Determination of the Preliminary Discriminating Concentration of Broflanilide Against Malaria Vector Mosquito Anopheles gambiae by Multi-Centre Susceptibility Testing

Determination of the Preliminary Discriminating Concentration of Broflanilide Against Malaria Vector Mosquito Anopheles gambiae by Multi-Centre Susceptibility Testing | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Determination of the Preliminary Discriminating Concentration of Broflanilide Against Malaria Vector Mosquito Anopheles gambiae by Multi-Centre Susceptibility Testing Yuki Ando, Kunizo Mori, John Bradley, Janneke Snetselaar, Graham Small This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7478645/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Mar, 2026 Read the published version in Malaria Journal → Version 1 posted 13 You are reading this latest preprint version Abstract Indoor residual spraying (IRS) of insecticides is widely used as an effective method to control malaria vector mosquitoes in sub-Saharan Africa. In 2023, a new IRS product, VECTRON™ T500 (Mitsui Chemicals Crop & Life Solutions, Inc.), was launched. This product contains broflanilide, a novel active ingredient for IRS, and has been confirmed to exhibit long-lasting insecticidal efficacy against malaria vector mosquitoes. However, the discriminating concentration to assess the susceptibility of wild Anopheles populations to broflanilide has not yet been determined. In this study, WHO bottle bioassays were conducted in nine research facilities to collect dose-response data on broflanilide against adult female mosquitoes of the insecticide susceptible Anopheles gambiae s.s. Kisumu strain. These data were statistically analysed and validated following WHO guidelines, and it was determined that the preliminary discriminating concentration of broflanilide for An. gambiae mosquitoes should be 18 μg/bottle. The results of this study will provide a useful benchmark for susceptibility monitoring of wild mosquito populations in regions of sub-Saharan Africa into which VECTRON™ T500 is being introduced for malaria vector control. Mosquito control Indoor residual spraying Broflanilide Discriminating concentration Figures Figure 1 1. Background Anopheline mosquitoes transmit malaria parasites to humans leading to malaria infections in sub-Saharan African countries [ 1 ]. To reduce and eliminate malaria in these countries, effective methods for the control of vector mosquitoes are essential. One of the most effective methods of mosquito vector control is indoor residual spraying (IRS) of insecticides, which has been widely used for several decades [ 2 , 3 ]. Killing blood-fed mosquitoes that rest on the indoor walls of houses, reduces the local vector population and prevents mosquitoes that have taken an infected blood meal from transmitting the malaria parasites in subsequent blood feeding. It is recommended that IRS products should have long-lasting residual efficacy, preferably of at least six months duration, as this is generally the main period of malaria transmission in sub-Saharan Africa [ 1 ]. A number of insecticides with several different modes of action (e.g., DDT, carbamates, organophosphates, and pyrethroids) have been used previously for IRS malaria vector control programmes, but overuse of the products into which these insecticides have been formulated has led to the evolution and spread of insecticide resistance in many vector populations [ 2 , 4 – 9 ]. This, in turn, has led to reduced effectiveness of some of the existing IRS products; therefore, the development and use of IRS products containing novel insecticides, to which there is currently no detected resistance, is essential to our fight against malaria [ 10 – 19 ]. VECTRON™ T500 (active ingredient broflanilide 50% wettable powder), developed by Mitsui Chemicals Crop & Life Solutions, Inc. (MCCLS), is a new IRS product that was evaluated and listed by the World Health Organization Prequalification Unit Vector Control Product Assessment Team (WHO PQT/VCP) in March 2023 and was released in December 2023. Previous studies have confirmed that this formulation exhibits high and long-term efficacy against Anopheline mosquitoes [ 20 – 28 ]. The active ingredient, broflanilide (tradename TENEBENAL™), is a novel meta-diamide insecticide compound developed by MCCLS. This compound is classified in IRAC group 30 (allosteric modulator of gamma-aminobutyric acid (GABA)-gated chloride ion channels) and has a novel mode of action [ 29 , 30 ] that differs from the active ingredients in existing IRS formulations. Previous studies have detected no cross-resistance to broflanilide in pyrethroid resistant mosquito strains or populations; therefore, the rotational use of this compound with compounds with different modes of action can be a viable option for controlling these malaria vectors and manage resistance development [ 20 – 28 ]. As part of any resistance management strategy, it is essential to establish the baseline susceptibility of wild mosquito populations in regions where the use of an insecticide is planned and to monitor for the possible development of resistance thereafter. For this purpose, insecticide susceptibility test methods have been developed which use a concentration of an insecticide that will discriminate between insecticide susceptible and resistant mosquitoes in terms of mortality (DC: Discriminating Concentration). For this susceptibility testing, the WHO recommends conducting a WHO bottle bioassay [ 31 , 32 ] with the DC for insecticide being used in mosquito vector control being determined via multi-centre studies led by WHO (e.g., the DCs of existing compounds for An. gambiae are as follows: clothianidin, 4 µg/bottle; flupyradifurone, 60 µg/bottle; transfluthrin, 2 µg/bottle; and chlorfenapyr, 100 µg/bottle) [ 32 , 33 ]. However, the DC of broflanilide has not been determined yet by WHO due to its novelty [ 32 ], while in African countries, there is a need for baseline susceptibility testing by implementing programmes as they introduce VECTRON™ T500 into their IRS campaigns [ 20 – 23 , 35 – 38 ]. Therefore, it is important to identify early on a preliminary DC (PDC) that can be used to monitor mosquito population susceptibility to broflanilide ahead of the introduction of this new insecticide and before WHO publishes its definitive DC. In this study, to determine the PDC of broflanilide, we followed as far as was possible the same process for DC establishment as described by WHO [ 32 ]. Previous studies have shown that the addition of the adjuvant Mero (81% rapeseed oil methyl ester; Bayer AG, Crop Science Division) can enhance and stabilize the compound's efficacy by inhibiting crystallization [ 31 – 34 ], and the DCs of broflanilide with 800 ppm Mero for An. gambiae have been proposed for use in association with large-scale community trials on VECTRON™ T500 in previous studies (6 µg/bottle and 17.82 µg/bottle) [ 35 , 36 ]. However, these PDCs were estimated using only one or a few datasets from dose response tests with An. gambiae . According to WHO guidelines, it is desirable to have three or more datasets to establish the DC [ 32 ]. Therefore, this study aimed to propose a more robust PDC by generating broflanilide does response data at multiple research facilities in Africa and the UK. Colonies of the insecticide susceptible An. gambiae s.s. Kisumu strain established at these research facilities were used for tests with a range of broflanilide concentrations and the data were statistically analysed to determine the PDC. This PDC will provide the necessary information for the stewardship of VECTRON™ T500 via broflanilide susceptibility monitoring at an early stage in in the roll out of this IRS product. 2. Materials & Methods 2.1. Mosquito Strain Colonies of the Anopheles gambiae s.s. Kisumu strain maintained by each trial facility were used in all the testing as an insecticide susceptible strain. This strain was originally collected in the Kisumu region of Kenya. All mosquitoes used were 2–5 day old, non-blood-fed adult females. 2.2. Susceptibility Testing with Broflanilide by WHO Bottle Bioassays at Multiple Study Facilities To obtain data on the dose response to broflanilide of Kisumu strain mosquitoes, WHO bottle bioassays were conducted [ 31 ]. The glass bottles (250 mL, Wheaton bottles) were treated with 1 mL of broflanilide dissolved in a mixture of acetone and 800 ppm Mero. Bottles treated with acetone + Mero only were also prepared as a negative control. Twenty-five (25) mosquitoes were introduced into each bottle and exposed for 1 hour. After exposure, the mosquitoes were aspirated into clean cups and provided with cotton wool soaked in a 10% sugar solution ad libitum . Mortality was recorded at 24 h intervals up to 72 h post exposure. The bioassays were conducted under laboratory conditions (27 ± 2℃, 80 ± 10% RH). There were some minor variations to the bioassays conducted by the 9 research facilities (i.e. the concentrations used, number of replicates and number of mosquitoes) which are detailed in Table 1 . The data generated by the Liverpool Insect Testing Establishment (LITE) facility (UK) were fully Good Laboratory Practice (GLP) compliant. The data from the African research facilities were generated during the conduct of non-GLP semi-field experimental hut or community VECTRON™ T500 product registration trials following the standard operating procedures implemented at the respective facilities. Data from 4 of the studies have previously been published elsewhere (Tanzania KCMUCo and NIMR [ 35 ], Benin [ 36 ], Burkina Faso [ 38 ]) but were included in our statistical analysis to provide further supporting information for the setting of the PDC. Table 1 Test conditions from each testing facility Country (facility) Location Concentrations (µg/bottle) Number of replicates Number of mosquitos Zambia (Tropical Disease Research Centre) Nchelenge 0.781, 1.563, 3.125, 6.25, 12.5, 25, 50 5 1037 Uganda (NMCD MOH) Nosako 1.563, 3.125, 4, 6.25, 12.5, 25 4 750 DRC (University of Kinshasa) Kinshasa 0.195, 0.39, 0.781, 1.563, 3.125, 6.25, 12.5, 25, 50 4 889 Burkina Faso (IRSS/DRO) Vallée du Kou 0.781, 1.563, 3.125, 6.25, 12.5, 25, 50 4 778 Ghana (NMIMR) Odumse 0.781, 1.563, 3.125, 6.25, 12.5, 25, 50 4 800 Benin (CREC/LSHTM) Cotonou 0.05, 0.1, 0.25, 0.5, 1, 2.2, 4.6, 10 4 800 Tanzania (KCMUCo-PAMVERC) Moshi 0.01, 0.0215, 0.0464, 0.1, 0.215, 1.0, 2.15, 4.64, 10 6 1661 Tanzania (NIMR Amani Reach Centre) Muheza 0.01, 0.0215, 0.0464, 0.1, 0.215, 1.0, 2.15, 4.64, 10 4 850 UK (LITE) Liverpool 0.3, 0.86, 1.42, 1.98, 2.54, 3.1 9 1571 2.3 Data Analysis All data analysis was undertaken using Stata 18. A probit model was used to estimate the lethal concentration that would induce 99% mortality in mosquitoes (LC 99 ). The independent variable was the logorith of the concentration of broflanilide. Abbott’s formula was used to correct mortality in test replicate for mortality in the negative controls (acetone and 800ppm Mero only). $$\:\text{C}\text{o}\text{r}\text{r}\text{e}\text{c}\text{t}\text{e}\text{d}\:\text{m}\text{o}\text{r}\text{t}\text{a}\text{l}\text{i}\text{t}\text{y}=\frac{\left(\text{%}\:\text{o}\text{b}\text{s}\text{e}\text{r}\text{v}\text{e}\text{d}\:\text{m}\text{o}\text{r}\text{t}\text{a}\text{l}\text{i}\text{t}\text{y}\:-\:\text{%}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\:\text{m}\text{o}\text{r}\text{t}\text{a}\text{l}\text{i}\text{t}\text{y}\right)}{\left(100\:-\:\text{%}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\:\text{m}\text{o}\text{r}\text{t}\text{a}\text{l}\text{i}\text{t}\text{y}\right)}\times\:100$$ Confidence intervals were estimated using the bootstrap, which took uncertainty in the control mortality into account. For each probit regression, a p-value for the Pearson’s Chi-squared test (goodness of fit) was calculated. 3. Results 3.1 Dose Response in Test Results for Each Facility Figure 1 shows the dose-response curves of the susceptible An. gambiae s.s. Kisumu strain mosquitoes to broflanilide obtained from the WHO bottle bioassays conducted by the 9 research facilities. In eight of the datasets, excluding those from the UK facility, 100% mortality was confirmed within the tested concentration ranges, but there was variability in the lowest LC 100 values (Zambia, DRC, and Ghana: 0.781 µg/bottle; Benin and Tanzania NIMR: 1.0 µg/bottle; Uganda: 1.562 µg/bottle; Tanzania KCMUCo: 2.15 µg/bottle; and Burkina Faso: 6.25 µg/bottle) (Table 2 ). Additionally, each dose-response curve rose very steeply which is typical for an insecticide susceptible strain (Fig. 1 ). In the bottle bioassay data from Zambia and Uganda, 100% mortality was observed at all broflanilide concentrations tested. Conversely, in the test results from the UK facility, the maximum mortality rate observed for the range of tested concentrations was 90.35%, and 100% mortality was not achieved. 3.2 Overview of Estimated Lethal Concentration (LC 99 ) Values for Each Facility and Determination of the Preliminary Discriminating Concentration for Broflanilide The data and statistical analysis outputs from the nine dose response studies (LC 99 and 95% CI, goodness of fit, number of data points with mortality rates neither 0% nor 100%, the lowest LC 100 if available, and 2x the LC99 or LC100) are summarised in Table 2 . The LC 99 of broflanilide for the susceptible An. gambiae s.s. Kisumu strain varied from 1.02 to 27.84 µg/bottle. The guidance from WHO on the validation of datasets [ 32 ] considers several aspects. First, to be considered valid, each dataset should have at least two concentrations that killed 50% of mosquitoes and one concentration that killed about 100% mosquitoes. After this, the validation of each dataset should consider the following: mortality of controls should be below the cut-off point of 20%; a minimum of six concentrations should be available to generate concentration–response curves and estimate lethal concentrations; the goodness of fit p-value should be > 0.05; and the lowest concentration that killed 100% of mosquitoes (LC 100 ), when available. Considering the datasets generated by the 9 research facilities, none could satisfy all the validation requirements stated by WHO. Therefore, we determined which datasets best matched WHO guidance. Generally, probit analysis represents the relationship between mortality and the logarithm of dose as a linear relationship [ 39 ]. Therefore, to perform linear regression, at least three data points are necessary in which the mortality rates of neither 0% nor 100% (if there are only two data points, the fit of the regression line will be perfect (R 2 = 1)).The number of data points used in the probit analysis for calculating the LC 99 was 0 or 2 in four datasets (Zambia, Uganda, DRC, and Ghana). Additionally, in three datasets, the p-value of the chi-squared test indicating the goodness of fit was less than 0.05 (Benin, Tanzania, and the UK). There were two results with a goodness of fit p-value greater than 0.05 and three or more valid data points (Tanzania NIMR and Burkina Faso). The LC99 values were 0.57 µg/bottle (Tanzania NIMR) and 8.42 µg/bottle (Burkina Faso). Therefore, taking into account WHO guidance on determining the DC of insecticides for monitoring resistance in mosquitoes in which the highest LC 99 is to be used [ 32 ], we took the 2×LC 99 for the Burkina Faso dataset (16.84 µg/bottle) to inform the appropriate PDC for susceptibility testing with broflanilide. Table 2 Summary of the data from multi-centre susceptibility testing of broflanilide against the insecticide susceptible An. gambiae s.s. Kisumu strain Country LC 99 ± 95%CI (µg/bottle) p-value of Chi-sq test 2×LC 99 (µg/bottle) Number of valid datapoint Observed lowest LC 100 (µg/bottle) 2×LC 100 (µg/bottle) Zambia NA NA NA 0 0.781 1.562 Uganda NA NA NA 0 1.563 3.126 Burkina Faso 8.42 [4.73–12.10] 0.3243 16.84 5 6.25 12.5 DRC 1.06 [0.91–1.21] 0.0636 2.12 2 0.781 1.562 Benin 1.02 [0.85–1.18] 0.0001 2.04 4 1 2 Ghana 1.08 [0.89–1.26] 0.0899 2.16 2 0.781 1.562 Tanzania (KCMUCo) 27.84 [17.75–37.90] <0.0001 55.68 7 2.15 4.3 Tanzania (NIMR) 0.57 [0.31–0.82] 0.4268 1.14 5 1 2 UK 13.44 [9.76–16.96] 0.0136 26.88 6 NA NA 4. Discussion 4.1 Establishment of the Preliminary Discriminating Concentration Most of the dose response studies were not specifically designed for the determination of the PDC for broflanilide but were part of non-GLP semi-field or community VECTRON™ T500 product registration trials. Therefore, it was not possible to meet all of the data validation criteria described by WHO [ 32 ]. Based on the calculation of LC 99 and statistical analysis results of each test shown in Table 2 , the PDC of broflanilide for the susceptible An. gambiae s.s. Kisumu strain was determined. Following the verification method for data as guided by WHO [ 32 ], for datasets with a goodness of fit p > 0.05 (Burkina Faso and Tanzania NIMR), the value twice the LC 99 was adopted for those tests. For datasets with a goodness of fit p < 0.05 (Benin, and Tanzania KCMUCo), the reliability of the LC 99 was regarded low. Additionally, the data with 100% mortality at all test concentrations (Zambia and Uganda) and the data where only two valid data points were obtained (DRC and Ghana), it was considered inappropriate to refer to the results of the probit analysis. For the results from the LITE facility in the UK, the goodness of fit p-value was less than 0.05 and no LC 100 was confirmed. Finally, among the nine LC 99 values determined through the above data verification, the highest was 8.42 µg/bottle (Burkina Faso). According to WHO guideline [ 32 ], twice this value, 16.84 µg/bottle, was determined to be the most appropriate for selecting the PDC for An. gambiae . Rounding this value to make it simpler results in 17 µg/bottle; however, since 17 is a prime number, it may pose difficulties in practical operations such as preparing stock solutions, working solutions, or performing serial dilutions. Additionally, several African countries intending to use VECTRON™ T500 are already conducting susceptibility tests with a provisional value of 18 µg/bottle (established from unpublished data). Following discussions with IRS programme implementers on the practicality of broflanilide susceptibility monitoring in sub-Saharan Africa and, therefore, to simplify the dilution of broflanilide for the treatment of bottles, we propose the PDC for broflanilide to be 18 µg/bottle (with 800 ppm Mero). 4.2 The Variation Between the Estimated LC Values Among Trial Facilities The results obtained in this research showed variations in the dose response of broflanilide for each test data set, even though the same strain ( An. gambiae s.s. Kisumu) was used. The mosquito colonies used in this study had been maintained and reared for a long time at the different testing facilities. Since the methods and conditions (e.g., temperature, humidity, density) for continuous rearing in each facility were not exactly the same, this might have led to variations in the general fitness and tolerance to exposure to broflanilide of the respective colonies. Ideally, cross-validation of the properties of the susceptible mosquito colonies and experimental methods across testing facilities should be conducted [ 32 ]. However, as mentioned previously, the eight bottle bioassays conducted in this study, apart from those conducted by the UK facility, were not specifically designed as dose-response studies to determine the PDC for broflanilide. Therefore, minor variations in the detailed procedures during the experiments (e.g., preparation and serial dilution of the broflanilide working solution, treatment of bottles, mosquito treatment when introducing into bottles or retrieval from bottles) or the storage conditions of mosquitoes after exposure to broflanilide (e.g., temperature, humidity, method of providing sugar solution) might also have influenced the dose-response to broflanilide in the mosquito populations at each testing facility. 5. Conclusion In this study, nine datasets of WHO bottle bioassays conducted at the multiple trial facilities were conducted, the DC (2xLC 99 ) of broflanilide for the An. gambiae s.s. Kisumu strain was determined to be 16.84 µg/bottle and a PDC of 18 µg/bottle is proposed for monitoring the susceptibility of wild populations of An. gambiae to broflanilide. However, there was considerable variability in the dose-response to broflanilide among the nine datasets used in this study. As a result of data validation, only two datasets were considered valid for the determination of the PDC. Data generated in further broflanilide dose-response studies are needed to establish a definitive DC for broflanilide, and the PDC proposed here may change based on the results of future studies sponsored by WHO to determine the definitive DC. However, for regions where the VECTRON™ T500 IRS product, containing broflanilide as an active ingredient, is already in use or will be used in the near future as part of malaria vector control campaigns, the PDC of broflanilide proposed by this study will serve as a benchmark for assessing the baseline susceptibility of wild populations of An. gambiae to broflanilide and to monitor for any changes in susceptibility with time. The data generated in susceptibility testing will facilitate rapid decision-making regarding the use of VECTRON™ T500 and, together with susceptibility data for insecticides used in other IRS products, will inform the implementation of insecticide resistance management through the rotation of IRS products containing insecticides with different modes of action. Abbreviations DC Discriminating Concentration GLP Good Laboratory Practice IRS Indoor Residual Spraying LC Lethal Concentration PDC Preliminary Discriminating Concentration Declarations Data availability All data associated with this study are present in the paper. All other relevant data are available from the corresponding author upon reasonable request. Acknowledgements In conducting this study, we received the broflanilide dose response data for the An. gambiae Kisumu strain. Some of them are from previously published studies. We would like to express our gratitude to the following data providers and authors. Dr. Natalie M. Portwood and Dr. Louisa Messenger [35], Dr. Corine Ngufor and Dr. Renaud Govoetchan [36], Prof. Abdoulaye Diabaté and Dr. Aristide Sawdetuo Hien [38], Jessica Williams, Amy Guy and Jennie Gibson (Liverpool Insect Testing Establishment, Liverpool, UK), Dr. Mbanga Muleba (Tropical Diseases Research Centre, Ndola, Zambia), Dr. Ntege Charles (National Malaria Control Division, Kampala, Uganda), Dr. Thierry Bobanga (Universite de Kinshasa, Kinshasa, Democratic Republic of the Congo) and Dr. Samuel Kweku Dadzie (Noguchi Memorial Institute for Medical Research, Accra, Ghana). Funding This study was funded through support from the Bill & Melinda Gates Foundation (grant: INV-007509), the Swiss Agency for Development and Cooperation (SDC) (grant: 81067480) and UK Aid (grant: 30041-105). The findings and conclusions contained within are those of the authors and do not necessarily reflect the positions or policies of the Bill & Melinda Gates Foundation, SDC, UK Aid or IVCC. Author information Authors and Affiliations Mitsui Chemicals Crop & Life Solutions, Inc., Tokyo, Japan Yuki Ando (ORCID iD : 0009-0005-4526-5082) Advisor to Mitsui Chemicals Crop & Life Solutions, Inc., Tokyo, Japan Kunizo Mori London School of Hygiene and Tropical Medicine, London, UK John Bradley (ORCID iD : 0000-0002-9449-4608) Innovative Vector Control Consortium, Liverpool, UK Janneke Snetselaar (ORCID iD : 0000-0002-5882-6855) & Graham Small Contributions YA, KM, JS and GS conceived and designed the study. JB analysed the study data. YA drafted the first version of the manuscript. All authors contributed to the manuscript and approved the submitted version. Corresponding author Correspondence to Yuki Ando Ethics declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. References World Health Organization. Indoor residual spraying: an operational manual for indoor residual spraying (IRS) for malaria transmission control and elimination . World Health Organization, 2015. ISBN: 978 92 4 150894 0 https://iris.who.int/bitstream/handle/10665/177242/9789241508940_eng.pdf Ngufor, Corine, et al. "Indoor spraying with chlorfenapyr (a pyrrole insecticide) provides residual control of pyrethroid-resistant malaria vectors in southern Benin." Malaria journal 19 (2020): 1-11. ISSN: 1475-2875 https://doi.org/10.1186/s12936-020-03325-2 Oxborough, Richard M. "Trends in US President’s Malaria Initiative-funded indoor residual spray coverage and insecticide choice in sub-Saharan Africa (2008–2015): urgent need for affordable, long-lasting insecticides." Malaria journal 15.1 (2016): 146. ISSN: 1475-2875 https://doi.org/10.1186/s12936-016-1201-1 Killeen, Gerry F., and Hilary Ranson. "Insecticide-resistant malaria vectors must be tackled." The Lancet 391.10130 (2018): 1551-1552. ISSN: 0140-6736 http://dx.doi.org/10.1016/S0140-6736(18)30844-4 Mathias, Derrick K., et al. "Spatial and temporal variation in the kdr allele L1014S in Anopheles gambiae ss and phenotypic variability in susceptibility to insecticides in Western Kenya." Malaria journal 10 (2011): 1-13. ISSN: 1475-2875 http://www.malariajournal.com/content/10/1/10 Ahoua Alou, Ludovic P., et al. "Distribution of ace-1 R and resistance to carbamates and organophosphates in Anopheles gambiae ss populations from Côte d'Ivoire." Malaria Journal 9 (2010): 1-7. ISSN: 1475-2875 http://www.malariajournal.com/content/9/1/167 Main, Bradley J., et al. "Complex genome evolution in Anopheles coluzzii associated with increased insecticide usage in Mali." Molecular ecology 24.20 (2015): 5145-5157. ISSN: 0962-1083 https://onlinelibrary.wiley.com/doi/full/10.1111/mec.13382 Barnes, Kayla G., et al. "Genomic footprints of selective sweeps from metabolic resistance to pyrethroids in African malaria vectors are driven by scale up of insecticide-based vector control." PLoS genetics 13.2 (2017): e1006539. ISSN: 1553-7404 https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1006539 World Health Organization. "Global report on insecticide resistance in malaria vectors: 2010–2016." (2018). ISBN: 978 92 4 151405 https://iris.who.int/bitstream/handle/10665/272533/9789241514057-eng.pdf Rowland, Mark, et al. "A new long-lasting indoor residual formulation of the organophosphate insecticide pirimiphos methyl for prolonged control of pyrethroid-resistant mosquitoes: an experimental hut trial in Benin." PloS one 8.7 (2013): e69516. ISSN: 1932-6203 https://doi.org/10.1371/journal.pone.0069516 Chanda, Emmanuel, et al. "Preventing malaria transmission by indoor residual spraying in Malawi: grappling with the challenge of uncertain sustainability." Malaria journal 14 (2015): 1-7. ISSN: 1475-2875 https://link.springer.com/article/10.1186/s12936-015-0759-3 Haji, Khamis A., et al. "Efficacy, persistence and vector susceptibility to pirimiphos-methyl (Actellic ® 300CS) insecticide for indoor residual spraying in Zanzibar." Parasites & vectors 8 (2015): 1-7. ISSN: 1756-3305 https://link.springer.com/article/10.1186/s13071-015-1239-x Ngufor, Corine, et al. "Indoor residual spraying with a mixture of clothianidin (a neonicotinoid insecticide) and deltamethrin provides improved control and long residual activity against pyrethroid resistant Anopheles gambiae sl in Southern Benin." PloS one 12.12 (2017): e0189575. ISSN: 1932-6203 https://doi.org/10.1371/journal.pone.0189575 Mashauri, Fabian M., et al. "Indoor residual spraying with micro-encapsulated pirimiphos-methyl (Actellic 300CS) against malaria vectors in the Lake Victoria basin, Tanzania." PloS one 12.5 (2017): e0176982. ISSN: 1932-6203 https://www.nature.com/articles/s41598-020-61350-2 Kweka, Eliningaya, et al. "Novel indoor residual spray insecticide with extended mortality effect: a case of SumiShield 50WG against wild resistant populations of Anopheles arabiensis in Northern Tanzania." Global Health: Science and Practice 6.4 (2018): 758-765. ISSN: 2169-575X https://doi.org/10.9745/GHSP-D-18-00213 Agossa, Fiacre R., et al. "Efficacy of a novel mode of action of an indoor residual spraying product, SumiShield ® 50WG against susceptible and resistant populations of Anopheles gambiae (sl) in Benin, West Africa." Parasites & vectors 11 (2018): 1-13. ISSN: 1756-3305 https://doi.org/10.1186/s13071-018-2869-6 Abong’o, Bernard, et al. "Impact of indoor residual spraying with pirimiphos-methyl (Actellic 300CS) on entomological indicators of transmission and malaria case burden in Migori County, western Kenya." Scientific reports 10.1 (2020): 4518. ISSN: 2045-2322 https://doi.org/10.1038/s41598-020-61350-2 Chabi, Joseph, et al. "Efficacy of partial spraying of SumiShield, Fludora Fusion and Actellic against wild populations of Anopheles gambiae sl in experimental huts in Tiassalé, Côte d'Ivoire." Scientific Reports 13.1 (2023): 11364. ISSN: 2045-2322 https://doi.org/10.1038/s41598-023-38583-y Thiomela, Riccado F., et al. "Indoor residual spraying of experimental huts in Cameroon highlights the potential of Fludora ® Fusion to control wild pyrethroid-resistant malaria vectors." BMC Infectious Diseases 24.1 (2024): 733. ISSN: 1471-2334 https://doi.org/10.1186/s12879-024-09630-4 Lees, Rosemary Susan, et al. "Tenebenal: a meta-diamide with potential for use as a novel mode of action insecticide for public health." Malaria Journal 19 (2020): 1-17. ISSN: 1475-2875 https://doi.org/10.1186/s12936-020-03466-4 Ngufor, Corine, et al. "Efficacy of broflanilide (VECTRON T500), a new meta-diamide insecticide, for indoor residual spraying against pyrethroid-resistant malaria vectors." Scientific reports 11.1 (2021): 7976. ISSN: 2045-2322 https://doi.org/10.1038/s41598-021-86935-3 Govoetchan, Renaud, et al. "VECTRON™ T500, a new broflanilide insecticide for indoor residual spraying, provides prolonged control of pyrethroid-resistant malaria vectors." Malaria Journal 21.1 (2022): 324. ISSN: 1475-2875 https://doi.org/10.1186/s12936-022-04336-x Ngufor, Corine, et al. "Community evaluation of VECTRON™ T500, a broflanilide insecticide, for indoor residual spraying for malaria vector control in central Benin; a two arm non-inferiority cluster randomised trial." Scientific reports 13.1 (2023): 17852. ISSN: 2045-2322 https://doi.org/10.1038/s41598-023-45047-w Bayili, Koama, et al. "Laboratory and experimental hut trial evaluation of VECTRON™ T500 for indoor residual spraying (IRS) against insecticide resistant malaria vectors in Burkina Faso." Gates Open Research 6 (2022). ISSN: 2572-4754 https://pmc.ncbi.nlm.nih.gov/articles/PMC9326088/#abstract1 Snetselaar, Janneke, et al. "Efficacy of indoor residual spraying with broflanilide (TENEBENAL), a novel meta-diamide insecticide, against pyrethroid-resistant anopheline vectors in northern Tanzania: An experimental hut trial." PloS One 16.3 (2021): e0248026. ISSN: 1932-6203 https://doi.org/10.1371/journal.pone.0248026 Mbewe, Njelembo J., et al. "A non-inferiority and GLP-compliant study of broflanilide IRS (VECTRON™ T500), a novel meta-diamide insecticide against Anopheles arabiensis." Frontiers in Tropical Diseases 4 (2023): 1126869. ISSN: 2673-7515 https://www.frontiersin.org/journals/tropical-diseases/articles/10.3389/fitd.2023.1126869/full#h4 Kweka, Eliningaya J., et al. "The bioefficacy of a novel VECTRON™ T500 indoor residual spray formulation in an experimental huts trial against Anopheles gambiae sl populations." Acta Tropica 259 (2024): 107376. ISSN: 1873-6254 https://doi.org/10.1016/j.actatropica.2024.107376. Simma, Eba Alemayehu, et al. "Determination of the Residual Efficacy of Broflanilide (VECTRONTM T500) Insecticide for Indoor Residual Spraying in a Semi-field Setting in Ethiopia." (2024). [preprint] https://doi.org/10.21203/rs.3.rs-4773038/v1 Nakao, Toshifumi, and Shinichi Banba. "Broflanilide: A meta-diamide insecticide with a novel mode of action." Bioorganic & medicinal chemistry 24.3 (2016): 372-377. ISSN: 0968-0896 https://doi.org/10.1016/j.bmc.2015.08.008. Umetsu, Noriharu, and Yuichi Shirai. "Development of novel pesticides in the 21st century." Journal of Pesticide Science 45.2 (2020): 54-74. ISSN: 1348-589X https://www.jstage.jst.go.jp/article/jpestics/45/2/45_D20-201/_article/-char/ja/ World Health Organization. "Standard operating procedure for testing insecticide susceptibility of adult mosquitoes in WHO bottle bioassays." (2022). ISBN: 978-92-4-004377-0 World Health Organization. Determining discriminating concentrations of insecticides for monitoring resistance in mosquitoes: report of a multi-centre laboratory study and WHO expert consultations. World Health Organization, 2022. ISBN: 978-92-4-004520-0 https://www.who.int/publications/i/item/9789240045200 Tchouakui, Magellan, et al. "Comparative study of the effect of solvents on the efficacy of neonicotinoid insecticides against malaria vector populations across Africa." Infectious Diseases of Poverty 11.02 (2022): 23-31. ISSN: 2095-5162 https://doi.org/10.1186/s40249-022-00962-4 Lees, Rosemary, et al. "A testing cascade to identify repurposed insecticides for next-generation vector control tools: screening a panel of chemistries with novel modes of action against a malaria vector." Gates open research 3 (2019). ISSN: 2572-4754 https://doi.org/10.12688/gatesopenres.12957.2 Portwood, Natalie M., et al. "Multi-centre discriminating concentration determination of broflanilide and potential for cross-resistance to other public health insecticides in Anopheles vector populations." Scientific Reports 12.1 (2022): 22359. ISSN: 2045-2322 https://www.nature.com/articles/s41598-022-26990-6 Govoetchan, Renaud, et al. "Investigating discriminating concentrations for monitoring susceptibility to broflanilide and cross resistance to other insecticide classes in Anopheles gambiae sensu lato, using the new WHO bottle bioassay method." Plos one 18.3 (2023): e0276246. ISSN: 1932-6203 https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0276246 Snetselaar, Janneke, et al. "Laboratory evaluation of broflanilide (TENEBENAL™) against Anopheles gambiae in Moshi, Tanzania–delayed mortality, cross-resistance, and residual efficacy." Frontiers in Tropical Diseases 4 (2023): 1097189. ISSN: 2673-7515 https://www.frontiersin.org/journals/tropical-diseases/articles/10.3389/fitd.2023.1097189/full Hien, Aristide Sawdetuo, et al. "Long-lasting residual efficacy of a new indoor residual spraying product, VECTRON™ T500 (broflanilide), against pyrethroid-resistant malaria vectors and its acceptance in a community trial in Burkina Faso." Parasites & Vectors 17.1 (2024): 484. ISSN: 1756-3305 https://link.springer.com/article/10.1186/s13071-024-06577-y Bliss, Chester Ittner. "The calculation of the dosage‐mortality curve." Annals of Applied Biology 22.1 (1935): 134-167. ISSN: 0003-4746 https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1744-7348.1935.tb07713.x Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 09 Mar, 2026 Read the published version in Malaria Journal → Version 1 posted Editorial decision: Revision requested 25 Sep, 2025 Reviews received at journal 24 Sep, 2025 Reviews received at journal 22 Sep, 2025 Reviews received at journal 19 Sep, 2025 Reviewers agreed at journal 09 Sep, 2025 Reviewers agreed at journal 07 Sep, 2025 Reviewers agreed at journal 02 Sep, 2025 Reviewers agreed at journal 02 Sep, 2025 Reviewers agreed at journal 02 Sep, 2025 Reviewers invited by journal 02 Sep, 2025 Editor assigned by journal 29 Aug, 2025 Submission checks completed at journal 29 Aug, 2025 First submitted to journal 28 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7478645","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":513975568,"identity":"92fcd936-8d60-4165-a70b-641775c9aa66","order_by":0,"name":"Yuki Ando","email":"data:image/png;base64,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","orcid":"","institution":"Mitsui Chemicals Crop \u0026 Life Solutions, Inc","correspondingAuthor":true,"prefix":"","firstName":"Yuki","middleName":"","lastName":"Ando","suffix":""},{"id":513975569,"identity":"62edb580-98ff-45d7-8542-b522fbf9a444","order_by":1,"name":"Kunizo Mori","email":"","orcid":"","institution":"Advisor to Mitsui Chemicals Crop \u0026 Life Solutions, Inc","correspondingAuthor":false,"prefix":"","firstName":"Kunizo","middleName":"","lastName":"Mori","suffix":""},{"id":513975570,"identity":"befb2654-80a2-446f-9283-4c834283fd36","order_by":2,"name":"John Bradley","email":"","orcid":"","institution":"London School of Hygiene and Tropical Medicine","correspondingAuthor":false,"prefix":"","firstName":"John","middleName":"","lastName":"Bradley","suffix":""},{"id":513975571,"identity":"47c5ff90-4267-473d-9ba8-979784f7daf4","order_by":3,"name":"Janneke Snetselaar","email":"","orcid":"","institution":"Innovative Vector Control Consortium","correspondingAuthor":false,"prefix":"","firstName":"Janneke","middleName":"","lastName":"Snetselaar","suffix":""},{"id":513975572,"identity":"c2b24f5e-1e3a-4dd6-b696-474d8b88c6d9","order_by":4,"name":"Graham Small","email":"","orcid":"","institution":"Innovative Vector Control Consortium","correspondingAuthor":false,"prefix":"","firstName":"Graham","middleName":"","lastName":"Small","suffix":""}],"badges":[],"createdAt":"2025-08-28 09:24:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7478645/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7478645/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12936-025-05728-5","type":"published","date":"2026-03-09T15:59:47+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91270454,"identity":"53627eac-e0f3-41e1-a31c-c558db59ef9e","added_by":"auto","created_at":"2025-09-14 10:12:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":185402,"visible":true,"origin":"","legend":"\u003cp\u003eDose response curves of broflanilide with the insecticide susceptible \u003cem\u003eAn. gambiae \u003c/em\u003es.s. Kisumu strain\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7478645/v1/ab299600879e8e052b86b53f.png"},{"id":104739510,"identity":"8fd35a32-9040-4e72-8e6b-2eca300e4b5d","added_by":"auto","created_at":"2026-03-16 16:08:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1063001,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7478645/v1/fa037e3d-ad8d-474a-b1c7-78a8386eba32.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Determination of the Preliminary Discriminating Concentration of Broflanilide Against Malaria Vector Mosquito Anopheles gambiae by Multi-Centre Susceptibility Testing","fulltext":[{"header":"1. Background","content":"\u003cp\u003eAnopheline mosquitoes transmit malaria parasites to humans leading to malaria infections in sub-Saharan African countries [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. To reduce and eliminate malaria in these countries, effective methods for the control of vector mosquitoes are essential. One of the most effective methods of mosquito vector control is indoor residual spraying (IRS) of insecticides, which has been widely used for several decades [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Killing blood-fed mosquitoes that rest on the indoor walls of houses, reduces the local vector population and prevents mosquitoes that have taken an infected blood meal from transmitting the malaria parasites in subsequent blood feeding. It is recommended that IRS products should have long-lasting residual efficacy, preferably of at least six months duration, as this is generally the main period of malaria transmission in sub-Saharan Africa [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eA number of insecticides with several different modes of action (e.g., DDT, carbamates, organophosphates, and pyrethroids) have been used previously for IRS malaria vector control programmes, but overuse of the products into which these insecticides have been formulated has led to the evolution and spread of insecticide resistance in many vector populations [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR5 CR6 CR7 CR8\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This, in turn, has led to reduced effectiveness of some of the existing IRS products; therefore, the development and use of IRS products containing novel insecticides, to which there is currently no detected resistance, is essential to our fight against malaria [\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14 CR15 CR16 CR17 CR18\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eVECTRON\u0026trade; T500 (active ingredient broflanilide 50% wettable powder), developed by Mitsui Chemicals Crop \u0026amp; Life Solutions, Inc. (MCCLS), is a new IRS product that was evaluated and listed by the World Health Organization Prequalification Unit Vector Control Product Assessment Team (WHO PQT/VCP) in March 2023 and was released in December 2023. Previous studies have confirmed that this formulation exhibits high and long-term efficacy against Anopheline mosquitoes [\u003cspan additionalcitationids=\"CR21 CR22 CR23 CR24 CR25 CR26 CR27\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The active ingredient, broflanilide (tradename TENEBENAL\u0026trade;), is a novel meta-diamide insecticide compound developed by MCCLS. This compound is classified in IRAC group 30 (allosteric modulator of gamma-aminobutyric acid (GABA)-gated chloride ion channels) and has a novel mode of action [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] that differs from the active ingredients in existing IRS formulations. Previous studies have detected no cross-resistance to broflanilide in pyrethroid resistant mosquito strains or populations; therefore, the rotational use of this compound with compounds with different modes of action can be a viable option for controlling these malaria vectors and manage resistance development [\u003cspan additionalcitationids=\"CR21 CR22 CR23 CR24 CR25 CR26 CR27\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAs part of any resistance management strategy, it is essential to establish the baseline susceptibility of wild mosquito populations in regions where the use of an insecticide is planned and to monitor for the possible development of resistance thereafter. For this purpose, insecticide susceptibility test methods have been developed which use a concentration of an insecticide that will discriminate between insecticide susceptible and resistant mosquitoes in terms of mortality (DC: Discriminating Concentration). For this susceptibility testing, the WHO recommends conducting a WHO bottle bioassay [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] with the DC for insecticide being used in mosquito vector control being determined via multi-centre studies led by WHO (e.g., the DCs of existing compounds for \u003cem\u003eAn. gambiae\u003c/em\u003e are as follows: clothianidin, 4 \u0026micro;g/bottle; flupyradifurone, 60 \u0026micro;g/bottle; transfluthrin, 2 \u0026micro;g/bottle; and chlorfenapyr, 100 \u0026micro;g/bottle) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. However, the DC of broflanilide has not been determined yet by WHO due to its novelty [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], while in African countries, there is a need for baseline susceptibility testing by implementing programmes as they introduce VECTRON\u0026trade; T500 into their IRS campaigns [\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan additionalcitationids=\"CR36 CR37\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Therefore, it is important to identify early on a preliminary DC (PDC) that can be used to monitor mosquito population susceptibility to broflanilide ahead of the introduction of this new insecticide and before WHO publishes its definitive DC.\u003c/p\u003e\u003cp\u003eIn this study, to determine the PDC of broflanilide, we followed as far as was possible the same process for DC establishment as described by WHO [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Previous studies have shown that the addition of the adjuvant Mero (81% rapeseed oil methyl ester; Bayer AG, Crop Science Division) can enhance and stabilize the compound's efficacy by inhibiting crystallization [\u003cspan additionalcitationids=\"CR32 CR33\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], and the DCs of broflanilide with 800 ppm Mero for \u003cem\u003eAn. gambiae\u003c/em\u003e have been proposed for use in association with large-scale community trials on VECTRON\u0026trade; T500 in previous studies (6 \u0026micro;g/bottle and 17.82 \u0026micro;g/bottle) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. However, these PDCs were estimated using only one or a few datasets from dose response tests with \u003cem\u003eAn. gambiae\u003c/em\u003e. According to WHO guidelines, it is desirable to have three or more datasets to establish the DC [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Therefore, this study aimed to propose a more robust PDC by generating broflanilide does response data at multiple research facilities in Africa and the UK. Colonies of the insecticide susceptible \u003cem\u003eAn. gambiae\u003c/em\u003e s.s. Kisumu strain established at these research facilities were used for tests with a range of broflanilide concentrations and the data were statistically analysed to determine the PDC. This PDC will provide the necessary information for the stewardship of VECTRON\u0026trade; T500 via broflanilide susceptibility monitoring at an early stage in in the roll out of this IRS product.\u003c/p\u003e"},{"header":"2. Materials \u0026 Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Mosquito Strain\u003c/h2\u003e\u003cp\u003eColonies of the \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.s. Kisumu strain maintained by each trial facility were used in all the testing as an insecticide susceptible strain. This strain was originally collected in the Kisumu region of Kenya. All mosquitoes used were 2\u0026ndash;5 day old, non-blood-fed adult females.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Susceptibility Testing with Broflanilide by WHO Bottle Bioassays at Multiple Study Facilities\u003c/h2\u003e\u003cp\u003eTo obtain data on the dose response to broflanilide of Kisumu strain mosquitoes, WHO bottle bioassays were conducted [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The glass bottles (250 mL, Wheaton bottles) were treated with 1 mL of broflanilide dissolved in a mixture of acetone and 800 ppm Mero. Bottles treated with acetone\u0026thinsp;+\u0026thinsp;Mero only were also prepared as a negative control. Twenty-five (25) mosquitoes were introduced into each bottle and exposed for 1 hour. After exposure, the mosquitoes were aspirated into clean cups and provided with cotton wool soaked in a 10% sugar solution \u003cem\u003ead libitum\u003c/em\u003e. Mortality was recorded at 24 h intervals up to 72 h post exposure. The bioassays were conducted under laboratory conditions (27\u0026thinsp;\u0026plusmn;\u0026thinsp;2℃, 80\u0026thinsp;\u0026plusmn;\u0026thinsp;10% RH).\u003c/p\u003e\u003cp\u003eThere were some minor variations to the bioassays conducted by the 9 research facilities (i.e. the concentrations used, number of replicates and number of mosquitoes) which are detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The data generated by the Liverpool Insect Testing Establishment (LITE) facility (UK) were fully Good Laboratory Practice (GLP) compliant. The data from the African research facilities were generated during the conduct of non-GLP semi-field experimental hut or community VECTRON\u0026trade; T500 product registration trials following the standard operating procedures implemented at the respective facilities.\u003c/p\u003e\u003cp\u003eData from 4 of the studies have previously been published elsewhere (Tanzania KCMUCo and NIMR [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], Benin [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], Burkina Faso [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]) but were included in our statistical analysis to provide further supporting information for the setting of the PDC.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTest conditions from each testing facility\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCountry\u003c/p\u003e\u003cp\u003e(facility)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLocation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eConcentrations (\u0026micro;g/bottle)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNumber of replicates\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNumber of\u003c/p\u003e\u003cp\u003emosquitos\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eZambia\u003c/p\u003e\u003cp\u003e(Tropical Disease Research Centre)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNchelenge\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.781, 1.563, 3.125, 6.25, 12.5, 25, 50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1037\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUganda\u003c/p\u003e\u003cp\u003e(NMCD MOH)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNosako\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.563, 3.125, 4, 6.25, 12.5, 25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e750\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDRC\u003c/p\u003e\u003cp\u003e(University of Kinshasa)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eKinshasa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.195, 0.39, 0.781, 1.563, 3.125, 6.25, 12.5, 25, 50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e889\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBurkina Faso\u003c/p\u003e\u003cp\u003e(IRSS/DRO)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eVall\u0026eacute;e du Kou\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.781, 1.563, 3.125, 6.25, 12.5, 25, 50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e778\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGhana\u003c/p\u003e\u003cp\u003e(NMIMR)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOdumse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.781, 1.563, 3.125, 6.25, 12.5, 25, 50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e800\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBenin\u003c/p\u003e\u003cp\u003e(CREC/LSHTM)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCotonou\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.05, 0.1, 0.25, 0.5, 1, 2.2, 4.6, 10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e800\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTanzania\u003c/p\u003e\u003cp\u003e(KCMUCo-PAMVERC)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMoshi\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.01, 0.0215, 0.0464, 0.1, 0.215, 1.0, 2.15, 4.64, 10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1661\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTanzania\u003c/p\u003e\u003cp\u003e(NIMR Amani Reach Centre)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMuheza\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.01, 0.0215, 0.0464, 0.1, 0.215, 1.0, 2.15, 4.64, 10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e850\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUK\u003c/p\u003e\u003cp\u003e(LITE)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLiverpool\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.3, 0.86, 1.42, 1.98, 2.54, 3.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1571\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Data Analysis\u003c/h2\u003e\u003cp\u003eAll data analysis was undertaken using Stata 18. A probit model was used to estimate the lethal concentration that would induce 99% mortality in mosquitoes (LC\u003csub\u003e99\u003c/sub\u003e). The independent variable was the logorith of the concentration of broflanilide. Abbott\u0026rsquo;s formula was used to correct mortality in test replicate for mortality in the negative controls (acetone and 800ppm Mero only).\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\text{C}\\text{o}\\text{r}\\text{r}\\text{e}\\text{c}\\text{t}\\text{e}\\text{d}\\:\\text{m}\\text{o}\\text{r}\\text{t}\\text{a}\\text{l}\\text{i}\\text{t}\\text{y}=\\frac{\\left(\\text{%}\\:\\text{o}\\text{b}\\text{s}\\text{e}\\text{r}\\text{v}\\text{e}\\text{d}\\:\\text{m}\\text{o}\\text{r}\\text{t}\\text{a}\\text{l}\\text{i}\\text{t}\\text{y}\\:-\\:\\text{%}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}\\:\\text{m}\\text{o}\\text{r}\\text{t}\\text{a}\\text{l}\\text{i}\\text{t}\\text{y}\\right)}{\\left(100\\:-\\:\\text{%}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}\\:\\text{m}\\text{o}\\text{r}\\text{t}\\text{a}\\text{l}\\text{i}\\text{t}\\text{y}\\right)}\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eConfidence intervals were estimated using the bootstrap, which took uncertainty in the control mortality into account. For each probit regression, a p-value for the Pearson\u0026rsquo;s Chi-squared test (goodness of fit) was calculated.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Dose Response in Test Results for Each Facility\u003c/h2\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the dose-response curves of the susceptible \u003cem\u003eAn. gambiae\u003c/em\u003e s.s. Kisumu strain mosquitoes to broflanilide obtained from the WHO bottle bioassays conducted by the 9 research facilities. In eight of the datasets, excluding those from the UK facility, 100% mortality was confirmed within the tested concentration ranges, but there was variability in the lowest LC\u003csub\u003e100\u003c/sub\u003e values (Zambia, DRC, and Ghana: 0.781 \u0026micro;g/bottle; Benin and Tanzania NIMR: 1.0 \u0026micro;g/bottle; Uganda: 1.562 \u0026micro;g/bottle; Tanzania KCMUCo: 2.15 \u0026micro;g/bottle; and Burkina Faso: 6.25 \u0026micro;g/bottle) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Additionally, each dose-response curve rose very steeply which is typical for an insecticide susceptible strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In the bottle bioassay data from Zambia and Uganda, 100% mortality was observed at all broflanilide concentrations tested. Conversely, in the test results from the UK facility, the maximum mortality rate observed for the range of tested concentrations was 90.35%, and 100% mortality was not achieved.\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.2 Overview of Estimated Lethal Concentration (LC\u003c/b\u003e\u003csub\u003e\u003cb\u003e99\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e) Values for Each Facility and Determination of the Preliminary Discriminating Concentration for Broflanilide\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe data and statistical analysis outputs from the nine dose response studies (LC\u003csub\u003e99\u003c/sub\u003e and 95% CI, goodness of fit, number of data points with mortality rates neither 0% nor 100%, the lowest LC\u003csub\u003e100\u003c/sub\u003e if available, and 2x the LC99 or LC100) are summarised in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The LC\u003csub\u003e99\u003c/sub\u003e of broflanilide for the susceptible \u003cem\u003eAn. gambiae s.s.\u003c/em\u003e Kisumu strain varied from 1.02 to 27.84 \u0026micro;g/bottle.\u003c/p\u003e\u003cp\u003eThe guidance from WHO on the validation of datasets [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] considers several aspects. First, to be considered valid, each dataset should have at least two concentrations that killed\u0026thinsp;\u0026lt;\u0026thinsp;50% of mosquitoes, one concentration that killed about 50%, two concentrations that killed\u0026thinsp;\u0026gt;\u0026thinsp;50% of mosquitoes and one concentration that killed about 100% mosquitoes. After this, the validation of each dataset should consider the following: mortality of controls should be below the cut-off point of 20%; a minimum of six concentrations should be available to generate concentration\u0026ndash;response curves and estimate lethal concentrations; the goodness of fit p-value should be \u0026gt;\u0026thinsp;0.05; and the lowest concentration that killed 100% of mosquitoes (LC\u003csub\u003e100\u003c/sub\u003e), when available. Considering the datasets generated by the 9 research facilities, none could satisfy all the validation requirements stated by WHO. Therefore, we determined which datasets best matched WHO guidance.\u003c/p\u003e\u003cp\u003eGenerally, probit analysis represents the relationship between mortality and the logarithm of dose as a linear relationship [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Therefore, to perform linear regression, at least three data points are necessary in which the mortality rates of neither 0% nor 100% (if there are only two data points, the fit of the regression line will be perfect (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;1)).The number of data points used in the probit analysis for calculating the LC\u003csub\u003e99\u003c/sub\u003e was 0 or 2 in four datasets (Zambia, Uganda, DRC, and Ghana). Additionally, in three datasets, the p-value of the chi-squared test indicating the goodness of fit was less than 0.05 (Benin, Tanzania, and the UK).\u003c/p\u003e\u003cp\u003eThere were two results with a goodness of fit p-value greater than 0.05 and three or more valid data points (Tanzania NIMR and Burkina Faso). The LC99 values were 0.57 \u0026micro;g/bottle (Tanzania NIMR) and 8.42 \u0026micro;g/bottle (Burkina Faso). Therefore, taking into account WHO guidance on determining the DC of insecticides for monitoring resistance in mosquitoes in which the highest LC\u003csub\u003e99\u003c/sub\u003e is to be used [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], we took the 2\u0026times;LC\u003csub\u003e99\u003c/sub\u003e for the Burkina Faso dataset (16.84 \u0026micro;g/bottle) to inform the appropriate PDC for susceptibility testing with broflanilide.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSummary of the data from multi-centre susceptibility testing of broflanilide against the insecticide susceptible \u003cem\u003eAn. gambiae\u003c/em\u003e s.s. Kisumu strain\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCountry\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLC\u003csub\u003e99\u003c/sub\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;95%CI\u003c/p\u003e\u003cp\u003e(\u0026micro;g/bottle)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ep-value of\u003c/p\u003e\u003cp\u003eChi-sq test\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2\u0026times;LC\u003csub\u003e99\u003c/sub\u003e\u003c/p\u003e\u003cp\u003e(\u0026micro;g/bottle)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNumber of\u003c/p\u003e\u003cp\u003evalid datapoint\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eObserved\u003c/p\u003e\u003cp\u003elowest LC\u003csub\u003e100\u003c/sub\u003e\u003c/p\u003e\u003cp\u003e(\u0026micro;g/bottle)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2\u0026times;LC\u003csub\u003e100\u003c/sub\u003e\u003c/p\u003e\u003cp\u003e(\u0026micro;g/bottle)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eZambia\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.781\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.562\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUganda\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.563\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3.126\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBurkina Faso\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8.42\u003c/p\u003e\u003cp\u003e[4.73\u0026ndash;12.10]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.3243\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e12.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDRC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.06\u003c/p\u003e\u003cp\u003e[0.91\u0026ndash;1.21]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0636\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.781\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.562\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBenin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.02\u003c/p\u003e\u003cp\u003e[0.85\u0026ndash;1.18]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGhana\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.08\u003c/p\u003e\u003cp\u003e[0.89\u0026ndash;1.26]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0899\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.781\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.562\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTanzania (KCMUCo)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e27.84\u003c/p\u003e\u003cp\u003e[17.75\u0026ndash;37.90]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e55.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e4.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTanzania (NIMR)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.57\u003c/p\u003e\u003cp\u003e[0.31\u0026ndash;0.82]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.4268\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUK\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13.44\u003c/p\u003e\u003cp\u003e[9.76\u0026ndash;16.96]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0136\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e26.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e4.1 Establishment of the Preliminary Discriminating Concentration\u003c/h2\u003e\u003cp\u003eMost of the dose response studies were not specifically designed for the determination of the PDC for broflanilide but were part of non-GLP semi-field or community VECTRON\u0026trade; T500 product registration trials. Therefore, it was not possible to meet all of the data validation criteria described by WHO [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBased on the calculation of LC\u003csub\u003e99\u003c/sub\u003e and statistical analysis results of each test shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the PDC of broflanilide for the susceptible \u003cem\u003eAn. gambiae\u003c/em\u003e s.s. Kisumu strain was determined. Following the verification method for data as guided by WHO [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], for datasets with a goodness of fit \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05 (Burkina Faso and Tanzania NIMR), the value twice the LC\u003csub\u003e99\u003c/sub\u003e was adopted for those tests. For datasets with a goodness of fit p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (Benin, and Tanzania KCMUCo), the reliability of the LC\u003csub\u003e99\u003c/sub\u003e was regarded low. Additionally, the data with 100% mortality at all test concentrations (Zambia and Uganda) and the data where only two valid data points were obtained (DRC and Ghana), it was considered inappropriate to refer to the results of the probit analysis.\u003c/p\u003e\u003cp\u003eFor the results from the LITE facility in the UK, the goodness of fit p-value was less than 0.05 and no LC\u003csub\u003e100\u003c/sub\u003e was confirmed.\u003c/p\u003e\u003cp\u003eFinally, among the nine LC\u003csub\u003e99\u003c/sub\u003e values determined through the above data verification, the highest was 8.42 \u0026micro;g/bottle (Burkina Faso). According to WHO guideline [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], twice this value, 16.84 \u0026micro;g/bottle, was determined to be the most appropriate for selecting the PDC for \u003cem\u003eAn. gambiae\u003c/em\u003e. Rounding this value to make it simpler results in 17 \u0026micro;g/bottle; however, since 17 is a prime number, it may pose difficulties in practical operations such as preparing stock solutions, working solutions, or performing serial dilutions. Additionally, several African countries intending to use VECTRON\u0026trade; T500 are already conducting susceptibility tests with a provisional value of 18 \u0026micro;g/bottle (established from unpublished data). Following discussions with IRS programme implementers on the practicality of broflanilide susceptibility monitoring in sub-Saharan Africa and, therefore, to simplify the dilution of broflanilide for the treatment of bottles, we propose the PDC for broflanilide to be 18 \u0026micro;g/bottle (with 800 ppm Mero).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e4.2 The Variation Between the Estimated LC Values Among Trial Facilities\u003c/h2\u003e\u003cp\u003eThe results obtained in this research showed variations in the dose response of broflanilide for each test data set, even though the same strain (\u003cem\u003eAn. gambiae\u003c/em\u003e s.s. Kisumu) was used.\u003c/p\u003e\u003cp\u003eThe mosquito colonies used in this study had been maintained and reared for a long time at the different testing facilities. Since the methods and conditions (e.g., temperature, humidity, density) for continuous rearing in each facility were not exactly the same, this might have led to variations in the general fitness and tolerance to exposure to broflanilide of the respective colonies. Ideally, cross-validation of the properties of the susceptible mosquito colonies and experimental methods across testing facilities should be conducted [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. However, as mentioned previously, the eight bottle bioassays conducted in this study, apart from those conducted by the UK facility, were not specifically designed as dose-response studies to determine the PDC for broflanilide. Therefore, minor variations in the detailed procedures during the experiments (e.g., preparation and serial dilution of the broflanilide working solution, treatment of bottles, mosquito treatment when introducing into bottles or retrieval from bottles) or the storage conditions of mosquitoes after exposure to broflanilide (e.g., temperature, humidity, method of providing sugar solution) might also have influenced the dose-response to broflanilide in the mosquito populations at each testing facility.\u003c/p\u003e\u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn this study, nine datasets of WHO bottle bioassays conducted at the multiple trial facilities were conducted, the DC (2xLC\u003csub\u003e99\u003c/sub\u003e) of broflanilide for the \u003cem\u003eAn. gambiae\u003c/em\u003e s.s. Kisumu strain was determined to be 16.84 \u0026micro;g/bottle and a PDC of 18 \u0026micro;g/bottle is proposed for monitoring the susceptibility of wild populations of \u003cem\u003eAn. gambiae\u003c/em\u003e to broflanilide. However, there was considerable variability in the dose-response to broflanilide among the nine datasets used in this study. As a result of data validation, only two datasets were considered valid for the determination of the PDC. Data generated in further broflanilide dose-response studies are needed to establish a definitive DC for broflanilide, and the PDC proposed here may change based on the results of future studies sponsored by WHO to determine the definitive DC. However, for regions where the VECTRON\u0026trade; T500 IRS product, containing broflanilide as an active ingredient, is already in use or will be used in the near future as part of malaria vector control campaigns, the PDC of broflanilide proposed by this study will serve as a benchmark for assessing the baseline susceptibility of wild populations of \u003cem\u003eAn. gambiae\u003c/em\u003e to broflanilide and to monitor for any changes in susceptibility with time. The data generated in susceptibility testing will facilitate rapid decision-making regarding the use of VECTRON\u0026trade; T500 and, together with susceptibility data for insecticides used in other IRS products, will inform the implementation of insecticide resistance management through the rotation of IRS products containing insecticides with different modes of action.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eDC\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDiscriminating Concentration\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eGLP\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGood Laboratory Practice\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eIRS\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eIndoor Residual Spraying\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eLC\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLethal Concentration\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003ePDC\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePreliminary Discriminating Concentration\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data associated with this study are present in the paper. All other relevant data are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn conducting this study, we received the broflanilide dose response data for the An. gambiae Kisumu strain. Some of them are from previously published studies. We would like to express our gratitude to the following data providers and authors.\u003c/p\u003e\n\u003cp\u003eDr. Natalie M. Portwood and Dr. Louisa Messenger [35], Dr. Corine Ngufor and Dr. Renaud Govoetchan [36], Prof. Abdoulaye Diabaté and Dr. Aristide Sawdetuo Hien [38], Jessica Williams, Amy Guy and Jennie Gibson (Liverpool Insect Testing Establishment, Liverpool, UK), Dr. Mbanga Muleba (Tropical Diseases Research Centre, Ndola, \u0026nbsp;Zambia), Dr. Ntege Charles (National Malaria Control Division, Kampala, Uganda), Dr. Thierry Bobanga (Universite de Kinshasa, Kinshasa, Democratic Republic of the Congo) and Dr. Samuel Kweku Dadzie (Noguchi Memorial Institute for Medical Research, Accra, Ghana).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded through support from the Bill \u0026amp; Melinda Gates Foundation (grant: INV-007509), the Swiss Agency for Development and Cooperation (SDC) (grant: 81067480) and UK Aid (grant: 30041-105). The findings and conclusions contained within are those of the authors and do not necessarily reflect the positions or policies of the Bill \u0026amp; Melinda Gates Foundation, SDC, UK Aid or IVCC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMitsui Chemicals Crop \u0026amp; Life Solutions, Inc., Tokyo, Japan\u003c/p\u003e\n\u003cp\u003eYuki Ando (ORCID iD : 0009-0005-4526-5082)\u003c/p\u003e\n\u003cp\u003eAdvisor to Mitsui Chemicals Crop \u0026amp; Life Solutions, Inc., Tokyo, Japan\u003c/p\u003e\n\u003cp\u003eKunizo Mori\u003c/p\u003e\n\u003cp\u003eLondon School of Hygiene and Tropical Medicine, London, UK\u003c/p\u003e\n\u003cp\u003eJohn Bradley (ORCID iD : 0000-0002-9449-4608)\u003c/p\u003e\n\u003cp\u003eInnovative Vector Control Consortium, Liverpool, UK\u003c/p\u003e\n\u003cp\u003eJanneke Snetselaar (ORCID iD : 0000-0002-5882-6855) \u0026amp; Graham Small\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYA, KM, JS and GS conceived and designed the study. JB analysed the study data. YA drafted the first version of the manuscript. All authors contributed to the manuscript and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Yuki Ando\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWorld Health Organization. \u003cem\u003eIndoor residual spraying: an operational manual for indoor residual spraying (IRS) for malaria transmission control and elimination\u003c/em\u003e. World Health Organization, 2015. ISBN: 978 92 4 150894 0 https://iris.who.int/bitstream/handle/10665/177242/9789241508940_eng.pdf\u003c/li\u003e\n\u003cli\u003eNgufor, Corine, et al. \u0026quot;Indoor spraying with chlorfenapyr (a pyrrole insecticide) provides residual control of pyrethroid-resistant malaria vectors in southern Benin.\u0026quot; \u003cem\u003eMalaria journal\u003c/em\u003e 19 (2020): 1-11. ISSN: 1475-2875 https://doi.org/10.1186/s12936-020-03325-2\u003c/li\u003e\n\u003cli\u003eOxborough, Richard M. \u0026quot;Trends in US President\u0026rsquo;s Malaria Initiative-funded indoor residual spray coverage and insecticide choice in sub-Saharan Africa (2008\u0026ndash;2015): urgent need for affordable, long-lasting insecticides.\u0026quot; \u003cem\u003eMalaria journal\u003c/em\u003e 15.1 (2016): 146. ISSN: 1475-2875 https://doi.org/10.1186/s12936-016-1201-1\u003c/li\u003e\n\u003cli\u003eKilleen, Gerry F., and Hilary Ranson. \u0026quot;Insecticide-resistant malaria vectors must be tackled.\u0026quot; \u003cem\u003eThe Lancet\u003c/em\u003e 391.10130 (2018): 1551-1552. ISSN: 0140-6736 http://dx.doi.org/10.1016/S0140-6736(18)30844-4\u003c/li\u003e\n\u003cli\u003eMathias, Derrick K., et al. \u0026quot;Spatial and temporal variation in the kdr allele L1014S in Anopheles gambiae ss and phenotypic variability in susceptibility to insecticides in Western Kenya.\u0026quot; \u003cem\u003eMalaria journal\u003c/em\u003e 10 (2011): 1-13. ISSN: 1475-2875 http://www.malariajournal.com/content/10/1/10\u003c/li\u003e\n\u003cli\u003eAhoua Alou, Ludovic P., et al. \u0026quot;Distribution of ace-1 R and resistance to carbamates and organophosphates in Anopheles gambiae ss populations from C\u0026ocirc;te d\u0026apos;Ivoire.\u0026quot; \u003cem\u003eMalaria Journal\u003c/em\u003e 9 (2010): 1-7. ISSN: 1475-2875 http://www.malariajournal.com/content/9/1/167\u003c/li\u003e\n\u003cli\u003eMain, Bradley J., et al. \u0026quot;Complex genome evolution in Anopheles coluzzii associated with increased insecticide usage in Mali.\u0026quot; \u003cem\u003eMolecular ecology\u003c/em\u003e 24.20 (2015): 5145-5157. ISSN: 0962-1083 https://onlinelibrary.wiley.com/doi/full/10.1111/mec.13382\u003c/li\u003e\n\u003cli\u003eBarnes, Kayla G., et al. \u0026quot;Genomic footprints of selective sweeps from metabolic resistance to pyrethroids in African malaria vectors are driven by scale up of insecticide-based vector control.\u0026quot; \u003cem\u003ePLoS genetics\u003c/em\u003e 13.2 (2017): e1006539. ISSN: 1553-7404 https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1006539\u003c/li\u003e\n\u003cli\u003eWorld Health Organization. \u0026quot;Global report on insecticide resistance in malaria vectors: 2010\u0026ndash;2016.\u0026quot; (2018). ISBN: 978 92 4 151405 https://iris.who.int/bitstream/handle/10665/272533/9789241514057-eng.pdf\u003c/li\u003e\n\u003cli\u003eRowland, Mark, et al. \u0026quot;A new long-lasting indoor residual formulation of the organophosphate insecticide pirimiphos methyl for prolonged control of pyrethroid-resistant mosquitoes: an experimental hut trial in Benin.\u0026quot; \u003cem\u003ePloS one\u003c/em\u003e 8.7 (2013): e69516. ISSN: 1932-6203 https://doi.org/10.1371/journal.pone.0069516\u003c/li\u003e\n\u003cli\u003eChanda, Emmanuel, et al. \u0026quot;Preventing malaria transmission by indoor residual spraying in Malawi: grappling with the challenge of uncertain sustainability.\u0026quot; \u003cem\u003eMalaria journal\u003c/em\u003e 14 (2015): 1-7. ISSN: 1475-2875 https://link.springer.com/article/10.1186/s12936-015-0759-3\u003c/li\u003e\n\u003cli\u003eHaji, Khamis A., et al. \u0026quot;Efficacy, persistence and vector susceptibility to pirimiphos-methyl (Actellic\u003csup\u003e\u0026reg;\u003c/sup\u003e 300CS) insecticide for indoor residual spraying in Zanzibar.\u0026quot; \u003cem\u003eParasites \u0026amp; vectors\u003c/em\u003e 8 (2015): 1-7. ISSN: 1756-3305 https://link.springer.com/article/10.1186/s13071-015-1239-x\u003c/li\u003e\n\u003cli\u003eNgufor, Corine, et al. \u0026quot;Indoor residual spraying with a mixture of clothianidin (a neonicotinoid insecticide) and deltamethrin provides improved control and long residual activity against pyrethroid resistant Anopheles gambiae sl in Southern Benin.\u0026quot; \u003cem\u003ePloS one\u003c/em\u003e 12.12 (2017): e0189575. ISSN: 1932-6203 https://doi.org/10.1371/journal.pone.0189575\u003c/li\u003e\n\u003cli\u003eMashauri, Fabian M., et al. \u0026quot;Indoor residual spraying with micro-encapsulated pirimiphos-methyl (Actellic 300CS) against malaria vectors in the Lake Victoria basin, Tanzania.\u0026quot; \u003cem\u003ePloS one\u003c/em\u003e 12.5 (2017): e0176982. ISSN: 1932-6203 https://www.nature.com/articles/s41598-020-61350-2\u003c/li\u003e\n\u003cli\u003eKweka, Eliningaya, et al. \u0026quot;Novel indoor residual spray insecticide with extended mortality effect: a case of SumiShield 50WG against wild resistant populations of Anopheles arabiensis in Northern Tanzania.\u0026quot; \u003cem\u003eGlobal Health: Science and Practice\u003c/em\u003e 6.4 (2018): 758-765. ISSN: 2169-575X https://doi.org/10.9745/GHSP-D-18-00213\u003c/li\u003e\n\u003cli\u003eAgossa, Fiacre R., et al. \u0026quot;Efficacy of a novel mode of action of an indoor residual spraying product, SumiShield\u003csup\u003e\u0026reg;\u003c/sup\u003e 50WG against susceptible and resistant populations of Anopheles gambiae (sl) in Benin, West Africa.\u0026quot; \u003cem\u003eParasites \u0026amp; vectors\u003c/em\u003e 11 (2018): 1-13. ISSN: 1756-3305 https://doi.org/10.1186/s13071-018-2869-6\u003c/li\u003e\n\u003cli\u003eAbong\u0026rsquo;o, Bernard, et al. \u0026quot;Impact of indoor residual spraying with pirimiphos-methyl (Actellic 300CS) on entomological indicators of transmission and malaria case burden in Migori County, western Kenya.\u0026quot; \u003cem\u003eScientific reports\u003c/em\u003e 10.1 (2020): 4518. ISSN: 2045-2322 https://doi.org/10.1038/s41598-020-61350-2\u003c/li\u003e\n\u003cli\u003eChabi, Joseph, et al. \u0026quot;Efficacy of partial spraying of SumiShield, Fludora Fusion and Actellic against wild populations of Anopheles gambiae sl in experimental huts in Tiassal\u0026eacute;, C\u0026ocirc;te d\u0026apos;Ivoire.\u0026quot; \u003cem\u003eScientific Reports\u003c/em\u003e 13.1 (2023): 11364. ISSN: 2045-2322 https://doi.org/10.1038/s41598-023-38583-y\u003c/li\u003e\n\u003cli\u003eThiomela, Riccado F., et al. \u0026quot;Indoor residual spraying of experimental huts in Cameroon highlights the potential of Fludora\u003csup\u003e\u0026reg;\u003c/sup\u003e Fusion to control wild pyrethroid-resistant malaria vectors.\u0026quot; \u003cem\u003eBMC Infectious Diseases\u003c/em\u003e 24.1 (2024): 733. ISSN: 1471-2334 https://doi.org/10.1186/s12879-024-09630-4\u003c/li\u003e\n\u003cli\u003eLees, Rosemary Susan, et al. \u0026quot;Tenebenal: a meta-diamide with potential for use as a novel mode of action insecticide for public health.\u0026quot; \u003cem\u003eMalaria Journal\u003c/em\u003e 19 (2020): 1-17. ISSN: 1475-2875 https://doi.org/10.1186/s12936-020-03466-4\u003c/li\u003e\n\u003cli\u003eNgufor, Corine, et al. \u0026quot;Efficacy of broflanilide (VECTRON T500), a new meta-diamide insecticide, for indoor residual spraying against pyrethroid-resistant malaria vectors.\u0026quot; \u003cem\u003eScientific reports\u003c/em\u003e 11.1 (2021): 7976. ISSN: 2045-2322 https://doi.org/10.1038/s41598-021-86935-3\u003c/li\u003e\n\u003cli\u003eGovoetchan, Renaud, et al. \u0026quot;VECTRON\u0026trade; T500, a new broflanilide insecticide for indoor residual spraying, provides prolonged control of pyrethroid-resistant malaria vectors.\u0026quot; \u003cem\u003eMalaria Journal\u003c/em\u003e 21.1 (2022): 324. ISSN: 1475-2875 https://doi.org/10.1186/s12936-022-04336-x\u003c/li\u003e\n\u003cli\u003eNgufor, Corine, et al. \u0026quot;Community evaluation of VECTRON\u0026trade; T500, a broflanilide insecticide, for indoor residual spraying for malaria vector control in central Benin; a two arm non-inferiority cluster randomised trial.\u0026quot; \u003cem\u003eScientific reports\u003c/em\u003e 13.1 (2023): 17852. ISSN: 2045-2322 https://doi.org/10.1038/s41598-023-45047-w\u003c/li\u003e\n\u003cli\u003eBayili, Koama, et al. \u0026quot;Laboratory and experimental hut trial evaluation of VECTRON\u0026trade; T500 for indoor residual spraying (IRS) against insecticide resistant malaria vectors in Burkina Faso.\u0026quot; \u003cem\u003eGates Open Research\u003c/em\u003e 6 (2022). ISSN: 2572-4754 https://pmc.ncbi.nlm.nih.gov/articles/PMC9326088/#abstract1\u003c/li\u003e\n\u003cli\u003eSnetselaar, Janneke, et al. \u0026quot;Efficacy of indoor residual spraying with broflanilide (TENEBENAL), a novel meta-diamide insecticide, against pyrethroid-resistant anopheline vectors in northern Tanzania: An experimental hut trial.\u0026quot; \u003cem\u003ePloS One\u003c/em\u003e 16.3 (2021): e0248026. ISSN: 1932-6203 https://doi.org/10.1371/journal.pone.0248026\u003c/li\u003e\n\u003cli\u003eMbewe, Njelembo J., et al. \u0026quot;A non-inferiority and GLP-compliant study of broflanilide IRS (VECTRON\u0026trade; T500), a novel meta-diamide insecticide against Anopheles arabiensis.\u0026quot; \u003cem\u003eFrontiers in Tropical Diseases\u003c/em\u003e 4 (2023): 1126869. ISSN: 2673-7515 https://www.frontiersin.org/journals/tropical-diseases/articles/10.3389/fitd.2023.1126869/full#h4\u003c/li\u003e\n\u003cli\u003eKweka, Eliningaya J., et al. \u0026quot;The bioefficacy of a novel VECTRON\u0026trade; T500 indoor residual spray formulation in an experimental huts trial against Anopheles gambiae sl populations.\u0026quot; \u003cem\u003eActa Tropica\u003c/em\u003e 259 (2024): 107376. ISSN: 1873-6254 https://doi.org/10.1016/j.actatropica.2024.107376.\u003c/li\u003e\n\u003cli\u003eSimma, Eba Alemayehu, et al. \u0026quot;Determination of the Residual Efficacy of Broflanilide (VECTRONTM T500) Insecticide for Indoor Residual Spraying in a Semi-field Setting in Ethiopia.\u0026quot; (2024). [preprint] https://doi.org/10.21203/rs.3.rs-4773038/v1\u003c/li\u003e\n\u003cli\u003eNakao, Toshifumi, and Shinichi Banba. \u0026quot;Broflanilide: A meta-diamide insecticide with a novel mode of action.\u0026quot; \u003cem\u003eBioorganic \u0026amp; medicinal chemistry\u003c/em\u003e 24.3 (2016): 372-377. ISSN: 0968-0896 https://doi.org/10.1016/j.bmc.2015.08.008.\u003c/li\u003e\n\u003cli\u003eUmetsu, Noriharu, and Yuichi Shirai. \u0026quot;Development of novel pesticides in the 21st century.\u0026quot; \u003cem\u003eJournal of Pesticide Science\u003c/em\u003e 45.2 (2020): 54-74. ISSN: 1348-589X https://www.jstage.jst.go.jp/article/jpestics/45/2/45_D20-201/_article/-char/ja/\u003c/li\u003e\n\u003cli\u003eWorld Health Organization. \u0026quot;Standard operating procedure for testing insecticide susceptibility of adult mosquitoes in WHO bottle bioassays.\u0026quot; (2022). ISBN: 978-92-4-004377-0\u003c/li\u003e\n\u003cli\u003eWorld Health Organization. \u003cem\u003eDetermining discriminating concentrations of insecticides for monitoring resistance in mosquitoes: report of a multi-centre laboratory study and WHO expert consultations.\u003c/em\u003e World Health Organization, 2022. ISBN: 978-92-4-004520-0 https://www.who.int/publications/i/item/9789240045200\u003c/li\u003e\n\u003cli\u003eTchouakui, Magellan, et al. \u0026quot;Comparative study of the effect of solvents on the efficacy of neonicotinoid insecticides against malaria vector populations across Africa.\u0026quot; \u003cem\u003eInfectious Diseases of Poverty\u003c/em\u003e 11.02 (2022): 23-31. ISSN: 2095-5162 https://doi.org/10.1186/s40249-022-00962-4\u003c/li\u003e\n\u003cli\u003eLees, Rosemary, et al. \u0026quot;A testing cascade to identify repurposed insecticides for next-generation vector control tools: screening a panel of chemistries with novel modes of action against a malaria vector.\u0026quot; \u003cem\u003eGates open research\u003c/em\u003e 3 (2019). ISSN: 2572-4754 https://doi.org/10.12688/gatesopenres.12957.2\u003c/li\u003e\n\u003cli\u003ePortwood, Natalie M., et al. \u0026quot;Multi-centre discriminating concentration determination of broflanilide and potential for cross-resistance to other public health insecticides in Anopheles vector populations.\u0026quot; \u003cem\u003eScientific Reports\u003c/em\u003e 12.1 (2022): 22359. ISSN: 2045-2322 https://www.nature.com/articles/s41598-022-26990-6\u003c/li\u003e\n\u003cli\u003eGovoetchan, Renaud, et al. \u0026quot;Investigating discriminating concentrations for monitoring susceptibility to broflanilide and cross resistance to other insecticide classes in Anopheles gambiae sensu lato, using the new WHO bottle bioassay method.\u0026quot; \u003cem\u003ePlos one\u003c/em\u003e 18.3 (2023): e0276246. ISSN: 1932-6203 https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0276246\u003c/li\u003e\n\u003cli\u003eSnetselaar, Janneke, et al. \u0026quot;Laboratory evaluation of broflanilide (TENEBENAL\u0026trade;) against Anopheles gambiae in Moshi, Tanzania\u0026ndash;delayed mortality, cross-resistance, and residual efficacy.\u0026quot; \u003cem\u003eFrontiers in Tropical Diseases\u003c/em\u003e 4 (2023): 1097189. ISSN: 2673-7515 https://www.frontiersin.org/journals/tropical-diseases/articles/10.3389/fitd.2023.1097189/full\u003c/li\u003e\n\u003cli\u003eHien, Aristide Sawdetuo, et al. \u0026quot;Long-lasting residual efficacy of a new indoor residual spraying product, VECTRON\u0026trade; T500 (broflanilide), against pyrethroid-resistant malaria vectors and its acceptance in a community trial in Burkina Faso.\u0026quot; \u003cem\u003eParasites \u0026amp; Vectors\u003c/em\u003e 17.1 (2024): 484. ISSN: 1756-3305 https://link.springer.com/article/10.1186/s13071-024-06577-y\u003c/li\u003e\n\u003cli\u003eBliss, Chester Ittner. \u0026quot;The calculation of the dosage‐mortality curve.\u0026quot; \u003cem\u003eAnnals of Applied Biology\u003c/em\u003e 22.1 (1935): 134-167. ISSN: 0003-4746 https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1744-7348.1935.tb07713.x\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Mosquito control, Indoor residual spraying, Broflanilide, Discriminating concentration","lastPublishedDoi":"10.21203/rs.3.rs-7478645/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7478645/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIndoor residual spraying (IRS) of insecticides is widely used as an effective method to control malaria vector mosquitoes in sub-Saharan Africa. In 2023, a new IRS product, VECTRON™ T500 (Mitsui Chemicals Crop \u0026amp; Life Solutions, Inc.), was launched. This product contains broflanilide, a novel active ingredient for IRS, and has been confirmed to exhibit long-lasting insecticidal efficacy against malaria vector mosquitoes. However, the discriminating concentration to assess the susceptibility of wild \u003cem\u003eAnopheles\u003c/em\u003e populations to broflanilide has not yet been determined.\u003c/p\u003e\n\u003cp\u003eIn this study, WHO bottle bioassays were conducted in nine research facilities to collect dose-response data on broflanilide against adult female mosquitoes of the insecticide susceptible \u003cem\u003eAnopheles gambiae \u003c/em\u003es.s. Kisumu strain. These data were statistically analysed and validated following WHO guidelines, and it was determined that the preliminary discriminating concentration of broflanilide for \u003cem\u003eAn. gambiae\u003c/em\u003e mosquitoes should be 18 μg/bottle. The results of this study will provide a useful benchmark for susceptibility monitoring of wild mosquito populations in regions of sub-Saharan Africa into which VECTRON™ T500 is being introduced for malaria vector control.\u003c/p\u003e","manuscriptTitle":"Determination of the Preliminary Discriminating Concentration of Broflanilide Against Malaria Vector Mosquito Anopheles gambiae by Multi-Centre Susceptibility Testing","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-14 10:12:06","doi":"10.21203/rs.3.rs-7478645/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-25T21:30:20+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-24T07:54:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-22T17:02:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-19T13:00:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"292413853047473550977319829695751491793","date":"2025-09-09T04:53:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"143056368108946459188506074300236357872","date":"2025-09-07T08:28:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"273480238801967953230049016362441405405","date":"2025-09-02T17:01:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"67657894783700158034404214289964464270","date":"2025-09-02T10:03:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"131341567487803462542973271397866774662","date":"2025-09-02T07:17:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-02T05:06:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-29T08:02:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-29T08:01:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Malaria Journal","date":"2025-08-28T09:19:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c5762831-d700-4895-ab4c-5155cbe0039a","owner":[],"postedDate":"September 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-16T16:03:42+00:00","versionOfRecord":{"articleIdentity":"rs-7478645","link":"https://doi.org/10.1186/s12936-025-05728-5","journal":{"identity":"malaria-journal","isVorOnly":false,"title":"Malaria Journal"},"publishedOn":"2026-03-09 15:59:47","publishedOnDateReadable":"March 9th, 2026"},"versionCreatedAt":"2025-09-14 10:12:06","video":"","vorDoi":"10.1186/s12936-025-05728-5","vorDoiUrl":"https://doi.org/10.1186/s12936-025-05728-5","workflowStages":[]},"version":"v1","identity":"rs-7478645","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7478645","identity":"rs-7478645","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}