Background: With insecticide resistance in malaria vectors spreading in geographical range and intensity, there is a need for compounds with novel modes of action to maintain the successes achieved to date by long-lasting insecticidal nets and indoor residual sprays, used as part of an insecticide resistance management strategy. Screening existing registered pesticides, predominantly those developed for use in agriculture, may provide a more rapid and less logistically challenging route to identifying active ingredients of value to public health than screening and chemical synthesis programmes for novel compounds. Methods: Insecticides and acaricides from all IRAC classes, including those with unclassified modes of action, were assessed for inclusion in a laboratory bioassay testing cascade against adult female Anopheles gambiae mosquitoes. A longlist of representative candidate compounds was selected, excluding those with safety concerns, unsuitable physiochemical properties, and likely hurdles to registration for public health use. An initial screen using topical application eliminated compounds with insufficient intrinsic activity, and a tarsal contact assay identified those with activity at an appropriate concentration. Compounds of interest were ranked by relative potency using dose response assays and discriminating dose calculations. Results: Inclusion of an adjuvant enhanced the tarsal efficacy of several compounds, facilitating the promotion of chemistries with great potential, given suitable formulation, which would not progress based on activity of compound alone. Comparison of data between stages in the testing cascade suggest that a more streamlined approach, topical application to test for intrinsic activity and determining the discriminating dose to compare relative potency of compounds, may be sufficient to identify compounds with potential value for use in long lasting insecticidal nets and indoor residual spray products. Conclusions: Identified were 11 compounds of interest as vector control agents (in descending order of potency): clothianidin, spinetoram, metaflumizone, dinotefuran, indoxacarb, abamectin, sulfoxaflor, oxazosulfyl, triflumezopyrim, fenpyroximate, and tolfenpyrad.

Pyrethroid resistance is widespread in malaria vectors. However, differential mortality in discriminating dose assays to different pyrethroids is often observed in wild populations. When this occurs, it is unclear if this differential mortality should be interpreted as an indication of differential levels of susceptibility within the pyrethroid class, and if so, if countries should consider selecting one specific pyrethroid for programmatic use over another. A review of evidence from molecular studies, resistance testing with laboratory colonies and wild populations, and mosquito behavioural assays were conducted to answer these questions. Evidence suggested that in areas where pyrethroid resistance exists, different results in insecticide susceptibility assays with specific pyrethroids currently in common use (deltamethrin, permethrin, α-cypermethrin, and λ-cyhalothrin) are not necessarily indicative of an operationally relevant difference in potential performance. Consequently, it is not advisable to use rotation between these pyrethroids as an insecticide-resistance management strategy. Less commonly used pyrethroids (bifenthrin and etofenprox) may have sufficiently different modes of action, though further work is needed to examine how this may apply to insecticide resistance management.

Accurately monitoring insecticide resistance in target mosquito populations is important for combating malaria and other vector-borne diseases, and robust methods are key. The “WHO susceptibility bioassay” has been available from the World Health Organization for 60+ years: mosquitoes of known physiological status are exposed to a discriminating concentration of insecticide. Several changes to the test procedures have been made historically, which may seem minor but could impact bioassay results. The published test procedures and literature for this method were reviewed for methodological details. Areas where there was room for interpretation in the test procedures or where the test procedures were not being followed were assessed experimentally for their impact on bioassay results: covering or uncovering of the tube end during exposure; the number of mosquitoes per test unit; and mosquito age. Many publications do not cite the most recent test procedures; methodological details are reported which contradict the test procedures referenced, or methodological details are not fully reported. As a result, the precise methodology is unclear. Experimental testing showed that using fewer than the recommended 15–30 mosquitoes per test unit significantly reduced mortality, covering the exposure tube had no significant effect, and using mosquitoes older than 2–5 days old increased mortality, particularly in the resistant strain. Recommendations are made for improved reporting of experimental parameters

Insecticides with novel modes of action are required to complement the pyrethroids currently relied upon for controlling malaria vectors. One example of this is the neonicotinoid clothianidin, the active ingredient in the indoor residual spray (IRS) SumiShield™ 50WG. In a preliminary experiment, the mortality of insecticide-susceptible and resistant An. gambiae adults exposed to filter papers treated with this IRS product reached 80% by 3 days post-exposure and 100% by 6 days post-exposure. Next, cement, wood, and mud tiles were treated with the clothianidin or a deltamethrin-based IRS formulation (K-Othrine WG250). Insecticide resistant and susceptible Anopheles and Aedes were exposed to these surfaces periodically for up to 18 months. Pyrethroid resistant Cx. quinquefasciatus was also exposed at 9 months. Between exposures, tiles were stored in heat and relative humidity conditions reflecting those found in the field. On these surfaces, the clothianidin IRS was effective at killing both susceptible and resistant An. gambiae for 18 months post-treatment, while mortality amongst the resistant strains when exposed to the deltamethrin IRS was not above that of the negative control. Greater efficacy of clothianidin was also demonstrated against insecticide resistant strains of An. funestus compared to deltamethrin, though the potency was lower when compared with An. gambiae. In general, higher efficacy of the clothianidin IRS was observed on cement and mud compared to wood, though it demonstrated poorer residual activity against Ae.aegypti and Cx. quinquefasciatus.

Background Resistance monitoring is a key element in controlling vector-borne diseases. The World Health Organization (WHO) and Centres for Disease Control and Prevention (CDC) have each developed bottle bioassay methods for determining insecticide susceptibility in mosquito vectors which are used globally. Methods This study aimed to identify variations in bottle bioassay methodologies and assess the potential impact on the data that is generated. Our approach involved a systematic examination of existing literature and protocols from WHO and CDC, with a focus on the specifics of reported methodologies, variation between versions, and reported outcomes. Building on this, we experimentally evaluated the impact of several variables on bioassay results. Results Our literature review exposed a significant inconsistency in the how bioassay methods are reported, hindering reliable interpretation of data and the ability to compare results between studies. The experimental research provided further insight by specifically identifying two key factors that influence the outcomes of bioassays: mosquito dry weight and relative humidity (RH). This finding not only advances our comprehension of these assays but also underscores the importance of establishing precisely defined methodologies for resistance monitoring. The study also demonstrates the importance of controlling bioassay variables, noting the significant influence of wing length, as an indicator of mosquito size, on mortality rates in standardized bioassays. Conclusions Generating data with improved protocol consistency and precision will not only deepen our understanding of resistance patterns but also better inform vector control measures. We call for continued research and collaboration to refine and build consensus on bioassay techniques, to help bolster the global effort against vector-borne diseases like malaria.

Background The WHO tube bioassay is a method for exposing mosquitos to determine susceptibility to insecticides, with mortality to discriminating doses <98% indicating possible resistance and <90% confirming resistance. This bioassay is also used for synergism testing to assess if susceptibility is restored by pre-exposure to the synergist piperonyl butoxide. Methods Here we perform testing with pyrethroid-susceptible and pyrethroid-resistant An. gambiae to quantify the variability of the WHO tube bioassay and identify its sources. These estimates of within and between day variability are then used to evaluate the power of the bioassay to detect a mortality difference between pyrethroid-only and pyrethroid-PBO. Results We show that approximately two-thirds of variation occurs between days, with the pyrethroid-susceptible strain twice as variable as the pyrethroid-resistant strain. The total number of mosquitoes in the tube and their bodyweight contributes to approximately 10% of this variability. Changes in temperature and humidity, within a climate-controlled insectary, didn’t impact mortality. Using a simulation-based framework, we show that the current synergism guidelines, using a 4x4 design, can reliably detect a difference between 90% and 100% mortality (>90% power). However, as the mortality of either group gets closer to 50%, a 10% difference between groups is more difficult to reliably detect. In the worst-case scenario where the mortality of either group is 50%, the mortality difference must be >22.5% to be detected with 80% power. We provide an R shiny application to assess power for other comparisons. Conclusions Our findings indicate that detecting synergism with the WHO tube assay is more difficult than assumed by the current WHO guidelines. Additionally, we demonstrate the value of using a Shiny application to make the outputs of simulation-based power analysis readily available to end-users, allowing them to determine the number of tubes needed to detect a given mortality difference.