A multi-country non-inferiority experimental hut evaluation of DuraNet® Plus, an alpha-cypermethrin and piperonyl butoxide treated net, for control of pyrethroid-resistant malaria vectors in West and Central Africa

A multi-country non-inferiority experimental hut evaluation of DuraNet® Plus, an alpha-cypermethrin and piperonyl butoxide treated net, for control of pyrethroid-resistant malaria vectors in West and Central Africa | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A multi-country non-inferiority experimental hut evaluation of DuraNet® Plus, an alpha-cypermethrin and piperonyl butoxide treated net, for control of pyrethroid-resistant malaria vectors in West and Central Africa Abel Agbevo, Judicael Nounagnon, Benjamin Menze, Loudovic Ahoua Alou, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6848690/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 14 You are reading this latest preprint version Abstract Background Pyrethroid-piperonyl butoxide (PBO) nets enhance malaria vector control by counteracting metabolic resistance mechanisms in malaria vectors through the synergistic action of PBO. DuraNet® Plus is an alpha-cypermethrin and PBO incorporated net developed Shobikaa Impex Private Limited. This study assessed its entomological efficacy relative to a standard pyrethroid-only net (DuraNet®) and an established pyrethroid-PBO net (Olyset® Plus), in support of WHO prequalification. Methods Experimental hut trials were conducted at three ecologically and entomologically distinct sites with pyrethroid-resistant vector populations: Covè, Benin ( Anopheles gambiae s.l.), Mibellon, Cameroon ( An. funestus ), and M’bé, Côte d’Ivoire ( An. gambiae s.l.). Each net type was tested unwashed and after 20 standardized washes. Primary outcomes included 24-hour mosquito mortality and blood-feeding inhibition. DuraNet® Plus was evaluated for non-inferiority to Olyset® Plus and superiority over DuraNet® using combined washed and unwashed data, in line with WHO guidelines. WHO bioassays confirmed pyrethroid resistance and assessed the role of cytochrome P450 enzymes. Chemical analyses measured pyrethroid and PBO retention after washing. Results DuraNet® Plus consistently induced higher mosquito mortality than Olyset® Plus across all sites (Benin: 29.5% vs. 14.9%, OR = 2.81, 95% CI: 2.34–3.38, NIM = 0.468; Cameroon: 27.8% vs. 22.2%, OR = 1.81, 95% CI: 1.32–2.49, NIM = 0.619; Côte d’Ivoire: 19.5% vs. 12.0%, OR = 2.28, 95% CI: 1.85–2.80, NIM = 0.373), with all odds ratios exceeding the WHO-defined non-inferiority margins. DuraNet® Plus also met non-inferiority criteria for blood-feeding inhibition compared to Olyset® Plus (Benin: OR = 0.23, 95% CI: 0.18–0.28, NIM = 1.345; Cameroon: OR = 0.66, 95% CI: 0.50–0.87, NIM = 1.324; Côte d’Ivoire: OR = 0.58, 95% CI: 0.48–0.69, NIM = 1.404). In addition, DuraNet® Plus was superior to DuraNet® in both mosquito mortality and blood-feeding inhibition across all study sites (p < 0.05). Susceptibility bioassays confirmed high frequencies of pyrethroid resistance across all three sites, with varying levels of P450 enzyme involvement. Chemical analysis showed higher retention of alpha-cypermethrin and PBO in DuraNet® Plus after 20 washes compared to Olyset® Plus. Conclusion DuraNet® Plus showed strong entomological efficacy and wash durability against pyrethroid-resistant malaria vectors across varied settings in West and Central Africa. It met WHO non-inferiority criteria compared to Olyset® Plus and was superior to a pyrethroid-only ITN, supporting its inclusion among WHO-prequalified products. These findings underscore its potential role in vector control strategies in areas affected by metabolic pyrethroid resistance. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Insecticide-treated nets (ITNs) have been a cornerstone of malaria control efforts in sub-Saharan Africa, accounting for an estimated 68% of the malaria cases averted globally since the year 2000 [ 1 ]. By providing both a physical barrier and an insecticidal effect, ITNs have substantially reduced malaria transmission, morbidity, and mortality. However, the long-term effectiveness of ITNs is increasingly threatened by the widespread emergence and intensification of Anopheles mosquito resistance to pyrethroids, the most widely used insecticide on ITNs [ 2 ]. Pyrethroid resistance has undermined the impact of pyrethroid ITNs, contributing to a stall in global progress against malaria [ 3 ]. To address this threat, the World Health Organization (WHO) has endorsed the development and deployment of a new generation of insecticide-treated nets (ITNs) that combine a pyrethroid insecticide with either a synergist or an additional insecticidal compound [ 4 ]. Among these innovations are pyrethroid-PBO nets, which incorporate piperonyl butoxide (PBO) alongside a pyrethroid [ 5 , 6 ]. PBO functions as a synergist by inhibiting cytochrome P450 enzymes responsible for the metabolic detoxification of pyrethroids in resistant Anopheles mosquitoes. By blocking these resistance mechanisms, PBO enhances the toxicity of pyrethroids and helps to restore the efficacy of the insecticidal component of the net. Multiple studies, conducted across various malaria-endemic regions, have demonstrated that pyrethroid-PBO nets achieve improved entomological outcomes such as increased mosquito mortality and reduced blood-feeding rates compared to standard pyrethroid-only nets [ 5 , 7 – 10 ]. Moreover, in certain settings, these benefits have translated into a measurable public health impact, with pyrethroid-PBO nets providing greater reductions in malaria incidence and prevalence relative to pyrethroid-only ITNs in large-scale cluster-randomized controlled trials (cRCT) [ 11 , 12 ]. The WHO’s endorsement of pyrethroid-PBO nets in 2017, was followed by a significant shift in procurement patterns across Africa, with increased uptake of these next-generation nets over standard pyrethroid-only ITNs [ 13 ]. Between 2022 and 2024, pyrethroid-PBO nets accounted for nearly 50% of all ITNs delivered to sub-Saharan Africa, underscoring widespread recognition of their enhanced performance against pyrethroid-resistant Anopheles vectors. To ensure a stable supply pipeline and foster healthy market competition, it is essential to expand the portfolio of WHO-prequalified pyrethroid-PBO nets available for large-scale procurement. DuraNet® Plus, a pyrethroid-PBO net combining alpha-cypermethrin with PBO, was developed by Shobikaa Impex Private Limited in response to growing procurement demands for effective pyrethroid-PBO ITNs. To obtain WHO prequalification, new ITN product types must undergo rigorous semi-field evaluations to demonstrate entomological superiority over standard pyrethroid-only nets [ 14 ]. In addition, current WHO guidelines require that pyrethroid-PBO nets show non-inferiority to an existing pyrethroid-PBO ITN with proven public health impact, as demonstrated through cRCT [ 15 ]. These comparative studies are expected to be conducted across a range of ecological settings representative of the diverse vector populations and resistance profiles where the nets are intended for deployment, ensuring that efficacy claims are broadly generalizable. To support the WHO prequalification of DuraNet® Plus, a series of experimental hut trials was conducted to evaluate its entomological efficacy at three sites with documented pyrethroid resistance in West and Central Africa; Benin, Côte d’Ivoire, and Cameroon. The study was designed in accordance with existing WHO guidelines, with the primary objective of assessing the non-inferiority of DuraNet® Plus compared to Olyset® Plus, a WHO-prequalified pyrethroid-PBO net that has demonstrated improved epidemiological impact against malaria. In addition, the study aimed to confirm the entomological superiority of DuraNet® Plus over DuraNet®, a standard WHO-prequalified pyrethroid-only ITN. Key outcomes measured included mosquito mortality and blood-feeding inhibition against wild populations of pyrethroid-resistant Anopheles vectors. WHO susceptibility bioassays were conducted to characterize the resistance profile of the vector populations at each site during the study period. Materials and methods Experimental hut sites The experimental hut trials were conducted between September 2018 and March 2019 at three sites: Covè (Benin), Mibellon (Cameroon), and Bouaké (Côte d’Ivoire). At each site, seven West African-style experimental huts were used. The huts were constructed from brick, plastered with cement, and topped with corrugated iron roofs. Each hut featured four narrow window slits (1 cm wide) on the walls to allow mosquito entry and was built on a raised concrete plinth surrounded by a water-filled moat to deter entry of predators such as ants and lizards. To eliminate any potential contamination from previous evaluations, all hut walls were re-plastered, and the cement was allowed to cure for one month prior to the start of the trials. A detailed description of the local vector populations at each study site is provided below: Benin The study was conducted at the CREC/LSHTM experimental hut site situated in irrigated rice fields in Covè, southern Benin, where the malaria vector population is highly resistant to pyrethroids. The vector species comprised a combination of Anopheles coluzzii and Anopheles gambiae s.s.. Resistance is mediated by a high frequency (89%) of the L1014F kdr mutation and overexpression of metabolic enzymes such as CYP6P3, known to metabolize pyrethroids efficiently [ 16 ]. Cameroon The trial was conducted at the CRID experimental hut station in Mibellon, Adamawa region, where permanent water bodies support high year-round mosquito densities. The local vector population is predominantly An. funestus (80%) with smaller proportions of An. gambiae s.s. (20%) with both species exhibiting high levels of resistance to pyrethroids and organochlorides [ 17 ]. Cote d’Ivoire The trial was conducted at the M’bé field station near Bouaké city in central Côte d’Ivoire. The vector species is also composed of a combination of Anopheles coluzzii and Anopheles gambiae s.s. though An. coluzzii predominates year-round in the surrounding irrigated rice fields. The mosquito population is highly resistant to multiple insecticide classes, with documented L1014F and Ace-1 mutations and elevated metabolic enzyme activity [ 18 ]. Resistance to pyrethroids is extremely high, with over 1700-fold resistance to deltamethrin reported. Insecticide susceptibility bioassays: To confirm the presence of pyrethroid resistance and investigate the potential role of cytochrome P450-mediated metabolic mechanisms, WHO cylinder bioassays were conducted at each study site (Covè, Mibellon, and M’bé) during the hut trial period. The assays were performed on 2–5-day-old adult F1 female mosquitoes reared from larvae collected near the experimental huts. For each insecticide treatment, approximately 100 female mosquitoes were tested in batches of 25 using filter papers treated with alpha-cypermethrin (0.05%) or permethrin (0.75%), with and without pre-exposure to 4% piperonyl butoxide (PBO). Mosquitoes were exposed for one hour, with mortality assessed after a 24-hour holding period. Experimental hut treatments DuraNet® Plus is a pyrethroid-PBO ITN manufactured by Shobikaa Impex Private Ltd, composed of 150 denier high-density polyethylene (HDPE) monofilament yarn. The net is uniformly treated on all panels with a combination of alpha-cypermethrin (6.0 g/kg) and PBO (2.2 g/kg). It has a mesh size of 20 holes/cm² and a fabric weight of 45 g/m². DuraNet® Plus was compared to two types of WHO prequalified ITNs: Olyset® Plus, a WHO-prequalified pyrethroid-PBO ITN developed by Sumitomo Chemical Company. Olyset® Plus is made from 150 denier monofilament HDPE and is treated with a combination of permethrin (20 g/kg) and PBO (10 g/kg) incorporated into the fibres. The net has a mesh size of 6.3 holes/cm² and a fabric weight of 40 g/m². DuraNet®, a WHO-prequalified pyrethroid-only ITN also manufactured by Shobikaa Impex Private Ltd. DuraNet® is made from 150 denier HDPE monofilament yarn and is treated with alpha-cypermethrin at a concentration of 5.8 g/kg. The net has a mesh size of 20 holes/cm² and a fabric weight of 45 g/m². All net types were evaluated in both unwashed and 20-times washed conditions following WHO standard procedures [ 14 ]. The following 7 treatments were thus assessed at each hut site: Untreated net Unwashed DuraNet® DuraNet® washed 20 times Unwashed Olyset® Plus Olyset® Plus washed 20 times Unwashed DuraNet® Plus DuraNet® Plus washed 20 times To simulate natural wear and tear, all nets including untreated control, were deliberately holed following WHO guidelines, with six 4 × 4 cm holes cut into each net (two on each long side and one on each short side). Net washing was performed according to WHO standard procedures. Each net was washed in an aluminium basin containing 10 litres of water and 2 g/L of Savon de Marseille , with agitation for a total of 10 minutes. The nets were then rinsed in clean water using the same method, dried horizontally in the shade, and stored at ambient temperature between washes. Washing intervals were based on regeneration time studies: daily for DuraNet® Plus and DuraNet® and every two days for Olyset® Plus. Pre-trial WHO cone bioassays To verify the quality of the test items prior to the hut trials, a series of pre-trial WHO cone bioassays were conducted using 30 × 30 cm net samples cut from unwashed nets of each ITN type at each study site. Bioassays were performed with both the susceptible An. gambiae Kisumu strain and the local field strain at each study site. Approximately 200 mosquitoes per strain were exposed for 3 minutes on five replicate net pieces per ITN type in groups of five per cone. After exposure, mosquitoes were held at 27 ± 2°C and 75 ± 10% relative humidity, with access to a 10% glucose solution. Delayed mortality was recorded 24 hours post-exposure. Hut trial design Treatments were initially assigned to the seven experimental huts at random. To minimise positional bias, a randomized Latin Square Design was used to rotate the seven treatments weekly across the huts. The core trial lasted for seven weeks to complete the Latin square cycle, after which mosquito collections continued for an additional two weeks to increase sample size. Data were collected over six nights each week, with the seventh day reserved for thorough cleaning of the huts in preparation for the next rotation. Six replicate nets were tested per treatment and rotated daily within each treatment arm. The entire trial spanned nine weeks, yielding a total of 54 nights of data collection. At each site, seven consenting adult volunteers slept in the huts each night from 9:00 p.m. to 5:00 a.m. To control for variation in individual attractiveness to mosquitoes, sleepers were also rotated daily between huts using a Latin Square Design. Each morning, mosquitoes were collected from inside the net, the hut room, and the veranda trap, then transferred to the laboratory for morphological identification and assessment of blood-feeding status and immediate mortality. Surviving female An. gambiae s.l. were maintained at 27 ± 2°C and 75 ± 10% relative humidity with access to 10% glucose solution, and delayed mortality was recorded after 24 hours. The following entomological outcomes were used to evaluate the efficacy of each treatment in the experimental huts: Mortality: The proportion of mosquitoes found dead after a 24-hour holding period following collection from each treatment arm. Blood-feeding rate: The proportion of mosquitoes that successfully blood-fed in each treatment group. Deterrence: The proportional reduction in the number of mosquitoes entering huts with treated nets compared to huts with untreated nets. Induced exophily: The proportion of mosquitoes exiting to the veranda in huts with treated nets, used to estimate insecticide-induced exiting relative to control huts. Blood-feeding inhibition: The proportional reduction in blood-feeding in huts with insecticide-treated nets compared to huts with untreated nets. Ethical considerations: Ethical approval for the study was obtained from the relevant institutional review boards in each participating country. Written informed consent was obtained from all volunteer sleepers prior to their enrolment, with the study procedures and consent forms explained in their local language by a trained interpreter. A standby nurse was present throughout the trial to monitor the health of participants and manage any cases of fever or persistent headache. Any sleeper who tested positive for malaria during the study was immediately withdrawn and provided with appropriate treatment in accordance with national public health guidelines. Statistical analysis Proportional outcomes (mosquito mortality, blood-feeding, and exophily) were analysed using logistic regression, while count data such as mosquito entry were assessed with negative binomial regression. Each outcome was modelled separately, with fixed effects included for hut, sleeper, and daily variation. Non-inferiority analyses comparing DuraNet® Plus to Olyset® Plus followed WHO guidelines. DuraNet® Plus was considered non-inferior for mortality if the lower bound of the 95% confidence interval (CI) for the odds ratio exceeded the non-inferiority margin (NIM), and for blood-feeding if the upper bound was below the NIM. The NIM represented a 7% difference in efficacy relative to Olyset® Plus. Superiority of DuraNet® Plus over DuraNet® was determined by a significantly higher mortality and lower blood-feeding rates (p < 0.05). Analyses for non-inferiority and superiority were primarily conducted using pooled data for unwashed and washed nets to assess overall efficacy across the net’s lifespan. All analyses were performed in Stata version 18. Results Pre-trial cone bioassays results All ITN types induced 80–100% mortality in the Kisumu strain in pretrial cone bioassays across the three sites (Fig. 1 ), confirming high insecticidal activity and appropriate storage prior to hut trials. In contrast, mortality rates among the local wild strains in the hut stations were considerably lower, highlighting varying degrees of pyrethroid resistance. DuraNet® induced minimal mortality (< 10%) against the wild strains across all three locations. In Benin and Cameroon, Olyset® Plus induced the highest mortality in wild mosquitoes (73% and 70%, respectively), followed by DuraNet® Plus (45% and 40%). In Côte d’Ivoire, mortality among the wild strain were lower (< 25%) though DuraNet® Plus achieved the highest mortality rate. The results therefore showed improved mortality with the pyrethroid-PBO nets compared to the pyrethroid-only net with the local strains which confirmed the suitability of the vector populations for the study. WHO cylinder bioassay results Mortality following exposure to pyrethroids alone was consistently low in cylinder bioassays across all sites, confirming a high frequency of resistance in the local vector populations (Fig. 2 ). For permethrin (0.75%), mortality ranged from approximately 15% in Côte d’Ivoire to 30% in Cameroon. Alpha-cypermethrin (0.05%) induced slightly higher mortality, ranging from 17% in Benin to 41% in Cameroon. Pre-exposure to PBO (4%) prior to pyrethroid exposure significantly increased mortality in most settings, indicating the involvement of P450-mediated metabolic resistance. In Cameroon, PBO fully restored susceptibility to alpha-cypermethrin, with mortality reaching 100%, and partially restored alpha-cypermethrin susceptibility in Côte d’Ivoire (73%) and Benin (55%). A similar improvement was observed for permethrin in Côte d’Ivoire and Cameroon, where mortality rose to over 60%. In contrast, pre-exposure to PBO had no effect on permethrin-induced mortality in Benin; mortality remained the same. Overall, these results highlight intense pyrethroid resistance across all sites and support the role of PBO in partially restoring susceptibility, particularly to alpha-cypermethrin. Mosquito entry rates A total of 18,075 wild pyrethroid-resistant malaria vector mosquitoes were collected across all sites and treatments during the experimental hut trials in Benin, Cameroon, and Côte d’Ivoire (Table 1 , Table S1 ). In Benin, mosquito entry remained high, with no deterrence observed for DuraNet® or Olyset® Plus. DuraNet® Plus demonstrated modest deterrence of 17.2% (unwashed) and 6.8% (washed), which was significantly higher than both DuraNet® and Olyset® Plus (p < 0.01). In Cameroon, all nets showed strong deterrent effects, with DuraNet® and Olyset® Plus reducing entry by 41.3–53.7%. DuraNet® Plus again achieved the highest deterrence (54.2% when unwashed and 51.9% when washed), with a significantly greater effect than DuraNet® (p = 0.03) and comparable to Olyset® Plus (p = 0.08). In Côte d’Ivoire, deterrence across treatments ranged from 24.2–46.9%, with DuraNet® Plus showing strong and consistent reductions in mosquito entry (42.5% unwashed; 37.5% washed), significantly higher than Olyset® Plus (p < 0.05). Overall, DuraNet® Plus consistently reduced mosquito entry across all sites and this effect was sustained after 20 washes, demonstrating superior or comparable deterrence to existing WHO-prequalified nets. Table 1 Mosquito entry rates in experimental huts Net type Control DuraNet Net Olyset Plus DuraNet® Plus Wash status - Unwashed Washed 20X Unwashed Washed 20X Unwashed Washed 20X Benin Total females caught 723 814 866 1139 1200 599 674 Average catch per night 13.38 15 16 21 22.22 11 12.48 % Deterrence - 0 0 0 0 17.2 6.8 Cameroon Total females caught 618 286 342 306 363 283 297 Average catch per night 11 5 6 6 7 5 6 % Deterrence - 53.7 44.7 50.5 41.3 54.2 51.9 Cote D'Ivoire Total females caught 1366 725 1017 908 1036 785 854 Average catch per night 16.6 9.3 10.9 12.5 15.1 10.4 10.3 % Deterrence - 46.9 25.5 33.5 24.2 42.5 37.5 Mortality rates in experimental huts: Across all three study sites, DuraNet® Plus consistently induced the highest mosquito mortality, both before and after washing (Table 2 , Fig. 3 ). In Covè, Benin ( An. gambiae s.l., Fig. 3 , Panel A), unwashed DuraNet® Plus resulted in significantly higher mortality (31.2%) compared to unwashed Olyset® Plus (16.0%, p = 0.025) and unwashed DuraNet® (20.0%, p < 0.001). This superiority was maintained following 20 washes, with washed DuraNet® Plus achieving significantly higher mortality than both washed Olyset® Plus (27.7% vs. 13.9%, p = 0.010) and washed DuraNet® (27.7% vs. 20.1%, p < 0.001). Within-product comparisons indicated no significant reduction in efficacy due to washing for DuraNet® Plus ( p = 0.228) or DuraNet® ( p = 0.743), whereas Olyset® Plus showed a significant decline ( p = 0.025). In Mibellon, Cameroon ( An. funestus , Fig. 3 , Panel B), unwashed DuraNet® Plus induced the highest mortality (32.8%), followed by Olyset® Plus (29.7%), although the difference between the two was not statistically significant ( p = 0.551). Both nets induced significantly higher mortality than unwashed DuraNet® (13.9%, p < 0.001). After 20 washes, DuraNet® Plus remained significantly more effective (22.8%) than both Olyset® Plus (14.6%, p = 0.001) and DuraNet® (11.9%, p < 0.001). The difference between washed Olyset® Plus and DuraNet® was not significant ( p = 0.103). Washing significantly reduced mortality for DuraNet® Plus ( p = 0.014), but not for Olyset® Plus ( p = 0.103) or DuraNet® ( p = 0.361). In M’bé, Côte d’Ivoire ( An. gambiae s.l., Fig. 3 , Panel C), DuraNet® Plus again achieved the highest mortality among unwashed nets (24.5%), significantly exceeding that of Olyset® Plus (15.3%, p < 0.001) and DuraNet® (12.3%, p < 0.001). The difference between Olyset® Plus and DuraNet® was not significant ( p = 0.316). Following 20 washes, DuraNet® Plus maintained superior efficacy (14.4%) compared to Olyset® Plus (8.6%, p = 0.001) and DuraNet® (7.0%, p < 0.001), while the difference between Olyset® Plus and DuraNet® remained non-significant ( p = 0.753). Washing led to a statistically significant reduction in mortality for all ITN types ( p < 0.001); however, DuraNet® Plus consistently retained the highest performance across wash conditions. Table 2 Mortality (24h) of wild free-flying malaria vectors exposed to ITNs in experimental huts in Benin, Cameroon, and Côte d’Ivoire . Treatment Untreated Net DuraNet Olyset Plus DuraNet Plus Number of washes n/a Unwashed 20 washes Unwashed 20 washes Unwashed 20 washes Covè, Benin ( An. gambiae s.l.) Total females caught 723 814 866 1139 1200 599 674 N dead 24 h 9 162 178 182 150 187 187 24 h mortality (%) 1.2 19.9 20.6 16.0 12.5 31.2 27.7 24 h mortality 95% Cls (0.4-2.0) (17.1–22.6) (17.8–23.2) (13.8–18.1) (10.6–14.3) (27.5–34.9) (24.3–31.1) Mibellon, Cameroon ( An funestus ) Total females caught 618 286 342 306 363 283 297 N dead 24 h 27 40 41 91 53 93 68 24 h mortality (%) 4.4 14.0 12.0 29.7 14.6 32.9 22.9 24 h mortality 95% Cls (2.7–5.9) (9.9–18.0) (8.5–15.4) (24.6–34.8) (10.9–18.2) (27.3–38.3) (18.1–27.6) M’bé, Côte d’Ivoire ( An. gambiae s.l.) Total females caught 1366 725 1017 908 1036 785 854 N dead 24 h 58 89 71 139 89 192 123 24 h mortality (%) 4.2 12.3 7.0 15.3 8.6 24.5 14.4 24 h mortality 95% Cls (3.1–5.3) (9.8–14.6) (5.4–8.5) (12.9–17.6) (6.8–10.3) (21.4–27.4) (12.0-16.7) When data from unwashed and washed nets were combined, DuraNet® Plus consistently induced higher mean mosquito mortality than Olyset® Plus across all study sites: 29.5% vs. 14.9% in Benin, 27.8% vs. 22.2% in Cameroon, and 19.5% vs. 12.0% in Côte d’Ivoire. In Benin, the odds ratio (OR) for mortality with DuraNet® Plus compared to Olyset® Plus was 2.809 (95% CI: 2.339–3.375), exceeding the non-inferiority margin (NIM) of 0.468 (Fig. 4 , Table S2). Similar findings were observed in Cameroon (OR = 1.811, 95% CI: 1.319–2.488, NIM = 0.619) and Côte d’Ivoire (OR = 2.277, 95% CI: 1.853–2.799, NIM = 0.373), confirming that DuraNet® Plus met the WHO criteria for non-inferiority to Olyset® Plus with respect to the primary endpoint of mosquito mortality in all three countries. Further analysis of superiority indicated that DuraNet® Plus was also significantly more effective than DuraNet® for mosquito mortality across all sites ( p < 0.05, Table S3). Detailed non-inferiority outcomes by wash status are provided in the supplementary information (Table S2). Blood-feeding rates in experimental huts: Blood-feeding rates were generally lower with all treated nets compared to the untreated control (Table 3 , Fig. 5 ). In Covè, Benin, DuraNet® Plus achieved significantly lower blood-feeding rates than both Olyset® Plus and DuraNet®, both in the unwashed condition (10.5% vs. 25.8% and 22.5%, respectively; p < 0.001) and after 20 washes (14.8% vs. 43.6% and 27.4%; p < 0.001). These results highlight the enhanced personal protection provided by DuraNet® Plus over Olyset® Plus in this setting. In Mibellon, Cameroon, differences in blood-feeding rates among ITNs were less pronounced, although DuraNet® Plus showed modest improvements in blood-feeding inhibition compared to both Olyset® Plus and DuraNet® in both unwashed and washed conditions (Table 3 ). In M’bé, Côte d’Ivoire, both pyrethroid-PBO nets—DuraNet® Plus and Olyset® Plus—significantly reduced blood-feeding compared to DuraNet® when unwashed (Fig. 5 ). However, the superior protection offered by Olyset® Plus diminished after washing (65.2–6.2% blood-feeding inhibition, Table 3 ), while DuraNet® Plus retained its protective efficacy. Among unwashed nets, Olyset® Plus achieved the highest blood-feeding inhibition (65.2%), whereas among washed nets, DuraNet® Plus provided the greatest inhibition (54.8%). Table 3 Blood-feeding of wild free-flying malaria vectors exposed to ITNs in experimental huts in Benin, Cameroon, and Côte d’Ivoire . Treatment Untreated Net DuraNet Olyset Plus DuraNet Plus Number of washes n/a Unwashed 20 washes Unwashed 20 washes Unwashed 20 washes Covè, Benin ( An. gambiae s.l.) Total females caught 723 814 866 1139 1200 599 674 N Blood-feeding 366 183 237 294 523 63 100 Blood-feeding (%) 50.6 22.5 27.4 25.8 43.6 10.5 14.8 Blood-feeding 95% Cls (47.0-54.2) (19.6–25.3) (24.4–30.3) (23.2–28.3) (40.7–46.3) (8.0–13) (12.1–17.5) Blood-feeding inhibition (%) - 55.6 45.9 49.0 13.9 79.2 70.7 Mibellon, Cameroon ( An funestus ) Total females caught 618 286 342 306 363 283 297 N Blood-feeding 410 118 147 133 164 97 105 Blood-feeding (%) 66.3 41.3 43.0 43.5 45.2 34.3 35.4 Blood-feeding 95% Cls (62.6–70.0) (35.5–46.9) (37.7–48.2) (37.9–49.0) (40.0-50.3) (28.7–39.8) (29.9–40.7) Blood-feeding inhibition (%) - 37.8 35.2 34.4 31.9 48.3 46.7 M’bé, Côte d’Ivoire ( An. gambiae s.l.) Total females caught 1366 725 1017 908 1036 785 854 N Blood-feeding 532 189 273 123 378 139 150 Blood-feeding (%) 38.9 26.1 26.8 13.5 36.5 17.7 17.6 Blood-feeding 95% Cls (36.3–41.5) (22.8–29.2) (24.1–29.5) (11.3–15.7) (33.5–39.4) (15.0-20.3) (15.0-20.1) Blood-feeding inhibition (%) - 33.0 31.0 65.2 6.2 54.5 54.8 When data from unwashed and washed nets were pooled, DuraNet® Plus demonstrated non-inferiority to Olyset® Plus in terms of protection against mosquito blood-feeding across all study sites (Fig. 6 ). In Benin, the OR for blood-feeding with DuraNet® Plus compared to Olyset® Plus was 0.225 (95% CI: 0.184–0.275), well below the non-inferiority margin (NIM) of 1.345. Similarly, in Cameroon, the OR was 0.657 (95% CI: 0.497–0.869), below the NIM of 1.324, and in Côte d’Ivoire, the OR was 0.579 (95% CI: 0.482–0.694), also below the NIM of 1.404. These findings confirm that DuraNet® Plus met WHO non-inferiority criteria for the secondary endpoint of blood-feeding inhibition in all three countries. Full non-inferiority results by wash status are provided in the supplementary information (Table S2). Chemical analysis results: Chemical analysis of the ITNs showed that all three products (DuraNet®, Olyset® Plus, and DuraNet® Plus) contained insecticide levels consistent with their respective target doses prior to washing (Table 4 ). For alpha-cypermethrin-treated DuraNet®, the average content was close to the target dose of 5.8 g/kg in all three countries, with retention after 20 washes ranging from 89.88–90.03%, indicating high wash durability. Olyset® Plus, also met its target dose initially (19.1 g/kg permethrin and 9.0 g/kg PBO), but retention after 20 washes was lower, particularly for PBO. Across all countries, permethrin retention was approximately 73.5%, while PBO retention dropped to ~ 44.7%, indicating significant loss of the synergist with washing. In contrast, DuraNet® Plus demonstrated higher wash durability for both active ingredients. Alpha-cypermethrin retention after 20 washes remained consistently high (89.8–90.0%), and PBO retention averaged around 81.8%, substantially higher than that observed for Olyset® Plus. These results highlight the superior chemical retention profile of DuraNet® Plus, especially for PBO, suggesting enhanced potential for sustained efficacy after repeated washing. Table 4 Chemical analysis results of net samples from hut trials in Covè, Mbè and Mibellon Average alphacypermethrin/Permethrin Content (g/kg) Average Piperonyl butoxide Content (g/kg) ITN brand Country Target dose Unwashed Washed 20X % retention Target dose Unwashed Washed 20X % retention DuraNet Benin 5.8g/kg 5.83 5.24 89.88 n/a - - - Cameroon 5.82 5.24 89.97 - - - Cote D'Ivoire 5.82 5.24 90.03 - - - Olyset Plus Benin 20g/kg 19.15 14.07 73.47 10g/kg 9.02 4.03 44.68 Cameroon 19.12 14.07 73.59 9.03 4.05 44.85 Cote D'Ivoire 19.15 14.07 73.47 9.02 4.03 44.68 DuraNet Plus Benin 6g/kg 5.99 5.39 89.98 2.2g/kg 2.22 1.81 81.53 Cameroon 5.99 5.38 89.82 2.21 1.81 81.90 Cote D'Ivoire 5.99 5.39 89.98 2.21 1.81 81.90 Discussion This multi-country experimental hut study assessed the entomological efficacy of DuraNet® Plus, an ITN incorporating alpha-cypermethrin and PBO, for the control of pyrethroid-resistant malaria vectors in West and Central Africa. Conducted across three ecologically and entomologically distinct settings (Benin, Cameroon, and Côte d’Ivoire), the study evaluated the net's performance under semi-field experimental hut conditions that closely reflect their operational use in households. These settings varied in vector species composition, resistance intensity, and behavioural characteristics, offering a robust test of product performance across a range of vector characteristics and resistance profiles. The findings demonstrate that DuraNet® Plus is both effective and wash-resistant across diverse pyrethroid-resistant vector populations, achieving non-inferiority to a WHO-prequalified pyrethroid-PBO net with demonstrated public health value, Olyset® Plus, in all study sites in accordance with WHO evaluation criteria. Importantly, DuraNet® Plus met WHO criteria for non-inferiority to Olyset® Plus for both mosquito mortality and blood-feeding protection across all three study sites. This was demonstrated using data for both unwashed and 20-times washed nets, indicating that DuraNet® Plus retains its entomological efficacy over time and with repeated washing. In Benin and Côte d’Ivoire, DuraNet® Plus not only met the non-inferiority criteria but also demonstrated superior efficacy to Olyset® Plus in reducing mosquito mortality and preventing blood-feeding. In Cameroon, while DuraNet® Plus achieved non-inferiority, the differences between the two pyrethroid-PBO nets were smaller. This may be explained by the predominance of An. funestus , a vector species characterised by complex and high-intensity resistance mechanisms [ 17 ]. Across Benin, Cameroon, and Côte d’Ivoire, DuraNet® Plus consistently outperformed the pyrethroid-only net (DuraNet®) in terms of both mosquito mortality and blood-feeding inhibition, maintaining superior efficacy in both unwashed and 20-times washed conditions in most cases. This enhanced performance highlights the role of PBO in improving net efficacy by inhibiting cytochrome P450 enzymes; key mediators of metabolic resistance that are commonly overexpressed in malaria vector populations across sub-Saharan Africa [ 19 ]. However, in Benin, Olyset® Plus did not induce higher mosquito mortality than DuraNet®, and in fact, mortality with Olyset® Plus was significantly lower than with DuraNet® both before and after washing. This difference likely stems from the type of pyrethroid used in each net: Olyset® Plus incorporates permethrin, while DuraNet® Plus and DuraNet® use alpha-cypermethrin. WHO susceptibility bioassays conducted in Benin revealed that pre-exposure to PBO partially restored susceptibility to alpha-cypermethrin but had no such effect on permethrin, confirming earlier observations in the local vector population [ 20 ]. These results suggest that permethrin resistance in this region may involve additional mechanisms beyond P450 enzyme activity which require further investigation. Taken together, these results provide strong evidence that DuraNet® Plus is a viable and effective pyrethroid-PBO ITN for use in regions facing widespread pyrethroid resistance mediated by over-expressed cytochrome P450 enzymes. Its superior entomological performance compared to pyrethroid-only nets, along with its demonstrated non-inferiority to an established pyrethroid-PBO product, supports its inclusion among WHO-prequalified ITNs for procurement and deployment in malaria-endemic settings. Based on the outcomes of these trials, DuraNet® Plus was added to the WHO list of prequalified products in 2020 [ 21 ]. Subsequently, in 2023, WHO extended its policy recommendation on pyrethroid-PBO nets to include DuraNet® Plus following a technical consultation to assess data on second-in-class vector control products [ 15 ]. While entomological outcomes from experimental hut studies are considered valid proxies for predicting the community-level performance of ITNs [ 22 ], further operational evidence is essential for informing policies for their deployment and long-term use by householders. Ongoing longitudinal studies in Benin, Cameroon, and Tanzania are currently assessing the durability of DuraNet® Plus under real-world conditions, including net attrition, fabric integrity, residual bioefficacy, and chemical retention over a three-year period [ 23 ]. The results from these evaluations will provide critical insights for guiding decisions on net deployment schedules and replacement cycles under large-scale use. Conclusion This multi-country experimental hut study provides robust entomological evidence that DuraNet® Plus, An alphacypermethrin-PBO ITN, is a highly effective intervention for controlling pyrethroid-resistant malaria vectors. DuraNet® Plus demonstrated superior efficacy to a pyrethroid-only ITN and met WHO non-inferiority criteria in comparison with an established pyrethroid-PBO net across diverse resistance settings in West and Central Africa. These findings support its use as a valuable addition to the current portfolio of WHO-prequalified ITNs and underscore its potential to contribute meaningfully to malaria vector control strategies in regions burdened by metabolic resistance to pyrethroids. Further field studies on product durability and operational performance will strengthen the evidence base for its broader programmatic adoption. Abbreviations AI Active ingredient CI Confidence interval ITN Insecticide–treated nets Kdr Knockdown resistance HDPE high–density polyethylene CREC Centre de Recherches Entomologiques de Cotonou PAMVERC Pan–African Malaria Vector Research Consortium AIRID African Institute for Research in Infectious Diseases LSHTM London School of Hygiene & Tropical Medicine PBO Piperonyl butoxide NIM Non inferiority margin PQT/VCP Prequalification Unit Vector Control Product Assessment Team P450 Cytochrome P450 monooxygenase OR Odds ratio cRCT cluster randomised controlled trial WHO World Health Organisation Declarations Ethical approval and consent to participate Ethical approval for conduct of was obtained from the ethics review board of the Ministry of Health in Benin. Informed written consent was obtained from all human volunteers before their participation. All volunteers were offered a free course of chemoprophylaxis to mitigate the risk of malaria infection, and a stand-by nurse was available throughout the trials to assess any volunteers presenting with febrile symptoms or an adverse reaction to the test items. The methods described in this paper followed relevant guidelines and regulations. Approval for using guinea pigs for tunnel tests was granted by LSHTM Animal Welfare Ethics Review Board (AWERB) (2020-01B). Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Funding This study was supported by a research grant from the Shobikaa Impex Private Limited to Corine Ngufor. The funders had no role in the study design, data collection and analysis and decision to publish this manuscript. Author Contribution CN designed the methods, acquired funding and had overall responsibility for the conduct of the study across trial sites. AA, BM, BN and LAA performed the hut trials and bioassays under the supervision of CN, CW and RN. AA and JN supported coordination across sites and analysed the study data. CN, AA and JN prepared the figures and tables. CN drafted the manuscript with input from AA and JN. All authors reviewed the final manuscript. Acknowledgement We gratefully acknowledge Palaniappan Meyyappan of Shobikaa Impex Private Limited for supplying the study nets. We also extend our sincere thanks to the rice farmers of Covè for their participation in the study. Special appreciation is due to the technical and administrative teams at CREC in Benin, IPR in Côte d’Ivoire, and CRID in Cameroon for their invaluable support. We further thank the team at IIBAT, India, for carrying out the chemical analyses. Data Availability Data is provided within the manuscript or supplementary information files References Bhatt S, Weiss D, Cameron E, Bisanzio D, Mappin B, Dalrymple U, Battle K, Moyes C, Henry A, Eckhoff P. The effect of malaria control on Plasmodium falciparum in Africa between 2000 and 2015. Nature. 2015;526:207–11. WHO: WHO Malaria Threats Map. World Health Organisation, Geneva. 2025, https://apps.who.int/malaria/maps/threats/ WHO. World Malaria report. World Health Organisation, Geneva 2023. WHO. Guidelines for malaria vector control. Geneva, Switzerland: World Health Organization 2025. Gleave K, Lissenden N, Richardson M, Choi L, Ranson H. Piperonyl butoxide (PBO) combined with pyrethroids in insecticide-treated nets to prevent malaria in Africa. Cochrane Database Syst Rev. 2018;11:CD012776. WHO: List of WHO prequalified vector control products. Geneva, Switzerland: World Health Organization. 2024, https://www.who.int/pq-vector-control/prequalified-lists/PrequalifiedProducts27January2020.pdf?ua=1 Ngufor C, Fagbohoun J, Agbevo A, Ismail H, Challenger JD, Churcher TS, Rowland M. Comparative efficacy of two pyrethroid-piperonyl butoxide nets (Olyset Plus and PermaNet 3.0) against pyrethroid resistant malaria vectors: a non-inferiority assessment. Malar J. 2022;21:20. N’Guessan R, Asidi A, Boko P, Odjo A, Akogbeto M, Pigeon O, Rowland M. An experimental hut evaluation of PermaNet® 3.0, a deltamethrin—piperonyl butoxide combination net, against pyrethroid-resistant Anopheles gambiae and Culex quinquefasciatus mosquitoes in southern Benin. Trans R Soc Trop Med Hyg. 2010;104:758–65. Oumbouke WA, Rowland M, Koffi AA, Alou L, Camara S, N’Guessan R. Evaluation of an alpha-cypermethrin + PBO mixture long-lasting insecticidal net VEERALIN® LN against pyrethroid resistant Anopheles gambiae ss: an experimental hut trial in M’bé, central Côte d’Ivoire. Parasites vectors. 2019;12:1–10. Tungu P, Magesa S, Maxwell C, Malima R, Masue D, Sudi W, Myamba J, Pigeon O, Rowland M. 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Lancet. 2020;395:1292–303. AMP. Alliance for malaria prevention; mass campagn tracker. https://allianceformalariapreventioncom/mass-campaign-tracker/?_sfm_mc_date_of_import=20221015 2024, Accessed 28th October 2024. WHO Guideline. for the prequalification assessment of insecticide-treated nets. WHO. Technical consultation to assess comparative efficacy of vector control products Meeting report, 5 and 9 June 2023. World Health Organisation; 2023. Ngufor C, N'Guessan R, Fagbohoun J, Subramaniam K, Odjo A, Fongnikin A, Akogbeto M, Weetman D, Rowland M. Insecticide resistance profile of Anopheles gambiae from a phase II field station in Cove, southern Benin: implications for the evaluation of novel vector control products. Malar J. 2015;14:464. Menze BD, Wondji MJ, Tchapga W, Tchoupo M, Riveron JM, Wondji CS. Bionomics and insecticides resistance profiling of malaria vectors at a selected site for experimental hut trials in central Cameroon. Malar J. 2018;17:317. Koffi AA, Ahoua Alou LP, Adja MA, Chandre F, Pennetier C. Insecticide resistance status of Anopheles gambiae s.s population from M’Bé: a WHOPES-labelled experimental hut station, 10 years after the political crisis in Côte d’Ivoire. Malar J. 2013;12:151. Ranson H, Lissenden N. Insecticide Resistance in African Anopheles Mosquitoes: A Worsening Situation that Needs Urgent Action to Maintain Malaria Control. Trends Parasitol. 2016;32:187–96. Syme T, Gbegbo M, Obuobi D, Fongnikin A, Agbevo A, Todjinou D, Ngufor C. Pyrethroid-piperonyl butoxide (PBO) nets reduce the efficacy of indoor residual spraying with pirimiphos-methyl against pyrethroid-resistant malaria vectors. Sci Rep. 2022;12:6857. WHO: WHO list of prequalified vector control products. DuraNet Plus https : //extranetwhoint/prequal/vector-control-products/duranet- plus 2020, accessed 3rd May 2025. Sherrard-Smith E, Ngufor C, Sanou A, Guelbeogo MW, N’Guessan R, Elobolobo E, Saute F, Varela K, Chaccour CJ, Zulliger R, et al. Inferring the epidemiological benefit of indoor vector control interventions against malaria from mosquito data. Nat Commun. 2022;13:3862. Agbevo A, Ahogni I, Menze B, Tungu P, Kemibala EE, Govoetchan R, Wondji C, Padonou GG, Ngufor C. Community evaluation of the physical and insecticidal durability of DuraNet® Plus, an alpha-cypermethrin and piperonyl butoxide incorporated mosquito net: protocol for a multi-country study in West, Central and East Africa. Archives Public Health. 2023;81:202. Additional Declarations No competing interests reported. Supplementary Files Supplementaryinformation08.06.25.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 01 Mar, 2026 Reviews received at journal 29 Aug, 2025 Reviews received at journal 21 Jul, 2025 Reviews received at journal 17 Jul, 2025 Reviews received at journal 11 Jul, 2025 Reviewers agreed at journal 06 Jul, 2025 Reviewers agreed at journal 03 Jul, 2025 Reviewers agreed at journal 01 Jul, 2025 Reviewers agreed at journal 01 Jul, 2025 Reviewers agreed at journal 01 Jul, 2025 Reviewers invited by journal 30 Jun, 2025 Editor assigned by journal 10 Jun, 2025 Submission checks completed at journal 10 Jun, 2025 First submitted to journal 08 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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17:46:49","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":25034,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6848690/v1/0860b932e5b266356c93ee43.png"},{"id":92537222,"identity":"aab7032d-51ee-41cc-a065-ec6cbed12e6c","added_by":"auto","created_at":"2025-09-30 17:38:49","extension":"xml","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":138351,"visible":true,"origin":"","legend":"","description":"","filename":"0c8c3c5de50d4db38211f3f5cedaf9a21structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-6848690/v1/6ceec2063bdcd507c374a99b.xml"},{"id":92537565,"identity":"135b3951-6532-4732-82b1-e3154eefa161","added_by":"auto","created_at":"2025-09-30 17:46:49","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":152366,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-6848690/v1/96691e4c6c399e0641ca40d7.html"},{"id":92537208,"identity":"7103c9a5-7723-4795-8f25-0d4433cc2199","added_by":"auto","created_at":"2025-09-30 17:38:49","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":245825,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eCone bioassay mortality of susceptible Anopheles gambiae (Kisumu strain) and local wild pyrethroid-resistant Anopheles strains exposed to study nets prior to experimental hut trials in Benin, Cameroon, and Côte d’Ivoire.\u003c/em\u003e Mean 24-hour mortality rates of \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.s. (Kisumu susceptible strain) and local wild \u003cem\u003eAnopheles\u003c/em\u003e strains following 3-minute WHO cone bioassay exposure to unwashed net samples of DuraNet®, Olyset® Plus, and DuraNet® Plus. Bioassays were conducted prior to the start of experimental hut trials to verify product quality in each country. Green bars represent mortality in the susceptible Kisumu strain; pink bars represent mortality in local wild strains. Error bars indicate 95% confidence intervals. Results show consistently high efficacy of pyrethroid-PBO ITNs against the susceptible strain, and improved efficacy against wild resistant mosquito populations compared to the pyrethroid-only net.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6848690/v1/f9a0192760ad8d2df85dd598.jpg"},{"id":92538227,"identity":"0d015875-0f46-4cf1-8437-56e5f58b2897","added_by":"auto","created_at":"2025-09-30 17:54:49","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":212404,"visible":true,"origin":"","legend":"\u003cp\u003eWHO cylinder bioassay mortality (%) of wild pyrethroid resistant \u003cem\u003eAnopheles gambiae \u003c/em\u003es.l. (Benin \u0026amp; Cote d'Ivoire) and \u003cem\u003eAnopheles funestus\u003c/em\u003e (Cameroon) from experimental hut stations. Mean 24-hour mortality of wild \u003cem\u003eAnopheles\u003c/em\u003epopulations from Covè, Benin (pink bar), Mibellon, Cameroon (green bar), and M’bé, Côte d’Ivoire (orange bar) exposed for 1-hour to diagnostic concentrations of permethrin and alpha-cypermethrin in WHO tube bioassays, with and without PBO pre-exposure. Bars represent mean mortality with error bars indicating 95% confidence intervals.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6848690/v1/3f57d896709160598ffde13b.jpg"},{"id":92537203,"identity":"8fc9d033-0605-4d1e-a756-7a50f48578fb","added_by":"auto","created_at":"2025-09-30 17:38:49","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":34216,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eMortality of wild free-flying pyrethroid-resistant malaria vectors in experimental huts in Benin, Cameroon, and Côte d’Ivoire.\u003c/em\u003e Mean 24-hour mortality rates (%) of wild free-flying pyrethroid-resistant \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. in Covè, Benin (A), \u003cem\u003eAnopheles funestus\u003c/em\u003e in Mibellon, Cameroon (B), and \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. in M’bé, Côte d’Ivoire (C) entering experimental huts treated with DuraNet®, Olyset® Plus, and DuraNet® Plus, either unwashed (blue bars) or washed 20 times (light-blue bars). Error bars represent 95% confidence intervals of the mean. Different letters above bars indicate statistically significant differences in mosquito mortality between treatment groups within each country (p \u0026lt; 0.05, logistic regression).\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6848690/v1/b1fad8257c38d6948e25dac6.jpg"},{"id":92537560,"identity":"9f49002e-7267-43e4-9682-781c57f87559","added_by":"auto","created_at":"2025-09-30 17:46:49","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":18514,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eOdds ratios for mortality wild pyrethroid-resistant Anopheles vectors comparing DuraNet® Plus to Olyset® Plus in experimental hut trials in Benin, Cameroon, and Côte d’Ivoire.\u003c/em\u003e Odds ratios and 95% confidence intervals for wild mosquito mortality comparing DuraNet® Plus to Olyset® Plus in experimental hut trials conducted in Bénin (\u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l.), Cameroon (\u003cem\u003eAnopheles funestus\u003c/em\u003e), and Côte d’Ivoire (\u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l.). Diamonds represent the point estimates for each country, and horizontal lines indicate 95% confidence intervals. The black dashed vertical line indicates the line of equivalence (OR = 1). Red dashed lines show the country-specific non-inferiority margins (NIM): OR = 0.468 for Bénin, OR = 0.619 for Cameroon, and OR = 0.373 for Côte d’Ivoire. Odds ratios to the right of the NIM indicate that DuraNet® Plus is non-inferior to Olyset® Plus.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6848690/v1/b5f577b21c7134b78e53e1f8.jpg"},{"id":92537562,"identity":"cdd46cb4-4c42-4a1f-befd-0607dad0968f","added_by":"auto","created_at":"2025-09-30 17:46:49","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":40984,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eBlood-feeding rates of wild free-flying pyrethroid resistant malaria vectors in experimental huts in Benin, Cameroon and Cote D’Ivoire.\u003c/em\u003e Percentage of blood-fed wild free-flying pyrethroid-resistant \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. in Covè, Benin (A), \u003cem\u003eAnopheles funestus\u003c/em\u003e in Mibellon, Cameroon (B), and \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. in M’bé, Côte d’Ivoire (C) collected in experimental huts treated with DuraNet®, Olyset® Plus, and DuraNet® Plus, either unwashed (red bars) or washed 20 times (light beige bars). Error bars represent 95% confidence intervals of the mean. Different letters above bars indicate statistically significant differences in blood-feeding rates between treatment groups within each country (p \u0026lt; 0.05, logistic regression).\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6848690/v1/4c3a570be0a4bba3220d847c.jpg"},{"id":92537210,"identity":"7d183e44-0291-4889-ad14-523b894accab","added_by":"auto","created_at":"2025-09-30 17:38:49","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":17426,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eOdds ratios for blood-feeding comparing DuraNet® Plus to Olyset® Plus in experimental hut trials in Benin, Cameroon, and Côte d’Ivoire. \u003c/em\u003eOdds ratios (ORs) and 95% confidence intervals for blood-feeding in wild free-flying pyrethroid-resistant \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. in Benin, \u003cem\u003eAnopheles funestus\u003c/em\u003ein Cameroon, and \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. in Côte d’Ivoire, comparing DuraNet® Plus to Olyset® Plus in experimental hut trials. Diamonds represent the point estimates; horizontal lines show 95% confidence intervals. The black dashed vertical line indicates the line of equivalence (OR = 1). Red dashed lines represent the country-specific non-inferiority margins (NIM): OR = 1.35 for Benin, OR = 1.37 for Cameroon, and OR = 1.41 for Côte d’Ivoire. Odds ratios to the left of the NIM line indicate that DuraNet® Plus is non-inferior to Olyset® Plus for blood-feeding protection.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6848690/v1/8498c589c30bdb62459b239a.jpg"},{"id":92538493,"identity":"9edc06a1-d775-4151-b5c3-3a3d7aabffe7","added_by":"auto","created_at":"2025-09-30 18:02:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1860037,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6848690/v1/479438c5-3d9a-4015-b060-0683d271e03a.pdf"},{"id":92537204,"identity":"bab69599-f100-41c3-998c-4b59c88b52d7","added_by":"auto","created_at":"2025-09-30 17:38:49","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":26620,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation08.06.25.docx","url":"https://assets-eu.researchsquare.com/files/rs-6848690/v1/019de766fc811df6f494dc87.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A multi-country non-inferiority experimental hut evaluation of DuraNet® Plus, an alpha-cypermethrin and piperonyl butoxide treated net, for control of pyrethroid-resistant malaria vectors in West and Central Africa","fulltext":[{"header":"Background","content":"\u003cp\u003eInsecticide-treated nets (ITNs) have been a cornerstone of malaria control efforts in sub-Saharan Africa, accounting for an estimated 68% of the malaria cases averted globally since the year 2000 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. By providing both a physical barrier and an insecticidal effect, ITNs have substantially reduced malaria transmission, morbidity, and mortality. However, the long-term effectiveness of ITNs is increasingly threatened by the widespread emergence and intensification of \u003cem\u003eAnopheles\u003c/em\u003e mosquito resistance to pyrethroids, the most widely used insecticide on ITNs [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Pyrethroid resistance has undermined the impact of pyrethroid ITNs, contributing to a stall in global progress against malaria [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo address this threat, the World Health Organization (WHO) has endorsed the development and deployment of a new generation of insecticide-treated nets (ITNs) that combine a pyrethroid insecticide with either a synergist or an additional insecticidal compound [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Among these innovations are pyrethroid-PBO nets, which incorporate piperonyl butoxide (PBO) alongside a pyrethroid [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. PBO functions as a synergist by inhibiting cytochrome P450 enzymes responsible for the metabolic detoxification of pyrethroids in resistant \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes. By blocking these resistance mechanisms, PBO enhances the toxicity of pyrethroids and helps to restore the efficacy of the insecticidal component of the net. Multiple studies, conducted across various malaria-endemic regions, have demonstrated that pyrethroid-PBO nets achieve improved entomological outcomes such as increased mosquito mortality and reduced blood-feeding rates compared to standard pyrethroid-only nets [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Moreover, in certain settings, these benefits have translated into a measurable public health impact, with pyrethroid-PBO nets providing greater reductions in malaria incidence and prevalence relative to pyrethroid-only ITNs in large-scale cluster-randomized controlled trials (cRCT) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe WHO\u0026rsquo;s endorsement of pyrethroid-PBO nets in 2017, was followed by a significant shift in procurement patterns across Africa, with increased uptake of these next-generation nets over standard pyrethroid-only ITNs [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Between 2022 and 2024, pyrethroid-PBO nets accounted for nearly 50% of all ITNs delivered to sub-Saharan Africa, underscoring widespread recognition of their enhanced performance against pyrethroid-resistant \u003cem\u003eAnopheles\u003c/em\u003e vectors. To ensure a stable supply pipeline and foster healthy market competition, it is essential to expand the portfolio of WHO-prequalified pyrethroid-PBO nets available for large-scale procurement.\u003c/p\u003e\u003cp\u003eDuraNet\u0026reg; Plus, a pyrethroid-PBO net combining alpha-cypermethrin with PBO, was developed by Shobikaa Impex Private Limited in response to growing procurement demands for effective pyrethroid-PBO ITNs. To obtain WHO prequalification, new ITN product types must undergo rigorous semi-field evaluations to demonstrate entomological superiority over standard pyrethroid-only nets [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In addition, current WHO guidelines require that pyrethroid-PBO nets show non-inferiority to an existing pyrethroid-PBO ITN with proven public health impact, as demonstrated through cRCT [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. These comparative studies are expected to be conducted across a range of ecological settings representative of the diverse vector populations and resistance profiles where the nets are intended for deployment, ensuring that efficacy claims are broadly generalizable.\u003c/p\u003e\u003cp\u003eTo support the WHO prequalification of DuraNet\u0026reg; Plus, a series of experimental hut trials was conducted to evaluate its entomological efficacy at three sites with documented pyrethroid resistance in West and Central Africa; Benin, C\u0026ocirc;te d\u0026rsquo;Ivoire, and Cameroon. The study was designed in accordance with existing WHO guidelines, with the primary objective of assessing the non-inferiority of DuraNet\u0026reg; Plus compared to Olyset\u0026reg; Plus, a WHO-prequalified pyrethroid-PBO net that has demonstrated improved epidemiological impact against malaria. In addition, the study aimed to confirm the entomological superiority of DuraNet\u0026reg; Plus over DuraNet\u0026reg;, a standard WHO-prequalified pyrethroid-only ITN. Key outcomes measured included mosquito mortality and blood-feeding inhibition against wild populations of pyrethroid-resistant \u003cem\u003eAnopheles\u003c/em\u003e vectors. WHO susceptibility bioassays were conducted to characterize the resistance profile of the vector populations at each site during the study period.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eExperimental hut sites\u003c/h2\u003e\u003cp\u003eThe experimental hut trials were conducted between September 2018 and March 2019 at three sites: Cov\u0026egrave; (Benin), Mibellon (Cameroon), and Bouak\u0026eacute; (C\u0026ocirc;te d\u0026rsquo;Ivoire). At each site, seven West African-style experimental huts were used. The huts were constructed from brick, plastered with cement, and topped with corrugated iron roofs. Each hut featured four narrow window slits (1 cm wide) on the walls to allow mosquito entry and was built on a raised concrete plinth surrounded by a water-filled moat to deter entry of predators such as ants and lizards. To eliminate any potential contamination from previous evaluations, all hut walls were re-plastered, and the cement was allowed to cure for one month prior to the start of the trials. A detailed description of the local vector populations at each study site is provided below:\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eBenin\u003c/strong\u003e\u003cp\u003eThe study was conducted at the CREC/LSHTM experimental hut site situated in irrigated rice fields in Cov\u0026egrave;, southern Benin, where the malaria vector population is highly resistant to pyrethroids. The vector species comprised a combination of \u003cem\u003eAnopheles coluzzii\u003c/em\u003e and \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.s.. Resistance is mediated by a high frequency (89%) of the L1014F \u003cem\u003ekdr\u003c/em\u003e mutation and overexpression of metabolic enzymes such as CYP6P3, known to metabolize pyrethroids efficiently [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCameroon\u003c/strong\u003e\u003cp\u003eThe trial was conducted at the CRID experimental hut station in Mibellon, Adamawa region, where permanent water bodies support high year-round mosquito densities. The local vector population is predominantly \u003cem\u003eAn. funestus\u003c/em\u003e (80%) with smaller proportions of \u003cem\u003eAn. gambiae\u003c/em\u003e s.s. (20%) with both species exhibiting high levels of resistance to pyrethroids and organochlorides [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCote d\u0026rsquo;Ivoire\u003c/strong\u003e\u003cp\u003eThe trial was conducted at the M\u0026rsquo;b\u0026eacute; field station near Bouak\u0026eacute; city in central C\u0026ocirc;te d\u0026rsquo;Ivoire. The vector species is also composed of a combination of \u003cem\u003eAnopheles coluzzii\u003c/em\u003e and \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.s. though \u003cem\u003eAn. coluzzii\u003c/em\u003e predominates year-round in the surrounding irrigated rice fields. The mosquito population is highly resistant to multiple insecticide classes, with documented L1014F and Ace-1 mutations and elevated metabolic enzyme activity [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Resistance to pyrethroids is extremely high, with over 1700-fold resistance to deltamethrin reported.\u003c/p\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eInsecticide susceptibility bioassays:\u003c/h3\u003e\n\u003cp\u003eTo confirm the presence of pyrethroid resistance and investigate the potential role of cytochrome P450-mediated metabolic mechanisms, WHO cylinder bioassays were conducted at each study site (Cov\u0026egrave;, Mibellon, and M\u0026rsquo;b\u0026eacute;) during the hut trial period. The assays were performed on 2\u0026ndash;5-day-old adult F1 female mosquitoes reared from larvae collected near the experimental huts. For each insecticide treatment, approximately 100 female mosquitoes were tested in batches of 25 using filter papers treated with alpha-cypermethrin (0.05%) or permethrin (0.75%), with and without pre-exposure to 4% piperonyl butoxide (PBO). Mosquitoes were exposed for one hour, with mortality assessed after a 24-hour holding period.\u003c/p\u003e\n\u003ch3\u003eExperimental hut treatments\u003c/h3\u003e\n\u003cp\u003eDuraNet\u0026reg; Plus is a pyrethroid-PBO ITN manufactured by Shobikaa Impex Private Ltd, composed of 150 denier high-density polyethylene (HDPE) monofilament yarn. The net is uniformly treated on all panels with a combination of alpha-cypermethrin (6.0 g/kg) and PBO (2.2 g/kg). It has a mesh size of 20 holes/cm\u0026sup2; and a fabric weight of 45 g/m\u0026sup2;. DuraNet\u0026reg; Plus was compared to two types of WHO prequalified ITNs:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eOlyset\u0026reg; Plus, a WHO-prequalified pyrethroid-PBO ITN developed by Sumitomo Chemical Company. Olyset\u0026reg; Plus is made from 150 denier monofilament HDPE and is treated with a combination of permethrin (20 g/kg) and PBO (10 g/kg) incorporated into the fibres. The net has a mesh size of 6.3 holes/cm\u0026sup2; and a fabric weight of 40 g/m\u0026sup2;.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eDuraNet\u0026reg;, a WHO-prequalified pyrethroid-only ITN also manufactured by Shobikaa Impex Private Ltd. DuraNet\u0026reg; is made from 150 denier HDPE monofilament yarn and is treated with alpha-cypermethrin at a concentration of 5.8 g/kg. The net has a mesh size of 20 holes/cm\u0026sup2; and a fabric weight of 45 g/m\u0026sup2;.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eAll net types were evaluated in both unwashed and 20-times washed conditions following WHO standard procedures [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The following 7 treatments were thus assessed at each hut site:\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eUntreated net\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eUnwashed DuraNet\u0026reg;\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eDuraNet\u0026reg; washed 20 times\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eUnwashed Olyset\u0026reg; Plus\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eOlyset\u0026reg; Plus washed 20 times\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eUnwashed DuraNet\u0026reg; Plus\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eDuraNet\u0026reg; Plus washed 20 times\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003e To simulate natural wear and tear, all nets including untreated control, were deliberately holed following WHO guidelines, with six 4 \u0026times; 4 cm holes cut into each net (two on each long side and one on each short side). Net washing was performed according to WHO standard procedures. Each net was washed in an aluminium basin containing 10 litres of water and 2 g/L of \u003cem\u003eSavon de Marseille\u003c/em\u003e, with agitation for a total of 10 minutes. The nets were then rinsed in clean water using the same method, dried horizontally in the shade, and stored at ambient temperature between washes. Washing intervals were based on regeneration time studies: daily for DuraNet\u0026reg; Plus and DuraNet\u0026reg; and every two days for Olyset\u0026reg; Plus.\u003c/p\u003e\n\u003ch3\u003ePre-trial WHO cone bioassays\u003c/h3\u003e\n\u003cp\u003eTo verify the quality of the test items prior to the hut trials, a series of pre-trial WHO cone bioassays were conducted using 30 \u0026times; 30 cm net samples cut from unwashed nets of each ITN type at each study site. Bioassays were performed with both the susceptible \u003cem\u003eAn. gambiae\u003c/em\u003e Kisumu strain and the local field strain at each study site. Approximately 200 mosquitoes per strain were exposed for 3 minutes on five replicate net pieces per ITN type in groups of five per cone. After exposure, mosquitoes were held at 27\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and 75\u0026thinsp;\u0026plusmn;\u0026thinsp;10% relative humidity, with access to a 10% glucose solution. Delayed mortality was recorded 24 hours post-exposure.\u003c/p\u003e\n\u003ch3\u003eHut trial design\u003c/h3\u003e\n\u003cp\u003eTreatments were initially assigned to the seven experimental huts at random. To minimise positional bias, a randomized Latin Square Design was used to rotate the seven treatments weekly across the huts. The core trial lasted for seven weeks to complete the Latin square cycle, after which mosquito collections continued for an additional two weeks to increase sample size. Data were collected over six nights each week, with the seventh day reserved for thorough cleaning of the huts in preparation for the next rotation. Six replicate nets were tested per treatment and rotated daily within each treatment arm. The entire trial spanned nine weeks, yielding a total of 54 nights of data collection.\u003c/p\u003e\u003cp\u003eAt each site, seven consenting adult volunteers slept in the huts each night from 9:00 p.m. to 5:00 a.m. To control for variation in individual attractiveness to mosquitoes, sleepers were also rotated daily between huts using a Latin Square Design. Each morning, mosquitoes were collected from inside the net, the hut room, and the veranda trap, then transferred to the laboratory for morphological identification and assessment of blood-feeding status and immediate mortality. Surviving female \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. were maintained at 27\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and 75\u0026thinsp;\u0026plusmn;\u0026thinsp;10% relative humidity with access to 10% glucose solution, and delayed mortality was recorded after 24 hours.\u003c/p\u003e\u003cp\u003eThe following entomological outcomes were used to evaluate the efficacy of each treatment in the experimental huts:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eMortality: The proportion of mosquitoes found dead after a 24-hour holding period following collection from each treatment arm.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eBlood-feeding rate: The proportion of mosquitoes that successfully blood-fed in each treatment group.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eDeterrence: The proportional reduction in the number of mosquitoes entering huts with treated nets compared to huts with untreated nets.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eInduced exophily: The proportion of mosquitoes exiting to the veranda in huts with treated nets, used to estimate insecticide-induced exiting relative to control huts.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eBlood-feeding inhibition: The proportional reduction in blood-feeding in huts with insecticide-treated nets compared to huts with untreated nets.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eEthical considerations:\u003c/h2\u003e\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003cp\u003e for the study was obtained from the relevant institutional review boards in each participating country. Written informed consent was obtained from all volunteer sleepers prior to their enrolment, with the study procedures and consent forms explained in their local language by a trained interpreter. A standby nurse was present throughout the trial to monitor the health of participants and manage any cases of fever or persistent headache. Any sleeper who tested positive for malaria during the study was immediately withdrawn and provided with appropriate treatment in accordance with national public health guidelines.\u003c/p\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eProportional outcomes (mosquito mortality, blood-feeding, and exophily) were analysed using logistic regression, while count data such as mosquito entry were assessed with negative binomial regression. Each outcome was modelled separately, with fixed effects included for hut, sleeper, and daily variation.\u003c/p\u003e\u003cp\u003e Non-inferiority analyses comparing DuraNet\u0026reg; Plus to Olyset\u0026reg; Plus followed WHO guidelines. DuraNet\u0026reg; Plus was considered non-inferior for mortality if the lower bound of the 95% confidence interval (CI) for the odds ratio exceeded the non-inferiority margin (NIM), and for blood-feeding if the upper bound was below the NIM. The NIM represented a 7% difference in efficacy relative to Olyset\u0026reg; Plus. Superiority of DuraNet\u0026reg; Plus over DuraNet\u0026reg; was determined by a significantly higher mortality and lower blood-feeding rates (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Analyses for non-inferiority and superiority were primarily conducted using pooled data for unwashed and washed nets to assess overall efficacy across the net\u0026rsquo;s lifespan.\u003c/p\u003e\u003cp\u003eAll analyses were performed in Stata version 18.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003ePre-trial cone bioassays results\u003c/h2\u003e\u003cp\u003eAll ITN types induced 80\u0026ndash;100% mortality in the Kisumu strain in pretrial cone bioassays across the three sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), confirming high insecticidal activity and appropriate storage prior to hut trials. In contrast, mortality rates among the local wild strains in the hut stations were considerably lower, highlighting varying degrees of pyrethroid resistance. DuraNet\u0026reg; induced minimal mortality (\u0026lt;\u0026thinsp;10%) against the wild strains across all three locations. In Benin and Cameroon, Olyset\u0026reg; Plus induced the highest mortality in wild mosquitoes (73% and 70%, respectively), followed by DuraNet\u0026reg; Plus (45% and 40%). In C\u0026ocirc;te d\u0026rsquo;Ivoire, mortality among the wild strain were lower (\u0026lt;\u0026thinsp;25%) though DuraNet\u0026reg; Plus achieved the highest mortality rate. The results therefore showed improved mortality with the pyrethroid-PBO nets compared to the pyrethroid-only net with the local strains which confirmed the suitability of the vector populations for the study.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eWHO cylinder bioassay results\u003c/h2\u003e\u003cp\u003eMortality following exposure to pyrethroids alone was consistently low in cylinder bioassays across all sites, confirming a high frequency of resistance in the local vector populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). For permethrin (0.75%), mortality ranged from approximately 15% in C\u0026ocirc;te d\u0026rsquo;Ivoire to 30% in Cameroon. Alpha-cypermethrin (0.05%) induced slightly higher mortality, ranging from 17% in Benin to 41% in Cameroon. Pre-exposure to PBO (4%) prior to pyrethroid exposure significantly increased mortality in most settings, indicating the involvement of P450-mediated metabolic resistance. In Cameroon, PBO fully restored susceptibility to alpha-cypermethrin, with mortality reaching 100%, and partially restored alpha-cypermethrin susceptibility in C\u0026ocirc;te d\u0026rsquo;Ivoire (73%) and Benin (55%). A similar improvement was observed for permethrin in C\u0026ocirc;te d\u0026rsquo;Ivoire and Cameroon, where mortality rose to over 60%. In contrast, pre-exposure to PBO had no effect on permethrin-induced mortality in Benin; mortality remained the same. Overall, these results highlight intense pyrethroid resistance across all sites and support the role of PBO in partially restoring susceptibility, particularly to alpha-cypermethrin.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eMosquito entry rates\u003c/h2\u003e\u003cp\u003eA total of 18,075 wild pyrethroid-resistant malaria vector mosquitoes were collected across all sites and treatments during the experimental hut trials in Benin, Cameroon, and C\u0026ocirc;te d\u0026rsquo;Ivoire (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). In Benin, mosquito entry remained high, with no deterrence observed for DuraNet\u0026reg; or Olyset\u0026reg; Plus. DuraNet\u0026reg; Plus demonstrated modest deterrence of 17.2% (unwashed) and 6.8% (washed), which was significantly higher than both DuraNet\u0026reg; and Olyset\u0026reg; Plus (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In Cameroon, all nets showed strong deterrent effects, with DuraNet\u0026reg; and Olyset\u0026reg; Plus reducing entry by 41.3\u0026ndash;53.7%. DuraNet\u0026reg; Plus again achieved the highest deterrence (54.2% when unwashed and 51.9% when washed), with a significantly greater effect than DuraNet\u0026reg; (p\u0026thinsp;=\u0026thinsp;0.03) and comparable to Olyset\u0026reg; Plus (p\u0026thinsp;=\u0026thinsp;0.08). In C\u0026ocirc;te d\u0026rsquo;Ivoire, deterrence across treatments ranged from 24.2\u0026ndash;46.9%, with DuraNet\u0026reg; Plus showing strong and consistent reductions in mosquito entry (42.5% unwashed; 37.5% washed), significantly higher than Olyset\u0026reg; Plus (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Overall, DuraNet\u0026reg; Plus consistently reduced mosquito entry across all sites and this effect was sustained after 20 washes, demonstrating superior or comparable deterrence to existing WHO-prequalified nets.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMosquito entry rates in experimental huts\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNet type\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eDuraNet Net\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eOlyset Plus\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003eDuraNet\u0026reg; Plus\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWash status\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eUnwashed\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eWashed 20X\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eUnwashed\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003eWashed 20X\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003eUnwashed\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003eWashed 20X\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"8\" nameend=\"c8\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eBenin\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal females caught\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e723\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e814\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e866\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1139\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e599\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e674\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAverage catch per night\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e22.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e12.48\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e% Deterrence\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e17.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e6.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"8\" nameend=\"c8\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCameroon\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal females caught\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e618\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e286\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e342\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e306\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e363\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e283\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e297\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAverage catch per night\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e% Deterrence\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e53.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e44.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e50.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e41.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e54.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e51.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"8\" nameend=\"c8\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCote D'Ivoire\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal females caught\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1366\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e725\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1017\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e908\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1036\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e785\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e854\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAverage catch per night\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e12.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e15.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e10.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e% Deterrence\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e46.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e25.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e33.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e24.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e42.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e37.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eMortality rates in experimental huts:\u003c/h2\u003e\u003cp\u003eAcross all three study sites, DuraNet\u0026reg; Plus consistently induced the highest mosquito mortality, both before and after washing (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn Cov\u0026egrave;, Benin (\u003cem\u003eAn. gambiae\u003c/em\u003e s.l., Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Panel A), unwashed DuraNet\u0026reg; Plus resulted in significantly higher mortality (31.2%) compared to unwashed Olyset\u0026reg; Plus (16.0%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.025) and unwashed DuraNet\u0026reg; (20.0%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). This superiority was maintained following 20 washes, with washed DuraNet\u0026reg; Plus achieving significantly higher mortality than both washed Olyset\u0026reg; Plus (27.7% vs. 13.9%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.010) and washed DuraNet\u0026reg; (27.7% vs. 20.1%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Within-product comparisons indicated no significant reduction in efficacy due to washing for DuraNet\u0026reg; Plus (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.228) or DuraNet\u0026reg; (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.743), whereas Olyset\u0026reg; Plus showed a significant decline (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.025).\u003c/p\u003e\u003cp\u003eIn Mibellon, Cameroon (\u003cem\u003eAn. funestus\u003c/em\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Panel B), unwashed DuraNet\u0026reg; Plus induced the highest mortality (32.8%), followed by Olyset\u0026reg; Plus (29.7%), although the difference between the two was not statistically significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.551). Both nets induced significantly higher mortality than unwashed DuraNet\u0026reg; (13.9%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). After 20 washes, DuraNet\u0026reg; Plus remained significantly more effective (22.8%) than both Olyset\u0026reg; Plus (14.6%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001) and DuraNet\u0026reg; (11.9%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The difference between washed Olyset\u0026reg; Plus and DuraNet\u0026reg; was not significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.103). Washing significantly reduced mortality for DuraNet\u0026reg; Plus (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.014), but not for Olyset\u0026reg; Plus (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.103) or DuraNet\u0026reg; (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.361).\u003c/p\u003e\u003cp\u003eIn M\u0026rsquo;b\u0026eacute;, C\u0026ocirc;te d\u0026rsquo;Ivoire (\u003cem\u003eAn. gambiae\u003c/em\u003e s.l., Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Panel C), DuraNet\u0026reg; Plus again achieved the highest mortality among unwashed nets (24.5%), significantly exceeding that of Olyset\u0026reg; Plus (15.3%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and DuraNet\u0026reg; (12.3%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The difference between Olyset\u0026reg; Plus and DuraNet\u0026reg; was not significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.316). Following 20 washes, DuraNet\u0026reg; Plus maintained superior efficacy (14.4%) compared to Olyset\u0026reg; Plus (8.6%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001) and DuraNet\u0026reg; (7.0%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while the difference between Olyset\u0026reg; Plus and DuraNet\u0026reg; remained non-significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.753). Washing led to a statistically significant reduction in mortality for all ITN types (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001); however, DuraNet\u0026reg; Plus consistently retained the highest performance across wash conditions.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cem\u003eMortality (24h) of wild free-flying malaria vectors exposed to ITNs in experimental huts in Benin, Cameroon, and C\u0026ocirc;te d\u0026rsquo;Ivoire\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUntreated Net\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eDuraNet\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eOlyset Plus\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003eDuraNet Plus\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of washes\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003en/a\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eUnwashed\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20 washes\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eUnwashed\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e20 washes\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eUnwashed\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003e20 washes\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"8\" nameend=\"c8\" namest=\"c1\"\u003e\u003cp\u003eCov\u0026egrave;, Benin (\u003cem\u003eAn. gambiae\u003c/em\u003e s.l.)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal females caught\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e723\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e814\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e866\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1139\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e599\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e674\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN dead 24 h\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e162\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e178\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e182\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e150\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e187\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e187\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e24 h mortality (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e19.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e16.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e12.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e31.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e27.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e24 h mortality 95% Cls\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e(0.4-2.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(17.1\u0026ndash;22.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(17.8\u0026ndash;23.2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(13.8\u0026ndash;18.1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e(10.6\u0026ndash;14.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e(27.5\u0026ndash;34.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e(24.3\u0026ndash;31.1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"8\" nameend=\"c8\" namest=\"c1\"\u003e\u003cp\u003eMibellon, Cameroon (\u003cem\u003eAn funestus\u003c/em\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal females caught\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e618\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e286\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e342\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e306\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e363\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e283\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e297\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN dead 24 h\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e68\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e24 h mortality (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e14.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e12.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e29.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e14.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e32.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e22.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e24 h mortality 95% Cls\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e(2.7\u0026ndash;5.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(9.9\u0026ndash;18.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(8.5\u0026ndash;15.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(24.6\u0026ndash;34.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e(10.9\u0026ndash;18.2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e(27.3\u0026ndash;38.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e(18.1\u0026ndash;27.6)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"8\" nameend=\"c8\" namest=\"c1\"\u003e\u003cp\u003eM\u0026rsquo;b\u0026eacute;, C\u0026ocirc;te d\u0026rsquo;Ivoire (\u003cem\u003eAn. gambiae\u003c/em\u003e s.l.)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal females caught\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1366\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e725\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1017\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e908\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1036\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e785\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e854\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN dead 24 h\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e139\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e192\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e123\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e24 h mortality (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e15.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e24.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e14.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e24 h mortality 95% Cls\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e(3.1\u0026ndash;5.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(9.8\u0026ndash;14.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(5.4\u0026ndash;8.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(12.9\u0026ndash;17.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e(6.8\u0026ndash;10.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e(21.4\u0026ndash;27.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e(12.0-16.7)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWhen data from unwashed and washed nets were combined, DuraNet\u0026reg; Plus consistently induced higher mean mosquito mortality than Olyset\u0026reg; Plus across all study sites: 29.5% vs. 14.9% in Benin, 27.8% vs. 22.2% in Cameroon, and 19.5% vs. 12.0% in C\u0026ocirc;te d\u0026rsquo;Ivoire. In Benin, the odds ratio (OR) for mortality with DuraNet\u0026reg; Plus compared to Olyset\u0026reg; Plus was 2.809 (95% CI: 2.339\u0026ndash;3.375), exceeding the non-inferiority margin (NIM) of 0.468 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Table S2). Similar findings were observed in Cameroon (OR\u0026thinsp;=\u0026thinsp;1.811, 95% CI: 1.319\u0026ndash;2.488, NIM\u0026thinsp;=\u0026thinsp;0.619) and C\u0026ocirc;te d\u0026rsquo;Ivoire (OR\u0026thinsp;=\u0026thinsp;2.277, 95% CI: 1.853\u0026ndash;2.799, NIM\u0026thinsp;=\u0026thinsp;0.373), confirming that DuraNet\u0026reg; Plus met the WHO criteria for non-inferiority to Olyset\u0026reg; Plus with respect to the primary endpoint of mosquito mortality in all three countries.\u003c/p\u003e\u003cp\u003eFurther analysis of superiority indicated that DuraNet\u0026reg; Plus was also significantly more effective than DuraNet\u0026reg; for mosquito mortality across all sites (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Table S3). Detailed non-inferiority outcomes by wash status are provided in the supplementary information (Table S2).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eBlood-feeding rates in experimental huts:\u003c/h2\u003e\u003cp\u003eBlood-feeding rates were generally lower with all treated nets compared to the untreated control (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In Cov\u0026egrave;, Benin, DuraNet\u0026reg; Plus achieved significantly lower blood-feeding rates than both Olyset\u0026reg; Plus and DuraNet\u0026reg;, both in the unwashed condition (10.5% vs. 25.8% and 22.5%, respectively; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and after 20 washes (14.8% vs. 43.6% and 27.4%; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). These results highlight the enhanced personal protection provided by DuraNet\u0026reg; Plus over Olyset\u0026reg; Plus in this setting. In Mibellon, Cameroon, differences in blood-feeding rates among ITNs were less pronounced, although DuraNet\u0026reg; Plus showed modest improvements in blood-feeding inhibition compared to both Olyset\u0026reg; Plus and DuraNet\u0026reg; in both unwashed and washed conditions (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In M\u0026rsquo;b\u0026eacute;, C\u0026ocirc;te d\u0026rsquo;Ivoire, both pyrethroid-PBO nets\u0026mdash;DuraNet\u0026reg; Plus and Olyset\u0026reg; Plus\u0026mdash;significantly reduced blood-feeding compared to DuraNet\u0026reg; when unwashed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). However, the superior protection offered by Olyset\u0026reg; Plus diminished after washing (65.2\u0026ndash;6.2% blood-feeding inhibition, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), while DuraNet\u0026reg; Plus retained its protective efficacy. Among unwashed nets, Olyset\u0026reg; Plus achieved the highest blood-feeding inhibition (65.2%), whereas among washed nets, DuraNet\u0026reg; Plus provided the greatest inhibition (54.8%).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cem\u003eBlood-feeding of wild free-flying malaria vectors exposed to ITNs in experimental huts in Benin, Cameroon, and C\u0026ocirc;te d\u0026rsquo;Ivoire\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUntreated Net\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eDuraNet\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eOlyset Plus\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003eDuraNet Plus\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of washes\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003en/a\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eUnwashed\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20 washes\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eUnwashed\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e20 washes\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eUnwashed\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003e20 washes\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"8\" nameend=\"c8\" namest=\"c1\"\u003e\u003cp\u003eCov\u0026egrave;, Benin (\u003cem\u003eAn. gambiae\u003c/em\u003e s.l.)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal females caught\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e723\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e814\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e866\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1139\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e599\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e674\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN Blood-feeding\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e366\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e183\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e237\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e294\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e523\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBlood-feeding (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e50.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e22.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e27.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e25.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e43.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e14.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBlood-feeding 95% Cls\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e(47.0-54.2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(19.6\u0026ndash;25.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(24.4\u0026ndash;30.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(23.2\u0026ndash;28.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e(40.7\u0026ndash;46.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e(8.0\u0026ndash;13)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e(12.1\u0026ndash;17.5)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBlood-feeding inhibition (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e55.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e45.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e49.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e13.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e79.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e70.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"8\" nameend=\"c8\" namest=\"c1\"\u003e\u003cp\u003eMibellon, Cameroon (\u003cem\u003eAn funestus\u003c/em\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal females caught\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e618\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e286\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e342\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e306\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e363\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e283\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e297\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN Blood-feeding\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e410\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e118\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e147\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e133\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e164\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e105\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBlood-feeding (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e66.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e41.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e43.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e43.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e45.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e34.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e35.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBlood-feeding 95% Cls\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e(62.6\u0026ndash;70.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(35.5\u0026ndash;46.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(37.7\u0026ndash;48.2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(37.9\u0026ndash;49.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e(40.0-50.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e(28.7\u0026ndash;39.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e(29.9\u0026ndash;40.7)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBlood-feeding inhibition (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e37.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e35.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e34.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e31.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e48.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e46.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"8\" nameend=\"c8\" namest=\"c1\"\u003e\u003cp\u003eM\u0026rsquo;b\u0026eacute;, C\u0026ocirc;te d\u0026rsquo;Ivoire (\u003cem\u003eAn. gambiae\u003c/em\u003e s.l.)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal females caught\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1366\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e725\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1017\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e908\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1036\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e785\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e854\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN Blood-feeding\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e532\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e189\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e273\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e123\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e378\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e139\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e150\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBlood-feeding (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e38.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e26.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e26.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e13.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e36.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e17.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e17.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBlood-feeding 95% Cls\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e(36.3\u0026ndash;41.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(22.8\u0026ndash;29.2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(24.1\u0026ndash;29.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(11.3\u0026ndash;15.7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e(33.5\u0026ndash;39.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e(15.0-20.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e(15.0-20.1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBlood-feeding inhibition (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e33.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e31.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e65.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e54.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e54.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWhen data from unwashed and washed nets were pooled, DuraNet\u0026reg; Plus demonstrated non-inferiority to Olyset\u0026reg; Plus in terms of protection against mosquito blood-feeding across all study sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In Benin, the OR for blood-feeding with DuraNet\u0026reg; Plus compared to Olyset\u0026reg; Plus was 0.225 (95% CI: 0.184\u0026ndash;0.275), well below the non-inferiority margin (NIM) of 1.345. Similarly, in Cameroon, the OR was 0.657 (95% CI: 0.497\u0026ndash;0.869), below the NIM of 1.324, and in C\u0026ocirc;te d\u0026rsquo;Ivoire, the OR was 0.579 (95% CI: 0.482\u0026ndash;0.694), also below the NIM of 1.404. These findings confirm that DuraNet\u0026reg; Plus met WHO non-inferiority criteria for the secondary endpoint of blood-feeding inhibition in all three countries. Full non-inferiority results by wash status are provided in the supplementary information (Table S2).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eChemical analysis results:\u003c/h2\u003e\u003cp\u003eChemical analysis of the ITNs showed that all three products (DuraNet\u0026reg;, Olyset\u0026reg; Plus, and DuraNet\u0026reg; Plus) contained insecticide levels consistent with their respective target doses prior to washing (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). For alpha-cypermethrin-treated DuraNet\u0026reg;, the average content was close to the target dose of 5.8 g/kg in all three countries, with retention after 20 washes ranging from 89.88\u0026ndash;90.03%, indicating high wash durability. Olyset\u0026reg; Plus, also met its target dose initially (19.1 g/kg permethrin and 9.0 g/kg PBO), but retention after 20 washes was lower, particularly for PBO. Across all countries, permethrin retention was approximately 73.5%, while PBO retention dropped to ~\u0026thinsp;44.7%, indicating significant loss of the synergist with washing. In contrast, DuraNet\u0026reg; Plus demonstrated higher wash durability for both active ingredients. Alpha-cypermethrin retention after 20 washes remained consistently high (89.8\u0026ndash;90.0%), and PBO retention averaged around 81.8%, substantially higher than that observed for Olyset\u0026reg; Plus. These results highlight the superior chemical retention profile of DuraNet\u0026reg; Plus, especially for PBO, suggesting enhanced potential for sustained efficacy after repeated washing.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eChemical analysis results of net samples from hut trials in Cov\u0026egrave;, Mb\u0026egrave; and Mibellon\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"10\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e\u003cp\u003eAverage alphacypermethrin/Permethrin Content (g/kg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c10\" namest=\"c7\"\u003e\u003cp\u003eAverage Piperonyl butoxide Content (g/kg)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eITN brand\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCountry\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTarget dose\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUnwashed\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eWashed 20X\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e% retention\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eTarget dose\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eUnwashed\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eWashed 20X\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003e% retention\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eDuraNet\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBenin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e5.8g/kg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e89.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003en/a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCameroon\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e89.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCote D'Ivoire\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e90.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eOlyset Plus\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBenin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e20g/kg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e19.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e14.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e73.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e10g/kg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e9.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e4.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e44.68\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCameroon\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e19.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e14.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e73.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e9.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e4.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e44.85\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCote D'Ivoire\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e19.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e14.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e73.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e9.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e4.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e44.68\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eDuraNet Plus\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBenin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e6g/kg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e89.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e2.2g/kg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e81.53\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCameroon\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e89.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e81.90\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCote D'Ivoire\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e89.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e81.90\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis multi-country experimental hut study assessed the entomological efficacy of DuraNet\u0026reg; Plus, an ITN incorporating alpha-cypermethrin and PBO, for the control of pyrethroid-resistant malaria vectors in West and Central Africa. Conducted across three ecologically and entomologically distinct settings (Benin, Cameroon, and C\u0026ocirc;te d\u0026rsquo;Ivoire), the study evaluated the net's performance under semi-field experimental hut conditions that closely reflect their operational use in households. These settings varied in vector species composition, resistance intensity, and behavioural characteristics, offering a robust test of product performance across a range of vector characteristics and resistance profiles. The findings demonstrate that DuraNet\u0026reg; Plus is both effective and wash-resistant across diverse pyrethroid-resistant vector populations, achieving non-inferiority to a WHO-prequalified pyrethroid-PBO net with demonstrated public health value, Olyset\u0026reg; Plus, in all study sites in accordance with WHO evaluation criteria.\u003c/p\u003e\u003cp\u003eImportantly, DuraNet\u0026reg; Plus met WHO criteria for non-inferiority to Olyset\u0026reg; Plus for both mosquito mortality and blood-feeding protection across all three study sites. This was demonstrated using data for both unwashed and 20-times washed nets, indicating that DuraNet\u0026reg; Plus retains its entomological efficacy over time and with repeated washing. In Benin and C\u0026ocirc;te d\u0026rsquo;Ivoire, DuraNet\u0026reg; Plus not only met the non-inferiority criteria but also demonstrated superior efficacy to Olyset\u0026reg; Plus in reducing mosquito mortality and preventing blood-feeding. In Cameroon, while DuraNet\u0026reg; Plus achieved non-inferiority, the differences between the two pyrethroid-PBO nets were smaller. This may be explained by the predominance of \u003cem\u003eAn. funestus\u003c/em\u003e, a vector species characterised by complex and high-intensity resistance mechanisms [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAcross Benin, Cameroon, and C\u0026ocirc;te d\u0026rsquo;Ivoire, DuraNet\u0026reg; Plus consistently outperformed the pyrethroid-only net (DuraNet\u0026reg;) in terms of both mosquito mortality and blood-feeding inhibition, maintaining superior efficacy in both unwashed and 20-times washed conditions in most cases. This enhanced performance highlights the role of PBO in improving net efficacy by inhibiting cytochrome P450 enzymes; key mediators of metabolic resistance that are commonly overexpressed in malaria vector populations across sub-Saharan Africa [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, in Benin, Olyset\u0026reg; Plus did not induce higher mosquito mortality than DuraNet\u0026reg;, and in fact, mortality with Olyset\u0026reg; Plus was significantly lower than with DuraNet\u0026reg; both before and after washing. This difference likely stems from the type of pyrethroid used in each net: Olyset\u0026reg; Plus incorporates permethrin, while DuraNet\u0026reg; Plus and DuraNet\u0026reg; use alpha-cypermethrin. WHO susceptibility bioassays conducted in Benin revealed that pre-exposure to PBO partially restored susceptibility to alpha-cypermethrin but had no such effect on permethrin, confirming earlier observations in the local vector population [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. These results suggest that permethrin resistance in this region may involve additional mechanisms beyond P450 enzyme activity which require further investigation.\u003c/p\u003e\u003cp\u003eTaken together, these results provide strong evidence that DuraNet\u0026reg; Plus is a viable and effective pyrethroid-PBO ITN for use in regions facing widespread pyrethroid resistance mediated by over-expressed cytochrome P450 enzymes. Its superior entomological performance compared to pyrethroid-only nets, along with its demonstrated non-inferiority to an established pyrethroid-PBO product, supports its inclusion among WHO-prequalified ITNs for procurement and deployment in malaria-endemic settings. Based on the outcomes of these trials, DuraNet\u0026reg; Plus was added to the WHO list of prequalified products in 2020 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Subsequently, in 2023, WHO extended its policy recommendation on pyrethroid-PBO nets to include DuraNet\u0026reg; Plus following a technical consultation to assess data on second-in-class vector control products [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWhile entomological outcomes from experimental hut studies are considered valid proxies for predicting the community-level performance of ITNs [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], further operational evidence is essential for informing policies for their deployment and long-term use by householders. Ongoing longitudinal studies in Benin, Cameroon, and Tanzania are currently assessing the durability of DuraNet\u0026reg; Plus under real-world conditions, including net attrition, fabric integrity, residual bioefficacy, and chemical retention over a three-year period [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The results from these evaluations will provide critical insights for guiding decisions on net deployment schedules and replacement cycles under large-scale use.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis multi-country experimental hut study provides robust entomological evidence that DuraNet\u0026reg; Plus, An alphacypermethrin-PBO ITN, is a highly effective intervention for controlling pyrethroid-resistant malaria vectors. DuraNet\u0026reg; Plus demonstrated superior efficacy to a pyrethroid-only ITN and met WHO non-inferiority criteria in comparison with an established pyrethroid-PBO net across diverse resistance settings in West and Central Africa. These findings support its use as a valuable addition to the current portfolio of WHO-prequalified ITNs and underscore its potential to contribute meaningfully to malaria vector control strategies in regions burdened by metabolic resistance to pyrethroids. Further field studies on product durability and operational performance will strengthen the evidence base for its broader programmatic adoption.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eActive ingredient\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eConfidence interval\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eITN\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInsecticide\u0026ndash;treated nets\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eKdr\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eKnockdown resistance\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHDPE\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ehigh\u0026ndash;density polyethylene\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCREC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCentre de Recherches Entomologiques de Cotonou\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePAMVERC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePan\u0026ndash;African Malaria Vector Research Consortium\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAIRID\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAfrican Institute for Research in Infectious Diseases\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLSHTM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLondon School of Hygiene \u0026amp; Tropical Medicine\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePBO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePiperonyl butoxide\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNIM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNon inferiority margin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePQT/VCP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePrequalification Unit Vector Control Product Assessment Team\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eP450\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCytochrome P450 monooxygenase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eOR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eOdds ratio\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ecRCT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ecluster randomised controlled trial\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eWHO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eWorld Health Organisation\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eEthical approval and consent to participate\u003c/h2\u003e\u003cp\u003e Ethical approval for conduct of was obtained from the ethics review board of the Ministry of Health in Benin. Informed written consent was obtained from all human volunteers before their participation. All volunteers were offered a free course of chemoprophylaxis to mitigate the risk of malaria infection, and a stand-by nurse was available throughout the trials to assess any volunteers presenting with febrile symptoms or an adverse reaction to the test items. The methods described in this paper followed relevant guidelines and regulations. Approval for using guinea pigs for tunnel tests was granted by LSHTM Animal Welfare Ethics Review Board (AWERB) (2020-01B).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis study was supported by a research grant from the Shobikaa Impex Private Limited to Corine Ngufor. The funders had no role in the study design, data collection and analysis and decision to publish this manuscript.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eCN designed the methods, acquired funding and had overall responsibility for the conduct of the study across trial sites. AA, BM, BN and LAA performed the hut trials and bioassays under the supervision of CN, CW and RN. AA and JN supported coordination across sites and analysed the study data. CN, AA and JN prepared the figures and tables. CN drafted the manuscript with input from AA and JN. All authors reviewed the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe gratefully acknowledge Palaniappan Meyyappan of Shobikaa Impex Private Limited for supplying the study nets. We also extend our sincere thanks to the rice farmers of Cov\u0026egrave; for their participation in the study. Special appreciation is due to the technical and administrative teams at CREC in Benin, IPR in C\u0026ocirc;te d\u0026rsquo;Ivoire, and CRID in Cameroon for their invaluable support. We further thank the team at IIBAT, India, for carrying out the chemical analyses.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData is provided within the manuscript or supplementary information files\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBhatt S, Weiss D, Cameron E, Bisanzio D, Mappin B, Dalrymple U, Battle K, Moyes C, Henry A, Eckhoff P. The effect of malaria control on Plasmodium falciparum in Africa between 2000 and 2015. Nature. 2015;526:207\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWHO: WHO Malaria Threats Map. World Health Organisation, Geneva. 2025, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://apps.who.int/malaria/maps/threats/\u003c/span\u003e\u003cspan address=\"https://apps.who.int/malaria/maps/threats/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWHO. World Malaria report. \u003cem\u003eWorld Health Organisation, Geneva\u003c/em\u003e 2023.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWHO. Guidelines for malaria vector control. \u003cem\u003eGeneva, Switzerland: World Health Organization\u003c/em\u003e 2025.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGleave K, Lissenden N, Richardson M, Choi L, Ranson H. Piperonyl butoxide (PBO) combined with pyrethroids in insecticide-treated nets to prevent malaria in Africa. Cochrane Database Syst Rev. 2018;11:CD012776.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWHO: List of WHO prequalified vector control products. Geneva, Switzerland: World Health Organization. 2024, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.who.int/pq-vector-control/prequalified-lists/PrequalifiedProducts27January2020.pdf?ua=1\u003c/span\u003e\u003cspan address=\"https://www.who.int/pq-vector-control/prequalified-lists/PrequalifiedProducts27January2020.pdf?ua=1\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNgufor C, Fagbohoun J, Agbevo A, Ismail H, Challenger JD, Churcher TS, Rowland M. Comparative efficacy of two pyrethroid-piperonyl butoxide nets (Olyset Plus and PermaNet 3.0) against pyrethroid resistant malaria vectors: a non-inferiority assessment. Malar J. 2022;21:20.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eN\u0026rsquo;Guessan R, Asidi A, Boko P, Odjo A, Akogbeto M, Pigeon O, Rowland M. An experimental hut evaluation of PermaNet\u0026reg; 3.0, a deltamethrin\u0026mdash;piperonyl butoxide combination net, against pyrethroid-resistant Anopheles gambiae and Culex quinquefasciatus mosquitoes in southern Benin. Trans R Soc Trop Med Hyg. 2010;104:758\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOumbouke WA, Rowland M, Koffi AA, Alou L, Camara S, N\u0026rsquo;Guessan R. Evaluation of an alpha-cypermethrin\u0026thinsp;+\u0026thinsp;PBO mixture long-lasting insecticidal net VEERALIN\u0026reg; LN against pyrethroid resistant Anopheles gambiae ss: an experimental hut trial in M\u0026rsquo;b\u0026eacute;, central C\u0026ocirc;te d\u0026rsquo;Ivoire. Parasites vectors. 2019;12:1\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTungu P, Magesa S, Maxwell C, Malima R, Masue D, Sudi W, Myamba J, Pigeon O, Rowland M. Evaluation of PermaNet 3.0 a deltamethrin-PBO combination net against Anopheles gambiae and pyrethroid resistant Culex quinquefasciatus mosquitoes: an experimental hut trial in Tanzania. Malar J. 2010;9:21.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eProtopopoff N, Mosha JF, Lukole E, Charlwood JD, Wright A, Mwalimu CD, Manjurano A, Mosha FW, Kisinza W, Kleinschmidt I, Rowland M. Effectiveness of a long-lasting piperonyl butoxide-treated insecticidal net and indoor residual spray interventions, separately and together, against malaria transmitted by pyrethroid-resistant mosquitoes: a cluster, randomised controlled, two-by-two factorial design trial. Lancet. 2018;391:1577\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStaedke SG, Gonahasa S, Dorsey G, Kamya MR, Maiteki-Sebuguzi C, Lynd A, Katureebe A, Kyohere M, Mutungi P, Kigozi SP, et al. Effect of long-lasting insecticidal nets with and without piperonyl butoxide on malaria indicators in Uganda (LLINEUP): a pragmatic, cluster-randomised trial embedded in a national LLIN distribution campaign. Lancet. 2020;395:1292\u0026ndash;303.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAMP. Alliance for malaria prevention; mass campagn tracker. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://allianceformalariapreventioncom/mass-campaign-tracker/?_sfm_mc_date_of_import=20221015\u003c/span\u003e\u003cspan address=\"https://allianceformalariapreventioncom/mass-campaign-tracker/?_sfm_mc_date_of_import=20221015\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e 2024, Accessed 28th October 2024.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWHO Guideline. for the prequalification assessment of insecticide-treated nets.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWHO. Technical consultation to assess comparative efficacy of vector control products Meeting report, 5 and 9 June 2023. World Health Organisation; 2023.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNgufor C, N'Guessan R, Fagbohoun J, Subramaniam K, Odjo A, Fongnikin A, Akogbeto M, Weetman D, Rowland M. Insecticide resistance profile of Anopheles gambiae from a phase II field station in Cove, southern Benin: implications for the evaluation of novel vector control products. Malar J. 2015;14:464.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMenze BD, Wondji MJ, Tchapga W, Tchoupo M, Riveron JM, Wondji CS. Bionomics and insecticides resistance profiling of malaria vectors at a selected site for experimental hut trials in central Cameroon. Malar J. 2018;17:317.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKoffi AA, Ahoua Alou LP, Adja MA, Chandre F, Pennetier C. Insecticide resistance status of Anopheles gambiae s.s population from M\u0026rsquo;B\u0026eacute;: a WHOPES-labelled experimental hut station, 10 years after the political crisis in C\u0026ocirc;te d\u0026rsquo;Ivoire. Malar J. 2013;12:151.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRanson H, Lissenden N. Insecticide Resistance in African Anopheles Mosquitoes: A Worsening Situation that Needs Urgent Action to Maintain Malaria Control. Trends Parasitol. 2016;32:187\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSyme T, Gbegbo M, Obuobi D, Fongnikin A, Agbevo A, Todjinou D, Ngufor C. Pyrethroid-piperonyl butoxide (PBO) nets reduce the efficacy of indoor residual spraying with pirimiphos-methyl against pyrethroid-resistant malaria vectors. Sci Rep. 2022;12:6857.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWHO: WHO list of prequalified vector control products. DuraNet Plus \u003cem\u003ehttps\u003c/em\u003e:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e//extranetwhoint/prequal/vector-control-products/duranet-\u003c/span\u003e\u003cspan address=\"http:////extranetwhoint/prequal/vector-control-products/duranet-\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cem\u003eplus\u003c/em\u003e 2020, accessed 3rd May 2025.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSherrard-Smith E, Ngufor C, Sanou A, Guelbeogo MW, N\u0026rsquo;Guessan R, Elobolobo E, Saute F, Varela K, Chaccour CJ, Zulliger R, et al. Inferring the epidemiological benefit of indoor vector control interventions against malaria from mosquito data. Nat Commun. 2022;13:3862.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAgbevo A, Ahogni I, Menze B, Tungu P, Kemibala EE, Govoetchan R, Wondji C, Padonou GG, Ngufor C. Community evaluation of the physical and insecticidal durability of DuraNet\u0026reg; Plus, an alpha-cypermethrin and piperonyl butoxide incorporated mosquito net: protocol for a multi-country study in West, Central and East Africa. Archives Public Health. 2023;81:202.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6848690/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6848690/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003ePyrethroid-piperonyl butoxide (PBO) nets enhance malaria vector control by counteracting metabolic resistance mechanisms in malaria vectors through the synergistic action of PBO. DuraNet\u0026reg; Plus is an alpha-cypermethrin and PBO incorporated net developed Shobikaa Impex Private Limited. This study assessed its entomological efficacy relative to a standard pyrethroid-only net (DuraNet\u0026reg;) and an established pyrethroid-PBO net (Olyset\u0026reg; Plus), in support of WHO prequalification.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eExperimental hut trials were conducted at three ecologically and entomologically distinct sites with pyrethroid-resistant vector populations: Cov\u0026egrave;, Benin (\u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l.), Mibellon, Cameroon (\u003cem\u003eAn. funestus\u003c/em\u003e), and M\u0026rsquo;b\u0026eacute;, C\u0026ocirc;te d\u0026rsquo;Ivoire (\u003cem\u003eAn. gambiae\u003c/em\u003e s.l.). Each net type was tested unwashed and after 20 standardized washes. Primary outcomes included 24-hour mosquito mortality and blood-feeding inhibition. DuraNet\u0026reg; Plus was evaluated for non-inferiority to Olyset\u0026reg; Plus and superiority over DuraNet\u0026reg; using combined washed and unwashed data, in line with WHO guidelines. WHO bioassays confirmed pyrethroid resistance and assessed the role of cytochrome P450 enzymes. Chemical analyses measured pyrethroid and PBO retention after washing.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eDuraNet\u0026reg; Plus consistently induced higher mosquito mortality than Olyset\u0026reg; Plus across all sites (Benin: 29.5% vs. 14.9%, OR\u0026thinsp;=\u0026thinsp;2.81, 95% CI: 2.34\u0026ndash;3.38, NIM\u0026thinsp;=\u0026thinsp;0.468; Cameroon: 27.8% vs. 22.2%, OR\u0026thinsp;=\u0026thinsp;1.81, 95% CI: 1.32\u0026ndash;2.49, NIM\u0026thinsp;=\u0026thinsp;0.619; C\u0026ocirc;te d\u0026rsquo;Ivoire: 19.5% vs. 12.0%, OR\u0026thinsp;=\u0026thinsp;2.28, 95% CI: 1.85\u0026ndash;2.80, NIM\u0026thinsp;=\u0026thinsp;0.373), with all odds ratios exceeding the WHO-defined non-inferiority margins. DuraNet\u0026reg; Plus also met non-inferiority criteria for blood-feeding inhibition compared to Olyset\u0026reg; Plus (Benin: OR\u0026thinsp;=\u0026thinsp;0.23, 95% CI: 0.18\u0026ndash;0.28, NIM\u0026thinsp;=\u0026thinsp;1.345; Cameroon: OR\u0026thinsp;=\u0026thinsp;0.66, 95% CI: 0.50\u0026ndash;0.87, NIM\u0026thinsp;=\u0026thinsp;1.324; C\u0026ocirc;te d\u0026rsquo;Ivoire: OR\u0026thinsp;=\u0026thinsp;0.58, 95% CI: 0.48\u0026ndash;0.69, NIM\u0026thinsp;=\u0026thinsp;1.404). In addition, DuraNet\u0026reg; Plus was superior to DuraNet\u0026reg; in both mosquito mortality and blood-feeding inhibition across all study sites (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Susceptibility bioassays confirmed high frequencies of pyrethroid resistance across all three sites, with varying levels of P450 enzyme involvement. Chemical analysis showed higher retention of alpha-cypermethrin and PBO in DuraNet\u0026reg; Plus after 20 washes compared to Olyset\u0026reg; Plus.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eDuraNet\u0026reg; Plus showed strong entomological efficacy and wash durability against pyrethroid-resistant malaria vectors across varied settings in West and Central Africa. It met WHO non-inferiority criteria compared to Olyset\u0026reg; Plus and was superior to a pyrethroid-only ITN, supporting its inclusion among WHO-prequalified products. These findings underscore its potential role in vector control strategies in areas affected by metabolic pyrethroid resistance.\u003c/p\u003e","manuscriptTitle":"A multi-country non-inferiority experimental hut evaluation of DuraNet® Plus, an alpha-cypermethrin and piperonyl butoxide treated net, for control of pyrethroid-resistant malaria vectors in West and Central Africa","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-30 17:38:44","doi":"10.21203/rs.3.rs-6848690/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-02T03:28:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-29T05:15:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-21T07:33:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-17T10:04:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-11T07:26:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"39733753249895164263232065520677722655","date":"2025-07-06T14:32:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"245000087001539981657134307103726230410","date":"2025-07-03T11:31:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"49967738377235280355590209012800654571","date":"2025-07-01T07:07:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"132186555069482610245782342447043329306","date":"2025-07-01T06:39:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"143056368108946459188506074300236357872","date":"2025-07-01T05:43:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-01T02:32:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-10T15:46:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-10T15:46:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"Malaria Journal","date":"2025-06-08T16:59:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5a2d6eda-d622-48b3-8b3e-a69ec4116274","owner":[],"postedDate":"September 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-07T17:08:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-30 17:38:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6848690","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6848690","identity":"rs-6848690","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}