Papua New Guinea (PNG) has the highest malaria transmission outside of Africa. Longlasting insecticidal nets (LLINs) are believed to have helped to reduce average malaria prevalence in PNG from 16% in 2008 to 1% in 2014. Since 2015 malaria in PNG has resurged significantly. Here, we present observations documenting decreased bioefficacy of unused LLINs with manufacturing dates between 2013 and 2019 collected from villages and LLIN distributors in PNG. Specifically, we show that of n = 167 tested LLINs manufactured after 2013, only 17% are fulfilling the required World Health Organisation bioefficacy standards of ≥ 80% 24 h mortality or ≥ 95% 60 min knockdown in bioassays with pyrethroid susceptible Anopheles farauti mosquitoes. In contrast, all (100%, n = 25) LLINs with manufacturing dates prior to 2013 are meeting these bioefficacy standards. These results suggest that decreased bioefficacy of LLINs is contributing to the malaria resurgence in PNG and increased scrutiny of LLIN quality is warranted.

Background: Papua New Guinea (PNG) has the highest malaria transmission outside of Africa and long-lasting insecticidal nets (LLIN) are the only vector-control tool distributed country-wide. LLIN were introduced into PNG in about 2005 and have been attributed to have had a huge impact on malaria transmission, with reductions in malaria prevalence observed from 15.7% (2008) to 1% (2014). Since 2015, malaria indicators in PNG have risen significantly. Similar trends have been observed in several African nations. In the present study, we observed a drastic reduction in bioefficacy of LLIN collected both from communities (used nets) and prior to use (new nets). We hypothesise that decreased bioefficacy of LLINs is a major contributor to the observed malaria resurgence in PNG and possibly in other parts of the world. Methods: New LLIN in original and unopened packaging (n=192) with a range of manufacturing dates from 2007-2019 were collected either directly from consignments or from households in 15 PNG provinces. Used LLIN (n=40) were collected in 2 provinces, with manufacturing dates ranging from 2008-2017. LLIN were subjected to standard WHO cone bioassays using fully susceptible An. farauti mosquitoes. A subset of LLIN was re-tested using fully susceptible An. gambiae mosquitoes in order to ensure reproducibility of results. Results: Only 7% (95% CI 4-12%) of new LLIN manufactured between 2013-2019 exhibited 100% mortality. However, 84% (95%CI: 65-84%) new nets manufactured in 2012 or before exhibited 100% mortality. Only 29 % of used LLIN less than 3 years old exhibited > 80% 24h-mortality. Results obtained in tests using An. farauti corresponded well with confirmatory tests conducted using An. gambiae. Discussion: Bioefficacy of LLIN in PNG appears to have been highly variable since 2013, with few nets manufactured since 2013 meeting WHO standards. This time-frame coincides with malaria resurgence in the country. These results may have ramifications for LLIN-based malaria control that go beyond the local PNG scenario.

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.

The authors recently reported that long-lasting insecticidal nets (LLINs) distributed in Papua New Guinea (PNG) between 2013 and 2019, exhibited severely diminished efficacy to knock down and kill susceptible Anopheles mosquitoes. This coincided with a rise in malaria observed in PNG since 2015. Here, the authors show that LLIN bioefficacy is increased by heating LLINs prior to WHO cone bioassays. Unused LLINs with low bioefficacy, delivered to PNG in 2019, were heated to 120°C for 5 minutes. Cone bioassays were performed before and at 1 hour, 7 days, and 30 days after heating. This led to a significant increase in 24-hour mortality (17–61%) and 60-minute knock down (31–72%). The effect was sustained over 30 days. Bioassays are crucial in quality assurance of LLIN products. Our findings indicate that bioefficacy of LLINs can be increased by heating. This may have implications for quality assurance procedures used to assess LLINs.

Background Long-lasting insecticidal nets (LLINs) play a key role in reducing malaria transmission in endemic countries. In a previous study, the authors demonstrated a substantial decrease in the bioefficacy of LLINs for malaria prevention delivered to Papua New Guinea (PNG) between 2013 and 2019. This coincided with a rise in malaria cases in the country. The present study was aimed at determining the underlying cause of the reduced bioefficacy observed in these LLINs. The main hypothesis was that a change in the coating formulation of the respective LLIN product was responsible, and had led to significantly altered product properties and performance. Methods A set of PermaNet® 2.0 LLIN samples (n = 12) manufactured between 2007 and 2019 was subjected to combustion ion chromatography in order to understand the chemistry of the LLIN polymer coating formulation. In addition, World Health Organization (WHO) LLIN standard wash tests and cone bioassays were conducted to further characterize the change in product performance that occurred between 2012 and 2013. Results High polymer fluorine content (average 3.2 g/kg) was measured in PermaNet® 2.0 manufactured up to 2012, whereas nets which were manufactured after 2012 contained very little polymer fluorine (average 0.04 g/kg) indicating a coating formulation change from a fluorocarbon (FC)-based to a non-FC-based formulation. The coating formulation change as part of the manufacturing process thus resulted in a significant reduction in bioefficacy. In addition, the manufacturing change affected wash resistance leading to a faster reduction in 24 h mosquito mortality in the non-FC-coated product with consecutive washes. Conclusion A change in coating formulation of PermaNet® 2.0 resulted in reduced product performance in PNG. Post-2012 PermaNet® 2.0 LLINs should not be considered to be the same product as PermaNet® 2.0 LLINs produced prior to and in 2012. Coating formulation changes should be validated to not impact LLIN product performance.

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.

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.

In a study published in Nature Communications in August 2020, we demonstrated an abrupt decrease in the bioefficacy of long-lasting insecticidal nets (LLINs) for malaria prevention delivered to Papua New Guinea (PNG) between 2012 and 2013. This coincided with a rise in malaria cases in the country. At the time of publication of the original article, we were unable to pinpoint the exact reasons for the observed shift towards inferior product performance and stated that "further studies are required to determine the underlying cause of the observed reduced bioefficacy of these LLINs" 1. Due to the potentially significant public health implications (hundreds of millions of this specific LLIN product had been distributed globally), our study led to discussions and speculation among stakeholders. Here, we present data unequivocally showing that the observed reduction in the ability to kill mosquitoes of these LLINs is a direct result of a manufacturing change that occurred at the same time.