A novel tetra-primer ARMS-PCR approach for the molecular karyotyping of chromosomal inversion 2Ru in the main malaria vectors Anopheles gambiae and Anopheles coluzzii.

BACKGROUND: Chromosomal inversion polymorphisms have been associated with adaptive behavioral, physiological, morphological and life history traits in the two main Afrotropical malaria vectors, Anopheles coluzzii and Anopheles gambiae. The understanding of the adaptive value of chromosomal inversion systems is constrained by the feasibility of cytological karyotyping. In recent years in silico and molecular approaches have been developed for the genotyping of most widespread inversions (2La, 2Rb and 2Rc). The 2Ru inversion, spanning roughly 8% of chromosome 2R, is commonly polymorphic in West African populations of An. coluzzii and An. gambiae and shows clear increases in frequency with increasing rainfall seasonally and geographically. The aim of this work was to overcome the constraints of currently available cytological and high-throughput molecular assays by developing a simple PCR assay for genotyping the 2Ru inversion in individual specimens of both mosquito species. METHODS: We designed tetra-primer amplification refractory mutation system (ARMS)-PCR assays based on five tag single-nucleotide polymorphisms (SNPs) previously shown to be strongly correlated with 2Ru inversion orientation. The most promising assay was validated against laboratory and field samples of An. coluzzii and An. gambiae karyotyped either cytogenetically or molecularly using a genotyping-in-thousands by sequencing (GT-seq) high-throughput approach that employs targeted sequencing of multiplexed PCR amplicons. RESULTS: A successful assay was designed based on the tag SNP at position 2R, 31710303, which is highly predictive of the 2Ru genotype. The assay, which requires only one PCR, and no additional post-PCR processing other than electrophoresis, produced a clear banding pattern for 98.5% of the 454 specimens tested, which is a 96.7% agreement with established karyotyping methods. Sequences were obtained for nine of the An. coluzzii specimens manifesting 2Ru genotype discrepancies with GT-seq. Possible sources of these discordances are discussed. CONCLUSIONS: The tetra-primer ARMS-PCR assay represents an accurate, streamlined and cost-effective method for the molecular karyotyping of the 2Ru inversion in An. coluzzii and An. gambiae. Together with approaches already available for the other common polymorphic inversions, 2La, 2Rb and 2Rc, this assay will allow investigations of the adaptive value of the complex set of inversion systems observed in the two major malaria vectors in the Afrotropical region.

Strengthening adult mosquito surveillance in Africa for disease control: learning from the present.

Mosquito surveillance is essential to successfully control and eliminate mosquito-borne diseases. Yet, it is often done by numerous organizations with little collaboration, incomplete understanding of existing gaps, and limited long-term vision. There is a clear disconnect between entomological and epidemiological indices, with entomological data informing control efforts inadequately. Here, we discuss current mosquito surveillance practises across the heterogeneous disease landscape in Africa. We advocate for the development of mosquito surveillance strategic plans to increase the impact and functionality of mosquito surveillance. We urge for a proactive approach to set up centralized mosquito data systems under the custodian of national governments, focus on epidemiologically relevant mosquito data, and increase the robustness of mosquito surveillance using a more spatially explicit sampling design.

Characterising the intensity of insecticide resistance: A novel framework for analysis of intensity bioassay data.

Insecticide resistance is a growing problem that risks harming the progress made by vector control tools in reducing the malaria burden globally. New methods for quantifying the extent of resistance in wild populations are urgently needed to guide deployment of interventions to improve disease control. Intensity bioassays measure mosquito mortality at a range of insecticide doses and characterise phenotypic resistance in regions where resistance is already detected. These data are increasingly being collected but tend to exhibit high measurement error and there is a lack of formal guidelines on how they should be analysed or compared. This paper introduces a novel Bayesian framework for analysing intensity bioassay data, which uses a flexible statistical model able to capture a wide variety of relationships between mortality and insecticide dose. By accounting for background mortality of mosquitoes, our approach minimises the impact of this source of measurement noise resulting in more precise quantification of resistance. It outputs a range of metrics for describing the intensity and variability in resistance within the sample and quantifies the level of measurement error in the assay. The functionality is illustrated with data from laboratory-reared mosquitoes to show how the lethal dose varies within and between different strains. The framework can also be used to formally test hypotheses by explicitly considering the high heterogeneity seen in these types of data in field samples. Here we show that the intensity of resistance (as measured by the median lethal dose (LC50) of insecticide) increases over 7 years in mosquitoes from one village in Burkina Faso but remains constant in another. This work showcases the benefits of statistically rigorous analysis of insecticide bioassay data and highlights the additional information available from this and other dose-response data.

Assessing the variability in experimental hut trials evaluating insecticide-treated nets against malaria vectors.

Experimental hut trials (EHTs) are used to evaluate indoor vector control interventions against malaria vectors in a controlled setting. The level of variability present in the assay will influence whether a given study is well powered to answer the research question being considered. We utilised disaggregated data from 15 previous EHTs to gain insight into the behaviour typically observed. Using simulations from generalised linear mixed models to obtain power estimates for EHTs, we show how factors such as the number of mosquitoes entering the huts each night and the magnitude of included random effects can influence study power. A wide variation in behaviour is observed in both the mean number of mosquitoes collected per hut per night (ranging from 1.6 to 32.5) and overdispersion in mosquito mortality. This variability in mortality is substantially greater than would be expected by chance and should be included in all statistical analyses to prevent false precision of results. We utilise both superiority and non-inferiority trials to illustrate our methodology, using mosquito mortality as the outcome of interest. The framework allows the measurement error of the assay to be reliably assessed and enables the identification of outlier results which could warrant further investigation. EHTs are increasingly playing an important role in the evaluation and regulation of indoor vector control interventions so it is important to ensure that these studies are adequately powered.

Inferring the epidemiological benefit of indoor vector control interventions against malaria from mosquito data.

The cause of malaria transmission has been known for over a century but it is still unclear whether entomological measures are sufficiently reliable to inform policy decisions in human health. Decision-making on the effectiveness of new insecticide-treated nets (ITNs) and the indoor residual spraying of insecticide (IRS) have been based on epidemiological data, typically collected in cluster-randomised control trials. The number of these trials that can be conducted is limited. Here we use a systematic review to highlight that efficacy estimates of the same intervention may vary substantially between trials. Analyses indicate that mosquito data collected in experimental hut trials can be used to parameterize mechanistic models for Plasmodium falciparum malaria and reliably predict the epidemiological efficacy of quick-acting, neuro-acting ITNs and IRS. Results suggest that for certain types of ITNs and IRS using this framework instead of clinical endpoints could support policy and expedite the widespread use of novel technologies.

Multiple insecticide resistance and first evidence of V410L kdr mutation in Aedes (Stegomyia) aegypti (Linnaeus) from Burkina Faso.

The response to recent dengue outbreaks in Burkina Faso was insecticide-based, despite poor knowledge of the vector population's susceptibility to the insecticides used. Here, we report on the susceptibility to the main insecticide classes and identify important underlying mechanisms in Aedes aegypti populations in Ouagadougou and Banfora, in 2019 and 2020. Wild Ae. aegypti were tested as adults in WHO bioassays and then screened in real time melting curve qPCR analyses to genotype the F1534C, V1016I, and V410L Aedes kdr mutations. Ae. aegypti showed moderate resistance to 0.1% bendiocarb (80-95% survival post-exposure), 0.8% Malathion (60-100%), 0.21% pirimiphos-methyl (75% - 97%), and high resistance to 0.03% deltamethrin (20-70%). PBO pre-exposure partially restored pyrethroid susceptibility. Genotyping detected high frequency of 1534C allele (0.92) and moderate 1016I (0.1-0.32). The V410L mutation was detected in Burkina Faso for the first time (frequency 0.1-0.36). Mosquitoes surviving 4 h exposure to 0.03% deltamethrin had significantly higher frequencies of the F1534C mutation than dead mosquitoes (0.70 vs. 0.96, p < 0.0001) and mosquitoes surviving 2 - 4 h exposure had a significantly reduced life span. Ae. aegypti from Burkina Faso are resistant to multiple insecticide classes with multiple mechanisms involved, demonstrating the essential role of insecticide resistance monitoring within national dengue control programmes.

Optimising the deployment of vector control tools against malaria: a data-informed modelling study.

BACKGROUND: Concern that insecticide resistant mosquitoes are threatening malaria control has driven the development of new types of insecticide treated nets (ITNs) and indoor residual spraying (IRS) of insecticide. Malaria control programmes have a choice of vector control interventions although it is unclear which controls should be used to combat the disease. The study aimed at producing a framework to easily compare the public health impact and cost-effectiveness of different malaria prevention measures currently in widespread use. METHODS: We used published data from experimental hut trials conducted across Africa to characterise the entomological effect of pyrethroid-only ITNs versus ITNs combining a pyrethroid insecticide with the synergist piperonyl butoxide (PBO). We use these estimates to parameterise a dynamic mathematical model of Plasmodium falciparum malaria which is validated for two sites by comparing simulated results to empirical data from randomised control trials (RCTs) in Tanzania and Uganda. We extrapolated model simulations for a series of potential scenarios likely across the sub-Saharan African region and include results in an online tool (Malaria INtervention Tool [MINT]) that aims to identify optimum vector control intervention packages for scenarios with varying budget, price, entomological and epidemiological factors. FINDINGS: Our model indicates that switching from pyrethroid-only to pyrethroid-PBO ITNs could averted up to twice as many cases, although the additional benefit is highly variable and depends on the setting conditions. We project that annual delivery of long-lasting, non-pyrethroid IRS would prevent substantially more cases over 3-years, while pyrethroid-PBO ITNs tend to be the most cost-effective intervention per case averted. The model was able to predict prevalence and efficacy against prevalence in both RCTs for the intervention types tested. MINT is applicable to regions of sub-Saharan Africa with endemic malaria and provides users with a method of designing intervention packages given their setting and budget. INTERPRETATION: The most cost-effective vector control package will vary locally. Models able to recreate results of RCTs can be used to extrapolate outcomes elsewhere to support evidence-based decision making for investment in vector control. FUNDING: Medical Research Council, IVCC, Wellcome Trust. TRANSLATION: For the French translation of the abstract see Supplementary Materials section.

Risk of Plasmodium falciparum infection in south-west Burkina Faso: potential impact of expanding eligibility for seasonal malaria chemoprevention.

Burkina Faso has one of the highest malaria burdens in sub-Saharan Africa despite the mass deployment of insecticide-treated nets (ITNs) and use of seasonal malaria chemoprevention (SMC) in children aged up to 5 years. Identification of risk factors for Plasmodium falciparum infection in rural Burkina Faso could help to identify and target malaria control measures. A cross-sectional survey of 1,199 children and adults was conducted during the peak malaria transmission season in the Cascades Region of south-west Burkina Faso in 2017. Logistic regression was used to identify risk factors for microscopically confirmed P. falciparum infection. A malaria transmission dynamic model was used to determine the impact on malaria cases averted of administering SMC to children aged 5-15 year old. P. falciparum prevalence was 32.8% in the study population. Children aged 5 to < 10 years old were at 3.74 times the odds (95% CI = 2.68-5.22, P < 0.001) and children aged 10 to 15 years old at 3.14 times the odds (95% CI = 1.20-8.21, P = 0.02) of P. falciparum infection compared to children aged less than 5 years old. Administration of SMC to children aged up to 10 years is predicted to avert an additional 57 malaria cases per 1000 population per year (9.4% reduction) and administration to children aged up to 15 years would avert an additional 89 malaria cases per 1000 population per year (14.6% reduction) in the Cascades Region, assuming current coverage of pyrethroid-piperonyl butoxide ITNs. Malaria infections were high in all age strata, although highest in children aged 5 to 15 years, despite roll out of core malaria control interventions. Given the burden of infection in school-age children, extension of the eligibility criteria for SMC could help reduce the burden of malaria in Burkina Faso and other countries in the region.

Design and methods for a quasi-experimental pilot study to evaluate the impact of dual active ingredient insecticide-treated nets on malaria burden in five regions in sub-Saharan Africa.

BACKGROUND: Vector control tools have contributed significantly to a reduction in malaria burden since 2000, primarily through insecticidal-treated bed nets (ITNs) and indoor residual spraying. In the face of increasing insecticide resistance in key malaria vector species, global progress in malaria control has stalled. Innovative tools, such as dual active ingredient (dual-AI) ITNs that are effective at killing insecticide-resistant mosquitoes have recently been introduced. However, large-scale uptake has been slow for several reasons, including higher costs and limited evidence on their incremental effectiveness and cost-effectiveness. The present report describes the design of several observational studies aimed to determine the effectiveness and cost-effectiveness of dual-AI ITNs, compared to standard pyrethroid-only ITNs, at reducing malaria transmission across a variety of transmission settings. METHODS: Observational pilot studies are ongoing in Burkina Faso, Mozambique, Nigeria, and Rwanda, leveraging dual-AI ITN rollouts nested within the 2019 and 2020 mass distribution campaigns in each country. Enhanced surveillance occurring in select study districts include annual cross-sectional surveys during peak transmission seasons, monthly entomological surveillance, passive case detection using routine health facility surveillance systems, and studies on human behaviour and ITN use patterns. Data will compare changes in malaria transmission and disease burden in districts receiving dual-AI ITNs to similar districts receiving standard pyrethroid-only ITNs over three years. The costs of net distribution will be calculated using the provider perspective including financial and economic costs, and a cost-effectiveness analysis will assess incremental cost-effectiveness ratios for Interceptor® G2, Royal Guard®, and piperonyl butoxide ITNs in comparison to standard pyrethroid-only ITNs, based on incidence rate ratios calculated from routine data. CONCLUSIONS: Evidence of the effectiveness and cost-effectiveness of the dual-AI ITNs from these pilot studies will complement evidence from two contemporary cluster randomized control trials, one in Benin and one in Tanzania, to provide key information to malaria control programmes, policymakers, and donors to help guide decision-making and planning for local malaria control and elimination strategies. Understanding the breadth of contexts where these dual-AI ITNs are most effective and collecting robust information on factors influencing comparative effectiveness could improve uptake and availability and help maximize their impact.

In vivo functional validation of the V402L voltage gated sodium channel mutation in the malaria vector An. gambiae.

BACKGROUND: Pyrethroids are the most widely used insecticides for the control of malaria transmitting Anopheles gambiae mosquitoes and rapid increase in resistance to this insecticide class is of major concern. Pyrethroids target the Voltage Gated Sodium Channels (VGSCs), that have a key role in the normal function of the mosquitoes' nervous system. VGSC mutations L995F and L995S have long been associated with pyrethroid resistance and screening for their presence is routine in insecticide resistance management programs. Recently, a VGSC haplotype containing two amino acid substitutions associated with resistance in other species, V402L and I1527T, was identified. These two VGSC mutations are found in tight linkage and are mutually exclusive to the classical L995F/S mutations. RESULTS: We identify the presence of the V402L-I1527T haplotype in resistant An. coluzzii colonized strains and in field populations from Burkina Faso, at frequencies higher than previously reported; in some cases almost reaching fixation. Functional validation of V402L in insecticide resistance using a CRISPR/Cas9 genome modified line showed that it confers reduced mortality after exposure to all tested pyrethroids and DDT, but at lower levels compared to L995F. In contrast to L995F however, no fitness costs were identified for mosquitoes carrying V402L under laboratory conditions. CONCLUSION: The V402L substitution confers pyrethroid resistance in An. gambiae in the absence of any other VGSC substitution and/or alternative resistance mechanisms. The lower fitness cost associated with this kdr mutation may provide a selective advantage over the classical kdr in some settings and genotyping at this locus should be added in the list of resistant alleles for routine screening.

Risk factors associated with house entry of malaria vectors in an area of Burkina Faso with high, persistent malaria transmission and high insecticide resistance.

BACKGROUND: In rural Burkina Faso, the primary malaria vector Anopheles gambiae sensu lato (s.l.) primarily feeds indoors at night. Identification of factors which influence mosquito house entry could lead to development of novel malaria vector control interventions. A study was therefore carried out to identify risk factors associated with house entry of An. gambiae s.l. in south-west Burkina Faso, an area of high insecticide resistance. METHODS: Mosquitoes were sampled monthly during the malaria transmission season using CDC light traps in 252 houses from 10 villages, each house sleeping at least one child aged five to 15 years old. Potential risk factors for house entry of An. gambiae s.l. were measured, including socio-economic status, caregiver's education and occupation, number of people sleeping in the same part of the house as the child, use of anti-mosquito measures, house construction and fittings, proximity of anopheline aquatic habitats and presence of animals near the house. Mosquito counts were compared using a generalized linear mixed-effect model with negative binomial and log link function, adjusting for repeated collections. RESULTS: 20,929 mosquitoes were caught, of which 16,270 (77.7%) were An. gambiae s.l. Of the 6691 An. gambiae s.l. identified to species, 4101 (61.3%) were An. gambiae sensu stricto and 2590 (38.7%) Anopheles coluzzii. Having a metal-roof on the child's sleeping space (IRR = 0.55, 95% CI 0.32-0.95, p = 0.03) was associated with fewer malaria vectors inside the home. CONCLUSION: This study demonstrated that the rate of An. gambiae s.l. was 45% lower in sleeping spaces with a metal roof, compared to those with thatch roofs. Improvements in house construction, including installation of metal roofs, should be considered in endemic areas of Africa to reduce the burden of malaria.

Insecticide resistance and behavioural adaptation as a response to long-lasting insecticidal net deployment in malaria vectors in the Cascades region of Burkina Faso.

The decline in malaria across Africa has been largely attributed to vector control using long-lasting insecticidal nets (LLINs). However, this intervention has prompted widespread insecticide resistance (IR) and been associated with changes in mosquito behaviour that reduce their contact with LLINs. The relative importance and rate at which IR and behavioural adaptations emerge are poorly understood. We conducted surveillance of mosquito behaviour and IR at 12 sites in Burkina Faso to assess the magnitude and temporal dynamics of insecticide, biting and resting behaviours in vectors in the 2-year period following mass LLIN distribution. Insecticide resistance was present in all vector populations and increased rapidly over the study period. In contrast, no longitudinal shifts in LLIN-avoidance behaviours (earlier or outdoor biting and resting) were detected. There was a moderate but statistically significant shift in vector species composition from Anopheles coluzzii to Anopheles gambiae which coincided with a reduction in the proportion of bites preventable by LLINs; possibly driven by between-species variation in behaviour. These findings indicate that adaptations based on insecticide resistance arise and intensify more rapidly than behavioural shifts within mosquito vectors. However, longitudinal shifts in mosquito vector species composition were evident within 2 years following a mass LLIN distribution. This ecological shift was characterized by a significant increase in the exophagic species (An. gambiae) and coincided with a predicted decline in the degree of protection expected from LLINs. Although human exposure fell through the study period due to reducing vector densities and infection rates, such ecological shifts in vector species along with insecticide resistance were likely to have eroded the efficacy of LLINs. While both adaptations impact malaria control, the rapid increase of the former indicates this strategy develops more quickly in response to selection from LLINS. However, interventions targeting both resistance strategies will be needed.

Laboratory and field evaluation of MAÏA®, an ointment containing N,N-diethyl-3-methylbenzamide (DEET) against mosquitoes in Burkina Faso.

BACKGROUND: Malaria vector control relies upon the use of insecticide-treated nets and indoor residual spraying. However, as the emergency of insecticide resistance in malaria vectors grows, the effectiveness of these measures could be limited. Alternative tools are needed. In this context, repellents can play an important role against exophagic and exophilic mosquitoes. This study evaluated the efficacy of MAÏA®, a novel repellent ointment, in laboratory and field conditions in Burkina Faso. METHODS: For laboratory and field assessment, 20 volunteers were enrolled and trained for nocturnal collection of mosquitoes using human landing catches (HLC). In the laboratory tests, 2 mg/sq cm of treatment (either MAIA® or 20 % DEET) were used to assess median complete protection time (CPT) against two species: Anopheles gambiae and Aedes aegypti, following WHO guidelines. For both species, two strains consisting of susceptible and local strains were used. The susceptible strains were Kisumu and Bora Bora for An. gambiae and Ae. aegypti, respectively. For the field test, the median CPT of MAÏA® was compared to that of a negative (70 % ethanol) and positive (20 % DEET) after carrying out HLCs in rural Burkina Faso in both indoor and outdoor settings. RESULTS: Laboratory tests showed median Kaplan-Meier CPT of 6 h 30 min for An. gambiae (Kisumu), 5 h 30 min for An. gambiae (Goden, local strain), and 4 h for Ae. aegypti for both the local and sensitive strain. These laboratory results suggest that MAÏA® is a good repellent against the three mosquito species. During these field tests, a total of 3979 mosquitoes were caught. In this population, anophelines represented 98.5 %, with culicines (Aedes) making up the remaining 1.5 %. Among anopheline mosquitoes, 95 % belonged to the An. gambiae complex, followed by Anopheles funestus and Anopheles pharoensis. The median CPT of 20 % DEET and MAÏA® were similar (8 h) and much longer than that of the negative control (2 h). CONCLUSIONS: Results from the present studies showed that MAÏA® offers high protection against anophelines biting indoors and outdoors and could play an important role in malaria prevention in Africa.

Quantifying individual variability in exposure risk to mosquito bites in the Cascades region, Burkina Faso.

BACKGROUND: The Cascades region, Burkina Faso, has a high malaria burden despite reported high insecticide-treated mosquito net (ITN) use. Human and vector activities outside the hours when indoor interventions offer direct protection from infectious bites potentially increase exposure risk to bites from malaria-transmitting Anopheles mosquitoes. This work investigated the degree of variation in human behaviour both between individuals and through time (season) to quantify how it impacts exposure to malaria vectors. METHODS: Patterns in human overnight activity (18:00-06:00) to quantify time spent using an ITN across 7 successive nights in two rural communities, Niakore (N = 24 participants) and Toma (71 participants), were observed in the dry and rainy seasons, between 2017 and 2018. Hourly human landing Anopheles mosquito catches were conducted in Niakore specifically, and Cascades region generally, between 2016 and 2017. Data were statistically combined to estimate seasonal variation in time spent outdoors and Anopheles bites received per person per night (bpppn). RESULTS: Substantial variability in exposure to outdoor Anopheles bites was detected within and between communities across seasons. In October, when Anopheles densities are highest, an individual's risk of Anopheles bites ranged from 2.2 to 52.2 bites per person per night (bpppn) within the same week with variable risk dependent on hours spent indoors. Comparably higher outdoor human activity was observed in April and July but, due to lower Anopheles densities estimated, bpppn were 0.2-4.7 and 0.5-32.0, respectively. Males and people aged over 21 years were predicted to receive more bites in both sentinel villages. CONCLUSION: This work presents one of the first clear descriptions of the degree of heterogeneity in time spent outdoors between people and across the year. Appreciation of sociodemographic, cultural and entomological activities will help refine approaches to vector control.

A cohort study to identify risk factors for Plasmodium falciparum infection in Burkinabe children: implications for other high burden high impact countries.

BACKGROUND: Progress in controlling malaria has stalled in recent years. Today the malaria burden is increasingly concentrated in a few countries, including Burkina Faso, where malaria is not declining. A cohort study was conducted to identify risk factors for malaria infection in children in southwest Burkina Faso, an area with high insecticide-treated net (ITN) coverage and insecticide-resistant vectors. METHODS: Incidence of Plasmodium falciparum infection was measured in 252 children aged 5 to 15 years, using active and passive detection, during the 2017 transmission season, following clearance of infection. Demographic, socio-economic, environmental, and entomological risk factors, including use of ITNs and insecticide resistance were monitored. RESULTS: During the six-month follow-up period, the overall incidence of P. falciparum infection was 2.78 episodes per child (95% CI = 2.66-2.91) by microscopy, and 3.11 (95% CI = 2.95-3.28) by polymerase chain reaction (PCR). The entomological inoculation rate (EIR) was 80.4 infective bites per child over the six-month malaria transmission season. At baseline, 80.6% of children were reported as sleeping under an ITN the previous night, although at the last survey, 23.3% of nets were in poor condition and considered no longer protective. No association was found between the rate of P. falciparum infection and either EIR (incidence rate ratio (IRR): 1.00, 95% CI: 1.00-1.00, p = 0.08) or mortality in WHO tube tests when vectors were exposed to 0.05% deltamethrin (IRR: 1.05, 95% CI: 0.73-1.50, p = 0.79). Travel history (IRR: 1.52, 95% CI: 1.45-1.59, p < 0.001) and higher socio-economic status were associated with an increased risk of P. falciparum infection (IRR: 1.05, 95% CI: 1.00-1.11, p = 0.04). CONCLUSIONS: Incidence of P. falciparum infection remains overwhelmingly high in the study area. The study findings suggest that because of the exceptionally high levels of malaria transmission in the study area, malaria elimination cannot be achieved solely by mass deployment of ITNs and additional control measures are needed.

Evaluation of mosquito electrocuting traps as a safe alternative to the human landing catch for measuring human exposure to malaria vectors in Burkina Faso.

BACKGROUND: Measuring human exposure to mosquito bites is a crucial component of vector-borne disease surveillance. For malaria vectors, the human landing catch (HLC) remains the gold standard for direct estimation of exposure. This method, however, is controversial since participants risk exposure to potentially infected mosquito bites. Recently an exposure-free mosquito electrocuting trap (MET) was developed to provide a safer alternative to the HLC. Early prototypes of the MET performed well in Tanzania but have yet to be tested in West Africa, where malaria vector species composition, ecology and behaviour are different. The performance of the MET relative to HLC for characterizing mosquito vector population dynamics and biting behaviour in Burkina Faso was evaluated. METHODS: A longitudinal study was initiated within 12 villages in Burkina Faso in October 2016. Host-seeking mosquitoes were sampled monthly using HLC and MET collections over 14 months. Collections were made at 4 households on each night, with METs deployed inside and outside at 2 houses, and HLC inside and outside at another two. Malaria vector abundance, species composition, sporozoite rate and location of biting (indoor versus outdoor) were recorded. RESULTS: In total, 41,800 mosquitoes were collected over 324 sampling nights, with the major malaria vector being Anopheles gambiae sensu lato (s.l.) complex. Overall the MET caught fewer An. gambiae s.l. than the HLC (mean predicted number of 0.78 versus 1.82 indoors, and 1.05 versus 2.04 outdoors). However, MET collections gave a consistent representation of seasonal dynamics in vector populations, species composition, biting behaviour (location and time) and malaria infection rates relative to HLC. As the relative performance of the MET was somewhat higher in outdoor versus indoor settings, this trapping method slightly underestimated the proportion of bites preventable by LLINs compared to the HLC (MET = 82.08%; HLC = 87.19%). CONCLUSIONS: The MET collected proportionately fewer mosquitoes than the HLC. However, estimates of An. gambiae s.l. density in METs were highly correlated with HLC. Thus, although less sensitive, the MET is a safer alternative than the HLC. Its use is recommended particularly for sampling vectors in outdoor environments where it is most sensitive.

Anopheline species composition and the 1014F-genotype in different ecological settings of Burkina Faso in relation to malaria transmission.

BACKGROUND: A three-year longitudinal study was conducted in four sentinel sites from different ecological settings in Burkina Faso, between 2008 and 2010 to identify longitudinal changes in insecticide resistance within Anopheles gambiae complex species based on larval collection. During this study, adult mosquitoes were also collected indoor and outdoor using several methods of collection. The present study reports the diversity of malaria vectors and the 1014F-genotype from this adult collection and investigates the association between this 1014F-genotype and sporozoite rate. METHODS: Adult mosquitoes were collected from July to August (corresponding to the start of rainy season) and October to November (corresponding to the end of rainy season) over 3 years (2008-2010) at four sites across the country, using pyrethrum spray catches (PSC), exit traps and pit shelters. Anopheles gambiae complex mosquitoes were identified to species and genotyped for the L1014F kdr mutation by PCR using genomic DNA. The circumsporozoite antigen of Plasmodium falciparum was detected in mosquitoes using sandwich ELISA. RESULTS: Overall 9212 anopheline mosquitoes were collected during the study period. Of those, 6767 mosquitoes were identified as Anopheles gambiae sensu lato (s.l.). Anopheles arabiensis, Anopheles coluzzii, Anopheles gambiae and or Anopheles funestus were incriminated as vectors of P. falciparum in the study area with an average sporozoite rate of 5%, (95% CI 4.14-5.99%). The kdr1014F-genotype frequencies were 11.44% (95% CI 2.5-39.85%), 19.2% (95% CI 4.53-53.73%) and 89.9 (95% CI 63.14-97.45%), respectively for An. arabiensis, An. coluzzii and An. gambiae. The proportion of the 1014F-genotype varied between sporozoite-infected and uninfected An. gambiae s.l. group. There was no significant difference in the 1014F-genotype frequency between infected and uninfected mosquitoes. CONCLUSION: The current study shows the diversity of malaria vectors and significant interaction between species composition and kdr1014F-genotype in An. gambiae complex mosquitoes from Burkina Faso. In this study, no associations were found between the 1014F-genotype and P. falciparum infection in the major malaria vector An. gambiae s.l.

Molecular Characterization of Diarrheagenic Escherichia Coli in Children Less Than 5 Years of Age with Diarrhea in Ouagadougou, Burkina Faso.

Diarrheagenic Escherichia coli (DEC) is important bacteria of children's endemic and epidemic diarrhea worldwide. The aim of this study was to determine the prevalence of DEC isolated from stool samples collected from children with acute diarrhea living in Ouagadougou, Burkina Faso. From August 2013 to October 2015, stool samples were collected from 315 children under 5 years of age suffering from diarrhea in the "Centre Médical avec Antenne Chirurgicale (CMA)" Paul VI and the CMA of Schiphra. E. coli were isolated and identified by standard microbiological methods, and the 16-plex PCR method was used to further characterize them. Four hundred and nineteen (419) E. coli strains were characterized, of which 31 (7.4%) DEC pathotypes were identified and classified in five E. coli pathotypes: 15 enteroaggregative E. coli (EAEC) (48.4%), 8 enteropathogenic E. coli (EPEC) (25.8%) with 4 typical EPEC and 4 atypical EPEC, 4 enteroinvasive E. coli (EIEC) (12.9%), 3 enterohemorrhagic E. coli (EHEC) 9.67%, and 1 enterotoxigenic E. coli (ETEC) 3.2%. The use of multiplex PCR as a routine in clinical laboratory for the detection of DEC would be a useful mean for a rapid management of an acute diarrhea in children.

The Sticky Resting Box, a new tool for studying resting behaviour of Afrotropical malaria vectors.

BACKGROUND: Monitoring densities of adult mosquito populations is a major challenge in efforts to evaluate the epidemiology of mosquito-borne diseases, and their response to vector control interventions. In the case of malaria, collection of outdoor-resting Anophelines is rarely incorporated into surveillance and control, partially due to the lack of standardized collection tools. Such an approach, however, is increasingly important to investigate possible changes in mosquito behaviour in response to the scale up of Insecticide Treated Nets and Indoor Residual Spraying. In this study we evaluated the Sticky Resting Box (SRB) - i.e. a sticky variant of previously investigated mosquito Resting Box, which allows passive collection of mosquitoes entering the box - and compared its performance against traditional methods for indoor and outdoor resting mosquito sampling. METHODS: Daily collections were carried out in two neighbouring villages of Burkina Faso during rainy season 2011 and dry season 2012 by SRB located indoors and outdoors, and by Back-Pack aspiration inside houses (BP) and in ad hoc built outdoor pit-shelters (PIT). RESULTS: Overall, almost 20,000 Culicidae specimens belonging to 16 species were collected and morphologically identified. Malaria vectors included Anopheles coluzzii (53%), An. arabiensis (12%), An. gambiae s.s. (2.0%) and An. funestus (4.5%). The diversity of species collected in the two villages was similar for SRB and PIT collections outdoors, and significantly higher for SRB than for BP indoors. The population dynamics of An. gambiae s.l. mosquitoes, as obtained by SRB-collections was significantly correlated with those obtained by the traditional methods. The predicted mean estimates of An. gambiae s.l. specimens/sampling-unit/night-of-collections was 6- and 5-times lower for SRB than for BP indoors and PIT outdoors, respectively. CONCLUSIONS: Overall, the daily performance of SRB in terms of number of malaria vectors/trap was lower than that of traditionally used approaches for in- and outdoor collections. However, unlike these methods, SRB could be set up to collect mosquitoes passively over at least a week. This makes SRB a promising tool for passively monitoring anopheline resting populations, with data presented here providing guidance for how to set up SRB-based collections to acquire information comparable to those obtained with other methods.

Equivalent susceptibility of Anopheles gambiae M and S molecular forms and Anopheles arabiensis to Plasmodium falciparum infection in Burkina Faso.

BACKGROUND: The Anopheles gambiae sensu lato (s.l.) species complex in Burkina Faso consists of Anopheles arabiensis, and molecular forms M and S of Anopheles gambiae sensu stricto (s.s.). Previous studies comparing the M and S forms for level of infection with Plasmodium falciparum have yielded conflicting results. METHODS: Mosquito larvae were sampled from natural pools, reared to adulthood under controlled conditions, and challenged with natural P. falciparum by experimental feeding with blood from gametocyte carriers. Oocyst infection prevalence and intensity was determined one week after infection. DNA from carcasses was genotyped to identify species and molecular form. RESULTS: In total, 7,400 adult mosquitoes grown from wild-caught larvae were challenged with gametocytes in 29 experimental infections spanning four transmission seasons. The overall infection prevalence averaged 40.7% for A. gambiae M form, 41.4% for A. gambiae S form, and 40.1% for A. arabiensis. There was no significant difference in infection prevalence or intensity between the three population groups. Notably, infection experiments in which the population groups were challenged in parallel on the same infective blood displayed less infection difference between population groups, while infections with less balanced composition of population groups had lower statistical power and displayed apparent differences that fluctuated more often from the null average. CONCLUSION: The study clearly establishes that, at the study site in Burkina Faso, there is no difference in genetic susceptibility to P. falciparum infection between three sympatric population groups of the A. gambiae s.l. complex. Feeding the mosquito groups on the same infective blood meal greatly increases statistical power. Conversely, comparison of the different mosquito groups between, rather than within, infections yields larger apparent difference between mosquito groups, resulting from lower statistical power and greater noise, and could lead to false-positive results. In making infection comparisons between population groups, it is more accurate to compare the different groups after feeding simultaneously upon the same infective blood.