The single water-surface sweep estimation method accurately estimates very low (n = 4) to low-moderate (n = 25-100) and high (n > 100) Aedes aegypti (Diptera: Culicidae) pupae numbers in large water containers up to 13 times faster than the exhaustive sweep and total count method and without any sediment contamination.

OBJECTIVE: To confirm that a single water-surface sweep-net collection coupled with three calibration factors (2.6, 3.0 and 3.5 for 1/3, 2/3 and 3/3 water levels, respectively) (WSCF) could accurately estimate very low to high Aedes aegypti pupae numbers in water containers more rapidly than the exhaustive 5-sweep and total count (ESTC) method recommended by WHO. METHODS: Both methods were compared in semi-field trials using low (n = 25) to moderate (n = 50-100) pupae numbers in a 250-l drum at 1/3, 2/3 and 3/3 water levels, and by their mean-time determinations using 200 pupae in three 220- to 1024-l water containers at these water levels. Accuracy was further assessed using 69.1% (393/569) of the field-based drums and tanks which contained <100 pupae. RESULTS: The WSCF method accurately estimated total populations in the semi-field trials up to 13.0 times faster than the ESTC method (all P < 0.001); no significant differences (all P-values ≥ 0.05) were obtained between the methods for very low (n = 4) to low-moderate (n = 25-100) and high (n > 100) pupae numbers/container and without sediment disturbance. CONCLUSION: The simple WSCF method sensitively, accurately and robustly estimated total pupae numbers in their principal breeding sites worldwide, containers with >20 l water volumes, significantly (2.7- to 13.0-fold: all P-values <0.001) faster than the ESTC method for very low to high pupae numbers/container without contaminating the clean water by sediment disturbance which is generated using the WHO-recommended ESTC method. The WSCF method seems ideal for global community-based surveillance and control programmes.

Three calibration factors, applied to a rapid sweeping method, can accurately estimate Aedes aegypti (Diptera: Culicidae) pupal numbers in large water-storage containers at all temperatures at which dengue virus transmission occurs.

The ability of a simple sweeping method, coupled to calibration factors, to accurately estimate the total numbers of Aedes aegypti (L.) (Diptera: Culicidae) pupae in water-storage containers (20-6412-liter capacities at different water levels) throughout their main dengue virus transmission temperature range was evaluated. Using this method, one set of three calibration factors were derived that could accurately estimate the total Ae. aegypti pupae in their principal breeding sites, large water-storage containers, found throughout the world. No significant differences were obtained using the method at different altitudes (14-1630 m above sea level) that included the range of temperatures (20-30 degrees C) at which dengue virus transmission occurs in the world. In addition, no significant differences were found in the results obtained between and within the 10 different teams that applied this method; therefore, this method was extremely robust. One person could estimate the Ae. aegypti pupae in each of the large water-storage containers in only 5 min by using this method, compared with two people requiring between 45 and 90 min to collect and count the total pupae population in each of them. Because the method was both rapid to perform and did not disturb the sediment layers in these domestic water-storage containers, it was more acceptable by the residents, and, therefore, ideally suited for routine surveillance purposes and to assess the efficacy of Ae. aegypti control programs in dengue virus-endemic areas throughout the world.

Sample sizes for identifying the key types of container occupied by dengue-vector pupae: the use of entropy in analyses of compositional data.

A method has been developed for estimating the sample sizes needed to identify categories that comprise a large proportion of a compositional data-set. The method is to be used in the design of surveys of mosquito pupae, for identifying the key container types from which the majority of adult dengue vectors emerge. Although a finite-population correction was devised for estimating the mean of a negative binomial distribution, other complications of parametric approaches make them unlikely to yield methods simple enough to be practically applicable. The Shannon-Wiener index was therefore investigated as a more useful alternative, at the cost of theoretical generalizability, in an approach based on re-sampling methods in conjunction with the use of entropy. This index can be used to summarize the degree to which pupae are either concentrated in a few container types, or dispersed among many. An empirical relationship between the index and the repeatability of surveys of differing sample sizes was observed. A step-wise rule, based on the entropy of the cumulative data, was devised for determining the sample size, in terms of the number of houses positive for pupae, at which a pupal survey might reasonably be stopped.

Pupal-productivity surveys to identify the key container habitats of Aedes aegypti (L.) in Barranquilla, the principal seaport of Colombia.

Surveys were conducted in three neighbourhoods of Barranquilla, the main seaport of Colombia, to identify, using counts of pupae in water containers during the wet and dry seasons, the most productive Aedes aegypti breeding sites. Overall, 3,433 premises were investigated in the wet season and 3,563 in the dry, representing, respectively, 82.3% and 84.6% of the total numbers of premises in the study areas. Despite a reasonably reliable supply of piped water, there were still some large storage containers for domestic water (cement ground tanks and plastic, metal and cement drums) in the area. Although such containers represented only 1.8%-16.3% of the total number of containers observed, they contributed 72.0%-78.2% and 65.0%-95.8% of the total Ae. aegypti pupal population in the three study neighbourhoods during the wet and dry seasons, respectively. In contrast, bottles represented 23.0%-88.9% of the total number of containers but produced no more than 0.1% of the total Ae aegypti pupal populations in these neighbourhoods. Other containers (tyres, vases, 'other discarded' and 'other used') generally produced only low numbers of pupae. In some settings, however, containers in the 'other discarded' category could contribute up to 19% of the total pupal population, and in one survey of one neighbourhood a single container in this category held 9.1% of all the pupae collected. These results, from a city where dengue fever is endemic, will help to focus local campaigns for Ae. aegypti source-reduction on the most productive categories of container.

The use of direct sequencing of dengue virus cDNA from individual field-collected Aedes aegypti for surveillance and epidemiological studies.

The relative efficiencies of four methods to extract viral RNA from individual dengue-2 virus (D-2V)-infected mosquitoes, Aedes aegypti (L.) (Diptera: Culicidae), were compared. The most efficient of these methods was then used to extract viral RNA for the preparation of cDNA from the abdomens of six engorged D-2V-infected mosquitoes and sera from three dengue fever (DF) patients collected in an isolated rural town in Colombia. Comparisons of viral envelope (E) gene sequences from each of these strongly suggested that the D-2V population which circulated in this study area was a homogeneous genotype which was unrelated to any of the D-2 viruses isolated from elsewhere in the world. When coupled with our rapid method to identify viruses in individual mosquitoes (Romero-Vivas et al. (1998) Medical and Veterinary Entomology, 12, 101-105), the methodology we describe should be useful for epidemiological and surveillance studies of dengue viruses and other arboviruses.

Determination of dengue virus serotypes in individual Aedes aegypti mosquitoes in Colombia.

Adult Aedes aegypti mosquitoes were collected in Puerto Triunfo, central Colombia, where dengue is endemic, during a six month period. Viral infection within the head of each individual mosquito was identified by an immunofluorescent assay (IFA) using a flavivirus-specific monoclonal antibody. The dengue virus serotype, present in each flavivirus-positive specimen, was then determined in portions of the remaining thorax using IFAs with serotype-specific monoclonal antibodies. Among 2065 female Aedes aegypti collected and tested, twenty-four flavivirus-positive individuals were found (minimum infection rate 11.6%), three identified as dengue type-1 and twenty-one as dengue type-2 virus. This was consistent with the isolation of only these two serotypes of dengue virus from dengue fever patients within this town. No vertical transmission of dengue virus could be detected in 1552 male Aedes aegypti collected. This method is inexpensive, simple, rapid to perform and suitable for use in developing countries to identify and distinguish different serotypes of dengue virus in their vectors during eco-epidemiological investigations.