The frequency of kdr and ace-1 alleles in Anopheles gambiae s.l. before and during indoor residual spraying (IRS) implementation and four years after IRS withdrawal in three districts in Atacora, Benin.

BACKGROUND: Indoor residual spraying (IRS) was first implemented in the Atacora department, Benin from 2011 to 2012 using bendiocarb (carbamate) followed by annual spraying with pirimiphos-methyl (organophosphate) from 2013 to 2018. Before and after IRS implementation in Atacora, standard pyrethroid insecticide-treated bed nets were the main method of vector control in the area. This study investigated the knockdown resistance (kdr) gene (L1014F) and the acetylcholinesterase (ace-1) gene (G119S), before and during IRS implementation, and 4-years after IRS withdrawal from Atacora. This was done to assess how changes in insecticide pressure from indoor residual spraying may have altered the genotypic resistance profile of Anopheles gambiae s.l. METHOD: Identification of sibling species of An. gambiae s.l. and detection of the L1014F mutation in the kdr gene and G119S mutation in ace-1 genes was done using molecular analysis. Allelic and genotypic frequencies were calculated and compared with each other before and during IRS implementation and 4 years after IRS withdrawal. The Hardy-Weinberg equilibrium and genetic differentiation within and between populations were assessed. RESULTS: Prevalence of the L1014F mutation in all geographic An. gambiae s.l. (An. gambiae s.s., Anopheles. coluzzii, Anopheles. arabiensis, and hybrids of "An. gambiae s.s. and An. coluzzii") populations increased from 69% before IRS to 87% and 90% during and after IRS. The G119S allele frequency during IRS (20%) was significantly higher than before IRS implementation (2%). Four years after IRS withdrawal, allele frequencies returned to similar levels as before IRS (3%). Four years after IRS withdrawal, the populations showed excess heterozygosity at the ace-1 gene and deficit heterozygosity at the kdr gene, whereas both genes had excess heterozygosity before and during IRS (FIS < 0). No genetic differentiation was observed within the populations. CONCLUSIONS: This study shows that the withdrawal of IRS with bendiocarb and pirimiphos-methyl may have slowed down the selection of individual mosquitoes with ace-1 resistance alleles in contrast to populations of An. gambiae s.l. with the L1014F resistance allele of the kdr gene. This may suggest that withdrawing the use of carbamates or organophosphates from IRS or rotating alternative insecticides with different modes of action may slow the development of ace-1 insecticide-resistance mutations. The increase in the prevalence of the L1014F mutation of the kdr gene in the population, despite the cessation of IRS, could be explained by the growing use of pyrethroids and DDT in agriculture and for other domestic use. More observational studies in countries where carbamates or organophosphates are still being used as public health insecticides may provide additional insights into these associations.

First report of natural infection of Anopheles gambiae s.s. and Anopheles coluzzii by Wolbachia and Microsporidia in Benin: a cross-sectional study.

BACKGROUND: Recently, bacterial endosymbiont, including Wolbachia and Microsporidia were found to limit the infection of Anopheles mosquitoes with Plasmodium falciparum. This study aimed to investigate the natural presence of key transmission-blocking endosymbionts in Anopheles gambiae and Anopheles coluzzii in Southern Benin. METHODS: The present study was conducted in seven communes (Cotonou, Porto-Novo, Aguégués, Ifangni, Pobè Athiémé, and Grand-Popo) of Southern Benin. Anopheles were collected using indoor/outdoor Human Landing Catches (HLCs) and Pyrethrum Spray Catches (PSCs). Following morphological identification, PCR was used to identify An. gambiae sensu lato (s.l.) to species level and to screen for the presence of both Wolbachia and Microsporidia. Plasmodium falciparum sporozoite infection was also assessed using ELISA. RESULTS: Overall, species composition in An. gambiae s.l. was 53.7% An. coluzzii, while the remainder was An. gambiae sensu stricto (s.s.). Combined data of the two sampling techniques revealed a mean infection prevalence with Wolbachia of 5.1% (95% CI 0.90-18.6) and 1.3% (95% CI 0.07-7.8) in An. gambiae s.s. and An. coluzzii, respectively. The mean infection prevalence with Microsporidia was 41.0% (95% CI 25.9-57.8) for An. gambiae s.s. and 57.0% (95% CI 45.4-67.9) for An. coluzzii. Wolbachia was only observed in Ifangni, Pobè, and Cotonou, while Microsporidia was detected in all study communes. Aggregated data for HLCs and PSCs showed a sporozoite rate (SR) of 0.80% (95% CI 0.09-2.87) and 0.69% (95% CI 0.09-2.87) for An. gambiae and An. coluzzii, respectively, with a mean of 0.74% (95% CI 0.20-1.90). Of the four individual mosquitoes which harboured P. falciparum, none were also infected with Wolbachia and one contained Microsporidia. CONCLUSIONS: The present study is the first report of natural infections of field-collected An. gambiae s.l. populations from Benin with Wolbachia and Microsporidia. Sustained efforts should be made to widen the spectrum of bacteria identified in mosquitoes, with the potential to develop endosymbiont-based control tools; such interventions could be the game-changer in the control of malaria and arboviral disease transmission.

Malaria transmission potential of Anopheles gambiae s.l. in indoor residual spraying areas with clothianidin 50 WG in northern Benin.

The study objective was to assess the frequency of the kdr-L995F and ace-1 G280S genetic mutations in Anopheles gambiae s.l. mosquitoes and examine their ability to transmit Plasmodium falciparum in areas where indoor residual spraying (IRS) was implemented with Clothianidin 50 WG. The study was conducted in six communes in the Alibori and Donga departments of which four were IRS-treated and two were untreated and served as control. Post-IRS monthly samples of adult mosquitoes were collected in study communes using human landing catches (HLC). An. gambiae s.l. specimens were processed to detect kdr-L995F and ace-1 G280S mutations via PCR as well as Plasmodium falciparum infectivity through CSP ELISA. Our data revealed a high and similar allelic frequency for the kdr-L995F mutation in both treated and control communes (79% vs. 77%, p = 0.14) whilst allelic frequency of the ace-1 G280S mutation was lower across the study area (2-3%, p = 0.58). The sporozoite rate was 2.6% and 2.4% respectively in treated and untreated communes (p = 0.751). No association was found between Plasmodium falciparum infection in Anopheles gambiae s.l. vectors and carriage of kdr-L995F and ace-1 G280S mutations regardless of genotypes. The study findings underline the need for an integrated approach to malaria control, combining different control methods to effectively target transmission. Regular monitoring of insecticide resistance and genetic mutations is essential to guide control strategies.

Effectiveness of pyriproxyfen-pyrethroid and chlorfenapyr-pyrethroid long-lasting insecticidal nets (LLINs) compared with pyrethroid-only LLINs for malaria control in the third year post-distribution: a secondary analysis of a cluster-randomised controlled trial in Benin.

BACKGROUND: Malaria continues to kill approximately 650 000 people each year. There is evidence that some second-generation insecticide-treated nets, which combine insecticide formulations with different modes of action, are protective against malaria while the nets are new; however, evidence for their impact over 3 years is scarce. In this study, we report the third-year results of a cluster-randomised controlled trial assessing the long-term effectiveness of dual-active ingredient long-lasting insecticidal nets (LLINs). METHODS: This is a secondary analysis of a cluster-randomised controlled trial, carried out between May 23, 2019, and April 30, 2023, in southern Benin. Restricted randomisation was used to assign 60 clusters (villages or groups of villages with a minimum of 100 households) to the three study groups (1:1:1) to evaluate the efficacy of pyriproxyfen-pyrethroid LLINs and chlorfenapyr-pyrethroid LLINs compared with pyrethroid-only LLINs (reference) against malaria transmission. The study staff and communities were masked to the group allocation. The primary outcome was malaria incidence measured over the third year after LLIN distribution, in a cohort of children aged 6 months to 9 years at the time of enrolment, in the intention-to-treat population. Here, we present the data of the third year post-LLIN distribution. The trial was registered with ClinicalTrials.gov, NCT03931473. FINDINGS: Study net use declined over the 3 years and was consistently lowest in the pyriproxyfen-pyrethroid LLIN group (at 36 months: 889 [39·4%] of 2257 participants vs 1278 [52·2%] of 2450 participants for the chlorfenapyr-pyrethroid LLIN group and 1400 [57·6%] of 2430 participants for the pyrethroid-only LLIN group). The cohort of children for the third year of follow-up (600 per group) were enrolled between April 9 and 30, 2022. Mean malaria incidence during the third year after distribution was 1·19 cases per child-year (95% CI 1·09-1·29) in the pyrethroid-only LLIN reference group, 1·21 cases per child-year (1·12-1·31) in the pyriproxyfen-pyrethroid LLIN group (hazard ratio [HR] 1·02, 95% CI 0·71-1·44; p=0·92), and 0·96 cases per child-year (0·88-1·05) in the chlorfenapyr-pyrethroid LLIN group (HR 0·80, 0·56-1·17; p=0·25). No adverse events related to study nets were reported by participants. INTERPRETATION: During the third year, as was also observed during the first 2 years, the pyriproxyfen-pyrethroid LLIN group did not have superior protection against malaria cases compared with the standard LLIN group. In the third year, people living in the chlorfenapyr-pyrethroid LLIN group no longer benefited from greater protection against malaria cases and infections than those living in the pyrethroid-only LLIN group. This was probably influenced by lower study net use than previous years and the declining concentration of partner insecticides in the nets. FUNDING: UNITAID, The Global Fund. TRANSLATION: For the French translation of the abstract see Supplementary Materials section.

Health systems impacts of the COVID-19 pandemic on malaria control program implementation and malaria burden in Benin: A mixed-method qualitative and mathematical modelling and study.

The COVID-19 pandemic has sent ripple effects across health systems and impacted the burden of many other diseases, such as malaria in sub-Saharan Africa. This study takes a mixed method approach to assess the impact of COVID-19 on malaria control programs in three rural communes in Benin. We conducted individual semi-structured interviews with key informants who play important roles in malaria control in Benin at three levels of the health system-national, health zone, and commune. Using a purposive sampling technique, informants were interviewed regarding their roles in malaria control, the impact of the pandemic on their activities, and the mitigation strategies adopted. Relevant themes were identified by content analysis. We then formulated an agent-based model of malaria epidemiology to assess the impacts of treatment disruption on malaria burden. The key informant interviews revealed that essential aspects of malaria control were upheld in Benin due to the close collaboration of public health practitioners and health care providers at all levels of the health system. There were some disruptions to case management services for malaria at the start of the pandemic due to the public avoiding health centers and a brief shortage of malaria treatment that may not be entirely attributable to the pandemic. Results from the agent-based model suggest that duration, severity, and timing of treatment disruption can impact malaria burden in a synergistic manner, though the effects are small given the relatively mild disruptions observed. This study highlights the importance of top-down leadership in health emergencies, as well as the critical role of community health workers in preventing negative health outcomes for their communities. We also showcased the integration of qualitative research and mathematical models-an underappreciated form of mixed methods research that offer immense value in the continued evaluation of rapidly evolving health emergencies.

The impact of pyrethroid-pyriproxyfen and pyrethroid-chlorfenapyr long-lasting insecticidal nets on density of primary malaria vectors Anopheles gambiae s.s. and Anopheles coluzzii in Benin: a secondary analysis of a cluster randomised controlled trial.

BACKGROUND: Long-lasting insecticidal nets (LLINs) may have different impacts on distinct mosquito vector species. We assessed the efficacy of pyrethroid-pyriproxyfen and pyrethroid-chlorfenapyr LLINs on the density of Anopheles gambiae s.s. and An. coluzzii compared to pyrethroid-only nets in a three-arm cluster randomised control trial in Benin. METHODS: Indoor and outdoor collections of adult mosquitoes took place in 60 clusters using human landing catches at baseline and every 3 months for 2 years. After morphological identification, around 15% of randomly selected samples of An. gambiae s.l. were dissected to determine parity, species (using PCR). RESULTS: Overall, a total of 46,613 mosquito specimens were collected at baseline and 259,250 in the eight quarterly collections post-net distribution. Post-net distribution, approximately 70% of the specimens of An. gambiae s.l. speciated were An. coluzzii, while the rest were mostly composed of An. gambiae s.s. with a small proportion (< 1%) of hybrids (An. gambiae/coluzzii). There was no evidence of a significant reduction in vector density indoors in either primary vector species [An. coluzzii: DR (density ratio) = 0.62 (95% CI 0.21-1.77), p = 0.3683 for the pyrethroid-pyriproxyfen LLIN and DR = 0.56 (95% CI 0.19-1.62), p = 0.2866 for the pyrethroid-chlorfenapyr LLIN, An. gambiae s.s.: DR = 0.52 (95% CI 0.18-1.46), p = 0.2192 for the pyrethroid-pyriproxyfen LLIN and DR = 0.53 (95% CI 0.19-1.46), p = 0.2222 for the pyrethroid-chlorfenapyr]. The same trend was observed outdoors. Parity rates of An. gambiae s.l. were also similar across study arms. CONCLUSIONS: Compared with pyrethroid-only LLINs, pyrethroid-chlorfenapyr LLINs and pyrethroid-pyriproxyfen LLINs performed similarly against the two primary mosquito species An. gambiae s.s. and An. coluzzii in Benin.

Presence of Plasmodium vivax in Anopheles gambiae and absence in other malaria vectors in Cove-Zagnanando-Ouinhi health zone in southern Benin, West Africa.

BACKGROUND: Malaria remains a major public health problem in sub-Saharan Africa, particularly in Benin. The present study aims to evaluate the different Plasmodium species transmitted by malaria vectors in the communes of Cove, Zagnanado and Ouinhi, Southern Benin. METHODS: The study was conducted between December 2021 and October 2022 in 60 villages spread over the three study communes. Adult mosquitoes were collected from four houses in each village using human landing catches (HLCs). After morphological identification, a subsample of Anopheles gambiae, Anopheles funestus and Anopheles nili was analysed by PCR to test for their infection to the different Plasmodium species. RESULTS: Anopheles gambiae was collected at higher frequency in all the three study communes, representing 93.5% (95% CI 92.9-94) of all collected mosquitoes (n = 10,465). In total, five molecular species were found, An. gambiae sensu stricto (s.s.) and Anopheles coluzzii of the Gambiae complex, An. funestus and Anopheles leesoni of the Funestus group, and An. nili s.s., the sole species of the Nili group. From the five molecular species, four (An. gambiae s.s., An. coluzzii, An. funestus s.s. and An. nili s.s.) were found to be infected. Plasmodium falciparum was the main Plasmodium species in the study area, followed by Plasmodium vivax and Plasmodium ovale. Only An. gambiae s.s. was infected with all three Plasmodium species, while An. coluzzii was infected with two species, P. falciparum and P. vivax. CONCLUSIONS: Plasmodium falciparum was the only species tested for in malaria vectors in Benin, and remains the only one against which most control tools are directed. It is, therefore, necessary that particular attention be paid to secondary Plasmodium species for an efficient control of the disease. The presence of P. vivax emphasizes the need for an update of case management for malaria.

["Farmer Field School", a participatory educational approach for improving the fight against malaria vectors in irrigated rice-growing areas in Benin]. / « Champ-École Paysan ¼, une approche pédagogique participative pour l'amélioration de la lutte contre les vecteurs du paludisme en zone de riziculture irriguée au Bénin.

Background & rationale: Malaria is a major health problem in Benin where it is the main cause of morbidity and mortality, particularly among children under 5 and pregnant women. Although the vast majority of malaria cases occurs in rural and agricultural areas and are often associated with development projects, very few interventions target the agro-ecosystem. In Benin, irrigated rice growing is expanding to meet the increasing demand of the population. However, continuous flooding and tillage systems induce the development and proliferation of malaria and other diseases vectors. Intermittent flooding of rice plots and minimal tillage can reduce significantly the proliferation of mosquitoes including Anopheles in rice fields. However, the dissemination and implementation of these agricultural practices require community-wide action for greater effectiveness. As part of strengthening the capacity of farming communities in the fight against malaria vectors, the "Farmer Field School" appears to be an innovative approach. This learning by doing method promotes interactions between groups of producers to disseminate proven technologies. This study aims to disseminate among rice producers the agricultural practices of intermittent flooding and minimal tillage, likely to reduce the proliferation of malaria vectors in the rice fields. Materials & methods: This study was carried out in the rice-growing perimeter of Malanville, Benin (11° 52' 5" North, 3° 22' 59" East) which covers an area of 516 hectares. Farmer Field Schools were set up after a basic survey at producer level. This survey was carried out through in-depth interview, focus group discussions and direct field observation with producers. Focus groups discussions and interviews made it possible to understand the perception of rice farmers on the link between rice production and the transmission of malaria. In order to disseminate new agricultural practices such as intermittent flooding and minimal tillage among producers, twelve plots have been set up. Farmer Field Schools were monitored weekly with rice producers accompanied by a facilitator and a medical entomologist (learning facilitator or moderator) helping the farmers with the collection and identification of mosquito larvae. According to the different stages of rice development (transplanting, tillering, maturation), the mosquito larvae were collected in the test and control plots from 10 a.m. to 2 p.m. by the dipping method. Then the water in the test compartments (intermittent flooding) was emptied. A cycle of 7 days of flooding and 2 days of drying was carried out for intermittent flooding. Mosquito larvae were identified morphologically using the identification key and Anopheles genus larvae were isolated in plastic cups. The impacts of intermittent flooding and minimum tillage in reducing breeding sites and larval densities were established by determining and comparing the larval densities of mosquitoes and of Anopheles between the test and control plots. Results: Direct observations in the field allowed to identify three tillage systems, which include the use of tiller (28%), plow (66%) and hoe (6%) on the rice field. Continuous flooding was the only irrigation system used by farmers. The water used for irrigation comes either from boreholes installed individually or from the Niger River. The volume of water used varies with the seasons, the size of the farms and the variety of rice grown. Farmers observe that the nuisance of mosquitoes increases during the rice production period with an outbreak of malaria cases, especially among children, which leads to crowds in health centers. The preventive measures against malaria among farmers are the use of impregnated mosquito nets distributed free of charge by the national program against malaria, and of insecticide bombs or spirals. Considering the development stages of the rice, the larval densities varied according to the treatments. Overall, minimal tillage applied to intermittent flooding significantly reduced the density of mosquito larvae of all species. The reduction rates were 10.5, 5.4 and 2.5 during transplanting, tillering and maturation, respectively. Considering only the Anopheles larvae, minimal tillage applied to intermittent flooding reduced their density by 16, 5.5 and 4 respectively during transplanting, tillering and maturation. Discussion/conclusion: The rice-growing area of Malanville has many favorable conditions for rice production, including the presence of water supply sources such as the Niger River located near the rice field and numerous boreholes. The availability of water pockets for mosquito breeding during irrigation appeared to contribute to the extension of malaria transmission. The present study showed that the intermittent flooding coupled with minimal tillage could reduce the proliferation of malaria vectors. The results suggested that with technical support to farmers through the "Farmer Field School", the malaria incidence could be reduced in the farming community.

Distribution and Abundance of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in Benin, West Africa.

Updated information on the distribution and abundance of Aedes aegypti and Aedes albopictus is crucial to prepare African countries, such as Benin, for possible arboviral disease outbreaks. This study aims to evaluate the geographical distribution, abundance and biting behaviour of these two vectors in Benin. Three sampling techniques were used in this study. The collection of Aedes spp. adults were made through human landing catch (HLC), immatures were captured with the use of ovitraps, and a dipping technique was used for the collection of Aedes spp. in 23 communes located along the North-South and East-West transect of Benin. Adult Aedes mosquitoes were collected indoors and outdoors using HLC. Mosquito eggs, larvae and pupae were collected from containers and ovitraps. The adult mosquitoes were morphologically identified, then confirmed using a polymerase chain reaction (PCR). Overall, 12,424 adult specimens of Aedes spp. were collected, out of which 76.53% (n = 9508) and 19.32% (n = 2400) were morphologically identified as Ae. aegypti and Ae. albopictus, respectively. Geographically, Ae. aegypti was found across the North-South transect unlike Ae. albopictus, which was only encountered in the southern part of the country, with a great preponderance in Avrankou. Furthermore, an exophagic behaviour was observed in both vectors. This updated distribution of Aedes mosquito species in Benin will help to accurately identify areas that are at risk of arboviral diseases and better plan for future vector control interventions.

Evaluating the attrition, fabric integrity and insecticidal durability of two dual active ingredient nets (Interceptor® G2 and Royal® Guard): methodology for a prospective study embedded in a cluster randomized controlled trial in Benin.

BACKGROUND: Following the World Health Organization (WHO) endorsement of dual active ingredient (AI) nets, an increased uptake of pyrethroid-chlorfenapyr and pyrethroid-pyriproxyfen nets is expected. Studies evaluating their physical and insecticidal durability are essential for making programmatic and procurement decisions. This paper describes the methodology for a prospective study to evaluate the attrition, fabric integrity, insecticidal durability of Interceptor® G2 (alpha-cypermethrin-chlorfenapyr) and Royal Guard® (alpha-cypermethrin-pyriproxyfen), compared to Interceptor® (alpha-cypermethrin), embedded in a 3-arm cluster randomized controlled trial (cRCT) in the Zou Department of Benin. METHODS: Ten clusters randomly selected from each arm of the cRCT will be used for the study. A total of 750 ITNs per type will be followed in 5 study clusters per arm to assess ITN attrition and fabric integrity at 6-, 12-, 24- and 36-months post distribution, using standard WHO procedures. A second cohort of 1800 nets per type will be withdrawn every 6 months from all 10 clusters per arm and assessed for chemical content and biological activity in laboratory bioassays at each time point. Alpha-cypermethrin bioefficacy in Interceptor® and Royal Guard® will be monitored in WHO cone bioassays and tunnel tests using the susceptible Anopheles gambiae Kisumu strain. The bioefficacy of the non-pyrethroid insecticides (chlorfenapyr in Interceptor® G2 and pyriproxyfen in Royal Guard®) will be monitored using the pyrethroid-resistant Anopheles coluzzii Akron strain. Chlorfenapyr activity will be assessed in tunnel tests while pyriproxyfen activity will be assessed in cone bioassays in terms of the reduction in fertility of blood-fed survivors observed by dissecting mosquito ovaries. Nets withdrawn at 12, 24 and 36 months will be tested in experimental hut trials within the cRCT study area against wild free-flying pyrethroid resistant An. gambiae sensu lato to investigate their superiority to Interceptor® and to compare them to ITNs washed 20 times for experimental hut evaluation studies. Mechanistic models will also be used to investigate whether entomological outcomes with each dual ITN type in experimental hut trials can predict their epidemiological performance in the cRCT. CONCLUSION: This study will provide information on the durability of two dual AI nets (Interceptor® G2 and Royal Guard®) in Benin and will help identify suitable methods for monitoring the durability of their insecticidal activity under operational conditions. The modelling component will determine the capacity of experimental hut trials to predict the epidemiological performance of dual AI nets across their lifespan.

Assessing the susceptibility and efficacy of traditional neurotoxic (pyrethroid) and new-generation insecticides (chlorfenapyr, clothianidin, and pyriproxyfen), on wild pyrethroid-resistant populations of Anopheles gambiae from southern Benin.

BACKGROUND: The objective of this study was to determine the susceptibility of wild Anopheles gambiae sensu lato (s.l.) from southern Benin to the new insecticides (chlorfenapyr (CFP), pyriproxyfen (PPF), and clothianidin (CTD)) and assess the efficacy of insecticide-treated bed nets (ITNs) that contain these new products. METHODS: Wild An. gambiae from the Benin communes of Allada, Ifangni, Akpro-Missérété, and Porto-Novo were tested for their susceptibility to CFP and PPF using the WHO bottle tests, and pyrethroids (alpha-cypermethrin, deltamethrin, and permethrin) and CTD using WHO tube tests. WHO cone tests were used to evaluate the efficacy of Interceptor® (which contains alpha-cypermethrin (ACM) only), Interceptor® G2, (CFP + ACM), and Royal Guard® nets (PPF + ACM). The ovaries of blood-fed An. gambiae from Ifangni exposed to a new PPF net were dissected, and egg development status was examined using Christopher's stages to determine the fertility status of the mosquitoes. Using a standardized protocol, the oviposition rate and oviposition inhibition rate were calculated from live blood-fed An. gambiae placed in oviposition chambers after exposure to PPF. RESULTS: In all four mosquito populations, pyrethroid mortality ranged from 5 to 80%, while chlorfenapyr and clothianidin mortality ranged from 98 to 100%. At Ifangni, all mosquitoes exposed to Royal Guard® nets were infertile (100%) while the majority (74.9%) of mosquitoes exposed to Interceptor® nets had fully developed their eggs to Christopher's stage V. The oviposition inhibition rate after exposure of the mosquitoes to the PPF was 99% for the wild population of An. gambiae s.l. and the susceptible laboratory strain, An. gambiae sensu stricto (Kisumu). CONCLUSIONS: The results of this study suggest that pyrethroid-resistant An. gambiae from the selected communes in southern Benin are susceptible to chlorfenapyr, clothianidin, and pyriproxyfen. In addition, based on bioassay results, new and unused Interceptor® G2 and Royal Guard® nets were effective on Ifangni's mosquito populations. Despite the availability of new effective insecticides, continued vigilance is needed in Benin. Therefore, monitoring of resistance to these insecticides will continue to periodically update the Benin national insecticide resistance database and management plan.

Attitudes and prevention towards malaria in the context of COVID-19 pandemic in urban community in Benin, West Africa.

BACKGROUND: The COVID-19 pandemic and its damages have severely impacted the global healthcare system even in countries with the best systems. In sub-Saharan Africa (SSA), it could worsen the malaria situation in endemic countries such as Benin. This study was conducted to describe the potential effects of the pandemic on urban dwellers attitudes, prevention and treatment against malaria in four major cities of Benin. METHODS: A cross-sectional questionnaire survey was conducted in Cotonou, Bohicon, Parakou and Natitingou, four urban cities of Benin. A total of 800 randomly selected households were interviewed. The questionnaire consisted of four parts: sociodemographic characteristics, knowledge, attitude, and practice. Descriptive statistics and binomial logistic regression analysis were used in the statistical analysis. RESULTS: More than 90% of the participants interviewed had a good level of knowledge about the transmission and prevention of malaria in the cities surveyed. In contrast, low proportions of participants reported visiting health-care facilities when they suspected malaria. Compared to the proportions observed at Parakou and Natitingou, the low proportion of participants was statistically significant at Cotonou (Parakou: X2 = 31.345, df = 1, P < 0.0001; Natitingou: X2 = 17.471, df = 1, P < 0.0001). Among the reasons for not seeking care, these related to COVID-19 were the most mentioned. Moreover, the good education level of the participants was one of the factors associated with the non-use of healthcare facilities due to over-knowledgeable about Covid-19, which might have increased the fear to go to the health facilities. Finally, high proportions of self-medication practice were mentioned with high use of malaria drugs to treat both malaria and to protect against COVID-19. CONCLUSIONS: The data show a negative impact of COVID-19 on visits to healthcare facilities for malarial treatment and malaria drugs usage by the population. It is, therefore, necessary to rebuild malaria programmes by integrating measures adapted to health crises such as the COVID-19 pandemic.

Field performance of three mosquito collection methods for assessing the entomological efficacy of dual-active ingredient long-lasting insecticidal nets.

Selection of mosquito collection methods is of crucial importance to evaluate the impact of vector control tools on entomological outcomes. During a cluster randomised control trial evaluating the relative efficacy of two dual-active ingredient (a.i.) long-lasting insecticidal nets (LLINs) compared to pyrethroid-only LLINs, we assessed the performance of different mosquito collection methods: Human landing catches (HLC), Centers for Disease Control and Prevention (CDC) light traps, and pyrethrum spray catches (PSC). Anopheles mosquitoes were collected using three collection methods in 4 houses, in each of the 60 trial clusters at baseline and every quarter for 24 months using PSCs and HLCs, while CDC light traps were performed during two quarters only. Mean density of vectors collected per method per night was the highest with HLCs (15.9), followed by CDC light traps (6.8); with PSCs (1.1) collecting 10 times less mosquitoes than HLCs. All three collection methods collected fewer mosquitoes in the Interceptor G2® dual a.i. arm, compared to the other trial arms, although only HLCs and PSCs demonstrated strong evidence of this due to a greater number of collection rounds undertaken, than CDC light traps. The broadly similar results regarding the differential impact of the two dual a.i. LLINs showed by the three collection methods suggest that the more ethically acceptable, cheaper, and logistically simpler methods such as CDC light traps could be prioritised for use in large community trials for measuring the efficacy of vector control tools.

What can be learned from the residual efficacy of three formulations of insecticides (pirimiphos-methyl, clothianidin and deltamethrin mixture, and clothianidin alone) in large-scale in community trial in North Benin, West Africa?

BACKGROUND: In Alibori and Donga, two departments of high malaria incidence of Northern Benin, pirimiphos-methyl, mixture deltamethrin + clothianidin, as well as clothianidin were used at large scale for IRS. The present study aimed to assess the residual efficacy of these products. METHODS: Immatures of Anopheles gambiae sensu lato (s.l.) collected in the communes of Kandi and Gogounou (Department of Alibori), Djougou and Copargo (Department of Donga) were reared until adulthood. Females aged 2-5 days were used for susceptibility tube tests following the WHO protocol. The tests were conducted with deltamethrin (0.05%), bendiocarb (0.1%), pirimiphos-methyl (0.25%) and clothianidin (2% weight per volume). For cone tests performed on cement and mud walls, the An. gambiae Kisumu susceptible strain was used. After the quality control of the IRS performed 1-week post-campaign, the evaluation of the residual activity of the different tested insecticides/mixture of insecticides was conducted on a monthly basis. RESULTS: Over the three study years, deltamethrin resistance was observed in all the communes. With bendiocarb, resistance or possible resistance was observed. In 2019 and 2020, full susceptibility to pirimiphos-methyl was observed, while possible resistance to the same product was detected in 2021 in Djougou, Gogounou and Kandi. With clothianidin, full susceptibility was observed 4-6 days post-exposure. The residual activity lasted 4-5 months for pirimiphos-methyl, and 8-10 months for clothianidin and the mixture deltamethrin + clothianidin. A slightly better efficacy of the different tested products was observed on cement walls compared to the mud walls. CONCLUSION: Overall, An. gambiae s.l. was fully susceptible to clothianidin, while resistance/possible resistance was observed the other tested insecticides. In addition, clothianidin-based insecticides showed a better residual activity compared to pirimiphos-methyl, showing thus their ability to provide an improved and prolonged control of pyrethroid resistant vectors.

Efficacy of Pyrethroid-Pyriproxyfen and Pyrethroid-Chlorfenapyr Long-Lasting Insecticidal Nets (LLINs) for the Control of Non-Anopheles Mosquitoes: Secondary Analysis from a Cluster Randomised Controlled Trial (cRCT).

The efficacy of a vector control tool in reducing mosquito biting is crucial for its acceptability. The present study compared the vector density of Culex spp. And Mansonia spp. across clusters, which received two dual-active ingredient (a.i.) long-lasting insecticidal nets (LLINs) and a standard pyrethroid-only LLIN, and assessed the seasonality of these mosquito genera. A total of 85,723 Culex spp. and 144,025 Mansonia spp. were caught over the study period. The density of Culex and Mansonia was reduced in all three arms over the study period. There was no evidence of a significant reduction in the indoor or outdoor density of Culex spp. in either dual-a.i. LLIN arm as compared to the standard pyrethroid-only net arm. A similar trend was observed with Mansonia spp. A high density of Culex spp. was found both in rainy and dry seasons, while for Mansonia spp., this was mainly observed during the rainy season. These results suggest that the novel insecticides in the dual-a.i. LLINs did not have an additional impact on these species and that pyrethroids might still be effective on them. Further work is required to determine whether these species of mosquitoes have resistance to the insecticides tested in this trial.

Community-level impacts of the coronavirus pandemic on malaria prevention and health-seeking behaviours in rural Benin: A mixed methods study.

Globally, negative impacts of the COVID-19 pandemic on malaria prevention and control efforts have been caused by delayed distributions of long-lasting insecticidal nets (LLIN), decreased outpatient attendance, and disruptions to malaria testing and treatment. Using a mixed methods approach, we aimed to evaluate the impact of COVID-19 on community-level malaria prevention and health-seeking practices in Benin more than one year after the start of the COVID-19 pandemic. We collected data through community-based cross-sectional surveys with 4200 households and ten focus group discussions (FGDs). Mixed effect logistic regression models accounting for a clustered sampling design were used to identify variables associated with main outcomes (good COVID-19 knowledge, LLIN usage and access, and avoidance of health centres). Consistent with the experiences of FGD participants, receiving information from radios or televisions was significantly associated with good COVID-19 knowledge and avoiding health centres because of the pandemic (p<0.001 for both). Qualitative findings also revealed varying and polarizing changes in health-seeking behaviours with participants noting that they either did not change their health-seeking behaviours or went to health centres less or more often because of the pandemic. LLIN usage and access did not decrease in the study area because of the pandemic (LLIN usage: 88% in 2019 to 99.9% in 2021; LLIN access: 62% in 2019 to 73% in 2021). An unexpected change and unintended challenge for sustained malaria prevention included families socially distancing in their homes, resulting in a shortage of LLINs. Our findings showed that there were minimal community-level impacts of the coronavirus pandemic on malaria prevention and health seeking behaviours in rural Benin, which highlights the importance of efforts to sustain malaria prevention and control interventions in the context of the COVID-19 pandemic.

Insecticide resistance status of Aedes aegypti and Aedes albopictus mosquitoes in southern Benin, West Africa.

BACKGROUND: The emergence of insecticide resistance in Aedes mosquitoes could undermine efforts to control arboviruses. The present study aims to assess in some communes of Southern Benin, the susceptibility level of Aedes aegypti (Linnaeus, 1762) and Aedes albopictus (Skuse, 1894) to insecticides commonly used in public health, as well as mechanisms involved. METHODS: Females Ae. albopictus and Ae. aegypti collected in Ifangni, Porto-Novo, Avrankou, Adjarra and Kétou from June 2021 to October 2022, were exposed to: deltamethrin 0.05%, permethrin 0.75%, alpha-cypermethrin 0.05%, pirimiphos methyl 0.25% and bendiocarb 0.1%, following the standard WHO susceptibility tube test protocol. In some sites, pre-exposure to the synergist PBO was used to verify if pyrethroid resistance of populations of Aedes was mediated by oxidases. RESULTS: Full susceptibility to deltamethrin and permethrin was observed in all tested populations of Ae. albopictus. However, with alphacypermethrin, a suspected resistance was observed in Adjarra (94.67%), Ifangni (93%) and Porto-Novo (94%), and a resistance in Avrankou (83%). The PBO-alphacypermethrin tests performed, led to a full susceptibility (100%) in all four sites, which confirms the full involvement of oxidases in resistance of all tested populations of Ae. albopictus to alphacypermethrin. At the opposite, Aedes aegypti was either resistant or suspected of being resistant to all tested pyrethroids in all four sites, except in Ifangni where a full susceptibility to alphacypermethrin was observed. The full susceptibility of Ae. aegypti to bendiocarb and pirimiphos-methyl in all communes suggests that these two insecticides can be good candidates for an effective control of pyrethroid-resistant Aedes vector populations. Use of permethrin and deltamethrin could also be considered for controlling populations of Ae. albopictus. CONCLUSION: Results of the present study will help guide strategy to implement for an effective control of Aedes vector populations in Benin.

Trophic preferences of Anopheles coluzzii (Diptera: Culicidae): what implications for malaria vector control in Benin?

The main objective of the present study is to assess the preferences in terms of vertebrate hosts of Anopheles coluzzii, the main malaria vector in the pastoral area of Malanville, Benin, where rice cultivation and livestock are the main source of income for the populations. Adult mosquitoes were collected through pyrethrum spray catch, and human landing catch in two communes in Benin: Malanville, a pastoral area, and Porto-Novo, a nonpastoral area. Molecular species identification was performed through PCR within the Anopheles gambiae complex. Blood meal origin and P. falciparum sporozoite infection were determined using ELISA blood meal and circumsporozoite protein tests, respectively. Overall, 97% of females of An. gambiae s.l. were An. coluzzii, with biting behavior more pronounced outdoors in the pastoral area. In Malanville, the main vertebrate hosts on which females An. coluzzii blood fed were goats (44%), humans (24.29%), bovines (22%), and pigs (1.4%). Our results also showed that single-host blood meals (human: 24.29% or animal: 68%) were mostly observed compared to mixed blood meals (8.58%). The human biting rate (HBR) and P. falciparum sporozoite rate (SR) of An. coluzzii were 66.25 bites/man/night and 0.77%, respectively. However, in the nonpastoral zone (Porto-novo), 93.98% of samples were An. coluzzii. The latter blood-fed mostly (86.84%) on humans, with an estimated HBR of 21.53 b/m/n and SR of 5.81%. The present study revealed an opportunistic and zoophagic behavior of An. coluzzii in the Malanville area with an overall low mean SR.

Evidence of Transmission of Plasmodium vivax 210 and Plasmodium vivax 247 by Anopheles gambiae and An. coluzzii, Major Malaria Vectors in Benin/West Africa.

Current diagnostic and surveillance systems in Benin are not designed to accurately identify or report non-Plasmodium falciparum (Pf) human malaria infections. This study aims to assess and compare the prevalence of circumsporozoite protein (CSP) antibodies of Pf and P. vivax (Pv) in Anopheles gambiae s.l. in Benin. For that, mosquito collections were performed through human landing catches (HLC) and pyrethrum spray catches (PSC). The collected mosquitoes were morphologically identified, and Pf, Pv 210, and Pv 247 CSP antibodies were sought in An. gambiae s.l. through the ELISA and polymerase chain reaction (PCR) techniques. Of the 32,773 collected mosquitoes, 20.9% were An. gambiae s.l., 3.9% An. funestus gr., and 0.6% An. nili gr. In An. gambiae s.l., the sporozoite rate was 2.6% (95% CI: 2.1-3.1) for Pf, against 0.30% (95% CI: 0.1-0.5) and 0.2% (95% CI: 0.1-0.4), respectively, for Pv 210 and Pv 247. P. falciparum sporozoite positive mosquitoes were mostly An. gambiae (64.35%), followed by An. coluzzii (34.78%) and An. arabiensis (0.86%). At the opposite, for the Pv 210 sporozoite-positive mosquitoes, An. coluzzii and An. gambiae accounted for 76.92% and 23.08%, respectively. Overall, the present study shows that P. falciparum is not the only Plasmodium species involved in malaria cases in Benin.

Entomological Characteristics of Malaria Transmission across Benin: An Essential Element for Improved Deployment of Vector Control Interventions.

Entomological surveillance in Benin has historically been limited to zones where indoor residual spraying was performed or where long-standing sentinel surveillance sites existed. However, there are significant country-wide gaps in entomological knowledge. The National Malaria Control Program (NMCP) assessed population dynamics of Anopheles vectors and malaria transmission in each of Benin's 12 departments to create an entomological risk profile. Two communes per department (24/77 communes) were chosen to reflect diverse geographies, ecologies and malaria prevalence. Two villages per commune were selected from which four households (HH) per village were used for human landing catches (HLCs). In each HH, an indoor and outdoor HLC occurred between 7 p.m. and 7 a.m. on two consecutive nights between July−September 2017. Captured Anopheles were identified, and ovaries were dissected to determine parous rate. Heads and thoraces were tested for Plasmodium falciparum sporozoites by ELISA. The Entomological Inoculation Rate (EIR) was calculated as the product of mosquito bite rate and sporozoite index. Bite rates from An. gambiae s.l., the primary vector species complex, differed considerably between communes; average sporozoite infection index was 3.5%. The EIR ranged from 0.02 infectious bites (ib) per human per night in the departments of Ouémé and Plateau to 1.66 ib/human/night in Collines. Based on transmission risk scales, Avrankou, Sakété and Nikki are areas of low transmission (0 < EIR < 3 ib/human/year), Adjarra, Adja Ouèrè, Zè, Toffo, Bopa, Pehunco, Pèrèrè and Kandi are of medium transmission (3 < EIR < 30 ib/human/year), and the other remaining districts are high transmission (EIR > 30 ib/human/year). The heterogeneous and diverse nature of malaria transmission in Benin was not readily apparent when only assessing entomological surveillance from sentinel sites. Prospectively, the NMCP will use study results to stratify and deploy targeted vector control interventions in districts with high EIRs to better protect populations most at-risk.