Does washing insecticide-treated nets 20 times for experimental hut evaluations provide a suitable proxy for their end-of-life performance under household conditions?

Abstract

Background: Insecticide-treated nets (ITNs) are washed 20 times as part of experimental hut trials to simulate the loss of active ingredient (AI) occurring over their intended 3-year lifespan and estimate insecticidal durability. The ability of the 20-wash method to predict the end-of-life performance of ITNs has not been empirically validated.

Methods: We performed an experimental hut trial to compare the efficacy of new ITNs unwashed and washed 20 times to field-aged ITNs withdrawn from households 3 years post-distribution against a pyrethroid-resistant vector population in Covè, Benin. Four products from pyrethroid-only (Interceptor^®), pyrethroid-piperonyl butoxide (PermaNet^® 3.0), pyrethroid-pyriproxyfen (Royal Guard^®) and pyrethroid-chlorfenapyr (Interceptor^® G2) ITN types were tested. Net pieces were tested in bioassays and sent for chemical analysis to assess differences in surface AI bioavailability and total chemical content between washed and field-aged nets. Susceptibility bioassays were also performed to assess insecticide resistance in the Covè vector population.

Results: Mosquito mortality in experimental huts was similar or slightly higher with field-aged nets than washed nets with Interceptor^® (11% vs. 10%, p = 0.339, OR = 1.19, 95% CIs [0.84, 1.69]), PermaNet^® 3.0 (12% vs. 18%, p < 0.001, OR = 1.78, 95% CIs [1.34, 2.38]) and Royal Guard^® (9% vs. 14%, p = 0.076, OR = 1.33, 95% CIs: [0.97, 1.83]). Likewise, field-aged Royal Guard^® induced a similar reduction in fertility to washed Royal Guard^® (22% vs. 29%, p = 0.066). In contrast, mortality was significantly lower with field-aged nets Interceptor^® G2 compared to washed nets (54% vs. 19%, p < 0.001, OR = 0.18, 95% CIs [0.14, 0.24]). Blood-feeding inhibition was higher with field-aged nets than washed nets across all ITN types. Retention of non-pyrethroid AIs was lower than for the pyrethroid, particularly with field-aged nets (PermaNet^® 3.0 (roof): 25% vs. 68%, p < 0.001, Royal Guard^®: 27% vs. 53%, p < 0.001, Interceptor^® G2: 14% vs. 39%, p < 0.001).

Conclusions: In this setting, the 20-wash method provided a suitable proxy for the end-of-life killing and sterilising performance of Interceptor^®, PermaNet^® 3.0 and Royal Guard® in experimental huts. In contrast, washing overestimated the end-of-life performance of Interceptor^® G2 for mortality and underestimated the personal protection of all field-aged ITNs.

Keywords: Insecticide-treated nets; Long-lasting insecticidal nets; Malaria; Mosquitoes; Vector control.

Fig. 1

Blood-feeding inhibition of wild, pyrethroid-resistant Anopheles gambiae sensu lato entering experimental huts in Covè southern Benin. According to logistic regression, bars bearing the same letter do not differ at the 5% level. Errors bars represent 95% confidence intervals

Fig. 2

Mortality rates after 72 h of wild, pyrethroid-resistant Anopheles gambiae sensu lato entering experimental huts in Covè southern Benin. Bars bearing the same letter do not differ at the 5% level, according to logistic regression. Errors bars represent 95% confidence intervals

Fig. 3

Odds ratios describing the difference in mortality rates after 72 h of wild, pyrethroid-resistant Anopheles gambiae sensu lato with nets washed 20 times to field-aged nets withdrawn from households 3 years post-distribution to nets washed 20 times. The dashed line represents an odds ratio of 1, indicating no difference in mortality between field-aged and washed nets. Odds ratios < 1 indicate higher mortality with washed nets. Error bars represent 95% confidence intervals

Fig. 4

Reduction in fertility of wild, pyrethroid-resistant Anopheles gambiae sensu lato entering experimental huts in Covè southern Benin. Bars bearing the same letter do not differ at the 5% level according to logistic regression. Errors bars represent 95% confidence intervals

Fig. 5

Supplementary laboratory bioassay results showing the bioavailability and potency of active ingredients on net pieces obtained from new unwashed nets, nets washed 20 times and nets withdrawn after 3 years of operational use. Each panel presents results with a different net type and test method. Error bars represent 95% confidence intervals

Fig. 6

Chemical analysis results showing total active ingredient content (g/kg) in net pieces obtained from new unwashed nets, nets washed 20 times and nets withdrawn after 3 years of operational use. Each panel presents results with a different net type and active ingredient. In each panel, boxes bearing the same letter do not differ significantly at the 5% level, according to Tukey's honest significant difference post hoc tests

Fig. 7

Proportional retention of the pyrethroid and non-pyrethroid components in washed and field-aged PermaNet® 3.0 (roof) (a), Royal Guard® (b) and Interceptor® G2 (c). Retention was calculated relative to the mean active ingredient content with new unwashed nets. Brackets between bars indicate pairwise comparisons between groups, with p-values representing the statistical significance. P-values < 0.05 were considered significant. Error bars represent 95% confidence intervals

Fig. 8

Susceptibility bioassay results with wild Anopheles gambiae sensu lato collected near the experimental hut site in Covè, southern Benin. Mortality was recorded after 24 h for pyrethroid exposures and after 72 h for chlorfenapyr. Pyriproxyfen susceptibility was assessed based on the reduction in fertility of surviving blood-fed mosquitoes relative to the untreated control observed via ovary dissection. Red dashed line represents the standard 98% susceptibility cut-off, while the grey dashed line represents the provisional 90% cut-off to confirm chlorfenapyr resistance. Error bars represent 95% confidence intervals