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Long-lasting insecticidal bed nets (LLINs) are a key measure for preventing malaria and their evaluation is coordinated by the World Health Organization Pesticide Evaluation Scheme (WHOPES). LifeNet® was granted WHOPES time-limited interim recommendation in 2011 after successful Phase I and Phase II evaluations. Here, we evaluated the durability and community acceptance of LifeNet® in a Phase III trial from June 2014 to June 2017 in Benin rural area. A prospective longitudinal, cluster-randomized, controlled trial with households as the unit of observation was designed to assess the performance of LifeNet® over a three-year period, using a WHOPES fully recommended LLIN (PermaNet® 2.0) as a positive control. The primary outcomes were the bioassay performance using WHO cone assays and tunnel tests, the insecticide content and physical integrity. At baseline, 100% of LLINs were within the tolerance limits of their target deltamethrin concentrations. By 36 months only 17.3% of LifeNet® and 8.5% of PermaNet® LLINs still were within their target deltamethrin concentrations. Despite these low rates, 100% of both LLINs meet WHO efficacy criteria (≥ 80% mortality or ≥ 95% knockdown or tunnel test criteria of ≥ 80% mortality or ≥ 90% blood-feeding inhibition) after 36 months using WHO cone bio-assays and tunnel tests. The proportion of LLINs in good physical condition was 33% for LifeNet® and 29% for PermaNet® after 36 months. After 36 M the survivorship was 21% and 26% for LifeNet® and PermaNet® respectively. Although both LLINs were well accepted by the population, complaints of side effects were significantly higher among LifeNet® users than PermaNet® ones. LifeNet® LLINs did meet WHO criteria for bio-efficacy throughout the study period and were well accepted by the population. This is an important step towards getting a full WHO recommendation for use in malaria endemic countries.

Twenty-seven villages were selected in southwest Burkina Faso to implement new vector control strategies in addition to long lasting insecticidal nets (LLINs) through a Randomized Controlled Trial (RCT). We conducted entomological surveys in the villages during the dry cold season (January 2017), dry hot season (March 2017) and rainy season (June 2017) to describe malaria vectors bionomics, insecticide resistance and transmission prior to this trial. We carried out hourly catches (from 17:00 to 09:00) inside and outside 4 houses in each village using the Human Landing Catch technique. Mosquitoes were identified using morphological taxonomic keys. Specimens belonging to the Anopheles gambiae complex and Anopheles funestus group were identified using molecular techniques as well as detection of Plasmodium falciparum infection and insecticide resistance target-site mutations. Eight Anopheles species were detected in the area. Anopheles funestus s.s was the main vector during the dry cold season. It was replaced by Anopheles coluzzii during the dry hot season whereas An. coluzzii and An. gambiae s.s. were the dominant species during the rainy season. Species composition of the Anopheles population varied significantly among seasons. All insecticide resistance mechanisms (kdr-w, kdr-e and ace-1 target site mutations) investigated were found in each members of the An. gambiae complex but at different frequencies. We observed early and late biting phenotypes in the main malaria vector species. Entomological inoculation rates were 2.61, 2.67 and 11.25 infected bites per human per month during dry cold season, dry hot season and rainy season, respectively. The entomological indicators of malaria transmission were high despite the universal coverage with LLINs. We detected early and late biting phenotypes in the main malaria vector species as well as physiological insecticide resistance mechanisms. These data will be used to evaluate the impact of complementary tools to LLINs in an upcoming RCT.

Insecticide resistance in malaria vectors poses a significant threat to effective control of malaria vectors across sub-Saharan Africa. In Côte d'Ivoire, Anopheles gambiae s.l. exhibits considerable variability in species distribution and insecticide resistance levels across distinct ecological areas. This study reports on insecticide resistance profiles within An. gambiae complex as well as species composition dynamics in various ecological settings in Côte d'Ivoire. From July to October 2020, larval and adult mosquitoes were collected across three ecological settings (savannah, pre-forest and forest) in Côte d'Ivoire. Larvae were reared to adult stage for susceptibility bioassays. Diagnostic concentrations (DCs) applied against An. gambiae s.l. were deltamethrin 0.05%, permethrin 0.75% and pirimiphos methyl 0.25% using WHO susceptibility test kits. When increased survival to diagnostic concentrations (DCs) was observed, intensity bioassays were conducted using 5x and 10x DCs. In addition, synergist assays were performed at the DC level with pre-exposure to 4% piperonyl butoxide (PBO). Together with adults directly collected from the field, female samples from the bioassays outcome were speciated and resistance target site mechanisms (Kdr L1014F and Ace-1R G119S) were determined using PCR. Species distribution and insecticide resistance were analyzed across ecological areas. Significant variations in insecticide resistance phenotypes and mechanisms in An. gambiae s.l. were found across the three ecological areas in Côte d'Ivoire. Mortality rates, following pyrethroid exposure, were significantly higher in An. gambiae s.l. collected in the savannah zone compared to those collected from the forest zone (p < 0.05). An. gambiae was more predominant in the savannah area (98.8%, 95% confidence of interval (CI) [93.5-100.0]), whereas An. coluzzii dominated in the pre-forest (92.9%, 95% CI [88.9-95.8]) and forest zones (61.3%, 95% CI [53.4-68.9]). Such predominance of An. coluzzii was associated with high intensity of pyrethroid resistance in these areas. Overall, the allelic frequencies of the resistance mutations in An. gambiae were higher than those in An. coluzzii regardless of the area. Kdr L1014F frequency in the forest zone was 78.1% (95% CI [68.0-88.2]) in An. gambiae larvae, 51.8% (95% CI [33.3-70.4]) in adults, and 63.1% (95% CI [52.4-73.8]) in An. coluzzii larvae. Variations in species distribution and insecticide resistance in ecological areas in Côte d'Ivoire should be carefully considered when developing and implementing vector control strategies.

Background Widespread Insecticide resistance among malaria vector populations poses a significant threat to the efficacy of current vector control strategies. This highlights the urgent need for innovative approaches that integrate existing tools with those under development. The primary objective of this study was to characterize the malaria epidemiological profile in the Korhogo region prior to the implementation of complementary vector control tools alongside long-lasting insecticidal nets (LLINs). Both passive and active case detection methods were employed. Methods Passive case detection involved data collection from heath care facilities between July 2016 and July 2017, covering a total of 398,424 person-weeks from 26 villages in the Korhogo health district, northern Côte d’Ivoire. Active case detection was carried out through three cross-sectional surveys (CSS) conducted in September 2016, December 2016 and March 2017. During these surveys, individuals under 21 years of age were invited to attend clinical consultations. Those presenting with fever (> 37,5 °C) were tested for malaria with rapid diagnostic test (RDT), and individuals with a positive result were treated with artemisinin-based combination therapy (ACT). Active case detection during the surveys enabled the estimation of malaria case prevalence, defined as the proportion of population with symptoms and a positive RDT at the time of the survey. Additionally, in a randomly selected sub-sample of six villages, thick blood smears were analysed—regardless of symptoms—to assess the prevalence of malaria infection in the general population during CSS. Results The mean weekly malaria incidence throughout the year in the overall population was 0.25 cases per 100 person-weeks, corresponding to 130 cases per 1000 person-years. During CSS, malaria cases prevalence ranged from 13.4% during the dry season to 43.4% in the late rainy season. The highest malaria cases’ prevalence was observed in children under 5 years of age, with significant decline in older age groups. Thick blood smears from six villages revealed parasite prevalence between 85.4% and 100%. Plasmodium falciparum accounted for 97% of single infection, although mixed infections with Plasmodium malariae and Plasmodium ovale were also detected. Conclusion Active case detection conducted through three transversal surveys indicates that the study area exhibits characteristics of malaria transmission hotspot. The region’s dense hydrographic network provides a favourable ecological and epidemiological context for assessing additional vector control strategies to supplement universal LLINs coverage in the fight against malaria.

Background Although larviciding may be a valuable tool to supplement long-lasting insecticide nets (LLINs) in West Africa in different ecological settings, its actual impact on malaria burden and transmission has yet to be demonstrated. A randomized controlled trial was therefore undertaken to assess the effectiveness of larviciding using Bacillus thuringiensis israeliensis ( Bti ) in addition to the use of LLINs. In order to optimally implement such a larviciding intervention, we first aimed to identify and to characterize the breeding habitats of Anopheles spp. in the entire study area located in the vicinity of Korhogo in northern Côte d’Ivoire. Methods We conducted two surveys during the rainy and the dry season, respectively, in the thirty villages around Korhogo involved in the study. In each survey, water bodies located within a 2 km radius around each village were identified and assessed for the presence of mosquito larvae. We morphologically identified the larvae to the genus level and we characterized all of the habitats positive for Anopheles spp. larvae based on a predefined set of criteria. Results Overall, 620 and 188 water bodies positive for Anopheles spp. larvae were sampled in the rainy and the dry season, respectively. A broad range of habitat types were identified. Rice paddies accounted for 61% and 57% of the habitats encountered in the rainy and the dry season, respectively. In the rainy season, edges of rivers and streams (12%) were the second most abundant habitats for Anopheles spp. larvae. More than 90% of the Anopheles spp. breeding habitats were surrounded by green areas. Dams, ponds and drains produced higher numbers of Anopheles spp. larvae per square meter than rice paddies (RR = 1.51; 95% CI: 1.18–1.94; P  = 0.0010). The density of Anopheles spp. larvae was significantly higher in habitats surrounded by low-density housing (RR = 4.81; 95% CI: 1.84–12.60; P  = 0.0014) and green areas (RR = 3.96; 95% CI: 1.92–8.16; P  = 0.0002] than habitats surrounded by high-density housing. Turbid water [RR = 1.42 (95% CI: 1.15–1.76; P  = 0.0012) was associated with higher densities of Anopheles spp. larvae. The likelihood of finding mosquito pupae in Anopheles spp. breeding habitats was higher in the dry season (OR = 5.92; 95% CI: 2.11–16.63; P  = 0.0007) than in the rainy season. Conclusions Rice paddies represented the most frequent habitat type for Anopheles spp. larvae in the Korhogo area during both the rainy and the dry seasons. Anopheles spp. breeding habitats covered a very large and dynamic area in the rainy season whereas they were fewer in number in the dry season. In this context, implementing a larviciding strategy from the end of the rainy season to the dry season is presumably the most cost-effective strategy.

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-1 R 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 ; χ 2  = 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%; χ 2  = 0.011; P = 0.9197) and for An. funestus (7.89 vs 8.16%; χ 2  = 2.58 e−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-1 R were higher in An. gambiae (0.97 for kdr and 0.19 for ace-1 R ) than in An. coluzzii (0.86 for kdr and 0.10 for ace-1 R ) (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.

In sub-Saharan Africa, despite the implementation of multiple control interventions, the prevalence of malaria infection and clinical cases remains high. The primary tool for vector control against malaria in this region is the use of long-lasting insecticide-treated nets (LLINs) combined or not with indoor residual spraying (IRS) to achieve a synergistic effect in protection. The objective of this study was to assess the effectiveness of LLINs, with or without IRS, protected against Plasmodium falciparum infection and uncomplicated clinical cases (UCC) of malaria in Benin. A case-control study was conducted, encompassing all age groups, in the urban area of Djougou and the rural area of Cobly. A cross-sectional survey was conducted that included 2080 individuals in the urban area and 2770 individuals in the rural area. In the urban area, sleeping under LLINs did not confer significant protection against malaria infection and UCC when compared to no intervention. However, certain neighbourhoods benefited from a notable reduction in infection rates ranging from 65% to 85%. In the rural area, the use of LLINs alone, IRS alone, or their combination did not provide additional protection compared to no intervention. IRS alone and LLINs combined with IRS provided 61% and 65% protection against malaria infection, respectively, compared to LLINs alone. The effectiveness of IRS alone and LLINs combined with IRS against UCC was 52% and 54%, respectively, when compared to LLINs alone. In both urban and rural areas, the use of LLINs alone, IRS alone, and their combination did not demonstrate significant individual protection against malaria infection and clinical cases when compared to no intervention. In the conditions of this study, LLINs combined or not with IRS are not effective enough to eliminate malaria. In addition to the interventions, this study identified factors associated with malaria in Benin as housing design, neglected social groups like gender-marginalised individuals and adolescents, and socio-economic conditions acting as barriers to effective malaria prevention. Addressing these factors is crucial in order to facilitate malaria elimination efforts in sub-Saharan Africa.

Background There is evidence that the knockdown resistance gene ( Kdr ) L1014F and acetylcholinesterase-1 gene ( Ace-1 R ) 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-1 R 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-1 R G119S mutations did not vary significantly between study arms. However, the frequencies of the Kdr L1014F and Ace-1 R 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-1 R ]. For both species, there were no significant differences in Kdr L1014F or Ace-1 R G119S genotypic and allelic frequency distributions between infected and uninfected specimens ( P  > 0.05). Conclusions Either alone or in combination, Kdr L1014F and Ace-1 R 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

Background Biotic and abiotic factors have been reported to affect the larvicidal efficacy of Bacillus thuringiensis israelensis ( Bti ) and Bacillus sphaericus ( Bs ), although the extent to which they are affected has been poorly documented. This paper studies the effect of sunlight exposure on the efficacy of a new larvicide formulation based on both Bti and Bs , herein after referred to as BTBSWAX, applied against two different larval stages. Methods The emergence of inhibition exhibited by BTBSWAX at three different dosages (1 g/m 2 , 1.5 g/m 2 , and 2 g/m 2 ) was monitored under semi-field conditions using a total of 32 containers comprising 16 that were covered and 16 that were uncovered. Two experiments were conducted using first- and second-instar larvae of Anopheles gambiae , respectively. Results BTBSWAX at 2 g/m 2 in covered containers exhibited high emergence inhibition (> 80%) when larvae were exposed from 1st instar on day-6 post-treatment, whereas the emergence inhibition was only 28% in uncovered containers. For larvae exposed from 1st instar on day-12 post-treatment, the emergence inhibition was moderate (70%) in covered containers but was low (< 20%) in uncovered containers. For larvae exposed from 2nd instar on day-10 post-treatment, the emergence inhibition was moderate (31%) in covered containers but was very low (< 10%) in uncovered containers. Moreover, the residual efficacy of BTBSWAX was markedly affected by environmental stresses, including sunlight exposure (Hazard ratio (HR) = 0.12, p < 0.001 and HR = 0.63, p = 0.033 for BTBSWAX at 2 g/m 2 against 1st and 2nd instar larvae, respectively). Conclusion These findings emphasize the impact of environmental variables (e.g., sunlight exposure) on the residual efficacy of Bti and Bs biolarvicides in the field. They hence highlight the need to take these factors into account for larvicide formulation development processes. Moreover, studies of the ecology of Anopheles larvae in targeted areas are also crucial for the integration of larval control strategies into malaria transmission plans devised by national malaria control programmes of endemic countries.

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. Graphical Abstract