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Long-lasting insecticidal nets (LLINs) are the primary malaria prevention tool, but their effectiveness is threatened by pyrethroid resistance. We embedded a pragmatic cluster-randomised trial into Uganda's national LLIN campaign to compare conventional LLINs with those containing piperonyl butoxide (PBO), a synergist that can partially restore pyrethroid susceptibility in mosquito vectors. 104 health sub-districts, from 48 districts in Uganda, were randomly assigned to LLINs with PBO (PermaNet 3.0 and Olyset Plus) and conventional LLINs (PermaNet 2.0 and Olyset Net) by proportionate randomisation using an iterative process. At baseline 6, 12, and 18 months after LLIN distribution, cross-sectional surveys were done in 50 randomly selected households per cluster (5200 per survey); a subset of ten households per cluster (1040 per survey) were randomly selected for entomological surveys. The primary outcome was parasite prevalence by microscopy in children aged 2–10 years, assessed in the as-treated population at 6, 12, and 18 months. This trial is registered with ISRCTN, ISRCTN17516395. LLINs were delivered to households from March 25, 2017, to March 18, 2018, 32 clusters were randomly assigned to PermaNet 3.0, 20 to Olyset Plus, 37 to PermaNet 2.0, and 15 to Olyset Net. In the as-treated analysis, three clusters were excluded because no dominant LLIN was received, and four clusters were reassigned, resulting in 49 PBO LLIN clusters (31 received PermaNet 3.0 and 18 received Olyset Plus) and 52 non-PBO LLIN clusters (39 received PermaNet 2.0 and 13 received Olyset Net). At 6 months, parasite prevalence was 11% (386/3614) in the PBO group compared with 15% (556/3844) in the non-PBO group (prevalence ratio [PR] adjusted for baseline values 0·74, 95% CI 0·62–0·87; p=0·0003). Parasite prevalence was similar at month 12 (11% vs 13%; PR 0·73, 95% CI 0·63–0·85; p=0·0001) and month 18 (12% vs 14%; PR 0·84, 95% CI 0·72–0·98; p=0·029). In Uganda, where pyrethroid resistance is high, PBO LLINs reduced parasite prevalence more effectively than did conventional LLINs for up to 18 months. This study provides evidence needed to support WHO's final recommendation on use of PBO LLINs. The Against Malaria Foundation, UK Department for International Development, Innovative Vector Control Consortium, and Bill and Melinda Gates Foundation.

Long-lasting insecticidal nets (LLINs) are the foundation of malaria control but resistance of mosquito vectors to pyrethroids threatens their effectiveness. We embedded a cluster-randomised trial into Uganda's 2017–18 campaign to distribute LLINs. LLINs with piperonyl butoxide (PBO) reduced parasite prevalence more effectively than conventional LLINs (without PBO) for 18 months. Here, we report the final 25-month survey results. LLINEUP was a cluster-randomised trial conducted in 48 districts in eastern and western Uganda. 104 health subdistricts (clusters) without ongoing or planned indoor residual spraying with pirimiphos-methyl (Actellic, Basel, Switzerland) were eligible for inclusion in the trial. Clusters were randomly assigned to PBO LLINs (PermaNet 3.0 or Olyset Plus) and conventional LLINs (PermaNet 2.0 or Olyset Net) with proportionate randomisation using STATA version 14.2. LLINs were delivered from March 25, 2017, to March 18, 2018. Between April 23, 2019, and Sept 13, 2019, community surveys were conducted in 50 randomly selected households per cluster; ten households per cluster were randomly selected for entomology surveys. Mosquitoes were collected in the morning from indoor surfaces of households using Prokopack aspirators. Due to COVID-19 restrictions, only 90 of the 104 clusters were surveyed at 25 months. The primary outcome was parasite prevalence by microscopy in children aged 2–10 years, assessed in the as-treated population, determined using the results from the 6-month household survey on the type of LLINs received in each cluster. This trial is registered with ISRCTN, ISRCTN17516395, and is now completed. In the as-treated analysis, two clusters were excluded (no predominant LLIN received) and four were reassigned; 40 PBO LLIN clusters (30 PermaNet 3.0, ten Olyset Plus) and 48 non-PBO LLIN (36 PermaNet 2.0, 12 Olyset Net) were included. Parasite prevalence was 17·1% (506 of 2958 participants) in the PBO group and 19·8% (701 of 3534) in the non-PBO group (prevalence ratio adjusted for baseline 0·80 [95% CI 0·69–0·93], p=0·0048). Comparing within-treatment group parasite prevalence to baseline, parasite prevalence ratios were lower in the PBO groups at all timepoints, but the difference was greatest at 6 months (PBO LLINs parasite prevalence at baseline 28·8% [1001 of 3472, 95% CI 27·3–30·4] vs at 6 months 12·0% [361 of 3009, 10·9–13·2], prevalence ratio [PR] 0·43 [95% CI 0·36–0·52], p<0·0001; non-PBO LLINs parasite prevalence at baseline 25·4% [1015 of 4004, 24·0–26·7] vs 6 months 14·8% [526 of 3551, 13·7–16·0], PR 0·60 [0·54–0·68], p<0·0001) and 25 months (PBO LLINs parasite prevalence at 25 months 17·1% [506 of 2958, 15·8–18·5], PR 0·63 [95% CI 0·57–0·71], p<0·0001; non-PBO LLINs parasite prevalence at 25 months 19·8% [701 of 3534, 18·5–21·2], PR 0·79 [0·73–0·86], p<0·0001). In Uganda, PBO LLINs outperformed pyrethroid-only LLINs for 25 months. WHO concluded that PBO LLINs are more effective against malaria than non-PBO LLINs when resistance to pyrethroids is high and issued a conditional recommendation suggesting PBO LLINs should be deployed in areas of pyrethroid resistance. The Against Malaria Foundation, UK Department for International Development, Innovative Vector Control Consortium, and Bill and Melinda Gates Foundation.

Background After decades of success in reducing malaria through the scale-up of pyrethroid long-lasting insecticidal nets (LLINs), the decline in the malaria burden has stalled, coinciding with the rapid spread of pyrethroid resistance. In a previously reported study, nets treated with a pyrethroid and a synergist, piperonyl butoxide (PBO), demonstrated superior efficacy compared to standard pyrethroid LLINs (std-LLINs) against malaria. Evidence was used to support the public health recommendation of PBO-Pyrethroid-LLIN by the World Health Organization in 2018. This study looks at the third year of rollout of these nets in Muleba district, Tanzania to inform whether policy guidelines need to be updated. Methods A four-group cluster randomized trial (CRT) using a two-by-two factorial design was carried out between January 2014 and December 2017. A total of 48 clusters, were randomized in a 1:1:1:1 ratio to the following treatment groups, each intervention being provided once in 2015: 1/std-LLIN; 2/PBO-pyrethroid LLIN; 3/std-LLIN + Indoor Residual Spraying (IRS) and 4/PBO-Pyrethroid-LLIN + IRS. During the third year follow-up, malaria infection prevalence in 80 children per cluster, aged 6 months to 14 years, was measured at 28- and 33-months post-intervention and analysed as intention-to-treat (ITT) and per protocol (PP). Mosquito collections were performed monthly in all clusters, using CDC light traps in 7 randomly selected houses per cluster. Results At 28 and 33 months, study net usage among household participants was only 47% and 31%, respectively. In ITT analysis, after 28 months malaria infection prevalence among 7471 children was 80.9% in the two std-LLIN groups compared to 69.3% in the two PBO-Pyrethroid-LLIN (Odds Ratio: 0.45, 95% Confidence Interval: 0.21–0.95, p-value: 0.0364). After 33 months the effect was weaker in the ITT analysis (prevalence 59.6% versus 49.9%, OR: 0.60, 95%CI:0.32–1.13, p-value: 0.1131) but still evident in the PP analysis (57.2% versus 44.2%, OR: 0.34, 95%CI: 0.16–0.71, p-value: 0.0051). Mean number of Anopheles per night collected per house was similar between PBO-Pyrethroid-LLIN groups (5.48) and std-LLIN groups (5.24) during the third year. Conclusions Despite low usage of PBO- Pyrethroid LLIN, a small impact of those nets on malaria infection prevalence was still observed in the 3rd year with the most protection offered to children still using them. To maximize impact, it is essential that net re-distribution cycles are aligned with this LLIN lifespan to maintain maximum coverage. Trial registration : The trial was registered with ClinicalTrials.gov (registration number NCT02288637).

Background Mass distributions of long-lasting insecticidal nets (LLINs) have contributed to large reductions in the malaria burden. However, this success is in jeopardy due in part to the increasing pyrethroid-resistant mosquito population as well as low LLINs coverage in various areas because the lifespan of LLINs is often shorter than the interval between replenishment campaigns. New insecticide-treated nets (ITNs) containing pyrethroid and piperonyl-butoxide (PBO) have shown a greater reduction in the incidence of malaria than pyrethroid LLINs in areas with pyrethroid-resistant mosquitoes. However, the durability (attrition, bio-efficacy, physical integrity and chemical retainment) of pyrethroid-PBO ITNs under operational settings has not been fully characterized. This study will measure the durability of pyrethroid-PBO ITNs to assess whether they meet the World Health Organization (WHO) three years of operational performance criteria required to be categorized as “long-lasting”. Methods A prospective household randomized controlled trial will be conducted simultaneously in Tanzania, India and Côte d’Ivoire to estimate the field durability of three pyrethroid-PBO ITNs (Veeralin®, Tsara® Boost, and Olyset® Plus) compared to a pyrethroid LLIN: MAGNet®. Durability monitoring will be conducted up to 36 months post-distribution and median survival in months will be calculated. The proportion of ITNs: (1) lost (attrition), (2) physical integrity, (3) resistance to damage score, (4) meeting WHO bio-efficacy (≥ 95% knockdown after 1 h or ≥ 80% mortality after 24 h for WHO cone bioassay, or ≥ 90% blood-feeding inhibition or ≥ 80% mortality after 24 h for WHO Tunnel tests) criteria against laboratory-reared resistant and susceptible mosquitoes, and insecticidal persistence over time will be estimated. The non-inferiority of Veeralin® and Tsara® Boost to the first-in-class, Olyset® Plus will additionally be assessed for mortality, and the equivalence of 20 times washed ITNs compared to field aged ITNs will be assessed for mortality and blood-feeding inhibition endpoints in the Ifakara Ambient Chamber Test, Tanzania. Conclusion This will be the first large-scale prospective household randomized controlled trial of pyrethroid-PBO ITNs in three different countries in East Africa, West Africa and South Asia, simultaneously. The study will generate information on the replenishment intervals for PBO nets.

Pyrethroid bednets treated with the synergist piperonyl butoxide (PBO) offer the possibility of improved vector control in mosquito populations with metabolic resistance. In 2017–2019, we conducted a large-scale, cluster-randomised trial (LLINEUP) to evaluate long-lasting insecticidal nets (LLINs) treated with a pyrethroid insecticide plus PBO (PBO LLINs), as compared to conventional, pyrethroid-only LLINs across 104 health sub-districts (HSDs) in Uganda. In LLINEUP, and similar trials in Tanzania, PBO LLINs were found to provide greater protection against malaria than conventional LLINs, reducing parasitaemia and vector density. In the LLINEUP trial, we conducted cross-sectional household entomological surveys at baseline and then every 6 months for two years, which we use here to investigate longitudinal changes in mosquito infection rate and genetic markers of resistance. Overall, 5395 female Anopheles mosquitoes were collected from 5046 households. The proportion of mosquitoes infected (PCR-positive) with Plasmodium falciparum did not change significantly over time, while infection with non- falciparum malaria decreased in An. gambiae s.s., but not An. funestus . The frequency of genetic markers associated with pyrethroid resistance increased significantly over time, but the rate of change was not different between the two LLIN types. The knock-down resistance ( kdr ) mutation Vgsc -995S declined over time as Vgsc -995F, the alternative resistance mutation at this codon, increased. Vgsc -995F appears to be spreading into Uganda. Distribution of LLINs in Uganda was previously found to be associated with reductions in parasite prevalence and vector density, but here we show that the proportion of infective mosquitoes remained stable across both PBO and non-PBO LLINs, suggesting that the potential for transmission persisted. The increased frequency of markers of pyrethroid resistance indicates that LLIN distribution favoured the evolution of resistance within local vectors and highlights the potential benefits of resistance management strategies. Trial registration : This study is registered with ISRCTN, ISRCTN17516395. Registered 14 February 2017, http://www.isrctn.com/ISRCTN17516395 .

Background The Dual-Active Ingredient long-lasting insecticidal nets (Dual-AI LLIN) have been developed to counteract the reduced efficacy of pyrethroid (PY)-only nets due to widespread pyrethroid insecticide resistance in malaria vector mosquitoes. They constitute half of the nets distributed in sub-Saharan Africa between 2022 and 2024. However, their effectiveness once they develop holes is unclear, particularly in pyrethroid-resistant settings. This study evaluates the textile integrity of three dual- AI LLINs compared to standard PY LLN, over 3 years of use in a community in Tanzania and the associated impact on malaria prevalence and incidence. Methods A secondary analysis of data from a randomized controlled trial (RCT) in North-western Tanzania was conducted to evaluate the effectiveness of α-cypermethrin only; pyriproxyfen and α-cypermethrin (PPF-PY); chlorfenapyr and α-cypermethrin (chlorfenapyr-PY); and the synergist piperonyl butoxide and permethrin (PBO-PY) LLINs on malaria infection prevalence and case incidence. The association between the net textile condition and 1/malaria prevalence over 3 years of use between 2019 and 2022, and 2/malaria case incidence in a cohort of children over 2 years of follow-up was assessed between 2019 and 2021. Results There was no significant association between damaged (OR 0.98, 95% CI 0.71–1.37, p-value  = 0.655) and too-torn (OR 1.07, 95% CI 0.77–1.47, p-value = 0.694) compared to intact nets on malaria prevalence for all net types. However, there were reduced rates of malaria case incidence in children sleeping under a net in good condition compared to too-torn nets (incidence rate ratio (IRR) 0.76 [95% CI 0.63–0.92], p = 0.005). Malaria incidence was also consistently lower in too-torn PBO-PY LLIN (IRR = 0.37 [95% CI 0.19–0.72], p = 0.003) and chlorfenapyr-PY LLIN (IRR = 0.45 [95% CI 0.33–0.97], p = 0.053) compared to an intact PY-only LLIN during the first year of follow up. In year 2, the incidence was only significantly lower in intact chlorfenapyr-PY LLIN (IRR = 0.49 [95% CI 0.29–0.81], p = 0.006) compared to intact PY LLIN. Conclusion The study confirmed that sleeping under a chlorfenapyr-PY LLIN or PBO-PY LLIN offered superior protection to pyrethroid-only nets even when torn. Preventing the development of holes is essential as they impact the level of protection offered against malaria infection. Trial registration : ClinicalTrials.gov, number (NCT03554616)

Background Long-lasting insecticide nets (LLINs) are a core malaria intervention. LLINs should retain efficacy against mosquito vectors for a minimum of three years. Efficacy and durability of Olyset ® Plus, a permethrin and piperonyl butoxide (PBO) treated LLIN, was evaluated versus permethrin treated Olyset ® Net. In the absence of WHO guidelines of how to evaluate PBO nets, and considering the manufacturer’s product claim, Olyset ® Plus was evaluated as a pyrethroid LLIN. Methods This was a household randomized controlled trial in a malaria endemic rice cultivation zone of Kirinyaga County, Kenya between 2014 and 2017. Cone bioassays and tunnel tests were done against Anopheles gambiae Kisumu. The chemical content, fabric integrity and LLIN survivorship were monitored. Comparisons between nets were tested for significance using the Chi-square test. Exact binomial distribution with 95% confidence intervals (95% CI) was used for percentages. The WHO efficacy criteria used were ≥ 95% knockdown and/or ≥ 80% mortality rate in cone bioassays and ≥ 80% mortality and/or ≥ 90% blood-feeding inhibition in tunnel tests. Results At 36 months, Olyset ® Plus lost 52% permethrin and 87% PBO content; Olyset ® Net lost 24% permethrin. Over 80% of Olyset ® Plus and Olyset ® Net passed the WHO efficacy criteria for LLINs up to 18 and 12 months, respectively. At month 36, 91.2% Olyset ® Plus and 86.4% Olyset ® Net survived, while 72% and 63% developed at least one hole. The proportionate Hole Index (pHI) values representing nets in good, serviceable and torn condition were 49.6%, 27.1% and 23.2%, respectively for Olyset ® Plus, and 44.9%, 32.8% and 22.2%, respectively for Olyset ® Net but were not significantly different. Conclusions Olyset ® Plus retained efficacy above or close to the WHO efficacy criteria for about 2 years than Olyset ® Net (1–1.5 years). Both nets did not meet the 3-year WHO efficacy criteria, and showed little attrition, comparable physical durability and survivorship, with 50% of Olyset ® Plus having good and serviceable condition after 3 years. Better community education on appropriate use and upkeep of LLINs is essential to ensure effectiveness of LLIN based malaria interventions. Graphical Abstract

Insecticide resistance poses a serious threat to insecticide-based interventions in Africa. There is a fear that resistance escalation could jeopardize malaria control efforts. Monitoring of cases of aggravation of resistance intensity and its impact on the efficacy of control tools is crucial to predict consequences of resistance. The resistance levels of an Anopheles funestus population from Palmeira, southern Mozambique, were characterized and their impact on the efficacy of various insecticide-treated nets established. A dramatic loss of efficacy of all long-lasting insecticidal nets (LLINs), including piperonyl butoxide (PBO)-based nets (Olyset Plus), was observed. This An. funestus population consistently (2016, 2017, and 2018) exhibited a high degree of pyrethroid resistance. Molecular analyses revealed that this resistance escalation was associated with a massive overexpression of the duplicated cytochrome P450 genes CYP6P9a and CYP6P9b, and also the fixation of the resistance CYP6P9a_R allele in this population in 2016 (100%) in contrast to 2002 (5%). However, the low recovery of susceptibility after PBO synergist assay suggests that other resistance mechanisms could be involved. The loss of efficacy of pyrethroid-based LLINs with and without PBO is a concern for the effectiveness of insecticide-based interventions, and action should be taken to prevent the spread of such super-resistance.

Long-lasting insecticidal nets (LLINs) are vital for malaria control in sub-Saharan Africa, but their durability is challenged by fabric decay and pyrethroid resistance. This study assessed the physical integrity and bioefficacy of piperonyl butoxide-LLINs (PBO-LLINs) and pyrethroid-only LLINs (pyrethroid-LLINs) after 1.5 years of use in western Kenya, where resistance is widespread. A survey on net integrity and insecticide efficacy was conducted in randomly selected households (101-107 per group per visit) from three villages per net type group in Muhoroni Sub-County, Kisumu County. Physical integrity surveys were done after every six months while residual bio-efficacy was after every three months for 18 months. Physical integrity and residual bio-efficacy studies were conducted following WHO guidelines. PBO-LLINs exhibited higher physical integrity than pyrethroid-LLINs over time. At 18 months, 45.2% (61/135) of pyrethroid-LLINs and 21.8% (31/142) of PBO-LLINs were torn, with pHI values of 2494.1 ± 1696.4 and 1618.6 ± 1056.7, respectively. Net type, net age and house wall structures significantly influenced net integrity (p < 0.05). Torn nets were significantly more common in pyrethroid-LLIN households with mud-unplastered [OR=5.323 (95% CI = 1.685-16.816), p = 0.004] and corrugated iron walls [OR=6.31 (95% CI = 2.10-18.93), p < 0.001] and in PBO-LLIN households with mud-unplastered walls [OR=9.823 (95% CI = 1.487-64.898), p = 0.018]. Against the Kisumu susceptible Anopheles gambiae s.s, both net types decreased in mortality at baseline (when new) from 97.6% to 18.4% and 98.6% to 18.5% for pyrethroid and PBO-LLINs respectively at 18 months. Against a Bungoma pyrethroid-resistant Anopheles gambiae s.s, mosquito mortality with pyrethroid-LLINs declined from 36.9% when new to 6.8% at 18 months, while PBO-LLINs dropped from 55.6% to 11.8%. Both physical integrity and bioefficacy of LLINs declined significantly within 18 months. The findings demonstrate that not all nets in the field offer maximum protection by this time point, calling for net care education and further evaluation of PBO-LLINs especially in pyrethroid-resistant regions.

Pirimiphos-methyl is a pro-insecticide requiring activation by mosquito cytochrome P450 enzymes to induce toxicity while PBO blocks activation of these enzymes in pyrethroid-resistant vector mosquitoes. PBO may thus antagonise the toxicity of pirimiphos-methyl IRS when combined with pyrethroid-PBO ITNs. The impact of combining Olyset Plus and PermaNet 3.0 with Actellic 300CS IRS was evaluated against pyrethroid-resistant Anopheles gambiae s.l. in two parallel experimental hut trials in southern Benin. The vector population was resistant to pyrethroids and PBO pre-exposure partially restored deltamethrin toxicity but not permethrin. Mosquito mortality in experimental huts was significantly improved in the combinations of bendiocarb IRS with pyrethroid-PBO ITNs (33–38%) compared to bendiocarb IRS alone (14–16%, p < 0.001), demonstrating an additive effect. Conversely, mortality was significantly reduced in the combinations of pirimiphos-methyl IRS with pyrethroid-PBO ITNs (55–59%) compared to pirimiphos-methyl IRS alone (77–78%, p < 0.001), demonstrating evidence of an antagonistic effect when both interventions are applied in the same household. Mosquito mortality in the combination was significantly higher compared to the pyrethroid-PBO ITNs alone (55–59% vs. 22–26% p < 0.001) showing potential of pirimiphos-methyl IRS to enhance vector control when deployed to complement pyrethroid-PBO ITNs in an area where PBO fails to fully restore susceptibility to pyrethroids.