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Background  Following agricultural use and large-scale distribution of insecticide-treated nets (ITNs), malaria vector resistance to pyrethroids is widespread in sub-Saharan Africa. Interceptor® G2 is a new dual active ingredient (AI) ITN treated with alpha-cypermethrin and chlorfenapyr for the control of pyrethroid-resistant malaria vectors. In anticipation of these new nets being more widely distributed, testing was conducted to develop a chlorfenapyr susceptibility bioassay protocol and gather susceptibility information.    Methods  Bottle bioassay tests were conducted using five concentrations of chlorfenapyr at 12.5, 25, 50, 100, and 200 µg AI/bottle in 10 countries in sub-Saharan Africa using 13,639 wild-collected  Anopheles gambiae  sensu lato ( s.l .) (56 vector populations per dose) and 4,494 pyrethroid-susceptible insectary mosquitoes from 8 colonized strains. In parallel, susceptibility tests were conducted using a provisional discriminating concentration of 100 µg AI/bottle in 16 countries using 23,422 wild-collected, pyrethroid-resistant  An. gambiae s.l.  (259 vector populations). Exposure time was 60 min, with mortality recorded at 24, 48 and 72 h after exposure.    Results  Median mortality rates (up to 72 h after exposure) of insectary colony mosquitoes was 100% at all five concentrations tested, but the lowest dose to kill all mosquitoes tested was 50 µg AI/bottle. The median 72-h mortality of wild  An. gambiae s.l.  in 10 countries was 71.5, 90.5, 96.5, 100, and 100% at concentrations of 12.5, 25, 50, 100, and 200 µg AI/bottle, respectively. Log-probit analysis of the five concentrations tested determined that the LC 95  of wild  An. gambiae  s .l.  was 67.9 µg AI/bottle (95% CI: 48.8–119.5). The discriminating concentration of 203.8 µg AI/bottle (95% CI: 146–359) was calculated by multiplying the LC 95  by three. However, the difference in mortality between 100 and 200 µg AI/bottle was minimal and large-scale testing using 100 µg AI/bottle with wild  An. gambiae s.l.  in 16 countries showed that this concentration was generally suitable, with a median mortality rate of 100% at 72 h.    Conclusions  This study determined that 100 or 200 µg AI/bottle chlorfenapyr in bottle bioassays are suitable discriminating concentrations for monitoring susceptibility of wild  An. gambiae s.l ., using mortality recorded up to 72 h. Testing in 16 countries in sub-Saharan Africa demonstrated vector susceptibility to chlorfenapyr, including mosquitoes with multiple resistance mechanisms to pyrethroids.    Supplementary Information  The online version contains supplementary material available at 10.1186/s12936-021-03847-3.

malaria journal

Determination of the discriminating concentration of chlorfenapyr (pyrrole) and Anopheles gambiae sensu lato susceptibility testing in preparation for distribution of Interceptor® G2 insecticide-treated nets