Timing is everything: a simple chemical method to determine the bioavailable surface concentration of insecticide for insecticide-treated net evaluation

(2025) 24:362 Skovmand et al. Malaria Journal https://doi.org/10.1186/s12936-025-05602-4 Malaria Journal Open Access RESEARCH Timing is everything: a simple chemical method to determine the bioavailable surface concentration of insecticide for insecticide‑treated net evaluation Ole Skovmand1, Duoc M. Dang2, Trung Q. Tran2,3, Rune Bosselmann4 and Sarah J. Moore5,6,7,8* Abstract Background 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 bioefficacy testing. Methods 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 FlameIonization 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. Results and recommendations 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 soapwash 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. Keywords Insecticide-Treated Bednet (ITN), Long-Lasting Insecticidal Nets (LLIN), Test methods, Bioefficacy, Regeneration time, Wash interval, Median knock down time (MKDT) *Correspondence: Sarah J. Moore Full list of author information is available at the end of the article © The Author(s) 2025. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/. Skovmand et al. Malaria Journal (2025) 24:362 Background Most malaria cases are in Africa [1] where the main malaria transmitting mosquito species mostly bite humans indoors during sleeping hours [2]. Therefore, insecticide-treated bed nets (ITNs) are effective means of malaria prevention [3]. More than 3 billion ITNs have been distributed in malaria endemic regions, mainly Africa, but also Latin America and Asia [1]. Since around the year 2000, most ITNs have been treated in a way that the insecticide is not easily washed off, so-called long-lasting insecticidal nets (LLIN). Two technologies are currently used to make LLINs [4]: (i) polyester nets coated with insecticide in a foulard process, and (ii) polyethylene or polypropylene nets with active ingredient (AI) incorporated during yarn extrusion. To be qualified for the donor financed market, a candidate ITN must pass tests defined by the World Health Organization (WHO) [5]. These tests comprise chemical and physical tests to define ITN product specifications and biological assays to test if the ITN remains sufficiently efficacious against mosquitoes following standard procedures intended to simulate use conditions [6]. LLINs differ from earlier dip-treated nets by retaining the insecticide after many washes, due to better adherence of insecticide on the surface and a reservoir within coatings or yarn polymers from which insecticide can migrate to the surface. WHO has defined evaluation criteria to predict whether an ITN can withstand repeated washing and use over several years, assessed in artificial aging assays that for practical reasons last only a few months [6]. An ITN can only be functionally long lasting if it maintains sufficient surface concentration of insecticide despite losses from washing, abrasion, evaporation or photodegradation. Because wash-off was the main limitation of dipped nets, wash resistance is among the key parameters currently measured. ITNs must maintain efficacy after 20 standard washes with continued mosquito mortality and protection against mosquito bites [7]. Wash resistance is defined as the number of washes for which the point estimate for the primary endpoint (usually mosquito mortality) remains within 5% of the endpoint result for the unwashed ITN, in short, retaining similar efficacy after 20 washes as before washing [8]. The critical parameter for evaluating insecticide loss to wash-off is the speed at which the net recovers insecticidal activity after 3 repeated soap washings in a day (Box 1). The regeneration time (RT) is defined as the time required (in days) to reach the plateau of bio-efficacy which is usually measured by either 24 h mortality (M24) or sublethal incapacitation i.e. knock down after 60 min (KD60) [6, 8]. RT depends on the release rate (RR) of insecticide from the yarn or coating to the surface. Page 2 of 21 RR gradually declines as the surface saturates with the molecular form of insecticide. Howeve (...truncated)