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

doi:10.21203/rs.3.rs-2751835/v1

Download PDF

Research Article

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

https://doi.org/10.21203/rs.3.rs-2751835/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

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.

Insecticide-Treated Bednet

ITN

Long-Lasting Insecticidal Nets

LLIN

test methods

bioefficacy

regeneration time

wash interval

Median knock down time

MKDT

Most malaria cases are in Africa [1] and the main African malaria transmitting mosquito species mostly bite humans indoors during sleeping hours [2]. Therefore, Insecticide Treated bed Nets (ITNs) are an effective means of malaria prevention [3] and more than 2 billion ITNs have been distributed in malaria endemic countries especially in Africa, but also Latin America, Southern and South-East 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) the insecticide is mixed with a coating and applied onto the surface of polyester nets in a foulard process, or (ii) the insecticide is mixed with polyethylene or polypropylene and incorporated into yarns made in an extrusion process, which are then knitted to nets.

To be qualified for the donor financed market, candidate ITN must pass a set of 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 is sufficiently efficacious against mosquitoes following standard procedures intended to simulate use conditions [6]. LLINs distinguished themselves from previous nets, that were simply dipped in insecticide solutions, by retaining the insecticide after many washes, due to better adherence of the insecticide on the surface and to unexposed insecticide reservoir in the coating or within the yarn polymer from where the insecticide can migrate to the surface.

WHO has defined preliminary evaluation criteria designed to predict if an ITN can withstand repeated washing and use over a number of years, in assays that for practical reasons, are conducted over the course of a few months [6]. An ITN can only be functionally long lasting if it provides a sufficient surface concentration of insecticide to kill mosquitoes over its lifespan despite wash off, rub off, evaporation or other inactivation of the insecticide. Since insecticide loss from washing was considered the biggest problem for dipped nets, resistance to wash off is among the key parameters currently measured. Therefore, according to current guidance, ITNs must provide a barrier against mosquito bites and continue to kill mosquitoes even after they have been repeatedly washed by users up to 20 times [7].

The key parameter for evaluating insecticide loss to wash-off is the speed at which the net regenerates insecticidal activity after 3 repeated washings with soap in a day: the “regeneration time” (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 hour mortality (M24) or sublethal incapacitation i.e. knock down after 60 minutes (KD60) [6].

RT depends on several factors. The Release Rate (RR) determines how fast insecticide is regenerated from the storage (coating or yarn polymer) in a molecular form to the surface of the yarn to become bioavailable. RR gradually declines as the surface is saturated. However, several insecticides have crystal form at room temperature, so the crystallisation is a concurrent process that absorbs the molecular form. Therefore, for such insecticides, RR never becomes 0, but it can be expected that after a certain time, the amount generated from storage and gone into crystals become stable and identical and that time will then be an ideal RT.

Once determined, the RT is used as the “wash interval” (Box 1) i.e., the time interval between serial wash-and-bioassays cycles to observe if the net maintains bioefficacy up to 20 washes [6]. The bioassay data for the washing cycles are supplemented with chemical analysis of the nets using standard Collaborative International Pesticides Analytical Council Limited (CIPAC) methods for determining insecticide content. Washing nets with too-short intervals results in either 1) less insecticide washed off per wash allowing a product to withstand more washes under laboratory testing than it might under user conditions or 2) insufficient insecticide becomes available and a product may fail bio-efficacy testing. Therefore, ITNs with a slow insecticide RR will be underestimated and ITNs with a high RR will be overestimated.

The current process for determining the RT [6] is based on 3 assumptions: (i), that the 3 washes in soap water in a day removes all insecticide from the net surface. (ii), that when bio-efficacy criteria e.g., M24 and KD60, reaches 100%, the net no longer continues to regenerate. Using susceptible mosquito strains this generally happens in one or two days and nets are therefore often designated a too-short regeneration time [8], though it is accepted that using more resistant strains, a longer RT is found (9). Thus deliberately ignoring that the only way the resistant strain can show a longer RT is that regeneration had not ended (iii), the sampling methods used for determining insecticide content at start and during the washing process to 20 washes is based on the assumption that net variation from start is less than 5% between samples used [9] although tolerance for ITNs is 20% [10]. Mean wash off for most nets is 0.5-2% per wash, therefore differences in dosages between two ITN samples from a net can be much bigger than the wash off and some tests may thus show a negative wash off which of course is meaningless.

An alternative bioassay method to determine the regeneration time of pyrethroid ITN or pyrethroid with piperonyl butoxide (PBO) ITN is based on Median Knock Down Time, MKDT [8]. This method gives a continuous value and does not go into saturation, but it was only briefly used in ITN evaluation by WHO [11]. MKDT is proportional to surface concentration and can follow the regeneration time [8].

Bioassays continue to be used as a means to measure ITN regeneration time since it was agreed that is was not possible to measure the surface concentration chemically [6]. However, it is generally agreed that using mosquitoes has several disadvantages. If an ITN contains two insecticides, they will not migrate at the same speed due to many factors including solubility, molecular size, shape and polarity. Bioassays can only measure the compound effect without knowing how the surface concentrations of the two components develop over time. Further, the phenotypic expression of insecticide resistance varies between mosquito species and strains. Even in the same mosquito strain, phenotypic resistance is influenced by insect rearing [12, 13] and therefore subject to variability over time and between test facilities. Consequently, mosquitoes must be carefully characterised and efforts made to standardise the strains [14].

Here, three methods are used to measure the development of the surface concentrations of one or two active ingredients (AI) released from incorporated ITNs. The first method directly measures the concentration of the AI in soap solutions used to wash an ITN (Soap Wash Analysis Method, SWAM) to establish the wash interval for wash resistance evaluations that underpin all ITN evaluations. The second method measures total content of a net sample before and after standard washing, the difference assumed to be what is washed off (Before and After Method, BAM). The third method measures the AI(s) extracted by cold acetone from an immersed net that has first been washed (Cold Acetone Method (CAM)) either using soap or acetone thus measuring what resurfaced. The results of these are compared to those obtained by the MKDT method [8] that also measure the surface concentration and to the Mortality/Knock down method used by WHO [6]. By following surface concentrations development this way in a regeneration test, the methods aim to provide a standardised test parameter for the wash interval to minimize bias in evaluations between different laboratories and products.

All tests were conducted at Biolytrics Vietnam between September 2018 and June 2020. Five experiments were performed. 1) validation of the GC-FID compared to standard CIPAC HPLC method 2) evaluation of CAM and MKDT, 3) evaluation of MKDT with mosquito strains that were pyrethroid sensitive or resistant, 4) comparison of SWAM and BAM after washing a net 3 times in a day, 5) demonstrate the significance of correctly identifying the wash interval for incorporated nets, 6) measuring surface concentrations of two commercial nets using SWAM and CAM. For all evaluations deltamethrin-PBO ITNs were used except one that used a deltamethrin net.

Test Nets

Polyethylene (PE) nets were provided by Intelligent Insect Control and produced at Sunpack, Jiangsu, China. Yarns were extruded by mixing High Density PE with Low Density PE with masterbatches with active ingredients and antioxidants [4,12]. Two types of nets were provided. One net had all yarns with 2 g of the pyrethroid deltamethrin (DM). The other type of net had 15 g DM plus 0.5 g PBO in one yarn and 13 g Piperonyl butoxide (PBO) in the other yarn. Yarns were warp knitted into mosquito nets where the ratio of the yarns determines the dosages of the DM-PBO net. A net with 3 g DM/kg and 11 g PBO/kg was made by having a yarn ratio DM-yarn to PBO yarn of 20:80. Experimental nets were also made with the high dosage DM yarn only, thus 15 g DM/kg net used for EM scanning to have more DM at the surface.

Two commercial nets combining PBO and DM were also compared: 6 months old Tsara Boost batch number 187 produced on 08/2018 at Sunpack (specification: 3 g DM/kg net, 11 g PBO /kg net) and 2.5 years old at the time of testing. PermaNet 3.0 produced in Vietnam (specification: 4 g DM/kg net, 25 g PBO /kg net on the roof panel) batch number 1062(1)16 produced 07/2016. The two commercial nets were in unopened packages.

Net sampling

Principle for net sampling: Series of samples were cut from nets along 30 cm wide panels that correspond to the yarns from two bins sitting opposite to each other on a knitting machine and knitted into the panel [4]. Before and after samples were taken from a single sample by cutting 5 cm wide top sample for initial chemical analysis of PE nets and 5 cm from the top and side from Polyester (PE) nets, then running bioassay on the remaining piece followed by chemical analysis (Figure 1). This method assures that samples are as homogenous as possible. If regeneration is to be conducted a further 5 cm strip is cut before testing for chemical analysis and a 15 x 15 cm sample is used for bioassay followed by chemical analysis

Net washing

For regeneration tests, an initial wash off was conducted either with acetone or by 3 repeated soap washing in a day as used in WHO method for determining the regeneration time [6]. Samples from a DM+PBO net were determined for total content by taking a subsample of 5 cm from the top cut off before the washing, then the washing water of the 3 washes were sampled for insecticide content and the total content determined for the rest of the sample after the 3^rd washing. The regeneration of insecticide after initial 3 soap washes or one acetone dipping and comparison to MKDT is summarised in Figure 2.

Determining surface regeneration time after initial soap or acetone washing and with bioassays using Cold Acetone Method (CAM): To chemically follow the accumulated effect of insecticide(s) release after the initial wash in the bioassays, at least one sample per time interval is needed since chemical assessments are sample destructive. Two pieces of 25x30 cm samples were cut from the sides of each of two PE ITNs drawn from one bale. Since nets are knitted from a multiple of 30 cm wide panels, panels can be identified by measuring the distance from the border (Figure 1). The 25 cm wide test panel was cut inside such a panel to be sure all yarns were from the same set of bins. Samples 25 cm long were cut from these panels.

In total, 20 samples were cut, each of these were divided in 2 (top piece of 5 cm length and bottom piece of 20 cm length). MKDT were double determined from the bottom piece and 2 chemical analyses were made for all samples and wash solutions as detailed below.

One 25cm sample was randomly selected from each panel and stored in foil at 5ºC. This sample was later exposed to cone bioassay for mortality and KD. A subsample was extracted for total content and the rest acetone extracted for surface content (Figure 1). Another sample was washed 3 times, regenerated for 1 day and exposed to cone bioassay. The remaining were either washed in soap water (10 samples) or in acetone (10 samples). The regeneration of surface concentrations after the 3 soap washes or the acetone wash was followed by storing the samples at 30°C for a selected time. The samples were further divided in a 15 cm length and of 10 cm length, thus containing identical yarns (Figure 1). The 15 cm sample was used for determining the MKDT followed by analysis of total content by GC-FID. The 10 cm long sample was dipped in cold acetone (20°C) for 10 min and the extract analysed by HPLC. For each day for each wash type 2 replicates were conducted for 1) initial wash with acetone or 2) initial 3 washes with soap. At each time point: 1, 3, 5, 7, 10, 15, 20 days, the net samples were tested using MKDT bioassays, total content, and total surface content as extracted by cold acetone.

Soap washing: Net washing was performed using soap solution and a “shaker” water bath [6]. Samples were washed three times at the start of the experiment to remove insecticide on the ITN surface. Net samples were individually introduced into 1 L glass bottles containing 500ml (Collaborative International Pesticides Analytical Council (CIPAC) MT 195 soap solution (2g/L), the bottle was capped with a steel lid and placed in a 30°C ± 2°C water bath (Julabo SW22) in an upright position and shaken for 10 minutes after which the piece of netting was removed with tweezers and excess fluid removed by gently shaking. After washing, the piece of netting was rinsed twice in deionised water. For each rinse, the piece of netting was added to a 1 L glass bottle containing 500 ml of de-ionized water at 30°C ± 2°C. The bottle was capped with a steel lid, placed in a water bath and shaken for 10 minutes after which the net sample was removed using tweezers. This procedure was repeated, and after the second rinse, excess water was removed from the net sample and allowed to dry on a line for 30 minutes at room temperature (27°C ± 5°C) out of direct sunlight. Net pieces were dried for two hours on a line in the dark. Once dry, the net sample was wrapped in aluminium foil and stored at 30°C in an incubator (Memmert UFE400/G410.2367) until bioassay and single soap washing.

Initial Acetone wash: Net samples were individually introduced into 1 L glass bottles containing 500ml cold acetone (20ºC) for 10 minutes after which the piece of netting was removed with tweezers and excess fluid removed by gently shaking into the bottle.

Wash resistance testing: To test wash resistance, washes were conducted on the same netting pieces at and analysed after 1, 3, 5, 10 and 15 washes.

Chemical analysis

Standard chemical analysis: Biolytrics Laboratories in Hanoi (BLH) used an ISO confirmed method for determining total content of insecticides developed by the WHO reference laboratory for quality control of pesticides, the Walloon Agricultural Research Centre in Gembloux, Belgium, for determining deltamethrin content in the nets (method CRA-W RESSM015). In short, the net piece is cut into very small pieces with an acetone cleaned scissor. 3 analytical portions of about 500 mg each are put into 3 conical flasks with 24 ml xylene and 1 ml of the internal standard solution mixed thoroughly at 20°C. The flasks are connected to a reflux condenser system and heated to reflux at 130°C for 60 minutes for completely dissolution of the samples. The solutions are cooled down to 25°C and the flasks are filled up to volume with xylene. The solutions are filtered through a 0.2μm nylon filter and 2 ml are transferred to injection vial and injected into the Gas Chromatograph with flame ionization detector (GC-FID 7890A Agilent) for analysing. To validate the GC-FID method and know the net variations, analysis and repeated analysis were carried out on nets and compared to results of using the CIPAC defined HPLC method, CIPAC method 333/LN/(M2)/ (HPLC-DAD 100 Agilent).

Chemical analysis of cold acetone method (CAM) “wash off”: Wash in cold acetone (20°C) was chosen to remove surfaced insecticides and used for a “clean start”, to determine surface concentrations and to estimate the ideal or “real regeneration time”, meaning stabilized level of insecticide after a clean start. Cold acetone was chosen because it dissolves DM (s=500 g/l at 20°C) and piperonyl butoxide (s=900 g/l at 20°C) but does not swell polyethylene and should therefore not extract from the PE matrix. For washing of a net sample (25 x 25 cm), the sample was immersed in 20°C acetone in a bottle hold in a temperature regulated bath (Julabo SW22) for 10 minutes. To determine amount extracted by cold acetone extraction, 1 g samples were cut from the test piece and immersed in Erlenmeyer cone bottles for 10 min at 20°C in acetone. BLH tests showed that to be sure to have all surface PBO dissolved, 99 g acetone had to be used. For DM, no change in extracted DM was found between 10 and 50 g acetone for a 1 g sample.

Chemical analysis of soap washing method (SWAM): DM and PBO were analysed in soap water used for washing samples. They were extracted with dichloromethane and determined by HPLC-UV using an external calibration standard. In short, 15 g sodium chloride is added to 100 ml of the soap solution with insecticides and extracted with dichloromethane. The extract is filtered through an anhydrous sodium sulphate, the solvent is dried off under vacuum and redissolved with 2 ml ES. 10 µl is injected into HPLC after the injection of the calibration solution. Peaks of PBO and DM are read on the same graphs together with peak of the calibration and the concentration in the solution is calculated. Dosage per kg net is then calculated from the volume soap water and weight of the net sample.

Surface concentration using before and after method (BAM): For each sample used in wash tests or bioassays (Figure 1), the upper 5 cm net (PE netting) or the L-shaped 5 cm wide (PET netting) was cut off to determine the total dosage (=surface + inside yarn or inside coating) of the insecticide before the first wash (C₀). Dosage of the rest of the net after the wash was (C₁). The difference between the concentrations (C₀-C₁) corresponds to the amount lost in the washing process. If the washing is an acetone washing (PE nets only), this amount is the insecticide at the surface of the yarn before the washing. The results are given as

Comparison of pyrethroid susceptible and pyrethroid resistant strains by MKDT: the samples from the acetone or soap wash test series from two deltamethrin PBO ITNs were tested for each day with both the pyrethroid susceptible and the pyrethroid resistant strains. MKDT was determined on day 0 (before washing) then on days 1, 2, 3, 5, 7 and 10 post-wash.

Comparing the two chemical methods to determine surface concentrations: 3 samples of Tsara Boost had total content of DM and PBO determined, then washed 3 times and the total content was determined again, with 3 washes between the before and after determination (BAM) of chemical content. The content of DM and PBO in the 3 soap water solutions were determined (SWAM) and the residual DM and PBO were re-determined after the first extraction from the wash water to measure if the process was complete.

Wash resistance using MKDT and surface concentrations: 2 DM+PBO nets had their regeneration time measured by MKDT. This interval was then applied for a standard laboratory wash/bioassay and chemical analysis following the standard WHO protocol [6] with analysis before wash, after 1, 3, 5, 10, and 15 washes (20 wash omitted in error). Bioassays were performed with both the pyrethroid susceptible and the pyrethroid resistant strains on the same pieces of nets the same day.

Mosquito Strains

Mosquitoes: Anopheles dirus received from NIMPE (National Institute of Malaria, Parasitology and Entomology) in 2008 and Culex quinquefasciatus-S-Lab originating from the university of California and transferred from IRD in Montpellier (Ref V.3.2.1.L.FR.4) to NIMPE and then to Biolytrics Laboratories, Hanoi. They were reared according to the guidelines of the Malaria Research and Reference Reagent Resource Centre, MR4 [15] for Anopheles. Larvae are fed a powdered mix of 6 g bread flower, 2 g shrimp powder, 2 g chickpea flour, 1 vit B pill B1, 110 g Aquafin red flakes for aquarium fish. Adults are fed 10 % glucose on cotton wool. Females are blood-fed on live White Swiss female mice, weight 21-24 g. An. dirus was confirmed fully susceptible at the time of testing [16]. The Cx. quinquefasciatus strain has low susceptibility to pyrethroids partially restored by PBO [16]. For all bioassays, 2-5 days old, non-blood fed females with access to sugar water were used.

Bioassays

WHO cone bioassays: Five mosquitoes were introduced into a cone fixed on a test ITN sample held at a 45º angle, removed after three minutes and transferred to a cup with sugar water and kept at 25-27°C, 80 % RH. Knock-down was observed after 60 min (KD60) and mortality after 24 hr (M24). This was repeated ten times so that 50 mosquitoes exposed per net sample, and 2-4 samples were tested per panel.

Median Knock Down Time: for MKDT data, 4 groups of 11 mosquitoes were placed under petri-dishes fixed on a test ITN sample and the knock down time was followed from the first to the 9th mosquito using a stopwatch. Median knock down was measured as the time of number 6 to be knocked down or calculated from the linear correlation curve of KDT to number of mosquitoes. This was repeated 2 times per net sample, and 2-4 samples were tested per panel.

EM scanning: DM and DM-PBO net samples were analysed by EM scanning before and after soap wash or acetone dipping. The samples were coated with carbon graphite and scanned with electron microscope (Quanta 450, FEI, USA). This allowed to see surface particles including DM crystal particles. Secondary electronic imaging was obtained by collecting back scattered electrons (BSE) that informs on the distribution of relatively heavy atoms like the halogene bromine. DM holds 2 bromine atoms. The back scanning thus reveals if there is bromine and therefore probably deltamethrin in areas of the yarn where there are no crystal particles, and thus find amorph deltamethrin. The analysis were carried out at the Centre for Excellence in Analysis and Experiment, Hanoi University, Vietnam (http://ceae.humg.edu.vn/do-sem/).

Data Analysis

Data were entered into Excel and analysed in Stata 17.0 statistical software StataCorp, USA. Comparison between GC FID and HPLC was conducted using Two-sample Wilcoxon rank-sum to test for a difference between methods for each component: DM, PBO and R-isomer of DM.

Median Knock Down Time (MKDT) for the 6^th mosquito was calculated from a linear regression of Knock down time for mosquito numbers 1 to 9. The correlation between 1) surface concentration by SWAM and MKDT for An. dirus and 2) correlation between MKDT for An. dirus and Cx. quinquefasciatus was calculated with Pearsons correlation coefficient. The regeneration time was calculated as the first day that MKDT was within 20% of the 20-day MKDT for the net in test or the normalized change in surface concentration change reach 80 % of the change. Comparison between MKDT for Tsara® Boost and PermaNet® 3.0 was conducted using Two-sample Wilcoxon rank-sum test.

1. Validation of the GC-FID compared to standard CIPAC HPLC method

The GC-FID measured almost identical to the standard CIPAC method 333/LN/(M2) (Figure 3).

Two-sample Wilcoxon rank-sum test showed that the two methods provided values that were not statistically different for DM for either polyester (PES) (z = 0.841, p = 0.435) or polyethylene (PE) (z = -0.404 p = 0.729). The R-isomer of DM was additionally checked in case GC-FID created R-isomer in the hot xylene used for extraction and injection, however there was no significant difference between R-isomer levels according to the two methods for either PES (z = 1.200 p = 0.244) or PE (z = 1.725 p = 0.196), (Figure 3). For PBO the two methods measured similar results. The mean PBO measured by GC FID was 11.06 (95% CI 10.95-11.18) g/kg and 11.34 (95% CI (10.86-11.83) g/kg measured by HPLC. However, the results were statistically different z = -2.085, p = 0.04.

2. Evaluation of cold acetone extracts compared to MKDT

2.1 Analysis of acetone surface extractions and total content

In total 20 samples from PE net #370 were analysed for total content of DM and PBO using GC-FID method before and after 3 soap washes in a day or an immersion in cold acetone. Starting 2 hours after these washings, net samples were extracted with cold acetone for analysis of surface content of the two biocides for 20 days to measure regeneration (Figure 2). These samples were also followed by MKDT (Table 1). Total content of AI was 12.27 g PBO/kg net and 3.74 g DM/kg net. Measured as the difference between net dosage before washing and the average of total content of all samples after either soap or acetone washing, the soap washing removed 79 mg DM/kg net and 679 mg PBO/kg net whereas acetone “wash” removed 422 mg DM/kg net and 688 mg PBO/kg net. The acetone extracts made 2 hrs after soap washing or acetone immersion (“washing”) showed that much more DM and PBO was left at the yarn surface after the soap wash than after the acetone “wash” (Table 1). With the assumption that the acetone wash removed both AIs from the yarn surface, the AI found after 2 hours is due to regeneration. Retracting the 2hr post acetone wash from the post soap wash, we thus find the residual AI that was not removed by the soap washing. This was 48 mg DM/kg net and 10 mg PBO/kg net. This matches well for the total content analysis for PBO (688-679= 9 mg/kg net) but not for DM (422-79= 343 mg/kg net). Soap wash thus removes nearly all PBO from the surface of the yarn but not all DM. However, loss due to soap washing seems strongly dependant on how it is measured as seen below where analysis of soap water and total content before and after soap washing is compared.

Table 1. Relationship between Median Knock Down Time (MKDT) measured with Anopheles dirus (pyrethroid sensitive) or surface concentrations determined by acetone extraction. ITNs were either subjected to an initial wash in soap water or immersion in cold acetone.

Time	After Soap Wash			After Acetone dip
after	PBO	DM	MKDT	PBO	DM	MKDT
washing	(mg/kg)	(mg/kg)	(sec)	(mg/kg)	(mg/kg)	(sec)
Unwashed			449			436
2 hrs	25.4	49.1	1206	15.8	1.7	1769
Day1	70.1	65.9	877	36.2	2.0	1090
Day2	92.1	59.8	963	47.7	2.5	1039
Day3	119.5	81.9	897	59.6	3.6	878
Day5	134.0	83.5	764	71.3	4.9	788
Day7	119.5	91.8	671	68.7	6.9	634
Day10	136.6	105.5	712	84.6	9.9	610
Day15	175.1	102.5	715	115.8	15.6	654
Day20	185.4	102.0	633	109.9	19.7	598

2.2 MKDT measurements

Median Knock Down Time (MKDT) was measured by following knock down time for mosquito 1 to 9 out of 11. The MKDT tests showed in general a linear relationship between time for knock down and mosquito number from number 1 to 9 with a correlation coefficient above 0.98. Therefore, it was determined that using the time for the median number 6 (out of 11) to be knocked down or determining this value from the regression curve resulted in similar estimates.

2.3 Comparison of MKDT after soap or acetone washing

MKDT of a commercial net Tsara Boost (DM+PBO) drawn from a bale was measured on the unwashed net used for either soap washing or acetone immersion in a double determination per group. Before washing or immersion, MKDT was 449 sec for the soap wash net group compared to 436 sec for the acetone wash group and thus not different. 2 hrs after the washings, MKDT was 1206 sec for the soap washed net compared to 1769 sec for the acetone washed nets. If it is assumed that the 3 soap washes effectively removes molecular DM for the yarn surface, but not all DM crystals (as seen in the EM scannings), this means that molecular DM regenerates faster from the remaining crystals at the surface than from the yarns. From day 3 to 20, MKDT values for the nettings washed by soap or dipped in acetone were nearly identical despite the fact that the amount of DM and PBO extracted from the net surface was very different between the two groups of nets (Table 1).

2.4 Correlation between surface content and MKDT

The acetone extracts after soap washing or acetone “washing” showed statistically significant correlation between the deltamethrin content and MTKD for pyrethroid susceptible An. dirus (Figure 4). There was greater correlation between the concentration of deltamethrin measured by GC-FID after an initial soap wash (r=-0.91, p=0.0006) compared to an initial acetone wash for deltamethrin (r=-0.65, p=0.056). There was similar correlation between PBO content and the MKDT after either initial soap (r=-0.88, p=0.001) or initial acetone wash (r=-0.85, p=0.003). However, it should be noted that the MKDT was measured with a pyrethroid susceptible mosquito strain and that PBO was engineered to have a similar release rate as deltamethrin.

3. Evaluation of MKDT with different strains

3.1 Relationship between MKDT for susceptible and resistant mosquito strains

The MKDT was highly correlated between susceptible Anopheles dirus and pyrethroid resistant Culex quinquefasciatus, r²= 0.96 (Figure 5). The regeneration time measured for the resistant and the susceptible strains was 5 days (Figure 6) using the time to the MKDT to be within 20% of the 20-day value. The median knock-down time was higher for Culex quinquefasciatus (843 seconds) than for Anopheles dirus (737 seconds) in accordance with their different level of pyrethroid sensitivity. The time to reach a stable level was about the same per product.

However, when the two species were used to determine regeneration time by cone bioassay according to the WHO method, An dirus showed 100 % mortality 1 day after the initial washing as well as before washing and thus one day regeneration time according to this method.

The regeneration time measured by cold acetone extracts was 5 days defined as the first time that values were recorded to be within 20% of the 20-day concentration of deltamethrin (Figure 6). Both cold acetone extracts and MKDT measured the same regeneration time, independent of resistance level of the mosquito strain used, although the trend line for the Cx quinquefasciatus shows a longer regeneration time. The ITNs were fully regenerated by day 10 - 20 measured by surface concentrations of DM or MKDT for either strain and 20 % off this final value at day 5 (Figure 7). Inter-sample variation was <2%. Surface concentration of PBO took slightly longer to stabilize than that of DM.

3.2 regeneration time with two species measured by MKDT compared to cone bioassay and chemical analysis.

MKDT tests with the fully susceptible An dirus and the less susceptible Culex quinquefasciatus S-Lab showed very different start values but ended with nearly the same after 20 days (Fig 6). Using the endpoints for creating normalized data as in Fig 7, regeneration time reached 20 % off from the end point between 3 and 5 days, and a net minimum regeneration time or “operational regeneration” of 3 days was estimated. This was repeated with a second set of nets that were used for estimating wash resistance.

Wash resistance

This interval was then used for wash/cone-bioassay/chemical content analysis up to 15 washes. The chemical analysis showed that more PBO than DM was lost in the first wash (0.3% vs 6.6%) and this increased to 8.5% vs 18.4% lost by wash 15. This is reflected in the sustained mortality of the pyrethroid sensitive An. dirus but rapid loss of efficacy of the ITN against the pyrethroid resistant Cx. quinquefasciatus.

4. Analysis of the 3 soap washes at start of the regeneration time study (Soap Wash Analysis Method (SWAM) compared to Before and After Method (BAM)

Regeneration time studies started with 3 soap washes with CIPAC defined soap [5]. The start dosage before washing was obtained from subsamples from the 5 cm long pieces cut from the top of a 30 x 30cm PE net sample (Fig 1). The rest was washed 3 times and the water from each wash was analysed (Fig 8). To be sure that the extraction from the first soap water was complete, a second extraction was made from this. Finally, the 3 times washed samples were analysed for total content after wash. The data show (Table 2) that PBO determined in this Before and After Method corresponds roughly to the sum of the 3 wash solutions, but the DM found in the wash solutions are lower. Further, standard deviations show much lower variations between determinations from soap water than from total content that even was negative from 1 of the 4 replicates. The first wash-off of PBO provided 90.7% of total washed off, whereas the first wash off for DM provided 66.1 % of total washed off.

Table 2. Three soap water solutions from washings of a Deltamethrin (DM) + Piperonyl Butoxide (PBO) net were analysed for total content before and after the 3 washes and the three soap wash solutions were analysed for content of the two biocides. % wash indicates the percentages of total wash off found in first, second and third wash solution. SWAM is the total amount found in wash waters. BAM is the difference in net dosages before and after the 3 washes. SWAM is lower than BAM. 66.1 % of the DM found in soap solutions were found in the first wash solution compared to 90.7 % of that of the PBO.

Sample	PBO (mg/kg)	% of wash	Deltamethrin (mg/kg)	% of wash
Net before wash	13,334.50 ± 95.01		3,608.83 ± 252.97
1st wash water	459.38 ± 71.17	90.7%	27.58 ± 3.78	66.1%
re-extract 1st	12.49 ± 1.53		0.69 ± 0.39
2nd wash water	30.82 ± 1.93	5.9%	7.66 ± 0.96	17.9%
3rd wash water	17.54 ± 2.54	3.4%	6.85 ± 0.53	16.0%
SWAM	520.23 ± 95.01	100.0%	42.78 ± 0.92	100.0%
Net after wash	12,603.25 ± 404.76		3,362.83 ± 325.95
BAM	731.25 ± 665.46		246.01 ± 578.21

5. The significance of correctly measuring the wash interval

Wash resistance is measured according to WHO by carrying out 20 laboratory or field washes of nets washed with intervals determined from the regeneration time study. The regeneration time is determined from the bioefficacy of the net after 3 soap washes using a fully susceptible Anopheles laboratory strain. Determined this way and using A. dirus, the commercial net Tsara Boost (DM + PBO) has a regeneration time of 1 day [17]. Our data shows that the regeneration time determined from MKDT bioassays was 3 days for the regeneration to begin to plateau and 10 days for full regeneration (Fig 7).

In order to evaluate the importance of the correct wash interval, samples of the PE DM-only net were washed on a 3-day wash interval but tested again after another 4 or 7 days after wash 10, 15 and 20. This improved both KD and M24 for Anopheles dirus and Culex quinquefasciatus; After 20 washes A dirus mortality increased from 68% to 95% by letting the net regenerate another 7 days (Figure 9). Thus, the 3-day interval was too short.

6. Measuring the surface concentrations of two commercial nets using BAM and SWAM

6.1 Comparing BAM and SWAM with MKDT

The two commercial PBO nets evaluated had very different start surface concentrations of biocides (Fig. 10). However, after washing this was more similar. Comparing the MKDT to what was removed by acetone dipping and soap washed ITNs, it is concluded that the 16-20 % of DM removed by the washings from PermaNet 3.0 had about the same biological effect as the 4-5 % DM removed from the Tsara Boost, since the MKDT values are not significantly different.

6.2 EM scannings of Tsara Boost after cold acetone or soap wash

Because the DM yarn in the Tsara Boost net only provides 20 % of the yarns used for the net, the dosage of this yarn is 4 times higher than in the final net. These yarns and yarns from DM-only net (thus low dosage) were exposed to EM scanning with the use of back scattering to observe for amorph deltamethrin identified by the presence of Bromine. The back scattering showed the presence of heavy atom, thus probably the Bromine also in yarns areas where no crystals could be seen. Acetone washing of these yarns complete removed crystallized DM, whereas soap washing did not (Fig 11).

The use of surface rinse with cold acetone to measure the bioavailable active ingredients is an important step in standardisation of ITN evaluations. The work presented here has shown that analysis of surface AI remaining after soap washing can be conducted via cold acetone rinse. The analytical method GC-FID used compared well to the standard CIPAC defined HPLC method, CIPAC method 333/LN/(M2)/ (HPLC-DAD 100 Agilent). The cold acetone extracts were used to quantify regeneration of DM and PBO and was used to provide a chemical method for measuring regeneration time (RT) of both biocides independently but simultaneously. The method correlates well with the development of surface concentration as measured by the bioassay Median Knock Down Time (MKDT). Loss due to washing of the ITN can be measured by total content analysis before and after the net washing (BAM) using already validated CIPAC methods. However, loss due to washing of insecticides that crystallize is not equivalent to loss of biological activity, since the crystal form is not all removed by washing and the crystal form has little biological activity. This also underlines why chemical content of field aged ITNs often has low agreement with traditional HPLC measurements of biological efficacy even with coated ITNs [18].

The GC-FID method using xylene or other injectable solvents shortened the process between extraction and injections because the same solvent is used for extraction as for injection, opposite to that for the HPLC method. However, when concentrations are very low, the HPLC method is more accurate. In total extractions from ITNs, insecticide concentrations are high.

The cold acetone extracts from the surface of a PE net quantifies the total surface concentration of insecticide on ITNs. This includes both bioactive deltamethrin (molecular form) and crystalline deltamethrin that is not bio-active. In a study of net regeneration after initial 3 soap washing on a day or one net immersion in cold acetone, the surface content was analysed by surface extractions with cold acetone from a series of samples tested from 2 hrs after the initial washing till day 20. EM scanning showed that the acetone extraction completely removed Deltamethrin (DM) from the yarn surface, but the soap washing did not (Fig. 10). Back scattered electrons showed that bromine is found in areas of the unwashed net without crystal DM, indicating that DM in amorph form is also present at the surface as also found by Gesta and Skovmand [15]. MKDT studies on the samples exposed to surface extraction in the regeneration time study showed that MKDT was linearly correlated to the total content of DM after initial 3 soap washes, but with lower correlation to the total surface content after initial acetone washing.

Further, that MKDT from day 3 to 20 were nearly identical independent of the initial “washing” method, but on day 3 the total surface content was much higher after soap washing (81.9 mg DM/kg net) than that after acetone washing (3.6 mg/kg net). The MKDT 2 hours after the soap washing was lower that the 2 hour sample after acetone washing. Provided that the soap wash removed molecular DM from the net surface and that only molecular from of DM is biologically active, this shows that molecular DM is quickly regenerated from the remaining crystals.

Therefore, it is concluded that large part of the insecticide removed by the acetone extraction is not biologically relevant and that most of the DM was probably present in its crystal form. This conclusion is supported by total content analysis after soap washing or acetone dipping of two commercial PBO nets that showed a similar trend (Fig. 4). Acetone immersion and soap washing removed equal parts of PBO that is an oil at room temperature and emulsified by the soap but removed different amounts of DM from both nets. Further, despite much more DM was removed from one net than from the other by both “washing” methods (thus two different measures of surface content), MKDT were not significantly different between the two nets before washing, meaning that the bio-active concentration was nearly the same. This again indicates that the total surface concentration of insecticide (consisting of crystallized and molecular DM) does not determine the biological activity that only depends on the fraction available in molecular form.

Analysis of the soap water solutions (SWAM) provided by 3 washes in a day in the start of a regeneration study showed that the first washing contained 66% of the DM washed of and nearly 88% of the PBO washed off over the 3 washes (Table 2). To confirm the extraction method, the first wash off was reanalysed in a second extraction. This only added 2% to the PBO of the first wash off, but 10% to the DM. Further, comparing the sum of the SWAM analysis of the 3-washing water to the BAM analyses of the net before and after the 3 washes, the SWAM recovered 88% of the PBO, but only 66% of the DM. A similar problem was found in the analysis that compared total extraction of a PBO-DM net to that of acetone extraction of the surface. Total extractions and acetone surface extractions matched, but those for DM did not.

In total extractions, soap water removed 79 mg DM/kg net compared to 422 mg DM/kg net by acetone immersion, thus a difference of 343 mg DM/kg net, but the remaining surface concentration of DM after the soap washings was just 48 mg DM/kg net measured by acetone extracts from the newly soap washed net (Table 1). It is possible that washing an ITN in soap water destroys part of the DM in a coating or yarn and is not just removing it. That would mean that the soap water penetrates a bit into these storages in line with that a patent describes reduced loss of DM from Polypropylene (PP) nets when citric acid is added to the PP before yarn extrusion [16]. This would only be possible if the soap water can penetrate into the yarn surface. PP is an olefine polymer like PE. This again is in line with the common experience that washing nets with highly alkalic soaps or detergents with chlorine increase losses of insecticide and reduce the biological effect and is therefore recommended against on ITN care labels. If soap water just removed all molecular DM from the net surface and a part of the crystal form, it would not matter if the soap was aggressive or not. These aspects need further clarification.

Single soap washes are used for measuring wash resistance of a net. Since most of the surface available PBO is washed off in a single wash, but only 2/3 of the DM (Table 2), a washing series will “reset” the PBO per wash but not the DM. If each of them regenerated to a stable level as a percentage of the total content, a washing series will remove relatively more PBO than DM even if they have the same release rates. Because of the different removal of DM and PBO by soap washing, a traditional series of soap washes will show higher wash off rate of PBO than of DM. Release rate impact what can be washed of but the wash method also influences what is measured as wash resistance and insecticide removal per wash. This is exactly what was found (Fig. 8). Had removal of DM been as effective per wash as for PBO, the DM removal rate would have been nearly identical to that of the PBO. The data are thus consistent, removal rate is not a measure for regeneration.

It was found that acetone immersion removed all DM from the net yarn including crystals. In a thesis study of migration of DM in PE yarns [15], Gesta found by modelling the surface extracts relative to total content in a diffusion model that the data only fitted if acetone removed DM from a very fine outer layer of the PE yarn. This may explain the apparently delayed regeneration of DM as measured by the surface extractions after the acetone wash compared to the regeneration after the soap wash (Table 1).

Determining surface concentration by acetone extraction is accurate and complete for insecticide soluble in acetone which most are. However, for insecticides that crystallize at the surface, a large part of this has no or very little biological relevance. Soap washes removes crystals and amorph DM to a smaller degree and several washes removes more than a single wash, but this is what a bed net is exposed to in normal use, not acetone immersions. The amount removed by soap washing may be used as proxy for the biologically relevant surface concentration if these are proportional. Even the acetone removal of surface DM included DM that was not biologically relevant, there was still a highly linear correlation between MKDT, and the amount removed by the acetone (Fig. 4) indicating that regenerated molecular DM as responsible for most of the increase in total content after the soap wash. Molecular DM is emulsified in soap water, but we still need to prove directly that there is a linear correlation between what is removed by the soap water and MKDT, preferably in an improved SWAM test.

When surface concentration as determined by MKDT or total extraction by cold acetone is used to determine regeneration of DM and PBO, the surface concentrations stabilised after 10 to 20 days, and the curves were parallel (Fig. 7). We tested to use a “minimum regeneration time” when these curves are 20% away from their final stable value corresponding to what laboratories use for the classic WHO test where curves in mortality flattens and are no longer significant different from the final value. In Fig. 7, MKDT and surface concentrations of PBO and DM are normalized by dividing all values by their endpoint (day 20) values. Applying the minimum regeneration time results in each washing cycle removing 20% less than if the full regeneration time was used, but it saves a lot of time in wash resistance tests, where 25 wash cycles are operated with the regeneration time as interval. However, modelling the consequence of doing so reveals that it has little impact when the wash off rate is 10% or higher, but high impact when the rate is 1.45% or lower (Fig. 12). In this model a threshold of 1 mg DM/m² net for 80% mortality was defined. This threshold is based on analysis of all WHOPES test with DM (Skovmand, pers. Comm). These wash off rates are typical for PET and PE nets respectively [17]. Replacing the full regeneration time with the minimum regeneration time is therefore neither neutral nor accurate, but it is pragmatic.

Using the traditional mortality defined regeneration time based on cone bioassay mortality for the PBO net, the regeneration time would have been 1 day because cone mortality day 1 was 100% after soap washing. That corresponded to 66 mg DM plus 70 mg PBO found by the acetone extraction day one which was 65% of the surface concentration for DM and 38% for PBO after real regeneration. Using the WHO defined wash interval thus leaves 35% DM and 62% PBO extra than if the full regeneration time was used. This amplifies further the problem discussed above.

The core problem with the mortality defined regeneration time is the use of mosquito strains that arrive at 100% mortality before the regeneration of insecticide has run at least to a stable level. Prolonging test intervals for PermaNet 2.0, a coated net, reduced measured wash resistance from 20 to 12 and 5 washes the longer the interval [18] and increased wash of per wash from 3.8% [18] to 7% [17]. These measurements were probably made on the same version of the net since the nets dated before 2013 [19]. Using the WHO mortality test method with fully susceptible Anopheles strains tends to estimate a shorter regeneration time than if a resistant strain is used.

Using a less sensitive mosquito strain that provides mortality well below 100% in the regeneration process can thus reduce the problem. When PermaNet 3.0 were exposed to regeneration tests with A gambiae Kis (a fully susceptible Anopheles reference strain) the regeneration time became 1 day but testing with Culex provided a longer regeneration time [20]. It should be noted that despite the Cx. quinquefasciatus S-lab strain is a fully susceptible strain of that species, it is more tolerant to insecticides than Anopheles and therefore define a much longer regeneration time for the nets than Anopheles does in the mortality test is used for determining regeneration time [19]. Regeneration time as measured according to the WHO method is therefore not a product characteristic but a mosquito (strain) characteristic. It is therefore important to consider the label claim of an ITN and select the correct strain for evaluation [14]. Contrary to that, the MKDT has an advantage because it has no upper or lower limits and is linearly correlated to surface concentrations just like the chemical methods for measuring it. The method has been used previously for ITN evaluation and should be reconsidered by product testing laboratories as it has multiple advantages over measuring mortality. However, it should be noted that the MKDT is only suitable for the evaluation of insecticide classes that induce rapid knock down and kill including pyrethroid ITNs and pyrethroid PBO ITNs. In dual-insecticide nets, it will thus measure the regeneration of the pyrethroid that all these nets marketed so far contains. Chemical methods as suggested here will be needed to measure the regeneration of both insecticides independently. Therefore, other means to quantify mosquito responses to insecticides on the surface of ITNs are needed for evaluation of AI with alternative modes of action.

Regeneration time as measured by the current WHO defined method is an ITN parameter that depends on the mosquito test strain more than of the product. It is thus not a net specification. This study confirms that the MKDT method can determine a product dependent regeneration time when at least one insecticide has a KD effect. It only measures the biologically available insecticide which is the most important information, and it allows for comparisons between nets with the same insecticide independent of formulations. Combining MTKD and chemical methods for surface concentrations could improve predictions of pyrethroid ITN performance.

The chemical method for measuring surface concentration determined from before and after total content analysis (BAM) can be applied for any insecticide incorporated net. However, to measure effects as before-after washings with sample destructive methods, samples must be cut from a single band on the ITN to minimise inter-sample variations. BAM can be used to measure loss of AI from the yarn surface following protocols currently recommended by WHO.

The surface wash off method (SWAM) for determining the surface concentration from analysis of wash off is a surrogate method that measures what was washed off and found in the soap water. When a sample is washed several times, it can thus follow the development of the surface concentration as can a MKDT bioassay because the methods are not sample destructive. However, at least for insecticides like deltamethrin that forms crystals at room temperature, soap washing does not remove all insecticide at the surface, but likely the biologically relevant part. For insecticides that are oils at room temperature, soap washings and acetone removal are nearly identical. Surface extraction by cold acetone measures accurately all (acetone soluble) insecticides at the yarn surface but includes crystal forms of insecticides that have recrystallized at the yarn surface. The crystal form has little biological impact as shown by the MKDT data. The SWAM method needs some improvements to provide the same results as the BAM method. However, it is possible that soap washing not only removes insecticide from the yarn surface but also destroys insecticide in the storage inside yarns or coatings. The loss is thus bigger than the wash off. Combining MKDT and CAM after soap-wash could improve predictions of ITN performance.

Further exploration of means to better characterise ITN regeneration time is a critical component of ITN product characterisation and evaluation. We have shown that ITNs will continue to regenerate for extended periods beyond the regeneration time. To develop longer lasting products that continue to provide protection from malaria transmitting mosquitoes and nuisance bites for several years after deployment it is crucial that we focus on these issues and develop more accurate methodologies to determine ITN surface concentrations. This may mean that we need to use longer regeneration times than at present although this can be overcome through careful advance planning of evaluation trials. Getting the regeneration time right is a precondition for making net testing relevant. Getting it too short will overestimate product performance for ITNs that rapidly release their AIs and will underestimate the performance of ITNs that release their AIs more slowly. Timing is everything.

Authors' contributions

OS conceived the study. OS, DMD, TQT conducted experiments. OS drafted the manuscript. DMD, TQT, RB, SJM contributed to data analysis and edited the manuscript. All authors read and approved the final manuscript.

Acknowledgements

The authors thank Olivier Pigeon for informative discussions on the topics raised in the manuscript.

Ethical Approval

The study was non-human subjects research. Ethical approval was not required.

Competing interests

DMD and TQT conduct product evaluations for a number of vector control product manufacturers through Biolytrics. SJM conducts product evaluations for a number of vector control product manufacturers through Ifakara Health Institute. OS and RB consult on product design and evaluation for a number of vector control product manufacturers.

Funding

No funding was received for this study.

Availability of data and materials

Data is available from OS at reasonable request.

WHO: The World Malaria Report 2021. Geneva: World Health Organization; 2021.
Sinka ME, Bangs MJ, Manguin S, Coetzee M, Mbogo CM, Hemingway J, Patil AP, Temperley WH, Gething PW, Kabaria CW, et al: The dominant Anopheles vectors of human malaria in Africa, Europe and the Middle East: occurrence data, distribution maps and bionomic precis. Parasit Vectors 2010, 3:117.
WHO: WHO Guidelines for malaria WHO/UCN/GMP/2021.01 Rev.1. Geneva: World Health Organisation; 2021.
Skovmand O, Dang DM, Tran TQ, Bossellman R, Moore SJ: From the factory to the field: considerations of product characteristics for insecticide-treated net (ITN) bioefficacy testing. Malar J 2021, 20:363.
Global Fund: Guide to Global Fund Policies on Procurement and Supply Management of Health Products. Geneva: Global Fund; 2018.
WHOPES: Guidelines for laboratory and field testing of long-lasting insecticidal nets WHO/HTM/NTD/WHOPES/2013.3. Geneva: World Health Institution; 2013.
WHO: WHO malaria terminology 2021 update. Geneva: World Health Organisation; 2021.
Skovmand O, Bonnet J, Pigeon O, Corbel V: Median knock-down time as a new method for evaluating insecticide-treated textiles for mosquito control. Malaria Journal 2008, 7:114.
WHOPES: Report of the eleventh WHOPES working group meeting : WHO/HQ, Geneva, 10-13 December 2006 : review of Spinosad 7.48% DT, Netprotect, Duranet, Dawaplus, Icon Maxx. Geneva: World Health Organization; 2008
Amendment to the Manual on development and use of FAO and WHO specifications for pesticides - First edition - third revision - 2016 Revised specification template for Long-Lasting Insecticidal Nets or Nettings (LN) https://www.who.int/pq-vector-control/resources/WHO_LN_specification_templates.pdf
WHOPES: Report of the fifteenth WHOPES working group meeting : WHO/HQ, Geneva, 18-22 June 2012 : review of Olyset Plus, Interceptor LN, Malahion 44 EW, Vectobac GR. Geneva: World Health Organization; 2012.
Salinas WS, Feria-Arroyo TP, Vitek CJ: Temperatures Influence Susceptibility to Insecticides in Aedes aegypti and Aedes albopictus (Diptera: Culicidae) Mosquitoes. Pathogens 2021, 10.
Norris EJ, Bloomquist JR: Nutritional status significantly affects toxicological endpoints in the CDC bottle bioassay. Pest Manag Sci 2021.
Lees RS, Armistead J, Azizi S, Diabate A, Fornadel C, Gimnig J, Hemingway J, Imponivil D, Irish S, Maweje H, et al: Strain Characterisation for Monitoring Bioefficacy in ITNs Treated with Two Active Ingredients (Dual AI ITNs): Devel-oping a Robust Protocol by Building Consensus Insects 2021, 12.
MR4: Methods in Anopheles Research Manual 2015 edition. 2016.
WHO: Test procedures for insecticide resistance monitoring in malaria vector mosquitoes – 2nd ed. Geneva: World Health Organisation; 2016.
WHOPES: Report of the twentieth WHOPES working group meeting, WHO/HQ, Geneva, 20–24 March 2017: Review Of: Interceptor G2 Ln Dawaplus 3.0 Ln Dawaplus 4.0 Ln Sumilarv 2 Mr Chlorfenapyr 240 Sc WHO/HTM/NTD/WHOPES/2017.04. Geneva: WHO Pesticide Evaluation Scheme; 2017.
Castellanos ME, Rodas S, Juárez JG, Lol JC, Chanquin S, Morales Z, Vizcaino L, Smith SC, Vanden Eng J, Woldu HG, et al: Evaluation of the durability of long‐lasting insecticidal nets in Guatemala. Malaria Journal 2021, 20:219.
World Health Organization: Report of the seventh WHOPES working group meeting. Review of: VectoBac WG; Permanet; and Gokilaht-s 5EC. WHO/CDS/WHOPES/2004.8. pp. 82. Geneva, Switzerland: WHO Pesticide Evaluation Scheme; 2004:82.

Box 1 is available in the Supplementary Files section.

Competing interest reported. DMD and TQT conduct product evaluations for a number of vector control product manufacturers through Biolytrics. SJM conducts product evaluations for a number of vector control product manufacturers through Ifakara Health Institute. OS and RB consult on product design and evaluation for a number of vector control product manufacturers.

Box1Definitions.docx

Download PDF

Version 1

posted

You are reading this latest preprint version

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

Status:

Version 1

Abstract

Figures

Background

Methods

Test Nets

Net sampling

Net washing

Chemical analysis

Mosquito Strains

Bioassays

Data Analysis

Results

1. Validation of the GC-FID compared to standard CIPAC HPLC method

2. Evaluation of cold acetone extracts compared to MKDT

3. Evaluation of MKDT with different strains

4. Analysis of the 3 soap washes at start of the regeneration time study (Soap Wash Analysis Method (SWAM) compared to Before and After Method (BAM)

5. The significance of correctly measuring the wash interval

6. Measuring the surface concentrations of two commercial nets using BAM and SWAM

Discussion

Conclusions

Declarations

Authors' contributions

Acknowledgements

Ethical Approval

Competing interests

Funding

Availability of data and materials

References

Box

Additional Declarations

Supplementary Files

Status:

Version 1