Summary 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 β-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.

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. Graphical Abstract

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.

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.

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.

Background: A study was conducted prior to implementing a cluster randomised controlled trial (CRT) of a lethal house lure strategy in central Côte d’Ivoire and aimed to provide baseline information on malaria vectors in 40 village clusters. Methods: Human landing catches (HLC) was performed between November-December 2016, capturing mosquitoes indoor and outdoor between 18.00-08.00. Mosquitoes were processed for entomological indicators of malaria transmission (human biting rates, parity rates, sporozoite infection rates and the 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 s.l. and Anopheles funestus were the two malaria vectors found during the survey period, with predominance for Anopheles gambiae s.l. (66.2%) compared to Anopheles funestus (10.3%). The mean biting rate for An. gambiae s.l. was almost 5 times higher than that for An. funestus s.l.(19.8 bites per person per night for An. gambiae s. l. vs 4.3 bites per person per night for An. funestus s. l.) and this was evident indoor and outdoor. An. funestus was more competent to transmit malaria parasites in the study area, despite relatively lower number tested for sporozoite index (1.6% (1,373) for An. gambiae vs 4.7 % (722) for An. funestus s.l.). There was no significant difference between the proportion infected outdoor and indoor for An. gambiae s.l. (1.6% vs 1.5%; OR=1.11[0.65-1.9]; P=0.676), but for An. funestus, more mosquitoes were infected outdoor (6.4%) than indoor (3.5%) (OR=1.86 [1.07-3.23]; P=0.0249). The majority of both infected vectors with malaria parasites harboured P. falciparum (90.6% for An. gambiae s. l. and 97, 8% for An. funestus s. l.). The EIR for both vectors (0.43 infected bites per night) were similar and there were no significant differences for transmission occurring outdoor and indoor for both species. Of the An. gambiae s.l. analysed, only An. gambiae (14.1%) and An. 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 of long-lasting insecticidal nets (LLINs) in the area, there was an abundance of malaria vectors in the study in area in central Côte d’Ivoire, specifically highly resistant An. gambiae s.l. as well as An. funestus s.l.. The malaria sporozoite rate was higher in An. funestus s.l than An. gambiae s.l.. but EIR rates in these two species were similarly high, both indoor and outdoor. Novel tools or strategies are urgently needed to further reduce malaria transmission in this area.

Background There is evidence that the Kdr L1014F and 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 behavior, life history, vectorial competence and capacity. In the present study, performed as part of a two-armed cluster randomized controlled trial (CRT) evaluating the impact of household screening plus a novel insecticide delivery system (In2Care EaveTubes), we investigated the distribution of insecticide target site mutations and their association with the infection status in wild An. gambiae s.l populations. Methods Mosquitoes were captured in 40 villages around Bouaké by human landing catches (HLC), from May 2017 to April 2019. Randomly selected sample of infected and uninfected An.gambiae s.l. with Plasmodium sp. were identified to species and then genotyped for Kdr L1014F and Ace-1R G119S mutations using quantitative polymerase chain reaction (qPCR) assays. The frequencies of the two alleles were compared between An. coluzzii and An. 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. Both species seemed to transmit equally Plasmodium parasites. 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 frequency of the Kdr L1014F and Ace-1R G119S resistance alleles were significantly higher in An. gambiae than in An. coluzzii (OR [95%CI]: 59.64 [30.81-131.63] for Kdr and OR [95%CI]: 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 distribution 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 similar competence for Plasmodium transmission in Bouaké. Additional factors influencing competence in natural population and those outside allele measurements contributing to resistance should be consider when establishing link between insecticide resistance and vector competence.

Background Attractive targeted sugar bait (ATSB) is a novel approach to vector control, offering an alternative mode of insecticide delivery via the 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 disrupt mitochondria of 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.Method In this study, we compared the effect of ATSBs containing chlorfenapyr insecticide and, as a positive control, the antimalarial drugs artemether/lumefantrine (A/L) on Plasmodium falciparum development in wild pyrethroid-resistant Anopheles gambiae s.s. for their capacity to reduce vector competence. To do this, we exposed females mosquitoes to ATSB containing 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 infective blood meals. The impact of their component compounds on the prevalence and intensity of P. falciparum infection were compared between treatments.Results We showed that both prevalence and intensity of infection were significantly reduced in mosquitoes exposed to either A/L or CFP, compared to unexposed negative control mosquitoes. The A/L dose (2.4/14.4 mg per 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 CFP (0.25%) 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 CFP, 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.