Complete series method (CSM): a convenient method to reduce daily heterogeneity when evaluating the regeneration time (RT) of insecticide-treated nets (ITNs).

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.

Temperature, mosquito feeding status and mosquito density influence the measured bio-efficacy of insecticide-treated nets in cone assays.

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.