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 In the past, this has relied on morphological and cytogenetic methods but these have been largely superseded by a robust allele-specific PCR (AS-PCR).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

Discrimination of 15 Amazonian Anopheline Mosquito Species by Polymerase Chain Reaction—Restriction Fragment Length Polymorphism

International audiencePrecise identification of anopheline species is paramount for incrimination of malaria vectors and implementation of a sustainable control program. Anopheline mosquitoes are routinely identified morphologically, a technique that is time-consuming, needs high level of expertise, and prone to misidentifications especially when considering Amazonian species. The aim of this study was therefore to develop a DNA-based identification technique to supplement traditional morphological identification methods for the discrimination of anopheline mosquitoes collected in French Guiana. The internal transcribed spacer 2 (ITS2) region of ribosomal DNA (rDNA) for anopheline species was amplified by polymerase chain reaction (PCR), and digested with AluI/MspI restriction enzymes. PCR-restriction fragments length polymorphism (RFLP) assay was compared to sequencing of the ITS2 region for validation. Fifteen Anopheles species have shown distinct PCR-RFLP profiles. A concordance of 100% was obtained when identification by PCR-RFLP was compared to sequencing of ITS2. A high throughput, fast, and cost-effective PCR-RFLP assay has been developed for unambiguous discrimination of fifteen anopheline mosquito species from French Guiana including primary and suspected secondary malaria vectors

Characterization of the Anopheles funestus group, including Anopheles funestus-like, from Northern Malawi

Background Limited information is available on malaria vector composition, feeding habits and malaria transmission in northern Malawi. Evidence of mosquito species diversity in this area was established in 2009, when Anopheles funestus-like, a new member of the An. funestus group was described. Additional biological information is needed to identify this species and to understand its role in malaria transmission. Methods Anopheline mosquitoes were collected in northern Malawi and analyzed for Plasmodium species infection, blood meal source and susceptibility to insecticides. A new hydrolysis probe assay was designed to identify An. funestus-like. Results Anopheles funestus and An. rivulorum predominated in the indoor collections. Most An. funestus-like were collected indoors, mainly fed on animals and were uninfected with P. falciparum. Anopheles funestus showed insecticide resistance to deltamethrin and bendiocarb. A high-precision hydrolysis probe assay was successfully developed to identify An. funestus-like. Discussion Four species in the An. funestus group were collected in Karonga. Resistance to deltamethrin and bendiocarb was observed in An. funestus and further investigation is needed on the insecticide resistance mechanisms. Anopheles funestus-like, while collected indoors, is mainly zoophilic and most likely not a malaria vector. Accession numbers An. funestus (GenBank accession no. KC771136), An. funestus-like (GenBank accession no. KC771137), An. parensis GenBank accession no. KC771138) and An. vaneedeni GenBank accession no. KC771139)

Spatial and Seasonal Dynamics of Anopheles Mosquitoes in Saint-Georges de l'Oyapock, French Guiana: Influence of Environmental Factors

International audienceLittle is known about the Anopheles fauna of Saint-Georges de l'Oyapock, a persistent malaria-endemic municipality in French Guiana. This study aimed to update the knowledge of local Anopheles diversity, and their ecology and role in malaria transmission. Sampling sessions were implemented between September 2013 and October 2014. Four species were identified from the 3,450 specimens collected: Anopheles darlingi Root, An. braziliensis, An. triannulatus s.l., and An. nuneztovari s.l. Anopheles darlingi was the predominant species. Its involvement in malaria transmission was suspected due to 1) its abundance, 2) the presence of a density peak during the malaria emergence period, and 3) a dynamic correlated with malaria cases observed two months later. Present and past studies show that the influence of environmental conditions on malaria vector dynamics is high, and may vary drastically according to the local context. This supports evidence that control strategies must be designed at fine scales

Characterization of the Anopheles funestus group, including Anopheles funestus-like, from northern Malawi

Background Limited information is available on malaria vector composition, feeding habits and malaria transmission in northern Malawi. Evidence of mosquito species diversity in this area was established in 2009, when Anopheles funestus-like, a new member of the An. funestus group was described. Additional biological information is needed to identify this species and to understand its role in malaria transmission. Methods Anopheline mosquitoes were collected in northern Malawi and analyzed for Plasmodium species infection, blood meal source and susceptibility to insecticides. A new hydrolysis probe assay was designed to identify An. funestus-like. Results Anopheles funestus and An. rivulorum predominated in the indoor collections. Most An. funestus-like were collected indoors, mainly fed on animals and were uninfected with P. falciparum. Anopheles funestus showed insecticide resistance to deltamethrin and bendiocarb. A high-precision hydrolysis probe assay was successfully developed to identify An. funestus-like. Discussion Four species in the An. funestus group were collected in Karonga. Resistance to deltamethrin and bendiocarb was observed in An. funestus and further investigation is needed on the insecticide resistance mechanisms. Anopheles funestus-like, while collected indoors, is mainly zoophilic and most likely not a malaria vector. Accession numbers An. funestus (GenBank accession no. KC771136), An. funestus-like (GenBank accession no. KC771137), An. parensis GenBank accession no. KC771138) and An. vaneedeni GenBank accession no. KC771139)