Background Insecticide-treated net (ITN) durability, measured through physical integrity and bioefficacy, must be accurately assessed in order to plan the timely replacement of worn out nets and guide procurement of longer-lasting, cost-effective nets. World Health Organization (WHO) guidance advises that new intervention class ITNs be assessed 3 years after distribution, in experimental huts. In order to obtain information on whole-net efficacy cost-effectively and with adequate replication, a new bioassay, the Ifakara Ambient Chamber Test (I-ACT), a semi-field whole net assay baited with human host, was compared to established WHO durability testing methods. Methods Two experiments were conducted using pyrethroid-susceptible female adult Anopheles gambiae sensu stricto comparing bioefficacy of Olyset ® , PermaNet ® 2.0 and NetProtect ® evaluated by I-ACT and WHO cone and tunnel tests. In total, 432 nets (144/brand) were evaluated using I-ACT and cone test. Olyset ® nets (132/144) that did not meet the WHO cone test threshold criteria (≥ 80% mortality or ≥ 95% knockdown) were evaluated using tunnel tests with threshold criteria of ≥ 80% mortality or ≥ 90% feeding inhibition for WHO tunnel and I-ACT. Pass rate of nets tested by WHO combined standard WHO bioassays (cone/tunnel tests) was compared to pass in I-ACT only by net brand and time after distribution. Results Overall, more nets passed WHO threshold criteria when tested with I-ACT than with standard WHO bioassays 92% vs 69%, (OR: 4.1, 95% CI 3.5–4.7, p < 0.0001). The proportion of Olyset ® nets that passed differed if WHO 2005 or WHO 2013 LN testing guidelines were followed: 77% vs 71%, respectively. Based on I-ACT results, PermaNet ® 2.0 and NetProtect ® demonstrated superior mortality and non-inferior feeding inhibition to Olyset ® over 3 years of field use in Tanzania. Conclusion Ifakara Ambient Chamber Test may have use for durability studies and non-inferiority testing of new ITN products. It measures composite bioefficacy and physical integrity with both mortality and feeding inhibition endpoints, using fewer mosquitoes than standard WHO bioassays (cone and tunnel tests). The I-ACT is a high-throughput assay to evaluate ITN products that work through either contact toxicity or feeding inhibition. I-ACT allows mosquitoes to interact with a host sleeping underneath a net as encountered in the field, without risk to human participants. Electronic supplementary material The online version of this article (10.1186/s12936-019-2741-y) contains supplementary material, which is available to authorized users.

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.

Genetic analysis of molecular markers is critical in tracking the emergence and/or spread of artemisinin resistant parasites. Clinical isolates collected in western Kenya pre- and post- introduction of artemisinin combination therapies (ACTs) were genotyped at SNP positions in regions of strong selection signatures on chromosome 13 and 14, as described in Southeast Asia (SEA). Twenty five SNPs were genotyped using Sequenom MassArray and pfmdr1 gene copy number by real-time PCR. Parasite clearance half-life and in vitro drug sensitivity testing were performed using standard methods. One hundred twenty nine isolates were successfully analyzed. Fifteen SNPs were present in pre-ACTs isolates and six in post-ACTs. None of the SNPs showed association with parasite clearance half-life. Post-ACTs parasites had significantly higher pfmdr1 copy number compared to pre-ACTs. Seven of eight parasites with multiple pfmdr1 were post-ACTs. When in vitro IC50s were compared for parasites with single vs. multiple gene copies, only amodiaquine and piperaquine reached statistical significance. Data showed SNPs on chromosome 13 and 14 had different frequency and trend in western Kenya parasites compared SEA. Increase in pfmdr1 gene copy is consistent with recent studies in African parasites. Data suggests genetic signature of artemisinin resistance in Africa might be different from SEA.