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Sensitive and specific point-of-care diagnostics are needed for elimination of this disease. Recombinase polymerase amplification (RPA) assays meet these criteria, and an assay to diagnose <jats:italic>S. haematobium<\/jats:italic> has been developed (Sh-RPA). However, false-positive results can occur, and optimisation of reaction conditions to mitigate these is needed. Ease of use and compatibility of DNA extraction methods must also be considered.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Methods<\/jats:title>\n                <jats:p>Using synthetic DNA, <jats:italic>S. haematobium<\/jats:italic> genomic DNA (gDNA), and urine samples from clinical cases, Sh-RPA reactions incorporating different betaine concentrations (0\u00a0M, 1\u00a0M, 2.5\u00a0M, 12.5\u00a0M) and the sample-to-water ratios were tested to determine effects on assay specificity and sensitivity. In addition, five commercial DNA extraction kits suitable for use in resource-limited settings were used to obtain gDNA from single <jats:italic>S. haematobium<\/jats:italic> eggs and evaluated in terms of DNA quality, quantity, and compatibility with the Sh-RPA assay. All samples were also evaluated by quantitative polymerase chain reaction (qPCR) to confirm DNA acquisition.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Results<\/jats:title>\n                <jats:p>The analytical sensitivity of the Sh-RPA with all betaine concentrations was\u2009\u2265\u200910 copies of the synthetic <jats:italic>Dra1<\/jats:italic> standard and 0.1\u00a0pg of <jats:italic>S. haematobium<\/jats:italic> gDNA. The addition of betaine improved Sh-RPA assay specificity in all reaction conditions, and the addition of 2.5\u00a0M of betaine together with the maximal possible sample volume of 12.7\u00a0\u00b5l proved to be the optimum reaction conditions. DNA was successfully isolated from a single <jats:italic>S. haematobium<\/jats:italic> egg using all five commercial DNA extraction kits, but the Sh-RPA performance of these kits varied, with one proving to be incompatible with RPA reactions.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Conclusions<\/jats:title>\n                <jats:p>The addition of 2.5\u00a0M of betaine to Sh-RPA reactions improved reaction specificity whilst having no detrimental effect on sensitivity. This increases the robustness of the assay, advancing the feasibility of using the Sh-RPA assay in resource-limited settings. The testing of commercial extraction kits proved that crude, rapid, and simple methods are sufficient for obtaining DNA from single <jats:italic>S. haematobium<\/jats:italic> eggs, and that these extracts can be used with Sh-RPA in most cases. However, the observed incompatibility of specific kits with Sh-RPA highlights the need for each stage of a molecular diagnostic platform to be robustly tested prior to implementation.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Graphical Abstract<\/jats:title>\n                \n              <\/jats:sec>","DOI":"10.1186\/s13071-024-06380-9","type":"journal-article","created":{"date-parts":[[2024,7,28]],"date-time":"2024-07-28T02:01:20Z","timestamp":1722132080000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Refining the Schistosoma haematobium recombinase polymerase amplification (Sh-RPA) assay: moving towards point-of-care use in endemic settings"],"prefix":"10.1186","volume":"17","author":[{"given":"Owain","family":"Donnelly","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Silvia","family":"Mesquita","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John","family":"Archer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Said M.","family":"Ali","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zikmund","family":"Bartonicek","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elena B.","family":"Lugli","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bonnie L.","family":"Webster","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2024,7,28]]},"reference":[{"key":"6380_CR1","doi-asserted-by":"publisher","first-page":"2253","DOI":"10.1016\/S0140-6736(13)61949-2","volume":"383","author":"DG Colley","year":"2014","unstructured":"Colley DG, Bustinduy AL, Secor WE, King CH. 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Details on how urine samples were collected, screened for <i>S. haematobium<\/i> infection, and stored have been outlined previously []. ZEST received permission to store and share urine samples from study participants for research purposes and received ethical approval from the Zanzibar Medical Research Ethical Committee (ZAMREC; reference number: ZAMREC 0003\/Sept\/011); the Swiss Ethikkommission beider Basel (EKBB; reference number: EKBB 236\/11); and the Institutional Review Board of the University of Georgia (IRB UGA project number: 2012-10138-0). ZEST was also registered on the International Standard Randomised Controlled Trial Number Register (ISRCTN48837681). Written informed consent from parents or legal guardians of participating children in Zanzibar was obtained for urine collection and storage, including examination for <i>S. haematobium<\/i> infections with newly developed diagnostic techniques at a later point in time. All study participants were offered praziquantel (40\u00a0mg\/kg) to treat schistosomiasis free of charge during the ongoing biannual island-wide MDA campaign.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"Not applicable.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent for publication"}},{"value":"The authors declare that they have no competing interests.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"321"}}