Variation in Pyrethroid Resistance Phenotypes in Anopheles darlingi in an Area with Residual Malaria Transmission: Warning of Suspected Resistance in French Guiana.

Anopheles darlingi is the main vector of malaria in South America. In French Guiana, malaria transmission occurs inland and along the rivers with a regular reemergence in the lower Oyapock area. Control against malaria vectors includes indoor residual spraying of deltamethrin and the distribution of long-lasting impregnated bednets. In this context, the level of resistance to pyrethroids was monitored for 4 years using CDC bottle tests in An. darlingi populations. A loss of susceptibility to pyrethroids was recorded with 30-minute knock-down measured as low as 81%. However, no pyrethroid molecular resistance was found by sequencing a 170 base pair fragment of the S6 segment of domain II of the voltage-gated sodium channel gene. Fluctuation of resistance phenotypes may be influenced by the reintroduction of susceptible alleles from sylvatic populations or by other mechanisms of metabolic resistance.

High malaria transmission in a forested malaria focus in French Guiana: How can exophagic Anopheles darlingi thwart vector control and prevention measures?

In French Guiana, malaria vector control and prevention relies on indoor residual spraying and distribution of long lasting insecticidal nets. These measures are based on solid epidemiological evidence but reveal a poor understanding of the vector. The current study investigated the behaviour of both vectors and humans in relation to the ongoing prevention strategies. In 2012 and 2013, Anopheles mosquitoes were sampled outdoors at different seasons and in various time slots. The collected mosquitoes were identified and screened for Plasmodium infection. Data on human behaviour and malaria episodes were obtained from an interview. A total of 3,135 Anopheles mosquitoes were collected, of which Anopheles darlingi was the predominant species (96.2%). For the December 2012-February 2013 period, the Plasmodium vivax infection rate for An. darlingi was 7.8%, and the entomological inoculation rate was 35.7 infective bites per person per three-month span. In spite of high bednet usage (95.7%) in 2012 and 2013, 52.2% and 37.0% of the participants, respectively, had at least one malaria episode. An. darlingi displayed heterogeneous biting behaviour that peaked between 20:30 and 22:30; however, 27.6% of the inhabitants were not yet protected by bednets by 21:30. The use of additional individual and collective protective measures is required to limit exposure to infective mosquito bites and reduce vector densities.

High malaria transmission in a forested malaria focus in French Guiana: How can exophagic Anopheles darlingi thwart vector control and prevention measures?

In French Guiana, malaria vector control and prevention relies on indoor residual spraying and distribution of long lasting insecticidal nets. These measures are based on solid epidemiological evidence but reveal a poor understanding of the vector. The current study investigated the behaviour of both vectors and humans in relation to the ongoing prevention strategies. In 2012 and 2013, Anopheles mosquitoes were sampled outdoors at different seasons and in various time slots. The collected mosquitoes were identified and screened for Plasmodium infection. Data on human behaviour and malaria episodes were obtained from an interview. A total of 3,135 Anopheles mosquitoes were collected, of which Anopheles darlingi was the predominant species (96.2%). For the December 2012-February 2013 period, the Plasmodium vivax infection rate for An. darlingi was 7.8%, and the entomological inoculation rate was 35.7 infective bites per person per three-month span. In spite of high bednet usage (95.7%) in 2012 and 2013, 52.2% and 37.0% of the participants, respectively, had at least one malaria episode. An. darlingi displayed heterogeneous biting behaviour that peaked between 20:30 and 22:30; however, 27.6% of the inhabitants were not yet protected by bednets by 21:30. The use of additional individual and collective protective measures is required to limit exposure to infective mosquito bites and reduce vector densities.

Anopheles darlingi (Diptera: Culicidae) Dynamics in Relation to Meteorological Data in a Cattle Farm Located in the Coastal Region of French Guiana: Advantage of Mosquito Magnet Trap.

Information on dynamics of anopheline mosquitoes is limited in the coastal zone of French Guiana compared with inland endemic areas. Importantly, improvement of surveillance techniques for assessing malaria transmission indicators and comprehension of impact of meteorological factors on Anopheles darlingi Root, the main malaria vector, are necessary. Anopheline mosquitoes were collected continuously during 2012 and 2013 using Mosquito Magnet traps baited with octenol and human landing catches. The two methods were compared based on trends in abundance and parity rate of An. darlingi. Impact of meteorological factors on An. darlingi density estimates was investigated using Spearman's correlation and by binomial negative regression analysis. In all, 11,928 anopheline mosquitoes were collected, and 90.7% (n = 10,815) were identified consisting of four species, with An. darlingi making up 94.9% (n = 10,264). An. darlingi specimens collected by the two methods were significantly correlated, and no difference in parity rate was observed. The abundance of this species peaked in September (dry season) and variations along the years were influenced by relative humidity, temperature, rainfall, and wind speed. Number of mosquitoes collected during peak aggression period was influenced by wind speed and rainfall. Data gathered in this study provide fundamental information about An. darlingi, which can facilitate the design of vector control strategies and construction of models for predicting malaria risk.

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.

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).

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.