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Background: Studies evaluating the attrition, physical and insecticidal durability of dual active ingredient (AI) insecticide-treated nets (ITNs) are essential for making programmatic decisions regarding their deployment. We performed a prospective study embedded in a cluster randomised controlled trial (cRCT) to evaluate the attrition, fabric integrity and insecticidal durability of Interceptor® G2 (alpha-cypermethrin-chlorfenapyr) and Royal Guard® (alpha-cypermethrin-pyriproxyfen), compared to Interceptor® (alpha-cypermethrin) in Benin., Methods: A total of 2428 study nets in 1093 randomly selected households in five clusters per arm of the cRCT were monitored for ITN attrition and fabric integrity every 6-12 months post-distribution. Householders were further surveyed to investigate non-study net use and their preference for ITN fabric types used in the study nets. A second cohort of 120 nets per ITN type were withdrawn every 12 months and assessed for chemical content and insecticidal activity in laboratory bioassays. Alpha-cypermethrin bioefficacy was investigated using the susceptible Anopheles gambiae Kisumu strain, and chlorfenapyr and pyriproxyfen bioefficacy were investigated using the pyrethroid-resistant Anopheles coluzzii Akron strain. Net pieces were tested in WHO cone bioassays and tunnel tests for alpha-cypermethrin and in tunnel tests for chlorfenapyr; pyriproxyfen activity was assessed in cone bioassays as the reduction in fertility of blood-fed survivors using ovary dissection. Bioefficacy was expressed as the proportion of ITNs passing predetermined WHO criteria, namely knock-down ≥ 95% or 24/72 h mortality ≥ 80% or reduction in fertility ≥ 50%., Results: Overall ITN survivorship was 52% at 24 months and fell to 15% at 36 months. Median ITN survival time was lower with Royal Guard® relative to Interceptor® [1.6 vs 2.3 years; hazard ratio (HR) 1.49, 95% confidence interval (CI) 1.36-1.66; p < 0.001] and Interceptor® G2 (1.6 vs 2.1 years; HR 1.33, 95% CI 1.20-1.47; p < 0.001). Householders overwhelmingly preferred polyester nets over polyethylene nets (96%), and more Royal Guard® nets were replaced with spare polyester nets from previous campaigns. All Royal Guard® nets passed efficacy criteria for alpha-cypermethrin at all time points (100%) while ITN pass rates after 24 months had fallen to < 40% for pyriproxyfen and chlorfenapyr. The chemical content analysis showed a higher loss rate of the non-pyrethroid insecticides relative to the pyrethroids in each dual ingredient AI ITN; 74% vs 47% for Royal Guard® and 85% vs 63% for Interceptor® G2 at 36 months., Conclusions: The median ITN survival time for Interceptor® G2 (2.1 years) and Royal Guard® (1.6 years) in Benin is substantially lower than 3 years. Royal Guard® nets were discarded more quickly by householders, partly due to their low preference for polyethylene nets. The insecticidal activity of the non-pyrethroid insecticides in both dual AI ITNs was short-lived compared to alpha-cypermethrin. The results corroborate the findings from the cRCT conducted in Benin., (© 2024. The Author(s).)

Background: Spatial repellents can provide personal and household protection against biting vector mosquitoes by volatizing repellents into the air within a given area. Mosquito Shield™ is a transfluthrin passive emanator undergoing evaluation for malaria control. Studies evaluating its entomological impact against different local malaria vector populations would help guide its deployment in endemic countries., Methods: A two-arm single-blinded small-scale household randomised entomological trial was conducted to assess the impact of Mosquito Shield™ on the human landing rate of wild pyrethroid-resistant Anopheles gambiae sensu lato (s.l.) vector mosquitoes in houses in the Ganhoua village of the Zakpota District of central Benin. From a total of 30 houses, 15 were randomly allocated to receive Mosquito Shield™, while the remainder received a placebo product. The trial lasted through the life of the Mosquito Shield™ product (32 days). Mosquito sampling was performed by human landing catches at baseline and at 6 timepoints post-intervention (days 0-1, 7-8, 14-15, 21-22, 28-29 and 31-32). Collections were performed for 2 nights at each sampling time point. WHO cylinder bioassays were conducted during the trial with F1 An. gambiae s.l. mosquitoes that emerged from larvae from the study area to assess the intensity of resistance to pyrethroids in the wild vector population., Results: The vector population in the study area showed a high intensity of resistance to pyrethroids. Baseline An. gambiae s.l. human landing rates were similar in houses in both study arms before product application (11.53/person/night vs 11.67/person/night, p > 0.05). A total of 5736 mosquitoes were collected in the placebo control arm and 3862 in the Mosquito Shield™ arm post-intervention. Overall An. gambiae s.l. post-intervention human landing rates were significantly lower in houses in the Mosquito Shield™ arm (18.13/person/night) compared to the houses in the placebo control arm (26.84/person/night, IRR = 0.658, p < 0.001). Over the lifespan of the product, Mosquito Shield™ provided a significant protective efficacy of 34.2% (22.1-44.4%, p < 0.001) against wild pyrethroid-resistant An. gambiae s.l. vectors compared to the placebo. Human landing rates of other nuisance vector mosquito species (Culex and Mansonia) were also reduced in houses treated with Mosquito Shield™ compared to the placebo., Conclusion: Mosquito Shield™, a transfluthrin passive emanator, provided significant protection against pyrethroid-resistant malaria vectors to households in Benin. The spatial repellent shows potential to reduce malaria transmission by pyrethroid-resistant An. gambiae s.l. vector mosquitoes and cover gaps in malaria control when deployed to complement existing vector control interventions., (© 2024. The Author(s).)

Background: Malaria transmission is known to be perennial and heterogeneous in Benin. Studies assessing local malaria prevalence, transmission levels and vector characteristics are critical for designing, monitoring and evaluating new vector control interventions in community trials. We conducted a study in the Zakpota sub-district of central Benin to collect baseline data on household characteristics, malaria prevalence, vector characteristics and transmission dynamics in preparation for a randomised controlled trial to evaluate the community impact of VECTRON™ T500, a new broflanilide indoor residual spraying (IRS) product., Methods: A total of 480 children under 5 years of age from the 15 villages of the sub-district were tested for malaria by rapid diagnostic tests (RDTs). Mosquitoes were collected by human landing catches (HLCs), pyrethrum spray catches (PSCs) and Centers for Disease Control and Prevention miniature light traps (CDC-LTs) in selected houses in each village to assess vector density, composition, vector infectivity and prevalence of insecticide resistance markers. Bioassays were performed to detect vector susceptibility to pyrethroids, broflanilide (6 µg/bottle) and clothianidin (90 µg/bottle)., Results: A total of 9080 households were enumerated in the 15 study villages. Insecticide-treated net (ITN) usage was > 90%, with 1-2 ITNs owned per household. Houses were constructed mainly with cement (44%) and mud (38%) substrates or a mixture of cement and mud (18%), and 60% of them had open eaves. The overall prevalence of P. falciparum infection was 19% among surveyed children: 20% among females and 18% among males. The haemoglobin rate showed an anaemia (< 11 g/dl) prevalence of 66%. Anopheles coluzzii and An. gambiae sensu stricto (s.s.) were the two vector species present at an overall proportion of 46% versus 54%, respectively. The human biting rate was 2.3 bites per person per night (b/p/n) and biting occurred mostly indoors compared with outdoors (IRR = 0.776; P = 0.001). The overall proportion of outdoor biting was 44% and exceeded indoor biting in three villages. The sporozoite rate was 2% with a combined yearly entomological inoculation rate (EIR) of 16.1 infected bites per person per year (ib/p/y). There was great variability in malaria transmission risk across the villages, with EIR ranging from 0 to 29.3 ib/p/y. The vector population showed a high intensity of resistance to pyrethroids across the study villages but was largely susceptible to broflanilide and clothianidin., Conclusions: This study found high levels of malaria prevalence, vector density and transmission in the Zakpota sub-district despite the wide use of insecticide-treated nets. The vector population was mostly indoor resting and showed a high intensity of pyrethroid resistance but was generally fully susceptible to broflanilide. These findings demonstrated the suitability of the study area for the assessment of VECTRON™ T500 in a community randomised trial., (© 2024. The Author(s).)

Background: Nets containing pyriproxyfen, an insect growth regulator that sterilizes adult mosquitoes, have become available for malaria control. Suitable methods for investigating vector susceptibility to pyriproxyfen and evaluating its efficacy on nets need to be identified. The sterilizing effects of pyriproxyfen on adult malaria vectors can be assessed by measuring oviposition or by dissecting mosquito ovaries to determine damage by pyriproxyfen (ovary dissection)., Method: Laboratory bioassays were performed to compare the oviposition and ovary dissection methods for monitoring susceptibility to pyriproxyfen in wild malaria vectors using WHO bottle bioassays and for evaluating its efficacy on nets in cone bioassays. Blood-fed mosquitoes of susceptible and pyrethroid-resistant strains of Anopheles gambiae sensu lato were exposed to pyriproxyfen-treated bottles (100 μg and 200 μg) and to unwashed and washed pieces of a pyriproxyfen long-lasting net in cone bioassays. Survivors were assessed for the sterilizing effects of pyriproxyfen using both methods. The methods were compared in terms of their reliability, sensitivity, specificity, resources (cost and time) required and perceived difficulties by trained laboratory technicians., Results: The total number of An. gambiae s.l. mosquitoes assessed for the sterilizing effects of pyriproxyfen were 1745 for the oviposition method and 1698 for the ovary dissection method. Fertility rates of control unexposed mosquitoes were significantly higher with ovary dissection compared to oviposition in both bottle bioassays (99-100% vs. 34-59%, P < 0.05) and cone bioassays (99-100% vs. 18-33%, P < 0.001). Oviposition rates of control unexposed mosquitoes were lower with wild pyrethroid-resistant An. gambiae s.l. Cové, compared to the laboratory-maintained reference susceptible An gambiae sensu stricto Kisumu (18-34% vs. 58-76%, P < 0.05). Sterilization rates of the Kisumu strain in bottle bioassays with the pyriproxyfen diagnostic dose (100 μg) were suboptimal with the oviposition method (90%) but showed full susceptibility with ovary dissection (99%). Wild pyrethroid-resistant Cové mosquitoes were fully susceptible to pyriproxyfen in bottle bioassays using ovary dissection (> 99%), but not with the oviposition method (69%). Both methods showed similar levels of sensitivity (89-98% vs. 89-100%). Specificity was substantially higher with ovary dissection compared to the oviposition method in both bottle bioassays (99-100% vs. 34-48%) and cone tests (100% vs.18-76%). Ovary dissection was also more sensitive for detecting the residual activity of pyriproxyfen in a washed net compared to oviposition. The oviposition method though cheaper, was less reliable and more time-consuming. Laboratory technicians preferred ovary dissection mostly due to its reliability., Conclusion: The ovary dissection method was more accurate, more reliable and more efficient compared to the oviposition method for evaluating the sterilizing effects of pyriproxyfen on adult malaria vectors in susceptibility bioassays and for evaluating the efficacy of pyriproxyfen-treated nets., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

Background: Pyrethroid-PBO nets have demonstrated improved impact against clinical malaria transmitted by pyrethroid resistant mosquito vectors and are being scaled up across Africa. However very little is known about their physical and insecticidal durability under operational conditions. This study will investigate the attrition, fabric integrity, insecticide content and bioefficacy of DuraNet® Plus, a new WHO prequalified alphacypermethrin and PBO incorporated net developed by Shobikaa Impex Private Limited over 3 years of field use in communities in Benin, Cameroon and Tanzania., Methods: The study will be conducted in parallel in selected villages in Zakpota District in Benin, Mbalmayo, District in Cameroon and Muheza District in Tanzania. In each country, ~ 1800 households will be recruited and randomised to receive DuraNet® Plus or DuraNet® (a WHO prequalified alphacypermethrin-only ITN). Follow up surveys will be performed at 1 month post distribution to investigate adverse events and subsequently every 6-12 months to assess ITN attrition and fabric integrity following standard WHO procedures. A second cohort of nets will be withdrawn every 6-12 months and assessed for alpha-cypermethrin and PBO content and for entomological activity in laboratory bioassays (cone bioassays and tunnel tests). Alpha-cypermethrin bioefficacy will be monitored using the susceptible Anopheles gambiae Kisumu strain in cone bioassays while PBO bioefficacy will be monitored using pyrethroid resistant strains with overexpressed P450 enzymes in tunnel tests to determine the proportion of efficacious nets (≥ 95% knockdown, ≥ 80% mortality or ≥ 90% blood feeding inhibition in tunnels) at each time point. Nets withdrawn at 12, 24 and 36 months from each country will also be tested in experimental hut trials against wild free-flying pyrethroid resistant Anopheles gambiae sl in Côvè Benin to investigate the superiority of DuraNet® Plus over DuraNet® at each time point under semi field conditions., Conclusion: This large-scale multi country trial will provide useful information on the durability of a pyrethroid-PBO net (DuraNet® Plus) in 3 different regions in sub-Saharan Africa. The methods proposed for bioefficacy testing could also contribute towards the development of new standardised guidelines for monitoring the insecticidal efficacy of pyrethroid-PBO nets under operational conditions., (© 2023. The Author(s).)

VECTRON™ T500 is a wettable powder IRS formulation of broflanilide, a newly discovered insecticide. We performed a two-arm non-inferiority community randomised evaluation of VECTRON™ T500, compared to Fludora® Fusion against pyrethroid-resistant Anopheles gambiae s.l. in an area of high coverage with pyrethroid-only nets in the Za-Kpota District of central Benin. One round of IRS was applied in all consenting households in the study area. Sixteen clusters were randomised (1:1) to receive VECTRON™ T500 (100 mg/m 2 for broflanilide) or Fludora® Fusion (200 mg/m 2 for clothianidin and 25 mg/m 2 for deltamethrin). Surveys were performed to assess adverse events and the operational feasibility and acceptability of VECTRON™ T500 among spray operators and household inhabitants. Human landing catches were conducted in 6 households every 1-2 months for up to 18 months post-intervention to assess the impact on vector densities, sporozoite rates and entomological inoculation rates. Bottle bioassays were performed to monitor vector susceptibility to pyrethroids, broflanilide and clothianidin. Monthly wall cone bioassays were conducted for 24 months to assess the residual efficacy of the IRS formulations using susceptible and pyrethroid-resistant An. gambiae s.l. A total of 26,562 female mosquitoes were collected during the study, of which 40% were An. gambiae s.l., the main malaria vector in the study area. The vector population showed high intensity pyrethroid resistance but was susceptible to broflanilide (6 µg/bottle) and clothianidin (90 µg/bottle). Using a non-inferiority margin of 50%, vector density indicated by the human biting rate (bites/person/night) was non-inferior in the VECTRON™ T500 arm compared to the Fludora® Fusion arm both indoors (0.846 bites/p/n in Fludora® Fusion arm vs. 0.741 bites/p/n in VECTRON™ T500 arm, IRR 0.54, 95% CI 0.22-1.35, p = 0.150) and outdoors (0.691 bites/p/n in Fludora® Fusion arm vs. 0.590 bites/p/n in VECTRON™ T500 clusters, IRR 0.75, 95% CI 0.41-1.38, p = 0.297). Sporozoite rates and entomological inoculation rates did not differ significantly between study arms (sporozoite rate: 0.9% vs 1.1%, p = 0. 0.746, EIR: 0.008 vs 0.006 infective bites per person per night, p = 0.589). Cone bioassay mortality with both VECTRON™ T500 and Fludora® Fusion was 100% for 24 months post-IRS application on both cement and mud treated house walls with both susceptible and pyrethroid-resistant strains of An. gambiae s.l. Perceived adverse events reported by spray operators and householders were generally very low (< 6%) in both study arms. VECTRON™ T500 was non-inferior to Fludora® Fusion in reducing the risk of malaria transmission by pyrethroid resistant vectors when applied for IRS in communities in central Benin. The insecticide showed prolonged residual efficacy on house walls, lasting over 24 months and had a high acceptability with homeowners. Community application of VECTRON™ T500 for IRS provides improved and prolonged control of pyrethroid resistant malaria vectors and enhances our capacity to manage insecticide resistance., (© 2023. Springer Nature Limited.)

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., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: Broflanilide is a new insecticide being developed for malaria vector control. As new insecticide chemistries become available, strategies to preserve the susceptibility of local malaria vectors and extend their useful life need to be considered before large scale deployment. This requires the development of appropriate testing procedures and identification of suitable discriminating concentrations for monitoring susceptibility in wild vector populations to facilitate decision making by control programmes., Methods: Dose-response WHO bottle bioassays were conducted using the insecticide-susceptible Anopheles gambiae s.s. Kisumu strain to determine a discriminating concentration of broflanilide. Bioassays were performed without the adjuvant Mero® and with two concentrations of Mero® (500 ppm and 800 ppm) to investigate its impact on the discriminating concentration of the insecticide. Probit analysis was used to determine the lethal doses at 50% (LC50) and 99% (LC99) at 24-, 48- and 72-hours post-exposure. Cross-resistance to broflanilide and pyrethroids, DDT, dieldrin and carbamates, was investigated using An. gambiae s.l. Covè and An. coluzzii Akron strains. The susceptibility of wild pyrethroid-resistant mosquitoes from communities in Southern Benin to broflanilide was assessed using the estimated discriminating concentrations., Results: Broflanilide induced a dose-dependent and delayed mortality effect. Mortality rates in bottles treated without Mero® were <80% using the range of broflanilide doses tested (0-100 μg/bottle) leading to high and unreliable estimates of LC99 values. The discriminating concentrations defined as 2XLC99 at 72h post exposure were estimated to be 2.2 μg/bottle with 800 ppm of Mero® and 6.0 μg/bottle with 500 ppm of Mero®. Very low resistance ratios (0.6-1.2) were determined with the insecticide resistant An. gambiae s.l. Covè and An. coluzzii Akron strains suggesting the absence of cross-resistance via the mechanisms of resistance to pyrethroids, DDT, dieldrin and carbamates they possess. Bottle bioassays performed with broflanilide at both discriminating concentrations of 6 μg/bottle with 500 ppm of Mero® and 2.2 μg/bottle with 800 ppm of Mero®, showed susceptibility of wild highly pyrethroid-resistant An. gambiae s.l. from villages in Southern Benin., Conclusion: We determined discriminating concentrations for monitoring susceptibility to broflanilide in bottle bioassays, using susceptible An. gambiae vectors. Using the estimated discriminating concentrations, we showed that wild pyrethroid-resistant populations of An. gambiae s.l. from southern Benin were fully susceptible to the insecticide. Broflanilide also shows potential to be highly effective against An. gambiae s.l. vector populations that have developed resistance to other public health insecticides., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Govoetchan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Broflanilide is a newly discovered insecticide with a novel mode of action targeting insect γ-aminobutyric acid receptors. The efficacy of VECTRON ™ T500, a wettable powder formulation of broflanilide, was assessed for IRS against wild pyrethroid-resistant malaria vectors in experimental huts in Benin., Methods: VECTRON ™ T500 was evaluated at 100 mg/m 2 in mud and cement-walled experimental huts against wild pyrethroid-resistant Anopheles gambiae sensu lato (s.l.) in Covè, southern Benin, over 18 months. A direct comparison was made with Actellic® 300CS, a WHO-recommended micro-encapsulated formulation of pirimiphos-methyl, applied at 1000 mg/m 2 . The vector population at Covè was investigated for susceptibility to broflanilide and other classes of insecticides used for vector control. Monthly wall cone bioassays were performed to assess the residual efficacy of VECTRON ™ T500 using insecticide susceptible An. gambiae Kisumu and pyrethroid-resistant An. gambiae s.l. Covè strains. The study complied with OECD principles of good laboratory practice., Results: The vector population at Covè was resistant to pyrethroids and organochlorines but susceptible to broflanilide and pirimiphos-methyl. A total of 23,171 free-flying wild pyrethroid-resistant female An. gambiae s.l. were collected in the experimental huts over 12 months. VECTRON ™ T500 induced 56%-60% mortality in wild vector mosquitoes in both cement and mud-walled huts. Mortality with VECTRON ™ T500 was 62%-73% in the first three months and remained > 50% for 9 months on both substrate-types. By comparison, mortality with Actellic ® 300CS was very high in the first three months (72%-95%) but declined sharply to < 40% after 4 months. Using a non-inferiority margin defined by the World Health Organization, overall mortality achieved with VECTRON ™ T500 was non-inferior to that observed in huts treated with Actellic ® 300CS with both cement and mud wall substrates. Monthly in situ wall cone bioassay mortality with VECTRON ™ T500 also remained over 80% for 18 months but dropped below 80% with Actellic ® 300CS at 6-7 months post spraying., Conclusion: VECTRON ™ T500 shows potential to provide substantial and prolonged control of malaria transmitted by pyrethroid-resistant mosquito vectors when applied for IRS. Its addition to the current list of WHO-approved IRS insecticides will provide a suitable option to facilitate rotation of IRS products with different modes of action., (© 2022. The Author(s).)

The present study investigated in 8 villages of the Plateau region the coverage, usage, physical integrity, and bio-efficacy of the Olyset nets distributed nationwide by the Benin's National Malaria Control Programme in July 2011. The questionnaire administered as well as the observations made in the households allowed estimating the coverage and usage rates of the 2011 Olyset nets. While their physical integrity was assessed through standard WHO methodology, their bio-efficacy was evaluated through gas chromatography, and WHO cone testing performed with the Kisumu susceptible strain. Mosquito collections through human landing catches (HLCs) were also performed in torn nets to assess if a loss of protection of sleepers occurred as the nets fabric integrity got more damaged. Nine months postdistribution, the coverage and usage rates of the 2011 Olyset nets were 67.4% (95% CI: 65.8-68.9) and 73.3% (95% CI: 70.7-75.8) respectively. About 28% of the 2011 Olyset nets were torn. A drastic drop of the insecticide quantity on the fibers of the nets [from 7.08 µg (95% CI: 5.74-8.42) to 0.2 µg (95% CI: 0.01-0.38)] as well as mortality rates <80% were observed with most nets evaluated. Moreover, the biting rates of An. gambiae s.l. (Diptera: Culicidae) inside torn nets increased in line with their fabric integrity loss. These data support the conclusion that future deployment of nets in the field must be strengthened by community sensitization on their correct use in order to postpone as much as possible appearance of holes and loss of insecticidal activity and encourage repairing of torn nets., (© The Author(s) 2021. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The rotational use of insecticides with different modes of action for indoor residual spraying (IRS) is recommended for improving malaria vector control and managing insecticide resistance. Insecticides with new chemistries are urgently needed. Broflanilide is a newly discovered insecticide under consideration. We investigated the efficacy of a wettable powder (WP) formulation of broflanilide (VECTRON T500) for IRS on mud and cement wall substrates in laboratory and experimental hut studies against pyrethroid-resistant malaria vectors in Benin, in comparison with pirimiphos-methyl CS (Actellic 300CS). There was no evidence of cross-resistance to pyrethroids and broflanilide in CDC bottle bioassays. In laboratory cone bioassays, broflanilide WP-treated substrates killed > 80% of susceptible and pyrethroid-resistant An. gambiae sl for 6-14 months. At application rates of 100 mg/m 2 and 150 mg/m 2 , mortality of wild pyrethroid-resistant An. gambiae sl entering experimental huts in Covè, Benin treated with VECTRON T500 was similar to pirimiphos-methyl CS (57-66% vs. 56%, P > 0.05). Throughout the 6-month hut trial, monthly wall cone bioassay mortality on VECTRON T500 treated hut walls remained > 80%. IRS with broflanilide shows potential to significantly improve the control of malaria transmitted by pyrethroid-resistant mosquito vectors and could thus be a crucial addition to the current portfolio of IRS insecticides.

Background: Where resources are available, non-pyrethroid IRS can be deployed to complement standard pyrethroid LLINs with the aim of achieving improved vector control and managing insecticide resistance. The impact of the combination may however depend on the type of IRS insecticide deployed. Studies comparing combinations of pyrethroid LLINs with different types of non-pyrethroid IRS products will be necessary for decision making., Methods: The efficacy of combining a standard pyrethroid LLIN (DuraNet®) with IRS insecticides from three chemical classes (bendiocarb, chlorfenapyr and pirimiphos-methyl CS) was evaluated in an experimental hut trial against wild pyrethroid-resistant Anopheles gambiae s.l. in Cové, Benin. The combinations were also compared to each intervention alone. WHO cylinder and CDC bottle bioassays were performed to assess susceptibility of the local An. gambiae s.l. vector population at the Cové hut site to insecticides used in the combinations., Results: Susceptibility bioassays revealed that the vector population at Cové, was resistant to pyrethroids (<20% mortality) but susceptible to carbamates, chlorfenapyr and organophosphates (≥98% mortality). Mortality of wild free-flying pyrethroid resistant An. gambiae s.l. entering the hut with the untreated net control (4%) did not differ significantly from DuraNet® alone (8%, p = 0.169). Pirimiphos-methyl CS IRS induced the highest mortality both on its own (85%) and in combination with DuraNet® (81%). Mortality with the DuraNet® + chlorfenapyr IRS combination was significantly higher than each intervention alone (46% vs. 33% and 8%, p<0.05) demonstrating an additive effect. The DuraNet® + bendiocarb IRS combination induced significantly lower mortality compared to the other combinations (32%, p<0.05). Blood-feeding inhibition was very low with the IRS treatments alone (3-5%) but increased significantly when they were combined with DuraNet® (61% - 71%, p<0.05). Blood-feeding rates in the combinations were similar to the net alone. Adding bendiocarb IRS to DuraNet® induced significantly lower levels of mosquito feeding compared to adding chlorfenapyr IRS (28% vs. 37%, p = 0.015)., Conclusions: Adding non-pyrethroid IRS to standard pyrethroid-only LLINs against a pyrethroid-resistant vector population which is susceptible to the IRS insecticide, can provide higher levels of vector mosquito control compared to the pyrethroid net alone or IRS alone. Adding pirimiphos-methyl CS IRS may provide substantial improvements in vector control while adding chlorfenapyr IRS can demonstrate an additive effect relative to both interventions alone. Adding bendiocarb IRS may show limited enhancements in vector control owing to its short residual effect., Competing Interests: The authors have declared that no competing interests exist.

Background: Knowledge of insecticide resistance status in the main malaria vectors is an essential component of effective malaria vector control. This study presents the first evaluation of the status of insecticide resistance in Anopheles gambiae populations from Bangui, the Central African Republic., Methods: Anopheles mosquitoes were reared from larvae collected in seven districts of Bangui between September to November 2014. The World Health Organisation's bioassay susceptibility tests to lambda-cyhalothrin (0.05%), deltamethrin (0.05%), DDT (4%), malathion (5%), fenitrothion (1%) and bendiocarb (0.1%) were performed on adult females. Species and molecular forms as well as the presence of L1014F kdr and Ace-1 R mutations were assessed by PCR. Additional tests were conducted to assess metabolic resistance status., Results: After 1 h exposure, a significant difference of knockdown effect was observed between districts in all insecticides tested except deltamethrin and malathion. The mortality rate (MR) of pyrethroids group ranging from 27% (CI: 19-37.5) in Petevo to 86% (CI: 77.6-92.1) in Gbanikola; while for DDT, MR ranged from 5% (CI: 1.6-11.3) in Centre-ville to 39% (CI: 29.4-49.3) in Ouango. For the organophosphate group a MR of 100% was observed in all districts except Gbanikola where a MR of 96% (CI: 90-98.9) was recorded. The mortality induced by bendiocarb was very heterogeneous, ranging from 75% (CI: 62.8-82.8) in Yapele to 99% (CI: 84.5-100) in Centre-ville. A high level of kdr-w (L1014F) frequency was observed in all districts ranging from 93 to 100%; however, no kdr-e (L1014S) and Ace-1 R mutation were found in all tested mosquitoes. Data of biochemical analysis showed significant overexpression activities of cytochrome P450, GST and esterases in Gbanikola and Yapele (χ 2  = 31.85, df = 2, P < 0.001). By contrast, esterases activities using α and β-naphthyl acetate were significantly low in mosquitoes from PK10 and Ouango in comparison to Kisumu strain (χ 2  = 17.34, df = 2, P < 0.005)., Conclusions: Evidence of resistance to DDT and pyrethroids as well as precocious emergence of resistance to carbamates were detected among A. gambiae mosquitoes from Bangui, including target-site mutations and metabolic mechanisms. The co-existence of these resistance mechanisms in A. gambiae may be a serious obstacle for the future success of malaria control programmes in this region.

Background: Insecticides are widely used to control malaria vectors and have significantly contributed to the reduction of malaria-caused mortality. In addition, the same classes of insecticides were widely introduced and used in agriculture in Benin since 1980s. These factors probably contributed to the selection of insecticide resistance in malaria vector populations reported in several localities in Benin. This insecticide resistance represents a threat to vector control tool and should be monitored. The present study reveals observed insecticide resistance trends in Benin to help for a better management of insecticide resistance., Methods: Mosquito larvae were collected in eight sites and reared in laboratory. Bioassays were conducted on the adult mosquitoes upon the four types of insecticide currently used in public health in Benin. Knock-down resistance, insensitive acetylcholinesterase-1 resistance, and metabolic resistance analysis were performed in the mosquito populations based on molecular and biochemical analysis. The data were mapped using Geographical Information Systems (GIS) with Arcgis software., Results: Mortalities observed with Deltamethrin (pyrethroid class) were less than 90% in 5 locations, between 90-97% in 2 locations, and over 98% in one location. Bendiocarb (carbamate class) showed mortalities ranged 90-97% in 2 locations and were over 98% in the others locations. A complete susceptibility to Pirimiphos methyl and Fenitrothion (organophosphate class) was observed in all locations with 98-100% mortalities. Knock-down resistance frequencies were high (0.78-0.96) and similar between Anopheles coluzzii, Anopheles gambiae, Anopheles arabiensis, and Anopheles melas. Insensitive acetylcholinesterase-1 was rare (0.002-0.1) and only detected in Anopheles gambiae in concomitance with Knock-down resistance mutation. The maps showed a large distribution of Deltamethrin resistance, Knock-down mutation and metabolic resistance throughout the country, a suspected resistance to Bendiocarb and detection of insensitive acetylcholinesterase-1 from northern Benin, and a wide distribution of susceptible vectors to Pirimiphos methyl and Fenitrothion., Conclusion: This study showed a widespread resistance of malaria vectors to pyrethroid previously located in southern Benin, an early emergence of carbamates resistance from northern Benin and a full susceptibility to organophosphates. Several resistance mechanisms were detected in vectors with a potential cross resistance to pyrethroids through Knock-down and metabolic resistance mechanisms.

Background: To better control malaria, the clear and urgent need is for improved data to inform decision makers, but in several African countries, there is a lack of baseline data on vectors and variation in the intensity of malaria transmission. This has resulted in the implementation of vector control efforts that ignore variation in vector behaviour and intensity of transmission, an approach that is most often not cost-effective. This study presents a detailed entomological description of mosquito distribution and variation in potentially transmissible contacts of Plasmodium falciparum following a south to north transect in Benin., Method: The study was conducted in five locations where environmental parameters were different and malaria prevalence ranged between 14 and 51%. The locations represent the main eco-epidemiological malaria areas in Benin. Mosquitoes were collected using human landing catches, pyrethrum spray catches and windows traps. They were taxonomically and molecularly identified. Head-thoraces of Anopheles gambiae s.l. were tested by enzyme-linked immunosorbent assay. Entomological indicators were estimated following WHO guidelines., Results: The results showed variation between location and period in distribution of Anopheles coluzzii, An. gambiae, and Anopheles arabiensis (p < 0.05). An extension of the reported range of An. arabiensis was also observed. Densities of malaria vectors varied significantly between rural and urban sites, however, indoor/outdoor biting ratios remained constant. Proportions of malaria vectors with circumsporozoite protein of P. falciparum were similar between locations. The entomological inoculation rates ranged between zero and eight bites/man/night with significant variations between areas.Four profiles of human exposure to infectious malaria vector bites were observed and included location with one season of high transmission (June - August), two seasons of lower transmission (March-August; October-November), moderate continuous transmission season, and high continuous transmission season of P. falciparum., Conclusion: The study revealed several entomological patterns in transmission of P. falciparum in Benin. The data could be used for purposes of planning a more cost-effective vector control strategy, by stratifying the country into higher and lower transmission zones. The information could also be used to guide extension of indoor residual spray based on a targeted use of IRS at sites where the duration of insecticidal effect following spraying coincides with the peak transmission period.

Background: The dynamics of mosquito populations depends on availability of suitable surface water for oviposition. It is well known that suitable management of mosquito larval habitats in the sub-Saharan countries, particularly during droughts, could help to suppress vector densities and malaria transmission. We conducted a field survey to investigate the spatial and seasonal distribution of mosquito larval habitats and identify drought-refugia for anopheline larvae., Methods: A GIS approach was used to identify, geo-reference and follow up longitudinally from May 2012 to May 2013, all mosquito breeding sites in two rural sites (Yondarou and Thui), one urban (Kossarou), and one peri-urban (Pèdè) site at Kandi, a municipality in northeastern Benin. In Kandi, droughts are excessive with no rain for nearly six months and a lot of sunshine. A comprehensive record of mosquito larval habitats was conducted periodically in all sites for the identification of drought-refugia of anopheline larval stages. With geospatialisation data, seasonal larval distribution maps were generated for each study site with the software ArcGIS version 10.2., Results: Overall, 187 mosquito breeding sites were identified of which 29.95% were recorded during drought. In rural, peri-urban and urban sites, most of the drought-refugia of anopheline larvae were domestic in nature (61.54%). Moreover, in rural settings, anopheline larvae were also sampled in cisterns and wells (25% of larval habitats sampled during drought in Yondarou and 20% in Thui). The mapping showed a significant decrease in the spatial distribution of mosquito larval habitats in rural, peri-urban and urban sites during drought, except in Yondarou (rural) where the aridity did not seem to influence the distribution of larval habitats., Conclusion: Our data showed that the main drought-refugia of anopheline larvae were of a domestic nature as well as wells and cisterns. A suitable management of mosquito larvae in sub-Saharan countries, particularly during droughts, should target such larval habitats for a meaningful impact on the dynamics of mosquito populations and malaria transmission.

Background: In arid settings, droughts usually lead to periods of very low or no malaria transmission. However, in rural Kandi (Sonsoro) in northeastern Benin, several malaria cases are often diagnosed during dry seasons. The underlying factors accounting for this phenomenon remain unknown., Methods: The entomological profile of Sonsoro has been studied compared to a location in urban Kandi (Gansosso) for a period of one year. During this period, Anopheles larval habitats were investigated and populations of Anopheles gambiae s.l. were sampled by human landing catches in both areas. Enzyme-linked immunosorbent assays (ELISA) for Plasmodium falciparum circumsporozoite protein (CSP) were conducted on vector specimens and the entomological inoculation rates (EIR) were determined per season (wet versus dry) in each area. In addition, during the severe drought period, Rapid Diagnostic Tests (RDTs) were conducted on school children under the age 10 years in these areas to provide a global view of drought-malaria prevalence and to perform a crossing with entomological data in Kandi., Results: Overall, An. gambiae s.l. was particularly abundant in rural Kandi compared to the urban area with a significant decrease of vector density in both sites during the dry season. In this period, larval sampling data identified household water sources as potential breeding sites in urban and rural Kandi. We also observed a significant seasonal variation of the infectivity rate in both areas but for each period (season), the EIR was higher in the rural site than in the urban. Data of P. falciparum detection was the reflection of entomological findings. The drought-malaria prevalence was 5.5 times higher in rural Kandi as compared to urban Kandi. The presence of a permanent water site and the low level of urbanization in rural Kandi were identified as a risk factor., Conclusion: Our data showed a high level of malaria transmission in the municipality of Kandi. Household water source plays an important role in maintaining the breeding of anopheles larvae and the malaria transmission in Kandi. In rural settings, the proximity to permanent water sites could probably be the aggravating factor.