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Background This study provides detailed characteristics of vector populations in preparation for a three-arm cluster randomized controlled trial (RCT) aiming to compare the community impact of dual active-ingredient (AI) long-lasting insecticidal nets (LLINs) that combine two novel insecticide classes–chlorfenapyr or pyriproxifen–with alpha-cypermethrin to improve the prevention of malaria transmitted by insecticide-resistant vectors compared to standard pyrethroid LLINs. Methods The study was carried out in 60 villages across Cove, Zangnanando and Ouinhi districts, southern Benin. Mosquito collections were performed using human landing catches (HLCs). After morphological identification, a sub-sample of Anopheles gambiae s.l. were dissected for parity, analyzed by PCR for species and presence of L1014F kdr mutation and by ELISA-CSP to identify Plasmodium falciparum sporozoite infection. WHO susceptibility tube tests were performed by exposing adult An. gambiae s.l., collected as larvae from each district, to 0.05% alphacypermethrin, 0.75% permethrin, 0.1% bendiocarb and 0.25% pirimiphos-methyl. Synergist assays were also conducted with exposure first to 4% PBO followed by alpha-cypermethrin. Results An. gambiae s.l. (n = 10807) was the main malaria vector complex found followed by Anopheles funestus s.l. (n = 397) and Anopheles nili (n = 82). An. gambiae s.l. was comprised of An. coluzzii (53.9%) and An. gambiae s.s. (46.1%), both displaying a frequency of the L1014F kdr mutation >80%. Although more than 80% of people slept under standard LLIN, human biting rate (HBR) in An. gambiae s.l. was higher indoors [26.5 bite/person/night (95% CI: 25.2–27.9)] than outdoors [18.5 b/p/n (95% CI: 17.4–19.6)], as were the trends for sporozoite rate (SR) [2.9% (95% CI: 1.7–4.8) vs 1.8% (95% CI: 0.6–3.8)] and entomological inoculation rate (EIR) [21.6 infected bites/person/month (95% CI: 20.4–22.8) vs 5.4 (95% CI: 4.8–6.0)]. Parous rate was 81.6% (95%CI: 75.4–88.4). An. gambiae s.l. was resistant to alpha-cypermethrin and permethrin but, fully susceptible to bendiocarb and pirimiphos-methyl. PBO pre-exposure followed by alpha-cypermethrin treatment induced a higher 24 hours mortality compared to alphacypermethrin alone but not exceeding 40%. Conclusions Despite a high usage of standard pyrethroid LLINs, the study area is characterized by intense malaria transmission. The main vectors An. coluzzii and An. gambiae s.s. were both highly resistant to pyrethroids and displayed multiple resistance mechanisms, L1014F kdr mutation and mixed function oxidases. These conditions of the study area make it an appropriate site to conduct the trial that aims to assess the effect of novel dual-AI LLINs on malaria transmitted by insecticide-resistant vectors.

Malaria remains the main cause of morbidity and mortality in Benin despite the scale-up of long-lasting insecticidal nets (LLINs), indoor residual spraying, and malaria case management. This study aimed to determine the malaria burden and its associated risk factors in a rural area of Benin characterized by high net coverage and pyrethroid-resistant mosquito vectors. A community-based cross-sectional survey was conducted in three districts in southern Benin. Approximately 4,320 randomly selected participants of all ages were tested for malaria using rapid diagnostic tests within 60 clusters. Risk factors for malaria infection were evaluated using mixed-effect logistic regression models. Despite high population net use (96%), malaria infection prevalence was 43.5% (cluster range: 15.1–72.7%). Children (58.7%) were more likely to be infected than adults (31.2%), with a higher malaria prevalence among older children (5–10 years: 69.1%; 10–15 years: 67.9%) compared with young children (< 5 years: 42.1%); however, young children were more likely to be symptomatic. High household density, low socioeconomic status, young age (< 15 years), poor net conditions, and low net usage during the previous week were significantly associated with malaria infection. Malaria prevalence remains high in this area of intense pyrethroid resistance despite high net use. New classes of LLINs effective against resistant vectors are therefore crucial to further reduce malaria in this area.

Monitoring vector resistance to insecticides is a major component of resistance management. Adults of Anopheles gambiae s.l. from larvae collected at thirteen sites were tested with papers impregnated with bendiocarb, permethrin; deltamethrin; pirimiphos-methyl and induced bottles of insecticide including bendiocarb; deltamethrin, permethrin and pirimiphos-methyl. Molecular analyses were performed and detoxification enzyme levels were determined for each study site. This study revealed a generalized distribution of resistance of An. gambiae s.l. to pyrethroids (permethrin and deltamethrin) and a clear resistance to carbamates (bendiocarb) especially in the northern region of the country. This vector is still susceptible to organophosphates, especially pirimiphos-methyl, but resistance is a dynamic phenomenon and it would be necessary to monitor the susceptibility of these anopheles to pyrimiphos-methyl and to find alternative vector control methods to slow down the spread of resistance genes.

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