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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) (r s  = 0.6, p  = 0.002,n = 20) and 24-h mortality (M24) (r s  = 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.

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.

Anopheles stephensi (Diptera: Culicidae) is the vector of urban malaria in India and has a significant impact in transmitting infection in cities and towns. Further, WHO has also alarmed its invasive nature as a threat to African countries. Entomopathogenic fungi such as Beauveria bassiana and Metarhizium anisopliae have been found to be highly effective in controlling vector mosquito populations and therefore could be used in integrated vector control programs. Before employing the entomopathogenic fungi into the control programs, an effective isolate must be selected. Two separate experiments were conducted to evaluate the efficacy of Beauveria bassiana (Bb5a and Bb-NBAIR) and Metarhizium anisopliae (Ma4 and Ma-NBAIR) isolates against An. stephensi. Cement and mud panels were treated with fungal conidia with the concentration of 1 × 107 conidia/mL and adult An. stephensi mosquitoes were exposed to the treated panels (24 h after conidia were applied) by conducting WHO cone bioassay tests. The survival of the mosquitoes was monitored daily until the 10th day. In the second experiment, second instar larvae of An. stephensi were treated with fungal (Bb5a, Bb-NBAIR, Ma4 and Ma-NBAIR) conidia and blastospores with the spore concentration of 1 × 107 spores/mL. The survival of larvae was monitored until pupation. All the fungal isolates tested caused mortality in the adult mosquitoes, with varying median survival times. The Bb5a isolate reported lesser median survival times on both cement and mud panels (6 days). The treated mosquitoes showed similar survival rates for each fungal isolate irrespective of the panel type. There was no mortality in the treated larvae; however, a delay in larval development to pupae was observed compared with the untreated control larvae. Ma4-treated larvae took 11 days (95% CI = 10.7–11.2) to become pupae when compared with the untreated control larvae (6 days [95% CI = 5.6–6.3]). The findings of this study will be useful to consider EPF as a tool for the management of vector mosquitoes.

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. Graphical Abstract

Background The WHO cone bioassay is routinely used to evaluate the bioefficacy of insecticide-treated nets (ITNs) for product pre-qualification and confirmation of continued ITN performance during operational monitoring. Despite its standardized nature, variability is often observed between tests. We investigated the influence of temperature in the testing environment, mosquito feeding status and mosquito density on cone bioassay results. Methods Cone bioassays were conducted on MAGNet (alphacypermethrin) and Veeralin (alphacypermethrin and piperonyl butoxide (PBO)) ITNs, using laboratory-reared pyrethroid-resistant Anopheles funestus sensu stricto (FUMOZ strain) mosquitoes. Three experiments were conducted using standard cone bioassays following WHO-recommended test parameters, with one variable changed in each bioassay: (i) environmental temperature during exposure: 22–23 °C, 26–27 °C, 29–30 °C and 32–33 °C; (ii) feeding regimen before exposure: sugar starved for 6 h, blood-fed or sugar-fed; and (iii) mosquito density per cone: 5, 10, 15 and 20 mosquitoes. For each test, 15 net samples per treatment arm were tested with four cones per sample ( N  = 60). Mortality after 24, 48 and 72 h post-exposure to ITNs was recorded. Results There was a notable influence of temperature, feeding status and mosquito density on An. funestus mortality for both types of ITNs. Mortality at 24 h post-exposure was significantly higher at 32–33 °C than at 26–27 °C for both the MAGNet [19.33% vs 7%; odds ratio (OR): 3.96, 95% confidence interval (CI): 1.99–7.87, P  < 0.001] and Veeralin (91% vs 47.33%; OR: 22.20, 95% CI: 11.45–43.05, P  < 0.001) ITNs. Mosquito feeding status influenced the observed mortality. Relative to sugar-fed mosquitoes, The MAGNet ITNs induced higher mortality among blood-fed mosquitoes (7% vs 3%; OR: 2.23, 95% CI: 0.94–5.27, P  = 0.068) and significantly higher mortality among starved mosquitoes (8% vs 3%, OR: 2.88, 95% CI: 1.25–6.63, P  = 0.013); in comparison, the Veeralin ITNs showed significantly lower mortality among blood-fed mosquitoes (43% vs 57%; OR: 0.56, 95% CI: 0.38–0.81, P  = 0.002) and no difference for starved mosquitoes (58% vs 57%; OR: 1.05, 95% CI: 0.72–1.51, P  = 0.816). Mortality significantly increased with increasing mosquito density for both the MAGNet (e.g. 5 vs 10 mosquitoes: 7% vs 12%; OR: 1.81, 95% CI: 1.03–3.20, P  = 0.040) and Veeralin (e.g. 5 vs 10 mosquitoes: 58% vs 71%; OR 2.06, 95% CI: 1.24–3.42, P  = 0.005) ITNs. Conclusions The results of this study highlight that the testing parameters temperature, feeding status and mosquito density significantly influence the mortality measured in cone bioassays. Careful adherence to testing parameters outlined in WHO ITN testing guidelines will likely improve the repeatability of studies within and between product testing facilities. Graphical Abstract

Background PermaNet ® 3.0 is a deltamethrin-treated combination long-lasting insecticidal net with the addition of synergist piperonylbutoxide (PBO) on its roof section. It is designed to overcome the challenge posed by pyrethroid resistant vector populations against mainstream long-lasting insecticidal nets impregnated with pyrethroids only. The objective of this study was to determine insecticide resistance status of Anopheline and Culicine mosquitoes, to evaluate the bio-efficacy of PermaNet ® 3.0 nets and to assess household factors affecting the physical integrity of PermaNet ® 3.0 after 3 years of use. Methods Insecticide susceptibility test was conducted using the WHO tube test. Bio-activity of PermaNet ® 3.0 samples was evaluated using the WHO cone bioassay. Cross-sectional survey was conducted on 150 randomly selected households from two districts to determine household factors affecting net utilization. One hundred fifty PermaNet ® 3.0 nets were randomly collected from the community with replacement after 3 years of deployment and physical integrity of each net was assessed. Results Both Anopheles gambiae sensu lato and Culex quinquefasciatus developed resistance against permethrin and deltamethrin. However, following pre-exposure to synergist PBO the susceptibility of mosquito population increased to both permethrin (from 39% without to 92% with PBO against An. gambiae and from 28% without to 94% with PBO against Culex quinquefasciatus ) and deltamethrin (from 52% without to 99% with PBO against An. gambiae and from 43% without to 98% with PBO against Culex quinquefasciatus ). Eighty percent (80%) mortality was recorded in wild population of An. gambiae s.l. exposed to unused PermaNet ® 3.0, but its bioactivity subsequently declined as washing frequency increased from 0 to 20. The PBO coated roof section of unused PermaNet ® 3.0 resulted in higher mosquito mortality (100%) compared to the side panels without PBO (85%). House structure, cooking and washing habits, and damage due to household pests were cited as determinants associated with bed net deterioration. Bed net proportionate hole index (pHI) was ranged from 0 to 6064. Of the 150 PermaNet ® 3.0 nets assessed 80, 29 and 41 were considered as ‘good’, ‘acceptable’ and ‘too torn’, respectively. Conclusions The bio-efficacy evaluation of PermaNet ® 3.0 from Jimma area, southwestern Ethiopia showed moderate efficacy against pyrethroid resistant population of An. gambiae and Culex quinquefasciatus. Thus, NMCPs in parallel to deployment of LLINs, should implement timely insecticide resistance management and integrated vector management strategies to slowdown the evolution and further spread of insecticide resistance. Household factors such as, housing conditions, open flame fire used for cooking and rodent attack were identified as factors contributing to the observed reduced bed net physical integrity in the study area. Universal coverage of bed nets should be accompanied with community awareness creation and training on net utilization and handling.

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. Graphical Abstract

Determination of the residual activity of insecticides is an essential component in the selection of an appropriate insecticide for indoor residual spraying operations. This report presents the results of a laboratory study to evaluate the residual bio-efficacy of four insecticides sprayed on the most common house-wall surfaces that occur in Egypt (wood, mud, and cement) against Phlebotomus papatasi (Scopoli, 1786) (Diptera: Psychodidae) and Culex pipiens Linnaeus, 1758 (Diptera: Culicidae). In total, 28,050 P. papatasi females and 31,275 Cx. pipiens females were subjected to the WHO cone bioassay. Effective and extended control (≥80% mortality) was produced by lambda-cyhalothrin on indoor wood and cement surfaces. Lambda-cyhalothrin effectively controlled (>80% mortality) P. papatasi and Cx. pipiens for 10 and 12 wk postspray on wood surfaces, respectively. Deltamethrin effectively controlled Cx. pipiens for 8 wk on indoor wood, mud, and cement surfaces. Indoor and outdoor-kept surfaces treated with permethrin and malathion provided negligible efficacy against P. papatasi and Cx. pipiens. Phlebotomus papatasi was better able to survive bioassay exposure than Cx. pipiens against all insecticides investigated. The role surfaces might play in inhibiting IRS-based vector control endeavors in rural areas in developing countries was highlighted in this study. The current insecticide labeling system that includes both sand flies with mosquitoes under the same dosage category should be revised periodically.

BACKGROUND: Long-Lasting Insecticidal Nets (LLINs) efficacy could be compromised due to a lot of influences together with user compliance and vector population insecticide resistance status. Thus, this study was to assess the biological efficacy of DuraNet® with the help of the World Health Organization cone bioassay and field experimental hut. METHODS: A laboratory and a semi-field conditions experimental huts against Anopheles Mosquitoes were conducted in southwestern Ethiopia from September 2015 to January 2016. The bio efficacy of DuraNet® was evaluated using the WHO cone bioassay test and then its field efficacy was evaluated using experimental huts against the malaria vector population. RESULTS: World Health Organization cone bioassay tests against pyrethroid-resistant An. arabiensis led to mean percent mortality and knockdown of 78% and 93%, respectively. Washing of DuraNet® successively reduced its efficacy from 93% knockdown (0 wash) to 45% knockdown (20 washes). Similarly, mean mortality decreased from 84% (0 wash) to 47% (20 washes). A total of 1575 female mosquitoes were collected over 40 nights out of which 1373(87.8%) were An. gambiae s.l., 116 (7.4%) were Anopheles coustani and 107 (6.8%) were An. pharoensis. The mean blood-feeding rate was significantly lower (P < .001) in hut containing unwashed DuraNet® when compared to hut containing untreated DuraNet®. The mean mortality rate was significantly higher (P < .001) in hut containing DuraNet® when compared to hut containing untreated DuraNet®. Unwashed DuraNet® showed the highest personal protection 88.7% and 100% against An. Arabiensis and An. pharoensis, respectively. CONCLUSION: Both DuraNet® and PermaNet 2.0 moderate efficacy against a pyrethroid-resistant population of An. arabiensis from Ethiopia. The bio efficacy of DuraNet® was found below the WHO recommendation. Therefore, the real impact of the observed insecticide resistance against DuraNet® to be further studied under phase-III trials, the need for new alternative vector control tools remains critical.

Background Biting midges of the genus Culicoides Latreille, 1809 (Diptera: Ceratopogonidae) cause a significant biting nuisance to equines and are responsible for the biological transmission of African horse sickness virus (AHSV). While currently restricted in distribution to sub-Saharan Africa, AHSV has a history of emergence into southern Europe and causes one of the most lethal diseases of horses and other species of Equidae. In the event of an outbreak of AHSV, the use of insecticide treated nets (ITNs) to screen equine accomodation is recommended by competent authorities including the Office International des Épizooties (OIE) in order to reduce vector-host contact. Methods Seven commercially avaliable pyrethroid insecticides and three repellent compounds, all of which are licensed for amateur use, were assessed in modified World Health Organization (WHO) cone bioassay trials in the laboratory using a colony line of Culicoides nubeculosus (Meigen), 1830. Two field trials were subsequently conducted to test the efficiency of treated net screens in preventing entry of Culicoides . Results A formulation of cypermethrin (0.15 % w/w ) and pyrethrins (0.2 % w/w ) (Tri-Tec 14®, LS Sales (Farnham) Ltd, Bloxham, UK) applied to black polyvinyl-coated polyester insect screen (1.6 mm aperture; 1.6 mm thickness) inflicted 100 % mortality on batches of C. nubeculosus following a three minute exposure in the WHO cone bioassays at 1, 7 and 14 days post-treatment. Tri-Tec 14® outperformed all other treatments tested and was subsequently selected for use in field trials. The first trial demonstrated that treated screens placed around an ultraviolet light-suction trap entirely prevented Culicoides being collected, despite their collection in identical traps with untreated screening or no screening. The second field trial examined entry of Culicoides into stables containing horses and found that while the insecticide treated screens reduced entry substantially, there was still a small risk of exposure to biting. Conclusions Screened stables can be utilised as part of an integrated control program in the event of an AHSV outbreak in order to reduce vector-host contact and may also be applicable to protection of horses from Culicoides during transport.