Timing is everything: A simple chemical method to determine the bioavailable surface concentration of insecticide for insecticide treated net (ITN) evaluation

Abstract

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.

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

“…The regeneration time (RT) measured by both WHO LM and CSM using a pyrethroid resistant strain was 7 days and the RT measured by each method agreed for all tests. These results highlight the importance of conducting rigorous regeneration time evaluations with adequate sample sizes to ensure reproducible results as the incorrect regeneration time has large implications for downstream evaluations of ITNs [33] that rely on washing to arti cially age the ITN [10].…”

“…This increase in bioavailable AI is re ected by an increase in the mortality measured in bioassays. Once the regeneration curve " attens" it indicates stability, suggesting that the mortality rate and consequently AI has reached a plateau or equilibrium [33].…”