Nicolas Moiroux

Pyrethroid resistance in African anopheline mosquitoes: what are the implications for malaria control?

The use of pyrethroid insecticides in malaria vector control has increased dramatically in the past decade through the scale up of insecticide treated net distribution programmes and indoor residual spraying campaigns. Inevitably, the major malaria vectors have developed resistance to these insecticides and the resistance alleles are spreading at an exceptionally rapid rate throughout Africa. Although substantial progress has been made on understanding the causes of pyrethroid resistance, remarkably few studies have focused on the epidemiological impact of resistance on current malaria control activities. As we move into the malaria eradication era, it is vital that the implications of insecticide resistance are understood and strategies to mitigate these effects are implemented.

Combination of malaria vector control interventions in pyrethroid resistance area in Benin: a cluster randomised controlled trial

BACKGROUND Malaria control efforts and elimination in Africa are being challenged by the development of resistance of parasites to antimalarial drugs and vectors to insecticides. We investigated whether the combination of long-lasting insecticidal mosquito nets (LLINs) with indoor residual spraying (IRS) or carbamate-treated plastic sheeting (CTPS) conferred enhanced protection against malaria and better management of pyrethroid-resistance in vectors than did LLINs alone. METHODS We did a cluster randomised controlled trial in 28 villages in southern Benin, west Africa. Inclusion criteria of the villages were moderate level of pyrethroid resistance in malaria vectors and minimum distance between villages of 2 km. We assessed four malaria vector control interventions: LLIN targeted coverage to pregnant women and children younger than 6 years (TLLIN, reference group), LLIN universal coverage of all sleeping units (ULLIN), TLLIN plus full coverage of carbamate-IRS applied every 8 months (TLLIN+IRS), and ULLIN plus full coverage of CTPS lined up to the upper part of the household walls (ULLIN+CTPS). The interventions were allocated to villages by a block randomisation on the basis of preliminary surveys and children of each village were randomly selected to participate with computer-generated numbers. The primary endpoint was the incidence density rate of Plasmodium falciparum clinical malaria in children younger than 6 years as was analysed by Poisson regression taking into account the effect of age and the sampling design with a generalised estimating equation approach. Clinical and parasitological information were obtained by active case detection of malaria episodes during 12 periods of 6 consecutive days scheduled at six weekly intervals and by cross-sectional surveys of asymptomatic plasmodial infections. Children or study investigators were not masked to study group. This study is registered with Current Controlled Trials, number ISRCTN07404145. FINDINGS Of 58 villages assessed, 28 were randomly assigned to intervention groups. 413-429 children were followed up in each intervention group for 18 months. The clinical incidence density of malaria was not reduced in the children from the ULLIN group (incidence density rate 0·95, 95% CI 0·67-1·36, p=0·79), nor in those from the TLLIN+IRS group (1·32, 0·90-1·93, p=0·15) or from the ULLIN+CTPS group (1·05, 0·75-1·48, p=0·77) compared with the reference group (TLLIN). The same trend was observed with the prevalence and parasite density of asymptomatic infections (non significant regression coefficients). INTERPRETATION No significant benefit for reducing malaria morbidity, infection, and transmission was reported when combining LLIN+IRS or LLIN+CTPS compared with a background of LLIN coverage. These findings are important for national malaria control programmes and should help the design of more cost-effective strategies for malaria control and elimination. FUNDING Ministère Français des Affaires Etrangères et Européennes (FSP project 2006-22), Institut de Recherche pour le Développement, Presidents Malaria Initiative (PMI) of US Governement.

Insecticide resistance status in Anopheles gambiae in southern Benin

BackgroundThe emergence of pyrethroid resistance in Anopheles gambiae has become a serious concern to the future success of malaria control. In Benin, the National Malaria Control Programme has recently planned to scaling up long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) for malaria prevention. It is, therefore, crucial to monitor the level and type of insecticide resistance in An. gambiae, particularly in southern Benin where reduced efficacy of insecticide-treated nets (ITNs) and IRS has previously been reported.MethodsThe protocol was based on mosquito collection during both dry and rainy seasons across forty districts selected in southern Benin. Bioassay were performed on adults collected from the field to assess the susceptibility of malaria vectors to insecticide-impregnated papers (permethrin 0.75%, delthamethrin 0.05%, DDT 4%, and bendiocarb 0.1%) following WHOPES guidelines. The species within An. gambiae complex, molecular form and presence of kdr and ace- 1 mutations were determined by PCR.ResultsStrong resistance to permethrin and DDT was found in An. gambiae populations from southern Benin, except in Aglangandan where mosquitoes were fully susceptible (mortality 100%) to all insecticides tested. PCR showed the presence of two sub-species of An. gambiae, namely An. gambiae s.s, and Anopheles melas, with a predominance for An. gambiae s.s (98%). The molecular M form of An. gambiae was predominant in southern Benin (97%). The kdr mutation was detected in all districts at various frequency (1% to 95%) whereas the Ace-1 mutation was found at a very low frequency (≤ 5%).ConclusionThis study showed a widespread resistance to permethrin in An. gambiae populations from southern Benin, with a significant increase of kdr frequency compared to what was observed previously in Benin. The low frequency of Ace-1 recorded in all populations is encouraging for the use of bendiocarb as an alternative insecticide to pyrethroids for IRS in Benin.

Culicidae diversity, malaria transmission and insecticide resistance alleles in malaria vectors in Ouidah-Kpomasse-Tori district from Benin (West Africa): A pre-intervention study

BackgroundTo implement an Insecticide Resistance Management (IRM) strategy through a randomized controlled trial (phase III), 28 villages were selected in southern Benin. No recent entomological data being available in these villages, entomological surveys were performed between October 2007 and May 2008, before vector control strategies implementation, to establish baseline data.MethodsMosquitoes were sampled by human landing collection (16 person-nights per village per survey per village) during 5 surveys. Mosquitoes were identified morphologically and by molecular methods. The Plasmodium falciparum circumsporozoïte indexes were measured by ELISA, and the entomological inoculation rates (EIRs) were calculated. Molecular detection of pyrethroid knock down resistance (Kdr) and of insensitive acetylcholinesterase were performed.Results44,693 mosquitoes belonging to 28 different species were caught from October 2007 to May 2008. Among mosquitoes caught, 318 were An. gambiae s.s., 2 were An. nili, 568 were An. funestus s.s., and one individual was An. leesoni. EIR was 2.05 ± 1.28 infective bites per human per 100 nights on average, of which 0.67 ± 0.60 were from An. funestus and 1.38 ± 0.94 infective bites were from An. gambiae. Important variations were noted between villages considering mosquito density and malaria transmission indicating a spatial heterogeneity in the study area. The kdr allelic frequency was 28.86% in An. gambiae s.s. on average and significantly increases from October 2007 (10.26%) to May 2008 (33.87%) in M molecular form of An. gambiae s.s. Ace 1 mutation was found in S molecular of An. gambiae s.s at a low frequency (< 1%).ConclusionThis study updates information on mosquito diversity and malaria risk in rural villages from south Benin. It showed a high spatial heterogeneity in mosquito distribution and malaria transmission and underlines the need of further investigations of biological, ecological, and behavioral traits of malaria vectors species and forms. This study is a necessary prerequisite to cartography malaria risk and to improve vector control operations in southern Benin.

Human Exposure to Early Morning Anopheles funestus Biting Behavior and Personal Protection Provided by Long-Lasting Insecticidal Nets.

A shift towards early morning biting behavior of the major malaria vector Anopheles funestus have been observed in two villages in south Benin following distribution of long-lasting insecticidal nets (LLINs), but the impact of these changes on the personal protection efficacy of LLINs was not evaluated. Data from human and An. funestus behavioral surveys were used to measure the human exposure to An. funestus bites through previously described mathematical models. We estimated the personal protection efficacy provided by LLINs and the proportions of exposure to bite occurring indoors and/or in the early morning. Average personal protection provided by using of LLIN was high (≥80% of the total exposure to bite), but for LLIN users, a large part of remaining exposure occurred outdoors (45.1% in Tokoli-V and 68.7% in Lokohoué) and/or in the early morning (38.5% in Tokoli-V and 69.4% in Lokohoué). This study highlights the crucial role of LLIN use and the possible need to develop new vector control strategies targeting malaria vectors with outdoor and early morning biting behavior. This multidisciplinary approach that supplements entomology with social science and mathematical modeling illustrates just how important it is to assess where and when humans are actually exposed to malaria vectors before vector control program managers, policy-makers and funders conclude what entomological observations imply.

Dry Season Determinants of Malaria Disease and Net Use in Benin, West Africa

Background To achieve malaria eradication, control efforts have to be sustained even when the incidence of malaria cases becomes low during the dry season. In this work, malaria incidence and its determinants including bed net use were investigated in children of under 5 years of age in 28 villages in southern Benin during the dry season. Methods and Findings Mean malaria clinical incidence was measured in children aged 0–5 years by active case detection in 28 villages of the Ouidah-Kpomasse-Tori Bossito sanitary district between November 2007 and March 2008. Using Poisson mixed-effect models, malaria incidence was assessed according to the level of transmission by different vector species, and Long-Lasting Insecticide-treated mosquito Nets (LLIN) use and ownership. Then, a Binomial mixed-effect model was developed to assess whether nighttime temperature (derived from MODIS remote sensing data), biting nuisance and LLIN ownership are good predictors of LLIN use >60%. Results suggested that Anopheles funestus (Incidence Rates Ratio (IRR) = 3.38 [IC95 1.91–6]) rather than An. gambiae s.s. is responsible for malaria transmission. A rate of LLIN use >60% was associated with a lower risk of malaria (IRR = 0.6 [IC95 0.37–0.99]). Low nocturnal temperature and high biting nuisance were good predictors of LLIN use >60%. Conclusions As recommended by the Malaria Eradication (MalERA) Consultative Group on Modelling, there is a need to understand better the effects of seasonality on malaria morbidity. This study highlights the need to take into account the specificity of malaria epidemiology during the dry-hot season and get a better understanding of the factors that influence malaria incidence and net use. These findings should help National Malaria Control Programmes to implement more effective and sustainable malaria control strategies in Africa.

Optimized Pan-species and Speciation Duplex Real-time PCR Assays for Plasmodium Parasites Detection in Malaria Vectors

Background An accurate method for detecting malaria parasites in the mosquito’s vector remains an essential component in the vector control. The Enzyme linked immunosorbent assay specific for circumsporozoite protein (ELISA-CSP) is the gold standard method for the detection of malaria parasites in the vector even if it presents some limitations. Here, we optimized multiplex real-time PCR assays to accurately detect minor populations in mixed infection with multiple Plasmodium species in the African malaria vectors Anopheles gambiae and Anopheles funestus. Methods Complementary TaqMan-based real-time PCR assays that detect Plasmodium species using specific primers and probes were first evaluated on artificial mixtures of different targets inserted in plasmid constructs. The assays were further validated in comparison with the ELISA-CSP on 200 field caught Anopheles gambiae and Anopheles funestus mosquitoes collected in two localities in southern Benin. Results The validation of the duplex real-time PCR assays on the plasmid mixtures demonstrated robust specificity and sensitivity for detecting distinct targets. Using a panel of mosquito specimen, the real-time PCR showed a relatively high sensitivity (88.6%) and specificity (98%), compared to ELISA-CSP as the referent standard. The agreement between both methods was “excellent” (κ = 0.8, P<0.05). The relative quantification of Plasmodium DNA between the two Anopheles species analyzed showed no significant difference (P = 0, 2). All infected mosquito samples contained Plasmodium falciparum DNA and mixed infections with P. malariae and/or P. ovale were observed in 18.6% and 13.6% of An. gambiae and An. funestus respectively. Plasmodium vivax was found in none of the mosquito samples analyzed. Conclusion This study presents an optimized method for detecting the four Plasmodium species in the African malaria vectors. The study highlights substantial discordance with traditional ELISA-CSP pointing out the utility of employing an accurate molecular diagnostic tool for detecting malaria parasites in field mosquito populations.

Characterization of swarming and mating behaviour between Anopheles coluzzii and Anopheles melas in a sympatry area of Benin.

The swarm structure of two sibling species, Anopheles gambiae coluzzii and Anopheles melas, was characterize to explore the ecological and environmental parameters associated with the formation of swarms and their spatial distribution. Swarms and breeding sites were searched and sampled between January and December 2010, and larval and adult samples were identified by PCR. During the dry season, 456 swarms of An. gambiae s.l. were sampled from 38 swarm sites yielding a total of 23,274 males and 76 females. Of these 38 swarming sites, 18 were composed exclusively of An. gambiae coluzzii and 20 exclusively of An. melas, presenting clear evidence of reproductive swarm segregation. The species makeup of couples sampled from swarms also demonstrated assortative mating. The swarm site localization was close to human dwellings in the case of the An. gambiae coluzzii and on salt production sites for An. melas. At the peak of the rainy season, swarms of An. melas were absent. These findings offer evidence that the ecological speciation of these two sibling species of mosquitoes is associated with spatial swarm segregation and assortative mating, providing strong support for the hypothesis that mate recognition is currently maintaining adaptive differentiation and promoting ecological speciation. Further studies on the swarming and mating systems of An. gambiae, with the prospect of producing a predictive model of swarm distribution, are needed to inform any future efforts to implement strategies based on the use of GMM or SIT.

First attempt to validate human IgG antibody response to Nterm-34kDa salivary peptide as biomarker for evaluating exposure to Aedes aegypti bites.

Background Much effort is being devoted for developing new indicators to evaluate the human exposure to Aedes mosquito bites and the risk of arbovirus transmission. Human antibody (Ab) responses to mosquito salivary components could represent a promising tool for evaluating the human-vector contact. Methodology/Principal findings To develop a specific biomarker of human exposure to Aedes aegypti bites, we measured IgG Ab response to Ae. aegypti Nterm-34 kDa salivary peptide in exposed children in 7 villages of Southern Benin (West Africa). Results showed that specific IgG response presented high inter-individual heterogeneity between villages. IgG response was associated with rainfall and IgG level increased from dry (low exposure) to rainy (high exposure) seasons. These findings indicate that IgG Ab to Nterm-34 kDa salivary peptide may represent a reliable biomarker to detect variation in human exposure to Ae. aegypti bites. Conclusion/Significance This preliminary study highlights the potential use of Ab response to this salivary peptide for evaluating human exposure to Ae. aegypti. This biomarker could represent a new promising tool for assessing the risk of arbovirus transmission and for evaluating the efficacy of vector control interventions.

Spatio-temporal analysis of abundances of three malaria vector species in southern Benin using zero-truncated models

BackgroundA better understanding of the ecology and spatial-temporal distribution of malaria vectors is essential to design more effective and sustainable strategies for malaria control and elimination. In a previous study, we analyzed presence-absence data of An. funestus, An. coluzzii, and An. gambiae s.s. in an area of southern Benin with high coverage of vector control measures. Here, we further extend the work by analysing the positive values of the dataset to assess the determinants of the abundance of these three vectors and to produce predictive maps of vector abundance.MethodsPositive counts of the three vectors were assessed using negative-binomial zero-truncated (NBZT) mixed-effect models according to vector control measures and environmental covariates derived from field and remote sensing data. After 8-fold cross-validation of the models, predictive maps of abundance of the sympatric An. funestus, An. coluzzii, and An. gambiae s.s. were produced.ResultsCross-validation of the NBZT models showed a satisfactory predictive accuracy. Almost all changes in abundance between two surveys in the same village were well predicted by the models but abundances for An. gambiae s.s. were slightly underestimated. During the dry season, predictive maps showed that abundance greater than 1 bite per person per night were observed only for An. funestus and An. coluzzii. During the rainy season, we observed both increase and decrease in abundance of An. funestus, which are dependent on the ecological setting. Abundances of both An. coluzzii and An. gambiae s.s. increased during the rainy season but not in the same areas.ConclusionsOur models helped characterize the ecological preferences of three major African malaria vectors. This works highlighted the importance to study independently the binomial and the zero-truncated count processes when evaluating vector control strategies. The study of the bio-ecology of malaria vector species in time and space is critical for the implementation of timely and efficient vector control strategies.