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Background: "Regeneration time" (RT) denotes the time required to obtain a stable mortality rate for mosquitoes exposed to insecticide-treated nets (ITNs) after three consecutive washes of a net in a day. The RT informs the wash interval used to artificially age ITNs to simulate their lifetime performance under user conditions (20 washes). RT was estimated following World Health Organization (WHO) longitudinal method (LM) procedures. Longitudinal evaluation may introduce heterogeneity due to mosquito batch variability, complicating RT determination. To overcome this, nets at each stage of regeneration (i.e., 1, 2, 3, 5 and 7 days post wash) were prepared in advance and refrigerated; then, a complete regeneration series was tested with a single mosquito batch on 1 testing day, completing four series over 4 days. This study compared the complete series method (CSM) against the LM. Methods: The overall heterogeneity in the methods for estimating RT of one incorporated alpha-cypermethrin and piperonyl butoxide (PBO) and one incorporated permethrin with PBO ITNs was determined using laboratory-reared resistant Anopheles arabiensis under standard laboratory conditions. LM methods and CSM were compared in two experiments with refrigerated nets acclimated for (i) 2 h (test 1) and (ii) 3 h (test 2). Four regeneration replicates per day were tested per ITN product with 50 mosquitoes exposed per replicate (equivalent sample size to LM). The heterogeneity from these methods was compared descriptively. Results: The intra-method variability for unwashed pieces was minimal, with variance of 1.26 for CSM and 1.18 for LM. For unwashed nets, LM had substantially greater variance and ratio of LM:CSM was 2.66 in test 1 and 2.49 in test 2. The magnitude of mortality measured in bioassays depended on sample acclimation after refrigeration. Conclusions: The CSM is a convenient method for determining the regeneration times. ITNs are prepared in advance, reducing pressure to prepare all samples to start on a single day. A complete regeneration series of samples is removed from the refrigerator, defrosted and evaluated on a single day with one mosquito batch reducing the influence of mosquito batch heterogeneity on results. Replicates can be conducted over several days but do not have to be conducted on consecutive days, allowing easy facility scheduling. [ABSTRACT FROM AUTHOR]

Background: Increasing metabolic resistance in malaria vector mosquitoes resulted in the development of insecticide-treated nets (ITNs) with active ingredients (AI) that target them. Bioassays that accurately measure the mortality induced by these AIs on ITNs are needed. Mosquito metabolic enzyme expression follows a circadian rhythm. Thus, this study assessed (i) influence of the time of day of mosquito exposure and (ii) timing of assessment of mortality post exposure (24 and 72 h) to ITNs against vectors that are susceptible to pyrethroids and those with metabolic and knockdown resistance mechanisms. Methods: Two cone bioassay experiments were conducted following World Health Organization (WHO) guidelines. Firstly, on ITNs incorporated with 2 g AI/kg of deltamethrin (DM) alone, or combined with 8 g AI/kg piperonyl butoxide (PBO) synergist, during the day (9:00–14:00 h) and repeated in the evening (18:00–20:00 h). This was followed by a confirmatory experiment during the afternoon (12:00–14:00 h) and repeated in the night (22:00–24:00 h) using mosquitoes unexposed or pre-exposed to PBO for 1 h before exposure to DM ITNs. Each net piece was tested with a minimum of eight cones per time (N = 24). The outcome was mortality after 24 h (M24) or 72 h (M72) of holding. Results: The cone bioassays performed using metabolic resistant mosquitoes during the evening showed significantly lower M24 than those performed in the day for DM: odds ratio (OR) 0.14 [95% confidence interval (CI) 0.06–0.30, p < 0.0001] and DM PBO [OR 0.29 (95% CI 0.18—0.49, p < 0.0001). M72 was higher than M24 for metabolic resistant mosquitoes exposed to DM [OR 1.44 (95% CI 1.09–1.88), p = 0.009] and DM PBO [OR 1.82 (95% CI 1.42–2.34), p < 0.0001]. An influence of hour of experiment and time of assessment was not observed for mosquitoes that had knockdown resistance or that were pyrethroid-susceptible. Conclusions: Time of day of experiment and hour of assessment of delayed mortality after exposure of mosquitoes are important considerations in evaluating insecticides that interact with mosquito metabolism to counter metabolic resistant mosquitoes. This is important when evaluating field-aged ITNs that may have lower concentrations of AI. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Simple Summary: Membrane feeding assays have been widely used in malaria transmission research and insectary colony maintenance. Here, we investigate whether a membrane feeder can replace animal baits for evaluating insecticide-treated nets (ITNs) bio-efficacy in the World Health Organization (WHO) tunnel test. The effect of (1) alternative baits, (2) exposure time, and (3) mosquito density on the endpoints of mosquito mortality and feeding inhibition or feeding success was investigated. Our results show that similar mortality at 24-h (M24) or 72-h (M72) is estimated using either a membrane feeder or a rabbit bait with an overnight (12 h) exposure. However, the membrane measured higher blood feeding inhibition than the rabbit, likely due to the absence of host cues, notably carbon dioxide. Therefore, the membrane feeder may be used instead of an animal bait to test mortality endpoints in WHO tunnel tests and blood feeding rates need to be improved. Experimental results demonstrated that using 50 or 100 mosquitoes per replicate measure the same for mortality and feeding inhibition endpoints with an animal bait. Therefore, WHO tunnel tests may be run with lower mosquito densities. This will reduce strain on insectaries to produce sufficient mosquitoes to meet the large sample sizes needed for bio-efficacy durability monitoring of chlorfenapyr ITNs that must be evaluated in "free-flying" bioassays. The standard World Health Organization (WHO) tunnel test is a reliable laboratory bioassay used for "free-flying" testing of insecticide-treated nets (ITNs) bio-efficacy where mosquitoes pass through a ITN sample to reach a live animal bait. Multiple parameters (i.e., bait, exposure time, and mosquito density) may affect the outcomes measured in tunnel tests. Therefore, a comparison was conducted of alternative hosts, exposure time, and lower mosquito density against the current gold standard test (100 mosquitoes, animal bait, and 12-h exposure) as outlined in the WHO ITN evaluation guideline. This was done with the aim to make the tunnel test cheaper and with higher throughput to meet the large sample sizes needed for bio-efficacy durability monitoring of chlorfenapyr ITNs that must be evaluated in "free-flying" bioassays. Methods: A series of experiments were conducted in the WHO tunnel test to evaluate the impact of the following factors on bio-efficacy endpoints of mosquito mortality at 24-h (M24) and 72-h (M72) and blood-feeding success (BFS): (1) baits (rabbit, membrane, human arm); (2) exposure time in the tunnel (1 h vs. 12 h); and (3) mosquito density (50 vs. 100). Finally, an alternative bioassay using a membrane with 50 mosquitoes (membrane-50) was compared to the gold standard bioassay (rabbit with 100 mosquitoes, rabbit-100). Pyrethroid-resistant Anopheles arabiensis and pyrethroid susceptible Anopheles gambiae were used to evaluate Interceptor® and Interceptor® G2 ITNs. Results: Using a human arm as bait gave a very different BFS, which impacted measurements of M24 and M72. The same trends in M24, M72 and BFS were observed for both Interceptor® ITN and Interceptor® G2 unwashed and washed 20 times measured using the gold standard WHO tunnel test (rabbit-100) or rabbit with 50 mosquitoes (rabbit-50). M24, M72 and BFS were not statistically different when either 50 or 100 mosquitoes were used with rabbit bait in the tunnel bioassay for either the susceptible or resistant strains. No systematic difference was observed between rabbit-50 and rabbit-100 in the agreement by the Bland and Altman method (B&A). The mean difference was 4.54% (−22.54–31.62) in BFS and 1.71% (−28.71–32.12) in M72 for rabbit-50 versus rabbit-100. Similar M24, M72 and lower BFS was measured by membrane-50 compared to rabbit-100. No systematic difference was observed in the agreement between membrane-50 and rabbit-100, by B&A. The mean difference was 9.06% (−11.42–29.64) for BSF and −5.44% (−50.3–39.45) for M72. Both membrane-50, rabbit-50 and rabbit-100 predicted the superiority of Interceptor® G2 over Interceptor® ITN for the resistant strain on M72. Conclusion: These results demonstrate that WHO tunnel tests using rabbit bait may be run with 50 mosquitoes to increase sample sizes needed for bio-efficacy durability monitoring of ITNs in "free-flying" bioassays. Using a membrane feeder with 50 mosquitoes is a potential replacement for the WHO tunnel bioassay with animal bait if control blood feeding rates can be improved to 50% because blood feeding impacts mosquito survival after exposure to insecticides. [ABSTRACT FROM AUTHOR]

Simple Summary: New types of bed nets are being developed which contain a pyrethroid plus a second chemical because of the development and increased frequency of mosquito mechanisms to avoid being killed by pyrethroids. When insecticide-treated bed nets are being trialed for efficacy or released onto the market it is essential to measure how effective the net is in killing mosquitoes, which includes testing how quickly insecticide is lost or degraded due to routine wear and tear. Pyrethroid-resistant mosquitoes are needed to test the effectiveness and insecticidal persistence of the second chemical, because they are not all killed by the pyrethroid, allowing the killing effect of the two chemicals to be evaluated independently. However, because resistance status varies between populations of mosquitoes that possess different resistance mechanisms, and because resistance intensity in a population can change over time, a method is needed to characterise the resistant mosquitoes. A focus group of experts discussed how this should best be done, considering pros and cons of different approaches, and co-wrote a protocol, which will be valuable for malaria control programmes and stakeholders wanting to test the effective lifespan of a new bed net in terms of the active ingredient bioefficacy. Durability monitoring of insecticide-treated nets (ITNs) containing a pyrethroid in combination with a second active ingredient (AI) must be adapted so that the insecticidal bioefficacy of each AI can be monitored independently. An effective way to do this is to measure rapid knock down of a pyrethroid-susceptible strain of mosquitoes to assess the bioefficacy of the pyrethroid component and to use a pyrethroid-resistant strain to measure the bioefficacy of the second ingredient. To allow robust comparison of results across tests within and between test facilities, and over time, protocols for bioefficacy testing must include either characterisation of the resistant strain, standardisation of the mosquitoes used for bioassays, or a combination of the two. Through a series of virtual meetings, key stakeholders and practitioners explored different approaches to achieving these goals. Via an iterative process we decided on the preferred approach and produced a protocol consisting of characterising mosquitoes used for bioefficacy testing before and after a round of bioassays, for example at each time point in a durability monitoring study. We present the final protocol and justify our approach to establishing a standard methodology for durability monitoring of ITNs containing pyrethroid and a second AI. [ABSTRACT FROM AUTHOR]

Background: Insecticide-treated net (ITN) durability is evaluated using longitudinal bioefficacy and fabric integrity sampling post-distribution. Interceptor® G2 was developed for resistance management and contains two adulticides: alpha-cypermethrin and chlorfenapyr; it is a pro-insecticide that is metabolized into its active form by mosquito-detoxifying enzymes and may be enhanced when the mosquito is physiologically active. To elucidate the impact of bioassay modality, mosquito exposures of the alphacypermethrin ITN Interceptor® and dual adulticide Interceptor® G2 were investigated. Methods: This study evaluated the performance of Interceptor® G2 compared to Interceptor® against local strains of mosquitoes in Tanzania. Unwashed and 20× times washed nets were tested. Efficacy of ITNs was measured by four bioassay types: (1) World Health Organisation (WHO) cone test (cone), (2) WHO tunnel test (tunnel), (3) Ifakara ambient chamber test (I-ACT) and (4) the WHO gold standard experimental hut test (hut). Hut tests were conducted against free-flying wild pyrethroid metabolically resistant Anopheles arabiensis and Culex quinquefasciatus. Cone, tunnel and I-ACT bioassays used laboratory-reared metabolically resistant An. arabiensis and Cx. quinquefasciatus and pyrethroid susceptible Anopheles gambiae sensu stricto and Aedes aegypti. Results: Against resistant strains, superiority of Interceptor® G2 over Interceptor® was observed in all "free-flying bioassays". In cone tests (which restrict mosquito flight), superiority of Interceptor® over Interceptor® G2 was recorded. Mortality of unwashed Interceptor® G2 among An. arabiensis was lowest in hut tests at 42.9% (95% CI: 37.3–48.5), although this increased to 66.7% (95% CI: 47.1–86.3) by blocking hut exit traps so mosquitoes presumably increased frequencies of contact with ITNs. Higher odds of mortality were consistently observed in Interceptor® G2 compared to Interceptor® in "free-flying" bioassays using An. arabiensis: tunnel (OR = 1.42 [95% CI:1.19–1.70], p < 0.001), I-ACT (OR = 1.61 [95% CI: 1.05–2.49], p = 0.031) and hut (OR = 2.53 [95% CI: 1.96–3.26], p < 0.001). Interceptor® and Interceptor® G2 showed high blood-feeding inhibition against all strains. Conclusion: Both free-flying laboratory bioassays (WHO Tunnel and I-ACT) consistently measured similarly, and both predicted the results of the experimental hut test. For bioefficacy monitoring and upstream product evaluation of ITNs in situ, the I-ACT may provide an alternative bioassay modality with improved statistical power. Interceptor G2® outperformed Interceptor ® against pyrethroid-resistant strains, demonstrating the usefulness of chlorfenapyr in mitigation of malaria. [ABSTRACT FROM AUTHOR]

Background: Between 2000 and 2019, more than 1.8 billion long-lasting insecticidal nets (LLINs) were distributed in Africa. While the insecticidal durability of LLINs is around 3 years, nets are commonly discarded 2 years post distribution. This study investigated the factors associated with the decision of users to discard LLINs. Methods: A mixed-method sequential explanatory approach using a structured questionnaire followed by focus group discussions (FGDs) to collect information on experiences, views, reasons, how and when LLINs are discarded. Out of 6,526 households that responded to the questionnaire of LLINs durability trial, 160 households were randomly selected from the households in four villages in Bagamoyo Tanzania for FGDs but only 155 households participated in the FGDs. Five of the household representatives couldn't participate due to unexpected circumstances. A total of sixteen FGDs each comprising of 8–10 adults were conducted; older women (40–60 years), older men (40–60 years), younger women (18–39 years), younger men (18–39 years). During the FGDs, participants visually inspected seven samples of LLINs that were "too-torn" based on Proportionate Hole Index recommended by the World Health Organization (WHO) guidelines on LLIN testing, the nets were brought to the discussion and participants had to determine if such LLINs were to be kept or discarded. The study assessed responses from the same participants that attended FGD and also responded to the structured questionnaire, 117 participants fulfilled the criteria, thus data from only 117 participants are analysed in this study. Results: In FGDs, integrity of LLIN influenced the decision to discard or keep a net. Those of older age, women, and householders with lower income were more likely to classify a WHO "too-torn" net as "good". The common methods used to discard LLINs were burning and burying. The findings were seen in the quantitative analysis. For every additional hole, the odds of discarding a WHO "too-torn" LLIN increased [OR = 1.05 (95%CI (1.04–1.07)), p < 0.001]. Younger age group [OR = 4.97 (95%CI (3.25–7.32)), p < 0.001], male-headed households [OR = 6.85 (95%CI (4.44 –10.59)), p < 0.001], and wealthy households [OR = 3.88 (95%CI (2.33–6.46)), p < 0.001] were more likely to discard LLINs. Conclusion: Integrity of LLIN was the main determinant for discarding or keeping LLINs and the decision to discard the net is associated with socioeconomic status of the household, and the age and gender of respondents. WHO "too torn" nets are encouraged to be used instead of none until replacement, and disposal of nets should be based on recommendation. [ABSTRACT FROM AUTHOR]

Background: A clear understanding of mosquito biology is fundamental to the control efforts of mosquito-borne diseases such as malaria. Mosquito mark-release-recapture (MMRR) experiments are a popular method of measuring the survival and dispersal of disease vectors; however, examples with African malaria vectors are limited. Ethical and technical difficulties involved in carrying out MMRR studies may have held back research in this area and, therefore, a device that marks mosquitoes as they emerge from breeding sites was developed and evaluated to overcome the problems of MMRR. Methods: A modified self-marking unit that marks mosquitoes with fluorescent pigment as they emerge from their breeding site was developed based on a previous design for Culex mosquitoes. The self-marking unit was first evaluated under semi-field conditions with laboratory-reared Anopheles arabiensis to determine the marking success and impact on mosquito survival. Subsequently, a field evaluation of MMRR was conducted in Yombo village, Tanzania, to examine the feasibility of the system. Results: During the semi-field evaluation the self-marking units successfully marked 86% of emerging mosquitoes and there was no effect of fluorescent marker on mosquito survival. The unit successfully marked wild male and female Anopheles gambiae sensu lato (s.l.) in sufficiently large numbers to justify its use in MMRR studies. The estimated daily survival probability of An. gambiae s.l. was 0.87 (95% CI 0.69–1.10) and mean dispersal distance was 579 m (95% CI 521–636 m). Conclusions: This study demonstrates the successful use of a self-marking device in an MMRR study with African malaria vectors. This method may be useful in investigating population structure and dispersal of mosquitoes for deployment and evaluation of future vector control tools, such as gene drive, and to better parameterize mathematical models. [ABSTRACT FROM AUTHOR]

Background: Extensive employment of long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) has substantially reduced malaria morbidity and mortality in sub-Saharan Africa. These tools target indoor resting and biting vectors, and may select for vectors that bite and rest outdoors. Thus, to significantly impact this residual malaria transmission outdoors, tools targeting outdoor transmission are required. Repellents, used for personal protection, offer one solution. However, the effectiveness of this method hinges upon its community acceptability. This study assessed the feasibility of using repellents as a malaria prevention tool in Mbingu village, Ulanga, Southern Tanzania. Methodology: Change in knowledge, attitude and practice (KAP) in relation to repellent use was assessed before and after the implementation of a cluster randomized clinical trial on topical repellents in rural Tanzania where repellent and placebo lotion were provided free of charge to 940 households for a period of 14 months between July 2009 and August 2010. Compliance, defined as the number of evenings that participants applied the recommended dose of repellent every month during the study period, was assessed using questionnaires, administered monthly during follow up of participants in the clinical trial. Focus group discussions (FGDs) were conducted in the same community three years later to assess the community's KAP in relation to repellents and preference to different repellent formats. Results: At baseline, only 0.32% (n = 2) households in the intervention arm and no households in the control arm had ever used topical repellents. During follow-up surveys, significantly more households, 100% (n = 457) in intervention arm relative to the control, 84.03% (n = 379), (p = <0.001) perceived the repellent to be effective. Post-study, 99.78% (n = 462) and 99.78% (n = 463), (p = 0.999) in the intervention and control arms respectively, were willing to continue repellent use. Mosquito nuisance motivated repellent use. From the FGDs, it emerged that most respondents preferred bed nets to repellents because of their longevity and cost effectiveness. Conclusion: High repellent acceptability indicates their feasibility for malaria control in this community. However, to improve the community's uptake of repellents for use complimentary to LLINs for early evening and outdoor protection from mosquito bites, longer lasting and cheap formats are required. [ABSTRACT FROM AUTHOR]

Background: Insecticides targeting adult mosquitoes are the main way of controlling malaria. They work not only by killing mosquitoes, but also by repelling and irritating them. Indeed their repellent action gives valuable personal protection against biting mosquitoes. In the context of malaria control this personal protection is especially relevant when mosquitoes are infectious, whereas to protect the community we would prefer that the mosquitoes that are not yet infectious are killed (so, not repelled) by the insecticide. As the infectious stage of malaria parasites increases the motivation of mosquitoes to bite, we predicted that it would also change their behavioural response to insecticides. Results: With two systems, a laboratory isolate of the rodent malaria Plasmodium berghei infecting Anopheles gambiae and several isolates of P. falciparum obtained from schoolchildren in Tanzania that infected Anopheles arabiensis, we found that mosquitoes harbouring the infectious stage (the sporozoites) of the parasite were less repelled by permethrin-treated nets than uninfected ones. Conclusions: Our results suggest that, at least in the laboratory, malaria infection decreases the personal protection offered by insecticide-treated nets at the stage where the personal protection is most valuable. Further studies must investigate whether these results hold true in the field and whether the less effective personal protection can be balanced by increased community protection. [ABSTRACT FROM AUTHOR]