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Background The effectiveness of long-lasting insecticidal nets (LLIN), the primary method for preventing malaria in Africa, is compromised by evolution and spread of pyrethroid resistance. Further gains require new insecticides with novel modes of action. Chlorfenapyr is a pyrrole insecticide that disrupts mitochrondrial function and confers no cross-resistance to neurotoxic insecticides. Interceptor® G2 LN (IG2) is an insecticide-mixture LLIN, which combines wash-resistant formulations of chlorfenapyr and the pyrethroid alpha-cypermethrin. The objective was to determine IG2 efficacy under controlled household-like conditions for personal protection and control of wild, pyrethroid-resistant Anopheles funestus mosquitoes. Methods Experimental hut trials tested IG2 efficacy against two positive controls—a chlorfenapyr-treated net and a standard alpha-cypermethrin LLIN, Interceptor LN (IG1)—consistent with World Health Organization (WHO) evaluation guidelines. Mosquito mortality, blood-feeding inhibition, personal protection, repellency and insecticide-induced exiting were recorded after zero and 20 washing cycles. The trial was repeated and analysed using multivariate and meta-analysis. Results In the two trials held in NE Tanzania, An. funestus mortality was 2.27 (risk ratio 95% CI 1.13–4.56) times greater with unwashed Interceptor G2 than with unwashed Interceptor LN (p = 0.012). There was no significant loss in mortality with IG2 between 0 and 20 washes (1.04, 95% CI 0.83–1.30, p = 0.73). Comparison with chlorfenapyr treated net indicated that most mortality was induced by the chlorfenapyr component of IG2 (0.96, CI 0.74–1.23), while comparison with Interceptor LN indicated blood-feeding was inhibited by the pyrethroid component of IG2 (IG2: 0.70, CI 0.44–1.11 vs IG1: 0.61, CI 0.39–0.97). Both insecticide components contributed to exiting from the huts but the contributions were heterogeneous between trials (heterogeneity Q = 36, P = 0.02). WHO susceptibility tests with pyrethroid papers recorded 44% survival in An. funestus . Conclusions The high mortality recorded by IG2 against pyrethroid-resistant An. funestus provides first field evidence of high efficacy against this primary, anthropophilic, malaria vector.

Background A new generation of IRS insecticides which can provide improved and prolonged control of pyrethroid-resistant malaria vector populations are being developed. Fludora® Fusion is a new IRS insecticide containing a mixture of deltamethrin and clothianidin, a neonicotinoid. Methods The efficacy of Fludora® Fusion IRS was evaluated over 11–12 months on concrete and mud substrates in laboratory bioassays and experimental huts against wild free-flying pyrethroid-resistant Anopheles gambiae ( sensu lato ) in Cové, Benin. A comparison was made with the two active ingredients of the mixture; clothianidin and deltamethrin, applied alone. CDC bottle bioassays were also performed to investigate resistance to clothianidin in the wild vector population. Results Fludora® Fusion induced > 80% laboratory cone bioassay mortality with both susceptible and pyrethroid-resistant An. gambiae ( s.l. ) for 7–9 months on concrete block substrates and 12 months on mud block substrates. The vector population at the experimental hut site was fully susceptible to clothianidin in CDC bottle bioassays. Overall mortality rates of wild free-flying pyrethroid-resistant An. gambiae ( s.l. ) entering the experimental huts during the 11-month trial were < 15% with deltamethrin and significantly higher with Fludora® Fusion (69–71%) and clothianidin alone (72–78%). Initial high experimental hut mortality rates with Fludora® Fusion (> 80%) only declined by 50% after 8 months. Monthly in situ wall cone bioassay mortality of susceptible mosquitoes was > 80% for 9–12 months with Fludora® Fusion and clothianidin alone. Fludora® Fusion induced significantly higher levels of early exiting of mosquitoes compared to clothianidin alone (55–60% vs 37–38%, P  < 0.05). Conclusions Indoor residual spraying with Fludora® Fusion induced high and prolonged mortality of wild pyrethroid-resistant malaria vectors for 7–10 months mostly due to the clothianidin component and substantial early exiting of mosquitoes from treated huts due to the pyrethroid component. Fludora® Fusion is an important addition to the current portfolio of IRS insecticides with the potential to significantly reduce transmission of malaria by pyrethroid-resistant mosquito vectors.

Escalating insecticide resistance threatens the efficacy of LLINs, undermining malaria control in Africa. We conducted the first experimental hut trials in Uganda using highly resistant free-flying wild  Anopheles  mosquitoes and F2 hybrids of FANG and Uganda An. funestus  to evaluate the performance of bednets. The interceptor G2 (chlorfenapyr) bednet demonstrated superior efficacy compared to Interceptor (pyrethroid-only) net [mortality odds ratio (OR): 18.7 (8.05–48.6) P  < 0.0001], achieving an overall mortality rate of 70.6% and 63.2% against An. funestus and An. gambiae respectively. In contrast, PermaNet 3.0 and Olyset Plus (piperonyl butoxide (PBO)) and Royal Guard (pyriproxyfen (PPF)-treated) bednets exhibited significantly lower mortality against An. funestus [Olyset Plus: 36.1%, PermaNet 3.0: 31.0% and Royal Guard (37.6%], though performance against An. gambiae was moderate [PermaNet 3.0: 61.4%, Olyset Plus: 50.0%, Royal Guard: 51.6%]. Interceptor net produced the lowest mortality (~ 25%) against both species. Regarding blood-feeding inhibition (BFI), PBO nets, particularly Olyset Plus, outperformed Interceptor G2 and Royal Guard, while Interceptor produced minimal BFI (< 36%). Further evaluation of Royal Guard’s PPF effect on oviposition revealed no significant reduction in oviposition rates compared to controls with An. funestus (63.9% vs. 63.3%, P  > 0.05). Genetic analysis using the hybrid crosses revealed that pyrethroid resistance markers ( 4.3 Kb-SV and G454A-Cyp9K1 ) were significantly associated with mosquito survival and blood-feeding success against PermaNet 2.0 (pyrethroid-only) and PermaNet 3.0 but showed no significant association with Interceptor G2 net. These findings support Interceptor G2 as a promising intervention for regions dominated by both highly resistant  An. funestus  s.l. and An. gambiae s.l. Piperonyl butoxide and PPF nets emerge as a good alternative for areas mostly dominated by resistant  An. gambiae  s.l. populations. Critically, the demonstrated variable impact of insecticide resistance on bednet efficacy underscores the imperative need for a comprehensive vector distribution mapping, continuous field efficacy assessments, and systematic resistance monitoring. This evidence-based triad should guide strategic LLIN distribution and rotations to sustain malaria control efficacy in resistance-prone settings.

Background Anopheles darlingi is a primary malaria vector in the Peruvian Amazon, yet characterization of behavioural traits contributing to human-vector contact is limited. Additionally, studies comparing key behaviours of wild-type to colonized An. darlingi populations are minimal. This study compared resting and entry behaviour between these two types of populations. Specific objectives were to use experimental huts to (1) evaluate and compare indoor resting behaviours of wild-type and colonized An. darlingi populations; (2) quantify An. darlingi house entry rates into interception traps in relation to protected Human Landing collection (HLC). Methods The study was conducted in Zungarococha village, Loreto, Peru. Prior to hut evaluations, An. darlingi biting activity and population dynamics were evaluated using HLC outside local homes from June 2014 to May 2015. Indoor resting location (window, door, wall, roof) of wild caught and colonized An. darlingi was evaluated in three experimental huts. Controlled indoor releases were performed for 6 days each month from March to August 2015. Wild An. darlingi hourly house-entry rates were quantified using interception traps affixed to an experimental hut and indoor HLC from May to August 2015. Two collectors were positioned inside huts to generate host-seeking cues during evaluations. Results Anopheles darlingi had a bimodal outdoor biting pattern with two peaks at 1800 h and 2200 h. HLC densities were associated with Amazon River levels measured nearby Iquitos city. Colonized An. darlingi preferred to rest in lower parts of the door (29.8%), roof (12.7%), and window (11.8%) which was similar to wild caught An. darlingi which preferred to rest in low door (32.7%), window (14.0%), and roof (13.0%). Wild An. darlingi entry behaviour peaked from 2200 to 2300 h, this was clearly observed when collection densities increased, varying between 2300 and 2400 h at low collection densities. Capture rates from interception traps were lower compared to indoor HLC rates from adjacent experimental huts and local houses. Conclusions Results from this study provide useful insights into An. darlingi resting and entry behaviour in a malaria endemic area in the Peruvian Amazon and inform on the use of colonized mosquitos in vector behavioural studies. This information is relevant to malaria epidemiology and will be useful to evaluate new tools for malaria control programmes.

Background A three-dimensional window screen (3D-Screen) has been developed to create a window double-screen trap (3D-WDST), effectively capturing and preventing the escape of mosquitoes. A 2015 laboratory study demonstrated the 3D-Screen's efficacy, capturing 92% of mosquitoes in a double-screen setup during wind tunnel assays. To further evaluate its effectiveness, phase II experimental hut trials were conducted in Muheza, Tanzania. Methods Three experimental hut trials were carried out between 2016 and 2017. Trial I tested two versions of the 3D-WDST in huts with open or closed eaves, with one version using a single 3D-Screen and the other using two 3D-Screens. Trial II examined the 3D-WDST with two 3D-Screens in huts with or without baffles, while Trial III compared handmade and machine-made 3D structures. Mosquito capturing efficacy of the 3D-WDST was measured by comparing the number of mosquitoes collected in the test hut to a control hut with standard exit traps. Results Trial I showed that the 3D-WDST with two 3D-Screens used in huts with open eaves achieved the highest mosquito-capturing efficacy. This treatment captured 33.11% (CI 7.40–58.81) of female anophelines relative to the total collected in this hut (3D-WDST and room collections) and 27.27% (CI 4.23–50.31) of female anophelines relative to the total collected in the control hut (exit traps, room, and verandahs collections). In Trial II, the two 3D-Screens version of the 3D-WDST captured 70.32% (CI 56.87–83.77) and 51.07% (CI 21.72–80.41) of female anophelines in huts with and without baffles, respectively. Compared to the control hut, the capturing efficacy for female anophelines was 138.6% (37.23–239.9) and 42.41% (14.77–70.05) for huts with and without baffles, respectively. Trial III demonstrated similar performance between hand- and machine-made 3D structures. Conclusions The 3D-WDST proved effective in capturing malaria vectors under semi-field experimental hut conditions. Using 3D-Screens on both sides of the window openings was more effective than using a single-sided 3D-Screen. Additionally, both hand- and machine-made 3D structures exhibited equally effective performance, supporting the production of durable cones on an industrial scale for future large-scale studies evaluating the 3D-WDST at the community level. Graphical Abstract

Background East African style experimental huts have been used in Tanzania since 1963 to evaluate vector control interventions such as insecticide-treated nets and indoor residual spraying. Over time, these huts have been modified to include eave baffles to minimise mosquito escape. In this study, we evaluated the impact of increasing baffle size and using netting to funnel mosquito entry into the room and prevent escape. We also explored mosquito entry behaviour using a sticky trap positioned on the hut exterior to determine if this behaviour could be leveraged for vector control. Methods This study was conducted in Moshi, Tanzania and included two trials. In trial one, we compared the original huts with baffles with small exit holes (4 × 110 cm at the wide end nearest the eave gap and 2 × 2 cm at the narrow funnel end) to those with a larger size of baffle exit hole (20 × 120 cm at the wide end and 4 × 10 cm at the narrow end). In trial two, we compared huts with a sticky trap on the exterior wall to those without. Data analyses used logistic regression models to compare mosquito entry, blood-feeding rates and exophily, adjusting for variation between huts, cows (for blood-feeding) and days of the trial. Results Larger eave baffles significantly increased entry of Anopheles gambiae Kisumu mosquitoes into huts [ p  = 0.01, adjusted odds ratio (AOR) 2.1, 95% confidence interval (CI) 1.2–3.8]. Blood-feeding rates were also significantly higher in huts with the larger baffle size compared to those with the original baffle size [ p  = 0.001, AOR 16.9 95% CI 4.9–59.0]. In trial two, 16% (95 CI 13.3–19.6) of An. gambiae Kisumu and 8% (95 CI: 6.3–10.5) of Anopheles arabiensis populations were collected on the sticky traps, significantly reducing mosquito entry into huts. With the presence of sticky traps, blood-feeding was inhibited by 12.7% for An. arabiensis and 32.6% for An. gambiae Kisumu. Conclusions The results of this study support the use of larger baffle sizes in East African huts to capture larger numbers of mosquitoes and improve the evaluation of vector control tools. Although only a small proportion of mosquitoes were found to have had direct contact with the exterior of the hut before entry, the presence of sticky traps still reduced blood-feeding rates by limiting the entry of host-seeking mosquitoes. Graphical Abstract

Background Pyrethroid-PBO nets were conditionally recommended for control of malaria transmitted by mosquitoes with oxidase-based pyrethroid-resistance based on epidemiological evidence of additional protective effect with Olyset Plus compared to a pyrethroid-only net (Olyset Net). Entomological studies can be used to assess the comparative performance of other brands of pyrethroid-PBO ITNs to Olyset Plus. Methods An experimental hut trial was performed in Cové, Benin to compare PermaNet 3.0 (deltamethrin plus PBO on roof panel only) to Olyset Plus (permethrin plus PBO on all panels) against wild pyrethroid-resistant Anopheles gambiae sensu lato (s.l.) following World Health Organization (WHO) guidelines. Both nets were tested unwashed and after 20 standardized washes compared to Olyset Net. Laboratory bioassays were also performed to help explain findings in the experimental huts. Results With unwashed nets, mosquito mortality was higher in huts with PermaNet 3.0 compared to Olyset Plus (41% vs. 28%, P < 0.001). After 20 washes, mortality declined significantly with PermaNet 3.0 (41% unwashed vs. 17% after washing P < 0.001), but not with Olyset Plus (28% unwashed vs. 24% after washing P = 0.433); Olyset Plus induced significantly higher mortality than PermaNet 3.0 and Olyset Net after 20 washes. PermaNet 3.0 showed a higher wash retention of PBO compared to Olyset Plus. A non-inferiority analysis performed with data from unwashed and washed nets together using a margin recommended by the WHO, showed that PermaNet 3.0 was non-inferior to Olyset Plus in terms of mosquito mortality (25% with Olyset Plus vs. 27% with PermaNet 3.0, OR = 1.528, 95%CI = 1.02–2.29) but not in reducing mosquito feeding (25% with Olyset Plus vs. 30% with PermaNet 3.0, OR = 1.192, 95%CI = 0.77–1.84). Both pyrethroid-PBO nets were superior to Olyset Net. Conclusion Olyset Plus outperformed PermaNet 3.0 in terms of its ability to cause greater margins of improved mosquito mortality compared to a standard pyrethroid net, after multiple standardized washes. However, using a margin of non-inferiority defined by the WHO, PermaNet 3.0 was non-inferior to Olyset Plus in inducing mosquito mortality. Considering the low levels of mortality observed and increasing pyrethroid-resistance in West Africa, it is unclear whether either of these nets would demonstrate the same epidemiological impact observed in community trials in East Africa.

Background In Senegal, the main vector control strategies include indoor residual spraying (IRS) and the distribution of insecticide-treated nets (ITNs). However, drugs and insecticides resistance have become a major challenge in the fight against malaria transmission. Addressing the problem of escalating resistance is vital to maintaining progress towards malaria elimination, which has stalled in recent years. New formulations belonging to the neonicotinoid class, clothianidin, have been developed and is now being used for malaria vector control through IRS. Methods The residual efficacy of clothianidin-treated walls was assessed monthly using WHO cone bioassays. Five houses in each of the two treated villages were evaluated, while one untreated house served as a control. In the experimental huts, a total of six huts, three in banco (mud) and three in cement, were evaluated on a monthly basis. Three cones were installed on three walls of each sprayed house at heights of 0.5 m, 1 m and 1.5 m above the ground, and three additional cones were placed in the control house. Ten female Anopheles coluzzii mosquitoes, aged between 3 and 5 days and derived from a laboratory-susceptible strain, were exposed in each cone for 30 min. After exposure, the mosquitoes were transferred to cups and mortality rates were recorded up to four days after exposure. Results Results demonstrate high efficacy of SumiShield 50WG on mud and cement substrates, residual activity for up to twelve months in experimental huts and eight months under field conditions. In experimental huts, the 96 h mortality rate of the susceptible mosquito strain remained at 100% throughout the study, except in months ten and twelve for mud-walled huts, and months six and ten for cement-walled huts, where mortality rates were 98.33%, 99.16%, 95.68%, and 97.52%, respectively. In the field sites of Bandafassi and Tomboronkoto, the 72 h mortality rate of the susceptible strain remained consistently at 100% over the eight-month period. Conclusions Clothianidin, a neonicotinoid insecticide, has not yet shown resistance in malaria vectors in Senegal. SumiShield 50WG is effective for resistance management through a rotation strategy using insecticides with different modes of action across spray cycles.

Background Vector control using insecticides is a key prevention strategy against malaria. Unfortunately, insecticide resistance in mosquitoes threatens all progress in malaria control. In the perspective of managing this resistance, new insecticide formulations are being tested to improve the effectiveness of vector control tools. Methods The efficacy and residual activity of Pirikool® 300 CS was evaluated in comparison with Actellic® 300 CS in experimental huts at the Tiassalé experimental station on three substrates including cement, wood and mud. The mortality, blood-feeding inhibition, exiting behaviour and deterrency of free-flying wild mosquitoes was evaluated. Cone bioassay tests with susceptible and resistant mosquito strains were conducted in the huts to determine residual efficacy. Results A total of 20,505 mosquitoes of which 10,979 (53%) wild female Anopheles gambiae were collected for 112 nights. Residual efficacy obtained from monthly cone bioassay was higher than 80% with the susceptible, laboratory-maintained An. gambiae Kisumu strain, from the first to the tenth study period on all three types of treated substrate for both Actellic® 300CS and Pirikool® 300CS. This residual efficacy on the wild Tiassalé strain was over 80% until the 4th month of study on Pirikool® 300CS S treated substrates. Overall 24-h mortalities of wild free-flying An. gambiae sensu lato which entered in the experimental huts over the 8-months trial on Pirikool® 300CS treatment was 50.5%, 75.9% and 52.7%, respectively, on cement wall, wood wall and mud wall. The positive reference product Actellic® 300CS treatment induced mortalities of 42.0%, 51.8% and 41.8% on cement wall, wood wall and mud wall. Conclusion Pirikool® 300CS has performed really well against resistant strains of An. gambiae using indoor residual spraying method in experimental huts. It could be an alternative product for indoor residual spraying in response to the vectors' resistance to insecticides.

Background Pyrethroid-chlorfenapyr (CFP) and pyrethroid-piperonyl butoxide (PBO) nets are being scaled across endemic countries to improve control of malaria transmitted by pyrethroid-resistant mosquitoes. CFP is a pro-insecticide requiring activation by mosquito cytochrome P450 monooxygenase enzymes (P450s) while PBO improves pyrethroid potency by inhibiting the action of these enzymes in pyrethroid-resistant mosquitoes. The inhibitory action of PBO against P450s may thus reduce the efficacy of pyrethroid-CFP nets when applied inside the same household as pyrethroid-PBO nets. Methods Two experimental hut trials were performed to evaluate the entomological impact of two different types of pyrethroid-CFP ITN (Interceptor ® G2, PermaNet ® Dual) when applied alone and in combination with pyrethroid-PBO ITNs (DuraNet ® Plus, PermaNet ® 3.0) against a pyrethroid-resistant vector population in southern Benin. In both trials, all net types were tested as single and double net treatments. Bioassays were also performed to assess the resistance profile of the vector population at the hut site and investigate interactions between CFP and PBO. Results The vector population was susceptible to CFP but exhibited a high intensity of pyrethroid resistance that was overcame by PBO pre-exposure. Vector mortality was significantly lower in huts with combinations of pyrethroid-CFP nets plus pyrethroid-PBO nets compared to huts with two pyrethroid-CFP nets (74% vs. 85% for Interceptor ® G2 and 57% vs. 83% for PermaNet ® Dual, p < 0.001). PBO pre-exposure reduced the toxicity of CFP in bottle bioassays suggesting this effect may be partly attributable to antagonism between CFP and PBO. Higher levels of vector mortality were observed in huts with net combinations that included pyrethroid-CFP nets compared to those that did not and highest mortality was achieved when pyrethroid-CFP nets were applied alone as two nets together (83–85%). Conclusions This study shows evidence of a reduced performance of pyrethroid-CFP nets when combined with pyrethroid-PBO ITNs compared to when applied alone and higher efficacy with net combinations that included pyrethroid-CFP nets. These findings suggest that in similar contexts, prioritizing distribution of pyrethroid-CFP nets over other net types would maximize vector control impact.