Effects of next-generation, dual-active-ingredient, long- lasting insecticidal net deployment on insecticide resistance in malaria vectors in Tanzania: an analysis of a 3-year, cluster- randomised controlled trial

Background Insecticide resistance among malaria-vector species is a pervasive problem that might jeopardise global disease-control efforts. Novel vector-control tools with different modes of action, including long-lasting insecticidal nets (LLINs) incorporating new active ingredients, are urgently needed to delay the evolution and spread of insecticide resistance. We aimed to measure phenotypic and genotypic insecticide-resistance profiles among wild Anopheles collected over 3 years to assess the longitudinal effects of dual-active-ingredient LLINs on insecticide resistance. Methods For this analysis, data nested in a 3-year, four parallel-arm, superiority cluster-randomised controlled trial (cRCT) in Tanzania, collected from 84 clusters (39 307 households) formed of 72 villages in the Misungwi district, were used to measure insecticide-resistance profiles among female Anopheles mosquitoes via insecticide-resistance bioassays and quantitative RT-PCR of metabolic-resistance genes. Wild, blood-fed, indoor-resting mosquitoes were collected annually during the rainy seasons from house walls in clusters from all four trial groups. Mosquitoes were morphologically identified as An gambiae sensu lato (SL) or An funestus SL before separate bioassay testing. The primary outcomes were lethal-dose values for a-cypermethrin, permethrin, and piperonyl butoxide pre-exposure plus permethrin-resistance intensity bioassays, mortality 72 h after insecticidal exposure for chlorfenapyr bioassays, fertility reduction 72 h after insecticidal exposure for pyriproxyfen bioassays, and fold change in metabolic-enzyme expression relative to an insecticide-susceptible laboratory strain. All primary outcomes were measured in An funestus SL 1 year, 2 years, and 3 years after LLIN distribution. Primary outcomes were also assessed in An gambiae SL if enough mosquitoes were collected. The cRCT is registered with ClinicalTrials.gov (NCT03554616). Findings Between May 24, 2019, and Oct 25, 2021, 47 224 female Anopheles were collected for resistance monitoring. In the pyrethroid (PY)-LLIN group, there were significant increases in a-cypermethrin-resistance intensity (year 1 LD50=9 center dot 52 vs year 2 76 center dot 20, p < 0 center dot 0001) and permethrin-resistance intensity (year 1 13 center dot 27 vs year 2 35 center dot 83, p=0 center dot 0019) in An funestus SL. In the pyriproxyfen PY-LLIN group, there was similar increase in a-cypermethrin-resistance intensity (year 1 0 center dot 71 vs year 2 81 center dot 56, p < 0 center dot 0001) and permethrin-resistance intensity (year 1 5 center dot 68 vs year 2 50 center dot 14, p < 0 center dot 0001). In the piperonyl butoxide PY-LLIN group, a-cypermethrin-resistance intensity (year 1 33 center dot 26 vs year 3 70 center dot 22, p=0 center dot 0071) and permethrin-resistance intensity (year 1 47 center dot 09 vs year 3 2635 center dot 29, p < 0 center dot 0001) also increased over time. In the chlorfenapyr PY-LLIN group, there were no effects on a-cypermethrin-resistance intensity (year 1 0 center dot 42 vs year 3 0 center dot 99, p=0 center dot 54) or permethrin-resistance intensity (data were not estimable due to nearly 100% mortality). There were also minimal reductions in chlorfenapyr susceptibility. However, in the chlorfenapyr PY-LLIN group, a significant decline in piperonyl-butoxide synergy was seen by year 3 (year 1 0 center dot 02 vs year 3 0 center dot 26, p=0 center dot 020). Highly over-expressed detoxification enzymes showed dynamic patterns of selection throughout the trial. Interpretation Our phenotypic data supports trial epidemiological findings; chlorfenapyr PY-LLINs provided superior protection from malaria across multiple transmission seasons, with few effects on insecticide-resistance selection. Rapid pyrethroid-resistance intensification in the piperonyl butoxide PY-LLIN group and pre-existing tolerance of pyriproxyfen in vector populations might explain the poorer performance of these two interventions regarding malaria outcomes. Further work is required to elucidate the potential mechanisms driving cross-resistance between pyrethroids and novel active ingredients to better inform the design of pre-emptive resistance-management strategies. Funding UK Department for International Development; UK Medical Research Council; Wellcome Trust; UK Department of Health and Social Care; UK Foreign, Commonwealth and Development Office; and The Bill and Melinda Gates Foundation via the Innovative Vector Control Consortium. (c) 2023 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license.