Efficacy of Vector Guard®, a mosaic alpha-cypermethrin and piperonyl butoxide- treated net, for the control of pyrethroid resistant malaria vectors; a non- inferiority experimental hut evaluation in Benin

Efficacy of Vector Guard®, a mosaic alpha-cypermethrin and piperonyl butoxide- treated net, for the control of pyrethroid resistant malaria vectors; a non- inferiority experimental hut evaluation in Benin | 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 Efficacy of Vector Guard ® , a mosaic alpha-cypermethrin and piperonyl butoxide- treated net, for the control of pyrethroid resistant malaria vectors; a non- inferiority experimental hut evaluation in Benin Judicael Nounagnon, Martial Gbegbo, Abel Agbevo, Estelle Vigninou, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6556525/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Insecticide-treated nets (ITNs) combining pyrethroids with piperonyl butoxide (PBO) have improved the control of malaria transmitted by pyrethroid-resistant vectors compared to standard pyrethroid-only ITNs. To sustain malaria prevention efforts, a broader range of effective pyrethroid-PBO nets is needed to enhance market diversity and supply resilience. This study evaluated the entomological efficacy and wash durability of Vector Guard®, a new mosaic alpha-cypermethrin-PBO ITN, against pyrethroid-resistant Anopheles gambiae s.l. in southern Benin. Methods An experimental hut trial was conducted in Covè, Benin, against wild, free-flying An. gambiae s.l. Vector Guard® was tested unwashed and after 20 standardized washes, and compared to two WHO-prequalified ITNs: Olyset® Plus (a permethrin-PBO net) and Royal Sentry® 2.0 (an alpha-cypermethrin-only net). Primary outcomes were mosquito mortality and blood-feeding protection. Susceptibility bioassays were conducted to assess local resistance mechanisms. Laboratory cone and tunnel tests were also performed to help explain the finding in the experimental huts. Chemical content analyses was performed to investigate active ingredient wash retention. Vector Guard® was assessed for its non-inferiority to Olyset® Plus following WHO guidance. Results The wild Anopheles gambiae s.l. population at Covè exhibited high frequencies of pyrethroid resistance, with PBO pre-exposure restoring partial susceptibility to alpha-cypermethrin (34% vs. 4% mortality) but not to permethrin (2.0% vs. 2.1% mortality). A total of 6,799 females were collected in the experimental huts. Vector Guard® outperformed both Royal Sentry® 2.0 and Olyset® Plus across all entomological endpoints. Mortality with Vector Guard® was significantly higher than with Olyset® Plus when unwashed (36.4% vs. 17.5%, p < 0.001) and after 20 washes (17.2% vs. 8.7%, p < 0.001). Non-inferiority analysis with pooled data for unwashed and washed nets confirmed that Vector Guard® was non-inferior to Olyset® Plus for both mortality (OR 2.71, 95% CI: 2.26–3.24; NIM: 0.423) and blood-feeding protection (OR 0.53, 95% CI: 0.45–0.62; NIM: 1.359). These findings were supported by cone and tunnel tests. Chemical analysis showed higher wash retention of active ingredients in Vector Guard® (83% for PBO and >94% for alpha-cypermethrin) compared to Olyset® Plus (39.7% for PBO and 69.6% for permethrin). Conclusion Vector Guard® demonstrated superior entomological efficacy and wash durability compared to Royal Sentry® 2.0 and Olyset® Plus, and fulfilled WHO non-inferiority criteria for mosquito mortality and blood-feeding inhibition. These findings supported its addition to the WHO list of prequalified pyrethroid-PBO ITNs and its potential to provide improved malaria control when deployed on a large scale in areas with high levels of pyrethroid resistance. Insecticide-treated nets Vector Guard Olyset Plus Royal Sentry Piperonyl butoxide Pyrethroid resistance Experimental hut trial pyrethroid-PBO nets Anopheles gambiae sl Benin malaria Covè Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Insecticide-treated nets (ITNs) are the most widely used tool for malaria prevention in endemic regions and have been instrumental in the significant declines in malaria incidence and mortality over the past two decades [ 1 ]. However, the widespread emergence of pyrethroid resistance in major malaria vectors has threatened their effectiveness [ 2 ]. In response, a new generation of ITNs has been developed that combine pyrethroids with additional active ingredients—such as synergists or alternative insecticide classes—to enhance efficacy against pyrethroid-resistant vector populations [ 3 ]. One key innovation in this space is the addition of piperonyl butoxide (PBO) on ITNs, a synergist that inhibits mosquito enzymes responsible for metabolic resistance to pyrethroids [ 4 ]. Pyrethroid-PBO nets have demonstrated enhanced efficacy against insecticide-resistant malaria vector populations in both experimental and operational settings. Several experimental hut trials conducted across Africa have demonstrated their superiority to pyrethroid-only nets for the control of pyrethroid-resistant malaria vectors [ 5 ]. Additionally, two large-scale cluster-randomized controlled trials (cRCTs) conducted in Tanzania and Uganda showed that pyrethroid-PBO nets significantly reduced malaria incidence and parasite prevalence in children compared to standard pyrethroid-only ITNs [ 6 , 7 ]. These findings have established pyrethroid-PBO nets as a critical tool for malaria control in areas with confirmed pyrethroid resistance [ 8 ]. As a result, several brands of pyrethroid-PBO ITNs have been prequalified by the World Health Organisation (WHO) and are now in wide use by national malaria control programmes across sub-Saharan Africa [ 3 , 9 ]. However, increasing the diversity of available pyrethroid-PBO nets is important to improve market competition, meet procurement demands, and ensure continuity of supply. In this context, Vector Guard®, a new mosaic alphacypermethrin-PBO net developed by Disease Control Technologies, represents a promising addition to the next-generation ITN portfolio. To qualify for WHO prequalification and programmatic use, Vector Guard® must demonstrate entomological efficacy that is superior to pyrethroid-only nets and non-inferior to a WHO-prequalified pyrethroid-PBO net with demonstrated public health value, in accordance with WHO’s guidance [ 10 ]. This study reports the findings of an experimental hut trial conducted in Covè, southern Benin, to assess the efficacy and wash resistance of Vector Guard® under controlled semi-field conditions against wild free-flying pyrethroid resistant Anopheles gambiae sl. Vector Guard® was evaluated unwashed and after 20 standardized washes, and compared to two WHO prequalified ITNs; Olyset® Plus, a WHO-prequalified permethrin-PBO net with demonstrated public health value in cRCTs, and Royal Sentry®, a standard alpha-cypermethrin-only ITN. The trial followed existing WHO guidelines for evaluation of ITNs [ 10 ], and outcomes were analyzed using WHO’s criteria for non-inferiority and superiority in entomological endpoints of mosquito mortality and blood-feeding inhibition [ 11 ]. Susceptibility bioassays were conducted to characterise the susceptibility status of the wild vector population at the hut site during the trial. Materials and methods Experimental hut site and vector profile The experimental hut study was conducted at the CREC/LSHTM experimental hut station, located in a rice-growing area in Covè, southern Benin (7.21° N, 2.34° E). The local vector population comprises a mixture of Anopheles coluzzii and An. gambiae s.s., with An. gambiae s.s. representing approximately 23% of the population, predominantly during the dry season. The vector population is highly resistant to pyrethroids, with over 90% survival observed in WHO susceptibility bioassays using alpha-cypermethrin, deltamethrin, and permethrin [12, 13]. Resistance is primarily driven by a high frequency (>90%) of the L1014F knockdown resistance (kdr) mutation and the over-expression of metabolic detoxification enzymes [5, 14]. The trial was carried out using seven West African-style experimental huts. Susceptibility bioassays To assess the frequency of pyrethroid resistance in the wild mosquito population during the hut trial, WHO susceptibility bioassays were conducted using 2–5-day-old, unfed adult F1 female mosquitoes reared from larvae collected near the experimental huts [15]. Four replicates of 20–25 mosquitoes were exposed to filter papers treated with discriminating concentrations of alpha-cypermethrin (0.05%) and permethrin (0.75%), alongside untreated control papers. The susceptible An. gambiae Kisumu strain was tested in parallel as a reference. Knockdown was recorded after 60 minutes, and mortality after a 24-hour holding period. To evaluate the involvement of metabolic resistance mechanisms, particularly cytochrome P450 monooxygenases, additional synergist bioassays were performed using PBO (4%). Eight replicates of 25 mosquitoes were first exposed to PBO-treated papers for 60 minutes. Half of these (four replicates) were subsequently exposed to alpha-cypermethrin (0.05%), and the remaining four replicates to permethrin (0.75%) for an additional 60 minutes. Another four replicates were exposed to PBO alone to assess its independent effects, and untreated papers were used as negative controls. Experimental hut treatments: Vector Guard® is made of high-density polyethylene (HDPE) with a 120-denier roof panel incorporating a mixture of alphacypermethrin (5.8 g/kg ± 25%) and piperonyl butoxide (PBO, 23.2 g/kg ± 25%). The side panels are treated with alphacypermethrin alone at 5.8 g/kg ± 25%. The candidate net was compared to two WHO-prequalified ITNs: Olyset® Plus (by Sumitomo Chemical Company), a monofilament HDPE net made of 150-denier yarn and incorporating permethrin (20 g/kg) and PBO (10 g/kg); and Royal Sentry® 2.0 (by Disease Control Technologies), a 120-denier HDPE net treated with alphacypermethrin at 5.8 g/kg ± 25%. All net types were evaluated in both unwashed and 20-times washed conditions following WHO standard procedures [10]. The following 7 treatments were thus assessed in the experimental hut trial: 1. Untreated control polyethylene net. 2. Royal Sentry® 2.0 (alpha-cypermethrin-only) unwashed 3. Royal Sentry® 2.0 (alpha-cypermethrin-only) washed 20 times 4. Olyset® Plus (permethrin +PBO) unwashed 5. Olyset® Plus (permethrin +PBO) washed 20 times 6. Vector Guard® (alpha-cypermethrin + PBO) unwashed 7. Vector Guard® (alpha-cypermethrin + PBO) washed 20 times To simulate wear and tear, all nets—including untreated controls—were deliberately holed in accordance with WHO guidelines, using six 4 × 4 cm holes (two on each long side and one on each short side). Net washing procedures also followed WHO standard protocols. Each net was washed in an aluminium bowl containing 10 litres of water with 2 g/L of Savon de Marseille, agitated for a total of 10 minutes. Nets were then rinsed in clean water using the same method, dried horizontally in the shade, and stored at ambient temperature between washes. The washing intervals were set at 2 days for Vector Guard® and Olyset® Plus, and 1 day for Royal Sentry® 2.0 in line with regeneration studies. Hut trial design To minimize potential bias due to hut position, treatments were rotated weekly across the seven experimental huts using a randomized Latin Square Design to reduce carry-over effects. The hut trial was conducted over 42 nights between February and April 2022. Data collection occurred over six consecutive nights each week, with the seventh day reserved for cleaning and airing the huts in preparation for the next rotation. Six replicate nets were tested per treatment, with nets rotated daily within each week. Each night, from 21:00 to 06:00, seven consenting human volunteers slept in the huts to attract wild, free-flying mosquitoes. Each morning, volunteers collected mosquitoes from the hut compartments (under the net, in the room, and veranda) using a torch and aspirator, and placed them in labelled plastic cups. Collections were transferred to the field laboratory for morphological identification and assessed for immediate mortality and blood-feeding status. 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 efficacy of treatments in the experimental huts was evaluated using the following outcome measures: · Entry rate : Total number of mosquitoes collected per hut. · Deterrence (%) : Reduction in mosquito entry in treated huts compared to the untreated control. · Exophily (%) : Proportion of mosquitoes found in the veranda, indicating treatment-induced exiting behavior. · Inside net (%) : Proportion of mosquitoes collected inside the net. · Blood-feeding rate (%) : Proportion of mosquitoes that were blood-fed. · Blood-feeding inhibition (%) : Reduction in blood-feeding in treated huts relative to the control · Personal protection (%) : Reduction in the number of blood-fed mosquitoes in the treated hut compared to the control. · Mortality (%) : Proportion of mosquitoes that died within 24 hours post-collection. Supplementary cone and tunnel tests To supplement hut trial data, laboratory cone bioassays and tunnel tests were conducted to assess the bioavailability and potency of active ingredients in each ITN. Net samples were taken from both unwashed and 20-times washed nets of each ITN type. Cone bioassays were performed using the pyrethroid-susceptible An. gambiae s.s. Kisumu strain to evaluate the efficacy of the pyrethroid component, while tunnel tests used the pyrethroid-resistant An. gambiae s.l. Covè strain to assess the added effect of PBO in Vector Guard® and Olyset® Plus. All assays were conducted under controlled conditions (27 ± 2°C and 75 ± 10% relative humidity). In cone bioassays, 8–12 unfed, 2–5-day-old An. gambiae s.s. Kisumu mosquitoes were exposed in two batches of 4–6 per cone for 3 minutes on each ITN piece. After exposure, mosquitoes were transferred to labelled holding cups, supplied with 10% glucose solution, and observed for knockdown at 60 minutes and mortality at 24 hours. Tunnel tests were conducted on two randomly selected net pieces from each ITN type and wash status. For Vector Guard®, only roof panel samples were tested. The tunnel apparatus simulates natural host-seeking behavior and consists of a glass chamber divided into two sections by a wooden frame holding the net sample. A guinea pig bait was placed in a cage at one end of the tunnel, and approximately 100 unfed, 5–8-day-old An. gambiae s.l. Covè mosquitoes were released at dusk into the opposite end. Net samples were perforated with nine 1-cm diameter holes to allow mosquito passage. The following morning, mosquitoes were collected, and immediate mortality and blood-feeding status were recorded. Surviving mosquitoes were held in labelled cups with access to 10% glucose solution and monitored for delayed mortality at 24 hours. Chemical analysis of insecticide content To assess within- and between-net variation in active ingredient content, as well as wash-resistance, chemical analyses were conducted on Vector Guard® net samples by the reference laboratory CRA-W (Gembloux, Belgium). Net pieces (30 × 30 cm) taken from the experimental hut trial were analysed for alpha-cypermethrin and PBO content using CIPAC methods 54/LN/M/3.2, 454/LN/M3/3, and 33/LN/(M)/3. These methods involve extraction of the active ingredients in a water bath at 85–90 °C for 45 minutes using heptane and dicyclohexyl phthalate as the internal standard, followed by quantification via gas chromatography with flame ionisation detection (GC-FID). The identity of the active ingredients was confirmed through comparison with authentic standards. Each net sample was analysed individually, and average concentrations were calculated per treatment group. Wash-resistance was determined using the WHO-recommended formula for the wash-resistance index (WRI): Wash Resistance Index (WRI) = 100 × n√(tn / t₀) where tn is the total active ingredient content after n washes, and t₀ is the content before washing. Statistical analysis Proportional outcomes—including mosquito mortality, blood-feeding, and exophily—were compared across treatments using logistic regression, while numerical outcomes such as mosquito entry were analysed using negative binomial regression. Each outcome was modelled separately, with adjustments for variation between huts, sleepers, and trial weeks, included as fixed effects [14]. Non-inferiority analyses comparing Vector Guard® to Olyset® Plus were performed in line with WHO guidelines [15]. Vector Guard® was considered non-inferior for mosquito 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 of the 95% CI was below the NIM. The NIM was calculated to reflect a 7% difference in efficacy (mortality or blood-feeding) of Vector Guard® relative to Olyset® Plus. Superiority of Vector Guard® over Royal Sentry® 2.0 was assessed based on significantly higher mosquito mortality and lower blood-feeding rates at the 5% significance level ( p < 0.05). Analyses were conducted separately for unwashed and washed nets, as well as pooled, to assess overall product efficacy across the net’s lifespan. All statistical analyses were performed using Stata version 18. Ethical considerations Ethical approval for the study was obtained from the Research Ethics Committee of the Ministry of Health in Benin (CNERS; Approval No. 53, issued 17 November 2021) and the London School of Hygiene & Tropical Medicine (LSHTM) Ethics Committee (Ref: 26429). Written informed consent was obtained from all human volunteer sleepers prior to participation. To mitigate the risk of malaria infection, all volunteers were provided with a full course of chemoprophylaxis for the duration of the study and for four weeks following its completion. Approval for the use of guinea pigs in tunnel tests was granted by the LSHTM Animal Welfare and Ethics Review Board (Ref: 2020-01). Guinea pig colonies were housed and maintained at the CREC/LSHTM facility in accordance with standard operating procedures aligned with national and international regulations on the ethical use of animals in scientific research. Compliance with OECD principles of Good Laboratory Practice To ensure compliance with OECD Good Laboratory Practice (GLP) principles, all phases of the study—from protocol development to reporting—were conducted under strict quality control. Equipment was calibrated, ITNs were verified for expiry and certification, and mosquito strains were handled according to SOPs. The candidate net was sourced from three production batches and stored under monitored conditions. Validated systems were used for data collection and processing, and all procedures were documented. The quality assurance team at CREC/LSHTM inspected all critical phases and found no non-conformances. External GLP inspections by SANAS in 2022 also reported full compliance. Results WHO cylinder bioassay results The frequency of resistance to pyrethroids was very high in wild Anopheles gambiae s.l. from the Covè hut site, with mortality rates of only 2.1% with permethrin 0.75% and 4% with alpha-cypermethrin 0.05% (Table 1). Pre-exposure to PBO followed by alpha-cypermethrin increased mortality substantially to 34%, indicating partial restoration of susceptibility. In contrast, pre-exposure to PBO had no impact on mortality when with permethrin, which remained low at 2%. PBO alone and control treatments resulted in negligible mortality (0–3.1%). In comparison, both permethrin and alpha-cypermethrin induced 100% mortality against the susceptible An. gambiae Kisumu strain, confirming full susceptibility. Table 1: WHO susceptibility cylinder bioassay results with wild mosquitoes collected as larvae from experimental hut station during the trial. Mosquito strain Insecticide N exposed N KD 60 mins % KD mins 95% CI N dead % Mortality 95% CI Pyrethroid -resistant An. gambiae s.l. Covè Control 97 1 1 (0-3) 3 3.1 (0-7) Permethrin 0.75% 94 0 0 - 2 2.1 (0-5) Alpha-cypermethrin 0.05% 99 0 0 - 4 4 0-8 PBO 4% 98 0 0 - 0 0 0 PBO + Permethrin 98 0 0 - 2 2 0-5 PBO + alphacypermethrin 94 28 29.8 (21-39) 32 34 (24-44) Susceptible An. gambiae Kisumu Control 101 0 0 - 2 1.98 0-5 Permethrin 0.75% 100 100 100 0 100 100 0 Alpha-cypermethrin 0.05% 100 100 100 0 100 100 0 Experimental hut results Entry and exiting results A total of 6,799 female Anopheles gambiae s.l. were collected in the experimental huts in Covè during the study period. The average number of mosquitoes caught per night across treatments ranged from 20 to 26 (Table 2). Vector Guard® induced the strongest deterrent effect relative to the control, both when unwashed (21.1%) and after 20 washes (12.5%). In contrast, Royal Sentry® 2.0 showed minimal deterrence, particularly after 20 washes (1.5%). The proportion of mosquitoes exiting the hut was significantly higher in all insecticide-treated arms compared to the untreated control (37.9%). Among unwashed nets, exit rates were comparable, with 60.5% for Royal Sentry® 2.0, 64.8% for Olyset® Plus, and 65.3% for Vector Guard®. After 20 washes, Vector Guard® maintained a high exit rate (65.3% vs 62.8%, p=0.348), whereas exiting decreased more noticeably with Royal Sentry® 2.0 (54.6%) and Olyset® Plus (50.1%), suggesting better retention of excito-repellent activity with Vector Guard® after washing (p<0.05). Table 2 : Entry and exiting of wild, free-flying, pyrethroid-resistant Anopheles gambiae sensu lato entering experimental huts in Covè, southern Benin. Net type Control Royal Sentry 2.0 Olyset® Plus Vector Guard® Net status - Unwashed Washed 20x Unwashed Washed 20x Unwashed Washed 20x Total females caught 1055 a,b 936 c,d 1039 a 957 b,c 1083 a 832 d 923 c,d Average catch per night 25 22 25 23 26 20 22 % Deterrence - 11.2 1.5 9.2 0.0 21.1 12.5 Total Exiting 400 567 568 621 543 544 580 % Exiting 37.9 a 60.5 b 54.6 c 64.8 b 50.1 c 65.3 b 62.8 b 95% Conf Interval (34.9-40.8) (57.4-63.7) (51.6-57.6) (61.8-67.9) (47.1-53.1) (62.1-68.6) (59.7-65.9) * Values in the same column bearing the same letter do not differ significantly at the 5% level according to logistic regression analysis Blood-feeding results The blood-feeding rate with the control (untreated net) was 60.6% (Figure 1, Table 3). Among unwashed nets, Olyset® Plus and Vector Guard® achieved the lowest blood-feeding rates at 15.0% and 15.6%, respectively, both significantly lower than that of Royal Sentry® 2.0 (24.3%; p <0.05). After 20 washes, Vector Guard® maintained superior performance with a blood-feeding rate of 27.6%, significantly lower than Olyset® Plus (46.5%; p <0.001) and Royal Sentry® 2.0 (44.8%; p <0.001). Blood-feeding inhibition mirrored these trends: Vector Guard® and Olyset® Plus achieved 74.2% and 75.1% inhibition, respectively, when unwashed, compared to 59.7% with Royal Sentry® 2.0 ( p <0.001). After 20 washes, blood-feeding inhibition with Vector Guard® remained high at 54.3%, significantly greater than Olyset® Plus (23.1%) and Royal Sentry® 2.0 (25.9%; p <0.001 for both). In addition, personal protection levels with unwashed nets were highest with Vector Guard® (79.7%) and Olyset® Plus (77.5%) while Royal Sentry® 2.0 provided the lowest level of personal protection (64.3%). After 20 washes, the personal protection level remained high with Vector Guard® (60.0%) whilst this declined substantially with both ITN types Olyset® Plus (21.1%) and Royal Sentry® 2.0 (27.1%). These findings highlight the superior and more wash-resistant blood-feeding inhibition and personal protection provided by Vector Guard® against pyrethroid-resistant mosquitoes. Table 3: Blood-feeding of wild, free-flying, pyrethroid-resistant Anopheles gambiae sensu lato entering experimental huts in Covè, southern Benin. Net type Control Royal Sentry 2.0® Olyset® Plus Vector Guard® No of washes - Unwashed Washed 20x Unwashed Washed 20x Unwashed Washed 20x Total females caught 1055 936 1039 957 1083 832 923 Total Blood fed 639 228 466 144 504 130 255 Blood-feeding % 60.5 a 24.3 b 44.8 c 15.0 d 46.5 c 15.6 d 27.6 b 95% Conf Interval (57.6-63.5) (21.6-27.1) (41.8-47.9) (12.8-17.3) (43.6-49.5) (13.2-18.1) (24.7-30.5) Blood feeding Inhibition (%) - 59.7 25.9 75.1 23.1 74.2 54.3 95% Conf Interval - (56.6-62.9) (23.9-28.6) (72.4-77.9) (20.7-25.7) (71.2-77.2) (51.2-57.6) Personal protection (%) - 64.3 27.0 77.4 21.1 79.6 60.0 * Values along a row bearing the same letter label are not significantly different (P>0.05, logistic regression) Mortality results The mortality of wild pyrethroid-resistant An. gambiae s.l. with the control net was 1% (Figure 2 and Table 4). With unwashed nets, the highest mortality rate was achieved with Vector Guard® (36.4%) compared to Olyset® Plus (17.6%); p<0.001 and Royal Sentry® 2.0 (27.2%); p<0.001. A similar trend was observed with nets washed 20 times; mortality was significantly higher with Vector Guard® compared to Olyset® Plus (17.2% vs. 8.8%, P<0.001) and Royal Sentry® 2.0 (17.2% vs. 9.9 %, P<0.001). Table 4 : Mortality of wild, free-flying, pyrethroid-resistant Anopheles gambiae sensu lato entering experimental huts in Covè, southern Benin. Net type Control Royal Sentry 2.0 Olyset® Plus Vector Guard® No of washes - Unwashed Washed 20x Unwashed Washed 20x Unwashed Washed 20x Total females caught 1055 936 1039 957 1083 832 923 N dead after 24h 11 255 103 168 95 303 159 % dead after 24h 1.0 a 27.2 b 9.9 c 17.5 d 8.7 c 36.4 e 17.2 d 95% Conf Interval 0.4-1.7 (24.4-30.1) (8.1-11.7) (15.1-19.9) (7.1-10.5) (33.2-39.7) (14.8-19.7) * Values along a row bearing the same letter label are not significantly different (P>0.05, logistic regression) Non-inferiority assessment of Vector Guard® to Olyset® Plus Mortality outcomes: Vector Guard® met WHO non-inferiority criteria for mosquito mortality when compared to Olyset® Plus under both unwashed and washed conditions (Figure 3, Table 5). When unwashed, Vector Guard® induced significantly higher mortality (36.4%) than Olyset® Plus (17.5%, p<0.001), with an odds ratio (OR) of 3.003 (95% CI: 2.384–3.784). The lower bound of the confidence interval exceeded the non-inferiority margin (NIM = 0.554), indicating both non-inferiority and superiority. After 20 washes, mortality remained significantly higher with Vector Guard® (17.2%) compared to Olyset® Plus (8.7%, p<0.001), with an OR of 2.286 (95% CI: 1.727–3.026) and a corresponding NIM of 0.187. As the lower confidence limit was also well above the NIM, Vector Guard® was confirmed to be non-inferior and also superior to Olyset® Plus after 20 washes. When results were pooled across washing conditions, Vector Guard® maintained a higher overall mortality rate (26.3%) than Olyset® Plus (12.8%), with an OR of 2.706 (95% CI: 2.261–3.239) and a NIM of 0.423, again confirming non-inferiority and superiority. Vector Guard® also consistently outperformed Royal Sentry® in vector mortality (p<0.001). Blood-feeding outcomes: In terms of blood-feeding inhibition, Vector Guard® was non-inferior to Olyset® Plus under all conditions (Figure 4, Table 5). When unwashed, the blood-feeding rate was 15.6% for Vector Guard® and 15.0% for Olyset® Plus (OR = 1.001, 95% CI: 0.767–1.306), with the upper confidence limit well below the NIM of 1.596, confirming non-inferiority. After 20 washes, blood-feeding remained significantly lower with Vector Guard® (27.6%) compared to Olyset® Plus (46.5%), with an OR of 0.39 (95% CI: 0.312–0.463) and a NIM of 1.323. The upper bound of the confidence interval was well below the NIM, supporting both non-inferiority and superiority. When pooled across washing conditions, Vector Guard® also demonstrated lower blood-feeding rates (21.9%) than Olyset® Plus (31.7%), with an OR of 0.531 (95% CI: 0.453–0.622) and a NIM of 1.359, confirming sustained non-inferiority and superior performance. In all conditions tested, Vector Guard® also showed greater blood-feeding inhibition than Royal Sentry® (p<0.001). Table 5: Non-inferiority and superiority analyses comparing the effect of Vector Guard® to Olyset® Plus and Royal Sentry 2.0 for mosquito mortality and blood-feeding outcomes in experimental huts. To fulfill non-inferiority criteria, lower 95% CI of odds ratio must exceed NIM for mortality while upper 95% CI of odds ratio must not exceed NIM for blood-feeding. Vector Guard must also be superior to Royal Sentry® 2.0 at the 5% level Unwashed 20x Washed Pooled Non-inferiority assessment Odds ratio (95% CIs) NIM Odds ratio (95% CIs) NIM Odds ratio (95% CIs) NIM Mortality 3.003 0.554 2.286 0.187 2.706 0.423 (2.382-3.784) Non-Inferior (1.727-3.026) Non-Inferior (2.261-3.239) Non-Inferior Blood-feeding 1.001 1.596 0.38 1.323 0.531 1.359 (0.767-1.306) Non-Inferior (0.312-0.463) Non-Inferior (0.453-0.622) Non-Inferior Superiority assessment Odds ratio (95% CIs) p-value Odds ratio (95% CIs) p-value Odds ratio (95% CIs) p-value Mortality 1.563 ˂0.001 2.08 ˂0.001 1.741 ˂0.001 (1.252-1.951) Superior (1.576-2.741) Superior (1.467-2.066) Superior Blood-feeding 0.61 ˂0.001 0.424 ˂0.001 0.477 ˂0.001 (0.475-0.783) Superior (0.347-0.518) Superior (0.408-0.559) Superior Supplementary laboratory bioassay results Cone bioassay results: In cone bioassays with the susceptible Anopheles gambiae s.s. Kisumu strain, all ITNs—including Vector Guard®, Olyset® Plus, and Royal Sentry® 2.0—achieved high knockdown and mortality rates when unwashed (Figure 5). After 20 washes, Vector Guard® (both side and roof panels) and Royal Sentry® 2.0 maintained high mortality rates above 80%, while Olyset® Plus exhibited a substantial decline, with mortality dropping to around 60%. Tunnel test results: In tunnel tests against the pyrethroid-resistant Anopheles gambiae s.l. Covè strain, Vector Guard® demonstrated the highest efficacy among the ITNs tested (Table 6). When unwashed, Vector Guard® (roof panels) induced 99% mortality, outperforming Olyset® Plus (91.9%) and Royal Sentry® 2.0 (76.4%). Following 20 washes, mortality with Vector Guard® remained high at 72.1%, substantially greater than Olyset® Plus, which dropped markedly to 20.8%, while Royal Sentry® 2.0 maintained 77.4%. Blood-feeding inhibition remained above 90% for all three nets when unwashed, but after 20 washes, Vector Guard® continued to provide strong blood-feeding protection (93.8% BFI), comparable to Royal Sentry® 2.0 (95.5%), whereas Olyset® Plus experienced a sharp decline in BFI to 53.7%. Table 6 : Summary results of tunnel tests with pyrethroid-resistant Anopheles gambiae sensu lato Covè strain Washes Treatments N Exposed N Dead % Mortality (95%CI) N Blood-fed % Blood-fed (95%CI) % Blood-feeding inhibition Unwashed Control 173 2 1.2 (0-2.7) 113 65.3 (58.2-72.4) - Royal Sentry® 2.0 203 155 76.4 (70.5-82.2) 12 5.9 (2.7-9.2) 90.95 Olyset® Plus Net 184 169 91.9 (87.9-95.8) 2 1.1 (0-2.6) 98.3 Vector Guard® (roof) 208 206 99 (97.7-100) 0 0 100 Washed 20X Royal Sentry® 2.0 239 185 77.4 (72.1-82.7) 7 2.9 (0.8-5.1) 95.52 Olyset® Plus Net 192 40 20.8 (15.1-26.6) 58 30.2 (23.7-36.7) 53.7 Vector Guard® (roof) 197 142 72.1 (65.8-78.3) 8 4.1 (1.3-6.8) 93.78 Chemical analysis of net pieces results The active ingredient content in all unwashed ITNs was within defined specifications declared by the manufacturers. Retention of PBO after 20 washes was lowest with Olyset® Plus (39.7%) (Table 7). Side panels of Vector Guard® showed high wash-retention of alpha-cypermethrin the (94.3%). Between pyrethroid-PBO ITNs, Vector Guard® (roof) showed higher levels of wash-retention of both active ingredients (94.5% for alpha-cypermethrin and 83.4% for PBO) compared to Olyset® Plus (69.6% for permethrin and 39.7% for PBO). The wash-resistance index of PBO was thus higher with Vector Guard® (99.1%) than with Olyset® Plus (95.5%). Table 7: Chemical content of unwashed and washed net pieces taken before and after the experimental hut trial in Covè, Benin. ITN type Active ingredient(s) AI content (g/kg) AI retention (%) Unwashed Washed 20X Royal Sentry® 2.0 Alpha-cypermethrin 5.3 4.7 90.4 Olyset® Plus Permethrin 18.4 12.8 69.6 PBO 8.7 3.5 39.7 Vector Guard® Alpha-cypermethrin (roof) 5.5 5.2 94.5 PBO (roof) 16.3 13.6 83.4 Alpha-cypermethrin (side) 5.3 5.0 94.3 Discussion This experimental hut study provides robust entomological evidence on the efficacy and wash durability of Vector Guard®, a new mosaic ITN combining alpha-cypermethrin and PBO, against wild, free-flying, pyrethroid-resistant Anopheles gambiae s.l. in southern Benin. The results show that Vector Guard® offers superior and more wash resistant protection compared to Royal Sentry® 2.0, a standard pyrethroid-only ITN, and is non-inferior—and in many outcomes, superior—to Olyset® Plus, a WHO-prequalified PBO net with documented epidemiological impact. Vector Guard® consistently outperformed both comparators across key entomological indicators, including mosquito deterrence, exiting rates, blood-feeding inhibition, and mortality. Importantly, these effects were sustained after 20 washes, indicating good wash durability and potential for long-term protective performance under field conditions. The superior efficacy of Vector Guard® over Royal Sentry® 2.0 aligns with findings from multiple experimental hut and community trials across sub-Saharan Africa comparing pyrethroid-PBO to pyrethroid-only nets [ 4 – 7 , 16 , 17 ], and supports current WHO recommendations favouring prioritisation of pyrethroid-PBO nets over pyrethroid-only nets in areas with confirmed pyrethroid resistance [ 8 ]. While Vector Guard® induced higher mosquito mortality than Royal Sentry®, Olyset® Plus performed worse, with mortality rates even lower than those observed with Royal Sentry® 2.0. These differences likely reflect the type of pyrethroid used—alpha-cypermethrin in Vector Guard® versus permethrin in Olyset® Plus. Pre-exposure to PBO partially restored susceptibility to alpha-cypermethrin, raising mortality from 4–34%, but had little effect on permethrin, where mortality remained low (~ 2%). This reduced synergism between PBO and permethrin, though not fully understood, is consistent with findings from previous studies conducted in Benin [ 13 ] and other settings in West Africa.[ 18 ]. The non-inferiority analyses confirmed that Vector Guard® meets WHO criteria [ 11 ] for non-inferiority to Olyset® Plus for both mortality and blood-feeding inhibition under unwashed, washed, and pooled conditions. In fact, the performance of Vector Guard® exceeded Olyset® Plus across all endpoints both in the hut trial and in the supplementary bioassays. Vector Guard® was associated with significantly higher mosquito mortality and lower blood-feeding rates, confirming not only non-inferiority but also superiority. The superior efficacy of Vector Guard compared to Olyset® Plus can also be explained by the afore-mentioned improved synergistic interaction between PBO and alpha-cypermethrin compared to permethrin. These findings suggest that pairing PBO with alpha-cypermethrin on ITNs (rather than permethrin) can yield better entomological efficacy against metabolically resistant mosquito populations. Chemical analysis further substantiated these results. Vector Guard® exhibited high retention of both alpha-cypermethrin and PBO after 20 washes, with over 83% retention of PBO and > 94% for alpha-cypermethrin. This contrasted with Olyset® Plus, which retained only 39.7% of its PBO content and 69.6% of its permethrin. The higher wash-resistance index for PBO in Vector Guard® reflects its superior formulation and suggests greater durability of its insecticidal efficacy under household use. Vector Guard® was added to the WHO list of prequalified vector control products [ 3 ] based on the results of this study and supporting evidence from a similar trial conducted against pyrethroid-resistant Anopheles arabiensis in Tanzania [ 19 ]. Vector Guard® is now commercialised under the brand name SafeNet® Plus after its ownership was transferred to Mainpol GmbH in 2024 [ 20 ]. Given its demonstrated superiority over Olyset® Plus, the net represents a strong alternative for malaria control programmes seeking effective pyrethroid-PBO nets for use in areas with high intensity pyrethroid-resistance. Further operational research is warranted to assess its physical integrity and insecticidal durability under long-term community use. Conclusion Vector Guard® demonstrated superior entomological efficacy and wash durability compared to Royal Sentry® 2.0 and Olyset® Plus, and fulfilled WHO non-inferiority criteria for mosquito mortality and blood-feeding inhibition. These findings support the inclusion of Vector Guard® as a strong candidate pyrethroid-PBO ITNs for malaria control in areas with high levels of pyrethroid resistance. 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 Acknowledgements We are grateful to Rod Flinn and Andy Buttenhof of Disease Control Technologies LLC for providing the study nets. We also thank the rice farmers of Covè for their participation in the study. Special appreciation goes to the technical and administrative staff of the CREC/LSHTM/PAMVERC research programme in Benin for their valuable support. We also acknowledge the team at CRAW Gembloux, Belgium, for conducting the chemical analyses Funding This study was supported by a grant from the Gates Foundation (INV-023420) to the London School of Hygiene and Tropical Medicine and the Centre de Recherche Entomologique de Cotonou. The funders had no role in the study design, data collection and analysis and decision to publish this manuscript. Availability of data and materials All data supporting the findings of this study are available within the paper and its supplementary material. Authors’ contributions CN designed the methods, acquired funding and was responsible for the overall conduct of the study. MG and AA performed the hut trials. JN and BN performed the laboratory bioassays. JN analysed the data and prepared the figures and tables. CN and JN wrote the manuscript. All authors reviewed the manuscript. Ethical approval and consent to participate Ethical approval for conduct of the experimental hut trial and withdrawal of the field-aged nets was obtained from the ethics review boards of the Ministry of Health in Benin and the institutional review board of LSHTM. 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. 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: 207-211. WHO: WHO Malaria Threats Map. World Health Organisation, Geneva 2025, https://apps.who.int/malaria/maps/threats/ . WHO: WHO List of Prequalified In Vitro Diagnostic Products. World Health Organisation, Geneva 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. 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. Staedke 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: 1292-1303. Protopopoff 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: 1577-1588. WHO: Guidelines for malaria vector control. Geneva, Switzerland: World Health Organization 2025. 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 World Health Organisation: Technical consultation to assess comparative efficacy of vector control products Meeting report, 5 and 9 June 2023. World Health Organisation; 2023. Agbevo A, Syme T, Fagbohoun J, Fongnikin A, Ahoga J, Accrombessi M, Protopopoff N, Cook J, Churcher TS, Padonou GG, et al: The experimental hut efficacy of next-generation insecticide-treated nets against pyrethroid-resistant malaria vectors after 12, 24 and 36 months of household use in Benin. Malaria Journal 2024, 23: 388. 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. 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. WHO: Manual for monitoring insecticide resistance in mosquito vectors and selecting appropriate interventions. World Health Organisation, Geneva 2022, https://www.who.int/publications/i/item/9789240051089 . 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: 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. Dadzie SK, Chabi J, Asafu-Adjaye A, Owusu-Akrofi O, Baffoe-Wilmot A, Malm K, Bart-Plange C, Coleman S, Appawu MA, Boakye DA: Evaluation of piperonyl butoxide in enhancing the efficacy of pyrethroid insecticides against resistant Anopheles gambiae s.l. in Ghana. Malaria Journal 2017, 16: 342. Machange JJ, Mbuba E, Irish SR, Swai JK, Ntabaliba W, Makungwa NO, Ngonyani S, Mpelepele AB, Kibondo UA, Odufuwa OG, Moore SJ: Comparative efficacy of Vector Guard® to Olyset® Plus insecticide-treated nets against strongly pyrethroid-resistant Anopheles arabiensis in experimental huts in Tanzania. Frontiers in Malaria 2024, Volume 2 - 2024 . WHO: Transfer of ownership of Vector Guard to Mainpol. https://extranetwhoint/prequal/sites/default/files/doc_parts/20240715-transfer-of-ownership-mainpol-p-00210-p-00211-p-09284-pqc-vcp-2024-0020_1pdf 2024, accessed March 2025 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6556525","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":450601379,"identity":"6a450ddc-a9cf-4a41-accb-882e0a632c74","order_by":0,"name":"Judicael Nounagnon","email":"","orcid":"","institution":"Centre de Recherches Entomologiques de Cotonou (CREC)","correspondingAuthor":false,"prefix":"","firstName":"Judicael","middleName":"","lastName":"Nounagnon","suffix":""},{"id":450601380,"identity":"5601d6bb-3e7e-4386-92eb-15be1c61f865","order_by":1,"name":"Martial Gbegbo","email":"","orcid":"","institution":"Centre de Recherches Entomologiques de Cotonou (CREC)","correspondingAuthor":false,"prefix":"","firstName":"Martial","middleName":"","lastName":"Gbegbo","suffix":""},{"id":450601381,"identity":"90414332-0f19-45ce-8cfe-a36959d12a82","order_by":2,"name":"Abel Agbevo","email":"","orcid":"","institution":"Centre de Recherches Entomologiques de Cotonou (CREC)","correspondingAuthor":false,"prefix":"","firstName":"Abel","middleName":"","lastName":"Agbevo","suffix":""},{"id":450601382,"identity":"459d8703-9141-47ef-b56d-cd557dcc790c","order_by":3,"name":"Estelle Vigninou","email":"","orcid":"","institution":"Centre de Recherches Entomologiques de Cotonou (CREC)","correspondingAuthor":false,"prefix":"","firstName":"Estelle","middleName":"","lastName":"Vigninou","suffix":""},{"id":450601383,"identity":"a46f458c-c143-49e9-a00d-649cfc4a009c","order_by":4,"name":"Boris N’dombidjé","email":"","orcid":"","institution":"Centre de Recherches Entomologiques de Cotonou (CREC)","correspondingAuthor":false,"prefix":"","firstName":"Boris","middleName":"","lastName":"N’dombidjé","suffix":""},{"id":450601386,"identity":"be31c7e6-1849-4252-b613-c44a84e47e42","order_by":5,"name":"Corine Ngufor","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIiWNgGAWjYJCCAxCK8QEDQ4UFgwFMWIKwFmag4jMSDAZsRGhhgGthbCNCC39778PDBRUMefIRyYyPK+dJ2JvLdycw/KhhSJzZgF2LxJnjBodnnGEoNryRzGx4dptE4s423g2MPccYEmfjsMVAIo3hMG8bQ+LGGfnHJBu3SSQYHOPdwMDbwJA4D6+WfyAtyWySjXMk7EFaGP8S1AJUMF8CpKVBgnEDUAszSASXwyTOHGM4zHNMInEDz2NmwwYQ41juhsMyxySMcXmfv72N+TNPjU3i/PZkxocNNTb2BofPbnz4psZGdsYBHNZALWMwQFZwgKiIlMfhjFEwCkbBKBgFDAB3cFdDGKjDYgAAAABJRU5ErkJggg==","orcid":"","institution":"Centre de Recherches Entomologiques de Cotonou (CREC)","correspondingAuthor":true,"prefix":"","firstName":"Corine","middleName":"","lastName":"Ngufor","suffix":""}],"badges":[],"createdAt":"2025-04-29 12:38:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6556525/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6556525/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82021103,"identity":"929829b5-bfc9-467a-9666-25d21f2f410f","added_by":"auto","created_at":"2025-05-06 05:31:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":62636,"visible":true,"origin":"","legend":"\u003cp\u003eBlood-feeding of wild, free-flying pyrethroid-resistant \u003cem\u003eAnopheles gambiae\u003c/em\u003e entering experimental huts in Covè, southern Benin. Bars with the same letter label are not significantly different; logistic regression, P\u0026gt;0.05. Error bars represent 95% CI.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6556525/v1/fcfe45fc84df08d190f310e4.png"},{"id":82021116,"identity":"c6ab4c68-f891-4003-b47e-09cca13ed642","added_by":"auto","created_at":"2025-05-06 05:31:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":60378,"visible":true,"origin":"","legend":"\u003cp\u003eMortality (24 h) of wild, free-flying, pyrethroid-resistant \u003cem\u003eAnopheles gambiae sensu lato \u003c/em\u003eentering experimental huts in Covè, southern Benin. \u003cem\u003eBars with the same letter label are not significantly different; logistic regression, P\u0026gt;0.05. Error bars represent 95% CI.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6556525/v1/b17708676341e0fb671c80aa.png"},{"id":82021766,"identity":"f89fceab-e733-4fbe-a9ca-fed2b9dde584","added_by":"auto","created_at":"2025-05-06 05:39:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":51451,"visible":true,"origin":"","legend":"\u003cp\u003eOdds ratios describing the difference in mosquito mortality after 24 h between the candidate net (Vector Guard®) and the active comparator (Olyset® Plus). \u003cem\u003eError bars represent 95% confidence intervals. Black dashed line represents odds ratio of 1 indicating no difference between the candidate and active comparator. Red dashed line represents non-inferiority margin. Candidate is considered non-inferior to the active comparator if the lower 95% confidence interval of the odds ratio is higher than the non-inferiority margin.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6556525/v1/e9e4d1d413517b5762da619e.png"},{"id":82021104,"identity":"4a8f7714-896d-44c7-bc9b-d6ecef9a3625","added_by":"auto","created_at":"2025-05-06 05:31:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":55310,"visible":true,"origin":"","legend":"\u003cp\u003eOdds ratios describing the difference in mosquito blood-feeding between the candidate net (Vector Guard®) and the active comparator (Olyset® Plus). \u003cem\u003eError bars represent 95% confidence intervals. Black dashed line represents odds ratio of 1 indicating no difference between the candidate and active comparator. Red dashed line represents non-inferiority margin. Candidate is considered non-inferior to the active comparator if the lower 95% confidence interval of the odds ratio is higher than the non-inferiority margin.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6556525/v1/af601f51d253832a9369e2ca.png"},{"id":82021106,"identity":"fa86082f-d406-42c9-bc2e-2dda423738c1","added_by":"auto","created_at":"2025-05-06 05:31:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":20345,"visible":true,"origin":"","legend":"\u003cp\u003eKnock-down and mortality (24h) of susceptible \u003cem\u003eAnopheles gambiae\u003c/em\u003e sensu stricto Kisumu in supplementary cone bioassays. \u003cem\u003eApproximately 8 ̶ 12 mosquitoes were exposed to each of the 5 net pieces cut from unwashed and washed nets before and after the hut trial for 3 mins in two batches of 4 ̶ 6. Error bars represent 95% CIs.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6556525/v1/661bcf7228c98c408ada315e.png"},{"id":83714440,"identity":"6ca58911-65f1-4083-9815-e50bf5ee3c99","added_by":"auto","created_at":"2025-05-31 18:31:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2981631,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6556525/v1/72dc9937-43cd-40e5-a061-80345a87aecb.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eEfficacy of Vector Guard\u003csup\u003e®\u003c/sup\u003e, a mosaic alpha-cypermethrin and piperonyl butoxide- treated net, for the control of pyrethroid resistant malaria vectors; a non- inferiority experimental hut evaluation in Benin\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eInsecticide-treated nets (ITNs) are the most widely used tool for malaria prevention in endemic regions and have been instrumental in the significant declines in malaria incidence and mortality over the past two decades [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, the widespread emergence of pyrethroid resistance in major malaria vectors has threatened their effectiveness [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In response, a new generation of ITNs has been developed that combine pyrethroids with additional active ingredients\u0026mdash;such as synergists or alternative insecticide classes\u0026mdash;to enhance efficacy against pyrethroid-resistant vector populations [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOne key innovation in this space is the addition of piperonyl butoxide (PBO) on ITNs, a synergist that inhibits mosquito enzymes responsible for metabolic resistance to pyrethroids [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Pyrethroid-PBO nets have demonstrated enhanced efficacy against insecticide-resistant malaria vector populations in both experimental and operational settings. Several experimental hut trials conducted across Africa have demonstrated their superiority to pyrethroid-only nets for the control of pyrethroid-resistant malaria vectors [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Additionally, two large-scale cluster-randomized controlled trials (cRCTs) conducted in Tanzania and Uganda showed that pyrethroid-PBO nets significantly reduced malaria incidence and parasite prevalence in children compared to standard pyrethroid-only ITNs [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These findings have established pyrethroid-PBO nets as a critical tool for malaria control in areas with confirmed pyrethroid resistance [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs a result, several brands of pyrethroid-PBO ITNs have been prequalified by the World Health Organisation (WHO) and are now in wide use by national malaria control programmes across sub-Saharan Africa [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, increasing the diversity of available pyrethroid-PBO nets is important to improve market competition, meet procurement demands, and ensure continuity of supply. In this context, Vector Guard\u0026reg;, a new mosaic alphacypermethrin-PBO net developed by Disease Control Technologies, represents a promising addition to the next-generation ITN portfolio. To qualify for WHO prequalification and programmatic use, Vector Guard\u0026reg; must demonstrate entomological efficacy that is superior to pyrethroid-only nets and non-inferior to a WHO-prequalified pyrethroid-PBO net with demonstrated public health value, in accordance with WHO\u0026rsquo;s guidance [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study reports the findings of an experimental hut trial conducted in Cov\u0026egrave;, southern Benin, to assess the efficacy and wash resistance of Vector Guard\u0026reg; under controlled semi-field conditions against wild free-flying pyrethroid resistant \u003cem\u003eAnopheles gambiae\u003c/em\u003e sl. Vector Guard\u0026reg; was evaluated unwashed and after 20 standardized washes, and compared to two WHO prequalified ITNs; Olyset\u0026reg; Plus, a WHO-prequalified permethrin-PBO net with demonstrated public health value in cRCTs, and Royal Sentry\u0026reg;, a standard alpha-cypermethrin-only ITN. The trial followed existing WHO guidelines for evaluation of ITNs [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and outcomes were analyzed using WHO\u0026rsquo;s criteria for non-inferiority and superiority in entomological endpoints of mosquito mortality and blood-feeding inhibition [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Susceptibility bioassays were conducted to characterise the susceptibility status of the wild vector population at the hut site during the trial.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eExperimental hut site and vector profile\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental hut study was conducted at the CREC/LSHTM experimental hut station, located in a rice-growing area in Covè, southern Benin (7.21° N, 2.34° E). The local vector population comprises a mixture of \u003cem\u003eAnopheles coluzzii\u003c/em\u003e and \u003cem\u003eAn. gambiae\u003c/em\u003e s.s., with \u003cem\u003eAn. gambiae\u003c/em\u003e s.s. representing approximately 23% of the population, predominantly during the dry season. The vector population is highly resistant to pyrethroids, with over 90% survival observed in WHO susceptibility bioassays using alpha-cypermethrin, deltamethrin, and permethrin [12, 13]. Resistance is primarily driven by a high frequency (\u0026gt;90%) of the L1014F knockdown resistance (kdr) mutation and the over-expression of metabolic detoxification enzymes [5, 14]. The trial was carried out using seven West African-style experimental huts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSusceptibility bioassays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the frequency of pyrethroid resistance in the wild mosquito population during the hut trial, WHO susceptibility bioassays were conducted using 2–5-day-old, unfed adult F1 female mosquitoes reared from larvae collected near the experimental huts [15]. Four replicates of 20–25 mosquitoes were exposed to filter papers treated with discriminating concentrations of alpha-cypermethrin (0.05%) and permethrin (0.75%), alongside untreated control papers. The susceptible \u003cem\u003eAn. gambiae\u003c/em\u003e Kisumu strain was tested in parallel as a reference. Knockdown was recorded after 60 minutes, and mortality after a 24-hour holding period.\u003c/p\u003e\n\u003cp\u003eTo evaluate the involvement of metabolic resistance mechanisms, particularly cytochrome P450 monooxygenases, additional synergist bioassays were performed using PBO (4%). Eight replicates of 25 mosquitoes were first exposed to PBO-treated papers for 60 minutes. Half of these (four replicates) were subsequently exposed to alpha-cypermethrin (0.05%), and the remaining four replicates to permethrin (0.75%) for an additional 60 minutes. Another four replicates were exposed to PBO alone to assess its independent effects, and untreated papers were used as negative controls.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental hut treatments:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVector Guard® is made of high-density polyethylene (HDPE) with a 120-denier roof panel incorporating a mixture of alphacypermethrin (5.8 g/kg ± 25%) and piperonyl butoxide (PBO, 23.2 g/kg ± 25%). The side panels are treated with alphacypermethrin alone at 5.8 g/kg ± 25%. The candidate net was compared to two WHO-prequalified ITNs: Olyset® Plus (by Sumitomo Chemical Company), a monofilament HDPE net made of 150-denier yarn and incorporating permethrin (20 g/kg) and PBO (10 g/kg); and Royal Sentry® 2.0 (by Disease Control Technologies), a 120-denier HDPE net treated with alphacypermethrin at 5.8 g/kg ± 25%.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll net types were evaluated in both unwashed and 20-times washed conditions following WHO standard procedures [10].\u0026nbsp;The following 7 treatments were thus assessed in the experimental hut trial:\u003c/p\u003e\n\u003cp\u003e1. Untreated control polyethylene net.\u003c/p\u003e\n\u003cp\u003e2.\u0026nbsp; \u0026nbsp;\u0026nbsp;Royal Sentry® 2.0 (alpha-cypermethrin-only) unwashed\u003c/p\u003e\n\u003cp\u003e3.\u0026nbsp; \u0026nbsp;\u0026nbsp;Royal Sentry® 2.0 (alpha-cypermethrin-only) washed 20 times\u003c/p\u003e\n\u003cp\u003e4. Olyset® Plus (permethrin +PBO) unwashed\u003c/p\u003e\n\u003cp\u003e5. Olyset® Plus (permethrin +PBO) washed 20 times\u003c/p\u003e\n\u003cp\u003e6.\u0026nbsp; \u0026nbsp;\u0026nbsp;Vector Guard® (alpha-cypermethrin + PBO) unwashed\u003c/p\u003e\n\u003cp\u003e7.\u0026nbsp; \u0026nbsp; Vector Guard® (alpha-cypermethrin + PBO) washed 20 times\u003c/p\u003e\n\u003cp\u003eTo simulate wear and tear, all nets—including untreated controls—were deliberately holed in accordance with WHO guidelines, using six 4 × 4 cm holes (two on each long side and one on each short side). Net washing procedures also followed WHO standard protocols. Each net was washed in an aluminium bowl containing 10 litres of water with 2 g/L of Savon de Marseille, agitated for a total of 10 minutes. Nets were then rinsed in clean water using the same method, dried horizontally in the shade, and stored at ambient temperature between washes. The washing intervals were set at 2 days for Vector Guard® and Olyset® Plus, and 1 day for Royal Sentry® 2.0 in line with regeneration studies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHut trial design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo minimize potential bias due to hut position, treatments were rotated weekly across the seven experimental huts using a randomized Latin Square Design to reduce carry-over effects. The hut trial was conducted over 42 nights between February and April 2022. Data collection occurred over six consecutive nights each week, with the seventh day reserved for cleaning and airing the huts in preparation for the next rotation. Six replicate nets were tested per treatment, with nets rotated daily within each week. Each night, from 21:00 to 06:00, seven consenting human volunteers slept in the huts to attract wild, free-flying mosquitoes. Each morning, volunteers collected mosquitoes from the hut compartments (under the net, in the room, and veranda) using a torch and aspirator, and placed them in labelled plastic cups. Collections were transferred to the field laboratory for morphological identification and assessed for immediate mortality and blood-feeding status. Surviving female \u003cem\u003eAn. gambiae\u003c/em\u003e 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.\u003c/p\u003e\n\u003cp\u003eThe efficacy of treatments in the experimental huts was evaluated using the following outcome measures:\u003c/p\u003e\n\u003cp\u003e· \u003cstrong\u003eEntry rate\u003c/strong\u003e: Total number of mosquitoes collected per hut.\u003c/p\u003e\n\u003cp\u003e· \u003cstrong\u003eDeterrence (%)\u003c/strong\u003e: Reduction in mosquito entry in treated huts compared to the untreated control.\u003c/p\u003e\n\u003cp\u003e· \u003cstrong\u003eExophily (%)\u003c/strong\u003e: Proportion of mosquitoes found in the veranda, indicating treatment-induced exiting behavior.\u003c/p\u003e\n\u003cp\u003e· \u003cstrong\u003eInside net (%)\u003c/strong\u003e: Proportion of mosquitoes collected inside the net.\u003c/p\u003e\n\u003cp\u003e· \u003cstrong\u003eBlood-feeding rate (%)\u003c/strong\u003e: Proportion of mosquitoes that were blood-fed.\u003c/p\u003e\n\u003cp\u003e· \u003cstrong\u003eBlood-feeding inhibition (%)\u003c/strong\u003e: Reduction in blood-feeding in treated huts relative to the control\u003c/p\u003e\n\u003cp\u003e· \u003cstrong\u003ePersonal protection (%)\u003c/strong\u003e: Reduction in the number of blood-fed mosquitoes in the treated hut compared to the control.\u003c/p\u003e\n\u003cp\u003e· \u003cstrong\u003eMortality (%)\u003c/strong\u003e: Proportion of mosquitoes that died within 24 hours post-collection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary cone and tunnel tests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo supplement hut trial data, laboratory cone bioassays and tunnel tests were conducted to assess the bioavailability and potency of active ingredients in each ITN. Net samples were taken from both unwashed and 20-times washed nets of each ITN type. Cone bioassays were performed using the pyrethroid-susceptible \u003cem\u003eAn. gambiae\u003c/em\u003e s.s. Kisumu strain to evaluate the efficacy of the pyrethroid component, while tunnel tests used the pyrethroid-resistant \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. Covè strain to assess the added effect of PBO in Vector Guard® and Olyset® Plus. All assays were conducted under controlled conditions (27 ± 2°C and 75 ± 10% relative humidity).\u003c/p\u003e\n\u003cp\u003eIn cone bioassays, 8–12 unfed, 2–5-day-old \u003cem\u003eAn. gambiae\u003c/em\u003e s.s. Kisumu mosquitoes were exposed in two batches of 4–6 per cone for 3 minutes on each ITN piece. After exposure, mosquitoes were transferred to labelled holding cups, supplied with 10% glucose solution, and observed for knockdown at 60 minutes and mortality at 24 hours.\u003c/p\u003e\n\u003cp\u003eTunnel tests were conducted on two randomly selected net pieces from each ITN type and wash status. For Vector Guard®, only roof panel samples were tested. The tunnel apparatus simulates natural host-seeking behavior and consists of a glass chamber divided into two sections by a wooden frame holding the net sample. A guinea pig bait was placed in a cage at one end of the tunnel, and approximately 100 unfed, 5–8-day-old \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. Covè mosquitoes were released at dusk into the opposite end. Net samples were perforated with nine 1-cm diameter holes to allow mosquito passage. The following morning, mosquitoes were collected, and immediate mortality and blood-feeding status were recorded. Surviving mosquitoes were held in labelled cups with access to 10% glucose solution and monitored for delayed mortality at 24 hours.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChemical analysis of insecticide content\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo assess within- and between-net variation in active ingredient content, as well as wash-resistance, chemical analyses were conducted on Vector Guard® net samples by the reference laboratory CRA-W (Gembloux, Belgium). Net pieces (30 × 30 cm) taken from the experimental hut trial were analysed for alpha-cypermethrin and PBO content using CIPAC methods 54/LN/M/3.2, 454/LN/M3/3, and 33/LN/(M)/3. These methods involve extraction of the active ingredients in a water bath at 85–90 °C for 45 minutes using heptane and dicyclohexyl phthalate as the internal standard, followed by quantification via gas chromatography with flame ionisation detection (GC-FID).\u003c/p\u003e\n\u003cp\u003eThe identity of the active ingredients was confirmed through comparison with authentic standards. Each net sample was analysed individually, and average concentrations were calculated per treatment group. Wash-resistance was determined using the WHO-recommended formula for the wash-resistance index (WRI):\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWash Resistance Index (WRI) = 100 × n√(tn / t₀)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ewhere \u003cem\u003etn\u003c/em\u003e is the total active ingredient content after \u003cem\u003en\u003c/em\u003e washes, and \u003cem\u003et₀\u003c/em\u003e is the content before washing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProportional outcomes—including mosquito mortality, blood-feeding, and exophily—were compared across treatments using logistic regression, while numerical outcomes such as mosquito entry were analysed using negative binomial regression. Each outcome was modelled separately, with adjustments for variation between huts, sleepers, and trial weeks, included as fixed effects [14].\u003c/p\u003e\n\u003cp\u003eNon-inferiority analyses comparing Vector Guard® to Olyset® Plus were performed in line with WHO guidelines [15]. Vector Guard® was considered non-inferior for mosquito 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 of the 95% CI was below the NIM. The NIM was calculated to reflect a 7% difference in efficacy (mortality or blood-feeding) of Vector Guard® relative to Olyset® Plus.\u003c/p\u003e\n\u003cp\u003eSuperiority of Vector Guard® over Royal Sentry® 2.0 was assessed based on significantly higher mosquito mortality and lower blood-feeding rates at the 5% significance level (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). Analyses were conducted separately for unwashed and washed nets, as well as pooled, to assess overall product efficacy across the net’s lifespan. All statistical analyses were performed using Stata version 18.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical considerations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval for the study was obtained from the Research Ethics Committee of the Ministry of Health in Benin (CNERS; Approval No. 53, issued 17 November 2021) and the London School of Hygiene \u0026amp; Tropical Medicine (LSHTM) Ethics Committee (Ref: 26429). Written informed consent was obtained from all human volunteer sleepers prior to participation. To mitigate the risk of malaria infection, all volunteers were provided with a full course of chemoprophylaxis for the duration of the study and for four weeks following its completion. Approval for the use of guinea pigs in tunnel tests was granted by the LSHTM Animal Welfare and Ethics Review Board (Ref: 2020-01). Guinea pig colonies were housed and maintained at the CREC/LSHTM facility in accordance with standard operating procedures aligned with national and international regulations on the ethical use of animals in scientific research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with OECD principles of Good Laboratory Practice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo ensure compliance with OECD Good Laboratory Practice (GLP) principles, all phases of the study—from protocol development to reporting—were conducted under strict quality control. Equipment was calibrated, ITNs were verified for expiry and certification, and mosquito strains were handled according to SOPs. The candidate net was sourced from three production batches and stored under monitored conditions. Validated systems were used for data collection and processing, and all procedures were documented. The quality assurance team at CREC/LSHTM inspected all critical phases and found no non-conformances. External GLP inspections by SANAS in 2022 also reported full compliance.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eWHO cylinder bioassay results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe frequency of resistance to pyrethroids was very high in wild \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. from the Cov\u0026egrave; hut site, with mortality rates of only 2.1% with permethrin 0.75% and 4% with alpha-cypermethrin 0.05% (Table 1). Pre-exposure to PBO followed by alpha-cypermethrin increased mortality substantially to 34%, indicating partial restoration of susceptibility. In contrast, pre-exposure to PBO had no impact on mortality when with permethrin, which remained low at 2%. PBO alone and control treatments resulted in negligible mortality (0\u0026ndash;3.1%). In comparison, both permethrin and alpha-cypermethrin induced 100% mortality against the susceptible \u003cem\u003eAn. gambiae\u003c/em\u003e Kisumu strain, confirming full susceptibility.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1:\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003eWHO susceptibility cylinder bioassay results with wild mosquitoes collected as larvae from experimental hut station during the trial.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"738\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMosquito strain\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInsecticide\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eN exposed\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eN KD 60 mins\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% KD mins\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e95% CI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eN dead\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% Mortality\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e95% CI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"6\" valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePyrethroid -resistant\u003cem\u003e\u0026nbsp;An. gambiae s.l.\u003c/em\u003e\u0026nbsp; Cov\u0026egrave;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e(0-3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e3.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e(0-7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003ePermethrin 0.75%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e2.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e(0-5)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eAlpha-cypermethrin 0.05%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0-8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003ePBO 4%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003ePBO + Permethrin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0-5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003ePBO + alphacypermethrin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e29.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e(21-39)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e(24-44)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSusceptible\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eAn. \u0026nbsp;gambiae\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eKisumu\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e101\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e1.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0-5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003ePermethrin 0.75%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eAlpha-cypermethrin 0.05%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental hut results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEntry and exiting results\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 6,799 female \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. were collected in the experimental huts in Cov\u0026egrave; during the study period. The average number of mosquitoes caught per night across treatments ranged from 20 to 26 (Table 2). Vector Guard\u0026reg; induced the strongest deterrent effect relative to the control, both when unwashed (21.1%) and after 20 washes (12.5%). In contrast, Royal Sentry\u0026reg; 2.0 showed minimal deterrence, particularly after 20 washes (1.5%). The proportion of mosquitoes exiting the hut was significantly higher in all insecticide-treated arms compared to the untreated control (37.9%). Among unwashed nets, exit rates were comparable, with 60.5% for Royal Sentry\u0026reg; 2.0, 64.8% for Olyset\u0026reg; Plus, and 65.3% for Vector Guard\u0026reg;. After 20 washes, Vector Guard\u0026reg; maintained a high exit rate (65.3% vs 62.8%, p=0.348), whereas exiting decreased more noticeably with Royal Sentry\u0026reg; 2.0 (54.6%) and Olyset\u0026reg; Plus (50.1%), suggesting better retention of excito-repellent activity with Vector Guard\u0026reg; after washing (p\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;2\u003c/strong\u003e:\u0026nbsp;Entry and exiting of wild, free-flying, pyrethroid-resistant\u0026nbsp;\u003cem\u003eAnopheles gambiae sensu lato entering experimental huts in Cov\u0026egrave;, southern Benin.\u003c/em\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"707\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNet type\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRoyal Sentry 2.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOlyset\u0026reg; Plus\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVector Guard\u0026reg;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNet status\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUnwashed\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWashed 20x\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUnwashed\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWashed 20x\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUnwashed\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWashed 20x\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal females caught\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e1055\u003csup\u003ea,b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e936\u003csup\u003ec,d\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e1039\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e957\u003csup\u003eb,c\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e1083\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e832\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e923\u003csup\u003ec,d\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAverage catch per night\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% Deterrence\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e11.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e9.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e21.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e12.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal Exiting\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e400\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e567\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e568\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e621\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e543\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e544\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e580\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% Exiting\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e37.9\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e60.5\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e54.6\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e64.8\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e50.1\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e65.3\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e62.8\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e95% Conf Interval\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e(34.9-40.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e(57.4-63.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e(51.6-57.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e(61.8-67.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e(47.1-53.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e(62.1-68.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e(59.7-65.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*\u003cem\u003eValues in the same column bearing the same letter do not differ significantly at the 5% level according to logistic regression analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBlood-feeding results\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe blood-feeding rate with the control (untreated net) was 60.6% (Figure 1, Table 3). Among unwashed nets, Olyset\u0026reg; Plus and Vector Guard\u0026reg; achieved the lowest blood-feeding rates at 15.0% and 15.6%, respectively, both significantly lower than that of Royal Sentry\u0026reg; 2.0 (24.3%; \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05). After 20 washes, Vector Guard\u0026reg; maintained superior performance with a blood-feeding rate of 27.6%, significantly lower than Olyset\u0026reg; Plus (46.5%; \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001) and Royal Sentry\u0026reg; 2.0 (44.8%; \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBlood-feeding inhibition mirrored these trends: Vector Guard\u0026reg; and Olyset\u0026reg; Plus achieved 74.2% and 75.1% inhibition, respectively, when unwashed, compared to 59.7% with Royal Sentry\u0026reg; 2.0 (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001). After 20 washes, blood-feeding inhibition with Vector Guard\u0026reg; remained high at 54.3%, significantly greater than Olyset\u0026reg; Plus (23.1%) and Royal Sentry\u0026reg; 2.0 (25.9%; \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 for both). \u0026nbsp;In addition, personal protection levels with unwashed nets were highest with Vector Guard\u0026reg; (79.7%) and Olyset\u0026reg; Plus (77.5%) while Royal Sentry\u0026reg; 2.0 provided the lowest level of personal protection (64.3%). After 20 washes, the personal protection level remained high with Vector Guard\u0026reg; (60.0%) whilst this declined substantially with both ITN types Olyset\u0026reg; Plus (21.1%) and Royal Sentry\u0026reg; 2.0 (27.1%). These findings highlight the superior and more wash-resistant blood-feeding inhibition and personal protection provided by Vector Guard\u0026reg; against pyrethroid-resistant mosquitoes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3:\u0026nbsp;\u003c/strong\u003eBlood-feeding of wild, free-flying, pyrethroid-resistant \u003cem\u003eAnopheles gambiae sensu lato\u0026nbsp;\u003c/em\u003eentering experimental huts in Cov\u0026egrave;, southern Benin.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"763\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNet type\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRoyal Sentry 2.0\u0026reg;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOlyset\u0026reg; Plus\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVector Guard\u0026reg;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo of washes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUnwashed\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWashed 20x\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUnwashed\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWashed 20x\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUnwashed\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWashed 20x\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal females caught\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e1055\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e936\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e1039\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e957\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e1083\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e832\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e923\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal Blood fed\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e639\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e228\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e466\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e144\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e504\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e130\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e255\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBlood-feeding %\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e60.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e24.3\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e44.8\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e15.0\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e46.5\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e15.6\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e27.6\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e95% Conf Interval\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e(57.6-63.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e(21.6-27.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e(41.8-47.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e(12.8-17.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e(43.6-49.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e(13.2-18.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e(24.7-30.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBlood feeding Inhibition (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e59.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e25.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e75.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e23.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e74.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e54.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e95% Conf Interval\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e(56.6-62.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e(23.9-28.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e(72.4-77.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e(20.7-25.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e(71.2-77.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e(51.2-57.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePersonal protection (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e64.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e27.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e77.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e21.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e79.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e60.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*\u003cem\u003eValues along a row bearing the same letter label are not significantly different (P\u0026gt;0.05, logistic regression)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMortality results\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mortality of wild pyrethroid-resistant \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. with the control net was 1% (Figure 2 and Table 4). With unwashed nets, the highest mortality rate was achieved with Vector Guard\u0026reg; (36.4%) compared to Olyset\u0026reg; Plus (17.6%); p\u0026lt;0.001 and Royal Sentry\u0026reg; 2.0 (27.2%); p\u0026lt;0.001. A similar trend was observed with nets washed 20 times; mortality was significantly higher with Vector Guard\u0026reg; compared to Olyset\u0026reg; Plus (17.2% vs. 8.8%, P\u0026lt;0.001) and Royal Sentry\u0026reg; 2.0 (17.2% vs. 9.9 %, P\u0026lt;0.001).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;4\u003c/strong\u003e: Mortality of wild, free-flying, pyrethroid-resistant\u0026nbsp;\u003cem\u003eAnopheles gambiae\u0026nbsp;\u003c/em\u003esensu lato entering experimental huts in Cov\u0026egrave;, southern Benin.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"715\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNet type\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 163px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRoyal Sentry 2.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 163px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOlyset\u0026reg; Plus\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 163px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVector Guard\u0026reg;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo of washes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUnwashed\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWashed 20x\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUnwashed\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWashed 20x\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUnwashed\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWashed 20x\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal females caught\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e1055\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e936\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e1039\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e957\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e1083\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e832\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e923\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eN dead after 24h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e255\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e103\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e168\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e303\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e159\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% dead after 24h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e1.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e27.2\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e9.9\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e17.5\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e8.7\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e36.4\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e17.2\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e95% Conf Interval\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0.4-1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e(24.4-30.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e(8.1-11.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e(15.1-19.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e(7.1-10.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e(33.2-39.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e(14.8-19.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*\u003cem\u003eValues along a row bearing the same letter label are not significantly different (P\u0026gt;0.05, logistic regression)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eNon-inferiority assessment of\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eVector Guard\u0026reg; to Olyset\u0026reg; Plus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMortality outcomes:\u003c/em\u003e Vector Guard\u0026reg; met WHO non-inferiority criteria for mosquito mortality when compared to Olyset\u0026reg; Plus under both unwashed and washed conditions (Figure 3, Table 5). When unwashed, Vector Guard\u0026reg; induced significantly higher mortality (36.4%) than Olyset\u0026reg; Plus (17.5%, p\u0026lt;0.001), with an odds ratio (OR) of 3.003 (95% CI: 2.384\u0026ndash;3.784). The lower bound of the confidence interval exceeded the non-inferiority margin (NIM = 0.554), indicating both non-inferiority and superiority. After 20 washes, mortality remained significantly higher with Vector Guard\u0026reg; (17.2%) compared to Olyset\u0026reg; Plus (8.7%, p\u0026lt;0.001), with an OR of 2.286 (95% CI: 1.727\u0026ndash;3.026) and a corresponding NIM of 0.187. As the lower confidence limit was also well above the NIM, Vector Guard\u0026reg; was confirmed to be non-inferior and also superior to Olyset\u0026reg; Plus after 20 washes. When results were pooled across washing conditions, Vector Guard\u0026reg; maintained a higher overall mortality rate (26.3%) than Olyset\u0026reg; Plus (12.8%), with an OR of 2.706 (95% CI: 2.261\u0026ndash;3.239) and a NIM of 0.423, again confirming non-inferiority and superiority. Vector Guard\u0026reg; also consistently outperformed Royal Sentry\u0026reg; in vector mortality (p\u0026lt;0.001).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBlood-feeding outcomes:\u003c/em\u003e In terms of blood-feeding inhibition, Vector Guard\u0026reg; was non-inferior to Olyset\u0026reg; Plus under all conditions (Figure 4, Table 5). When unwashed, the blood-feeding rate was 15.6% for Vector Guard\u0026reg; and 15.0% for Olyset\u0026reg; Plus (OR = 1.001, 95% CI: 0.767\u0026ndash;1.306), with the upper confidence limit well below the NIM of 1.596, confirming non-inferiority. After 20 washes, blood-feeding remained significantly lower with Vector Guard\u0026reg; (27.6%) compared to Olyset\u0026reg; Plus (46.5%), with an OR of 0.39 (95% CI: 0.312\u0026ndash;0.463) and a NIM of 1.323. The upper bound of the confidence interval was well below the NIM, supporting both non-inferiority and superiority. When pooled across washing conditions, Vector Guard\u0026reg; also demonstrated lower blood-feeding rates (21.9%) than Olyset\u0026reg; Plus (31.7%), with an OR of 0.531 (95% CI: 0.453\u0026ndash;0.622) and a NIM of 1.359, confirming sustained non-inferiority and superior performance. In all conditions tested, Vector Guard\u0026reg; also showed greater blood-feeding inhibition than Royal Sentry\u0026reg; (p\u0026lt;0.001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5:\u003c/strong\u003e Non-inferiority and superiority analyses comparing the effect of Vector Guard\u0026reg; to Olyset\u0026reg; Plus \u0026nbsp;and Royal Sentry 2.0 for mosquito mortality and blood-feeding outcomes in experimental huts. \u003cem\u003eTo fulfill non-inferiority\u003c/em\u003e \u003cem\u003ecriteria, lower 95% CI of odds ratio must exceed NIM for mortality while upper 95% CI of odds ratio\u003c/em\u003e \u003cem\u003emust not exceed NIM for blood-feeding. Vector Guard must also be superior to Royal Sentry\u0026reg; 2.0\u0026nbsp;\u003c/em\u003e\u003cem\u003eat the 5% level\u003c/em\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"763\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUnwashed\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 227px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e20x Washed\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePooled\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" valign=\"top\" style=\"width: 763px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eNon-inferiority assessment\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eOdds ratio (95% CIs)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eNIM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eOdds ratio (95% CIs)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eNIM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 131px;\"\u003e\n \u003cp\u003eOdds ratio (95% CIs)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003eNIM\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMortality\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e3.003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.554\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e2.286\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e0.187\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 131px;\"\u003e\n \u003cp\u003e2.706\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e0.423\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e(2.382-3.784)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eNon-Inferior\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e(1.727-3.026)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cem\u003eNon-Inferior\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 131px;\"\u003e\n \u003cp\u003e(2.261-3.239)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cem\u003eNon-Inferior\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBlood-feeding\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e1.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e1.596\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e1.323\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 131px;\"\u003e\n \u003cp\u003e0.531\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e1.359\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e(0.767-1.306)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eNon-Inferior\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e(0.312-0.463)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cem\u003eNon-Inferior\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 131px;\"\u003e\n \u003cp\u003e(0.453-0.622)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cem\u003eNon-Inferior\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" valign=\"top\" style=\"width: 763px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eSuperiority assessment\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eOdds ratio (95% CIs)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eOdds ratio (95% CIs)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 131px;\"\u003e\n \u003cp\u003eOdds ratio (95% CIs)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMortality\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e1.563\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e˂0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e2.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e˂0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 131px;\"\u003e\n \u003cp\u003e1.741\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e˂0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e(1.252-1.951)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eSuperior\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e(1.576-2.741)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cem\u003eSuperior\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 131px;\"\u003e\n \u003cp\u003e(1.467-2.066)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cem\u003eSuperior\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBlood-feeding\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e0.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e˂0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e0.424\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e˂0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 131px;\"\u003e\n \u003cp\u003e0.477\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e˂0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e(0.475-0.783)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eSuperior\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e(0.347-0.518)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cem\u003eSuperior\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 131px;\"\u003e\n \u003cp\u003e(0.408-0.559)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cem\u003eSuperior\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary laboratory bioassay results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCone bioassay results:\u0026nbsp;\u003c/em\u003eIn cone bioassays with the susceptible \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.s. Kisumu strain, all ITNs\u0026mdash;including Vector Guard\u0026reg;, Olyset\u0026reg; Plus, and Royal Sentry\u0026reg; 2.0\u0026mdash;achieved high knockdown and mortality rates when unwashed (Figure 5). After 20 washes, Vector Guard\u0026reg; (both side and roof panels) and Royal Sentry\u0026reg; 2.0 maintained high mortality rates above 80%, while Olyset\u0026reg; Plus exhibited a substantial decline, with mortality dropping to around 60%.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTunnel test results:\u003c/em\u003e In tunnel tests against the pyrethroid-resistant \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. Cov\u0026egrave; strain, Vector Guard\u0026reg; demonstrated the highest efficacy among the ITNs tested (Table 6). When unwashed, Vector Guard\u0026reg; (roof panels) induced 99% mortality, outperforming Olyset\u0026reg; Plus (91.9%) and Royal Sentry\u0026reg; 2.0 (76.4%). Following 20 washes, mortality with Vector Guard\u0026reg; remained high at 72.1%, substantially greater than Olyset\u0026reg; Plus, which dropped markedly to 20.8%, while Royal Sentry\u0026reg; 2.0 maintained 77.4%. Blood-feeding inhibition remained above 90% for all three nets when unwashed, but after 20 washes, Vector Guard\u0026reg; continued to provide strong blood-feeding protection (93.8% BFI), comparable to Royal Sentry\u0026reg; 2.0 (95.5%), whereas Olyset\u0026reg; Plus experienced a sharp decline in BFI to 53.7%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 6\u003c/strong\u003e: Summary results of tunnel tests with pyrethroid-resistant\u0026nbsp;\u003cem\u003eAnopheles gambiae\u003c/em\u003e sensu lato Cov\u0026egrave; strain\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"767\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; Washes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTreatments\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eN Exposed\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eN Dead\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% Mortality (95%CI)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eN Blood-fed\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% Blood-fed (95%CI)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% Blood-feeding inhibition\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUnwashed\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e173\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e1.2 (0-2.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e113\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003e65.3 (58.2-72.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003eRoyal Sentry\u0026reg; 2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e203\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e155\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e76.4 (70.5-82.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003e5.9 (2.7-9.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e90.95\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003eOlyset\u0026reg; Plus\u0026nbsp;Net\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e184\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e169\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e91.9 (87.9-95.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003e1.1 (0-2.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e98.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003eVector Guard\u0026reg; (roof)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e208\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e206\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e99 (97.7-100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWashed 20X\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003eRoyal Sentry\u0026reg; 2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e239\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e185\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e77.4 (72.1-82.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003e2.9 (0.8-5.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e95.52\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003eOlyset\u0026reg;\u003csup\u003e\u0026nbsp;\u003c/sup\u003ePlus\u0026nbsp;Net\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e192\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e20.8 (15.1-26.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003e30.2 (23.7-36.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e53.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003eVector Guard\u0026reg; (roof)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e197\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e142\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e72.1 (65.8-78.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003e4.1 (1.3-6.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e93.78\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eChemical analysis of net pieces results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe active ingredient content in all unwashed ITNs was within defined specifications declared by the manufacturers. Retention of PBO after 20 washes was lowest with Olyset\u0026reg; Plus (39.7%) (Table 7). Side panels of Vector Guard\u0026reg; showed high wash-retention of alpha-cypermethrin the (94.3%). Between pyrethroid-PBO ITNs, Vector Guard\u0026reg; (roof) showed higher levels of wash-retention of both active ingredients (94.5% for alpha-cypermethrin and 83.4% for PBO) compared to Olyset\u0026reg; Plus (69.6% for permethrin and 39.7% for PBO). The wash-resistance index of PBO was thus higher with Vector Guard\u0026reg; (99.1%) than with Olyset\u0026reg; Plus (95.5%).\u003c/p\u003e\n\u003cp\u003eTable 7: Chemical content of unwashed and washed net pieces taken before and after the experimental hut trial in Cov\u0026egrave;, Benin.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"677\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eITN type\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 181px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eActive ingredient(s)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 241px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAI content (g/kg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAI retention (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUnwashed\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 130px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWashed 20X\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRoyal Sentry\u0026reg; 2.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 181px;\"\u003e\n \u003cp\u003eAlpha-cypermethrin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e5.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 130px;\"\u003e\n \u003cp\u003e4.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e90.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOlyset\u0026reg; Plus\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 181px;\"\u003e\n \u003cp\u003ePermethrin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e18.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 130px;\"\u003e\n \u003cp\u003e12.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e69.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 181px;\"\u003e\n \u003cp\u003ePBO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e8.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 130px;\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e39.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVector Guard\u0026reg;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 181px;\"\u003e\n \u003cp\u003eAlpha-cypermethrin (roof)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e5.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 130px;\"\u003e\n \u003cp\u003e5.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e94.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 181px;\"\u003e\n \u003cp\u003ePBO (roof)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e16.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 130px;\"\u003e\n \u003cp\u003e13.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e83.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 181px;\"\u003e\n \u003cp\u003eAlpha-cypermethrin (side)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e5.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 130px;\"\u003e\n \u003cp\u003e5.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e94.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis experimental hut study provides robust entomological evidence on the efficacy and wash durability of Vector Guard\u0026reg;, a new mosaic ITN combining alpha-cypermethrin and PBO, against wild, free-flying, pyrethroid-resistant \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. in southern Benin. The results show that Vector Guard\u0026reg; offers superior and more wash resistant protection compared to Royal Sentry\u0026reg; 2.0, a standard pyrethroid-only ITN, and is non-inferior\u0026mdash;and in many outcomes, superior\u0026mdash;to Olyset\u0026reg; Plus, a WHO-prequalified PBO net with documented epidemiological impact. Vector Guard\u0026reg; consistently outperformed both comparators across key entomological indicators, including mosquito deterrence, exiting rates, blood-feeding inhibition, and mortality. Importantly, these effects were sustained after 20 washes, indicating good wash durability and potential for long-term protective performance under field conditions.\u003c/p\u003e \u003cp\u003eThe superior efficacy of Vector Guard\u0026reg; over Royal Sentry\u0026reg; 2.0 aligns with findings from multiple experimental hut and community trials across sub-Saharan Africa comparing pyrethroid-PBO to pyrethroid-only nets [\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and supports current WHO recommendations favouring prioritisation of pyrethroid-PBO nets over pyrethroid-only nets in areas with confirmed pyrethroid resistance [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. While Vector Guard\u0026reg; induced higher mosquito mortality than Royal Sentry\u0026reg;, Olyset\u0026reg; Plus performed worse, with mortality rates even lower than those observed with Royal Sentry\u0026reg; 2.0. These differences likely reflect the type of pyrethroid used\u0026mdash;alpha-cypermethrin in Vector Guard\u0026reg; versus permethrin in Olyset\u0026reg; Plus. Pre-exposure to PBO partially restored susceptibility to alpha-cypermethrin, raising mortality from 4\u0026ndash;34%, but had little effect on permethrin, where mortality remained low (~\u0026thinsp;2%). This reduced synergism between PBO and permethrin, though not fully understood, is consistent with findings from previous studies conducted in Benin [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and other settings in West Africa.[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe non-inferiority analyses confirmed that Vector Guard\u0026reg; meets WHO criteria [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] for non-inferiority to Olyset\u0026reg; Plus for both mortality and blood-feeding inhibition under unwashed, washed, and pooled conditions. In fact, the performance of Vector Guard\u0026reg; exceeded Olyset\u0026reg; Plus across all endpoints both in the hut trial and in the supplementary bioassays. Vector Guard\u0026reg; was associated with significantly higher mosquito mortality and lower blood-feeding rates, confirming not only non-inferiority but also superiority. The superior efficacy of Vector Guard compared to Olyset\u0026reg; Plus can also be explained by the afore-mentioned improved synergistic interaction between PBO and alpha-cypermethrin compared to permethrin. These findings suggest that pairing PBO with alpha-cypermethrin on ITNs (rather than permethrin) can yield better entomological efficacy against metabolically resistant mosquito populations. Chemical analysis further substantiated these results. Vector Guard\u0026reg; exhibited high retention of both alpha-cypermethrin and PBO after 20 washes, with over 83% retention of PBO and \u0026gt;\u0026thinsp;94% for alpha-cypermethrin. This contrasted with Olyset\u0026reg; Plus, which retained only 39.7% of its PBO content and 69.6% of its permethrin. The higher wash-resistance index for PBO in Vector Guard\u0026reg; reflects its superior formulation and suggests greater durability of its insecticidal efficacy under household use.\u003c/p\u003e \u003cp\u003eVector Guard\u0026reg; was added to the WHO list of prequalified vector control products [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] based on the results of this study and supporting evidence from a similar trial conducted against pyrethroid-resistant \u003cem\u003eAnopheles arabiensis\u003c/em\u003e in Tanzania [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Vector Guard\u0026reg; is now commercialised under the brand name SafeNet\u0026reg; Plus after its ownership was transferred to Mainpol GmbH in 2024 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Given its demonstrated superiority over Olyset\u0026reg; Plus, the net represents a strong alternative for malaria control programmes seeking effective pyrethroid-PBO nets for use in areas with high intensity pyrethroid-resistance. Further operational research is warranted to assess its physical integrity and insecticidal durability under long-term community use.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eVector Guard\u0026reg; demonstrated superior entomological efficacy and wash durability compared to Royal Sentry\u0026reg; 2.0 and Olyset\u0026reg; Plus, and fulfilled WHO non-inferiority criteria for mosquito mortality and blood-feeding inhibition. These findings support the inclusion of Vector Guard\u0026reg; as a strong candidate pyrethroid-PBO ITNs for malaria control in areas with high levels of pyrethroid resistance.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAI – Active ingredient\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCI – Confidence interval\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eITN – Insecticide-treated nets\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eKdr\u003c/em\u003e – Knockdown resistance\u003c/p\u003e\n\u003cp\u003eHDPE – high-density polyethylene\u003c/p\u003e\n\u003cp\u003eCREC –\u0026nbsp;Centre de Recherches Entomologiques de Cotonou\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePAMVERC –\u0026nbsp;Pan-African Malaria Vector Research Consortium\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAIRID –\u0026nbsp;African Institute for Research in Infectious Diseases\u003c/p\u003e\n\u003cp\u003eLSHTM – London School of Hygiene \u0026amp; Tropical Medicine\u003c/p\u003e\n\u003cp\u003ePBO – Piperonyl butoxide\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNIM ­– Non inferiority margin\u003c/p\u003e\n\u003cp\u003ePQT/VCP – Prequalification Unit Vector Control Product Assessment Team\u003c/p\u003e\n\u003cp\u003eP450 – Cytochrome P450 monooxygenase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOR – Odds ratio\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ecRCT – cluster randomised controlled trial\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWHO – World Health Organisation\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Rod Flinn and Andy Buttenhof of Disease Control Technologies LLC for providing the study nets. We also thank the rice farmers of Covè for their participation in the study. Special appreciation goes to the technical and administrative staff of the CREC/LSHTM/PAMVERC research programme in Benin for their valuable support. We also acknowledge the team at CRAW Gembloux, Belgium, for conducting the chemical analyses\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by a grant from the Gates Foundation (INV-023420) to the London School of Hygiene and Tropical Medicine and the Centre de Recherche Entomologique de Cotonou. The funders had no role in the study design, data collection and analysis and decision to publish this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are available within the paper and its supplementary material.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCN designed the methods, acquired funding and was responsible for the overall conduct of the study. MG and AA performed the hut trials. JN and BN performed the laboratory bioassays. JN analysed the data and prepared the figures and tables. CN and JN wrote the manuscript. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval for conduct of the experimental hut trial and withdrawal of the field-aged nets was obtained from the ethics review boards of the Ministry of Health in Benin and the institutional review board of LSHTM. 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\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBhatt S, Weiss D, Cameron E, Bisanzio D, Mappin B, Dalrymple U, Battle K, Moyes C, Henry A, Eckhoff P: \u003cstrong\u003eThe effect of malaria control on Plasmodium falciparum in Africa between 2000 and 2015.\u003c/strong\u003e \u003cem\u003eNature\u0026nbsp;\u003c/em\u003e2015, \u003cstrong\u003e526:\u003c/strong\u003e207-211.\u003c/li\u003e\n \u003cli\u003eWHO: \u003cstrong\u003eWHO Malaria Threats Map.\u003c/strong\u003e \u003cem\u003eWorld Health Organisation, Geneva\u0026nbsp;\u003c/em\u003e2025, \u003cstrong\u003ehttps://apps.who.int/malaria/maps/threats/\u003c/strong\u003e.\u003c/li\u003e\n \u003cli\u003eWHO: \u003cstrong\u003eWHO List of Prequalified In Vitro Diagnostic Products.\u003c/strong\u003e \u003cem\u003eWorld Health Organisation, Geneva\u0026nbsp;\u003c/em\u003e2025.\u003c/li\u003e\n \u003cli\u003eGleave K, Lissenden N, Richardson M, Choi L, Ranson H: \u003cstrong\u003ePiperonyl butoxide (PBO) combined with pyrethroids in insecticide-treated nets to prevent malaria in Africa.\u003c/strong\u003e \u003cem\u003eCochrane Database Syst Rev\u0026nbsp;\u003c/em\u003e2018, \u003cstrong\u003e11:\u003c/strong\u003eCD012776.\u003c/li\u003e\n \u003cli\u003eNgufor C, Fagbohoun J, Agbevo A, Ismail H, Challenger JD, Churcher TS, Rowland M: \u003cstrong\u003eComparative efficacy of two pyrethroid-piperonyl butoxide nets (Olyset Plus and PermaNet 3.0) against pyrethroid resistant malaria vectors: a non-inferiority assessment.\u003c/strong\u003e \u003cem\u003eMalar J\u0026nbsp;\u003c/em\u003e2022, \u003cstrong\u003e21:\u003c/strong\u003e20.\u003c/li\u003e\n \u003cli\u003eStaedke SG, Gonahasa S, Dorsey G, Kamya MR, Maiteki-Sebuguzi C, Lynd A, Katureebe A, Kyohere M, Mutungi P, Kigozi SP, et al: \u003cstrong\u003eEffect 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.\u003c/strong\u003e \u003cem\u003eLancet\u0026nbsp;\u003c/em\u003e2020, \u003cstrong\u003e395:\u003c/strong\u003e1292-1303.\u003c/li\u003e\n \u003cli\u003eProtopopoff N, Mosha JF, Lukole E, Charlwood JD, Wright A, Mwalimu CD, Manjurano A, Mosha FW, Kisinza W, Kleinschmidt I, Rowland M: \u003cstrong\u003eEffectiveness 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.\u003c/strong\u003e \u003cem\u003eLancet\u0026nbsp;\u003c/em\u003e2018, \u003cstrong\u003e391:\u003c/strong\u003e1577-1588.\u003c/li\u003e\n \u003cli\u003eWHO: \u003cstrong\u003eGuidelines for malaria vector control.\u003c/strong\u003e \u003cem\u003eGeneva, Switzerland: World Health Organization\u0026nbsp;\u003c/em\u003e2025.\u003c/li\u003e\n \u003cli\u003eAMP: \u003cstrong\u003eAlliance for malaria prevention; 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LN against pyrethroid resistant Anopheles gambiae ss: an experimental hut trial in M\u0026rsquo;b\u0026eacute;, central C\u0026ocirc;te d\u0026rsquo;Ivoire.\u003c/strong\u003e \u003cem\u003eParasites \u0026amp; vectors\u0026nbsp;\u003c/em\u003e2019, \u003cstrong\u003e12:\u003c/strong\u003e1-10.\u003c/li\u003e\n \u003cli\u003eTungu P, Magesa S, Maxwell C, Malima R, Masue D, Sudi W, Myamba J, Pigeon O, Rowland M: \u003cstrong\u003eEvaluation of PermaNet 3.0 a deltamethrin-PBO combination net against Anopheles gambiae and pyrethroid resistant Culex quinquefasciatus mosquitoes: an experimental hut trial in Tanzania.\u003c/strong\u003e \u003cem\u003eMalar J\u0026nbsp;\u003c/em\u003e2010, \u003cstrong\u003e9:\u003c/strong\u003e21.\u003c/li\u003e\n \u003cli\u003eDadzie SK, Chabi J, Asafu-Adjaye A, Owusu-Akrofi O, Baffoe-Wilmot A, Malm K, Bart-Plange C, Coleman S, Appawu MA, Boakye DA: \u003cstrong\u003eEvaluation of piperonyl butoxide in enhancing the efficacy of pyrethroid insecticides against resistant Anopheles gambiae s.l. in Ghana.\u003c/strong\u003e \u003cem\u003eMalaria Journal\u0026nbsp;\u003c/em\u003e2017, \u003cstrong\u003e16:\u003c/strong\u003e342.\u003c/li\u003e\n \u003cli\u003eMachange JJ, Mbuba E, Irish SR, Swai JK, Ntabaliba W, Makungwa NO, Ngonyani S, Mpelepele AB, Kibondo UA, Odufuwa OG, Moore SJ: \u003cstrong\u003eComparative efficacy of Vector Guard\u0026reg; to Olyset\u0026reg; Plus insecticide-treated nets against strongly pyrethroid-resistant Anopheles arabiensis in experimental huts in Tanzania.\u003c/strong\u003e \u003cem\u003eFrontiers in Malaria\u0026nbsp;\u003c/em\u003e2024, \u003cstrong\u003eVolume 2 - 2024\u003c/strong\u003e.\u003c/li\u003e\n \u003cli\u003eWHO: \u003cstrong\u003eTransfer of ownership of Vector Guard to Mainpol.\u003c/strong\u003e \u003cem\u003ehttps://extranetwhoint/prequal/sites/default/files/doc_parts/20240715-transfer-of-ownership-mainpol-p-00210-p-00211-p-09284-pqc-vcp-2024-0020_1pdf\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e2024, \u003cstrong\u003eaccessed March 2025\u003c/strong\u003e.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Insecticide-treated nets, Vector Guard, Olyset Plus, Royal Sentry, Piperonyl butoxide, Pyrethroid resistance, Experimental hut trial, pyrethroid-PBO nets, Anopheles gambiae sl, Benin, malaria, Covè","lastPublishedDoi":"10.21203/rs.3.rs-6556525/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6556525/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Insecticide-treated nets (ITNs) combining pyrethroids with piperonyl butoxide (PBO) have improved the control of malaria transmitted by pyrethroid-resistant vectors compared to standard pyrethroid-only ITNs. To sustain malaria prevention efforts, a broader range of effective pyrethroid-PBO nets is needed to enhance market diversity and supply resilience. This study evaluated the entomological efficacy and wash durability of Vector Guard®, a new mosaic alpha-cypermethrin-PBO ITN, against pyrethroid-resistant Anopheles gambiae s.l. in southern Benin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003cbr\u003e\nAn experimental hut trial was conducted in Covè, Benin, against wild, free-flying \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. Vector Guard® was tested unwashed and after 20 standardized washes, and compared to two WHO-prequalified ITNs: Olyset® Plus (a permethrin-PBO net) and Royal Sentry® 2.0 (an alpha-cypermethrin-only net). Primary outcomes were mosquito mortality and blood-feeding protection. Susceptibility bioassays were conducted to assess local resistance mechanisms. Laboratory cone and tunnel tests were also performed to help explain the finding in the experimental huts. Chemical content analyses was performed to investigate active ingredient wash retention. Vector Guard® was assessed for its non-inferiority to Olyset® Plus following WHO guidance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003cbr\u003e\nThe wild \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. population at Covè exhibited high frequencies of pyrethroid resistance, with PBO pre-exposure restoring partial susceptibility to alpha-cypermethrin (34% vs. 4% mortality) but not to permethrin (2.0% vs. 2.1% mortality). A total of 6,799 females were collected in the experimental huts. Vector Guard® outperformed both Royal Sentry® 2.0 and Olyset® Plus across all entomological endpoints. Mortality with Vector Guard® was significantly higher than with Olyset® Plus when unwashed (36.4% vs. 17.5%, p \u0026lt; 0.001) and after 20 washes (17.2% vs. 8.7%, p \u0026lt; 0.001). Non-inferiority analysis with pooled data for unwashed and washed nets confirmed that Vector Guard® was non-inferior to Olyset® Plus for both mortality (OR 2.71, 95% CI: 2.26–3.24; NIM: 0.423) and blood-feeding protection (OR 0.53, 95% CI: 0.45–0.62; NIM: 1.359). These findings were supported by cone and tunnel tests. Chemical analysis showed higher wash retention of active ingredients in Vector Guard® (83% for PBO and \u0026gt;94% for alpha-cypermethrin) compared to Olyset® Plus (39.7% for PBO and 69.6% for permethrin).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003cbr\u003e\nVector Guard® demonstrated superior entomological efficacy and wash durability compared to Royal Sentry® 2.0 and Olyset® Plus, and fulfilled WHO non-inferiority criteria for mosquito mortality and blood-feeding inhibition. These findings supported its addition to the WHO list of prequalified pyrethroid-PBO ITNs and its potential to provide improved malaria control when deployed on a large scale in areas with high levels of pyrethroid resistance.\u003c/p\u003e","manuscriptTitle":"Efficacy of Vector Guard®, a mosaic alpha-cypermethrin and piperonyl butoxide- treated net, for the control of pyrethroid resistant malaria vectors; a non- inferiority experimental hut evaluation in Benin","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-06 05:31:39","doi":"10.21203/rs.3.rs-6556525/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5a2d6eda-d622-48b3-8b3e-a69ec4116274","owner":[],"postedDate":"May 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-31T18:23:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-06 05:31:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6556525","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6556525","identity":"rs-6556525","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}