Abstract The effectiveness of malaria vector control is increasingly threatened by widespread insecticide resistance, particularly to pyrethroids. In response, the World Health Organization recommends dual-active ingredients (dual-A.I.) insecticide-treated nets (ITNs) in resistant settings. This study evaluated the efficacy of several dual-A.I. ITNs in two experimental hut trial sites an area with high insecticide resistant malaria vectors in Tanzania with differing vector species composition and bionomics. Five ITNs were evaluated: a pyrethroid-only net (MAGNet®), three pyrethroid-piperonyl butoxide (PBO) nets (OlysetTM Plus, Veeralin®, and PermaNet® 3.0), one dual-A.I. net (Interceptor® G2; chlorfenapyr + alpha-cypermethrin), and an untreated control net. Mosquitoes were collected from huts, morphologically identified, and monitored for mortality up to 72 h. WHO cone and tunnel tests using susceptible An. gambiae s.s. were conducted for quality control, while field-collected mosquitoes were used for bio-efficacy monitoring. Molecular species identification was performed using PCR assays, and genomic DNA was extracted from An. funestus s.l. and An. gambiae s.l. collected from the trials. All ITNs met WHO bio-efficacy criteria in cone and tunnel tests. An. gambiae s.l. predominated in Mwagagala (77%) but was rare in Magu (4%), where An. funestus complex was dominant. In Magu, Interceptor® G2 and Veeralin® achieved significantly higher mortality than MAGNet® at 24 h (53% and 45% vs 24%) and 72 h (61% and 53% vs 24%). In contrast, no significant mortality differences were observed among ITNs in Mwagagala. High resistance intensity was detected in both vector groups (mortality <20% in bioassays). In An. funestus from Magu, the 4.3 kb-SV resistant genotype was fixed, while G454A-CYP9K1 resistance was highly prevalent (RR: 0.70– 1.00). Interceptor® G2 and Veeralin® outperformed the pyrethroid-only reference net against An. funestus complex but not against predominantly An. arabiensis populations.

Over-reliance on pyrethroid insecticides in insecticide-treated nets (ITNs) has imposed significant selection pressure for the evolution of insecticide resistance among major malaria vector species. In the Democratic Republic of Congo (DRC), the country with the second highest malaria burden globally, pyrethroid resistance is pervasive, but there is a paucity of information regarding the molecular mechanisms driving resistance. A clear understanding of the specificity of resistance mechanisms to individual insecticides and the likelihood of selecting for cross-resistance mechanisms is crucial for the development of new vector control tools. Anopheles gambiae s.l. populations from eight study sites across the DRC were phenotyped for resistance to alpha-cypermethrin, deltamethrin and permethrin, with and without pre-exposure to the synergist piperonyl butoxide (PBO), followed by multiplex amplicon sequencing. Phenotypic pyrethroid resistance and loss of PBO synergy was confirmed in all sites across the DRC. In An. gambiae s.s. four non-synonymous SNPs which have been previously associated with insecticide resistance were detected: gste2-L119V, vgsc-L995F, vgsc-L995S and rdl-A296G, while three were novel: gste2-T154S, ace1-N246T and ace1-P265L. Nationwide geographical trends in insecticide resistance mutation distribution, prevalence and selection were evident. In the West, near fixation of vgsc-L995F and almost complete absence of vgsc-L995S was observed, alongside low-moderate frequencies of rdl-A296G and gste2-L119V. Further East, the converse was apparent. Gste2-L119V was significantly associated with resistance to deltamethrin following PBO-pre-exposure, warranting functional validation to determine its putative role in reduced PBO synergy. Furthermore, gste2-T154S was implicated in deltamethrin and permethrin resistance but susceptibility to alpha-cypermethrin after PBO pre-exposure. Study findings comprise the most comprehensive overview of the prevalence of genetic markers of Anopheles insecticide resistance across the DRC and provide an important baseline for improved malaria vector control and the design of proactive insecticide resistance management strategies. Given the significant scale up in PBO-ITNs, with more than 58% of all ITNs delivered to sub-Saharan Africa in 2023 containing PBO, there is an urgent need to identify novel molecular markers to monitor changes in PBO synergy, which may be predicative of loss of intervention operational efficacy.

The presence of a community effect in cluster randomized trials of malaria vector control interventions has led to the implementation of “buffer zones” around clusters to limit the potential for contamination between interventions. No consensus has been reached on how large these buffers need to be to encapsulate the effect. Nested within a phase-III cluster randomized malaria vector control trial in Northwest Tanzania, this study aims to determine the presence and spatial range of community effects from long-lasting insecticidal net (LLIN) and indoor residual spraying (IRS) interventions on household-level malaria infection in trial clusters four months post-intervention. Effective spatial range estimates of intervention community effects were compared to the 300m buffer distance implemented to limit intervention spillover between clusters in the trial. Geographically-weighted adjusted odds of malaria infection in children aged 0.5–14 years were determined four months post community-level intervention with a randomized allocation comprising one of two LLIN products (OlysetTM LN: 1000mg/m2 permethrin or OlysetTM Plus LN: 400 + permethrin 800mg/m2) with either IRS (Actellic®300CS: 1000mg/m2 micro-encapsulated pirimiphos-methyl) or no IRS. Robust semivariances were calculated for each of 48 intervention clusters and fit to semivariogram models by Weighted Least Squares. 6440 children from 2785 households were included in the geographically-weighted logistic regression. Prevalence of Plasmodium falciparum infection was 45.9

The World Health Organization (WHO) recommends dual-active ingredient long-lasting insecticidal nets (dual-AI LLINs) for protection against malaria in areas with insecticide resistance. The effectiveness of LLINs, however, depends on user compliance, influenced by community perceptions of malaria, prevention methods, and the acceptability of LLINs. Understanding these factors is essential for the success of large-scale implementation. This study was part of the two cluster-randomized controlled trials (RCTs) evaluating the efficacy of dual-AI LLINs on malaria indicators in Muleba and Misungwi districts, Tanzania. Polyethylene and polyester rectangular LLINs were distributed in Muleba (Olyset Plus and Olyset Net) in 2015 and in Misungwi (Olyset Plus, Royal Guard, Interceptor G2, and Interceptor) in 2019. A mixed-methods approach was used to assess users’ acceptability, preferences, and perceptions, and identifying barriers to consistent use. Quantitative data were collected from 14,475 households, while qualitative data came from 36 focus group discussions and 44 in-depth interviews. A thematic analysis was applied using a deductive approach, guided by the study’s conceptual framework. Descriptive statistics were used for quantitative analysis. LLIN usage and acceptability were influenced by their availability in the households, the physical integrity, side-effects, nuisance from mosquito bites and perceived malaria risk. Olyset Plus was slightly favoured over Olyset net in Muleba due to the perception that the insecticide had a stronger effect (72

Abstract Background Long-lasting insecticidal nets (LLINs) were once fully effective for the prevention of malaria; however, mosquitoes have developed resistance to pyrethroids, the main class of insecticides used on nets. Dual active ingredient LLINs (dual-AI LLINs) have been rolled out as an alternative to pyrethroid (PY)-only LLINs to counteract this. Understanding the minimum community usage at which these LLINs elicit an effect that also benefits non-users against malaria infection is important. Methods We conducted a secondary analysis of a 3-year randomized controlled trial (RCT) in 84 clusters in North-western Tanzania to evaluate the effectiveness of three dual-AI LLINs: pyriproxyfen and alpha(α)-cypermethrin, chlorfenapyr and α-cypermethrin, and the piperonyl-butoxide (PBO) and permethrin compared to α-cypermethrin only LLINs. We measured malaria infection prevalence using 5 cross-sectional surveys between 2020 and 2022. We assessed net usage at the cluster level and malaria infection in children aged from 6 months to 14 years in 45 households per cluster. The trial was registered as a clinical trial on www.clinicaltrials.gov: ClinicalTrials.gov (NCT03554616) on 2018-06-13. Results A total of 22,479 children from 12,654 households were tested for malaria using rapid diagnostic tests in January 2020, 2021, & 2022 and July 2020 & 2021. Among non-users, community-level usage of > 40% of dual-AI LLIN was significantly associated with protection against malaria infection: chlorfenapyr arm (OR: 0.44 (95% CI: 0.27–0.71), p = 0.0009), PBO arm (OR: 0.55 (95% CI: 0.33–0.94), p = 0.0277) and pyriproxyfen arm (OR: 0.61 (95% CI: 0.37–0.99), p = 0.0470) compared with non-users in clusters with > 40% usage of pyrethroid-only LLINs. There were indications of some protection against malaria infection to non-users in the chlorfenapyr arm when community-level usage was ≤ 40% (OR: 0.65 (95% CI: 0.42–1.01), p = 0.0528) compared to those living in clusters with > 40% usage of pyrethroid-only LLINs. Conclusion Our study demonstrated that at a community usage of 40% or more of dual-AI LLINs non-users benefited from the presence of these nets. Noticeably, even when usage was ≤ 40% in the chlorfenapyr arm, non-users were better protected than non-users in the higher coverage pyrethroid-only arm. The greater difference in malaria risk observed between users and non-users indicates that LLINs play a crucial role in providing personal protection against malaria infection for the people using the net.

Pyrethroid-treated long-lasting insecticidal nets (LLINs) have been the main contributor to the reduction in malaria in the past two decades in sub-Saharan Africa. The development of pyrethroid insecticide resistance threatens the future of LLINs, especially when nets become holed and pyrethroid decays. In this study, three new classes of dual-active ingredient (AI) LLINs were evaluated for their physical durability: (1) Royal Guard, combining pyriproxyfen, which disrupts female fertility, and a pyrethroid, alpha-cypermethrin; (2) Interceptor G2, which combines the pyrrole chlorfenapyr and a pyrethroid (alpha-cypermethrin); (3) Olyset Plus, which incorporates the pyrethroid permethrin and the synergist piperonyl butoxide, to enhance the pyrethroid potency; and Interceptor, a reference net that contains alpha-cypermethrin as the sole active ingredient. About 40,000 nets of each type were distributed in February 2019 to different villages in Misungwi. A total of 3072 LLINs were followed up every 6–12 months up to 36 months to assess survivorship and fabric integrity. The median functional survival was less than three years with Interceptor, Interceptor G2, and Royal Guard showing 1.9 years each and Olyset Plus showing 0.9 years. After 36 months, 90% of Olyset Plus and Royal Guard and 87% of Interceptor G2 were no longer in use (discarded) due to wear and tear, compared to 79% for Interceptor. All dual-AI LLINs exhibited poor textile durability, with Olyset Plus being the worst.

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. 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. There was no significant association between damaged (OR 0.98, 95

New classes of long-lasting insecticidal nets (LLINs), have been recommended by the World Health Organization (WHO) to control malaria vectors resistant to pyrethroid insecticides. This study was nested in a large-scale cluster-randomized controlled trial conducted (cRCT) in Tanzania. A series of experimental hut trials (EHTs) aimed to evaluate the bio-efficacy of trial LLINs on mosquito indicators most pertinent to malaria transmission over 3 years of use in the community in order to better understand the outcomes of the cRCT. The following field-collected LLINs were assessed: 1) Olyset Plus (combining piperonyl butoxide synergist and permethrin), 2) Interceptor G2 (chlorfenapyr and alpha-cypermethrin), 3) Royal Guard (pyriproxyfen and alpha-cypermethrin), 4) Interceptor (alpha-cypermethrin only) conducted in parallel with 5) a new Interceptor, and 6) an untreated net. Thirty nets of each type were withdrawn from the community at 12, 24, and 36 months after distribution and used for the EHTs. Pre-specified outcomes were 72-hour mortality for Interceptor G2, 24-hour mortality for Olyset Plus, and fertility based on egg development stage for Royal Guard. Overall, Interceptor G2 LLINs induced higher 72-hour mortality compared to standard LLINs of the same age up to12 months (44% vs 21%, OR: 3.5, 95% CI: 1.9-6.6, p-value < 0.001), and 24-hour mortality was only significantly higher in Olyset Plus when new (OR: 13.6, 95%CI: 4.4-41.3, p-value < 0.001) compared to standard LLINs but not at 12 months (17% vs 13%; OR: 2.1, 95% CI: 1.0-4.3; p-value = 0.112). A small, non-significant effect of pyriproxyfen on Anopheles fertility was observed for Royal Guard up to 12 months (75% vs 98%, OR: 1.1, 95% CI: 0.0-24.9, p-value = 0.951). There was no evidence of a difference in the main outcomes for any of the new classes of LLINs at 24 and 36 months compared to standard LLINs. Interceptor G2 LLINs showed superior bio-efficacy compared to standard LLINs only up to 12 months, and the effect of Olyset Plus was observed when new for all species and 12 months for An. gambiae s.l. only. The pyriproxyfen component of Royal Guard had a short and limited effect on fertility. The decrease in effectiveness of Olyset Plus and Royal Guard LLINs in the EHTs aligns with findings from the cRCT, whereas efficacy of Interceptor G2 lasted for a longer period in the cRCT compared to the EHT. Further investigations are needed to understand the complete scope of chlorfenapyr mode of action. Additional EHT in various contexts will help confirm the residual efficacy of the dual active ingredient LLINs and support the development of longer-lasting nets.

BACKGROUND:Malaria continues to kill approximately 650 000 people each year. There is evidence that some second-generation insecticide-treated nets, which combine insecticide formulations with different modes of action, are protective against malaria while the nets are new; however, evidence for their impact over 3 years is scarce. In this study, we report the third-year results of a cluster-randomised controlled trial assessing the long-term effectiveness of dual-active ingredient long-lasting insecticidal nets (LLINs). METHODS:This is a secondary analysis of a cluster-randomised controlled trial, carried out between May 23, 2019, and April 30, 2023, in southern Benin. Restricted randomisation was used to assign 60 clusters (villages or groups of villages with a minimum of 100 households) to the three study groups (1:1:1) to evaluate the efficacy of pyriproxyfen-pyrethroid LLINs and chlorfenapyr-pyrethroid LLINs compared with pyrethroid-only LLINs (reference) against malaria transmission. The study staff and communities were masked to the group allocation. The primary outcome was malaria incidence measured over the third year after LLIN distribution, in a cohort of children aged 6 months to 9 years at the time of enrolment, in the intention-to-treat population. Here, we present the data of the third year post-LLIN distribution. The trial was registered with ClinicalTrials.gov, NCT03931473. FINDINGS:Study net use declined over the 3 years and was consistently lowest in the pyriproxyfen-pyrethroid LLIN group (at 36 months: 889 [39·4%] of 2257 participants vs 1278 [52·2%] of 2450 participants for the chlorfenapyr-pyrethroid LLIN group and 1400 [57·6%] of 2430 participants for the pyrethroid-only LLIN group). The cohort of children for the third year of follow-up (600 per group) were enrolled between April 9 and 30, 2022. Mean malaria incidence during the third year after distribution was 1·19 cases per child-year (95% CI 1·09-1·29) in the pyrethroid-only LLIN reference group, 1·21 cases per child-year (1·12-1·31) in the pyriproxyfen-pyrethroid LLIN group (hazard ratio [HR] 1·02, 95% CI 0·71-1·44; p=0·92), and 0·96 cases per child-year (0·88-1·05) in the chlorfenapyr-pyrethroid LLIN group (HR 0·80, 0·56-1·17; p=0·25). No adverse events related to study nets were reported by participants. INTERPRETATION:During the third year, as was also observed during the first 2 years, the pyriproxyfen-pyrethroid LLIN group did not have superior protection against malaria cases compared with the standard LLIN group. In the third year, people living in the chlorfenapyr-pyrethroid LLIN group no longer benefited from greater protection against malaria cases and infections than those living in the pyrethroid-only LLIN group. This was probably influenced by lower study net use than previous years and the declining concentration of partner insecticides in the nets. FUNDING:UNITAID, The Global Fund. TRANSLATION:For the French translation of the abstract see Supplementary Materials section.

Background: The extent to which insecticide resistance is affecting malaria vector control in community and home is not fully understood. This study assessed the implications of insecticide resistance for entomological efficacy of LLINs against wild free-flying field Anopheles gambiae s.l. and Anopheles funestuss.l. in experimental hut trials (EHT) in NE Tanzania before and after the evolution of pyrethroid-resistance. Methods: Evaluations of LLIN efficacy were conducted following World Health Organization (WHO) guidelines in a series of ten trials commissioned by WHO between 2006 and 2017, before and after resistance development.The evaluation criteria were based on mortality, blood feeding inhibition, induced exophily, personal protection, mass killing effect and deterrence. WHO bioassay methods were used to detect resistance while PCR molecular diagnostics detected resistance alleles and identified mosquitoes to species. Results: Anopheline mosquitoes were fully susceptible to pyrethroids until 2010-2013 when they began to show resistance. The VGSC L1014S point mutation kdr was detected in An. gambiae s.s. at the allelic frequency of 47%. Synergist tests with PBO restored efficacy, indicating added involvement of metabolic mechanisms. Meta-analysis of the 10 trials showed that mortality of susceptible An gambiae s.l.was 6.7 and 5.2-fold greater on zero-times (z = 6.6, p = 0.001) and 20-times washed LLINs (z = 2.3, p = 0.023) than on resistant An gambiae s.l. The mortality of unwashed and washed LLINs against susceptible An funestus s.l. was 3.3 (z = 2.8, p = 0.004) and 2.6 (z = 2.9, p = 0.004) fold greater than on resistant An funestus s.l. Resistant An gambiae s.l. were more likely to exit the huts as compared to susceptible An gambiae s.l. (z = 2.79, p = 0.005). The transition from susceptibility to resistance on changes to blood-feeding rates was non-significant for either species. Conclusions: Reduced mortality induced by LLINs after selection of pyrethroid resistance indicates that resistance may undermine household and community control of vector populations. Personal protection parameters seemed less affected by the transition to resistance. Meta-analysis, comparing the same net brands before and after selection of resistance, reveal which control parameters seem most affected by resistance.

Next-generation insecticide-treated bed nets (ITNs) combining two insecticides or an insecticide with a synergist are vital in combating malaria, especially in areas with pyrethroid-resistant mosquitoes where standard pyrethroid long-lasting insecticidal net (LLIN) may be less effective. A community durability study was conducted in Misungwi, Tanzania, during a cluster randomised controlled trial. This study assessed the bio-efficacy of three net brands combining a pyrethroid insecticide and either a synergist PBO for Olyset Plus, or a second insecticide pyriproxyfen for Royal Guard, and chlorfenapyr for Interceptor G2 over three years. These nets were compared to Interceptor, a standard pyrethroid-only net. A total of 1950 nets were enrolled across 10 clusters in each treatment arm. Thirty nets per type were collected every 6 months up to 30 months, with 50 nets sampled at 36 months. WHO cone bioassays and tunnel tests were performed at 0, 12, 24, 30 and 36 months. Both susceptible An. gambiae (s.s.) Kisumu strain and resistant An. gambiae (s.s.) Muleba-Kis strain were exposed. Over 80% of nets tested against the susceptible Kisumu strain met the WHO criteria after three years of community use. In tunnel tests, mortality (72 h) of the resistant Anopheles varied between 52% and 20%, in Interceptor G2 and was higher than standard Interceptor net up to 24 months. Olyset Plus mortality (24 h) ranged between 84% and 33% in tunnel tests with superior efficacy compared to Interceptor at 0, 24 and 36 months. Sterility effects in Royal Guard were higher when these nets were new and at six months but decreased to less than 10% after 12 months. Royal Guard consistently induced higher mortality compared to Interceptor up to 30 months while next-generation ITNs demonstrated higher efficacy in terms of mortality compared to standard LLINs against resistant strains; this superior bio-efficacy did not persist for the full three years. The impact of active ingredient (dual-AI) and PBO diminished relatively quickly. Aside from the initial period when the nets were new, the differences in mortality for Interceptor G2 and Olyset Plus and in sterility for Royal Guard, compared to the standard LLINs, were relatively small thereafter.

BACKGROUND:Insecticide treated nets (ITNs) are the most important malaria prevention tool in Africa but the rise of pyrethroid resistance in mosquitoes is likely impeding control. WHO has recommended a novel pyrethroid-pyrrole ITN following evidence of epidemiological benefit in two cluster-randomised, controlled trials (CRTs). It remains unclear how effective more costly pyrethroid-pyrrole ITNs are compared with other tools, or whether they should be deployed when budgets are limited. We aimed to compare the epidemiological impact and cost-effectiveness of the mass distribution of pyrethroid-pyrrole ITNs relative to other ITNs over 3 years in different African settings. METHODS:In this individual-based malaria transmission dynamics modelling study we characterise the entomological impact of ITNs using data from a systematic review of experimental hut trials from across Africa. This African entomological data was used to inform an individual-based malaria transmission dynamics model, which was validated against CRT results from Benin and Tanzania. The full impact of new ITNs was quantified for trial sites and simulation was used to project impact in different settings which were included within an accessible interface (the Malaria Intervention Tool) to support National Malaria Programmes to explore how vector control tools and budgets could be allocated across regions to avert the most cases. FINDINGS:The model projects that distributing pyrethroid-pyrrole ITNs averted 65% (95% credible interval 48-74) of cases over 3 years in Tanzania, and 75% (28-93) in Benin. The model indicates that trials might have underestimated the benefits of switching ITNs by 12-16% over 3 years because participants stopped using trial-allocated nets. In moderate endemicity non-trial settings, pyrethroid-pyrrole ITNs are projected to reduce malaria prevalence by 25-60% and switching from pyrethroid-only ITNs is probably highly cost-effective in most locations given current prices, averting an additional 10-30% of cases. INTERPRETATION:The benefit of pyrethroid-pyrrole ITNs varies by setting but is generally the most cost-effective indoor vector control intervention in Africa. National Malaria Programmes can strategise deployment to maximise impact. Entomological data could broadly predict epidemiological impact, although there are some inconsistencies, illustrating the challenge in capturing the dynamics across diverse settings. FUNDING:Unitaid, Bill & Melinda Gates Foundation, the UK Medical Research Council, Wellcome Trust, and the UK Foreign, Commonwealth & Development Office.

Background: Insecticide treated nets (ITNs) are the most important malaria prevention in Africa but the rise of pyrethroid resistance in mosquitoes is likely impeding control. The World Health Organisation have recommended a novel pyrethroid-pyrrole ITN following evidence of epidemiological benefit in cluster-randomised control trials (cRCTs) in Tanzania and Benin. It remains unclear how effective these more costly pyrethroid-pyrrole ITNs are compared to other tools and there is a need for guidance on use when budgets are limited. Methods: Entomological data are collated from across Africa to inform a malaria transmission dynamics model which is validated against cRCT results. The full impact of new ITNs is quantified for trial sites and simulations for different settings are included within a user-friendly interface MINT (https://mint.dide.ic.ac.uk/) to allow National Malaria Programmes (NMPs) to explore local impact and how budgets should be allocated across regions to avert most cases. Findings: The model projects that distributing pyrethroid-pyrrole ITNs averted 65% of cases over three-years in Tanzania, and 60% in Benin. It indicates that both cRCTs underestimated the benefit of switching to pyrethroid-pyrrole ITNs by at least 6-12%, as participants stopped using allocated nets. In non-trial settings pyrethroid-pyrrole ITNs are projected to reduce malaria prevalence by 20-65% over three-years and that switching from pyrethroid-only ITNs could avert an additional 10-30% more cases depending on where they were deployed. Interpretation: Pyrethroid-pyrrole ITNs are likely to be the most cost-effective malaria control intervention in Africa and NMPs can strategise deployment to maximise impact where budgets are restricted. Funding: Funding from the Bill & Melinda Gates Foundation (OPP1200155), Innovative Vector Control Consortium through the New Nets Project funded by Unitaid, and a UKRI Future Leaders Fellowship to ES-S from the Medical Research Council (MR/T041986/1). GSC was supported by the Wellcome Trust (220900/Z/20/Z) and TSC, IS, AH, DPD, JC, PW, GC, ER, RF and ES-S received support from the MRC Centre for Global Infectious Disease Analysis (MR/R015600/1), jointly funded by the UK Medical Research Council (MRC) and the UK Foreign, Commonwealth & Development Office (FCDO), under the MRC/FCDO Concordat agreement, the EDCTP2 programme supported by the European Union, and Community Jameel.Declaration of Interest: None.

BACKGROUND:New classes of long-lasting insecticidal nets (LLINs) containing two active ingredients have been recently recommended by WHO in areas where malaria vectors are resistant to pyrethroids. This policy was based on evidence generated by the first 2 years of our recently published trial in Tanzania. In this Article, we report the final third-year trial findings, which are necessary for assessing the long-term effectiveness of new classes of LLIN in the community and the replacement intervals required. METHODS:A third year of follow-up of a four-arm, single-blind, cluster-randomised controlled trial of dual active ingredient LLINs was conducted between July 14, 2021, and Feb 10, 2022, in Misungwi, Tanzania. Restricted randomisation was used to assign 84 clusters to the four LLIN groups (1:1:1:1) to receive either standard pyrethroid (PY) LLINs (reference), chlorfenapyr-PY LLINs, pyriproxyfen-PY LLINs, or piperonyl butoxide (PBO)-PY LLINs. All households received one LLIN for every two people. Data collection was done in consenting households in the cluster core area with at least one child between 6 months and 15 years of age who permanently resided in the selected household. Exclusion criteria were householders absent during the visit, living in the cluster buffer area, no adult caregiver capable of giving informed consent, or eligible children who were severely ill. Field staff and study participants were masked to allocation, and those analysing data were not. The primary 24-month endpoint was reported previously; here, we present the secondary outcome, malaria infection prevalence in children at 36 months post LLIN distribution, reported in the intention-to-treat analysis. The trial was registered with ClinicalTrials.gov (NCT03554616) and is now complete. FINDINGS:Overall usage of study nets was 1023 (22·3%) of 4587 people at 36 months post distribution. In the standard PY LLIN group, malaria infection was prevalent in 407 (37·4%) of 1088 participants, compared with 261 (22·8%) of 1145 in the chlorfenapyr-PY LLIN group (odds ratio 0·57, 95% CI 0·38-0·86; p=0·0069), 338 (32·2%) of 1048 in the PBO-PY LLIN group (0·95, 0·64-1·42; p=0·80), and 302 (28·8%) of 1050 in the pyriproxyfen-PY LLIN group (0·82, 0·55-1·23; p=0·34). None of the participants or caregivers reported side-effects. INTERPRETATION:Despite low coverage, the protective efficacy against malaria offered by chlorfenapyr-PY LLINs was superior to that provided by standard PY LLINs over a 3-year LLIN lifespan. Appropriate LLIN replacement strategies to maintain adequate usage of nets will be necessary to maximise the full potential of these nets. FUNDING:Department for International Development, UK Medical Research Council, Wellcome Trust, Department of Health and Social Care, and Bill & Melinda Gates Foundation via the Innovative Vector Control Consortium.

BACKGROUND:The optimal dosing of primaquine to prevent relapsing Plasmodium vivax malaria in South Asia remains unclear. We investigated the efficacy and safety of different primaquine regimens to prevent P. vivax relapse. METHODS:A systematic review identified P. vivax efficacy studies from South Asia published between 1 January 2000 and 23 August 2021. In a one-stage meta-analysis of available individual patient data, the cumulative risks of P. vivax recurrence at day 42 and 180 were assessed by primaquine total mg/kg dose and duration. The risk of recurrence by day 180 was also determined in a two-stage meta-analysis. Patients with a >25% drop in haemoglobin to <70 g/L, or an absolute drop of >50 g/L between days 1 and 14 were categorised by daily mg/kg primaquine dose. RESULTS:In 791 patients from 7 studies in the one-stage meta-analysis, the day 180 cumulative risk of recurrence was 61.1% (95% CI 42.2% to 80.4%; 201 patients; 25 recurrences) after treatment without primaquine, 28.8% (95% CI 8.2% to 74.1%; 398 patients; 4 recurrences) following low total (2 to <5 mg/kg) and 0% (96 patients; 0 recurrences) following high total dose primaquine (≥5 mg/kg). In the subsequent two-stage meta-analysis of nine studies (3529 patients), the pooled proportions of P. vivax recurrences by day 180 were 12.1% (95% CI 7.7% to 17.2%), 2.3% (95% CI 0.3% to 5.4%) and 0.7% (95% CI 0% to 6.1%), respectively. No patients had a >25% drop in haemoglobin to <70 g/L. CONCLUSIONS:Primaquine treatment led to a marked decrease in P. vivax recurrences following low (~3.5 mg/kg) and high (~7 mg/kg) total doses, with no reported severe haemolytic events. PROSPERO REGISTRATION NUMBER:CRD42022313730.

Pyriproxyfen (PPF) is an insect growth regulator used in the co-treatment of long-lasting insecticidal nets for its ability to sterilize female mosquitoes. To evaluate the efficacy of PPF-treated nets on mosquito reproductivity, most studies observe oviposition (egg-laying) rates in the laboratory. This technique has several technical disadvantages. Our study assessed if ovarial dissection could serve as an effective proxy for evaluating sterility in Anopheles gambiae mosquitoes. Blood-fed females were exposed to untreated or PPF-treated nets in cylinder assays and followed over several days to observe oviposition rates or egg development by dissection. For identifying PPF-exposed mosquitoes, both techniques demonstrated high sensitivity (oviposition: 99.1%; dissection: 100.0%), but for identifying non-exposed mosquitoes, specificity was significantly higher in the dissection group (52.5% vs. 18.9%). To assess whether dissection could be applied to nets treated with a pyrethroid or co-treated with a pyrethroid and PPF in tunnel tests, a blinded investigator performed dissections to predict the PPF exposure status across different treatment groups. The exposure status of dissected females was predicted with >90% accuracy. We report that dissection is a sensitive technique to assess sterility in female Anopheles gambiae mosquitoes and can be used as a predictor of PPF exposure.

Background After a decade of successful control, malaria is on the rise again. The prevalence of malaria in Tanzania has increased from 7% in 2017 to 8% in 2022 and reached 18% in Kagera region in the North West of Tanzania. Malaria vectors in Muleba district Kagera have high level of pyrethroid resistance. The aim of this paper is to explore factors associated with malaria infection prevalence in children aged 6 months to 14 years in Muleba, where Long Lasting Insecticidal Net (LLIN) combining a pyrethroid insecticide and synergist piperonyl butoxide (PBO) that counteract resistance in the mosquitoes, was first distributed under trial conditions in 2015. Methods The trial was a community randomized control in which there were two malaria prevalence cross-sectional household surveys each year (June and December) from 2015 to 2017 in Muleba. In this study we conducted a secondary data analysis of the December surveys only. Multilevel Poisson regression analysis was used to assess factors associated with malaria infection. Results A total of 10,941 children and 4,611 households were included in this study. Overall malaria prevalence was 35.8%, 53.3% and 54.4% in the year 2015, 2016 and 2017 respectively. Living in an area with standard LLIN as opposed to the novel PBO synergist LLIN, being a male child, above 5 years of age, living in a house with open eaves, living in house without IRS, having head of household with no formal education, lower socioeconomic status and survey year were associated with increased risk of malaria infection. Conclusions Using PBO LLIN reduced the risk of malaria infection. However, additional measures could further reduce malaria infection in areas of insecticide resistance such as housing improvement.

Background Attractive targeted sugar bait (ATSB) is a novel approach to vector control, offering an alternative mode of insecticide delivery via the insect alimentary canal, with potential to deliver a variety of compounds new to medical entomology and malaria control. Its potential to control mosquitoes was recently demonstrated in major field trials in Africa. The pyrrole chlorfenapyr is an insecticide new to malaria vector control, and through its unique mode of action—disruption of ATP mediated energy transfer in mitochondria—it may have direct action on energy transfer in the flight muscle cells of mosquitoes. It may also have potential to disrupt mitochondrial function in malarial parasites co-existing within the infected mosquito. However, little is known about the impact of such compounds on vector competence in mosquitoes responsible for malaria transmission. Methods In this study, ATSBs containing chlorfenapyr insecticide and, as a positive control, the anti-malarial drugs artemether/lumefantrine (A/L) were compared for their effect on Plasmodium falciparum development in wild pyrethroid-resistant Anopheles gambiae sensu stricto (s.s.) and for their capacity to reduce vector competence. Female mosquitoes were exposed to ATSB containing either sublethal dose of chlorfenapyr (CFP: 0.025%) or concentrations of A/L ranging from 0.4/2.4 mg/ml to 2.4/14.4 mg/ml, either shortly before or after taking infective blood meals. The impact of their component compounds on the prevalence and intensity of P. falciparum infection were compared between treatments. Results Both the prevalence and intensity of infection were significantly reduced in mosquitoes exposed to either A/L or chlorfenapyr, compared to unexposed negative control mosquitoes. The A/L dose (2.4/14.4 mg/ml) totally erased P. falciparum parasites: 0% prevalence of infection in female mosquitoes exposed compared to 62% of infection in negative controls (df = 1, χ 2 = 31.23 p < 0.001). The dose of chlorfenapyr (0.025%) that killed < 20% females in ATSB showed a reduction in oocyte density of 95% per midgut (0.18/3.43 per midgut). Conclusion These results are evidence that chlorfenapyr, in addition to its direct killing effect on the vector, has the capacity to block Plasmodium transmission by interfering with oocyte development inside pyrethroid-resistant mosquitoes, and through this dual action may potentiate its impact under field conditions.

Introduction Broflanilide is a novel meta-diamide insecticide, which has a distinct mode of action compared to other active ingredients used for Indoor Residual Spraying (IRS). This study details a laboratory evaluation of broflanilide, of putative discriminating concentrations, potential cross-resistance, and residual efficacy on two substrates. Methods Mosquitoes were exposed to broflanilide in bottle bioassays to determine lethal concentrations (LC50 and LC95). These were used to calculate resistance ratios between the susceptible Kisumu and the pyrethroid-resistant Muleba-Kis strains of Anopheles gambiae s.s. Prototype wettable powder formulations of broflanilide were applied to mud and concrete to determine the optimal observation period for determining delayed mortality, and a Cox proportional hazards model was fitted to determine a potential dose response effect. Subsequently, residual efficacy of application rates ranging between 25 mg/m2 and 300 mg/m2 were monitored monthly. Results LC values of tested strains were not significantly different; therefore, the polyfactorial resistance mechanisms possessed by the resistant strain did not confer resistance to broflanilide. A significant effect of concentration and time since spraying was found on mosquito survival, indicating that higher broflanilide concentrations are more effective and that this effect was strongest immediately after spraying. Knockdown at 60min post exposure was negligible, with on average only 1% of all mosquitoes knocked down. On the mud surface, but not on concrete, there was a delayed killing effect, with mortality increasing until 72 hours after exposure. The residual efficacy test indicated that on concrete the 100 mg/m2 and 200 mg/m2 concentrations of both broflanilide 50WP formulations remained efficacious for 9 months post spraying. On mud, there were large variations in mosquito mortality from month to month. Generally, higher concentrations resulted in higher mortality, despite variation over time. Discussion Cross-resistance to broflanilide was not detected in mosquitoes with multiple resistance mechanisms. This opens up the possibility for wide-scale use of broflanilide, especially in areas with established pyrethroid resistance. Like some other insecticide classes, delayed mortality up to 72 hours post exposure was found for broflanilide. Promising residual efficacy was found with broflanilide 50WP formulations on concrete. On mud, efficacy varied and further testing with a refined formulation is recommended.