A series of hut-based pilot trials were conducted in Khowe, southern Benin, to evaluate the biological efficacy of new and field-tested next-generation mosquito nets against pyrethrin-resistant malaria vectors. Field-aged nets were removed from households after 12, 24 and 36 months. Web pieces cut from whole ITNs were analysed for chemical composition and susceptibility bioassays were conducted during each trial to assess changes in insecticide resistance in the Khowe vector population.
Interceptor® G2 outperformed other ITNs, confirming the superiority of pyrethroid and chlorfenapyr nets over other net types. Among the new products, all next-generation ITNs demonstrated better bioefficacy than Interceptor®; however, the magnitude of this improvement was reduced after field aging due to the shorter durability of non-pyrethroid compounds. These results highlight the need to improve the insecticidal persistence of next-generation ITNs.
Insecticide-treated mosquito nets (ITNs) have played a critical role in reducing malaria morbidity and mortality over the past 20 years. Since 2004, more than 3 billion ITNs have been distributed worldwide , and modelling studies suggest that 68% of malaria cases in sub-Saharan Africa were averted between 2000 and 2015 . Unfortunately, resistance of malaria vector populations to pyrethroids (the standard class of insecticides used in ITNs) has increased significantly , threatening the effectiveness of this essential intervention. At the same time, progress in malaria control has slowed globally, with a number of high-burden countries experiencing an increase in malaria cases since 2015 . These trends have driven the development of a new generation of innovative ITN products aimed at addressing the threat of pyrethroid resistance and helping to reduce this burden and achieve ambitious global targets .
There are currently three new generation ITNs on the market, each combining a pyrethroid with another insecticide or synergist capable of overcoming pyrethroid resistance in malaria vectors. In recent years, a number of cluster randomised controlled trials (RCTs) have been conducted to assess the epidemiological effectiveness of these nets compared with standard pyrethroid-only nets and to provide the necessary evidence to support World Health Organization (WHO) recommendations. Bed nets combining pyrethroids with piperonyl butoxide (PBO), a synergist that enhances the effectiveness of pyrethroids by inhibiting mosquito detoxification enzymes, were the first to be recommended by WHO after two products (Olyset® Plus and PermaNet® 3.0) demonstrated superior epidemiological impact compared with pyrethroid-only bed nets in cluster randomised controlled trials in Tanzania and Uganda. However, more data are needed to determine the public health value of pyrethroid-PBO bed nets in West Africa, where severe pyrethroid resistance may reduce their benefits compared with pyrethroid-only bed nets .
The insecticidal persistence of ITNs is typically assessed by periodically collecting nets from communities and testing them in laboratory bioassays using insect-bred mosquito strains . While these assays are useful for characterizing the bioavailability and efficacy of insecticides on the surface of bednets over time, they provide limited information on the comparative effectiveness of different types of next-generation bednets because the methods and mosquito strains used must be adapted to the mode of action of the insecticides they contain . The experimental hut test is an alternative approach that can be used to comparatively evaluate the effectiveness of insecticide-treated nets in durability studies under conditions that mimic the natural interactions between wild mosquito hosts and household nets during use. Indeed, recent modelling studies using entomological surrogates for epidemiological data have shown that mosquito mortality and feeding rates measured in these trials can be used to predict the impact of ITNs on malaria incidence and prevalence in cluster RCTs . Thus, hut-based experimental trials in which field-collected insecticide-treated lymph nodes are included in cluster RCTs may provide valuable data on the comparative bioefficacy and insecticidal persistence of insecticide-treated lymph nodes over their expected lifespan, and help interpret the epidemiological results of these studies.
The experimental hut test is a standardized simulated human habitation recommended by the World Health Organization for evaluating the effectiveness of insecticide-treated mosquito nets. These tests replicate the real-world exposure conditions that mosquito hosts encounter when interacting with household bed nets and are therefore a highly appropriate approach for assessing the biological effectiveness of used bed nets over their expected service life.
This study assessed the entomological efficacy of three different types of new generation insecticidal mosquito nets (PermaNet® 3.0, Royal Guard® and Interceptor® G2) under field conditions in experimental barns and compared them with a standard pyrethrin-only net (Interceptor®). All of these insecticide-treated mosquito nets are included in the WHO prequalified list for vector control. Detailed characteristics of each mosquito net are provided below:
In March 2020, a large-scale distribution campaign of field-aged mosquito nets was carried out in hut villages in Zou Prefecture, southern Benin, for pilot trials in huts. Interceptor®, Royal Guard® and Interceptor® G2 bed nets were selected from randomly selected clusters in the municipalities of Kove, Zagnanado and Ouinhi as part of a durability observational study nested within a cluster RCT to assess the epidemiological effectiveness of dual insecticide-treated bed nets . PermaNet® 3.0 mosquito nets were collected in Avokanzun village near Jija and Bohicon townships (7°20′ N, 1°56′ E) and distributed simultaneously with RCT cluster mosquito nets during the 2020 mass campaign of the National Malaria Control Programme. Figure 1 shows the locations of the study clusters/villages where the different ITN types were collected relative to the experimental hut sites.
A pilot hut trial was conducted to compare the entomological performance of Interceptor®, PermaNet® 3.0, Royal Guard® and Interceptor® G2 ITNs when removed from households at 12, 24 and 36 months post-dissemination. At each annual time point, the performance of aged ITNs in the field was compared with new, unused nets of each type and untreated nets as a negative control. At each annual time point, a total of 54 replicate samples of field-aged ITNs and 6 new ITNs of each type were tested in 1 or 2 replicate hut trials with daily rotation of treatments. Before each hut trial, the average porosity index of the aged field nets of each ITN type was measured according to WHO recommendations . To simulate wear and tear from daily use, all new ITNs and untreated control nets were perforated with six 4 x 4 cm holes: two in each long side panel and one in each short side panel, in accordance with WHO recommendations. The mosquito net was installed inside the hut by tying the edges of the roof sheets with ropes to nails in the upper corners of the hut walls. The following treatments were evaluated in each hut trial:
Field-aged nets were evaluated in experimental huts in the same year as the nets were removed. Hut trials were conducted at the same site from May to September 2021, April to June 2022, and May to July 2023, with nets removed after 12, 24, and 36 months, respectively. Each trial lasted for one complete treatment cycle (54 nights over 9 weeks), except for 12 months, when two consecutive treatment cycles were conducted to increase the mosquito sample size. Following a Latin square design, treatments were rotated weekly between experimental huts to control for hut location effects, while volunteers were rotated daily to control for differences in mosquito attractiveness of individual hosts. Mosquitoes were collected 6 days per week; on day 7, before the next rotation cycle, huts were cleaned and ventilated to prevent infestation.
The primary efficacy endpoints for the experimental hut treatment against pyrethroid-resistant Anopheles gambiae mosquitoes and the comparison of the next generation ITN with the pyrethroid-only Interceptor® net were:
Secondary efficacy endpoints for the experimental hut treatment against pyrethroid-resistant Anopheles gambiae mosquitoes were as follows:
Containment (%) – reduction in entry rate into the treated group compared to the untreated group. The calculation is as follows:
where Tu is the number of mosquitoes included in the untreated control group, and Tt is the number of mosquitoes included in the treated group.
Churn Rate (%) – Churn rate due to potential irritation from treatment, expressed as a proportion of mosquitoes collected on the balcony.
. Bloodsucking suppression coefficient (%) is the reduction in the proportion of bloodsucking mosquitoes in the treated group compared to the untreated control group. The calculation method is as follows: where Bfu is the proportion of bloodsucking mosquitoes in the untreated control group, and Bft is the proportion of bloodsucking mosquitoes in the treated group.
Reduction in fertility (%) — the reduction in the proportion of fertile mosquitoes in the treated group compared to the untreated control. The calculation method is as follows: where Fu is the proportion of fertile mosquitoes in the untreated control group, and Ft is the proportion of fertile mosquitoes in the treated group.
To monitor changes in the resistance profile of Covè vector populations over time, WHO conducted in vitro and vial bioassays in the same year of each experimental hut trial (2021, 2022, 2023) to assess susceptibility to AI in the ITNs under study and to inform interpretation of the results. In the in vitro studies, mosquitoes were exposed to filter papers treated with defined concentrations of alpha-cypermethrin (0.05%) and deltamethrin (0.05%), and to bottles coated with defined concentrations of CFP (100 μg/bottle) and PPF (100 μg/bottle) to assess susceptibility to these insecticides. The intensity of pyrethroid resistance was investigated by exposing mosquitoes to 5-fold (0.25%) and 10-fold (0.50%) differential concentrations of α-cypermethrin and deltamethrin. Finally, the contribution of PBO synergy and cytochrome P450 monooxygenase (P450) overexpression to pyrethroid resistance was assessed by pre-exposing mosquitoes to differential concentrations of α-cypermethrin (0.05%) and deltamethrin (0.05%), and pre-exposure to PBO (4%). The filter paper used for the WHO tube test was purchased from Universiti Sains Malaysia. The WHO bioassay test vials using CFP and PPF were prepared according to WHO recommendations .
Mosquitoes used for bioassays were collected at the larval stage from breeding sites near the experimental huts and then reared to adults. At each time point, at least 100 mosquitoes were exposed to each treatment for 60 min, with 4 replicates per tube/bottle and approximately 25 mosquitoes per tube/bottle. For pyrethroid and CFP exposures, 3–5 day-old unfed mosquitoes were used, whereas for PPF, 5–7 day-old bloodsucking mosquitoes were used to stimulate oogenesis and assess the effect of PPF on mosquito reproduction. Parallel exposures were conducted using silicone oil-impregnated filter paper, neat PBO (4%), and acetone-coated bottles as controls. At the end of the exposure, mosquitoes were transferred to untreated containers and exposed to cotton wool soaked in 10% (w/v) glucose solution. Mortality was recorded 24 h after pyrethroid exposure and every 24 h for 72 h after CFP and PPF exposure. To assess susceptibility to PPF, surviving PPF-exposed mosquitoes and corresponding negative controls were dissected after delayed mortality was recorded, ovarian development was observed using a compound microscope, and fertility was assessed according to the Christophers stage of egg development [28, 30]. If the eggs fully developed to Christophers stage V, the mosquitoes were classified as fertile, and if the eggs were not fully developed and remained at stages I–IV, the mosquitoes were classified as sterile.
At each time point of the year, 30 × 30 cm pieces were cut from new and field-aged nets at the locations specified in the WHO recommendations [22]. After cutting, the nets were labelled, wrapped in aluminium foil and stored in a refrigerator at 4 ± 2 °C to prevent AI migration into the fabric. The nets were then sent to the Walloon Agricultural Research Centre in Belgium for chemical analysis to measure changes in total AI content during their service life. The analytical methods used (based on the methods recommended by the International Cooperative Committee for Pesticide Analysis) have been described previously [25, 31].
For the experimental hut trial data, the total numbers of live/dead, biting/non-biting, and fertile/sterile mosquitoes in the different hut compartments were summed for each treatment in each trial to calculate the various proportional outcomes (72-hour mortality, biting, ectoparasitism, net entrapment, fertility) and their corresponding 95% confidence intervals (CIs). Differences between treatments for these proportional binary outcomes were analyzed using logistic regression, while differences for count outcomes were analyzed using negative binomial regression. Because two treatment rotation cycles were conducted every 12 months and some treatments were tested across trials, mosquito penetration analyses were adjusted for the number of days each treatment was tested. The new ITN for each outcome was also analyzed to obtain a single estimate for all time points. In addition to the main explanatory variable of treatment, each model included hut, sleeper, trial period, ITN aperture index, and day as fixed effects to control for variation due to differences in individual sleeper and hut attractiveness, seasonality, mosquito net status, and excess dispersion. Regression analyses produced adjusted odds ratios (ORs) and corresponding 95% confidence intervals to estimate the effect of the new-generation ITN compared with the pyrethroid-only net, Interceptor®, on the primary outcomes of mosquito mortality and fecundity. P values from the models were also used to assign compact letters indicating statistical significance at the 5% level for all pairwise comparisons of the primary and secondary outcomes. All regression analyses were performed in Stata version 18.
Susceptibility of Covese vector populations was interpreted based on mortality and fecundity observed in vitro and bottle bioassays according to World Health Organization recommendations . Chemical analysis results provided total AI content in ITN fragments, which was used to calculate the AI retention rate in field-aged nets compared to new nets at each time point each year. All data were manually recorded on standardized forms and then double-entered into a Microsoft Excel database.
The Ethics Committees of the Ministry of Health of Benin (No. 6/30/MS/DC/DRFMT/CNERS/SA), the London School of Hygiene & Tropical Medicine (LSHTM) (No. 16237) and the World Health Organization (No. ERC.0003153) approved the conduct of a pilot hut trial involving volunteers. Written informed consent was obtained from all volunteers prior to participation in the study. All volunteers received free chemoprophylaxis to reduce the risk of malaria, and a nurse was on duty throughout the trial to assess any volunteer who developed symptoms of fever or an adverse reaction to the test product.
Full results from the experimental huts, summarizing the total numbers of live/dead, starved/blood-fed, and fertile/sterile mosquitoes for each experimental group, as well as descriptive statistics are presented as supplementary material (Table S1).
In an experimental hut in Kowa, Benin, blood feeding of wild pyrethroid-resistant Anopheles gambiae mosquitoes was suppressed. Data from untreated controls and novel nets were pooled across trials to provide a single efficacy estimate. By logistic regression analysis, columns with common letters were not significantly different at the 5% level (p > 0.05). Error bars represent 95% confidence intervals.
Mortality of wild pyrethroid-resistant Anopheles gambiae mosquitoes entering an experimental hut in Kowa, Benin. Data from untreated controls and novel nets were pooled across trials to provide a single estimate of efficacy. By logistic regression analysis, columns with common letters were not significantly different at the 5% level (p > 0.05). Error bars represent 95% confidence intervals.
The odds ratio describes the difference in mortality with new-generation mosquito nets compared with pyrethroid-only mosquito nets. The dotted line represents an odds ratio of 1, indicating no difference in mortality. An odds ratio > 1 indicates higher mortality with new-generation mosquito nets. Data for new-generation mosquito nets were pooled across trials to produce a single estimate of effectiveness. Error bars represent 95% confidence intervals.
Although Interceptor® demonstrated the lowest mortality of all ITNs tested, field aging did not negatively impact its impact on vector mortality. In fact, new Interceptor® resulted in 12% mortality, whereas field-aged nets showed a slight improvement at 12 months (17%, p=0.006) and 24 months (17%, p=0.004), before returning to levels similar to new nets at 36 months (11%, p=0.05). In contrast, mortality rates for the next generation of insecticide-treated nets gradually declined over time after deployment. The reduction was most pronounced with Interceptor® G2, where mortality decreased from 58% with the new meshes to 36% at 12 months (p < 0.001), 31% at 24 months (p < 0.001), and 20% at 36 months (p < 0.001). The new PermaNet® 3.0 resulted in a reduction in mortality to 37%, which also decreased significantly to 20% at 12 months (p < 0.001), 16% at 24 months (p < 0.001), and 18% at 36 months (p < 0.001). A similar trend was observed with Royal Guard®, with the new mesh resulting in a 33% reduction in mortality, followed by a significant reduction to 21% at 12 months (p < 0.001), 17% at 24 months (p < 0.001) and 15% at 36 months (p < 0.001).
Reduction in fecundity of wild pyrethroid-resistant Anopheles gambiae mosquitoes entering an experimental hut in Kwa, Benin. Data from untreated controls and novel nets were pooled across trials to provide a single estimate of efficacy. Bars with common letters were not significantly different at the 5% level (p > 0.05) by logistic regression analysis. Error bars represent 95% confidence intervals.
Odds ratios describe the difference in fertility with new-generation mosquito nets compared with pyrethroid-only mosquito nets. The dotted line represents a ratio of 1, indicating no difference in fertility. Odds ratios < 1 indicate a greater reduction in fertility with new-generation nets. Data for new-generation mosquito nets were pooled across trials to produce a single estimate of effectiveness. Error bars represent 95% confidence intervals.
Post time: Feb-17-2025