Comparison of cone bioassay estimates at two laboratories with different Anopheles mosquitoes 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. This study explored utility of cone bioassays for pre-delivery QA of pyrethroid ITNs to test the assumption that cone bioassays are consistent across locations, mosquito strains, and laboratories. Methods Double-blinded bioassays were conducted on twenty unused pyrethroid ITNs of 4 brands (100 nets, 5 subsamples per net) that had been delivered for mass distribution in Papua New Guinea (PNG) having passed predelivery inspections. Cone bioassays were performed on the same net pieces following World Health Organization (WHO) guidelines at the PNG Institute of Medical Research (PNGIMR) using pyrethroid susceptible Anopheles farauti sensu stricto (s.s.) and at Ifakara Health Institute (IHI), Tanzania using pyrethroid susceptible Anopheles gambiae s.s. Additionally, WHO tunnel tests were conducted at IHI on ITNs that did not meet cone bioefficacy thresholds. Results from IHI and PNGIMR were compared using Spearman’s Rank correlation, Bland–Altman (BA) analysis and analysis of agreement. Literature review on the use of cone bioassays for unused pyrethroid ITNs testing was conducted. Results In cone bioassays, 13/20 nets (65%) at IHI and 8/20 (40%) at PNGIMR met WHO bioefficacy criteria. All nets met WHO bioefficacy criteria on combined cone/tunnel tests at IHI. Results from IHI and PNGIMR correlated on 60-min knockdown (KD60) (rs = 0.6,p = 0.002,n = 20) and 24-h mortality (M24) (rs = 0.9,p < 0.0001,n = 20) but BA showed systematic bias between the results. 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. Including these as a pass, the agreement between the results to predict ITN failure was good with kappa = 0.79 (0.53–1.00) and 90% accuracy. Conclusions Based on these study findings, the WHO cone bioassay is a reproducible bioassay for ITNs with > 80% M24, and for all ITNs provided inherent stochastic variation and systematic bias are accounted for. The literature review confirms that WHO cone bioassay bioefficacy criteria have been previously achieved by all pyrethroid ITNs (unwashed), without the need for additional tunnel tests. The 80% M24 threshold remains the most reliable indicator of pyrethroid ITN quality using pyrethroid susceptible mosquitoes. In the absence of alternative tests, cone bioassays could be used as part of pre-delivery QA.

Coating formulation change leads to inferior performance of long-lasting insecticidal nets in Papua New Guinea

“…farauti mosquitoes in PNG when new, used in the field and also after 25 washes [ 5 ]. This was further confirmed in independent repetitions of the bioassays with different mosquito colonies [ 5 , 10 ]. Despite this, both types of PermaNet ® 2.0 (FC-coated and non-FC-coated) contained similar amounts of deltamethrin, meaning that bioefficacy was affected by the coating formulation change only, leading to a completely altered LLIN product with a significantly inferior ability to kill susceptible An.…”

“…Despite this, both types of PermaNet ® 2.0 (FC-coated and non-FC-coated) contained similar amounts of deltamethrin, meaning that bioefficacy was affected by the coating formulation change only, leading to a completely altered LLIN product with a significantly inferior ability to kill susceptible An. farauti and Anopheles gambiae (Ifakara) colony mosquitoes [ 5 , 10 ]. During the coating process, a polymer film forms around the polyester fibres and the insecticide crystals are bound in the polymer coating.…”

“…It was also suspected that the mosquito strain that had been used (a fully 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 LLIN performance. These possibilities were ruled out categorically, by subsequent multi-centre trials with the same LLIN samples, showing that the observations from PNG were reproducible in an African ‘Good Laboratory Practice’- accredited facility [ 10 ].…”

Coating formulation change leads to inferior bioefficacy of long-lasting insecticidal nets in Papua New Guinea

“…farauti mosquitoes in PNG when new, used in the eld and also after 25 washes [5]. This was further con rmed in independent repetitions of the bioassays with different mosquito colonies [5,9]. Despite this, both types of PermaNet® 2.0 (PFC and non-PFC) contain similar amounts of deltamethrin, meaning that bioe cacy was affected by the coating formulation change only.…”

“…It was also suspected that the mosquito strain that we had used (a fully 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 LLIN performance. We ruled out these possibilities categorically, by conducting a multi-centre trial with the same LLIN samples, which showed that our observations were reproducible in an African 'Good Laboratory Practice' accredited facility [9].…”