Modified cone bioassay methods for evaluating chlorfenapyr indoor residual spray

Chlorfenapyr-based indoor residual spraying (IRS) has been prequalified for malaria control. Residual efficacy (mosquito mortality) of IRS applied to houses is typically assessed using forced-contact cone bioassays. However, chlorfenapyr is a pro-insecticide that requires mosquito metabolic activity for conversion to its toxic metabolite, tralopyril. Cone bioassays consistently underestimate mortality compared to free-flying bioassays. This study evaluated whether increasing mosquito activity during cone bioassays could better capture chlorfenapyr-induced mortality on mud surface using scalable methods.

The bioefficacy of chlorfenapyr (Sylando® 240 SC) sprayed on mud surfaces was assessed in two experiments using cone bioassays against sugar-fed, laboratory-reared pyrethroid-resistant Anopheles arabiensis and An. funestus. Experiment 1 evaluated six exposure scenarios varying cone contact time (30 vs. 120 min) and post-exposure activity using clean CDC bottles (none, 1-hr vs. 12-hrs). Experiment 2 assessed the interaction between exposure duration (30-min vs. 12-hrs) and the presence or absence of a host (rabbit). Delayed mortality was monitored up to 120 hrs. An indicative mortality threshold was estimated using a random-effect meta-analysis of three free-flying semi-field studies.

Mortality remained low across all treatments in experiment 1 (7.8-15.4% at 72 hrs; 14.1-23.1% at 120 hrs), with no consistent improvement from post-exposure activity. In Experiment 2, mortality increased with 12-hrs exposure, particularly in the presence of a host (23.5% at 72 hrs; OR 2.25, 95% CI 1.71-2.97, p < 0.001). However, mortality remained well below the indicative value (52%).

Prolonged exposure and host presence modestly increased mortality, but did not produce values consistent with expected chlorfenapyr bioefficacy against free-flying mosquitoes. These findings suggest that modified cone bioassays are unlikely to provide reliable estimates of chlorfenapyr-IRS performance. Further validation is required to assess whether overnight cone tests in people's homes can provide a more realistic measure of residual bioefficacy.