{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T03:54:53Z","timestamp":1790049293488,"version":"4.0.1"},"reference-count":52,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2026,3,4]],"date-time":"2026-03-04T00:00:00Z","timestamp":1772582400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Research Institute for Tropical Medicine"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["TropicalMed"],"abstract":"<jats:p>Leptospirosis remains a public health concern in the Philippines. Conventional diagnostic methods, including the microscopic agglutination test (MAT) and qPCR, are routinely used for outbreak response and surveillance. However, these methods often yield discordant results due to cross-reactivity, limited sensitivity, or lack of species-level resolution. To address these diagnostic gaps, this study optimized the Boonsilp 16S rRNA PCR assay and applied Sanger sequencing for accurate species identification of Leptospira in 92 archived DNA samples collected between 2018 and 2020. The sensitivity and specificity of the optimized assay were compared with those of MAT and qPCR. Species-level identification was confirmed via sequencing, and a phylogenetic tree was constructed. Among the 92 samples, 46 (50.0%) tested positive by qPCR, 39 (42.4%) by MAT, and 67 (72.8%) by at least one of the two methods. The optimized Boonsilp assay detected Leptospira in 23 samples (25.0%), of which 22 were also qPCR positive. Twenty-one samples were confirmed as L. interrogans, one as L. borgpetersenii, and one as an unclassified Leptospira species. One sample undetected by both MAT and qPCR tested positive using the optimized assay. Compared to the composite reference, the Boonsilp assay showed 32.8% sensitivity and 96.0% specificity. Phylogenetic analysis revealed multiple L. interrogans strains, including those closely related to reference sequences of Copenhageni, Manilae, and Canicola. While the optimized Boonsilp PCR assay demonstrates diagnostic value as an adjunct molecular tool to qPCR and MAT supporting species-level identification during outbreak surveillance, this warrants further validation in freshly isolated DNA samples.<\/jats:p>","DOI":"10.3390\/tropicalmed11030069","type":"journal-article","created":{"date-parts":[[2026,3,4]],"date-time":"2026-03-04T08:51:33Z","timestamp":1772614293000},"page":"69","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Retrospective Molecular Detection and Characterization of Pathogenic Leptospira in the Philippines"],"prefix":"10.3390","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-9716-2423","authenticated-orcid":false,"given":"Joanna Ina G.","family":"Manalo","sequence":"first","affiliation":[{"name":"Advanced Molecular Technologies Laboratory (AMTL), Research Institute for Tropical Medicine (RITM), Muntinlupa 1781, Philippines"},{"name":"Graduate School, Centro Escolar University, Manila 1008, Philippines"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-3471-1780","authenticated-orcid":false,"given":"Adeliza Mae L.","family":"Realingo","sequence":"additional","affiliation":[{"name":"Advanced Molecular Technologies Laboratory (AMTL), Research Institute for Tropical Medicine (RITM), Muntinlupa 1781, Philippines"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0758-9968","authenticated-orcid":false,"given":"Lei Lanna M.","family":"Dancel","sequence":"additional","affiliation":[{"name":"Advanced Molecular Technologies Laboratory (AMTL), Research Institute for Tropical Medicine (RITM), Muntinlupa 1781, Philippines"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3359-4947","authenticated-orcid":false,"given":"Timothy John R.","family":"Dizon","sequence":"additional","affiliation":[{"name":"Advanced Molecular Technologies Laboratory (AMTL), Research Institute for Tropical Medicine (RITM), Muntinlupa 1781, Philippines"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3323-6303","authenticated-orcid":false,"given":"Amalea Dulcene","family":"Nicolasora","sequence":"additional","affiliation":[{"name":"Advanced Molecular Technologies Laboratory (AMTL), Research Institute for Tropical Medicine (RITM), Muntinlupa 1781, Philippines"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-3821-2873","authenticated-orcid":false,"given":"Kristine","family":"Alvarado-Dela Cruz","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Research Institute for Tropical Medicine (RITM), Muntinlupa 1781, Philippines"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-9384-3211","authenticated-orcid":false,"given":"Desiree D.","family":"Argana","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Research Institute for Tropical Medicine (RITM), Muntinlupa 1781, Philippines"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4987-3577","authenticated-orcid":false,"given":"Arjay Ni\u00f1o A.","family":"Digman","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Research Institute for Tropical Medicine (RITM), Muntinlupa 1781, Philippines"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-1822-3125","authenticated-orcid":false,"given":"Emarld Julian G.","family":"Medina","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Research Institute for Tropical Medicine (RITM), Muntinlupa 1781, Philippines"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-1920-3501","authenticated-orcid":false,"given":"Celine Bernice A.","family":"Roxas","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Research Institute for Tropical Medicine (RITM), Muntinlupa 1781, Philippines"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-3529-6053","authenticated-orcid":false,"given":"Rubelia A.","family":"Baterna","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Research Institute for Tropical Medicine (RITM), Muntinlupa 1781, Philippines"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0172-7602","authenticated-orcid":false,"given":"Julieta Z.","family":"Dungca","sequence":"additional","affiliation":[{"name":"Graduate School, Centro Escolar University, Manila 1008, Philippines"},{"name":"School of Science and Technology, Centro Escolar University, Manila 1008, Philippines"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,3,4]]},"reference":[{"key":"ref_1","unstructured":"CDC (2024, November 23). 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