Insufficient duration of insecticidal efficacy of Yahe® insecticide-treated nets in Papua New Guinea.

BACKGROUND: Insecticide-treated nets (ITNs) are the backbone of anti-malarial vector control in Papua New Guinea (PNG). Over recent years the quality and performance of ITNs delivered to PNG decreased, which has likely contributed to the stagnation in the malaria control effort in the country. The present study reports results from the first 24 months of a durability study with the ITN product Yahe LN® in PNG. METHODS: The durability study was conducted in four villages on the northern coast of PNG, in an area with high malaria parasite transmission, following WHO-recommended methodology adapted to the local scenario. A cohort of n = 500 individually identifiable Yahe® ITNs was distributed by the PNG National Malaria Control Programme from October to December 2021. Insecticidal efficacy of the ITNs was tested using cone bioassays with fully pyrethroid susceptible Anopheles farauti colony mosquitoes at baseline and at 6 months intervals, alongside evaluation of physical integrity and the proportion of ITNs lost to follow-up. A questionnaire was used to collect information on ITN end user behaviour, such as the frequency of use and washing. The observations from the durability study were augmented with simulated laboratory wash assays. RESULTS: Gradual uptake and replacement of previous campaign nets by the communities was observed, such that at 6 months 45% of all newly distributed nets were in use in their designated households. Insecticidal efficacy of the Yahe® nets, expressed as the percent 24 h mortality in cone bioassays decreased from 91 to 45% within the first 6 months of distribution, even though > 90% of study nets had never been washed. Insecticidal efficacy decreased further to < 20% after 24 months. ITNs accumulated physical damage (holes) at a rate similar to previous studies, and 35% were classified as 'too torn' by proportional hole index after 24 months. ITNs were lost to follow-up such that 61% of cohort nets were still present after 24 months. Laboratory wash assays indicated a rapid reduction in insecticidal performance with each consecutive wash such that average 24 h mortality was below 20% after 10 washes. CONCLUSION: Yahe® ITNs are not performing as per label claim in an area with fully pyrethroid susceptible vectors, and should be investigated more comprehensively and in other settings for compliance with currently recommended durability and efficacy thresholds. The mass distribution of low quality ITN products with variable performance is one of the major ongoing challenges for global malaria control in the last decade.

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

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.

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

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.

Comparison of cone bioassay estimates at two laboratories with different Anopheles mosquitoes for quality assurance of pyrethroid insecticide-treated nets.

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.

Effect of Short-Term Heating on Bioefficacy of Deltamethrin-Coated Long-Lasting Insecticidal Nets.

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 from 17% to 61% and 60-minute knock down from 31% to 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.

Decreased bioefficacy of long-lasting insecticidal nets and the resurgence of malaria in Papua New Guinea.

Papua New Guinea (PNG) has the highest malaria transmission outside of Africa. Long-lasting 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.