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Current understanding of these infections is limited in part by existing point-of-care diagnostics that fail to differentiate them and have poor sensitivity for low-density infections. Accurate diagnosis currently requires molecular assays performed in well-resourced laboratories. This report describes the development of a <jats:italic>P. malariae<\/jats:italic> diagnostic assay that uses rapid, isothermal recombinase polymerase amplification (RPA) and lateral-flow-strip detection.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Methods<\/jats:title>\n                <jats:p>Multiple combinations of custom RPA primers and probes were designed using publicly available <jats:italic>P. malariae<\/jats:italic> genomic sequences, and by modifying published primer sets. Based on manufacturer RPA reaction conditions (TwistDx nfo kit), an isothermal assay was optimized targeting the multicopy <jats:italic>P. malariae<\/jats:italic> 18S rRNA gene with 39\u00a0\u00b0C incubation and 30-min run time. RPA product was visualized using lateral strips (FAM-labeled, biotinylated amplicon detected by a sandwich immunoassay, visualized using gold nanoparticles). Analytical sensitivity was evaluated using 18S rRNA plasmid DNA, and clinical sensitivity determined using qPCR-confirmed samples collected from Tanzania, Ethiopia, and the Democratic Republic of the Congo.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Results<\/jats:title>\n                <jats:p>Using 18S rRNA plasmid DNA, the assay demonstrates a detection limit of 10 copies\/\u00b5L (~\u20091.7 genome equivalents) and 100% analytical specificity. Testing in field samples showed 95% clinical sensitivity and 88% specificity compared to qPCR. Total assay time was less than 40\u00a0min.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Conclusion<\/jats:title>\n                <jats:p>Combined with simplified DNA extraction methods, the assay has potential for future field-deployable, point-of-care use to detect <jats:italic>P. malariae<\/jats:italic> infection, which remains largely undiagnosed but a neglected cause of chronic malaria. The assay provides a rapid, simple readout on a lateral flow strip without the need for expensive laboratory equipment.<\/jats:p>\n              <\/jats:sec>","DOI":"10.1186\/s12936-024-04928-9","type":"journal-article","created":{"date-parts":[[2024,4,12]],"date-time":"2024-04-12T19:01:38Z","timestamp":1712948498000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Detection of P. malariae using a new rapid isothermal amplification lateral flow assay"],"prefix":"10.1186","volume":"23","author":[{"given":"Ashenafi","family":"Assefa","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kevin","family":"Wamae","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Christopher M.","family":"Hennelly","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Billy","family":"Ngasala","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Meredith","family":"Muller","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Albert","family":"Kalonji","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fernandine","family":"Phanzu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Clark H.","family":"Cunningham","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jessica T.","family":"Lin","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jonathan B.","family":"Parr","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2024,4,12]]},"reference":[{"key":"4928_CR1","doi-asserted-by":"publisher","first-page":"1056","DOI":"10.1016\/S0140-6736(19)31139-0","volume":"394","author":"RGA Feachem","year":"2019","unstructured":"Feachem RGA, Chen I, Akbari O, Bertozzi-Villa A, Bhatt S, Binka F, et al. 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