Ludovic Phamien Ahoua Alou

Background: Despite the significant impact of long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) on malaria, 597,000 deaths from malaria were still recorded in 2023. Additional measures are clearly needed to complement current tools. We assessed the efficacy of insecticide-treated window screens (a piece of PermaNet 3.0 containing deltamethrin + piperonyl butoxide) versus untreated window screening and eave tube inserts treated with β-cyfluthrin as household interventions against Anopheles gambiae sensu lato . Method: Between August and October 2022, five experimental houses in Kolongonouan village, central Côte d’Ivoire, were used under semi-field conditions to evaluate the following interventions: (i) standard control house with small openings in the eaves and no window screens (SCH); (ii) insecticide-treated screening (ITS) of windows with small openings in the eaves (ITS op); (iii) ITS of windows with eaves blocked to prevent mosquito entry via eaves (ITS blq); (iv) insecticide treatment applied to the eaves using In2Care eave tubes, without window screening (ET); and (v) untreated window screening together with insecticide-treated eave tubes (SET). The efficacy of these treatments on the indoor and outdoor density of naturally recruiting Anopheles gambiae mosquitoes was assessed by human landing catches from 6:00 pm to 8:00 am. The impact of these treatments on mosquito mortality and blood-feeding rates was further assessed using release–recapture experiments, in which large enclosures were built around individual houses and approximately 100 non-blood-fed female An. gambiae were released each night over 15 nights. In addition, for the ITS blq treatment, the importance of physical integrity was assessed by evaluating the effect of damaging the screening with two or four 4 cm × 4 cm holes in each screened window. Results: Insecticide-treated window screening plus eave blocking (ITS blq) significantly reduced by 31.8% the number of An. gambiae entering houses overnight relative to standard control houses (SCH) ( P < 0.001). The use of untreated screening + eave tubes (SET) induced a similar reduction (27.0%, P < 0.001). The house entry reduction magnitude for eave tubes alone (ET) was lower (23.5%), though it remained significant relative to the control ( P < 0.001). The impact of the ITS op (insecticide-treated window screening with open eaves) was lower and nonsignificant (18.1%; P = 0.4019). There were no significant differences in house entry rates between ET, ITS blq, and ITS op ( P > 0.05). The peak in mosquito abundance within houses occurred between 11:00 pm and 3:00 am. There were no significant differences between treatments in the numbers of mosquitoes collected outdoors. In the release–recapture experiments, all treatments significantly reduced the blood-feeding rate and increased the mortality of An. gambiae , with again a similar impact for ITS blq and SET. Damaging the ITS with two holes still led to a significant reduction in blood-feeding rate, but protection was lost with four holes. Mortality rate also declined with increasing levels of damage, although it remained significantly higher than that with the untreated window screening. Conclusions: This study demonstrated that the combination (insecticide-treated window screening and blocking of eave access) reduced mosquito entry, increased mortality, and lowered blood-feeding rates of An. gambiae to levels comparable to insecticide-treated eave tubes combined with untreated window screening (SET). A previous cluster randomized controlled trial of SET in this area demonstrated a significant reduction in malaria incidence and prevalence. Insecticide-treated window screens could therefore yield a similar epidemiological impact while potentially being simpler and less expensive to implement. Larger-scale epidemiological trials involving communities are needed to test this assumption and further optimize the approach. Graphical

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. 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. 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. 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.

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

Background: A better understanding of malaria transmission at a local scale is essential for developing and implementing effective control strategies. In the frame of a randomized control trial, we aimed to provide an updated description of malaria transmission in the Korhogo area, northern Côte d’Ivoire, and to get baseline data for the trial. Methods: We performed Human Landing Collections 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 Polymerase chain reaction techniques to ascertain the species of the An. gambiae complex, Plasmodium sp sporozoite infection and insecticide resistance mechanisms in a subset of Anopheles vectors. Results: 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. Conclusions: 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.

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. 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. 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. 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.

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. 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). 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. 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.

Insecticide resistance in malaria vectors is an increasing threat to vector control tools currently deployed in endemic countries. Resistance management must be an integral part of National Malaria Control Programmes’ (NMCPs) next strategic plans to alleviate the risk of control failure. This obviously will require a clear database on insecticide resistance to support the development of such a plan. The present work gathers original data on insecticide resistance between 2009 and 2015 across Côte d’Ivoire in West Africa. Two approaches were adopted to build or update the resistance data in the country. Resistance monitoring was conducted between 2013 and 2015 in 35 sentinel sites across the country using the WHO standard procedure of susceptibility test on adult mosquitoes. Four insecticide families (pyrethroids, organochlorides, carbamates and organophosphates) were tested. In addition to this survey, we also reviewed the literature to assemble existing data on resistance between 2009 and 2015. High resistance levels to pyrethroids, organochlorides and carbamates were widespread in all study sites whereas some Anopheles populations remained susceptible to organophosphates. Three resistance mechanisms were identified, involving high allelic frequencies of kdr L1014F mutation (range = 0.46–1), relatively low frequencies of ace-1 R (below 0.5) and elevated activity of insecticide detoxifying enzymes, mainly mixed function oxidases (MFO), esterase and glutathione S-transferase (GST) in almost all study sites. This detailed map of resistance highlights the urgent need to develop new vector control tools to complement current long-lasting insecticidal nets (LLINs) although it is yet unclear whether these resistance mechanisms will impact malaria transmission control. Researchers, industry, WHO and stakeholders must urgently join forces to develop alternative tools. By then, NMCPs must strive to develop effective tactics or plans to manage resistance keeping in mind country-specific context and feasibility.

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. 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 (<20%). The high mortality rate to deltamethrin, organophosphate and the non-detection of P450 activity in resistance observed in Ehania-V1 appears as a positive outcome for further control strategies as metabolic-based P450 resistance remains major challenge to manage. These results should help the National Malaria Control Programme when designing strategies for vector control in palm oil areas of Côte d’Ivoire.

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. 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. 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. 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.

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.