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 bioefficacy against malaria mosquitoes. The cone bioassay provides a simple evaluation of ITN bioefficacy 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 bioefficacy 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 bioefficacy criteria at IHI and 8/20 (40%) at PNGIMR. All nets met WHO bioefficacy 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 confidence 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 bioefficacy 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.

Coating formulation change is the root cause for inferior bioefficacy of long-lasting insecticidal nets in Papua New Guinea

“…It was also suspected that the mosquito strain that we had used (a pyrethroid susceptible strain of Anopheles farauti ) or technicalities related to conducting WHO cone bioassays at the PNG Institute of Medical Research were responsible for the observed inferior performance. We ruled out these possibilities categorically, by conducting a multi-centre trial with the same nets that showed that our observations from PNG were reproducible in an African ‘Good Laboratory Practice’ accredited facility 10 .…”

Quantitative analysis of insecticides in long-lasting insecticidal nets using liquid chromatography mass spectrometry (LC-MS) and X-ray fluorescence (XRF) spectroscopy

“…This is normal because when nets are washed or exposed to UV radiation (sun exposure), total active ingredient (AI) content decreases and once a threshold AI concentration is reached, 100% bioefficacy normally observed with fully susceptible strains in new and unwashed nets is not maintained. However, other studies have demonstrated that recently manufactured new and unused LLIN products may not be able to pass WHO cone bioassay performance criteria with susceptible mosquito colonies, even though their total AI content was determined to be adequate in predelivery inspections [7, 11]. This may be for different reasons including restricted bioavailability of AI on the net surface due to the nature of the LLIN coating, presence of AI in chemical or physical states that may be detrimental to its effectiveness (such as isomers with reduced potency or crystalline states that may reduce the uptake of AI by mosquitoes) [5, 6, 12, 13].…”