Small-scale field assessment against the dengue vector Aedes aegypti using the auto-dissemination approach in an urban area of Vientiane, Lao PDR.

BACKGROUND: In Lao PDR, dengue fever is the most important vector borne disease and vector control remains the principal method to fight against Aedes aegypti the primary transmitter mosquito species. Vector control management programs need new strategies in addition to conventional larviciding and adulticiding interventions in the country. In this study, we examined the In2Care® Mosquito Trap's efficacy using insecticide auto-dissemination strategy. The insecticide pyriproxyfen, present in powder form inside the trap station, contaminates the body of gravid female mosquitoes visiting the traps and is later on disseminated via the mosquitoes in breeding sites surrounding the traps. We tested the attractiveness of the Traps, their efficacy to reduce the larval and adult abundance, and the impact on emergence rates. Specifically, we tested if the servicing interval of the In2Care® Mosquito Trap could be extended to 12 weeks. METHODS: Two black plastic ovitrap buckets and two BG® sentinel traps were placed in the premises of the Science campus of Vientiane Capital located in an urban area to measure weekly the larval and adult relative abundance of Aedes mosquitoes from 2017 to 2019. Twenty-five In2Care® Mosquito Traps were evenly distributed in this area and two studies of 12 weeks were implemented during January and April 2018 and, July to October 2018 (dry and rainy season, respectively). Every 2 weeks, water samples from 5 In2Care® Traps were randomly selected and tested at the laboratory with Ae. aegypti larvae to measure the larval and pupal mortality. The relative abundance of Aedes mosquitoes in the BG traps® with the presence of In2Care® Traps in 2018, was compared with the surveillance results obtained in 2017 and 2019 without In2Care® Traps. Every week, water samples from the ovitrap buckets were tested for Emergence Inhibition (EI). RESULTS: The In2Care® Traps were very attractive to gravid Ae. aegypti mosquitoes specifically during the rainy seasons with 96% of the traps colonized with larvae/pupae within four weeks. The bioassays showed 100% mortality in the water samples from the traps during the twelve weeks studies showing the good efficacy over time of the pyriproxyfen without additional servicing in the 12 week period. In addition, the larvicide was successfully disseminated into the ovitrap buckets placed in the treated area where 100% of EI during all weeks of intervention was measured. There was no significant effect of the treatment on adult abundance reduction in the treated area, probably due to recolonization of adult mosquitoes surrounding the field experiment. CONCLUSIONS: The observed potential of the In2Care® Mosquito Trap using the auto-dissemination strategy could lead to the use of this new tool in combination with conventional control methods against Dengue vectors in urban tropical areas. Large scale field trials should be implemented in Lao PDR to prove its efficacy for Public Health programs.

Detection of pyrethroid resistance mutations and intron variants in the voltage-gated sodium channel of Aedes (Stegomyia) aegypti and Aedes (Stegomyia) albopictus mosquitoes from Lao People's Democratic Republic.

In Lao People's Democratic Republic, Aedes aegypti (Linnaeus 1762) and Aedes albopictus (Skuse 1894) mosquitoes (Diptera: Culicidae) are vectors of arboviral diseases such as dengue. As the treatment for these diseases is limited, control of the vectors with the use of pyrethroid insecticides is still essential. However, mutations in the voltage-gated sodium channel (vgsc) gene giving rise to pyrethroid resistance are threatening vector control programs. Here, we analysed both Ae. aegypti and Ae. albopictus mosquitoes, which were collected in different districts of Laos (Kaysone Phomvihane, Vangvieng, Saysettha and Xaythany), for vgsc mutations commonly found throughout Asia (S989P, V1016G and F1534C). Sequences of the vgsc gene showed that the F1534C mutation was prevalent in both Aedes species. S989P and V1016G mutations were detected in Ae. aegypti from each site and were always found together. In addition, the mutation T1520I was seen in Ae. albopictus mosquitoes from Saysettha district as well as in all Ae. aegypti samples. Thus, mutations in the vgsc gene of Ae. aegypti are prevalent in the four districts studied indicating growing insecticide resistance throughout Laos. Constant monitoring programmes and alternative strategies for controlling Aedes should be utilized in order to prolong the effectiveness of pyrethroids thereby maximizing vector control.

Malaria and Dengue Mosquito Vectors from Lao PDR Show a Lack of the rdl Mutant Allele Responsible for Cyclodiene Insecticide Resistance.

The gamma-aminobutyric acid (GABA) receptor, RDL, plays important roles in neuronal signaling and is the target of highly effective insecticides. A mutation in RDL, commonly A296S, underlies resistance to several insecticides such as cyclodienes. Even though the use of cyclodienes has been banned, the occurrence of mutations substituting A296 is notably high in mosquitoes from several countries. Here, we report a survey investigating the prevalence of the Rdl mutant allele in mosquitoes from Laos, a country where mosquito-borne diseases such as malaria and dengue fever are health concerns. Anopheles and Aedes mosquitoes were collected from 12 provinces in Laos. Adult bioassays on Aedes aegypti (Linnaeus) (Diptera: Culicidae) and Aedes albopictus (Skuse) (Diptera: Culicidae) showed that all the populations tested were susceptible to dieldrin (4%) following WHO protocols. Exon 7 from a total of 791 mosquitoes was sequenced to identify the amino acid encoded for at 296 of RDL. Only one of these mosquitoes, Anopheles maculatus rampae Harbach and Somboon (Diptera: Culicidae) from Attapeu, carried the mutant allele being heterozygous for A296S. We therefore found a general lack of the Rdl mutant allele indicating that mosquitoes from Laos are not exposed to insecticides that act on the GABA receptor compared to mosquitoes in several other countries. Identifying the prevalence of the Rdl mutation may help inform the potential use of alternative insecticides that act on the GABA receptor should there be a need to replace pyrethroids in order to prevent/manage resistance.

Distribution of insecticide resistance and mechanisms involved in the arbovirus vector Aedes aegypti in Laos and implication for vector control.

BACKGROUND: The yellow fever mosquito Aedes aegypti is the major vector of dengue, yellow fever, Zika, and Chikungunya viruses. Worldwide vector control is largely based on insecticide treatments but, unfortunately, vector control programs are facing operational challenges due to mosquitoes becoming resistant to commonly used insecticides. In Southeast Asia, resistance of Ae. aegypti to chemical insecticides has been documented in several countries but no data regarding insecticide resistance has been reported in Laos. To fill this gap, we assessed the insecticide resistance of 11 Ae. aegypti populations to larvicides and adulticides used in public health operations in the country. We also investigated the underlying molecular mechanisms associated with resistance, including target site mutations and detoxification enzymes putatively involved in metabolic resistance. METHODS AND RESULTS: Bioassays on adults and larvae collected in five provinces revealed various levels of resistance to organophosphates (malathion and temephos), organochlorine (DDT) and pyrethroids (permethrin and deltamethrin). Synergist bioassays showed a significant increased susceptibility of mosquitoes to insecticides after exposure to detoxification enzyme inhibitors. Biochemical assays confirmed these results by showing significant elevated activities of cytochrome P450 monooxygenases (P450), glutathione S-transferases (GST) and carboxylesterases (CCE) in adults. Two kdr mutations, V1016G and F1534C, were detected by qPCR at low and high frequency, respectively, in all populations tested. A significant negative association between the two kdr mutations was detected. No significant association between kdr mutations frequency (for both 1534C and 1016G) and survival rate to DDT or permethrin (P > 0.05) was detected. Gene Copy Number Variations (CNV) were detected for particular detoxification enzymes. At the population level, the presence of CNV affecting the carboxylesterase CCEAE3A and the two cytochrome P450 CYP6BB2 and CYP6P12 were significantly correlated to insecticide resistance. CONCLUSIONS: These results suggest that both kdr mutations and metabolic resistance mechanisms are present in Laos but their impact on phenotypic resistance may differ in proportion at the population or individual level. Molecular analyses suggest that CNV affecting CCEAE3A previously associated with temephos resistance is also associated with malathion resistance while CNV affecting CYP6BB2 and CYP6P12 are associated with pyrethroid and possibly DDT resistance. The presence of high levels of insecticide resistance in the main arbovirus vector in Laos is worrying and may have important implications for dengue vector control in the country.

Population genetics of Aedes albopictus (Diptera: Culicidae) in its native range in Lao People's Democratic Republic.

BACKGROUND: The Asian tiger mosquito, Aedes (Stegomyia) albopictus (Skuse) is an important worldwide invasive species and can be a locally important vector of chikungunya, dengue and, potentially, Zika. This species is native to Southeast Asia where populations thrive in both temperate and tropical climates. A better understanding of the population structure of Ae. albopictus in Lao PDR is very important in order to support the implementation of strategies for diseases prevention and vector control. In the present study, we investigated the genetic variability of Ae. albopictus across a north-south transect in Lao PDR. METHODS: We used variability in a 1337-bp fragment of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1), to assess the population structure of Ae. albopictus in Lao PDR. For context, we also examined variability at the same genetic locus in samples of Ae. albopictus from Thailand, China, Taiwan, Japan, Singapore, Italy and the USA. RESULTS: We observed very high levels of genetic polymorphism with 46 novel haplotypes in Ae. albopictus from 9 localities in Lao PDR and Thailand populations. Significant differences were observed between the Luangnamtha population and other locations in Lao PDR. However, we found no evidence of isolation by distance. There was overall little genetic structure indicating ongoing and frequent gene flow among populations or a recent population expansion. Indeed, the neutrality test supported population expansion in Laotian Ae. albopictus and mismatch distribution analyses showed a lack of low frequency alleles, a pattern often seen in bottlenecked populations. When samples from Lao PDR were analyzed together with samples from Thailand, China, Taiwan, Japan, Singapore, Italy and the USA, phylogenetic network and Bayesian cluster analysis showed that most populations from tropical/subtropical regions are more genetically related to each other, than populations from temperate regions. Similarly, most populations from temperate regions are more genetically related to each other, than those from tropical/subtropical regions. CONCLUSIONS: Aedes albopictus in Lao PDR are genetically related to populations from tropical/subtropical regions (i.e. Thailand, Singapore, and California and Texas in the USA). The extensive gene flow among locations in Lao PDR indicates that local control is undermined by repeated introductions from untreated sites.

Field evaluation of personal protection methods against outdoor-biting mosquitoes in Lao PDR.

BACKGROUND: Protecting people outdoors against mosquito-borne diseases is a major challenge. Here we compared commercially available personal protection methods to identify the most effective method for outdoor use in northern Lao PDR. METHODS: From June to August 2016 the protective efficacy of treatments were compared in a secondary forest during the afternoon and a village during the evening. Comparisons were made using a replicated Latin square design between: (i) short permethrin-treated overalls; (ii) long permethrin-treated overalls; (iii) short untreated overalls with para-menthane-3,8-diol (PMD) applied topically; (iv) short permethrin-treated overalls plus PMD applied topically; (v) short untreated overalls with metofluthrin coils in a metal casing worn on a belt; and (vi) long untreated overalls. Short untreated overalls served as the control. Cone tests were conducted on the treated and untreated fabric before and after field experiments. A questionnaire survey was used to measure social acceptability. RESULTS: Mosquito coils in a metal casing worn on a belt resulted in 92.3% (95% confidence interval, CI: 88.9-94.6%). landing protection from female mosquitoes in the afternoon and 68.8% (95% CI: 41.7-83.3%) protection in the evening compared to short untreated clothing. PMD was protective both when combined with short permethrin-treated overalls (afternoon, 68.2%, 95% CI: 52.6-78.7%; evening, 52.3%, 95% CI: 33.8-65.7%) and when used in combination with short untreated overalls (afternoon, 55.0%, 95% CI: 41.7-65.2%; evening, 25.2%, 95% CI: 9.4-38.2%). Whilst long permethrin-treated overalls were protective (afternoon, 61.1%, 95% CI: 51.4-68.8%; evening, 43.0%, 95% CI: 25.5-56.4%), short permethrin-treated overalls and long untreated overalls were not. Exposure to new permethrin-treated fabric in cone tests resulted in 25.0% (95% CI, 17.8-32.2%) and 26.2% (95% CI 16.7-35.8%) mortality for susceptible Ae. albopictus and susceptible Ae. aegypti, respectively. There was a loss of efficacy of permethrin-treated clothing after use in the field, with 3 min knockdown rates of Ae. albopictus and 1 h knockdown of Ae. aegypti decreasing over time. Participants considered all treatments acceptable. CONCLUSIONS: The portable mosquito coils were highly protective against outdoor biting mosquitoes, although there are safety concerns related to its use. The combination of permethrin-treated clothing and PMD repellent represent an alternative treatment for protection against outdoor-biting mosquitoes.

Alternative insecticides for larval control of the dengue vector Aedes aegypti in Lao PDR: insecticide resistance and semi-field trial study.

BACKGROUND: The mosquito Aedes aegypti is the primary vector of several arboviruses, such as dengue, chikungunya and Zika, and represents a major public health problem in Southeast Asia. In Laos, where dengue is reemerging, several Ae. aegypti populations from the capital Vientiane have shown resistance to the organophosphate temephos, a commonly-used larvicide for public health interventions. METHODS: Here, we tested the insecticide susceptibility of a wild larval population of Ae. aegypti against Bacillus thuringiensis israelensis (Bti), diflubenzuron, pyriproxyfen and spinosad. Residual efficacies of Bti (VectobacWG®), diflubenzuron (Killmos®) and temephos (Abate®) were then evaluated under simulated field conditions against the wild Ae. aegypti population. RESULTS: The larval bioassays showed that the wild Ae. aegypti strain was moderately resistant to temephos and spinosad (resistance ratio, RR < 5) and fully susceptible to the other insecticides (RR = 1). The simulated field trial bioassays showed that all of the insecticides tested remained above the WHO acceptable larvicide threshold after 28 weeks. CONCLUSIONS: These results suggest that Bti and diflubenzuron may be promising alternative larvicides for controlling dengue vectors in water-storage containers in Laos, especially against Ae. aegypti populations, in which resistance to temephos has been detected.

Bionomics and insecticide resistance of the arboviral vector Aedes albopictus in northern Lao PDR.

In the last four decades there has been a staggering increase in the geographical range of the arboviral vector Aedes albopictus (Skuse, 1894). This species is now found in every continent except Antarctica, increasing the distribution of arboviral diseases such as dengue and chikungunya. In Lao PDR dengue epidemics occur regularly, with cases of chikungunya also reported. As treatment methods for arboviral diseases is limited, the control of the vector mosquitoes are essential. There is a paucity of information on the bionomics and resistance status of this mosquito for successful vector control efforts. Here we describe the bionomics and insecticide resistance status of Ae. albopictus in Laos to identify opportunities for control. Adult Ae. albopictus were collected using human-baited double bed net (HDN) traps in forests, villages and rubber plantations and tested for alpha- and flaviviruses with RT-PCR. Surveys were also conducted to identify larval habitats. Seven adult and larval populations originating from Vientiane Capital and Luang Prabang province were tested against DDT, malathion, permethrin, deltamethrin and, temephos following WHO protocols. Aedes albopictus were found throughout the year, but were six-fold greater in the rainy season than the dry season. Adult females were active for 24 hours, with peak of behaviour at 18.00 h. The secondary forest and rubber plantation samples showed evidence of Pan-flaviviruses, while samples from the villages did not. More than half of the emerged Ae. albopictus were collected from mature rubber plantations (53.9%; 1,533/2,845). Most Ae. albopictus mosquitoes emerged from latex collection cups (19.7%; 562/2,845), small water containers (19.7%; 562/2,845) and tyres (17.4%; 495/2,845). Adult mosquitoes were susceptible to pyrethroids, apart from one population in Vientiane city. All populations were resistant to DDT (between 27-90% mortality) and all except one were resistant to malathion (20-86%). Three of the seven larval populations were resistant to temephos (42-87%), with suspected resistance found in three other populations (92-98%).This study demonstrates that rural areas in northern Laos are potential hot spots for arboviral disease transmission. Multiple-insecticide resistance was found. Aedes albopictus control efforts in villages need to expand to include secondary forests and rubber plantations, with larval source management and limited use of insecticides.

Risk of exposure to potential vector mosquitoes for rural workers in Northern Lao PDR.

BACKGROUND: One major consequence of economic development in South-East Asia has been a rapid expansion of rubber plantations, in which outbreaks of dengue and malaria have occurred. Here we explored the difference in risk of exposure to potential dengue, Japanese encephalitis (JE), and malaria vectors between rubber workers and those engaged in traditional forest activities in northern Laos PDR. METHODOLOGY/PRINCIPAL FINDINGS: Adult mosquitoes were collected for nine months in secondary forests, mature and immature rubber plantations, and villages. Human behavior data were collected using rapid participatory rural appraisals and surveys. Exposure risk was assessed by combining vector and human behavior and calculating the basic reproduction number (R0) in different typologies. Compared to those that stayed in the village, the risk of dengue vector exposure was higher for those that visited the secondary forests during the day (odds ratio (OR) 36.0), for those living and working in rubber plantations (OR 16.2) and for those that tapped rubber (OR 3.2). Exposure to JE vectors was also higher in the forest (OR 1.4) and, similar when working (OR 1.0) and living in the plantations (OR 0.8). Exposure to malaria vectors was greater in the forest (OR 1.3), similar when working in the plantations (OR 0.9) and lower when living in the plantations (OR 0.6). R0 for dengue was >2.8 for all habitats surveyed, except villages where R0≤0.06. The main malaria vector in all habitats was Anopheles maculatus s.l. in the rainy season and An. minimus s.l. in the dry season. CONCLUSIONS/SIGNIFICANCE: The highest risk of exposure to vector mosquitoes occurred when people visit natural forests. However, since rubber workers spend long periods in the rubber plantations, their risk of exposure is increased greatly compared to those who temporarily enter natural forests or remain in the village. This study highlights the necessity of broadening mosquito control to include rubber plantations.

Diversity of Mosquitoes (Diptera: Culicidae) Attracted to Human Subjects in Rubber Plantations, Secondary Forests, and Villages in Luang Prabang Province, Northern Lao PDR.

The impact of the rapid expansion of rubber plantations in South-East Asia on mosquito populations is uncertain. We compared the abundance and diversity of adult mosquitoes using human-baited traps in four typical rural habitats in northern Lao PDR: secondary forests, immature rubber plantations, mature rubber plantations, and villages. Generalized estimating equations were used to explore differences in mosquito abundance between habitats, and Simpson's diversity index was used to measure species diversity. Over nine months, 24,927 female mosquitoes were collected, including 51 species newly recorded in Lao PDR. A list of the 114 mosquito species identified is included. More mosquitoes, including vector species, were collected in the secondary forest than immature rubber plantations (rainy season, odds ratio [OR] 0.33, 95% confidence interval [CI] 0.31-0.36; dry season, 0.46, 95% CI 0.41-0.51), mature rubber plantations (rainy season, OR 0.25, 95% CI 0.23-0.27; dry season, OR 0.25, 95% CI 0.22-0.28), and villages (rainy season, OR 0.13, 95% CI 0.12-0.14; dry season, 0.20, 95% CI 0.18-0.23). All habitats showed high species diversity (Simpson's indexes between 0.82-0.86) with vectors of dengue, Japanese encephalitis (JE), lymphatic filariasis, and malaria. In the secondary forests and rubber plantations, Aedes albopictus (Skuse), a dengue vector, was the dominant mosquito species, while in the villages, Culex vishnui (Theobald), a JE vector, was most common. This study has increased the overall knowledge of mosquito fauna in Lao PDR. The high abundance of Ae. albopictus in natural and man-made forests warrants concern, with vector control measures currently only implemented in cities and villages.

Risk and Control of Mosquito-Borne Diseases in Southeast Asian Rubber Plantations.

Unprecedented economic growth in Southeast Asia (SEA) has encouraged the expansion of rubber plantations. This land-use transformation is changing the risk of mosquito-borne diseases. Mature plantations provide ideal habitats for the mosquito vectors of malaria, dengue, and chikungunya. Migrant workers may introduce pathogens into plantation areas, most worryingly artemisinin-resistant malaria parasites. The close proximity of rubber plantations to natural forest also increases the threat from zoonoses, where new vector-borne pathogens spill over from wild animals into humans. There is therefore an urgent need to scale up vector control and access to health care for rubber workers. This requires an intersectoral approach with strong collaboration between the health sector, rubber industry, and local communities.

The Human-Baited Double Net Trap: An Alternative to Human Landing Catches for Collecting Outdoor Biting Mosquitoes in Lao PDR.

Estimating the exposure of individuals to mosquito-borne diseases is a key measure used to evaluate the success of vector control operations. The gold standard is to use human landing catches where mosquitoes are collected off the exposed limbs of human collectors. This is however an unsatisfactory method since it potentially exposes individuals to a range of mosquito-borne diseases. In this study several sampling methods were compared to find a method that is representative of the human-biting rate outdoors, but which does not expose collectors to mosquito-borne infections. The sampling efficiency of four odour-baited traps were compared outdoors in rural Lao PDR; the human-baited double net (HDN) trap, CDC light trap, BG sentinel trap and Suna trap. Subsequently the HDN, the best performing trap, was compared directly with human landing catches (HLC), the 'gold standard', for estimating human-biting rates. HDNs collected 11-44 times more mosquitoes than the other traps, with the exception of the HLC. The HDN collected similar numbers of Anopheles (Rate Ratio, RR = 1.16, 95% Confidence Intervals, 95% CI = 0.61-2.20) and Culex mosquitoes (RR = 1.26, 95% CI = 0.74-2.17) as HLC, but under-estimated the numbers of Aedes albopictus (RR = 0.45, 95% CI = 0.27-0.77). Simpson's index of diversity was 0.845 (95% CI 0.836-0.854) for the HDN trap and 0.778 (95% CI 0.769-0.787) for HLC, indicating that the HDN collected a greater diversity of mosquito species than HLC. Both HLC and HDN can distinguish between low and high biting rates and are crude ways to measure human-biting rate. The HDN is a simple and cheap method to estimate the human-biting rate outdoors without exposing collectors to mosquito bites.