Background Insecticide-treated nets (ITNs) undergo a series of tests to obtain listing by World Health Organization (WHO) Prequalification. These tests characterize the bioefficacy, physical and chemical properties of the ITN. ITN procurers assume that product specifications relate to product performance. Here, ITN test methods and their underlying assumptions are discussed from the perspective of the ITN manufacturing process and product characteristics. Methods Data were extracted from WHO Pesticide Evaluation Scheme (WHOPES) meeting reports from 2003 to 2017, supplemented with additional chemical analysis to critically evaluate ITNs bioassays with a focus on sampling, washing and wash resistance, and bioefficacy testing. Production methods for ITNs and their impact on testing outcomes are described. Results and recommendations ITNs are not homogenous products. They vary within panels and between the sides and the roof. Running tests of wash resistance using a before/after tests on the same sample or band within a net reduces test variability. As mosquitoes frequently interact with ITN roofs, additional sampling of the roof when evaluating ITNs is advisable because in nets where roof and sides are of the same material, the contribution of roof sample (20–25%) to the average is less than the tolerance for the specification (25%). Mosquito mortality data cannot be reliably used to evaluate net surface concentration to determine regeneration time (RT) and resistance to washing as nets may regenerate beyond the insecticide concentrations needed to kill 100% of susceptible mosquitoes. Chemical assays to quantify surface concentration are needed. The Wash Resistance Index (WRI) averaged over the first four washes is only informative if the product has a log linear loss rate of insecticide. Using a WRI that excludes the first wash off gives more reliable results. Storage conditions used for product specifications are lower than those encountered under product shipping and storage that may exceed 50 °C, and should be reconsidered. Operational monitoring of new ITNs and linking observed product performance, such as bioefficacy after 2 or 3 years of use, with product characteristics, such as WRI, will aid the development of more robust test methods and product specifications for new products coming to market.

Background: Insecticide Treated Nets (ITNs) must meet bioefficacy performance standards after 20 washes to receive WHO prequalification. Days between washes (wash interval, WI) is determined using the regeneration time (RT) method. Mosquitoes are exposed to active ingredients (AI) on the net surface in a repeated measures experiment up to seven days after washing. RT is the number of days for mortality to reach a plateau where the surface insecticide concentration is assumed to be maximal and defines WI. However, mosquito mortality may reach 100% before all insecticide migrates to the ITN surface and thus RT is a function of test mosquitoes and may be underestimated. Using too short WI results in 1) less insecticide removed per wash allowing an ITN to withstand more washes than it might under user conditions or 2) insufficient insecticide becomes available and a product may fail bio-efficacy testing. Methods: Three methods were used to determine surface concentrations of deltamethrin and Piperonyl Butoxide (PBO) in commercial ITNs: 1) Before and After Method, (BAM) using Gas-Chromatography Flame-Ionization Detection (GC-FID) before and after a standard soap-wash; 2) Soap Wash and Analysis Method (SWAM) measuring the AI from the soap-washing water using High Pressure Liquid Chromatography (HPLC); 3) Cold Acetone Method (CAM) extracting AI in cold acetone before HPLC. RT of deltamethrin and PBO were estimated using SWAM or CAM and correlated biological efficacy measured by median knock-down time (MKDT) with pyrethroid susceptible and resistant mosquitoes. BAM and CAM were compared in a wash Resistance Index (WRI) test. Results and Recommendations: CAM dosages after soap-wash showed 90% correlation with MKDT (r=-0.91, p=0.0006) and measured the same regeneration time. Inter-sample variation was <2%. Wash intervals were longer than these determined by standard WHO method. Comparing acetone and soap washed ITNs, it is concluded that a part of the surface available pyrethroid has little biological activity, most likely the pyrethroid recrystallized on the yarn surface, confirmed by EM scanning. Combining MKDT and CAM after soap-wash could improve predictions of ITN performance. BAM can be used to measure loss of bio-active AI from the yarn surface following protocols currently recommended by WHO.