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Seminal Aedes aegypti control Case Study Published in PLOS

In September 2016, a much-anticipated research article was published in PLOS Neglected Tropical Diseases detailing a multi-phased study on container mosquito control in Cambodia. This long-term study evaluated the impact of biological larviciding on Aedes aegypti population density and dengue transmission utilizing of Bacillus thuringiensis subsp. israelensis (Bti) Strain AM65-52. The results add to mounting evidence supporting effective reduction of both Aedes aegypti populations and incidence of human dengue cases.

Spanning a seven-year period from 2005-2011, the study was the result of a public and private partnership between the Cambodia’s National Malaria Center (CNM) and Valent BioSciences Corporation (VBC). In search of new larvicide tools to address the onset of temephos resistance in the region, CNM evaluated VectoBac® WG, a water dispersible granular formulation of Bti that has been observed to perform favorably as a temephos substitute.

Focusing on the direct link between container mosquito control and vector management, the final phase of the study involved the incorporation of VectoBac WG in a large-scale operational program in Kandal Province, Cambodia, a temephos-resistant site, to measure impact. Over the period, six districts receiving Bti treatments showed a 47% reduction in dengue cases, whereas five non-treated districts showed a 351% increase in dengue cases over the same period of time.


Dengue infections throughout the world number around 400 million per year. Dengue transmission is prevalent in Cambodia, with the container breeder Ae aegypti acting as the primary vector. While the study was centered on use of VectoBac WG for dengue control, the recent outbreak of Zika virus in the Americas has added prominence to the study since Aedes aegypti is also the primary vector of Zika and the resulting cases of microcephaly

Ae aegypti thrives in all types of containers either man-made or natural. Like many developing countries, Cambodia is highly dependent upon agriculture and many of its communities lie in rural areas where piped water is not available. In these areas, storage containers are used to collect and hold water from rain, groundwater, and rivers for drinking, cooking, and other home uses.

Cambodia has a tropical climate where Ae aegypti-driven dengue transmission peaks during the wet and humid monsoon season between May and September. In 1995, Cambodia’s National Dengue Control Program was established and adopted the organophosphate temephos at its primary means of vector control, applying the larvicide twice each wet season to control dengue. Temephos resistance was subsequently reported in field populations as early as 2001, then later verified by the World Health Organization (WHO) Collaborating Centre at Institute for Medical Research, Malaysia. In the face of these resistance challenges, the CNM decided to evaluate Bti Strain AM65-52 in its search for new larviciding tools to control dengue. Use of Bti in temephos-resistant areas of Brazil yielded promising results dating back to 2001. In addition to its target specificity, Bti was also attractive given its 2007 approval for application into drinking water by WHOPES.


The initial phase of the multi-year study was to evaluate the field efficacy of Bti on Ae aegypti populations. Cambodia is divided into 24 provinces, which are then further divided into districts, communes, and villages. The 2005 – 2006 phase of the study measured the impact of Bti applications by recording the number of adult Ae aegypti mosquitoes present per household in two closely situated communes within the Kâmpóng Chhnang Province. Both communes contained the same number of villages (9) and roughly the same number of households and residents. The environments of both areas were also similar: farming communities surrounded by paddy fields.

Containers belonging to households in the Peani commune were treated with Bti while households in the Ou Ruessei commune served as untreated controls. A variety of container types were treated with direct application (using spoons) of VectoBac WG.

Over the two-year phase of the study, a total of 24 surveillance rounds (50 households per round) were conducted in each commune by a common entomology team. Pupal and adult mosquitos were counted in each household. In the two months following the Bti treatment in the Peani commune, 27% of the homes inspected had zero adult Ae aegypti mosquitoes and nearly 80% had five or fewer. In the untreated control, only 4% of homes had no adults and more than half (51%) had 11 or more adults (see table). Months after the treatment, the percentage of adult Aedes-free households in treated communes were nearly three times greater than untreated households. Likewise, the number of homes with 11 or more Ae aegypti adults was almost double that of treated homes (see table).


In 2007, the second phase of the study expanded into a pilot program that included 58 communes in 11 districts across the Kandal Province of Cambodia. Since Ae aegypti thrives in urban areas, the communes were selected based on population density and dengue incidence rate. Building on the field efficacy of the study’s initial phase, the second phase also included a post-treatment survey to gauge household attitudes about treatments as well as to assess applicators’ ability to recognize and treat the wide variety and number of containers within the province.

From a performance standpoint, Bti efficacy was comparable to the 2005-2006 trials. Moreover, 95% of the people surveyed in treated areas accepted the treatment because larvae died within two hours of the application without any changes to the color or odor of the treated water. The survey also showed that applicators were able to recognize and treat more than 92% of containers in the selected communes. With field efficacy, operational feasibility, and public acceptance of the treatments all established, the study was able to enter the large scale operational trial and dengue impact evaluation phase.


Once again, Kandal Province was selected for the third phase of the study given its widespread temephos resistance among Ae aegypti populations, its pronounced dengue incidence rate, and high human population density. In 2010, select communes within all 11 districts received two treatments of Bti and dengue incidence was recorded. In 2011, the six Kandal districts that had experienced the highest dengue incidence in 2010 were treated twice with Bti, this time in their entirety. The other five districts were left untreated. In all, more than three-quarters of a million containers in 85,239 households received treatments in the first Bti application alone. To measure impact, researchers measured 2011 dengue incidence in both treated and untreated districts during the peak dengue season (August – September) and compared the data to the same districts, all of which were only selectively treated, in 2010.

All told, the six districts treated with VectoBac WG in 2011 displayed a significant reduction of dengue cases vs. the prior year. Overall, a 47% reduction across the six districts was achieved. Three districts experienced a reduction of between 66-85%. Conversely, the five untreated districts saw a 351% increase in dengue cases vs. 2010 over the same two month period.

Source: Setha T, Chantha N, Benjamin S, Socheat D (2016). “Bacterial Larvicide, Bacillus thuringiensis israelensis Strain AM65-52 Water Dispersible Granule Formulation Impacts Both Dengue Vector, Aedes aegypti (L.) Population Density and Disease Transmission in Cambodia. PLoS Negl Trop Dis 10 (9): e0004973. doi:10.1371/journal.pntd.0004973, September 14, 2016