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

Abstract 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 of ITNs 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) bioassay procedures. However, longitudinal evaluation may introduce daily heterogeneity due to mosquito batch variability, complicating RT determination. To overcome this, nets at each stage of regeneration (i.e., 1, 2, 3, 5 &7 days post wash) were prepared in advance and refrigerated, then a complete regeneration series was tested with a single mosquito batch on one testing day over 4 days (N=1 per timepoint /day). This study validated the complete series method (CSM) against the LM. Methods: The RT of two incorporated alpha-cypermethrin and piperonyl butoxide (PBO) and one incorporated permethrin with PBO ITNs were determined using laboratory-reared resistant Anopheles arabiensis under standard laboratory conditions. LM methods and CSM were compared in two experiments 1) refrigerated nets acclimatized for two hours (Test 1), and 2) refrigerated nets acclimatized for three hours (Test 2). Four replicates per day of regeneration were tested per ITN product with 50 mosquitoes exposed per replicate to give an equivalent sample size to that of LM. The RTs and overall heterogeneity from these methods were compared descriptively. Results: The RT measured by the CSM showed 100% agreement with that measured by LM. 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, ratio of LM:CSM was 2.66 in test one and 2.49 in test two. The magnitude of mortality measured in bioassays depended on sample acclimation after refrigeration. Conclusion The CSM is a convenient method for determining the regeneration times of ITNs that are prepared in advance, and a complete series of samples is removed from the fridge, defrosted and evaluated on a single day allowing easy facility scheduling and reduced daily heterogeneity. The CSM gave comparable estimates of regeneration time as the LM.