Insecticide resistance of malaria mosquitoes from Guinea Conakry

Introduction: There is only limited information on malaria vector species composition, vector status and susceptibility status in Guinea Conakry. This work provides more information from three localities (Boffa, Siguiri and Mount Nimba) with very different ecological settings. Methods: Anopheles mosquitoes were collected resting inside houses. Field bioassays were performed on An. gambiae s.l using the standard WHO protocol. Further species identification by PCR, laboratory bioassay by WHO standards, biochemical enzyme analysis, sporozoite ELISA, kdr detection by PCR and pyrosequencing were performed. Results: Of 600 specimens, 494 (82.3%) were An. gambiae and 2 (0.33%) An. arabiensis. Plasmodium infection rates varied from 1.25 to 5%. Resistance to 4% dieldrin and 0.1% bendiocarb need confirmation in Boffa and to 0.05% deltamethrin and 5% malathion in Siguiri. Resistance to DDT, dieldrin and bendiocarb occurred in Siguiri and Mount Nimba. F1 progeny of wild An. gambiae s.s exposed to pirimiphos methyl indicated elevated esterase and monooxygenase enzymes involved in metabolic detoxification of insecticides. The Kdr-w mutation is present in both the M and S forms of An. gambiae. Kdr genotypes by pyrosequencing and sequencing were in agreement but the standard PCR method gave 28% false positives. Conclusion: Both molecular forms of An. gambiae are implicated in malaria transmission, occur in sympatry and exhibit some degree of resistance to insecticides at all localities. The mechanism of resistance is unclear. Pyrosequencing is more accurate for detecting kdr than the standard PCR method. Vector control interventions need to be tailored to each site based on the data collected by on-going monitoring and surveillance

Systematic study of the new Anopheles funestus-like species from Malawi

Thesis (Ph.D.(Virology))--University of the Witwatersrand, Faculty of Health Sciences, 2012Morphological similarity between malaria vectors and non-vectors occurring in sympatry has serious consequences if the killer diseases have to be controlled. Malaria in Malawi is transmitted by Anopheles gambiae, An. arabiensis and An. funestus. This vector diversity is further complicated by the recently discovered An. funestus-like species which is morphologically similar to An. funestus, and found in association with humans. Currently there is no single assay available that differentiates An. funestus-like from the other African members in the An. funestus group. The objective of this study was to investigate the biology and behavior of the newly discovered An. funestus-like species and its possible role in malaria transmission. This information will assist in the implementation of vector control programs. In addition to this, the study investigated the development of a DNA based assay to differentiate between the members of the An. funestus group and to morphologically described An. funestus-like species. Anopheles mosquitoes were collected resting indoors and outdoors from Karonga in Malawi. Specimens were identified morphologically and molecularly using chain reaction PCR. Identified samples were analyzed by ELISA for blood meal source and Plasmodium sporozoite infection. Anopheles funestus-like was morphologically compared with An. funestus. Real time based PCR was developed and compared to the current multiplex or allele-specific PCR (AS-PCR) assay for sensitivity and performance. The IGS region of the rDNA gene was investigated for development of AS-PCR. Phylogenetic relationship of mosquitoes was constructed from ITS2 and D3 sequences. Adult An. funestus mosquitoes (n = 391) were collected during April and September, 2010. Karonga contributed 63.9% and Likoma Island 36.1%. Of the identified specimens (n = 347) An. funestus-like comprised 10.4%, An. rivulorum 31.7%, An. funestus 57.3% and An. parensis 0.6%. Most of the An. funestus-like species were collected resting indoors 91.7% (33/36) compared to outdoors 8.3% (3/36). The species was predominant during the dry season 63.9% (23/36) compared to the wet season. A total of 19 An. funestus-like females were analyzed for blood meal source. Mixed blood meal from goat and bovine was found in 7 specimens and a single blood meal from goat in 3 specimens.. The rest of the An. funestus-like was negative for blood meal. An overall dry season infection rate of An. funestus-like species by Plasmodium vivax was 5% (1/20) in this study and 3.1% (2/64) from samples collected in 2009 was found. However, the possibility of false positivity could not be excluded and further study is urgently needed to investigate this. Real-time PCR for the identification of members of the An. funestus group was found to be more sensitive (0.02ng/μl) than AS-PCR (0.04ng/μl) and had performance comparable to AS-PCR. AS-PCR developed from the intergenic spacer region of rDNA discriminates An. funestus, An. rivulorum, An. vaneedeni and An. parensis. Of all assays developed in this study, the hydrolysis probe assay is the most reliable assay for identifying members in the An. funestus group. This study confirmed the existence of An. funestus-like species in sympatry with An. funestus group members. An. funestus-like was predominantly found resting indoors (endophilic) but preferring animal over human blood (zoophilic). No consistent morphological characters were found to discriminate between An. funestus and An. funestus-like based on morphological data, An. funestus-like is very similar and closely related to An. funestus which is supported by phylogenetic analysis. However, Restriction Fragment Length Polymorphism separates the two species

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

Mosquito magnet® liberty plus trap baited with octenol confirmed best candidate for Anopheles surveillance and proved promising in predicting risk of malaria transmission in French Guiana

BACKGROUND: In French Guiana, Mosquito Magnet(®) Liberty Plus trap baited with octenol (MMoct) has been proposed for sampling Anopheles darlingi after comparison with CDC light trap and Human landing catch (HLC). However, other available lures were not tested. The current study compared MMoct and MM baited with Lurex™ (MMlur) to HLC, and analysed entomological data from MMoct collection with malaria cases to facilitate malaria surveillance. METHODS: Two independent experiments were conducted during 2012 and 2013 in Saint-Georges town, French Guiana. The first experiment used Latin square design to compare MMoct and MMlur to HLC between 18:30 to 22:30 and 05:00 to 07:00. Parity rate was determined for An. darlingi from each sampling system. In the second experiment, a 24:00 hour collection was done for four consecutive days during the first week of each month and every four days for the rest of the month using MMoct. Portion of the 24 hour collection was dissected for parity rate. All anophelines were screened for Plasmodium infection by PCR. Data for number of malaria cases was analysed for association with density of An. darlingi. RESULTS: In the first experiment, 3,721 anopheline mosquitoes were collected over 21 nights. Of these, 95.7% was identified morphologically to five species and An. darlingi contributed 98.4%, mainly from HLC (75.1%, CI 95% [73.2-77.0]) than MMoct (14.1%, CI 95% [12.6-15.7]) and MMlur (10.8%, CI 95% [9.4-12.2]). Species richness was highest in HLC meanwhile species diversity index was greatest in MMoct. MMoct collected more parous An. darlingi than HLC (p < 0.0001) and MMlur (p = 0.0021). The second experiment amounted to 2035 females, 60.8% belonging to 10 species. Anopheles darlingi constituted 85.0% of the species and had parity rate of 52.3%. Specimens were uninfected with Plasmodium. Density of An. darlingi best correlated with malaria cases observed six weeks later (p = 0.0016; r = 0.4774). CONCLUSION: Though MMoct and MMlur performed well in sampling An. darlingi, MMoct captured more species and, therefore, would be useful for surveillance. Even if it collected mostly parous mosquitoes, MMoct proved useful in collecting entomological data required for predicting malaria emergence. It is a potential replacement for HLC

Resistance to DDT and Pyrethroids and Increased kdr Mutation Frequency in An. gambiae after the Implementation of Permethrin-Treated Nets in Senegal

Introduction: The aim of this study was to evaluate the susceptibility to insecticides of An. gambiae mosquitoes sampled in Dielmo (Senegal), in 2010, 2 years after the implementation of Long Lasting Insecticide-treated Nets (LLINs) and to report the evolution of kdr mutation frequency from 2006 to 2010. Methods: WHO bioassay susceptibility tests to 6 insecticides were performed on adults F0, issuing from immature stages of An. gambiae s.l., sampled in August 2010. Species and molecular forms as well as the presence of L1014F and L1014S kd

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)