Reducing costs and operational constraints of dengue vector control by targeting productive breeding places: a multi-country non-inferiority cluster randomized trial.

OBJECTIVES: To test the non-inferiority hypothesis that a vector control approach targeting only the most productive water container types gives the same or greater reduction of the vector population as a non-targeted approach in different ecological settings and to analyse whether the targeted intervention is less costly. METHODS: Cluster randomized trial in eight study sites (Venezuela, Mexico, Peru, Kenya, Thailand, Myanmar, Vietnam, Philippines), with each study area divided into 18-20 clusters (sectors or neighbourhoods) of approximately 50-100 households each. Using a baseline pupal-demographic survey, the most productive container types were identified which produced >or=55% of all Ae. aegypti pupae. Clusters were then paired based on similar pupae per person indices. One cluster from each pair was randomly allocated to receive the targeted vector control intervention; the other received the 'blanket' (non-targeted) intervention attempting to reach all water holding containers. RESULTS: The pupal-demographic baseline survey showed a large variation of productive container types across all study sites. In four sites the vector control interventions in both study arms were insecticidal and in the other four sites, non-insecticidal (environmental management and/or biological control methods). Both approaches were associated with a reduction of outcome indicators in the targeted and non-targeted intervention arm of the six study sites where the follow up study was conducted (PPI, Pupae per Person Index and BI, Breteau Index). Targeted interventions were as effective as non-targeted ones in terms of PPI. The direct costs per house reached were lower in targeted intervention clusters than in non-targeted intervention clusters with only one exception, where the targeted intervention was delivered through staff-intensive social mobilization. CONCLUSIONS: Targeting only the most productive water container types (roughly half of all water holding container types) was as effective in lowering entomological indices as targeting all water holding containers at lower implementation costs. Further research is required to establish the most efficacious method or combination of methods for targeted dengue vector interventions.

Molecular identification of mosquito species in the Anopheles annularis group in southern Asia.

The Anopheles annularis group of subgenus Cellia Theobald (Diptera: Culicidae) includes five currently recognized species in southern Asia: An. annularis Van der Wulp, Anopheles nivipes (Theobald) and Anopheles philippinensis Ludlow, which are widespread in the region, Anopheles pallidus Theobald, which is known in Sri Lanka, India and Myanmar, and Anopheles schueffneri Stanton, which occurs in Java and Sumatra. Identification of the four mainland species based on morphology is problematic. In view of the fact that the three widespread species are variously involved in malaria transmission in different parts of the region, we developed a species-specific polymerase chain reaction assay based on rDNA internal transcribed spacer 2 (ITS2) sequences to facilitate entomological and epidemiological studies of the four species. The method proved to be reliable when tested over a wide geographical area.

Population structure and population history of Anopheles dirus mosquitoes in Southeast Asia.

Separating the confounding effects of long-term population history from gene flow can be difficult. Here, we address the question of what inferences about gene flow can be made from mitochondrial sequence data in three closely related species of mosquitoes, Anopheles dirus species A, C, and D, from southeast Asia. A total of 84 sequences of 923 bp of the mitochondrial cytochrome oxidase I gene were obtained from 14 populations in Thailand, Myanmar, and Bangladesh. The genealogy of sequences obtained from two populations of AN: dirus C indicates no contemporary gene flow between them. The F(ST) value of 0.421 therefore probably represents a recent common history, perhaps involving colonization events. Anopheles dirus A and D are parapatric, yet no differentiation was seen either within or between species. The starlike genealogy of their haplotypes, smooth unimodal mismatch distributions, and excess of low frequency mutations indicate population expansion in An. dirus A and D. This, rather than widespread gene flow, explains their low within-species F(ST) values (0.018 and 0.022). The greater genetic diversity of An. dirus D suggests that expansion occurred first in species D and subsequently in species A. The current geographical separation and low hybrid fitness of these species also argue against ongoing interspecific gene flow. They suggest instead either historical introgression of mtDNA from An. dirus D into species A followed by independent range expansions, or a selective sweep of mtDNA that originated in An. dirus D. While not excluding contemporary gene flow, historical population processes are sufficient to explain the data in An. dirus A and D. The genealogical relationships between haplotypes could not be used to make inferences of gene flow because of extensive homoplasy due to hypervariable sites and possibly also recombination. However, it is concluded that this approach, rather than the use of fixation indices, is required in the future to understand contemporary gene flow in these mosquitoes. The implications of these results for understanding gene flow in another important and comparable group of malaria vector mosquitoes in Africa, the An. gambiae complex, are also discussed.

Effects of temperature and larval diet on development rates and survival of the dengue vector Aedes aegypti in north Queensland, Australia.

Immature development times, survival rates and adult size (wing-lengths) of the mosquito Aedes aegypti (L.) (Diptera: Culicidae) were studied in the laboratory at temperatures of 10-40 degrees C. The duration of development from egg eclosion (hatching of the first instar) to adult was inversely related to temperature, ranging from 7.2 +/- 0.2 days at 35 degrees C to 39.7 +/- 2.3 days at 15 degrees C. The minimum temperature threshold for development (t) was determined as 8.3 +/- 3.6 degrees C and the thermal constant (K) was 181.2 +/- 36.1 day-degrees above the threshold. Maximum survival rates of 88-93% were obtained between 20 and 30 degrees C. Wing-length was inversely related to temperature. The sex ratio (female:male) was 1:1 at all temperatures tested (15, 20, 25 and 35 degrees C) except 30 degrees C (4:3). Under field conditions at Townsville and Charters Towers, north Queensland, the duration of immature development varied according to the container position (i.e. shaded or exposed) and the availability of food resources, as well as inversely with temperature. These data indicate that containers with an abundance of organic matter (e.g. those used for striking plant cuttings) or those amongst foliage or under trees (e.g. discarded plastic tubs and tyres) tended to produce the largest adult Ae. aegypti, which had faster development and better immature survival. As such progeny have been linked to a greater risk of dengue transmission, it would seem important to focus on control of such containers.

Critical examination of Aedes aegypti indices: correlations with abundance.

The following immature stage indices for Aedes (Stegomyia) aegypti surveillance were evaluated in four north Queensland, Australia towns with respect to their relationship to immature and adult female densities: Breteau, House, Container, Larval Density, Stegomyia (and modifications thereof), and a newly created Adult Productivity Index. Spearman's correlations of indices that considered larval or immature (larvae and pupae) numbers had a better relationship with immature abundance but this was not necessarily the case against adult abundance. To examine the robustness of the indices, data from 758 premises in Townsville, Charters Towers, Ravenswood, and Mingela were pooled and 30 random subsamples, each consisting of 50 premises were taken. After each subsample was taken, the premises selected were reintroduced into the original data bank of 758 premises, and therefore, were available for further selection, i.e., sampling with replacement. Indices were calculated for each of the 30 subsamples and the coefficients of variation of each index were estimated from these. The Breteau, Adult Productivity, House, and Adult density indices proved to have the smallest coefficients compared with index size. No alternate index was regarded as being superior to the Breteau, including the Adult Productivity Index measuring both container type frequency and immature density. For this reason and in view of the labor intensiveness of estimating immature indices that incorporate productivity, it is recommended that new and cost-effective methods of adult surveillance be pursued.

The Premise Condition Index: a tool for streamlining surveys of Aedes aegypti.

Premise inspections and treatment of the larval habitats of container-breeding Aedes aegypti are extremely labor intensive. Since this means of control is the only one available in relation to dengue fever, this report presents an effective approach for streamlining premise surveys in north Queensland, Australia. From a survey of 877 premises in Townsville, Charters Towers, and Mingela/Ravenswood in 1990, occupier and premise variables were collected to examine any relationships with the presence of Ae. aegypti. Statistical modeling of these parameters using multiple and simple Poisson regression indicated that for both adjusted and unadjusted models respectively, the degree of shade and tidiness of the yard had strong correlations with both the proportion of positive premises and the numbers of infested containers therein. Condition of the house was also a significant variable in the unadjusted model. On this basis, the Premise Condition Index is proposed as a rapid assessment tool that can increase efficiency of detecting positive premises and containers by 270-370%. Although habitat selection by Aedes varies throughout the world, this approach could be used as a model for control of vectors of both dengue and yellow fever.

Understanding productivity, a key to Aedes aegypti surveillance.

The objective of this work was to define criteria that could be applied to achieve faster, more economical, and accurate assessment of vector populations for control of dengue viruses. During 1989-1990, 1,349 premises were surveyed in Townsville, Charters Towers and Mingela/Ravenswood, Queensland, Australia. In each locality, 1.9-8.4% of premises contained three or more containers with Aedes aegypti immature forms and were designated as key premises. Comparison of surveys in Townsville from 1989 to 1990 indicated that positive premises (i.e., those with at least one container with Ae. aegypti present) were 3.22 times more likely to remain positive than negative houses to become positive the following year. The Ae. aegypti population in Townsville was seen to be totally associated with garden receptacles, discarded household items, and trash but one well and one rainwater tank were responsible for 28% of all immature forms recorded in the 1,349 premises inspected. These breeding sites of high productivity were designated as key containers. At Charters Towers, Mingela, and Ravenswood, rainwater tanks were seen as the most important key container because although they constituted 13-29% of positive containers, they supported 60-63% of the immature forms. This study demonstrates that there is a certain degree of stability with regard to positive premises and that some of these, or some container types, contribute disproportionately to the Ae. aegypti population. Control programs could be made more efficient if efforts were concentrated on these sites of key vector productivity.

Hyperendemic malaria in a forested, hilly Myanmar village.

A 1-year longitudinal study of hyperendemic malaria was carried out at Tha-bye-wa village, Oktwin township, situated in the forested Bago mountain range in south-central Myanmar. Mosquito infectivity was assayed using specific, sporozoite enzyme-linked immunosorbent assays. Anopheles dirus was the predominant vector in the postmonsoon season (October); during the cool-dry season (January), both An. dirus and Anopheles minimus were vectors. Members of the Anopheles culicifacies complex were caught in the hot-dry season (April) but none was infective. The entomological inoculation rate was estimated to be at least 13.7 infective bites/person/year. Infective An. dirus were caught feeding on cattle as well as on humans. Three of the 4 positive An. dirus and both positive An. minimus were caught biting humans indoors in the second quarter of the night when most people were sleeping. This suggests that use of insecticide-impregnated bednets in this area could interrupt transmission.

Rapid and efficient removal of immature Aedes aegypti in metal drums by sweep net and modified sweeping method.

A modified sweeping method was developed using a cotton sweep net for control of Ae. aegypti immatures in 200 liter (44 gallon) metal drums which are major sources of breeding in Yangon. Laboratory experiments revealed that with only 4 sweeps (approximately 10 minutes duration), the mean % removal (+/- SD) of Ae. aegypti fourth stage larvae was 88.5% +/- 1.47. This was followed by a field study undertaken in Sanchaung township, Yangon. Twenty-eight drums with moderate (< 500 immatures) to high (> or = 500 immatures) Ae. aegypti density were tested in the field. It was found that with only 4 sweeps per drum, a total of 24,886 immatures were removed out of 29,155 immatures in these 28 drums, giving a mean % removal per drum of 85.36% +/- 10.74 (range = 55.54-98.62%). This sweeping method is simple, cost-effective, and readily accepted by the community. It could be an appropriate technology for control of the dengue hemorrhagic fever (DHF) vector Ae. aegypti.

Quantitative sampling of immature Aedes aegypti in metal drums using sweep net and dipping methods.

The behavior of aquatic stages of Aedes aegypti was evaluated in 200-liter metal drums to develop improved surveillance. When known numbers of larvae recovered with a rectangular net and with a standard dipper were compared, all stages except 1st instar were most numerous in the top 1/3 of the drum. Water temperature was the only significant variable affecting the vertical distribution of 4th-instar larvae; light intensity and pH were nonsignificant factors. Rust in the water was also thought to influence distribution towards the surface layer. When 100-400 immatures were released into a full drum, immatures were detected by sweeping and dipping the surface layer on 93.3 and 72.9% of trials, respectively. The correlation coefficients (r) of recoveries of known numbers of immatures by sweeping and dipping varied from 0.92 to 0.98 and from 0.38 to 0.89, respectively. The coefficients of variation were 2-5 times smaller for sweeping than for dipping. Recoveries from dipping were shown to be affected by the water volume in the drum. Counts of 4th-instar larvae from one sweep around the top are sufficient to assess productivity in drums.