taxonomy keys   under a compound microscope (Nikon EclipseÒ E100, Japan). Contents were examined for species identification until no newly emerged larvae were found.
2.5. WHO Wall Deposit Bioassay
The residual activity of deltamethrin SC-PE was evaluated following standard WHO wall deposit bioassay procedures with a few modifications . The susceptible laboratory-reared strains of Ae. aegypti and Ae. albopictus from insectary of Medical Entomology Unit, IMR were used in this bioassay. Wall bioassay was conducted biweekly until week 16 in each cycle and was performed on the treated wall surfaces of approximately five percent of the total houses. The houses for the bioassays were randomly selected. Standard transparent plastic cones (internal diameter 8.5 cm and internal height 5.5 cm) were fixed vertically onto the wall surfaces. Three bioassay cones, each containing 10 laboratory-reared mosquitoes (sucrose-fed, 2 - 5 days old) were prepared for each Aedes species. Mosquitoes were introduced gently into the bioassay cone using battery-operated aspirator. The aperture of the bioassay cone was plugged with a cotton ball. Mosquito knockdown was observed at the 1-minute interval for 30 min. After the exposure, the mosquitoes were aspirated out and transferred into clean paper cups. The mosquitoes were fed with 10% (w/v) sugar solution with the holding condition of 28˚C ± 2˚C and 75% ± 10% relative humidity. The mortality was recorded at 24 h post-exposure. The classification of adult mosquitoes as knocked down or dead was as described by WHO .
2.6. Meteorological Data
We set up a weather station in each study site. Temperature and relative humidity were recorded using thermohygrometer Tinytag View 2 (TV-4500, Gemini Data Loggers Ltd., Sussex, UK) while rain gauge (RGR126N, Wireless Rain Gauge, Oregon Scientific, Oregon, US) was used to closely monitor rainfalls.
2.7. Data Analysis
Ovitrap index (OI) was calculated to assess the density of the Aedes population. OI is defined as the percentage of positive ovitraps per the number of ovitraps recovered. We also calculated the mean number of larvae in each positive ovitrap for each Aedes species. Since the data was not normally distributed, a non-parametric test of Mann-Whitney Test (equations described in 1, 2 and 3) was used to compare the OI and mean number of larvae between unsprayed control and treated sites.
where S1 = sum of the ranks assigned to the sample observations from Population 1, S2 = sum of the ranks assigned to the sample observations from Population 2, n = number of individuals in the sample . Bioassay results of 30 min knockdown (KD30) and mortality rates were calculated and analysed according to . The association between time (months) and mortality were analysed using log-time probit regression  . Comparisons of mean mortality between the types of housing were analysed using Mann-Whitney Test as described above. All analyses were performed using the Statistical Package for Social Sciences (SPSS) version 25.0 .
3.1. Surveillance of the Aedes Population
The mean temperature recorded during the entire period studied was 29.29˚C ± 0.117˚C (26.38˚C - 32.20˚C) while the mean relative humidity was 73.45% ± 1.004% (65.46% - 81.44%). The mean precipitation recorded by the rain gauge was the highest during cycle 2 with 6.46 ± 1.089 mm precipitations in the low-rise residences and 7.47 ± 1.406 mm in the high-rise residences (Table 2). The rain amount was not statistically significant between the different housing types during the treatment period (Table 2).
Comparisons of the OI between unsprayed control and treated low-rise residences for semi-indoor ovitraps showed that there were no statistical differences (p-value > 0.05) during the treatment period (Table 3). Surprisingly, the OI at both control and treated sites decreased after spraying was conducted. Looking at the species-specific mean number of larvae per ovitrap, treated houses have unexpectedly higher mean number of Ae. aegypti larvae per ovitrap compared to unsprayed control (except in cycle 3). Interestingly, this contrasted with Ae. albopictus, which displayed a downward trend of larvae number per ovitrap with significant differences between treated houses and unsprayed control. This was reflected in the Ae. aegypti: Ae. albopictus ratio in which Ae. albopictus dominated during the baseline period and the predominant species changed to Ae. aegypti only in the treated area as seen in the treatment period (Table 3). Meanwhile,
Table 3. Comparisons of the ovitrap index and mean number of larvae between unsprayed control and treated low-rise residences for ovitraps placed in the semi-indoor environment.
*indicates that there is a significant difference between control and treated site.
analyses of OI for ovitraps placed in the outdoor environment revealed that the treated houses have slightly lower OI values compared to unsprayed control (Table 4). As with the semi-indoor, we observed the same decreasing trend for Ae. albopictus where the differences were statistically significant (p = 0.000) between control and treated area (Table 4). Altogether, these results suggested that ORS with deltamethrin SC-PE was reducing Ae. albopictus larvae number, indicating a decreased of Ae. albopictus density in the low-rise residences.
The OI of semi-indoor ovitraps at treated high-rise residences declined after the first round of spraying with a significant difference between unsprayed control and treated site (Table 5). The OI subsequently increased in cycle 2 and decreased in cycle 3 (Table 5). As opposed to the low-rise housing, we found that the ORS is effective in reducing Ae. aegypti abundance as evident from the significantly lower number of Ae. aegypti larvae in the treated site than the unsprayed control (Table 5). This was supported by the Ae. aegypti: Ae. albopictus ratio that shifted to Ae. albopictus dominance during cycle 1 and 3. The ORS, unfortunately, did not seem to influence the number of Ae. albopictus larvae per
Table 4. Comparisons of the ovitrap index and mean number of larvae between unsprayed control and treated low-rise residences for ovitraps placed in the outdoor environment.
*indicates that there is a significant difference between control and treated site.
Table 5. Comparisons of the ovitrap index and mean number of larvae between unsprayed control and treated high-rise residences for ovitraps placed in the semi-indoor environment.
*indicates that there is a significant difference between control and treated site.
ovitrap after spraying (Table 5). Both indices of OI and number of larvae for outdoor ovitraps in the high-rise residences did not differ considerably between unsprayed control and treated site (Table 6).
3.2. Residual Bio-Efficacy of Deltamethrin SC-PE on Cement Walls
Wall bioassays done on cement-painted walls in terrace houses displayed considerable variation in KD30 between the three spray cycles (Figure 1). The knockdown
Table 6. Comparisons of the ovitrap index and mean number of larvae between unsprayed control and treated high-rise residences for ovitraps placed in the outdoor environment.
*indicates that there is a significant difference between control and treated site.
Figure 1. Percentage knockdown (KD30) of laboratory-reared Ae. aegypti and Ae. albopictus post-exposure to deltamethrin-treated walls in three rounds of spraying at treated low rise residences.
rates in cycle 1 and 3 remained above 80% for 12 weeks in exposed Ae. aegypti, while for 6 weeks in cycle 2 (Figure 1). The KD30 values, however, did not appear to be reflected in the percentage mortality where cycle 2 exhibited high mortality rate (above the WHO 80% mortality threshold) until week 14 for both Aedes spp., as opposed to cycle 1 and 3 that lasted until week 12 (Figure 2). Overall, there was a downward trend of KD30 and mortality from week 2 to week 16, suggesting that the residual bio-efficacy of deltamethrin SC-PE was reducing over time.
Figure 2. Percentage mortality (24 h) of laboratory-reared Ae. aegypti and Ae. albopictus post-exposure to deltamethrin-treated walls in three rounds of spraying at treated low-rise residences.
Figure 3. Percentage knockdown (KD30) of laboratory-reared Ae. aegypti and Ae. albopictus after exposure to deltamethrin-treated walls in three rounds of spraying at high-rise residences.
above 80% until week 12 in cycle 3 (Figure 3). There were very high mortality rates (above 80% threshold) observed for both Aedes spp. in cycle 3, which lasted until week 16 (Figure 4). Looking at the mortality rates between the different accommodation types suggests that deltamethrin SC-PE has a higher residual efficacy in eliminating Aedes laboratory strains in the apartment (>80% for 16 weeks) compared to terrace houses (>80% for 14 weeks).
The time (months) needed to eliminate 50% of the Aedes population (LT50) and 90% of the population (LT90) of exposed mosquito was analyzed using log-time probit regression. The results of probit analysis revealed that Ae. aegypti exposed to deltamethrin SC-PE in terrace houses exhibited LT50 of 22.32 months and LT90 of 6.61 months (Table 6). In comparison, longer periods of LT50 of 34.44 months and LT90 of 9.52 months were seen for Ae. aegypti in the treated apartment (Table 7), suggesting that the residual bio-efficacy in apartment exceeded that of terrace houses. Ae. albopictus followed the same trend, at which treated apartment displayed a longer period of LT90 (6.26 months) compared to terrace houses (LT90 = 4.49 months) (Table 7). Post-hoc analyses supported probit regression results demonstrating that treated apartment has higher means of mortality rate for both Aedes spp. in comparison to treated terrace houses (Table 8).
Figure 4. Percentage mortality (24 h) of laboratory-reared Ae. aegypti and Ae. albopictus after exposure to deltamethrin-treated walls in three rounds of spraying at high-rise residences.
This was the first field study that evaluated the efficacy of deltamethrin SC-PE in an ORS intervention. The findings of this study provide initial evidence on its potential utilization as part of the national dengue vector control program. ORS can potentially be used during dengue outbreaks acting as an alternative to ULV or thermal fogging that only has transient residual activity. The study demonstrated that ORS had entomological impacts, with significant reductions in population densities of Ae. aegypti or Ae. albopictus—depending on the type of accommodation, up to 4 months of post-spraying. The results of this study contrasted with a previous ORS study performed using deltamethrin WG which did not show any effect on the number of larvae in the sprayed area . The study, however, was only carried out in a short period of 12 weeks. Our study used the improved formulation of deltamethrin and showed an impact on reducing Aedes population size.
The initial dominance of Ae. albopictus during the baseline in the semi-indoor and outdoor environments in low-rise residences could be elucidated through its behavior. It is worthwhile mentioning that in terrace houses, semi-indoor ovitraps were placed outside of houses, in which they were partly shielded by roofs. Ae. albopictus is largely considered as exophilic (outdoor resting) and exophagic (feeds outdoor), which breeds in natural habitats such as tree holes as well as man-made containers  . Moderate- or high-density vegetation such as shrubs and trees that were spotted in the terrace house yards are the preferred habitats for Ae. albopictus. Vector studies in other locations in Malaysia have also discovered that Ae. albopictus was the dominant species in the outdoor environment  . ORS using deltamethrin SC-PE appeared to produce a quantifiable impact on Ae. albopictus population size, but not on Ae. aegypti, depicted from the significant reduction in the number of larvae and the shifting of dominance to Ae. aegypti at post-spraying. We think that ORS may have affected Ae. aegypti abundance but the method that we used was not sensitive enough to capture this impact since Ae. aegypti was not the dominant species in this type of housing. Resistance to deltamethrin seemed unlikely since standard WHO susceptibility assay performed using another pyrethroid insecticide of permethrin 0.75% against wild Ae. aegypti in the treated area exhibited 100% mortality .
In the high-rise housing type, Ae. aegypti dominated in the semi-indoor environment during the baseline. The apartment semi-indoor was mostly sheltered walls that were nonetheless considered as indoor, and so the initial abundance of Ae. aegypti was highly anticipated due to its behavior. Ae. aegypti is primarily known as endophilic species (indoor resting), endophagy (feeds indoors) and prefers indoor artificial containers for breeding  . We discovered that the number of Ae. aegypti population size decreased in the treated site after spraying and that the dominance has changed to Ae. albopictus in cycle 1 and 3, suggesting ORS was effective in eliminating Ae. aegypti. ORS did not seem to have an effect on the outdoor environment. A likely explanation was that the outdoor ovitraps were placed quite far from the sprayed walls, offering a less residual effect of the ORS.
Several external factors could have influenced the OI and the number of larvae per ovitrap in both control and treated sites. A likely factor was the dengue control and preventive actions performed by the Health District Office if there is a reported dengue case in the areas, which might influence the Aedes population. Another factor that should be taken into consideration was the rainy season in Malaysia. During the second cycle, we observed an increase in OI in both types of accommodation. The second cycle coincided with the rainy season of the inter-monsoon period (October-November), in which the rainfall was higher than the rest of the cycles. Additionally, results should be interpreted with caution because measures of ovitraps in the study did not correlate with indoor adult abundance. The evaluation of efficacy was based solely on semi-indoor and outdoor ovitrap collection, which did not consider the probability of indoor cryptic breeding sites such as the toilet tanks .
The high percentage mortality of laboratory-reared strains of Ae. aegypti and Ae. albopictus (above the WHO 80% mortality threshold) was probably stemmed from the correct formulation of deltamethrin SC-PE used on the painted cement walls. The SC formulation was found to be more suitable on cement wall surfaces than another deltamethrin formulation of WG in a recent study . Furthermore, the higher mean of mortality for both Aedes spp. in the high-rise residences in comparison to the low-rise residences was most likely, in part, due to environmental influences. Since the walls in terrace houses were exposed to rainfalls and sunlight, the rate of insecticide removal from the wall surfaces of terrace houses was higher than the sheltered walls in the apartment. Besides, there appeared to be a gradual accumulation of insecticide on the wall going from spray cycle 1 to cycle 3, influencing the knockdown and mortality rates. It was observed that cycle 3 exhibited mortality of >80% until week 16 for both Aedes spp. in the high-rise residences in comparison to cycle 1 which lasted until week 14 for Ae. aegypti and week 12 for Ae. albopictus.
The impact of vector control on reducing Aedes density is well established , but the evidence of the efficacy of vector control in reducing the incidence of dengue is limited. Indeed, a key limitation was that our study was not designed to detect such a relationship. To our knowledge, only a few studies have reported a positive relationship between mosquitos abundance and the number of dengue cases  . Interestingly, Guo et al.  found that the increase in Ae. albopictus abundance with a lag time of one month is associated with dengue outbreak in the region. In their critical review, Wilson et al.  reported that shortcomings in the study design, conduct, analysis, and reporting of vector control studies are the common factors that limit the assessment of the efficacy of vector control programs on the epidemiological outcomes i.e. reduction of human cases. Recommendations on the design and conduct of vector-control studies that would provide evidence of such interventions on the epidemiology of the targeted vector-borne disease have been published recently  , which could be applied in the future studies.
In summary, we report on the initial evidence of deltamethrin SC-PE in reducing Aedes populations in the low-rise and high-rise housings. This study was useful in providing an estimation of the performance of deltamethrin SC-PE in ORS. We suggested that the ORS needs to be completed in three spray rounds of a four-month cycle to optimize its efficacy. Perhaps more well-designed randomized controlled studies with both entomological and epidemiological endpoints are warranted to better understand the true entomological effects of ORS and its impact on dengue incidence. Our results showed that ORS is a promising tool in the dengue vector control and like IRS in malaria control, it is a powerful and effective method if conducted correctly. However, similar to the IRS, several factors should be considered when conducting ORS. These include the requirement for skillful staffs to ensure uniform application of the insecticide, household’s accessibility, and suitability of wall surfaces and insecticides. This study did develop excellent community trust and support that could help pave the way for further trials aiming at evaluating the impact of ORS on the incidence of vector-borne diseases such as dengue, chikungunya, and Zika.
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