Efficacy of chlorfenapyr-pyrethroid and piperonyl butoxide-pyrethroid long-lasting insecticidal nets (LLINs) compared to pyrethroid-only LLINs for malaria control in Côte d'Ivoire: a three group, cluster randomised trial.

BACKGROUND: The massive scale-up of long-lasting insecticidal nets (LLIN) has led to a major reduction in malaria burden in many sub-Saharan African (SSA) countries. The World Health Organization (WHO) has recently issued a strong recommendation for the use of chlorfenapyr-pyrethroid LLINs compared to standard pyrethroid-only LLINs in areas of high insecticide resistance intensity. However, there is still a lack of conclusive evidence on the efficacy of piperonyl butoxide-pyrethroid (PBO-py) LLINs, especially in West Africa, where vector composition and resistance mechanisms may be different from vectors in East Africa. METHODS: This is a three-arm, superiority, triple-blinded, cluster randomised trial, with village as the unit of randomisation. This study conducted in Côte d'Ivoire will evaluate the efficacy on epidemiological and entomological outcomes of (1) the control arm: MAGNet® LN, which contains the pyrethroid, alpha-cypermethrin, (2) VEERALIN® LN, a net combining the synergist PBO and alpha-cypermethrin, and (3) Interceptor® G2 LN, which incorporates chlorfenapyr and alpha-cypermethrin, two adulticides with different mechanisms of action. A total of 33 villages with an average of 200 households per village will be identified, mapped, and randomised in a ratio of 1:1:1. Nets will be distributed at a central point following national guidelines with 1 net for every 2 people. The primary outcome of the trial will be incidence of malaria cases (confirmed by rapid diagnostic test (RDT)) in a cohort of 50 children aged 6 months to 10 years in each cluster, followed for 12 months (active case detection). Secondary outcomes are cross-sectional community prevalence of malaria infection (confirmed by RDT) in the study population at 6 and 12 months post-intervention (50 randomly selected persons per cluster), vector density, entomological inoculation rate (EIR), and phenotypic and genotypic insecticide resistance at baseline and 12 months post-intervention in 3 sentinel villages in each treatment arm. DISCUSSION: In addition to generating further evidence for next-generation LLINs, this study will also provide the first evidence for pyrethroid-PBO nets in a West African setting. This could further inform WHO recommendations on the pragmatic use of pyrethroid-PBO nets. TRIAL REGISTRATION: ClinicalTrials.gov NCT05796193. Registered on April 3, 2023.

Despite structural identity, ace-1 heterogenous duplication resistance alleles are quite diverse in Anopheles mosquitoes.

Anopheles gambiae s.l. has been the target of intense insecticide treatment since the mid-20th century to try and control malaria. A substitution in the ace-1 locus has been rapidly selected for, allowing resistance to organophosphate and carbamate insecticides. Since then, two types of duplication of the ace-1 locus have been found in An. gambiae s.l. populations: homogeneous duplications that are composed of several resistance copies, or heterogeneous duplications that contain both resistance and susceptible copies. The substitution induces a trade-off between resistance in the presence of insecticides and disadvantages in their absence: the heterogeneous duplications allow the fixation of the intermediate heterozygote phenotype. So far, a single heterogeneous duplication has been described in An. gambiae s.l. populations (in contrast with the multiple duplicated alleles found in Culex pipiens mosquitoes). We used a new approach, combining long and short-read sequencing with Sanger sequencing to precisely identify and describe at least nine different heterogeneous duplications, in two populations of An. gambiae s.l. We show that these alleles share the same structure as the previously identified heterogeneous and homogeneous duplications, namely 203-kb tandem amplifications with conserved breakpoints. Our study sheds new light on the origin and maintenance of these alleles in An. gambiae s.l. populations, and their role in mosquito adaptation.

Global malaria predictors at a localized scale.

Malaria is a life-threatening disease caused by Plasmodium parasites transmitted by Anopheles mosquitoes. In 2021, more than 247 million cases of malaria were reported worldwide, with an estimated 619,000 deaths. While malaria incidence has decreased globally in recent decades, some public health gains have plateaued, and many endemic hotspots still face high transmission rates. Understanding local drivers of malaria transmission is crucial but challenging due to the complex interactions between climate, entomological and human variables, and land use. This study focuses on highly climatically suitable and endemic areas in Côte d'Ivoire to assess the explanatory power of coarse climatic predictors of malaria transmission at a fine scale. Using data from 40 villages participating in a randomized controlled trial of a household malaria intervention, the study examines the effects of climate variation over time on malaria transmission. Through panel regressions and statistical modeling, the study investigates which variable (temperature, precipitation, or entomological inoculation rate) and its form (linear or unimodal) best explains seasonal malaria transmission and the factors predicting spatial variation in transmission. The results highlight the importance of temperature and rainfall, with quadratic temperature and all precipitation models performing well, but the causal influence of each driver remains unclear due to their strong correlation. Further, an independent, mechanistic temperature-dependent R 0 model based on laboratory data aligns well with observed malaria incidence rates, emphasizing the significance and predictability of temperature suitability across scales. By contrast, entomological variables, such as entomological inoculation rate, were not strong predictors of human incidence in this context. Finally, the study explores the predictors of spatial variation in malaria, considering land use, intervention, and entomological variables. The findings contribute to a better understanding of malaria transmission dynamics at local scales, aiding in the development of effective control strategies in endemic regions.

EaveTubes for control of vector-borne diseases in Côte d'Ivoire: study protocol for a cluster randomized controlled trial.

BACKGROUND: Vector control tools, long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS), have significantly contributed to malaria prevention efforts in sub-Saharan Africa. However, insecticide resistance has seriously hampered their efficacy in recent years and new tools are essential to further progress. In2Care® EaveTubes (ETs) are an inexpensive, new resistance-breaking vector control product under World Health Organization (WHO) evaluation informed by mosquito ecology to efficiently target malaria vectors. By installing ETs in the walls of the house at the eave level that funnel the natural airflow, mosquitoes are drawn in by the same heat and odor cues that typically attract them through open eaves. Once inside an ET, mosquitoes are exposed to insecticide-treated netting placed inside the ET. The aim of this study is to test whether ETs as stand-alone tool have an effect on the epidemiology of malaria in villages where houses have been modified with the ET intervention. METHODS: A two-armed, cluster randomized controlled trial will be conducted to evaluate the effect of ETs on clinical malaria incidence in children living in Côte d'Ivoire. Thirty-four villages will be selected based on population size and the proportion of houses suitable for modification with ETs (17 treatment arms (ETs + LLINs, 17 control arms (LLINs only)). Based on the population census, 55 households per cluster with eligible children (i.e., between the ages of 6 months to 8 years old at the start of the study) will be randomly selected for recruitment into the active detection cohorts. In the treatment arm, we will enroll eligible children who reside in ET-treated houses. The intervention and control cohorts will be followed for 4 months for baseline covariate measurements and 24 months with intervention. During case detection visits, blood samples will be taken from all febrile children and tested for malaria infection with rapid diagnostic tests (RDTs). All positive clinical malaria infections will be treated. To estimate the impact of the ET on malaria vector densities, entomological measurements (indoor sampling with CDC traps) will be conducted monthly in 20 clusters (10 ET, 10 Control) in 10 randomly selected households per cluster. To estimate the infectiousness of malaria vectors, sporozoite rates will be measured in subsets of the collected mosquito samples. DISCUSSION: Findings will serve as an efficacy trial of ETs and will be submitted to the WHO Vector Control Advisory Group (VCAG) for assessment of public health value. Entomological outcomes will also be measured as proxies of malaria transmission to help develop guidelines for the evaluation of future In2Care® ETs products. TRIAL REGISTRATION: ClinicalTrials.gov NCT05736679. Registered on 10 February 2023.

Attractive targeted sugar bait: the pyrrole insecticide chlorfenapyr and the anti-malarial pharmaceutical artemether-lumefantrine arrest Plasmodium falciparum development inside wild pyrethroid-resistant Anopheles gambiae s.s. mosquitoes.

BACKGROUND: Attractive targeted sugar bait (ATSB) is a novel approach to vector control, offering an alternative mode of insecticide delivery via the insect alimentary canal, with potential to deliver a variety of compounds new to medical entomology and malaria control. Its potential to control mosquitoes was recently demonstrated in major field trials in Africa. The pyrrole chlorfenapyr is an insecticide new to malaria vector control, and through its unique mode of action-disruption of ATP mediated energy transfer in mitochondria-it may have direct action on energy transfer in the flight muscle cells of mosquitoes. It may also have potential to disrupt mitochondrial function in malarial parasites co-existing within the infected mosquito. However, little is known about the impact of such compounds on vector competence in mosquitoes responsible for malaria transmission. METHODS: In this study, ATSBs containing chlorfenapyr insecticide and, as a positive control, the anti-malarial drugs artemether/lumefantrine (A/L) were compared for their effect on Plasmodium falciparum development in wild pyrethroid-resistant Anopheles gambiae sensu stricto (s.s.) and for their capacity to reduce vector competence. Female mosquitoes were exposed to ATSB containing either sublethal dose of chlorfenapyr (CFP: 0.025%) or concentrations of A/L ranging from 0.4/2.4 mg/ml to 2.4/14.4 mg/ml, either shortly before or after taking infective blood meals. The impact of their component compounds on the prevalence and intensity of P. falciparum infection were compared between treatments. RESULTS: Both the prevalence and intensity of infection were significantly reduced in mosquitoes exposed to either A/L or chlorfenapyr, compared to unexposed negative control mosquitoes. The A/L dose (2.4/14.4 mg/ml) totally erased P. falciparum parasites: 0% prevalence of infection in female mosquitoes exposed compared to 62% of infection in negative controls (df = 1, χ2 = 31.23 p < 0.001). The dose of chlorfenapyr (0.025%) that killed < 20% females in ATSB showed a reduction in oocyte density of 95% per midgut (0.18/3.43 per midgut). CONCLUSION: These results are evidence that chlorfenapyr, in addition to its direct killing effect on the vector, has the capacity to block Plasmodium transmission by interfering with oocyte development inside pyrethroid-resistant mosquitoes, and through this dual action may potentiate its impact under field conditions.

Efficacy of a 'lethal house lure' against Culex quinquefasciatus from Bouaké city, Côte d'Ivoire.

BACKGROUND: Eave tube technology is a novel method of insecticide application that uses an electrostatic coating system to boost insecticide efficacy against resistant mosquitoes. A series of previous experiments showed encouraging insecticidal effects against malaria vectors. This study was undertaken to assess the effects of the eave tube approach on other Culicidae, in particular Culex quinquefasciatus, under laboratory and semi-field conditions. METHODS: Larvae of Cx. quinquefasciatus from Bouaké were collected and reared to adult stage, and World Health Organization (WHO) cylinder tests were performed to determine their resistance status. WHO standard 3-min cone bioassays were conducted using PermaNet 2.0 netting versus eave tube-treated inserts. To assess the transient exposure effect on Cx. quinquefasciatus, eave tube assay utilizing smelly socks as attractant was performed with exposure time of 30 s, 1 min, and 2 min on 10% beta-cyfluthrin-treated inserts. Residual activity of these treated inserts was then monitored over 9 months. Field tests involving release-recapture of Cx. quinquefasciatus within enclosures around experimental huts fitted with windows and untreated or insecticide-treated eave tubes were conducted to determine house entry preference and the impact of tubes on the survival of this species. RESULTS: Bouaké Cx. quinquefasciatus displayed high resistance to three out of four classes of insecticides currently used in public health. After 3 min of exposure in cone tests, 10% beta-cyfluthrin-treated inserts induced 100% mortality in Cx. quinquefasciatus, whereas the long-lasting insecticidal net (LLIN) only killed 4.5%. With reduced exposure time on the eave tube insert, mortality was still 100% after 2 min, 88% after 1 min, and 44% after 30 s. Mortality following 1 h exposure on 10% beta-cyfluthrin-treated insert was > 80% continuously up to 7 months post-treatment. Data suggest that Cx. quinquefasciatus have a stronger preference for entering a house through the eaves than through windows. Beta-cyfluthrin-treated inserts were able to kill 51% of resistant Cx. quinquefasciatus released within the enclosure. CONCLUSIONS: Eave tubes are a novel method for delivery of insecticide to the house. They attract nuisance host-seeking Cx. quinquefasciatus mosquitoes and are as effective in controlling them as they are against pyrethroid-resistant Anopheles gambiae, despite the high level of resistance Cx. quinquefasciatus have developed.

Anopheles sampling collections in the health districts of Korhogo (Côte d'Ivoire) and Diébougou (Burkina Faso) between 2016 and 2018.

Characterizing the entomological profile of malaria transmission at fine spatiotemporal scales is essential for developing and implementing effective vector control strategies. Here, we present a fine-grained dataset of Anopheles mosquitoes (Diptera: Culicidae) collected in 55 villages of the rural districts of Korhogo (Northern Côte d'Ivoire) and Diébougou (South-West Burkina Faso) between 2016 and 2018. In the framework of a randomized controlled trial, Anopheles mosquitoes were periodically collected by Human Landing Catches experts inside and outside households, and analyzed individually to identify the genus and, for a subsample, species, insecticide resistance genetic mutations, Plasmodium falciparum infection, and parity status. More than 3,000 collection sessions were carried out, achieving about 45,000 h of sampling efforts. Over 60,000 Anopheles were collected (mainly A. gambiae s.s., A. coluzzii, and A. funestus). The dataset is published as a Darwin Core archive in the Global Biodiversity Information Facility, comprising four files: events, occurrences, mosquito characterizations, and environmental data.

Anopheles vector distribution and malaria transmission dynamics in Gbêkê region, central Côte d'Ivoire.

BACKGROUND: A better understanding of vector distribution and malaria transmission dynamics at a local scale is essential for implementing and evaluating effectiveness of vector control strategies. Through the data gathered in the framework of a cluster randomized controlled trial (CRT) evaluating the In2Care (Wageningen, Netherlands) Eave Tubes strategy, the distribution of the Anopheles vector, their biting behaviour and malaria transmission dynamics were investigated in Gbêkê region, central Côte d'Ivoire. METHODS: From May 2017 to April 2019, adult mosquitoes were collected monthly using human landing catches (HLC) in twenty villages in Gbêkê region. Mosquito species wereidentified morphologically. Monthly entomological inoculation rates (EIR) were estimated by combining the HLC data with mosquito sporozoite infection rates measured in a subset of Anopheles vectors using PCR. Finally, biting rate and EIR fluctuations were fit to local rainfall data to investigate the seasonal determinants of mosquito abundance and malaria transmission in this region. RESULTS: Overall, Anopheles gambiae, Anopheles funestus, and Anopheles nili were the three vector complexes found infected in the Gbêkê region, but there was a variation in Anopheles vector composition between villages. Anopheles gambiae was the predominant malaria vector responsible for 84.8% of Plasmodium parasite transmission in the area. An unprotected individual living in Gbêkê region received an average of 260 [222-298], 43.5 [35.8-51.29] and 3.02 [1.96-4] infected bites per year from An. gambiae, An. funestus and An. nili, respectively. Vector abundance and malaria transmission dynamics varied significantly between seasons and the highest biting rate and EIRs occurred in the months of heavy rainfall. However, mosquitoes infected with malaria parasites remained present in the dry season, despite the low density of mosquito populations. CONCLUSION: These results demonstrate that the intensity of malaria transmission is extremely high in Gbêkê region, especially during the rainy season. The study highlights the risk factors of transmission that could negatively impact current interventions that target indoor control, as well as the urgent need for additional vector control tools to target the population of malaria vectors in Gbêkê region and reduce the burden of the disease.

Housing modification for malaria control: impact of a "lethal house lure" intervention on malaria infection prevalence in a cluster randomised control trial in Côte d'Ivoire.

BACKGROUND: In recent years, the downward trajectory of malaria transmission has slowed and, in some places, reversed. New tools are needed to further reduce malaria transmission. One approach that has received recent attention is a novel house-based intervention comprising window screening (S) and general house repairs to make the house more mosquito proof, together with EaveTubes (ET) that provide an innovative way of targeting mosquitoes with insecticides as they search for human hosts at night. The combined approach of Screening + EaveTubes (SET) essentially turns the house into a 'lure and kill' device. METHODS: This study evaluated the impact of SET on malaria infection prevalence in Côte d'Ivoire and compares the result in the primary outcome, malaria case incidence. Malaria infection prevalence was measured in a cross-sectional survey in 40 villages, as part of a cluster-randomised trial evaluating the impact of SET on malaria case incidence. RESULTS: Infection prevalence, measured by rapid diagnostic test (RDT), was 50.4% and 36.7% in the control arm and intervention arm, respectively, corresponding to an odds ratio of 0.57 (0.45-0.71), p < 0.0001). There was moderate agreement between RDT and microscopy results, with a reduction in odds of infection of 36% recorded when infection was measured by microscopy. Prevalence measured by RDT correlated strongly with incidence at a cluster level. CONCLUSIONS: In addition to reducing malaria case incidence, house screening and EaveTubes substantially reduced malaria infection prevalence 18 months after installation. Infection prevalence may be a good metric to use for evaluating malaria interventions in areas of similar transmission levels to this setting. TRIAL REGISTRATION: ISRCTN18145556, registered 1 February 2017.

Exploring alternative insecticide delivery options in a "lethal house lure" for malaria vector control.

The In2Care EaveTube is a house modification designed to block and kill malaria mosquitoes using an electrostatic netting treated with insecticide powder. A previous study demonstrated prolonged duration of effective action of insecticide-treated electrostatic netting in a semi-field setting. As part of a cluster randomized controlled trial (CRT) of the EaveTube intervention in Côte d'Ivoire, we investigated the residual efficacy of a pyrethroid insecticide deployed in EaveTubes under village conditions of use. We also explored the scope of using existing malaria control technologies including LLINs and IRS as alternative methods to deliver insecticides in the lethal house lure. The efficacy of beta-cyfluthrin was monitored over time using the "eave tube bioassay" method. Mortality of beta-cyfluthrin exposed pyrethroid resistant Anopheles gambiae mosquitoes was > 80% after 4 months. The impact (mosquito mortality) of PVC tubes coated with pirimiphos methyl was similar to that of beta-cyfluthrin treated insert (66.8 vs. 62.8%) in release-recapture experiments in experimental huts. Efficacy was significantly lower with all the LLINs tested; however, the roof of PermaNet 3.0 induced significantly higher mosquito mortality (50.4%) compared to Olyset Plus (25.9%) and Interceptor G2 (21.6%) LLINs. The efficacy of the alternative delivery methods was short-lived with mortality decreasing below 50% within 2 months in residual activity bioassays. None of the products tested appeared superior to the powder treatments. Further research is therefore required to identify suitable insecticide delivery options in EaveTube for malaria vector control.

Entomological monitoring data driving decision-making for appropriate and sustainable malaria vector control in Côte d'Ivoire.

BACKGROUND: Entomological surveillance provides critical information on vectors for appropriate malaria vector control and strategic decision-making. The widely documented insecticide resistance of malaria vectors in Côte d'Ivoire requires that any vector control intervention deployment be driven by entomological data to optimize its effectiveness and appropriate resource allocations. To achieve this goal, this study documents the results of monthly vector surveillance and insecticide susceptibility tests conducted in 2019 and a review of all previous entomological monitoring data used to guide vector control decision making. Furthermore, susceptibility to pirimiphos-methyl and clothianidin was assessed in addition to chlorfenapyr and pyrethroids (intensity and piperonyl butoxide (PBO) synergism) tests previously reported. Vector bionomic data were conducted monthly in four sites (Sakassou, Béoumi, Dabakala and Nassian) that were selected based on their reported high malaria incidence. Adult mosquitoes were collected using human landing catches (HLCs), pyrethrum spray catches (PSCs), and human-baited CDC light traps to assess vector density, behaviour, species composition and sporozoite infectivity. RESULTS: Pirimiphos-methyl and clothianidin susceptibility was observed in 8 and 10 sites, respectively, while previous data reported chlorfenapyr (200 µg/bottle) susceptibility in 13 of the sites, high pyrethroid resistance intensity and increased mortality with PBO pre-exposure at all 17 tested sites. Anopheles gambiae sensu lato was the predominant malaria vector collected in all four bionomic sites. Vector density was relatively higher in Sakassou throughout the year with mean biting rates of 278.2 bites per person per night (b/p/n) compared to Béoumi, Dabakala and Nassian (mean of 48.5, 81.4 and 26.6 b/p/n, respectively). The mean entomological inoculation rate (EIR) was 4.44 infective bites per person per night (ib/p/n) in Sakassou, 0.34 ib/p/n in Beoumi, 1.17 ib/p/n in Dabakala and 1.02 ib/p/n in Nassian. The highest EIRs were recorded in October in Béoumi (1.71 ib/p/n) and Nassian (3.22 ib/p/n), in July in Dabakala (4.46 ib/p/n) and in May in Sakassou (15.6 ib/p/n). CONCLUSION: Based on all results and data review, the National Malaria Control Programme developed and implemented a stratified insecticide-treated net (ITN) mass distribution in 2021 considering new generation ITNs. These results also supported the selection of clothianidin-based products and an optimal spraying time for the first indoor residual spraying (IRS) campaign in Sakassou and Nassian in 2020.

Entomological indicators of malaria transmission prior to a cluster-randomized controlled trial of a 'lethal house lure' intervention in central Côte d'Ivoire.

BACKGROUND: A study was conducted prior to implementing a cluster-randomized controlled trial (CRT) of a lethal house lure strategy in central Côte d'Ivoire to provide baseline information on malaria indicators in 40 villages across five health districts. METHODS: Human landing catches (HLC) were performed between November and December 2016, capturing mosquitoes indoors and outdoors between 18.00 and 08.00 h. Mosquitoes were processed for entomological indicators of malaria transmission (human biting, parity, sporozoite, and entomological inoculation rates (EIR)). Species composition and allelic frequencies of kdr-w and ace-1R mutations were also investigated within the Anopheles gambiae complex. RESULTS: Overall, 15,632 mosquitoes were captured. Anopheles gambiae sensu lato (s.l.) and Anopheles funestus were the two malaria vectors found during the survey period, with predominance for An. gambiae (66.2%) compared to An. funestus (10.3%). The mean biting rate for An. gambiae was almost five times higher than that for An. funestus (19.8 bites per person per night for An. gambiae vs 4.3 bites per person per night for An. funestus) and this was evident indoors and outdoors. Anopheles funestus was more competent to transmit malaria parasites in the study area, despite relatively lower number tested for sporozoite index (4.14% (63/1521) for An. gambiae vs 8.01% (59/736) for An. funestus; χ2 = 12.216; P < 0.0001). There were no significant differences between the proportions infected outdoors and indoors for An. gambiae (4.03 vs 4.13%; χ2 = 0.011; P = 0.9197) and for An. funestus (7.89 vs 8.16%; χ2 = 2.58e-29; P = 1). The majority of both infected vectors with malaria parasites harboured Plasmodium falciparum (93.65% for An. gambiae and 98. 31% for An. funestus). Overall, the EIR range for both species in the different districts appeared to be high (0.35-2.20 infected bites per human per night) with the highest value observed in the district of North-Eastern-Bouaké. There were no significant differences between transmission occurring outdoor and indoor for both species. Of the An. gambiae s.l. analysed, only An. gambiae sensu stricto (14.1%) and Anopheles coluzzii (85.9%) were found. The allelic frequencies of kdr and ace-1R were higher in An. gambiae (0.97 for kdr and 0.19 for ace-1R) than in An. coluzzii (0.86 for kdr and 0.10 for ace-1R) (P < 0.001). CONCLUSION: Despite universal coverage with long-lasting insecticidal nets (LLINs) in the area, there was an abundance of the malaria vectors (An. gambiae and An. funestus) in the study area in central Côte d'Ivoire. Consistent with high insecticide resistance intensity previously detected in these districts, the current study detected high kdr frequency (> 85%), coupled with high malaria transmission pattern, which could guide the use of Eave tubes in the study areas.

Technical Workflow Development for Integrating Drone Surveys and Entomological Sampling to Characterise Aquatic Larval Habitats of Anopheles funestus in Agricultural Landscapes in Côte d'Ivoire.

Land-use practices such as agriculture can impact mosquito vector breeding ecology, resulting in changes in disease transmission. The typical breeding habitats of Africa's second most important malaria vector Anopheles funestus are large, semipermanent water bodies, which make them potential candidates for targeted larval source management. This is a technical workflow for the integration of drone surveys and mosquito larval sampling, designed for a case study aiming to characterise An. funestus breeding sites near two villages in an agricultural setting in Côte d'Ivoire. Using satellite remote sensing data, we developed an environmentally and spatially representative sampling frame and conducted paired mosquito larvae and drone mapping surveys from June to August 2021. To categorise the drone imagery, we also developed a land cover classification scheme with classes relative to An. funestus breeding ecology. We sampled 189 potential breeding habitats, of which 119 (63%) were positive for the Anopheles genus and nine (4.8%) were positive for An. funestus. We mapped 30.42 km2 of the region of interest including all water bodies which were sampled for larvae. These data can be used to inform targeted vector control efforts, although its generalisability over a large region is limited by the fine-scale nature of this study area. This paper develops protocols for integrating drone surveys and statistically rigorous entomological sampling, which can be adjusted to collect data on vector breeding habitats in other ecological contexts. Further research using data collected in this study can enable the development of deep-learning algorithms for identifying An. funestus breeding habitats across rural agricultural landscapes in Côte d'Ivoire and the analysis of risk factors for these sites.

Evaluation of the interaction between insecticide resistance-associated genes and malaria transmission in Anopheles gambiae sensu lato in central Côte d'Ivoire.

BACKGROUND: There is evidence that the knockdown resistance gene (Kdr) L1014F and acetylcholinesterase-1 gene (Ace-1R) G119S mutations involved in pyrethroid and carbamate resistance in Anopheles gambiae influence malaria transmission in sub-Saharan Africa. This is likely due to changes in the behaviour, life history and vector competence and capacity of An. gambiae. In the present study, performed as part of a two-arm cluster randomized controlled trial evaluating the impact of household screening plus a novel insecticide delivery system (In2Care Eave Tubes), we investigated the distribution of insecticide target site mutations and their association with infection status in wild An. gambiae sensu lato (s.l.) populations. METHODS: Mosquitoes were captured in 40 villages around Bouaké by human landing catch from May 2017 to April 2019. Randomly selected samples of An. gambiae s.l. that were infected or not infected with Plasmodium sp. were identified to species and then genotyped for Kdr L1014F and Ace-1R G119S mutations using quantitative polymerase chain reaction assays. The frequencies of the two alleles were compared between Anopheles coluzzii and Anopheles gambiae and then between infected and uninfected groups for each species. RESULTS: The presence of An. gambiae (49%) and An. coluzzii (51%) was confirmed in Bouaké. Individuals of both species infected with Plasmodium parasites were found. Over the study period, the average frequency of the Kdr L1014F and Ace-1R G119S mutations did not vary significantly between study arms. However, the frequencies of the Kdr L1014F and Ace-1R G119S resistance alleles were significantly higher in An. gambiae than in An. coluzzii [odds ratio (95% confidence interval): 59.64 (30.81-131.63) for Kdr, and 2.79 (2.17-3.60) for Ace-1R]. For both species, there were no significant differences in Kdr L1014F or Ace-1R G119S genotypic and allelic frequency distributions between infected and uninfected specimens (P > 0.05). CONCLUSIONS: Either alone or in combination, Kdr L1014F and Ace-1R G119S showed no significant association with Plasmodium infection in wild An. gambiae and An. coluzzii, demonstrating the similar competence of these species for Plasmodium transmission in Bouaké. Additional factors including behavioural and environmental ones that influence vector competence in natural populations, and those other than allele measurements (metabolic resistance factors) that contribute to resistance, should be considered when establishing the existence of a link between insecticide resistance and vector competence.

Spatio-temporal analysis and prediction of malaria cases using remote sensing meteorological data in Diébougou health district, Burkina Faso, 2016-2017.

Malaria control and prevention programs are more efficient and cost-effective when they target hotspots or select the best periods of year to implement interventions. This study aimed to identify the spatial distribution of malaria hotspots at the village level in Diébougou health district, Burkina Faso, and to model the temporal dynamics of malaria cases as a function of meteorological conditions and of the distance between villages and health centres (HCs). Case data for 27 villages were collected in 13 HCs. Meteorological data were obtained through remote sensing. Two synthetic meteorological indicators (SMIs) were created to summarize meteorological variables. Spatial hotspots were detected using the Kulldorf scanning method. A General Additive Model was used to determine the time lag between cases and SMIs and to evaluate the effect of SMIs and distance to HC on the temporal evolution of malaria cases. The multivariate model was fitted with data from the epidemic year to predict the number of cases in the following outbreak. Overall, the incidence rate in the area was 429.13 cases per 1000 person-year with important spatial and temporal heterogeneities. Four spatial hotspots, involving 7 of the 27 villages, were detected, for an incidence rate of 854.02 cases per 1000 person-year. The hotspot with the highest risk (relative risk = 4.06) consisted of a single village, with an incidence rate of 1750.75 cases per 1000 person-years. The multivariate analysis found greater variability in incidence between HCs than between villages linked to the same HC. The time lag that generated the better predictions of cases was 9 weeks for SMI1 (positively correlated with precipitation variables) and 16 weeks for SMI2 (positively correlated with temperature variables. The prediction followed the overall pattern of the time series of reported cases and predicted the onset of the following outbreak with a precision of less than 3 weeks. This analysis of malaria cases in Diébougou health district, Burkina Faso, provides a powerful prospective method for identifying and predicting high-risk areas and high-transmission periods that could be targeted in future malaria control and prevention campaigns.

Influence of Host-Related Factors and Exposure to Mosquito Bites on the Dynamics of Antibody Response to Plasmodium falciparum Antigens.

Humoral immunity to Plasmodium falciparum is acquired after repeated infections, and can lead to clinical protection. This study aimed to evaluate how human-, parasite-, and environment-related determinants can modulate the dynamics of IgG responses to Plasmodium falciparum after an infection. Individuals (n = 68, average age = 8.2 years) with uncomplicated malaria were treated with ACT and followed up for 42 days. IgG responses to P. falciparum merozoite antigens (PfMSP1, PfMSP3, PfAMA1, PfGLURP-R0), to whole schizont extract (PfSchz), and to Anopheles gSG6-P1 and Aedes Nterm-34 kDa salivary peptides were measured. Regression analyses were used to identify factors that influence the dynamics of IgG response to P. falciparum antigen between D0 and D42, including demographic and biological factors and the level of exposure to mosquito bites. The dynamics of IgG response to P. falciparum differed according to the antigen. According to multivariate analysis, IgG responses to PfSchz and to PfGLURP-R0 appear to be affected by exposure to Aedes saliva and are associated with age, parasite density, and anti-Plasmodium pre-existing immune response at study inclusion. The present work shows that human exposure to Aedes saliva may contribute, in addition to other factors, to the regulation of anti-Plasmodium immune responses during a natural infection.

Data-driven and interpretable machine-learning modeling to explore the fine-scale environmental determinants of malaria vectors biting rates in rural Burkina Faso.

BACKGROUND: Improving the knowledge and understanding of the environmental determinants of malaria vector abundance at fine spatiotemporal scales is essential to design locally tailored vector control intervention. This work is aimed at exploring the environmental tenets of human-biting activity in the main malaria vectors (Anopheles gambiae s.s., Anopheles coluzzii and Anopheles funestus) in the health district of Diébougou, rural Burkina Faso. METHODS: Anopheles human-biting activity was monitored in 27 villages during 15 months (in 2017-2018), and environmental variables (meteorological and landscape) were extracted from high-resolution satellite imagery. A two-step data-driven modeling study was then carried out. Correlation coefficients between the biting rates of each vector species and the environmental variables taken at various temporal lags and spatial distances from the biting events were first calculated. Then, multivariate machine-learning models were generated and interpreted to (i) pinpoint primary and secondary environmental drivers of variation in the biting rates of each species and (ii) identify complex associations between the environmental conditions and the biting rates. RESULTS: Meteorological and landscape variables were often significantly correlated with the vectors' biting rates. Many nonlinear associations and thresholds were unveiled by the multivariate models, for both meteorological and landscape variables. From these results, several aspects of the bio-ecology of the main malaria vectors were identified or hypothesized for the Diébougou area, including breeding site typologies, development and survival rates in relation to weather, flight ranges from breeding sites and dispersal related to landscape openness. CONCLUSIONS: Using high-resolution data in an interpretable machine-learning modeling framework proved to be an efficient way to enhance the knowledge of the complex links between the environment and the malaria vectors at a local scale. More broadly, the emerging field of interpretable machine learning has significant potential to help improve our understanding of the complex processes leading to malaria transmission, and to aid in developing operational tools to support the fight against the disease (e.g. vector control intervention plans, seasonal maps of predicted biting rates, early warning systems).

Operational Evaluation of the Effectiveness of Long-lasting Insecticidal Nets on Human-Vector Contact in an African Urban Malaria Context.

BACKGROUND: Malaria is still a major public health concern in Côte d'Ivoire despite mass distribution of long-lasting insecticidal nets (LLINs) as a key preventive strategy. This study intended to evaluate the operational effectiveness of LLINs on the level of human-vector contact using 1 antibody-based biomarker of exposure to Anopheles in urban areas. METHODS: This cross-sectional study collected socio-demographic data and use of LLINs from 9 neighborhoods in the city of Bouaké (Côte d'Ivoire). Dry blood spots performed in children aged >6 months and adults were used to evaluate immunoglobulin G (IgG) responses to the Anopheles gSG6-P1 salivary peptide. RESULTS: IgG response levels to the salivary peptide were significantly lower in individuals who declared having "always" (n = 270) slept under an LLIN compared with those who had "often" (n = 2087) and "never" (n = 88) slept under an LLIN (P < .0001). IgG response levels to gSG6-P1 between those who declared having "always" and "not always" slept under an LLIN varied according to neighborhood, socio-professional category, and age group. CONCLUSIONS: The human IgG level to this gSG6-P1 salivary peptide could be a useful tool to evaluate the actual effectiveness of LLINs and help design behavioral change interventions that are crucial for sustaining universal coverage.

Impact and cost-effectiveness of a lethal house lure against malaria transmission in central Côte d'Ivoire: a two-arm, cluster-randomised controlled trial.

BACKGROUND: New vector control tools are required to sustain the fight against malaria. Lethal house lures, which target mosquitoes as they attempt to enter houses to blood feed, are one approach. Here we evaluated lethal house lures consisting of In2Care (Wageningen, Netherlands) Eave Tubes, which provide point-source insecticide treatments against host-seeking mosquitoes, in combination with house screening, which aims to reduce mosquito entry. METHODS: We did a two-arm, cluster-randomised controlled trial with 40 village-level clusters in central Côte d'Ivoire between Sept 26, 2016, and April 10, 2019. All households received new insecticide-treated nets at universal coverage (one bednet per two people). Suitable households within the clusters assigned to the treatment group were offered screening plus Eave Tubes, with Eave Tubes treated using a 10% wettable powder formulation of the pyrethroid ß-cyfluthrin. Because of the nature of the intervention, treatment could not be masked for households and field teams, but all analyses were blinded. The primary endpoint was clinical malaria incidence recorded by active case detection over 2 years in cohorts of children aged 6 months to 10 years. This trial is registered with ISRCTN, ISRCTN18145556. FINDINGS: 3022 houses received screening plus Eave Tubes, with an average coverage of 70% across the intervention clusters. 1300 eligible children were recruited for active case detection in the control group and 1260 in the intervention group. During the 2-year follow-up period, malaria case incidence was 2·29 per child-year (95% CI 1·97-2·61) in the control group and 1·43 per child-year (1·21-1·65) in the intervention group (hazard ratio 0·62, 95% CI 0·51-0·76; p<0·0001). Cost-effectiveness simulations suggested that screening plus Eave Tubes has a 74·0% chance of representing a cost-effective intervention, compared with existing healthcare activities in Côte d'Ivoire, and is similarly cost-effective to other core vector control interventions across sub-Saharan Africa. No serious adverse events associated with the intervention were reported during follow-up. INTERPRETATION: Screening plus Eave Tubes can provide protection against malaria in addition to the effects of insecticide-treated nets, offering potential for a new, cost-effective strategy to supplement existing vector control tools. Additional trials are needed to confirm these initial results and further optimise Eave Tubes and the lethal house lure concept to facilitate adoption. FUNDING: The Bill & Melinda Gates Foundation.