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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 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.

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

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) (PConclusion: 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.

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 km(2) 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

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 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.

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

Background Indoor attractive toxic sugar bait (ATSB) has potential as a supplementary vector-control and resistance-management tool, offering an alternative mode of insecticide delivery to current core vector-control interventions, with potential to deliver novel insecticides. Given the high long-lasting insecticidal bed net (LLIN) coverage across Africa, it is crucial that the efficacy of indoor ATSB in combination with LLINs is established before it is considered for wider use in public health. Methods An experimental hut trial to evaluate the efficacy of indoor ATSB traps treated with 4% boric acid (BA ATSB) or 1% chlorfenapyr (CFP ATSB) in combination with untreated nets or LLINs (holed or intact), took place at the M’bé field station in central Côte d’Ivoire against pyrethroid resistant Anopheles gambiae sensu lato. Results The addition of ATSB to LLINs increased the mortality rates of wild pyrethroid-resistant An. gambiae from 19% with LLIN alone to 28% with added BA ATSB and to 39% with added CFP ATSB (p Anopheles gambiae mortality with combined ATSB and untreated net was similar to that of combined ATSB and LLIN regardless of which insecticide was used in the ATSB. The presence of holes in the LLIN did not significantly affect ATSB-induced An. gambiae mortality. Comparative tests against pyrethroid resistant and susceptible strains using oral application of ATSB treated with pyrethroid demonstrated 66% higher survival rate among pyrethroid-resistant mosquitoes. Conclusion Indoor ATSB traps in combination with LLINs enhanced the control of pyrethroid-resistant An. gambiae. However, many host-seeking An. gambiae entering experimental huts with indoor ATSB exited into the verandah trap without sugar feeding when restricted from a host by a LLIN. Although ATSB has potential for making effective use of classes of insecticide otherwise unsuited to vector control, it does not exempt potential selection of resistance via this route.

Background Long-lasting insecticidal nets (LLINs) are the primary method of malaria prevention. However, the widespread resistance to pyrethroids among major malaria vector species represents a significant threat to the continued efficacy of pyrethroid LLIN. Piperonyl butoxide (PBO) is a synergist that inhibits the activity of metabolic enzymes of the cytochrome P450 family known to detoxify insecticides including pyrethroids. Synergist LLIN incorporating PBO and a pyrethroid may provide improved control compared to pyrethroid-only LLIN. Methods The efficacy of VEERALIN® LN (VKA polymers Pvt Ltd, India), an alpha-cypermethrin PBO synergist net was evaluated in experimental huts in M’bé, central Côte d’Ivoire against wild pyrethroid resistant Anopheles gambiae s.s. Comparison was made with a standard alpha-cypermethrin-treated net (MAGNet® LN, VKA polymers Pvt Ltd, India). Nets were tested unwashed and after 20 standardized washes. Results VEERALIN® LN demonstrated improved efficacy compared to MAGNet® LN against wild free-flying pyrethroid-resistant An. gambiae s.s. Before washing, VEERALIN® LN produced mortality of An. gambiae s.s. (51%) significantly higher than the standard pyrethroid-only net (29%) (P vs 17%) (P P An. gambiae s.s. (62.6%) compared to MAGNet® LN (35.4%) (P Conclusions The addition of the synergist PBO to pyrethroid net greatly improved protection and control of pyrethroid-resistant An. gambiae s.s. The pyrethroid-PBO VEERALIN® LN has the potential to reduce transmission in areas compromised by pyrethroid resistance.

Routine monitoring of occurrence, levels and mechanisms of insecticide resistance informs effective management strategies, and should be used to assess the effect of new tools on resistance. As part of a cluster randomised controlled trial evaluating a novel insecticide-based intervention in central Côte d’Ivoire, we assessed resistance and its underlying mechanisms in Anopheles gambiae populations from a subset of trial villages. Resistance to multiple insecticides in An. gambiae s.s. and An. coluzzii was detected across villages, with dose–response assays demonstrating extremely high resistance intensity to the pyrethroid deltamethrin (> 1,500-fold), and mortality following exposure to pyrethroid-treated bednets was low (kdr mutation was almost fixed (≥ 90%) in all villages but the 1575Y kdr-amplifying mutation was relatively rare (Ace-1 G119S mutation was also detected at moderate frequencies (22–43%). Transcriptome analysis identified overexpression of P450 genes known to confer pyrethroid resistance (Cyp9K1, Cyp6P3, and Cyp6M2), and also a carboxylesterase (COEAE1F) as major candidates. Cyp6P3 expression was high but variable (up to 33-fold) and correlated positively with deltamethrin resistance intensity across villages (r2 = 0.78, P = 0.02). Tools and strategies to mitigate the extreme and multiple resistance provided by these mechanisms are required in this area to avoid future control failures.

International audience; A better understanding of malaria transmission at a local scale is essential for developing and implementing effective control strategies. In the framework of a randomized controlled trial (RCT), we aimed to provide an updated description of malaria transmission in the Korhogo area, northern Côte d'Ivoire, and to obtain baseline data for the trial. We performed human landing collections (HLCs) in 26 villages in the Korhogo area during the rainy season (September-October 2016, April-May 2017) and the dry season (November-December 2016, February-March 2017). We used PCR techniques to ascertain the species of the Anopheles gambiae complex, Plasmodium falciparum sporozoite infection, and insecticide resistance mechanisms in a subset of Anopheles vectors. Anopheles gambiae s.l. was the predominant malaria vector in the Korhogo area. Overall, more vectors were collected outdoors than indoors (p < 0.001). Of the 774 An. gambiae s.l. tested in the laboratory, 89.65% were An. gambiae s.s. and 10.35% were An. coluzzii. The frequencies of the kdr allele were very high in An. gambiae s.s. but the ace-1 allele was found at moderate frequencies. An unprotected individual living in the Korhogo area received an average of 9.04, 0.63, 0.06 and 0.12 infected bites per night in September-October, November-December, February-March, and April-May, respectively. These results demonstrate that the intensity of malaria transmission is extremely high in the Korhogo area, especially during the rainy season. Malaria control in highly endemic areas such as Korhogo needs to be strengthened with complementary tools in order to reduce the burden of the disease.; Une meilleure connaissance de la transmission du paludisme à l’échelle locale est essentielle pour élaborer et mettre en œuvre des stratégies de lutte efficaces. Dans le cadre d’un essai contrôlé randomisé, nous avons pour objectifs de fournir une description actualisée de la transmission du paludisme dans la zone de Korhogo, au nord de la Côte d’Ivoire, et de collecter les données de base pour l’essai. Nous avons capturé les moustiques sur des volontaires humains dans 26 villages de la zone de Korhogo pendant la saison pluvieuse (septembre–octobre 2016, avril–mai 2017) et la saison sèche (novembre–décembre 2016, février–mars 2017). À l’aide des techniques de PCR, nous avons déterminé les espèces au sein du complexe Anopheles gambiae, les infections par Plasmodium falciparum au stade sporozoïte et les mécanismes de résistance aux insecticides dans un sous-échantillon d’anophèles vecteurs. Anopheles gambiae s.l. est de loin le vecteur majoritaire du paludisme dans la zone de Korhogo. Au total, plus de vecteurs ont été collectés à l’extérieur des habitations qu’à l’intérieur (p < 0.001). Des 774 An. gambiae s.l. analysés au laboratoire, 89,65 % étaient An. gambiae s.s. et 10,35 % An. coluzzii. Les fréquences alléliques du gène kdr étaient très élevées chez An. gambiae s.s. alors que les fréquences alléliques du gène ace-1 étaient modérées. Une personne non protégée vivant à Korhogo reçoit chaque nuit en moyenne 9,04 piqûres infectantes (pi) en septembre–octobre, 0,63 pi en novembre–décembre, 0,06 pi en février-mars et 0,12 pi en avril–mai. Ces résultats démontrent que l’intensité de la transmission du paludisme est très élevée dans la zone de Korhogo, particulièrement en saison pluvieuse. La lutte contre le paludisme dans les zones de forte endémicité comme Korhogo doit être renforcée par des outils complémentaires afin de réduire le fardeau de la maladie.

Housing improvement such as blocking eaves and screening windows can help in reducing exposure to indoor biting mosquitoes. The impacts of physical barriers could potentially be boosted by the addition of a mechanism that kills mosquitoes as they attempt to enter the house. One example is to combine household screening with EaveTubes, which are insecticide-treated tubes inserted into closed eaves that attract and kill host-searching mosquitoes. The epidemiological impact of screening + EaveTubes is being evaluated in a large cluster randomized trial in Cote d'Ivoire. The study presented here is designed as a complement to this trial to help better understand the functional roles of screening and EaveTubes. We began by evaluating householder behaviour and household condition in the study villages. This work revealed that doors (and to some extent windows) were left open for large parts of the evening and morning, and that even houses modified to make them more ‘mosquito proof’ often had possible entry points for mosquitoes. We next built two realistic experimental houses in a village to enable us to explore how these aspects of behaviour and household quality affected the impact of screening and EaveTubes. We found that screening could have a substantial impact on indoor mosquito densities, even with realistic household condition and behaviour. By contrast, EaveTubes had no significant impact on indoor mosquito density, either as a stand-alone intervention or in combination with screening. However, there was evidence that mosquitoes recruited to the EaveTubes, and the resulting mortality could create a community benefit. These complementary modes of action of screening and EaveTubes support the rationale of combining the technologies to create a ‘Lethal House Lure’. This article is part of the theme issue ‘Novel control strategies for mosquito-borne diseases’.

BackgroundThere is evidence from experimental hut and household studies that the entomological efficacy of long lasting pyrethroid treated nets (LLINs) is compromised in areas of pyrethroid resistance. The rapid increase in resistance intensity in African malaria vectors could further undermine the performance of these nets. The pyrethroid resistance intensity in Anopheles gambiae s.l. M'bé from central Côte d'Ivoire is reported to be high (> 1700 fold). Whether this translates into an increase in entomological indicators of malaria transmission needs investigation.MethodThe efficacy of two long lasting insecticidal nets (LN) MiraNet and MagNet, both alpha-cypermethrin based was evaluated in experimental huts against pyrethroid resistant Anopheles gambiae in M'bé, central Côte d'Ivoire. All nets were deliberately holed to simulate wear-and-tear and were tested unwashed and after 20 standardized washes.ResultsThe entry rates of An. gambiae s.l. into huts with insecticide treated nets were 62-84% lower than entry into huts with untreated nets (p < 0.001). Exit rates of An. gambiae s.l. with unwashed MiraNet and MagNet LNs were significantly greater than with untreated nets (50-60% vs 26%) and this effect after washing 20 times nets did not decrease. Blood-feeding with both nets was significantly inhibited relative to the untreated reference net (31-55%) (p < 0.001). Washing MiraNet LN 20 times had no significant impact on protection against An. gambiae s.l. bites but it did cause a significant fall by 40% in protection with MagNet LN (p < 0.001). All insecticide treated nets induced higher mortality of An. gambiae s.l. than the untreated net (p < 0.05). The impact though significant was limited (14-30%). The personal protection against An. gambiae s.l. bites derived from all treatments was high (75-90%). The overall insecticidal effect was compromised by pyrethroid resistance and was not detectable in some treatments.ConclusionIn this area of high pyrethroid resistance intensity (over 1700 fold), both MiraNet and MagNet LNs still conferred appreciable personal protection against mosquito bites despite inducing only slightly greater mortality of pyrethroid resistant Anopheles mosquitoes than untreated nets. The impact is comparable to moderately intense Benin resistance area (207 fold) and Burkina Faso (over 1000 fold). This preserved level of protection plus the small but sensitive killing of mosquitoes may continue to impact vectorial capacity despite high intensity of resistance. Nevertheless, there is an obvious need for strategies and nets with novel mode of action to enhance vector control.

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/m2, 1.5 g/m2, and 2 g/m2) 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/m2 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 (

International audience; Background: Several studies were carried out in experimental hut station in areas surrounding the city of Bouaké, after the crisis in Côte d'Ivoire. They reported increasing resistance levels to insecticide for malaria transmiting mosquitoes. The present work aims to evaluate the current resistance level of An. gambiae (s.l.) in rural and urban areas in the city of Bouaké.Methods: Larvae of Anopheles gambiae (s.l.) were collected from five different study sites and reared to adult stages. The resistance status was assessed using the WHO bioassay test kits for adult mosquitoes, with eight insecticides belonging to pyrethroids, organochlorines, carbamates and organophosphates classes. Molecular assays were performed to identify the molecular forms of An. gambiae (s.l.), the L1014F kdr and the ace-1R alleles in individual mosquitoes. The synergist PBO was used to investigate the role of enzymes in resistance. Biochemical assays were performed to detect potential increased activities in mixed function oxidase (MFO) levels, non-specific esterases (NSE) and glutathione S-transferases (GST).Results: High resistance levels to pyrethroids, organochlorines, and carbamates were observed in Anopheles gambiae (s.l.) from Bouaké. Mortalities ranged between 0 and 73% for the eight tested insecticides. The pre-exposure to PBO restored full or partial susceptibility to pyrethroids in the different sites. The same trend was observed with the carbamates in five sites, but to a lesser extent. With DDT, pre-exposure to PBO did not increase the mortality rate of An. gambiae (s.l.) from the same sites. Tolerance to organophosphates was observed. An increased activity of NSE and higher level of MFO were found compared to the Kisumu susceptible reference strain. Two molecular forms, S form [(An. gambiae (s.s)] and M form (An. coluzzi) were identified. The kdr allele frequencies vary from 85.9 to 99.8% for An. gambiae (s.s.) and from 81.7 to 99.6% for An. coluzzii. The ace-1R frequencies vary between 25.6 and 38.8% for An. gambiae (s.s.) and from 28.6 to 36.7% for An. coluzzii.Conclusion: Resistance to insecticides is widespread within both An. gambiae (s.s.) and An. coluzzii. Two mechanisms of resistance, i.e. metabolic and target-site mutation seemed to largely explain the high resistance level of mosquitoes in Bouaké. Pyrethroid resistance was found exclusively due to the metabolic mechanism.

Background: The widespread insecticide resistance in malaria vector populations is a serious threat to the efficacy of vector control tools. As a result, the World Health Organization (WHO) supports the development of alternative tools that combine several insecticides with the aim of improving vector control and the management of insecticide resistance. In the present study, a long-lasting insecticidal net treated with a mixture of chlorfenapyr and alphacypermethrin was evaluated against wild pyrethroid-resistant Anopheles gambiae s.s in M’bé, Côte d’Ivoire. Centers for Disease Control and Prevention (CDC) bottle tests were carried out with resistant An. gambiae s.s. of M’bé and the susceptible strain, to assess the resistance level to chlorfenapyr and alphacypermethrin. Results: CDC bottle bioassays revealed a high level of resistance of An. gambiae s.s. population from M’bé to alphacypermethrin, whereas they revealed low resistance to chlorfenapyr. In experimental huts, Interceptor® G2 that was unwashed or washed 20 times killed 87% and 82% of An. gambiae s.s., respectively, whereas Interceptor® LN that was either unwashed or washed 20 times killed only about 10% of the mosquitoes. The blood-feeding inhibition induced by Interceptor® was not significantly different compared to untreated nets, whereas Interceptor® G2 that was unwashed or washed 20 times induced 42% and 34% inhibition of blood-feeding, respectively. Conclusion: Interceptor® G2 met the WHOPES criteria to undergo a phase III study. Investigation of its efficacy at a community level and the conduct of randomized controlled trials dealing with epidemiological outputs are warranted in order to study the potential of Interceptor® G2 to better protect communities.

Background The effectiveness of malaria control programmes through implementation of vector control activities is challenged by the emergence of insecticide resistance. In the South-Eastern region of Côte d’Ivoire, where palm oil plantations remain the predominant agricultural crop, the susceptibility of wild Anopheles gambiae sensu lato species is still unknown and thus requires a particular attention. The current study was carried out to address the gap by in-depth characterization of susceptibility level of An. gambiae mosquitoes from Ehania-V1 to WHO-recommended doses of six insecticides belonging to available classes and also to screen a subset for target site mutations and possible inhibition of P450 enzymes. Results Overall results showed variable resistance profile across WHO-recommended insecticides tested. Mortalities ranged from 8.3% (the lowest mortality was recorded with DDT) to 98% (the highest mortality was recorded with fenitrothion). Importantly, mortality to deltamethrin, an important pyrethroid used in public health for impregnation of mosquito nets was close to 98%, precluding a possible susceptibility to this insecticide, albeit further investigations are required. Pre-exposure of An. gambiae s.l. to PBO did not show any significant variation across insecticides (p = 0.002), although a partial increase was detected for alphacypermethrin and bendiocarb, suggesting a low of activity of cytochrome P450 enzymes (p = 0.277). High frequency of kdr L1014F was recorded in both Anopheles coluzzii (91%) and in An. gambiae (96%), associated with ace-1 R G119S mutation at low frequency (

Insecticide resistance constitutes a major threat that may undermine current gain in malaria control in most endemic countries. National Malaria Control Programmes (NMCPs) need as much information as possible on the resistance status of malaria vectors and underlying mechanisms in order to implement the most relevant and efficient control strategy. Bioassays, biochemical and molecular analysis were performed on An. gambiae collected in six sentinel sites in Côte d'Ivoire. The sites were selected on the basis of their bioclimatic status and agricultural practices. An. gambiae populations across sites showed high levels of resistance to organochloride, pyrethroid and carbamate insecticides. The kdr and ace-1(R) mutations were detected in almost all sentinel sites with mosquitoes on the coastal and cotton growing areas mostly affected by these mutations. At almost all sites, the levels of detoxifying enzymes (mixed-function oxidases (MFOs), non-specific esterases (NSE) and glutathione-S-transferases (GSTs)) in An. gambiae populations were significantly higher than the levels found in the susceptible strain Kisumu. Pre-exposure of mosquitoes to PBO, an inhibitor of MFOs and NSEs, significantly increased mortality rates to pyrethroids and carbamates in mosquitoes but resistance in most cases was not fully synergised by PBO, inferring a residual role of additional mechanisms, including kdr and ace-1 site insensitivity. The large distribution of resistance in Côte d'Ivoire raises an important question of whether to continue to deploy pyrethroid-based long-lasting insecticidal nets (LLINs) and insecticide residual spraying (IRS) towards which resistance continues to rise with no guarantee that the level of resistance would not compromise their efficacy. Innovative strategies that combine insecticide and synergists in LLINs or spatially LLIN and an effective non-pyrethroid insecticide for IRS could be in the short term the best practice for the NMCP to manage insecticide resistance in malaria vectors in Côte d'Ivoire and other endemic countries facing resistance.

International audience; Background: An experimental hut station built at M'Bé in 1998 was used for many years for the evaluation of insecticidal product for public health until the civil war broke out in 2002. Breeding sites of mosquitoes and selection pressure in the area were maintained by local farming practices and the West African Rice Development Association (WARDA, actually AfricaRice) in a large rice growing area. Ten years after the crisis, bioassays, molecular and biochemical analyses were conducted to update the resistance status and study the evolution of resistance mechanisms of Anopheles gambiae s.s population. Methods: Anopheles gambiae s.s larvae from M'Bé were collected in breeding sites and reared until emergence. Resistance status of this population to conventional insecticides was assessed using WHO bioassay test kits for adult mosquitoes, with 10 insecticides belonging to pyrethroids, pseudo-pyrethroid, organochlorides, carbamates and organophosphates with and without the inhibitor piperonyl butoxyde (PBO). Molecular and biochemical assays were carried out to identify the L1014F kdr, L1014S kdr and ace-1 R alleles in individual mosquitoes and to detect potential increase in mixed function oxidases (MFO) level, non-specific esterases (NSE) and glutathione S-transferases (GST) activities. Results and discussion: Anopheles gambiae s.s from M'Bé exerted high resistance levels to organochlorides, pyrethroids, and carbamates. Mortalities ranged from 3% to 21% for organochlorides, from 50% to 75% for pyrethroids, 34% for etofenprox, the pseudo-pyrethroid, and from 7% to 80% for carbamates. Tolerance to organophosphates was observed with mortalities ranging from 95% to 98%. Bioassays run with a pre-exposition of mosquitoes to PBO induced very high levels of mortalities compared to the bioassays without PBO, suggesting that the resistance to pyrethroid and carbamate relied largely on detoxifying enzymes' activities. The L1014F kdr allelic frequency was 0.33 in 2012 compared to 0.05 before the crisis in 2002. Neither the L1014S kdr nor ace-1 R mutations were detected. An increased activity of NSE and level of MFO was found relative to the reference strain Kisumu. This was the first evidence of metabolic resistance based resistance in An. gambiae s.s from M'Bé. Conclusion: The An. gambiae s.s population showed very high resistance to organochlorides, pyrethroids and carbamates. This resistance level relied largely on two major types of resistance: metabolic and target-site mutation. This multifactorial resistance offers a unique opportunity to evaluate the impact of both mechanisms and their interaction with the vector control tools currently used or in development.

Background At Yaokoffikro field site near Bouaké, in central Côte d'Ivoire, a group of experimental huts built in 1996 served over many years for the evaluation of insecticides against highly resistant mosquitoes. Breeding sites of mosquitoes and selection pressure in the area were maintained by local farming practices until a war broke out in September 2002. Six years after the crisis, we conducted bioassays and biochemical analysis to update the resistance status of Anopheles gambiae s.s. populations and detect other potential mechanisms of resistance that might have evolved. Methods An. gambiae s.s. larvae from Yaokoffikro were collected in breeding sites and reared to adults. Resistance status of this population to insecticides was assessed using WHO bioassay test kits for adult mosquitoes with seven insecticides: two pyrethroids, a pseudo-pyrethroid, an organochloride, two carbamates and an organophosphate. Molecular and biochemical assays were carried out to identify the L1014F kdr and ace-1R alleles in individual mosquitoes and to detect potential increase in mixed function oxidases (MFO), non-specific esterases (NSE) and glutathione S-transferases (GST) activity. Results High pyrethroids, DDT and carbamate resistance was confirmed in An. gambiae s.s. populations from Yaokoffikro. Mortality rates were less than 70% with pyrethroids and etofenprox, 12% with DDT, and less than 22% with the carbamates. Tolerance to fenitrothion was observed, with 95% mortality after 24 h. PCR analysis of samples from the site showed high allelic frequency of the L1014F kdr (0.94) and the ace-1R (0.50) as before the crisis. In addition, increased activity of NSE, GST and to a lesser extent MFO was found relative to the reference strain Kisumu. This was the first report detecting enhanced activity of these enzymes in An. gambiae s.s from Yaokoffikro, which could have serious implications in detoxification of insecticides. Their specific roles in resistance should be investigated using additional tools. Conclusion The insecticide resistance profile at Yaokoffikro appears multifactorial. The site presents a unique opportunity to evaluate its impact on the protective efficacy of insecticidal products as well as new tools to manage these complex mechanisms. It calls for innovative research on the behaviour of the local vector, its biology and genetics that drive resistance.

Background The spread of pyrethroid resistance in Anopheles gambiae s.s. is a critical issue for malaria vector control based on the use of insecticide-treated nets. Carbamates and organophosphates insecticides are regarded as alternatives or supplements to pyrethroids used in nets treatment. It is, therefore, essential to investigate on the susceptibility of pyrethroid resistant populations of An. gambiae s.s. to these alternative products. Methods In September 2004, a cross sectional survey was conducted in six localities in Côte d'Ivoire: Toumbokro, Yamoussoukro, Toumodi in the Southern Guinea savannah, Tiassalé in semi-deciduous forest, then Nieky and Abidjan in evergreen forest area. An. gambiae populations from these localities were previously reported to be highly resistant to pyrethroids insecticides. Anopheline larvae were collected from the field and reared to adults. Resistance/susceptibility to carbamates (0.4% carbosulfan, 0.1% propoxur) and organophosphates (0.4% chlorpyrifos-methyl, 1% fenitrothion) was assessed using WHO bioassay test kits for adult mosquitoes. Then, PCR assays were run to determine the molecular forms (M) and (S), as well as phenotypes for insensitive acetylcholinesterase (AChE1) due to G119S mutation. Results Bioassays showed carbamates (carbosulfan and propoxur) resistance in all tested populations of An. gambiae s.s. In addition, two out of the six tested populations (Toumodi and Tiassalé) were also resistant to organophosphates (mortality rates ranged from 29.5% to 93.3%). The M-form was predominant in tested samples (91.8%). M and S molecular forms were sympatric at two localities but no M/S hybrids were detected. The highest proportion of S-form (7.9% of An. gambiae identified) was in sample from Toumbokro, in the southern Guinea savannah. The G119S mutation was found in both M and S molecular forms with frequency from 30.9 to 35.2%. Conclusion This study revealed a wide distribution of insensitive acetylcholinesterase due to the G119S mutation in both M and S molecular forms of the populations of An. gambiae s.s. tested. The low cross-resistance between carbamates and organophosphates highly suggests involvement of other resistance mechanisms such as metabolic detoxification or F290V mutation.

In the context of the widespread distribution of pyrethroid resistance among malaria vectors, we did a release-recapture trial in experimental huts to investigate the insecticidal and sterilizing effects of a novel long-lasting net (LN), Olyset (R) Duo, incorporating a mixture of permethrin (PER) and the insect growth regulator (IGR), pyriproxyfen (PPF). An LN containing PPF alone and a classic Olyset (R) Net were tested in parallel as positive controls. The effect of progressive number of holes (6, 30, or 150) that may accrue in nets over time was simulated. We used two laboratory Anopheles gambiae s.s. strains: the susceptible Kisumu strain and the pyrethroid-resistant VK-Per strain having solely kdr as resistance mechanism. The effect of these nets on the reproductive success of blood-fed females that survived the different LNs conditions was recorded. Regardless of the mosquito strain, the LNs containing PPF alone with as many as 30 holes drastically reduced the number of eggs laid by females succeeding in feeding, i.e. fecundity by 98% and egg hatching rate (fertility) by 93% relative to untreated control net. Very few of the resistant females blood fed and survived under the Olyset (R) Duo with similar number of holes (up to 30) but of these few, the inhibition of reproductive success was 100%. There was no evidence that the Olyset (R) Duo LN with 150 holes impacted fecundity or fertility of the resistant colony. The efficacy of Olyset (R) Duo is encouraging and clearly illustrates that this new net might be a promising tool for malaria transmission control and resistance management.

Pyrethroid resistance in malaria vectors has spread across sub-Saharan Africa. Alternative tools and molecules are urgently needed for effective vector control. One of the most promising strategies to prevent or delay the development of resistance is to use at least two molecules having unrelated modes of action in combination in the same bed net. We evaluated in experimental huts in Côte d’Ivoire, a new polyethylene long-lasting insecticidal net (LN) product, Olyset® Duo, incorporating permethrin (PER) and pyriproxyfen (PPF), an insect growth regulator (IGR). PPF alone or in combination with permethrin had a significant impact on fertility (7–12% reduction relative to control) and no effect on fecundity of wild multi-resistant An. gambiae s.s. These results triggered crucial research questions on the behaviour of targeted mosquitoes around the LN. To maximize the sterilizing effect of PPF in the combination, there would be a need for a trade-off between the necessary contact time of the insect with PPF and the surface content of the pyrethroid insecticide that is bioavailable and induces excito-repellency.

Background The growing development of pyrethroid resistance constitutes a serious threat to malaria control programmes and if measures are not taken in time, resistance may compromise control efforts in the foreseeable future. Prior to Long Lasting Insecticidal Nets (LLINs) distribution in Eastern Cote d’Ivoire, we conducted bioassays to inform the National Malaria Control Programme of the resistance status of the main malaria vector, Anopheles gambiae s. s. and the need for close surveillance of resistance. Methods Larvae of An. gambiae s. s. were collected in two areas of Adzopé (Port-Bouët and Tsassodji) and reared to adults. WHO susceptibility tests with impregnated filter papers were carried out to detect resistance to three pyrethroids commonly used to develop LLINs: permethrin 1%, deltamethrin 0.05% and lambda-cyhalothrin 0.05%. Molecular assays were conducted to detect M and S forms and the L1014F kdr allele in individual mosquitoes. Results Resistance, at various degrees was detected in both areas of Adzopé. Overall, populations of An. gambiae at both sites surveyed showed equivalent frequency of the L1014F kdr allele (0.67) but for all tested pyrethroids, there were significantly higher survival rates for mosquitoes from Tsassodji (32–58%) than those from Port-Bouët (3–32%) (p kdr alone. During the survey period (May–June) in this forested area of Côte d’Ivoire, An. gambiae s. s. found were exclusively of the M form and were apparently selected for pyrethroid resistance through agricultural and household usage of insecticides. Conclusion Prior to LLINs scaling up in Eastern Côte d’Ivoire, resistance was largely present at various levels in An. gambiae. Underlying mechanisms included the high frequency of the L1014F kdr mutation and other unidentified components, probably metabolic detoxifiers. Their impact on the efficacy of the planned strategy (LLINs) in the area should be investigated alongside careful monitoring of the trend in that resistance over time. The need for alternative insecticides to supplement or replace pyrethroids on nets must be stressed.