Rosemary Susan Lees

The increasing use of passive spatial emanators (SEs) for vector control has underscored the need for standardised laboratory methods for bioefficacy testing. Peet–Grady (PG) chamber assays are widely used to test volatile insecticides; however, key methodological parameters remain poorly characterised. Here, we investigated how airflow settings (fan speed and baffle setup), mosquito-holding cage design, and cage mesh structure influence mosquito responses to a transfluthrin (TF) passive emanator. Susceptible and pyrethroid-resistant Anopheles gambiae mosquitoes were exposed to TF, and effects were assessed using flight inhibition (FI) and 24-hour mortality as endpoints. The assay was highly reproducible, with 83% agreement in FI dynamics across replicate comparisons. Airflow settings were more strongly associated with FI than cage geometry or mesh structure, with 5.86- to 9.57-fold higher odds of FI without a baffle. In contrast, 24-hour mortality varied less across airflow settings, suggesting that FI may be a more sensitive assay endpoint. Time to FI thresholds was up to 4-fold shorter under the medium fan speed setting without a baffle than under the low fan speed setting with a baffle. Cage mesh structure influenced FI responses, with larger mesh apertures showing 10- to 20-fold higher odds of FI in certain configurations, whereas cage geometry had little effect, supporting the use of a higher-throughput dual-cage configuration. These findings highlight the importance of airflow setting, mosquito-holding cage design, and cage mesh structure in determining FI responses in PG-chamber assays and support their consideration when standardising passive spatial emanator bioassays.

Introduction: Discriminating concentrations (DCs) underpin routine monitoring of insecticide susceptibility, but none had been established for broflanilide or isocycloseram, two new active ingredients entering vector control. This study aimed to establish and validate DCs in WHO bottle bioassays for monitoring susceptibility to broflanilide and isocycloseram in Anopheles gambiae s.s., An. funestus, An. stephensi , and Aedes aegypti . Methods: In 2024–2025, a multi-centre study involving seven international laboratories was conducted with the support of the World Health Organization (WHO), applying the generic WHO protocol for establishing discriminating concentrations to susceptible reference strains of each species. Results: The following values are recommended for adoption as DCs for broflanilide: 10 μg/bottle for An. gambiae and Ae. aegypti , 15 μg/bottle for An. funestus , and 25 μg/bottle for An. stephensi . The recommended DCs for isocycloseram are 15 μg/bottle for Ae. aegypti , 30 μg/bottle for An. gambiae , 50 μg/bottle for An. stephensi , and 60 μg/bottle for An. funestus . Discussion: Based on the experience gained from conducting this study, technical recommendations are made to support the generation and analysis of DC data for insecticides in the future.

The effectiveness of malaria vector control is increasingly threatened by widespread insecticide resistance, particularly to pyrethroids. In response, the World Health Organization recommends dual-active ingredients (dual-A.I.) insecticide-treated nets (ITNs) in resistant settings. This study evaluated the efficacy of several dual-A.I. ITNs in two experimental hut trial sites an area with high insecticide resistant malaria vectors in Tanzania with differing vector species composition and bionomics. Five ITNs were evaluated: a pyrethroid-only net (MAGNet®), three pyrethroid-piperonyl butoxide (PBO) nets (OlysetTM Plus, Veeralin®, and PermaNet® 3.0), one dual-A.I. net (Interceptor® G2; chlorfenapyr + alpha-cypermethrin), and an untreated control net. Mosquitoes were collected from huts, morphologically identified, and monitored for mortality up to 72 h. WHO cone and tunnel tests using susceptible An. gambiae s.s. were conducted for quality control, while field-collected mosquitoes were used for bio-efficacy monitoring. Molecular species identification was performed using PCR assays, and genomic DNA was extracted from An. funestus s.l. and An. gambiae s.l. collected from the trials. All ITNs met WHO bio-efficacy criteria in cone and tunnel tests. An. gambiae s.l. predominated in Mwagagala (77%) but was rare in Magu (4%), where An. funestus complex was dominant. In Magu, Interceptor® G2 and Veeralin® achieved significantly higher mortality than MAGNet® at 24 h (53% and 45% vs 24%) and 72 h (61% and 53% vs 24%). In contrast, no significant mortality differences were observed among ITNs in Mwagagala. High resistance intensity was detected in both vector groups (mortality An. funestus from Magu, the 4.3 kb-SV resistant genotype was fixed, while G454A-CYP9K1 resistance was highly prevalent (RR: 0.70– 1.00). Interceptor® G2 and Veeralin® outperformed the pyrethroid-only reference net against An. funestus complex but not against predominantly An. arabiensis populations.

Testing of insecticides and insecticide-based vector control tools relies on the use of bioassays whereby insects are exposed and mortality, or another relevant entomological endpoint, is measured. To be able to compare results between products and across time and space standard laboratory strains are used. Insecticide-susceptible strains are often used as comparators in studies involving insecticide-resistant populations. ‘Kisumu’ is a long-established, widely-used insecticide-susceptible strain of Anopheles gambiae . In this study, 16 facilities across different countries exposed their Kisumu colonies aged 3-5 days to four insecticides (permethrin, alpha-cypermethrin, DDT and pirimiphos-methyl) at a range of concentrations in WHO tube assays to assess susceptibility. Insecticide-treated filter papers were made at the Liverpool School of Tropical Medicine and sent to testing centres, along with a standard protocol and testing conditions requirements, to standardise testing as much as possible. A first observation was that DDT results were affected by the use of Risella oil, the WHO-recommended carrier oil but immiscible in acetone, leading to uneven coating of papers and increased variability in bioassay results (40x variance between colonies). Bioassay data generated by the testing facilities suggest a wide range of susceptibility to different insecticides between colonies of Kisumu: LC ₅₀ values between colonies differed, though not significantly, by up to 4x for permethrin, 6x for pirimiphos-methyl, and 15x for alpha-cypermethrin. Some colonies are classified as insecticide resistant based on the WHO definition of <98% mortality on exposure to a discriminating concentration. The assumption that Kisumu is a standard susceptible strain, consistent between facilities is challenged, which has implications when comparing results between studies. We propose genotypic analysis of the colonies to identify signs of resistance markers indicative of contamination from resistant strains or wild mosquitoes, and generation of guidance to support the maintenance and monitoring of susceptible mosquito strains.

Introduction: Progress against malaria has stalled since 2015, with insecticide resistance a key driver. Spatial emanators release volatile insecticides into the air, exposing mosquitoes through a route distinct from the tarsal contact used by treated nets, indoor residual spraying, and standard bioassays. Transfluthrin is currently monitored using the WHO bottle bioassay, which combines contact and vapour exposure and cannot isolate airborne effects. A scalable, vapour-only method is needed to characterise susceptibility to volatile pyrethroids. Methods: We adapted the WHO tube bioassay to deliver non-contact transfluthrin vapour exposure without contacting treated papers. Non-blood-fed female Anopheles were exposed to acetone control or transfluthrin papers at 0.005, 0.1 and 2 mg/paper. Knockdown was recorded over 60 minutes and mortality at 24 hours. Testing spanned three laboratories (LSTM, AIRID and KEMRI) using susceptible reference and resistant lab colonies. Dose response models estimated EC₅₀; for knockdown (EC₅₀KD) and mortality (EC₅₀Mort), with resistance ratios (RR) against the local susceptible reference. Results: The assay generated clear concentration- and time-dependent responses. At LSTM, Kisumu gave an EC₅₀KD of 0.052 and EC₅₀Mort of 0.054 mg/paper; Siaya was comparable (RR₅₀KD 1.17, RR₅₀Mort 0.89), whereas Tiassalé 13 and KDR showed reduced susceptibility (RR₅₀KD 2.42 and 2.85; RR₅₀Mort 5.56 and 6.65). At AIRID, the resistant Cové strain did not reach 50% knockdown or mortality. At KEMRI, Siaya showed reduced susceptibility (RR₅₀KD 3.36/RR₅₀Mort 5.65). Kisumu at AIRID and KEMRI was comparable to LSTM (EC₅₀KD 0.007/EC₅₀Mort 0.030; EC₅₀KD 0.044/EC₅₀Mort 0.023). Despite inter-laboratory variation, susceptible strains remained distinguishable from resistant ones. Lower RRs than for contact pyrethroids suggest contact-based phenotypes may not predict vapour susceptibility. Conclusion: The assay provides a practical benchtop method using standard WHO hardware, distinguishing susceptible from resistant strains across three laboratories. Twenty-four-hour mortality is the recommended endpoint, 60-minute knockdown secondary. Not yet validated, it provides a foundation for further validation and adaptation to other actives.

1 In 2024–2025 a multi-centre study involving seven international laboratories was conducted with the support of the World Health Organization (WHO). The aim of the study was to establish and validate discriminating concentrations (DCs) in WHO bottle bioassays for monitoring susceptibility to broflanilide and isocycloseram in Anopheles gambiae s . s ., An. funestus, An. stephensi and Aedes aegypti . The following values are recommended for adoption as DCs for broflanilide: 10 µg/bottle for An. gambiae and Ae. aegypti , 15 µg/bottle for An. funestus , and 25 µg/bottle for An. stephensi . The recommended DCs for isocycloseram are 15 µg/bottle for Ae. aegypti , 30 µg/bottle for An. gambiae , 50 µg/bottle for An. stephensi , and 60 µg/bottle for An. funestu s. Based on the experiences of conducting this study, which represents the application of a generic protocol for establishing discriminating concentrations produced by the WHO, technical recommendations are made on the generation and analysis of DC data for insecticides in future.

Dual-active-ingredient insecticide-treated nets (Dual-AI ITNs) combining pyrethroids with chlorfenapyr are increasingly being deployed across malaria-endemic areas. Laboratory evaluation of chlorfenapyr ITNs is challenging due to chlorfenapyr’s requirement for metabolic activation. Inspired by the tunnel test, the mini chamber test is smaller, has higher throughput, and is animal-free. We optimised the primary parameters using pyrethroid-susceptible and -resistant mosquito strains exposed to new pyrethroid-chlorfenapyr ITNs versus controls. With parameters defined, we used a draft standard operating procedure (SOP) to evaluate the mini chamber against the gold standard tunnel test in a comparator study. The generated data were used to finalise an SOP. The number of mosquitoes per bioassay had no effect on 72 hour mortality on exposure to the pyrethroid-chlorfenapyr net (10, 20, or 30 mosquitoes per mini chamber: 98% vs. 99% vs. 99%, p = 0.46), nor did the chamber orientation (horizontal vs. vertical: 70% vs. 74%, p = 0.35). Mean 72-hour mortality reduced with the length of exposure: 99% with overnight exposure, 74% with 4 hours and 35% with 2 hour exposure. In direct comparisons, the mini chamber gave 95% 72 hour mortality and the tunnel test 70% with overnight exposure. A trend was observed between chemical content (measured by high performance liquid chromatography (HPLC)) and mortality in ITN samples that had been washed 20 times or used in experimental hut trials, compared to unused comparators, when a 6 hour exposure length was used, showing potential for application to durability monitoring. A mini chamber SOP is shared (https://github.com/i2i-Data-Repository/Mini-Chamber-Test-paper-data-R-code-and-SOP), and further improvements may come from internal and multi-site validation and review of data generated. The method shows promise as an effective bioassay for use where tunnel tests are not available, for measuring the biological activity of chlorfenapyr in ITNs when measuring regeneration times, wash resistance, residual efficacy, and other studies.