Chlorfenapyr bednets effectively overcome pyrethroid resistance escalation in highly resistant Anopheles malaria vectors in Uganda

Escalating insecticide resistance threatens the efficacy of LLINs, undermining malaria control in Africa. We conducted the first experimental hut trials in Uganda using highly resistant free-flying wild Anopheles mosquitoes and F2 hybrids of FANG and Uganda An. funestus to evaluate the performance of bednets. The interceptor G2 (chlorfenapyr) bednet demonstrated superior efficacy compared to Interceptor (pyrethroid-only) net [mortality odds ratio (OR): 18.7 (8.05-48.6) P < 0.0001], achieving an overall mortality rate of 70.6% and 63.2% against An. funestus and An. gambiae respectively. In contrast, PermaNet 3.0 and Olyset Plus (piperonyl butoxide (PBO)) and Royal Guard (pyriproxyfen (PPF)-treated) bednets exhibited significantly lower mortality against An. funestus [Olyset Plus: 36.1%, PermaNet 3.0: 31.0% and Royal Guard (37.6%], though performance against An. gambiae was moderate [PermaNet 3.0: 61.4%, Olyset Plus: 50.0%, Royal Guard: 51.6%]. Interceptor net produced the lowest mortality (~ 25%) against both species. Regarding blood-feeding inhibition (BFI), PBO nets, particularly Olyset Plus, outperformed Interceptor G2 and Royal Guard, while Interceptor produced minimal BFI (< 36%). Further evaluation of Royal Guard's PPF effect on oviposition revealed no significant reduction in oviposition rates compared to controls with An. funestus (63.9% vs. 63.3%, P > 0.05). Genetic analysis using the hybrid crosses revealed that pyrethroid resistance markers (4.3 Kb-SV and G454A-Cyp9K1) were significantly associated with mosquito survival and blood-feeding success against PermaNet 2.0 (pyrethroid-only) and PermaNet 3.0 but showed no significant association with Interceptor G2 net. These findings support Interceptor G2 as a promising intervention for regions dominated by both highly resistant An. funestus s.l. and An. gambiae s.l. Piperonyl butoxide and PPF nets emerge as a good alternative for areas mostly dominated by resistant An. gambiae s.l. populations. Critically, the demonstrated variable impact of insecticide resistance on bednet efficacy underscores the imperative need for a comprehensive vector distribution mapping, continuous field efficacy assessments, and systematic resistance monitoring. This evidence-based triad should guide strategic LLIN distribution and rotations to sustain malaria control efficacy in resistance-prone settings.