Significant variations in tolerance to clothianidin and pirimiphos-methyl in and populations during a dramatic malaria resurgence despite sustained indoor residual spraying in Uganda.

A dramatic malaria resurgence occurred in areas of Uganda between 2020 and 2022 coincident with the switch to clothianidin-based formulations for indoor residual spraying. During the resurgence, numbers increased but when an alternative insecticide, pirimiphos methyl, was reintroduced in 2023, both malaria cases and mosquito density fell. In this study, we investigated possible causes of the resurgence by assessing; 1) whether sufficient quantities of insecticide were sprayed; 2) the residual insecticide bioefficacy against wild mosquitoes and; 3) the insecticide susceptibility of vector populations using standard test tube assays and wall cone assays. In 2023, after adjusting for extraction efficiency, 70-80% of the houses had optimal residual concentrations of insecticides (clothianidin >0.3g/m; pirimiphos methyl >0.5g/m) with significant variations between sampling rounds and wall types. Mud walls had the lowest residual concentration of insecticides, and the lowest observed mortality in wall cone assays, compared to fired bricks with plaster/cement/paint. In the studies of residual bio efficacy, by World Health Organization (WHO) definitions, showed resistance to clothianidin (<80% mortality) up to 11 months and susceptibility to pirimiphos methyl (>90% mortality) when exposed to wall surfaces up to 7 months post-spray. In WHO tube tests, variations were observed in susceptibility to clothianidin in populations using dose- and time-response assays (80-98% mortality). In 2022, was largely susceptible to the clothianidin-based formulation Sumishield (85-90% mortality) although the levels dropped slightly in 2023 (60-85% mortality) mainly in mud and pole houses. In contrast, was mildly susceptible to the pirimiphos methyl-based formulation Actellic (~80% mortality) and time response assays showed had very low knockdown and mortality at lower exposure time compared to . Regression models showed a positive association between residual insecticide concentration (RIC) and mortality in houses sprayed with Sumishield but not Actellic houses. Despite the possible variations observed in spray operations, the study revealed that . exhibited a higher tolerance to clothianidin-based formulations compared to , and this might have driven the malaria resurgence observed in Uganda. However, there are signals of resistance to pirimiphos-methyl which will require further investigation and monitoring.