Determinants of uptake, short-term and continued use of insecticide-treated curtains and jar covers for dengue control

Objective To evaluate the acceptance and long‐term use of insecticide‐treated (IT) materials for dengue vector control. Methods In 2007, IT jar covers and/or curtains (PermaNet®) were distributed under routine conditions to 4101 households (10 clusters) in Venezuela and to 2032 households (22 clusters) in Thailand. The use of IT tools was measured at distribution (uptake), at 5/6 months (short‐term use) and at 18/22 months (continued use) after distribution. Determinants of use were assessed with logistic regression analysis. Results The uptake of IT curtains was 76.7% in Venezuela and 92.3% in Thailand. It was associated with being a resident for >5 years (OR Venezuela 3.0 95% CI 2.0–4.4; OR Thailand 3.5 95% CI 1.7–7.3) and with pre‐intervention use of ordinary curtains (OR Venezuela 2.2 95% CI 1.4–3.6). The continued use decreased significantly to 38.4% of households in Venezuela and 59.7% in Thailand and was, conditional on short‐term use, only determined by the perceived effectiveness of IT curtains (OR Venezuela 13.0 95%CI 8.7–19.5; OR Thailand 4.9 95% CI 3.1–7.8). Disease knowledge and pre‐intervention perception of mosquito nuisance were not associated with IT curtains’ uptake or use. The uptake of IT jar covers in Venezuela was 21.5% and essentially determined by the presence of uncovered jars in the household (OR 32.5 95% CI 14.5–72.6). Their continued use, conditional on short‐time use, was positively associated with the household use of Abate® (OR 7.8 95% CI 2.1–28.9). Conclusion The use of IT curtains rapidly declines over time. Continued use is mainly determined by the perceived effectiveness of the tool. This poses a real challenge if IT curtains are to be introduced in dengue control programmes.     

The development of predictive tools for pre‐emptive dengue vector control: a study of Aedes aegypti abundance and meteorological variables in North Queensland,Australia

Objectives To describe the meteorological influences on adult dengue vector abundance in Australia for the development of predictive models to trigger pre‐emptive control operation. Methods Multiple linear regression analyses were performed using meteorological data and female Aedes aegypti collection data from BG‐Sentinel Mosquito traps placed at 11 monitoring sites in Cairns, north Queensland. Results Considerable regression coefficients (R² = 0.64 and 0.61) for longer‐ and shorter‐term factor models respectively were derived. Longer‐term factors significantly associated with abundance of adult vectors were mean minimum temperature (lagged 6 month) and mean daily temperature (lagged 4 month), explaining the predictable increase in abundance during the wet season. Factors explaining fluctuation in abundance in the shorter term were mean relative humidity over the previous 2 week and current daily average temperature. Rainfall variables were not found to be strong predictors of A. aegypti abundance in either longer‐ or shorter‐term models. Conclusions The implications of these findings for the development of useful predictive models for vector abundance risks are discussed. Such models can be used to guide the application of pre‐emptive dengue vector control, and thereby enhance disease management.     

Insecticide-treated bednets to control dengue vectors: preliminary evidence from a controlled trial in Haiti

Objectives Insecticide‐treated bednets (ITNs) are effective in preventing nocturnally transmitted vector‐borne diseases, but their effect on diurnally active dengue vectors has never been studied. We investigated the efficacy of ITNs in reducing Aedes aegypti populations and dengue transmission. Methods A cluster‐randomized trial was carried out in Leogane, Haiti between July 2003 and July 2004. The study area (1017 houses) was divided into 18 sectors (clusters): nine received ITNs (Olyset® long‐lasting insecticidal bednets) and nine were untreated controls. Entomological surveys [measuring Breteau (BI), house (HI), container (CI) and pupae per person (PPI) indices and oviposition activity] were undertaken at baseline and at 1 and 5 months post‐intervention. All houses were georeferenced to enable spatial analysis. Control sectors received ITNs at 6 months, and a final entomological and attitudinal survey was undertaken at 12 months after baseline. Anti‐dengue IgM seropositivity rates were measured at baseline and after 12 months. Efficacy of ITNs was assessed by WHO cone bioassays. Results At 1‐month post‐intervention, entomological indices fell in all sectors, with HI and BI in the bednet sectors reduced by 6.7 (95% CI ?10.6, ?2.7; P < 0.01) and 8.4 (95% CI ?14.1, ?2.6; P < 0.01) respectively. Moreover at 1 month, ovitraps in control sectors were significantly more likely to be positive than in bednet sectors (P < 0.01). By 5 months, all indices remained low and HI, CI and BI were also significantly lower than that of baseline in the control arm. Curiously, at 5 months, HI, CI and BI were lower in the control arm than that in the bednet arm. A final survey, 12 months after the initial baseline study (5 months after bednets had been given to all households) indicated that all indices were significantly lower than that at baseline (P < 0.001). Control houses located within 50 m of a bednet house had significantly lower CI (Z = ?2.67, P = 0.008) and PPI (Z = ?2.19, P = 0.028) at 1 month, an effect that extended to 100 m by 5 months (Z = ?2.03, P = 0.042 and Z = ?2.37, P = 0.018 respectively), suggesting a spill‐over effect of the bednets. An IgM serosurvey showed a 15.3% decrease (95% CI 5.0–25.5%, P < 0.01) in the number of IgM‐positive individuals from baseline to12 months later. Conclusions Insecticide‐treated bednets had an immediate effect on dengue vector populations after their introduction, and over the next 5–12 months, the presence of ITNs may have continued to affect vector populations and dengue transmission.     

Survival of Betta splendens fish (Regan, 1910) in domestic water containers and its effectiveness in controlling Aedes aegypti larvae (Linnaeus, 1762) in Northeast Brazil

Objective In Northeast Brazil, large domestic containers used to store water are important breeding sites of Aedes aegypti, the main vector of dengue fever. The objective of this study was to estimate the survival of Betta splendens (Perciformes: Osphronemidae) fish in domestic containers in Fortaleza (Ceará State), as well as its effectiveness in the control of premature A. aegypti stages. Methods The use of B. splendens was compared to Bacillus thuringiensis israelensis (Bti) in domestic containers. In a first home visit, B. splendens or Bti were applied to water containers. Two follow‐up visits were conducted after 3–4 and 5–6 months to assess the presence of viable fish in the containers and infestation by larvae. Betta splendens fish were still present in 97.6% of containers 45–60 days after application. When the fish was present, the infestation rate was significantly higher (P < 0.001) in the Bti group (IR ratio = 21.60; 95% CI: 6.46–72.28). In deposits where the fish remained, efficacy was 85% better than Bti. The permanence of fish was higher in concrete tanks (48.5%) located outside the house (47.5%) and at ground level (53.3%). We conclude that B. splendens may be suitable for biological control of A. aegypti larvae in large domestic water containers, but that appropriate measures should be taken to assure prolonged survival and the presence of fish in the containers.     

Costs of dengue prevention and incremental cost of dengue outbreak control in Guantanamo, Cuba

Objective To assess the economic cost of routine Aedes aegypti control in an at‐risk environment without dengue endemicity and the incremental costs incurred during a sporadic outbreak. Methods The study was conducted in 2006 in the city of Guantanamo, Cuba. We took a societal perspective to calculate costs in months without dengue transmission (January–July) and during an outbreak (August–December). Data sources were bookkeeping records, direct observations and interviews. Results The total economic cost per inhabitant (p.i.) per month. (p.m.) increased from 2.76 USD in months without dengue transmission to 6.05 USD during an outbreak. In months without transmission, the routine Aedes control programme cost 1.67 USD p.i. p.m. Incremental costs during the outbreak were mainly incurred by the population and the primary/secondary level of the healthcare system, hardly by the vector control programme (1.64, 1.44 and 0.21 UDS increment p.i. p.m., respectively). The total cost for managing a hospitalized suspected dengue case was 296.60 USD (62.0% direct medical, 9.0% direct non‐medical and 29.0% indirect costs). In both periods, the main cost drivers for the Aedes control programme, the healthcare system and the community were the value of personnel and volunteer time or productivity losses. Conclusions Intensive efforts to keep A. aegypti infestation low entail important economic costs for society. When a dengue outbreak does occur eventually, costs increase sharply. In‐depth studies should assess which mix of activities and actors could maximize the effectiveness and cost‐effectiveness of routine Aedes control and dengue prevention.     

The unbearable lightness of technocratic efforts at dengue control

Objective To identify key elements that should provide an added value and assure sustainable effects of the deployment of technical tools for Aedes aegypti control. Methods An observational study was conducted between April 2001 and March 2002 in 30 blocks (1574 houses) in the central zone of Guantanamo city. A trial that combined two complementary technical interventions, the distribution of new ground level water tanks and the intensive use of insecticide, was nested in May 2001. Another 30 blocks (1535 houses) were selected as control area. We assessed community perceptions and household risk behaviour at baseline and after 9 months, and measured the trial’s impact through entomological indices. Results Perceived self efficacy to solve A. aegypti infestation and prevent dengue was not modified. We found no changes in behaviour. In the study area the container indices decreased significantly from 0.7% before to 0.1% 1 month after the intervention. Six months later, they had increased to 2.7% and uncovered new water tanks constituted 75.9% of all breeding sites. Over the 9 months after the trial the average monthly house indices were similar in the study and control areas. A technical approach and lack of community involvement in the trial’s implementation were the main causes of these short‐lived results. Conclusions Top–down deployment of technical tools without active involvement of the community has a temporary effect and does not lead to the behavioural changes necessary for sustainable A. aegypti control.     

Association of human immune response to Aedes aegypti salivary proteins with dengue disease severity

Dengue viruses (DENV; family Flaviviridae, genus Flavivirus) are transmitted by Aedes aegypti mosquitoes and can cause dengue fever (DF), a relatively benign disease, or more severe dengue haemorrhagic fever (DHF). Arthropod saliva contains proteins delivered into the bite wound that can modulate the host haemostatic and immune responses to facilitate the intake of a blood meal. The potential effects on DENV infection of previous exposure to Ae. aegypti salivary proteins have not been investigated. We collected Ae. aegypti saliva, concentrated the proteins and fractionated them by nondenaturing polyacrylamide gel electrophoresis (PAGE). By the use of immunoblots, we analysed reactivity with the mosquito salivary proteins (MSP) of sera from 96 Thai children diagnosed with secondary DENV infections leading either to DF or DHF, or with no DENV infection, and found that different proportions of each patient group had serum antibodies reactive to specific Ae. aegypti salivary proteins. Our results suggest that prior exposure to MSP might play a role in the outcome of DENV infection in humans.     

Can piperonyl butoxide enhance the efficacy of pyrethroids against pyrethroid‐resistant Aedes aegypti?

Background Pyrethroid resistance can be considered the main threat to the continued control of many mosquito vectors of disease. Piperonyl butoxide (PBO) has been used as a synergist to help increase the efficacy of certain insecticides. This enhancement stems from its ability to inhibit two major metabolic enzyme systems, P450s and non‐specific esterases, and to enhance cuticular penetration of the insecticide. Objective To compare the mortality of a characterized resistant Aedes aegypti strain, Nha Trang, from Vietnam and the susceptible laboratory strain Bora Bora on netting with the pyrethroid deltamethrin (DM) alone and in combination with PBO. Methods Resistance mechanisms were characterized using molecular and bioassay techniques; standard PCR was used to test for the kdr target site mutation. Potential genes conferring metabolic resistance to DM were identified with microarray analysis using the Ae. aegypti‘detox chip’. These data were analysed alongside results from WHO susceptibility tests. P450, CYP9J32, was significantly overexpressed in the DM‐resistant strain compared with the susceptible Bora Bora strain. Another five genes involved with oxidative stress responses in mosquitoes were also significantly overexpressed. The Nha Trang strain was homozygous for two kdr mutations. WHO cone bioassays were used to investigate mortality with incorporated DM‐treated nets with and without PBO. PBO used in combination with DM resulted in higher mortality than DM alone. Conclusion Synergists may have an important role to play in the future design of vector control products in an era when alternatives to pyrethroids are scarce.     

First dengue virus detection in Aedes albopictus from Delhi, India: its breeding ecology and role in dengue transmission

Objective To report dengue virus and its disease transmission in Aedes albopictus in the National Capital Territory of Delhi, India. Methods Monthly Aedes surveys were carried out in 126 urban localities of Delhi in 2008 and 2009. Pools of all three species of Aedes mosquitoes were tested for Dengue virus (DENV) using an antigen‐capture enzyme‐linked immunosorbent assay. Results Aedes aegypti was the most prevalent species, breeding throughout the year. Aedes albopictus was found in 9.52% of surveyed localities including the central urban part of Delhi, in March and from August to October. Aedes albopictus and Aedes vittatus are adapting to breed in manmade containers in the urban areas of Delhi in addition to their natural habitats of bamboo bushes and rock pits. Of the 229 pools of Ae. aegypti and 34 pools of Ae. albopictus tested, 10.5% and 11.76% were positive for dengue virus, respectively. No dengue virus infection was recorded in Ae. vittatus. Conclusion This is the first report of dengue virus in Ae. albopictus from north India. Because DENV was detected in Ae. albopictus, which adapted to manmade containers, both its spread and transmission dynamics should be checked.     

Aedes aegypti in Jamaica,West Indies: container productivity profiles to inform control strategies

Objective To describe the Aedes aegypti container profile in the three parishes of Portland, St. Anns and St. Catherine, Jamaica. Method Traditional stegomyia and pupae per person indices. Results A total of 8855 containers were inspected. A. aegypti were breeding in 19.2% of the 4728 containers in Portland, in 6.7% of the 2639 containers in St. Ann, and in 27.2% of the 1488 containers in Tryhall Heights, St. Catherine. Container types differed between Portland (P > 0.02) on one hand and St. Ann and Tryhall Heights, St. Catherine on the other hand: there were with no vases or potted plants with water saucers in St. Ann and St. Catherine. A. aegypti were breeding in more containers in St. Catherine (38%) (38% in wet season and 21% in the dry season) than in Portland (19%) or St. Ann (6%), both of which had more containers but A. aegypti breeding in fewer: 17.7% and 11.2% in the wet and 20.4% and 3.5% in the dry seasons respectively. The daily production of adult mosquitoes in the three study sites was 1.51, 1.29 and 0.66 adult female mosquitoes per person in Portland, St. Ann and St. Catherine during the dry season and 1.12, 0.23 and 1.04 female mosquitoes per person in the wet season respectively. Conclusion All three communities are at risk for dengue outbreaks and vector control should concentrate on reducing the mosquito populations from the most productive containers before a new dengue virus serotype is introduced into Jamaica.     

Personal protection by long‐lasting insecticidal hammocks against the bites of forest malaria vectors

Objective In Southeast Asia, malaria vectors bite outside the houses before bedtime, and forest dwellers rarely use insecticide‐treated nets (ITNs). Thus, we tested the protection of long‐lasting insecticidal hammocks (LLIH) using Olyset? technology against exophagic vectors in two forest villages of Cambodia. Methods In each village, we conducted two entomological surveys (middle and end of the rainy season), each lasting 10 consecutive nights. These comprised human landing collections during the whole night by people sitting outside in the hammocks. Five households were selected per village, and for each household, two fixed positions were allocated: one for the control and one for the treated hammock. Results In total, 6449 mosquitoes were collected from control hammocks compared to 4481 in treated hammocks. Personal protection conferred by the hammocks was 46% (CI 95%: 35–55%) against the bites of Anopheles minimus. A significant reduction of An. dirus bites (46% CI 95%: 25–62%) was only observed at the end of the rainy season. An. maculatus and culicines bites were only reduced in one of the two study sites. Conclusion Even if this LLIH is not inducing full protection against the bites of malaria vectors, it could prove effective in protecting forest workers and villagers before sleeping time. LLIH can be an additional and valuable tool in eliminating artemisinin‐resistant malaria in the region.     

High susceptibility to Chikungunya virus of Aedes aegypti from the French West Indies and French Guiana

Objectives To estimate the vector competence of Aedes aegypti populations sampled from distinct anthropogenic environments in French Guiana, Guadeloupe and Martinique for the strain CHIKV 06.21. Methods F₁/F₂ females were orally infected at titres of 10⁶ and 10^7.5 pfu/ml in blood‐meals. Disseminated infection rates (DIR) of mosquitoes were estimated using indirect fluorescent antibody assay on heads’ squashes, 7 or 14 days post‐infection (pi). Results At a titre of 10^7.5 pfu/ml, DIR ranged from 88.9% to 100.0% and were not significantly different whether assessed at day 7 or 14 pi. At a titre of 10⁶ pfu/ml, DIR observed 7 days pi ranged from 37.6 to 62.0%. Conclusions Ae. aegypti from French Guiana and French West Indies are highly competent to transmit CHIKV. An evaluation of DIR 7 days rather than 14 days pi is adequate to estimate vector competence. The titre of 10⁶ pfu/ml allows us to distinguish Ae. aegypti populations originating from distinct environments (dense or diffuse housing) by their vector competence. This assessment is a prerequisite to better evaluate the potential risk of Chikungunya outbreaks once the virus is introduced from endemic regions.     

Is routine dengue vector surveillance in central Brazil able to accurately monitor the Aedes aegypti population? Results from a pupal productivity survey

Objective To assess how well the Aedes aegypti infestation rapid survey, Levantamento Rapido de Indice para Aedes aegypti (LIRAa), is able to accurately estimate dengue vector densities and target the most important breeding sites in Goiania, Brazil. Methods In February 2009, a pupal productivity survey was conducted in 2 districts of the city of Goiania, central Brazil. The results were compared to those of LIRAas conducted in the same districts during the months before and after the pupal productivity survey. Results In the pupal productivity survey, 2 024 houses were surveyed and 2 969 water‐holding containers were inspected. Discarded small water containers most frequently contained immature Ae. aegypti. The most pupal‐productive containers were elevated water tanks, roof gutters and water holding roofs. Combined, these three containers accounted for <40% of all positive containers but produced >70% of all pupae. In the two districts where the pupal productivity survey was conducted, the house indices were 5.1 and 4.6 and the Breteau indices were 5.9 and 6.0. In contrast, the two LIRAs conducted in the same two districts resulted in an average house index of 1.5 and Breteau index of 2.5, with discarded items identified as the most frequently infested container type. Conclusion Both the LIRAa and the pupal productivity survey identified discarded items as being most frequently infested with immature stages of Ae. aegypti, but the pupal productivity survey showed that elevated containers produced the greatest proportion of Ae. aegypti pupae (a proxy measure of adult vector density) and that the values of the Stegomyia indices were substantially underestimated by LIRAa. Although both surveys differ considerably in terms of sampling method and manpower, in the case of this study the LIRAa did not accurately identify or target the containers that were the most important to adult mosquito production.     

Field comparison of thermal and non‐thermal ultra‐low‐volume applications using water and diesel as solvents for managing dengue vector,Aedes aegypti

Objectives To compare the effectiveness on Aedes aegypti (Linneo) (Diptera: Culicidae) of a larvicide‐adulticide ULV formulation applied by a thermal or a cold fogger using different solvents. Methods We applied, in field conditions, a ULV formulation containing pyriproxyfen and permethrin, using a thermal and a cold fogger and water or diesel as solvent. We determined the effectiveness of these applications on Ae. aegypti adults and larvae by different bioassays and measuring Breteau, house and adult indices. Results When water was used as solvent, the treatments applied with the cold or the thermal foggers were equally effective on adult mortality (close to 90%) and adult emergence inhibition (% EI) (close to 70%). When the thermal fogger was used with water as solvent, the adult mortality outside the houses (85%) was higher, but not significantly different, than with diesel (65%). The contrary happens inside (22%vs. 58%), while there were no differences in %EI. Adult and larval indices behaved similarly in all areas, with a slight tendency for the treatments applied using water as solvent to be more effective. Conclusions Water‐based formulations are equally or more effective than the one applied with diesel as solvent. The use of water as solvent will not only improve the effectiveness of this formulation but also reduce the environmental impact and costs of spraying compared to the use of diesel.     

High-throughput assays for detection of the F1534C mutation in the voltage-gated sodium channel gene in permethrin-resistant Aedes aegypti and the distribution of this mutation throughout Thailand

Objectives To develop rapid monitoring tools to detect the F1534C permethrin‐resistance mutation in domain IIIS6 of the Aedes aegypti voltage‐gated sodium channel gene and determine the frequency and distribution of this mutation in Thailand. Methods A TaqMan SNP genotyping and an allele specific PCR (AS‐PCR) assay were developed and validated by comparison with DNA sequencing of homozygous susceptible and homozygous resistant laboratory strains, their reciprocal‐cross progenies, and field‐caught mosquitoes. To determine the resistance phenotype of wild‐caught A. aegypti, mosquitoes were exposed to 0.75% permethrin paper. The AS‐PCR assay was used to screen 619 individuals from 20 localities throughout Thailand. Results Overall, both assays gave results consistent with DNA sequencing for laboratory strains of known genotype and for wild‐caught A. aegypti. The only slight discrepancy was for the AS‐PCR method, which overestimated the mutant allele frequency by 1.8% in wild‐caught samples. AS‐PCR assays of permethrin‐exposed samples show that the mutant C1534 allele is very closely associated with the resistant phenotype. However, 19 permethrin‐resistant individuals were homozygous for the wild‐type F1534 allele. DNA sequencing revealed all these individuals were homozygous for two other mutations in domain II, V1016G and S989P, which are known to confer resistance ( Srisawat et al. 2010 ). The F1534C mutation is widespread in Thailand with mutant allele frequencies varying among populations from 0.20 to 1.00. Conclusions These assays can be used for the rapid detection of the F1534C resistance mutation in A. aegypti populations. The F1534C, and other, mutations underlie an extremely high prevalence of pyrethroid resistance in Thailand.     

Geographic Partitioning of Dengue Virus Transmission Risk in Florida

Dengue viruses (DENVs) cause the greatest public health burden globally among the arthropod-borne viruses. DENV transmission risk has also expanded from tropical to subtropical regions due to the increasing range of its principal mosquito vector, Aedes aegypti. Focal outbreaks of dengue fever (dengue) in the state of Florida (FL) in the USA have increased since 2009. However, little is known about the competence of Ae. aegypti populations across different regions of FL to transmit DENVs. To understand the effects of DENV genotype and serotype variations on vector susceptibility and transmission potential in FL, we orally infected a colony of Ae. aegypti (Orlando/ORL) with low passage or laboratory DENV-1 through -4. Low passage DENVs were more infectious to and had higher transmission potential by ORL mosquitoes. We used these same DENVs to examine natural Ae. aegypti populations to determine whether spatial distributions correlated with differential vector competence. Vector competence across all DENV serotypes was greater for mosquitoes from areas with the highest dengue incidence in south FL compared to north FL. Vector competence for low passage DENVs was significantly higher, revealing that transmission risk is influenced by virus/vector combinations. These data support a targeted mosquito-plus-pathogen screening approach to more accurately estimate DENV transmission risk.     

Long‐term effects of malaria prevention with insecticide‐treated mosquito nets on morbidity and mortality in African children: randomised controlled trial

Objective The objective is to investigate the effect of malaria control with insecticide‐treated mosquito nets (ITNs) regarding possible higher mortality in children protected during early infancy, due to interference with immunity development, and to assess long‐term effects on malaria prevalence and morbidity. Methods Between 2000 and 2002, a birth cohort was enrolled in 41 villages of a malaria holoendemic area in north‐western Burkina Faso. All neonates (n = 3387) were individually randomised to ITN protection from birth (group A) vs. ITN protection from age 6 months (group B). Primary outcome was all‐cause mortality. In 2009, a survey took place in six sentinel villages, and in 2010, a census was conducted in all study villages. Results After a median follow‐up time of 8.3 years, 443/3387 (13.1%) children had migrated out of the area and 484/2944 (16.4%) had died, mostly at home. Long‐term compliance with ITN protection was good. There were no differences in mortality between study groups (248 deaths in group A, 236 deaths in group B; rate ratio 1.05, 95% CI: 0.889–1.237, P = 0.574). The survey conducted briefly after the rainy season in 2009 showed that more than 80% of study children carried asexual malaria parasites and up to 20% had clinical malaria. Conclusion Insecticide‐treated mosquito net protection in early infancy is not a risk factor for mortality. Individual ITN protection does not sufficiently reduce malaria prevalence in high‐transmission areas. Achieving universal ITN coverage remains a major challenge for malaria prevention in Africa.     

Development and evaluation of a single-step multiplex PCR to differentiate the aquatic stages of morphologically similar Aedes (subgenus: Stegomyia) species

Objective To develop a single‐step multiplex PCR to differentiate the aquatic stages of Aedes aegypti, Aedes albopictus and Aedes vittatus collected from different breeding spots in arbovirus endemic/epidemic areas and to detect the most abundant species by the multiplex PCR. Methods Aquatic stages of different mosquito species were sampled by inspecting artificial and natural breeding sites in domestic and peridomestic areas. DNA was isolated from different stages of the three Aedes species. Using novel primers based on 18S rDNA sequence, a single‐step multiplex PCR was developed to clearly distinguish the three Aedes species. It was then evaluated in the aquatic stages of Aedes species collected from different areas. Results A total of 1150 aquatic stages were collected from 294 breeding spots, of which 156 contained Aedes species. Discarded tires were the major breeding spots of Aedes species. The aquatic stages were clustered into 230 pools; Ae. albopictus was detected in the largest number of pools, followed by Ae. aegypti and Ae. vittatus. Conclusions The Multiplex PCR clearly differentiated the aquatic stages of the three Aedes species and detected that Ae. albopictus was most profuse in different breeding spots surveyed, hence indicating to be the main vector in this region. So control measures can be designed against Ae. albopictus at an early stage to prevent any arboviral outbreak. This method is a convenient tool for precise identification of Aedes vectors during entomological surveys in arbovirus endemic/epidemic areas where several species coexist.