Combining Synthetic Human Odours and Low-Cost Electrocuting Grids to Attract and Kill Outdoor-Biting Mosquitoes: Field and Semi-Field Evaluation of an Improved Mosquito Landing Box.

BACKGROUND: On-going malaria transmission is increasingly mediated by outdoor-biting vectors, especially where indoor insecticidal interventions such as long-lasting insecticide treated nets (LLINs) are widespread. Often, the vectors are also physiologically resistant to insecticides, presenting major obstacles for elimination. We tested a combination of electrocuting grids with synthetic odours as an alternative killing mechanism against outdoor-biting mosquitoes. METHODS: An odour-baited device, the Mosquito Landing Box (MLB), was improved by fitting it with low-cost electrocuting grids to instantly kill mosquitoes attracted to the odour lure, and automated photo switch to activate attractant-dispensing and mosquito-killing systems between dusk and dawn. MLBs fitted with one, two or three electrocuting grids were compared outdoors in a malaria endemic village in Tanzania, where vectors had lost susceptibility to pyrethroids. MLBs with three grids were also tested in a large semi-field cage (9.6 × 9.6 × 4.5m), to assess effects on biting-densities of laboratory-reared Anopheles arabiensis on volunteers sitting near MLBs. RESULTS: Significantly more mosquitoes were killed when MLBs had two or three grids, than one grid in wet and dry seasons (P<0.05). The MLBs were highly efficient against Mansonia species and malaria vector, An. arabiensis. Of all mosquitoes, 99% were non-blood fed, suggesting host-seeking status. In the semi-field, the MLBs reduced mean number of malaria mosquitoes attempting to bite humans fourfold. CONCLUSION: The improved odour-baited MLBs effectively kill outdoor-biting malaria vector mosquitoes that are behaviourally and physiologically resistant to insecticidal interventions e.g. LLINs. The MLBs reduce human-biting vector densities even when used close to humans, and are insecticide-free, hence potentially antiresistance. The devices could either be used as surveillance tools or complementary mosquito control interventions to accelerate malaria elimination where outdoor transmission is significant.

Development of multiplex real-time PCR assays for identification of members of the Anopheles funestus species group.

BACKGROUND: The malaria vector and non-vector species of the Anopheles funestus group are morphologically very similar and accurate identification is required as part of effective control strategies. In the past, this has relied on morphological and cytogenetic methods but these have been largely superseded by a robust allele-specific PCR (AS-PCR). One disadvantage of AS-PCR is the requirement for post-PCR processing by gel electrophoresis of PCR products. In this study, three new high-throughput 'closed-tube' assays were developed and compared with the previously described AS-PCR technique. METHODS: Protocols for three fluorescence-based assays based on Melt Curve Analysis (MCA), High Resolution Melt (HRM) and TaqMan SNP genotyping were developed to detect and discriminate Anopheles parensis, Anopheles leesoni, Anopheles vaneedeni, Anopheles rivulorum and An. funestus s.s. The sensitivity and specificity of these assays were compared with the widely used AS-PCR in a blind trial using DNA extracted from wild-caught mosquitoes. RESULTS: The TaqMan assay proved to be the most sensitive and specific of the three new assays. The MCA and HRM assays initially gave promising results, but were more sensitive to both DNA quality and quantity and consequently showed a higher rate of incorrect identifications. CONCLUSION: The TaqMan assay proved to be the most robust of the three protocols tested in this study. This assay very effectively identified all five members of the An. funestus group using fluorescently-labeled probes with distinct emission and excitation spectra allowing their independent detection in a single reaction. This method is at least as sensitive and specific as the gold standard AS-PCR approach and because it has no requirement for post-PCR processing is simpler and more rapid to run. The one disadvantage of the TaqMan assay is the cost of this assay, both in terms of initial capital outlay and running cost per sample, which is higher than AS-PCR. However, the cost of both the real-time PCR machine and fluorescently labelled probes required is falling and in the future the cost of this assay is likely to become closer to that of standard PCR.