Bioefficacy Of Long-lasting Insecticidal Nets Research Articles

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

BackgroundThe 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.MethodsWHO 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.ResultsResults 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.ConclusionThe 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.

LLIN evaluation in Uganda project (LLINEUP): The fabric integrity, chemical content and bioefficacy of long-lasting insecticidal nets treated with and without piperonyl butoxide across two years of operational use in Uganda

Long-lasting insecticidal nets (LLINs) supplemented with the synergist piperonyl butoxide have been developed in response to growing pyrethroid resistance; however, their durability in the field remains poorly described. A pragmatic cluster-randomised trial was embedded into Ugandaʼs 2017–2018 LLIN distribution to compare the durability of LLINs with and without PBO. A total of 104 clusters (health sub-districts) were included with each receiving one of four LLIN products, two with pyrethroid + PBO (Olyset Plus and PermaNet 3.0) and two pyrethroid-only (Olyset Net and PermaNet 2.0). Nets were sampled at baseline, 12 and 25 months post-distribution to assess physical condition, chemical content, and bioefficacy. Physical condition was quantified using proportionate Hole Index and chemical content measured using high-performance liquid chromatography. Bioefficacy was assessed with three-minute World Health Organisation (WHO) Cone and Wireball assays using pyrethroid-resistant Anopheles gambiae, with 1-h knockdown and 24-h mortality recorded. There was no difference in physical durability between LLIN products assessed (P = 0.644). The pyrethroid content of all products remained relatively stable across time-points but PBO content declined by 55% (P < 0.001) and 58% (P < 0.001) for Olyset Plus and PermaNet 3.0 respectively. Both PBO LLINs were highly effective against pyrethroid-resistant mosquitoes when new, knocking down all mosquitoes. However, bioefficacy declined over time with Olyset Plus knocking down 45.72% (95% CI: 22.84–68.62%, P = 0.021) and Permanent 3.0 knocking down 78.57% (95% CI: 63.57–93.58%, P < 0.001) after 25 months. Here we demonstrate that both Olyset Plus and PermaNet 3.0 are as durable as their pyrethroid-only equivalents and had superior bioefficacy against pyrethroid-resistant An. gambiae. However, the superiority of PBO-LLINs decreased with operational use, correlating with a reduction in total PBO content. This decline in bioefficacy after just two years is concerning and there is an urgent need to assess the durability of PBO LLINs in other settings.

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.

High insecticide resistance intensity of Anopheles gambiae (s.l.) and low efficacy of pyrethroid LLINs in Accra, Ghana

BackgroundInsecticide resistance of Anopheles gambiae (s.l.) against public health insecticides is increasingly reported in Ghana and need to be closely monitored. This study investigated the intensity of insecticide resistance of An. gambiae (s.l.) found in a vegetable growing area in Accra, Ghana, where insecticides, herbicides and fertilizers are massively used for plant protection. The bioefficacy of long-lasting insecticidal nets (LLINs) currently distributed in the country was also assessed to delimitate the impact of the insecticide resistance intensity on the effectiveness of those nets.MethodsThree- to five-day-old adult mosquitoes that emerged from collected larvae from Opeibea, Accra (Ghana), were assayed using CDC bottle and WHO tube intensity assays against different insecticides. The Vgsc-L1014F and ace-1 mutations within the population were also characterized using PCR methods. Furthermore, cone bioassays against different types of LLINs were conducted to evaluate the extent and impact of the resistance of An. gambiae (s.l.) from Opeibea.ResultsAnopheles gambiae (s.l.) from Opeibea were resistant to all the insecticides tested with very low mortality observed against organochlorine, carbamates and pyrethroid insecticides using WHO susceptibility tests at diagnostic doses during three consecutive years of monitoring. The average frequencies of Vgsc-1014F and ace-1 in the An. gambiae (s.l.) population tested were 0.99 and 0.76, respectively. The intensity assays using both CDC bottle and WHO tubes showed high resistance intensity to pyrethroids and carbamates with survivals at 10× the diagnostic doses of the insecticides tested. Only pirimiphos methyl recorded a low resistance intensity with 100% mortality at 5× the diagnostic dose. The bioefficacy of pyrethroid LLINs ranged from 2.2 to 16.2% mortality while the PBO LLIN, PermaNet® 3.0, was 73%.ConclusionsWHO susceptibility tests using the diagnostic doses described the susceptibility status of the mosquito colony while CDC bottle and WHO tube intensity assays showed varying degrees of resistance intensity. Although both methods are not directly comparable, the indication of the resistance intensity showed the alarming insecticide resistance intensity in Opeibea and its surroundings, which could have an operational impact on the efficacy of vector control tools and particularly on pyrethroid LLINs.

Bio-efficacy of long-lasting insecticidal nets against &lt;i&gt;Anopheles gambiae&lt;/i&gt; mosquito after four years of usage in Mikang, Plateau State, Nigeria

Insecticide-treated nets (ITN) and Indoor residual spraying (IRS) are key components in malaria control through reliance on susceptibility of Anopheles vector mosquitoes to chemical insecticides in bed nets and for IRS. Bio- efficacy of long-lasting insecticidal nets (LLINs) of various years of usage on populations of An. gambiae was investigated in Mikang LGA of Plateau State, Nigeria. About 2-3 days old non-blood fed adult An. gambiae mosquitoes reared from larvae were tested with LLINs that had been in use for 1-4 years using the standard WHO cone test. Bio-efficacy of LLINs decreased with age of the nets tested. Knockdown of mosquitoes were 90 (100%), 87 (96.7%), 86 (95.6%), 56 (62.0%) and 36 (40.0%) in control nets ages 0, 1, 2, 3 and 4 years respectively. Significant difference was observed between the control and the other net age groups while no significant difference (p&lt;0.05)was observed between the knockdown rates for net age 1 and 2 and those in use for 3 and 4 years suggesting insecticide decay as nets age. In the current study 100% mortality after 24 hours post exposure was observed in adult mosquitoes from control nets and those in use for 1-4 years, indicating susceptibility. There was significant difference in the susceptibility of mosquitoes to different nets.LLINs should be monitored for pyrethroids resistance as they age and preferably, they should be replaced after every two years of use.Keywords: Malaria; pyrethroid; insecticide decay; bio-efficacy; susceptibility.

Bioefficacy of long-lasting insecticidal nets against pyrethroid-resistant populations of Anopheles gambiae s.s. from different malaria transmission zones in Uganda.

BackgroundThere are major concerns over sustaining the efficacy of current malaria vector control interventions given the rapid spread of resistance, particularly to pyrethroids. This study assessed the bioefficacy of five WHO-recommended long-lasting insecticidal nets (LLINs) against pyrethroid-resistant Anopheles gambiae field populations from Uganda.MethodsAdult An. gambiae from Lira, Tororo, Wakiso and Kanungu districts were exposed to permethrin (0.75%) or deltamethrin (0.05%) in standard WHO susceptibility tests. Cone bioassays were used to measure the bioefficacy of four mono-treated LLINs (Olyset®, Interceptor®, Netprotect® and PermaNet® 2.0) and one combination LLIN (PermaNet® 3.0) against the four mosquito populations. Wireball assays were similarly conducted to determine knockdown rates. Species composition and kdr mutation frequency were determined for a sample of mosquitoes from each population. Chemical assays confirmed that test nets fell within target dose ranges.ResultsAnopheles gambiae s.s. predominated at all four sites (86 - 99% of Anopheles spp.) with moderate kdr L1014S allelic frequency (0.34 – 0.37). Confirmed or possible resistance to both permethrin and deltamethrin was identified for all four test populations. Reduced susceptibility to standard LLINs was observed for all four populations, with mortality rates as low as 45.8% even though the nets were unused. The combination LLIN PermaNet®3.0 showed the highest overall bioefficacy against all four An. gambiae s.l. populations (98.5 - 100% mortality). Wireball assays provided a more sensitive indicator of comparative bioefficacy, and PermaNet 3.0 was again associated with the highest bioefficacy against all four populations (76.5 – 91.7% mortality after 30 mins).ConclusionsThe bioefficacy of mono-treated LLINs against pyrethroid-resistant field populations of An. gambiae varied by LLIN type and mosquito population, indicating that certain LLINs may be more suitable than others at particular sites. In contrast, the combination LLIN PermaNet 3.0 performed optimally against the four An. gambiae populations tested. The observed reduced susceptibility of malaria vectors to mono-treated LLINs is of particular concern, especially considering all nets were unused. With ongoing scale-up of insecticidal tools in the advent of increasing resistance, it is essential that those interventions with proven enhanced efficacy are given preference particularly in areas with high resistance.