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

Abstract

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 malaria 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 24 h mortality (M24). The present study tested this hypothesis in two facilities using two fully susceptible mosquito colonies. 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 whole net samples per brand were used (total of n = 20 whole nets). Five pieces per whole net sample were prepared in duplicate resulting in a total of n = 100 pairs. 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 colony. In particular, with An. farauti, a significantly higher M24 was observed 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.

Complete series method (CSM): a convenient method to reduce daily heterogeneity when evaluating the regeneration time (RT) of insecticide-treated nets (ITNs)

“…Cone bioassays were performed according to standard WHO 2013 procedures [ 12 ], with two modifications to standardize exposure: (i) the boards were held at 60 degrees [ 26 ] and (ii) holes were cut in the board to ensure that the mosquitoes rested only on the net [ 15 ]. On each netting sample, cones were placed and held in place using masking tape.…”

“…This was estimated following the WHO longitudinal process: net samples were cut from the sides and roof of ITNs following a specific pattern: washed, rinsed and dried three times in a single day to remove the insecticide from the surface, after which the net samples were tested using cone bioassay at days 1, 2, 3, 5 and 7 post-wash following the 2013 WHO guidelines [12]. Several factors influence bioassay results [13] including ITN preparation exposure temperature [14] and test procedures [15]; mosquitoes also affect the results by their age [16], blood-feeding status [17], density [18], species [19], resistance level [20] and fitness [6,21]. Mosquito heterogeneity and its impact on bioassays was first described in 1971 [22] and has been shown to impact insecticide susceptibility [21].…”

Complete series method (CSM): a convenient method to reduce daily heterogeneity when evaluating the regeneration time (RT) of insecticide-treated nets (ITNs)

“…Cone bioassays were performed according to standard WHO 2013 procedures [11], with two modi cations to standardize exposure: 1) the boards were held at 60 degrees [25] and 2) holes were cut in the board to ensure that the mosquitoes rest only on the net [14]. On each netting sample, the cones were placed and held in place using a masking tape.…”

“…This was estimated following the WHO longitudinal process: net samples are cut from the sides and roof of the ITN following a speci c pattern, washed three times a day to remove the insecticide from the surface, after which the net samples were bioassayed at days 1, 2, 3, 5 and 7 post wash [11]. Several factors in uence bioassay results [12] including ITN preparation exposure temperature [13] and test procedures [14]; Mosquitoes also affect the results including age [15], blood feeding status [16], density [17], species [18], resistance level [19], and tness [5,20]. Mosquito heterogeneity and its impact on bioassays was rst described in 1971 [21], and has been shown to impact insecticide susceptibility [20].…”