Timing is everything: a simple chemical method to determine the bioavailable surface concentration of insecticide for...

Insecticide-Treated Nets (ITNs) must retain bioefficacy after 20 washes to receive World Health Organization (WHO) prequalification. Days between washes (wash interval, WI),  are determined using the regeneration time (RT) method. Mosquitoes are exposed to surface insecticide in a repeated measures experiment after washing. RT is the number of days required for mosquito mortality to plateau, assumed to reflect maximal surface concentration. However, mosquito mortality may reach 100% before all active ingredient (AI) migrates to the ITN surface making RT dependent on test strain. Using too short WI results in (1) less AI removed per wash allowing an ITN to withstand more washes than it might under user conditions or (2) insufficient AI becomes available and a product may fail bio-efficacy testing.

Three methods were used to determine surface concentrations of deltamethrin and Piperonyl Butoxide (PBO) in commercial incorporated ITNs: (1) Before and After Method, (BAM) using Gas-Chromatography Flame-Ionization Detection (GC-FID) of ITN sample chemical content 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 with biological efficacy measured by median knock-down time (MKDT) using pyrethroid susceptible and resistant mosquitoes. BAM and CAM were compared in a wash Resistance Index (WRI) test.

CAM dosages correlated strongly with MKDT (r = - 0.91, p = 0.0006), inter-sample variation below 2%, and comparable RT estimates. CAM surface extracts also correlated well with MKDT. WIs derived by chemical methods were longer than these determined by standard WHO method. Comparison of acetone and soap-washed nets indicate that part of the surface available pyrethroid has little biological activity, likely due to recrystallization on the yarn surface, confirmed by electron microscopy. Combining MKDT and SWAM 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.