Bacterial Larvicide, Bacillus thuringiensis israelensis Strain AM 65-52 Water Dispersible Granule Formulation Impacts Both Dengue Vector, Aedes aegypti (L.) Population Density and Disease Transmission in Cambodia.

A multi-phased study was conducted in Cambodia from 2005-2011 to measure the impact of larviciding with the bacterial larvicide, Bacillus thuringiensis israelensis (Bti), a water dispersible granule (WG) formulation on the vector, Aedes aegypti (L.) and the epidemiology. In our studies, all in-use containers were treated at 8 g/1000 L, including smaller containers and animal feeders which were found to contribute 23% of Ae aegypti pupae. The treated waters were subjected to routine water exchange activities. Pupal production was suppressed by an average 91% for 8 weeks. Pupal numbers continued to remain significantly lower than the untreated commune (UTC) for 13 weeks post treatment in the peak dengue vector season (p<0.05). Suppression of pupal production was supported by very low adult numbers in the treated commune. An average 70% of the household harbored 0-5 Ae aegypti mosquitoes per home for 8 weeks post treatment, but in the same period of time >50% of the household in the UTC harbored ≥11 mosquitoes per home. The adult population continued to remain at significantly much lower numbers in the Bti treated commune than in the UTC for 10-12 weeks post treatment (p<0.05). In 2011, a pilot operational program was evaluated in Kandal Province, a temephos resistant site. It was concluded that 2 cycles of Bti treatment in the 6 months monsoon season with complete coverage of the target districts achieved an overall dengue case reduction of 48% in the 6 treated districts compared to the previous year, 2010. Five untreated districts in the same province had an overwhelming increase of 352% of dengue cases during the same period of time. The larvicide efficacy, treatment of all in-use containers at the start of the monsoon season, together with treatment coverage of entire districts interrupted disease transmission in the temephos resistant province.

Spatial epidemiology and climatic predictors of paediatric dengue infections captured via sentinel site surveillance, Phnom Penh Cambodia 2011-2012.

BACKGROUND: Dengue is a major contributor to morbidity in children aged twelve and below throughout Cambodia; the 2012 epidemic season was the most severe in the country since 2007, with more than 42,000 reported (suspect or confirmed) cases. METHODS: We report basic epidemiological characteristics in a series of 701 patients at the National Paediatric Hospital in Cambodia, recruited during a prospective clinical study (2011-2012). To more fully explore this cohort, we examined climatic factors using multivariate negative binomial models and spatial clustering of cases using spatial scan statistics to place the clinical study within a larger epidemiological framework. RESULTS: We identify statistically significant spatial clusters at the urban village scale, and find that the key climatic predictors of increasing cases are weekly minimum temperature, median relative humidity, but find a negative association with rainfall maximum, all at lag times of 1-6 weeks, with significant effects extending to 10 weeks. CONCLUSIONS: Our results identify clustering of infections at the neighbourhood scale, suggesting points for targeted interventions, and we find that the complex interactions of vectors and climatic conditions in this setting may be best captured by rising minimum temperature, and median (as opposed to mean) relative humidity, with complex and limited effects from rainfall. These results suggest that real-time cluster detection during epidemics should be considered in Cambodia, and that improvements in weather data reporting could benefit national control programs by allow greater prioritization of limited health resources to both vulnerable populations and time periods of greatest risk. Finally, these results add to the increasing body of knowledge suggesting complex interactions between climate and dengue cases that require further targeted research.

Genetic diversity and lineage dynamic of dengue virus serotype 1 (DENV-1) in Cambodia.

In Cambodia, dengue virus (DENV) was first isolated in 1963 and has become endemic with peak epidemic during raining season. Since 2000, the Dengue National Control Program has reported from 10,000 to 40,000 cases per year with fatality rates ranging from 0.7 to 1.7. All four dengue serotypes are found circulating in Cambodia with alternative predominance of serotypes DENV-2 and DENV-3. The DENV-1 represents from 5% to 20% of all circulating viruses, depending upon the year. In this work, 79 clinical strains of DENV-1 were isolated between 2000 and 2009 and their genome fully sequenced. Four distinct lineages with different dynamics were identified. The main evolutionary drive was negative selective pressure but each lineage was characterized by the presence of specific mutations acquired through evolution. Coexistence, extinction and replacement of lineages occurred over the 10-year period. Lineages 1, 2 and 3 were all detected since 2000-2002 and disappeared in 2003, 2004-2005 and 2007, respectively. Lineages 1 and 2 displayed different dynamics. Lineage 1 was very diverse whereas lineage 2 was very homogeneous. Lineage 4 which derived from lineage 3 in 2003 remained the only one at the end of the sampling period in 2008-2009 owing to a selective sweep. The lineages dynamic of DENV-1 viruses and consequences for molecular epidemiology are discussed.

Clinical characterization, diagnosis and socioeconomic impact of hospitalized dengue in Cambodia.

Dengue infection is endemic among children in Cambodia. Few studies have described the clinical characteristics, diagnoses and the socioeconomic impact of hospitalized dengue cases. As part of a multicountry prospective dengue study, we analysed interviews and record reviews of 123 consecutive cases of children hospitalized with confirmed dengue in a major public provincial hospital. About 80% of cases evidenced plasma leak. On average, a hospitalized dengue case lasted 6.3 days, with 3.3 days of inpatient care, and cost US$116 (Singapore dollar 48). The majority (88%) of these cases’ households reported a substantial adverse economic impact, perhaps explained by their inability to afford medical care (39%), or on account of loss of five or more days of income (91%), need to borrow money beyond family or friends (39%), being compelled to sell or sale of property (53%) to pay for treatment. Hospitalized dengue has major clinical and socioeconomic consequences in Cambodia.

Community-based use of the larvivorous fish Poecilia reticulata to control the dengue vector Aedes aegypti in domestic water storage containers in rural Cambodia.

A community-based study of the distribution of larvivorous fish, Poecilia reticulata (common name: guppy), in water storage containers for dengue control was undertaken in 14 villages and approximately 1,000 households in Cambodia. Community volunteers reared guppies and distributed them in water jars and tanks in households for which they were responsible. A nearby control area received no intervention. One year after project commencement, 56.9% of eligible containers contained guppies and there was a 79.0% reduction in Aedes infestation in the intervention community compared with the control. Smaller or discarded containers unsuitable for guppy distribution in the intervention area also had 51% less infestation than those in the control area, suggesting a "community-wide" protective effect. In addition, there was less infestation in villages with higher rates of fish uptake, suggesting that the presence of fish was responsible for a reduction in Aedes infestation. This applied vector control model was well-accepted, effective, efficient, and shows promise as a sustainable community-based, non-insecticidal intervention for dengue vector control in large domestic water storage containers in rural Cambodia and elsewhere.

The effect of long-lasting insecticidal water container covers on field populations of Aedes aegypti (L.) mosquitoes in Cambodia.

Dengue in Cambodia is mainly transmitted by Aedes aegypti (L.) mosquitoes that primarily breed in large, concrete jars (> or =200 liters) used for the storage of water for domestic use. Following a preliminary risk assessment, long-lasting insecticidal netting (LN) treated with deltamethrin was incorporated into the design of the covers for these jars. Their effect on immature and adult female populations of Ae. aegypti in six villages in a peri-urban area of Cambodia were compared with populations in six nearby control villages before and for 22 weeks after distribution of the jar covers. There were significantly fewer pupae per house in intervention villages than in control villages (6.6 and 31.9, respectively, p<0.01). Fewer pupae were recovered from intervention houses than from control houses at every post-intervention assessment. Two weeks after the intervention, the average number of indoor resting female Ae. aegypti per house in the intervention villages had declined approximately three-fold, whereas in the controls there was only a slight reduction (16%). The magnitude of the difference between the two areas diminished over time, which contact bioassays confirmed was likely due to a gradual reduction of insecticidal effect of the jar covers. In the study area, insecticide-treated covers for large concrete water storage jars were efficacious for controlling Ae. aegypti in the protected water jars and with a demonstrable effect on adult densities and survival. Further studies of this targeted container strategy in Cambodia, and elsewhere, are recommended. However, improvements in technology that would extend the duration of insecticidal effectiveness of LN materials may be needed for the development of cost-effective public health applications.

Cost-effectiveness of annual targeted larviciding campaigns in Cambodia against the dengue vector Aedes aegypti.

OBJECTIVE: To assess the cost-effectiveness (CE) of annual targeted larviciding campaigns from 2001 to 2005 against the dengue vector Aedes aegypti in two urban areas of Cambodia with a population of 2.9 million people. METHODS: The intervention under analysis consisted of annual larviciding campaigns targeting medium to large water storage containers in households and other premises. The CE compared the intervention against the hypothetical alternative of no intervention. The CE was calculated as the ratio of disability adjusted life years (DALYs) saved to the net cost of the intervention (in 2005 US dollars) by year. A sensitivity analysis explored the range of study parameters. RESULTS: The intervention reduced the number of dengue cases and deaths by 53%. It averted an annual average of 2980 dengue hospitalizations, 11,921 dengue ambulatory cases and 23 dengue deaths, resulting in a saving of 997 DALYs per year. The gross cost of the intervention was US $567,800 per year, or US $0.20 per person covered. As the intervention averted considerable medical care, the annual net cost of the intervention was US $312,214 (US $0.11 per person covered) from a public sector perspective and US $37,137 (US $0.01 per person covered) from a societal perspective. The resulting CE ratios were: US $313/DALY gained from the public perspective and US $37/DALY gained from the societal perspective. Even under the most conservative assumption, the intervention remained cost effective from both perspectives. CONCLUSIONS: Annual, targeted larviciding campaigns appear to have been effective and cost-effective medium-term interventions to reduce the epidemiologic and economic burden of dengue in urban areas of Cambodia.

Efficacy of Bacillus thuringiensis israelensis, VectoBac WG and DT, formulations against dengue mosquito vectors in cement potable water jars in Cambodia.

This study reports the evaluation of Bacillus thuringiensis israelensis (Bti), a biological larvicide, in cement jars holding river, well and rain water. Two Bti formulations, VectoBac WG and VectoBac DT, were evaluated in a village in Phnom Penh. Thirty-one households with cement jars supporting the colonization of Ae.aegypti immatures were chosen. In each house 3 jars were aligned next to each another and filled with the same type of water. One of the 3 jars was treated with VectoBac WG at 0.4 g per 50 liters, a second jar was treated with VectoBac DT at 1 tablet per 50 liters, and a third jar was an untreated control (UTC). The jars were not covered, kept outdoors and not subjected to water exchange activity. The efficacy of VectoBac to control natural Ae.aegypti infestation was measured by Ae.aegypti pupae surveillance, conducted 3 days per week for 3 months post-treatment (June-September 2004). All pupae were removed, allowed to emerge in the Cambodia National Malaria Center insectarium and the emerged adults were identified and counted. The VectoBac treatments were more effective in river water, followed by well and rain water. The VectoBac treatments significantly reduced the pupae numbers for a minimum of 3 months in the river water and 2.5 months in the well water (p < 0.05). In the rain water, the pupae densities in the VectoBac WG and DT treated jars were not significantly different from the untreated jars, although the treated jars yielded 2.0 to 5.2 fold less pupae, respectively, than in the untreated jars during the 3 months post-treatment. The efficacy of VectoBac WG to control Ae.aegypti was similar to the efficacy of VectoBac DT in the 3 water types (p > 0.05). It was also observed that VectoBac WG and DT were target specific, without any adverse effects on aquatic predatory insects common in well and rain water. VectoBac WG and DT were found to be easy-to-use formulations, with no need to repackage them prior to use in the containers. The amounts of VectoBac WG and DT used were 12.5 fold less by weight than temephos (Abate 1.0% SG).

Influence of breeding sites features on genetic differentiation of Aedes aegypti populations analyzed on a local scale in Phnom Penh Municipality of Cambodia.

This study analyzed genetic differentiation of 20 Aedes aegypti populations collected along a street in Phnom Penh Municipality of Cambodia. Using allozyme and microsatellite variations, we demonstrated that populations were differentiated and the pattern of differentiation was dependent on the type of breeding sites. Moreover, insecticide treatments with temephos mostly affect the population functioning of discarded containers. Low gene flow detected could limit the natural diffusion of resistant populations that might instead take advantage of human displacements to spread.

Human transportation influences Aedes aegypti gene flow in Southeast Asia.

Estimates of population genetic organization and gene flow of Aedes aegypti, the main vector of dengue viruses, have provided insights into dengue epidemiology. To assess the role of the vector in the changing pattern of dengue in Southeast Asia, extensive studies on the genetic differentiation of Aedes aegypti have been carried out. Among them, vector differentiation has been estimated for Cambodia, Thailand and South Vietnam using microsatellite markers. Analysis showed that there was less genetic differentiation between mosquito populations from Ho Chi Minh City and Phnom Penh than from either of them and Thailand, suggesting that passive migrations through human transportation help to explain this pattern of differentiation.

Variation over space and time of Aedes aegypti in Phnom Penh (Cambodia): genetic structure and oral susceptibility to a dengue virus.

We studied spatial and temporal variation in 20-23 Aedes aegypti samples collected in Phnom Penh and its suburbs to estimate the population genetic structure using allozymes and the susceptibility to a dengue-2 virus. Based on seven allozyme systems, we detected low levels of genetic exchanges (i.e. high, significant F(ST) values) between populations collected in the city centre, and different patterns of genetic structure for samples collected in the suburbs, depending on the type of environment and the date of collection. In the southern suburbs and the Chroy Chang Var Peninsula, differentiation became highly significant at the end of the dry season, whereas the opposite situation was observed for collections from the northern suburbs. Vector competence assessed by oral infections with a dengue-2 virus was lower for samples collected in the city centre than in the suburbs. A significant decrease of dengue susceptibility was observed in populations during the dry season. This study allows a model of Ae. aegypti population functioning in Phnom Penh to be suggested. Dynamics of dengue virus diffusion depend on the population genetic structure of the vector and its evolution over space and time.

Status of Dengue Control in Cambodia, 1997.

Since 1962, Cambodia has been facing regular dengue fever outbreaks which affect children in areas with concentrated populations, especially in Phnom Penh and Battambang. Identified areas at risk extend out from the south (Kampot & Kampong, Som povinces) to the north-west (Battambang and Banteay Meanchey provinces), encompassing 16 provinces. Epidemics were reported in 1983, 1985, 1987 and 1990, which were also peak years for dengue in Thailand. In comparison to neighbouring countries, the case fatality rate in Cambodia has been very high, ranging from 3.6 to 15%. However, improvement in health services has brought about a noticeable decrease in mortality due to DF/DHF.