The mini chamber test: a novel bioassay for bioefficacy testing of chlorfenapyr treated nets used in malaria control

Dual-active-ingredient insecticide-treated nets (Dual-AI ITNs) combining pyrethroids with chlorfenapyr are increasingly being deployed across malaria-endemic areas. Laboratory evaluation of chlorfenapyr ITNs is challenging due to chlorfenapyr's requirement for metabolic activation. Inspired by the tunnel test, the mini chamber test is smaller, has higher throughput, and is animal-free. We optimised the primary parameters using pyrethroid-susceptible and -resistant mosquito strains exposed to new pyrethroid-chlorfenapyr ITNs versus controls. With parameters defined, we used a draft standard operating procedure (SOP) to evaluate the mini chamber against the gold standard tunnel test in a comparator study. The generated data were used to finalise an SOP. The number of mosquitoes per bioassay had no effect on 72 hour mortality on exposure to the pyrethroid-chlorfenapyr net (10, 20, or 30 mosquitoes per mini chamber: 98% vs. 99% vs. 99%, p = 0.46), nor did the chamber orientation (horizontal vs. vertical: 70% vs. 74%, p = 0.35). Mean 72-hour mortality reduced with the length of exposure: 99% with overnight exposure, 74% with 4 hours and 35% with 2 hour exposure. In direct comparisons, the mini chamber gave 95% 72 hour mortality and the tunnel test 70% with overnight exposure. A trend was observed between chemical content (measured by high performance liquid chromatography (HPLC)) and mortality in ITN samples that had been washed 20 times or used in experimental hut trials, compared to unused comparators, when a 6 hour exposure length was used, showing potential for application to durability monitoring. A mini chamber SOP is shared ( https://github.com/i2i-Data-Repository/Mini-Chamber-Test-paper-data-R-code-and-SOP), and further improvements may come from internal and multi-site validation and review of data generated. The method shows promise as an effective bioassay for use where tunnel tests are not available, for measuring the biological activity of chlorfenapyr in ITNs when measuring regeneration times, wash resistance, residual efficacy, and other studies.