Cone Bioassays Provide Reproducible Bioefficacy Estimates with Different Anopheline Mosquitoes and Can Be Used for Quality Assurance of Pyrethroid Insecticide Treated Nets

Abstract

Background Quality assurance (QA) of insecticide-treated nets (ITNs) delivered to malaria-endemic countries is conducted by measuring physiochemical parameters, but not bioecacy against malaria mosquitoes. The cone bioassay provides a simple evaluation of ITN bioecacy and its conditions and parameters are prescribed by the World Health Organization (WHO). This study explored utility of cone bioassays for pre- delivery QA of pyrethroid ITNs in two test facilities using different mosquito species to test the assumption that cone bioassays are consistent and reproducible across locations, mosquito strains, and laboratories. Methods Double-blinded bioassays were conducted on unused pyrethroid ITNs of 4 brands (5 nets/brand, 5 subsamples/net) that had been delivered for mass distribution in Papua New Guinea (PNG) having passed physiochemical testing of chemical content. Cone bioassays were performed on adjacent net pieces following WHO guidelines at the PNG Institute of Medical Research (PNGIMR) using pyrethroid susceptible Anopheles farauti s.s. and at Ifakara Health Institute (IHI), Tanzania using pyrethroid susceptible Anopheles gambiae s.s. Additionally, WHO tunnel tests was conducted at IHI on ITNs that did not meet cone bioecacy thresholds. Results from IHI and PNGIMR were compared using Spearman’s Rank, Bland Altman and Cohen’s kappa. A literature review on the utility of cone bioassays for unused pyrethroid ITNs testing was also conducted. Results In cone bioassays, 13/20 nets (65%) met WHO bioecacy criteria at IHI and 8/20 (40%) at PNGIMR. All nets met WHO bioecacy criteria on combined cone/tunnel tests. Results from IHI and PNGIMR correlated on 60-minute knockdown (rs=0.6, p=0.002,n=20) and 24-hour mortality (rs=0.9, p<0.0001,n=20) but there was systematic bias between the results measured by Bland Altman. Of the 5 nets with discrepant result between IHI and PNGIMR, three had condence intervals overlapping the 80% mortality threshold, with averages within 1-3% of the threshold. The agreement between the results to predict ITN failure was good with kappa=0.79 (0.53-1.00) and 90% accuracy. Conclusions WHO cone is a reproducible means to measure pyrethroid ITN bioecacy using a combination of knockdown and mortality. In the absence of an alternative tests, cone tests could be used to assess the availability of active ingredients at the surface of ITN (where mosquitoes encounter it) as part of pre-delivery QA.