{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,9]],"date-time":"2026-07-09T11:24:13Z","timestamp":1783596253541,"version":"3.55.0"},"reference-count":31,"publisher":"F1000 Research Ltd","license":[{"start":{"date-parts":[[2026,4,8]],"date-time":"2026-04-08T00:00:00Z","timestamp":1775606400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000865","name":"Bill and Melinda Gates Foundation","doi-asserted-by":"publisher","award":["INV-050591"],"award-info":[{"award-number":["INV-050591"]}],"id":[{"id":"10.13039\/100000865","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["gatesopenresearch.org"],"crossmark-restriction":false},"short-container-title":["Gates Open Res"],"abstract":"<ns5:p>Dual-active-ingredient insecticide-treated nets (Dual-AI ITNs) combining pyrethroids with chlorfenapyr are increasingly being deployed across malaria-endemic areas. Laboratory evaluation of chlorfenapyr ITNs is challenging due to chlorfenapyr\u2019s requirement for metabolic activation. Inspired by the tunnel test, the mini chamber test is smaller, has higher throughput, and is animal-free. We optimised the primary parameters using pyrethroid-susceptible and -resistant mosquito strains exposed to new pyrethroid-chlorfenapyr ITNs versus controls. With parameters defined, we used a draft standard operating procedure (SOP) to evaluate the mini chamber against the gold standard tunnel test in a comparator study. The generated data were used to finalise an SOP. The number of mosquitoes per bioassay had no effect on 72 hour mortality on exposure to the pyrethroid-chlorfenapyr net (10, 20, or 30 mosquitoes per mini chamber: 98% vs. 99% vs. 99%, p = 0.46), nor did the chamber orientation (horizontal vs. vertical: 70% vs. 74%, p = 0.35). Mean 72-hour mortality reduced with the length of exposure: 99% with overnight exposure, 74% with 4 hours and 35% with 2 hour exposure. In direct comparisons, the mini chamber gave 95% 72 hour mortality and the tunnel test 70% with overnight exposure. A trend was observed between chemical content (measured by high performance liquid chromatography (HPLC)) and mortality in ITN samples that had been washed 20 times or used in experimental hut trials, compared to unused comparators, when a 6 hour exposure length was used, showing potential for application to durability monitoring. A mini chamber SOP is shared (https:\/\/github.com\/i2i-Data-Repository\/Mini-Chamber-Test-paper-data-R-code-and-SOP), and further improvements may come from internal and multi-site validation and review of data generated. The method shows promise as an effective bioassay for use where tunnel tests are not available, for measuring the biological activity of chlorfenapyr in ITNs when measuring regeneration times, wash resistance, residual efficacy, and other studies.<\/ns5:p>","DOI":"10.12688\/gatesopenres.16381.1","type":"journal-article","created":{"date-parts":[[2026,7,9]],"date-time":"2026-07-09T10:49:08Z","timestamp":1783594148000},"page":"14","update-policy":"https:\/\/doi.org\/10.12688\/gatesopenres.crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["The mini chamber test: a novel bioassay for bioefficacy testing of chlorfenapyr treated nets used in malaria control"],"prefix":"10.12688","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-4846-2335","authenticated-orcid":false,"given":"Amy","family":"Lewis","sequence":"first","affiliation":[{"name":"Liverpool England L3 5QA Liverpool School of Tropical Medicine"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rowan","family":"Jones-Brown","sequence":"additional","affiliation":[{"name":"Liverpool England L3 5QA Liverpool School of Tropical Medicine"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gemma","family":"Harvey","sequence":"additional","affiliation":[{"name":"Liverpool England L3 5QA Liverpool School of Tropical Medicine"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2601-2598","authenticated-orcid":false,"given":"Giorgio","family":"Praulins","sequence":"additional","affiliation":[{"name":"Liverpool England L3 5QA Liverpool School of Tropical Medicine"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emma","family":"Reid","sequence":"additional","affiliation":[{"name":"Liverpool England L3 5QA Liverpool School of Tropical Medicine"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aidi Galus","family":"Lugenge","sequence":"additional","affiliation":[{"name":"Bagamoyo P.O Box 74 Ifakara Health Institute"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Frank","family":"Mechan","sequence":"additional","affiliation":[{"name":"Liverpool England L3 5QA Liverpool School of Tropical Medicine"},{"name":"Liverpool England L3 5QA Liverpool School of Tropical Medicine"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katherine","family":"Gleave","sequence":"additional","affiliation":[{"name":"Liverpool England L3 5QA Liverpool School of Tropical Medicine"},{"name":"Liverpool England L3 5QA Liverpool School of Tropical Medicine"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4232-9125","authenticated-orcid":false,"given":"Rosemary Susan","family":"Lees","sequence":"additional","affiliation":[{"name":"Liverpool England L3 5QA Liverpool School of Tropical Medicine"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"2560","published-online":{"date-parts":[[2026,4,8]]},"reference":[{"key":"ref1","article-title":"World malaria report 2024: addressing inequity in the global malaria response.","year":"2024"},{"key":"ref2","doi-asserted-by":"publisher","first-page":"676","DOI":"10.1038\/s41467-023-36356-9","article-title":"Quantifying the direct and indirect protection provided by insecticide treated bed nets against malaria.","volume":"14","author":"H Unwin","year":"2023","journal-title":"Nat. Commun."},{"key":"ref3","doi-asserted-by":"publisher","first-page":"199-213","DOI":"10.1016\/B978-012286975-4\/50014-0","author":"A Farnham","year":"1999","journal-title":"Piperonyl Butoxide."},{"key":"ref4","doi-asserted-by":"publisher","first-page":"e0008669","DOI":"10.1371\/journal.pntd.0008669","article-title":"Molecular action of pyriproxyfen: Role of the Methoprene-tolerant protein in the pyriproxyfen-induced sterilization of adult female mosquitoes.","volume":"14","author":"T Ahmed","year":"2020","journal-title":"PLoS. Negl. Trop. Dis."},{"key":"ref5","doi-asserted-by":"crossref","first-page":"115-128","DOI":"10.1006\/pest.1994.1064","article-title":"Insecticidal action and mitochondrial uncoupling activity of ac-303,630 and related halogenated pyrroles.","volume":"50","author":"B Black","year":"1994","journal-title":"Pestic. Biochem. Physiol."},{"key":"ref6","doi-asserted-by":"publisher","first-page":"1227-1241","DOI":"10.1016\/S0140-6736(21)02499-5","article-title":"Effectiveness and cost-effectiveness against malaria of three types of dual-active-ingredient long-lasting insecticidal nets (Llins) compared with pyrethroid-only LLINs in Tanzania: a four-arm, cluster-randomised trial.","volume":"399","author":"J Mosha","year":"2022","journal-title":"Lancet."},{"key":"ref7","doi-asserted-by":"publisher","first-page":"435-446","DOI":"10.1016\/S0140-6736(22)02319-4","article-title":"Efficacy of pyriproxyfen-pyrethroid long-lasting insecticidal nets (Llins) and chlorfenapyr-pyrethroid LLINs compared with pyrethroid-only LLINs for malaria control in Benin: a cluster-randomised, superiority trial.","volume":"401","author":"M Accrombessi","year":"2023","journal-title":"Lancet."},{"key":"ref8","article-title":"World malaria report 2023.","year":"2023"},{"key":"ref9","doi-asserted-by":"publisher","first-page":"e1325-e1331","DOI":"10.1016\/S2214-109X(21)00216-3","article-title":"Threats to the effectiveness of insecticide-treated bednets for malaria control: thinking beyond insecticide resistance.","volume":"9","author":"S Lindsay","year":"2021","journal-title":"Lancet Glob. Health."},{"key":"ref10","article-title":"Vector control product list.","year":"2026 12\/01\/2026"},{"key":"ref11","article-title":"Bioassay methods for insecticide treated nets: Cone test.","year":"2024"},{"key":"ref12","article-title":"Manual for monitoring insecticide resistance in mosquito vectors and selecting appropriate interventions.","year":"2022"},{"key":"ref13","doi-asserted-by":"publisher","first-page":"342","DOI":"10.1186\/s13071-023-05963-2","article-title":"Sub-lethal exposure to chlorfenapyr reduces the probability of developing Plasmodium falciparum parasites in surviving Anopheles mosquitoes.","volume":"16","author":"P Kweyamba","year":"2023","journal-title":"Parasit. Vectors."},{"key":"ref14","article-title":"Bioassay methods for insecticide treated nets: Tunnel test.","year":"2024"},{"key":"ref15","doi-asserted-by":"publisher","first-page":"124","DOI":"10.1186\/s13071-022-05207-9","article-title":"Influence of testing modality on bioefficacy for the evaluation of Interceptor\u00ae G2 mosquito nets to combat malaria mosquitoes in Tanzania.","volume":"15","author":"U Kibondo","year":"2022","journal-title":"Parasit. Vectors."},{"key":"ref16","doi-asserted-by":"publisher","first-page":"289","DOI":"10.1186\/s12936-023-04717-w","article-title":"A bioassay method validation framework for laboratory and semi-field tests used to evaluate vector control tools.","volume":"22","author":"A Matope","year":"2023","journal-title":"Malar. J."},{"key":"ref17","doi-asserted-by":"publisher","first-page":"522","DOI":"10.1186\/s13071-019-3774-3","article-title":"Characterisation of Anopheles strains used for laboratory screening of new vector control products.","volume":"12","author":"J Williams","year":"2019","journal-title":"Parasit. Vectors."},{"key":"ref18","article-title":"MR4: methods in Anopheles research manual 2015 edition.","year":"2023"},{"key":"ref19","doi-asserted-by":"publisher","first-page":"434","DOI":"10.3390\/insects13050434","article-title":"Strain characterisation for measuring bioefficacy of itns treated with two active ingredients (Dual-ai itns): developing a robust protocol by building consensus.","volume":"13","author":"R Lees","year":"2022","journal-title":"Insects."},{"key":"ref20","doi-asserted-by":"publisher","first-page":"159","DOI":"10.1186\/s13071-024-06210-y","article-title":"Temperature, mosquito feeding status and mosquito density influence the measured bio-efficacy of insecticide-treated nets in cone assays.","volume":"17","author":"J Mseti","year":"2024","journal-title":"Parasit. Vectors."},{"key":"ref21","doi-asserted-by":"publisher","first-page":"544","DOI":"10.3390\/insects13060544","article-title":"Reviewing the who tube bioassay methodology: accurate method reporting and numbers of mosquitoes are key to producing robust results.","volume":"13","author":"G Praulins","year":"2022","journal-title":"Insects."},{"key":"ref22","doi-asserted-by":"publisher","first-page":"243","DOI":"10.1186\/s12936-016-1303-9","article-title":"How important is the angle of tilt in the WHO cone bioassay?","volume":"15","author":"H Owusu","year":"2016","journal-title":"Malar. J."},{"key":"ref23","doi-asserted-by":"publisher","first-page":"562","DOI":"10.3390\/insects13070562","article-title":"Modified world health organization (Who) tunnel test for higher throughput evaluation of insecticide-treated nets (Itns) considering the effect of alternative hosts, exposure time, and mosquito density.","volume":"13","author":"D Kamande","year":"2022","journal-title":"Insects."},{"key":"ref24","doi-asserted-by":"publisher","first-page":"9715","DOI":"10.1038\/s41598-022-13768-z","article-title":"A high-throughput HPLC method for simultaneous quantification of pyrethroid and pyriproxyfen in long-lasting insecticide-treated nets.","volume":"12","author":"K Walker","year":"2022","journal-title":"Sci. Rep."},{"key":"ref26","doi-asserted-by":"publisher","first-page":"e0165925","DOI":"10.1371\/journal.pone.0165925","article-title":"A chlorfenapyr mixture net interceptor\u00ae g2 shows high efficacy and wash durability against resistant mosquitoes in west africa.","volume":"11","author":"R N\u2019Guessan","year":"2016","journal-title":"PLoS One."},{"key":"ref27","doi-asserted-by":"publisher","first-page":"81","DOI":"10.1186\/s40249-023-01132-w","article-title":"High efficacy of chlorfenapyr-based net Interceptor\u00ae G2 against pyrethroid-resistant malaria vectors from Cameroon.","volume":"12","author":"M Tchouakui","year":"2023","journal-title":"Infect. Dis. Poverty."},{"key":"ref28","first-page":"129","article-title":"Maximising power with the minimum number of mosquitoes: Designing robust sample sizes for the WHO Cone Bioassay.","volume":"2","author":"G Harvey","year":"2025","journal-title":"VeriXiv."},{"key":"ref29","doi-asserted-by":"publisher","first-page":"12232","DOI":"10.1038\/s41598-023-39140-3","article-title":"PermaNet Dual, a new deltamethrin-chlorfenapyr mixture net, shows improved efficacy against pyrethroid-resistant Anopheles gambiae sensu lato in southern Benin.","volume":"13","author":"T Syme","year":"2023","journal-title":"Sci. Rep."},{"key":"ref30","doi-asserted-by":"publisher","first-page":"103","DOI":"10.1186\/s13071-024-06190-z","article-title":"Time of exposure and assessment influence the mortality induced by insecticides against metabolic resistant mosquitoes.","volume":"17","author":"O Odufuwa","year":"2024","journal-title":"Parasit. Vectors."},{"key":"ref31","doi-asserted-by":"publisher","first-page":"124","DOI":"10.1186\/s12936-015-0639-x","article-title":"The activity of the pyrrole insecticide chlorfenapyr in mosquito bioassay: towards a more rational testing and screening of non-neurotoxic insecticides for malaria vector control.","volume":"14","author":"R Oxborough","year":"2015","journal-title":"Malar. J."},{"key":"ref32","doi-asserted-by":"publisher","DOI":"10.5281\/zenodo.17780455","article-title":"Mini chamber test raw data.","author":"A Lewis","year":"2025","journal-title":"Zenodo."}],"container-title":["Gates Open Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/gatesopenresearch.org\/articles\/10-14\/v1\/xml","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/gatesopenresearch.org\/articles\/10-14\/v1\/pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/gatesopenresearch.org\/articles\/10-14\/v1\/iparadigms","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,9]],"date-time":"2026-07-09T10:49:22Z","timestamp":1783594162000},"score":1,"resource":{"primary":{"URL":"https:\/\/gatesopenresearch.org\/articles\/10-14\/v1"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,4,8]]},"references-count":31,"URL":"https:\/\/doi.org\/10.12688\/gatesopenres.16381.1","relation":{"has-preprint":[{"id-type":"doi","id":"10.12688\/verixiv.2828.1","asserted-by":"subject"}]},"ISSN":["2572-4754"],"issn-type":[{"value":"2572-4754","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,4,8]]},"assertion":[{"value":"Awaiting Peer Review","URL":"https:\/\/gatesopenresearch.org\/articles\/10-14\/v1#article-reports","order":0,"name":"referee-status","label":"Referee status","group":{"name":"current-referee-status","label":"Current Referee Status"}},{"value":"This study was supported by the Gates Foundation (INV-050591). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.","order":0,"name":"grant-information","label":"Grant Information"},{"value":"This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.","order":0,"name":"copyright-info","label":"Copyright"}]}}