Multiple insecticide resistance in Anopheles funestus from Mopeia, Central Mozambique

Background: The main malaria vector control methods implemented in Mozambique are insecticide-treated nets (ITN’s) and indoor residual spraying (IRS). These insecticide-based interventions are currently threatened by the rapidly spreading insecticide resistance in several major malaria vectors. Monitoring of insecticide resistance is necessary to inform the selection of insecticides by control programmes. This study describes the insecticide resistance profiles of the main malaria vector, Anopheles funestus sensu lato. in Mopeia district, a malaria holoendemic area of the Zambezia province of Mozambique. Methods: Anopheles adults and larvae were collected from 15 sentinel sites across the district between October 2021 and September 2022. Wild-caught, unfed female adults were collected using CDC-light traps and pooled over three days before exposure to the test insecticide. For mosquitoes collected as larvae, F0 adults aged 3–5 days post-emergence were used for insecticide susceptibility testing. Resistance to bendiocarb, DDT, deltamethrin and pirimiphos-methyl was evaluated using the standard WHO tube bioassay. The mechanism of resistance was probed using the PBO (piperonyl butoxide) synergistic bioassay. The presence and frequency of different genetic mutations associated with insecticide resistance was assessed using polymerase chain reaction, including A296S-Rdl, L119F-GSTe2 and 6.5 kb SV (structural variation) insertion. Results: A total of 1349 female Anopheles mosquitoes (controls included) were used for susceptibility tests with discriminating insecticide concentrations. Phenotypic resistance to bendiocarb, DDT, deltamethrin and pirimiphos-methyl was observed, with 37%, 79%, 14% and 67% mortality rate respectively. Pre-exposure to PBO partially restored susceptibility to deltamethrin to a mortality rate of 80%. The frequency of the insecticide resistance mutations was 0.49, 0.05 and 0.92, for A296S-Rdl, L119F-GSTe2 and 6.5 kb SV insertion, respectively. Conclusion: Malaria vectors in Mopeia exhibit resistance to all four major public health insecticide classes: pyrethroids, organophosphates, organochlorides and carbamates. This highlights the urgent need to adopt new insecticide classes for vector control interventions. The partial restoration of susceptibility by PBO suggests resistance is being driven by various mechanisms including the involvement of metabolic resistance through cytochrome P450 monooxygenase enzymes and glutathione S-transferases.