WHO cone bioassay boards with or without holes: Relevance for bioassay outcomes in long-lasting insecticidal net studies

Background: The World Health Organization (WHO) cone bioassay is a key method used to evaluate the bioefficacy of long-lasting insecticidal nets (LLINs) used for vector control. These tests also play an important role in LLIN product prequalification and longitudinal monitoring. Standardization of these assays is therefore important. While many parameters for WHO cone bioassays are defined in the respective WHO guidelines, others are not. One of these undefined parameters is the exact configuration of the bioassay boards. In cone bioassays, LLIN samples are pinned onto a bioassay board for testing. Anecdotal evidence suggests that bioassay boards with holes behind the LLIN samples lead to greater exposure to insecticide, as the mosquitoes are ‘forced to stand on the net material’. This may increase the key assay outcomes of 60 min knockdown (KD60) and 24h mortality (M24). Here, we test this hypothesis in two facilities using two fully susceptible mosquito strains. Methods: : WHO cone bioassays were performed using bioassay boards with holes and boards without holes in parallel following WHO guidelines . Five brands of LLINs with four new and unwashed samples per brand were used (total n=20). Knock-down (KD) was recorded in 10 min intervals within the first hour after exposure and mortality was recorded at 24 h. Assays with Anopheles farauti were done at the Papua New Guinea Institute of Medical Research (PNGIMR) and assays with Aedes aegypti were done at James Cook University, Australia. Results: : Results varied not only with bioassay board configuration but also with mosquito strain. In particular, with An. farauti we observed significantly higher M24 when boards with holes were used, while this was not observed with Ae. aegypti. WHO cone bioassay results were systematically biased between the two facilities such that the use of An. farauti at PNGIMR predicted higher KD60 and M24. Conclusion: The present study highlights the need for further harmonization of WHO cone bioassay methodology. Parameters such as bioassay board configuration and mosquito species systematically affect the observations, which impedes generalizability of WHO cone bioassay outcomes.