Use of alternative bioassays to explore the impact of pyrethroid resistance on LLIN efficacy

Abstract

Background: There is substantial concern that the spread of insecticide resistance will render long-lasting insecticide-treated nets (LLINs) ineffective. However, there is limited evidence supporting a clear association between insecticide resistance and malaria incidence or prevalence in the field. We suggest that one reason for this disconnect is that the standard WHO assays used in surveillance to classify mosquito populations as resistant are not designed to determine how resistance might impact LLIN efficacy. The standard assays expose young, unfed female mosquitoes to a diagnostic insecticide dose in a single, forced exposure, whereas in the field, mosquitoes vary in their age, blood-feeding status, and the frequency or intensity of LLIN exposure. These more realistic conditions could ultimately impact the capacity of "resistant" mosquitoes to transmit malaria. Methods: Here, we test this hypothesis using two different assays that allow female mosquitoes to contact a LLIN as they host-seek and blood-feed. We quantified mortality after both single and multiple exposures, using seven different strains of Anopheles ranging in pyrethroid resistance intensity. Results: We found that strains classified as 1×-resistant to the pyrethroid insecticide deltamethrin in the standard WHO assay exhibited > 90% mortality over 24 h following more realistic LLIN contact. Mosquitoes that were able to blood-feed had increased survival compared to their unfed counterparts, but none of the 1×-resistant strains survived for 12 days post-exposure (the typical period for malaria parasite development within the mosquito). Mosquitoes that were 5×-and 10×-resistant (i.e. moderate or high intensity resistance based on the WHO assays) survived a single LLIN exposure well. However, only about 2-3% of these mosquitoes survived multiple exposures over the course of 12 days and successfully blood-fed during the last exposure. Conclusions: These results suggest that the standard assays provide limited insight into how resistance might impact LLIN efficacy. In our laboratory setting, there appears little functional consequence of 1×-resistance and even mosquitoes with moderate (5×) or high (10×) intensity resistance can suffer substantial reduction in transmission potential. Monitoring efforts should focus on better characterizing intensity of resistance to inform resistance management strategies and prioritize deployment of next generation vector control products.

The fabric of life: what if mosquito nets were durable and widely available but insecticide-free?

“…Though these thresholds are difficult to determine in field settings, sub-lethal insecticides have been shown to remain marginally impactful either by reducing survival of older mosquitoes [ 163 – 166 ] or disrupting development of malaria parasites inside the mosquitoes [ 166 – 168 ]. Moreover, repeated contacts may also lead to higher mortality typically undetectable using standard resistance tests [ 169 ]. These effects probably also contribute to the observed continued effectiveness of ITNs in pyrethroid resistance settings.…”

The need for practical insecticide-resistance guidelines to effectively inform mosquito-borne disease control programs

“…Standardized phenotypic insecticide-resistance bioassays are unlikely to reflect the efficacy of vector control intervention on mosquito control in the field for the aforementioned reasons. Indeed, this gap between bioassay data and mosquito control is frequently discussed in the literature ( Churcher et al, 2016 ; Grossman et al, 2020 ; Hancock et al, 2018 ; Ranson and Lissenden, 2016 ; Sherrard-Smith et al, 2018 ; Venter et al, 2017 ; Vontas and Mavridis, 2019 ) and even acknowledged in the WHO test procedure guidelines ( World Health Organization, 2016b ). This disparity between the insecticide efficacy in a lab bioassay and in a field setting may explain the fact that the strong increase in pyrethroid resistance globally (as determined by laboratory bioassays) does not always translate well to mosquito control failure, nor to transmission control failure (as reviewed in Alout et al, 2017a ; Alout et al, 2017b ).…”

“…The complexities described above may explain some of the widely acknowledged discrepancies between laboratory resistance bioassay results and the observed nonfailure of vector control interventions ( Churcher et al, 2016 ; Grossman et al, 2020 ; Ranson and Lissenden, 2016 ; Sherrard-Smith et al, 2018 ; Vontas and Mavridis, 2019 ). The WHO introduced intensity bioassays by incorporating 5x and 10x discriminating concentrations in their resistance testing guidelines, mentioning that “resistance phenotypes detected using the discriminating concentrations do not necessarily provide information in terms of efficacy failure of that insecticide in the field” ( World Health Organization, 2016b ).…”

“…Furthermore, the cone test does not replicate the natural contact of the mosquito with interventions, as highlighted above. There are some tests (such as WHO’s tunnel test, tent assay, cup assay, and the Mosquito Contamination Device (MCD) bottle assay) that allow for more natural mosquito behavioral responses (including transient contact and avoidance) to insecticide-treated nets, thereby assessing if the efficacy of those nets is underestimated compared to standard cone bioassays ( Grossman et al, 2020 ; Sternberg et al, 2014 ; World Health Organization, 2013a ). This is particularly relevant for insecticides that have a high excito-repellent effect, such as permethrin.…”

Determination of the amount of insecticide picked up by mosquitoes from treated nets

“…Variability between individual insects has been observed in nature, caused by differences in mosquito age, blood-feeding status, the presence of different resistance mechanisms and any fitness costs they might incur, and differences in mosquito behaviour in relation to LLINs and frequency of exposure [49]. Some resistance genes, such as kdr, are only weakly related to survival and their presence also means that mosquitoes are less repelled by pyrethroids, so they tend to acquire a higher dose of insecticide which can be lethal [20].…”