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Sensitive environmental surveillance tools are needed to support transmission monitoring, particularly where conventional snail surveys are seasonally constrained. This study developed and evaluated a multiplex quantitative PCR (qPCR) assay for simultaneous detection of\n                      <jats:italic>S. mekongi<\/jats:italic>\n                      and its intermediate snail host,\n                      <jats:italic>Neotricula aperta<\/jats:italic>\n                      , in aquatic environmental DNA (eDNA) samples.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      Singleplex and multiplex qPCR assays targeting\n                      <jats:italic>S. mekongi<\/jats:italic>\n                      and\n                      <jats:italic>N. aperta<\/jats:italic>\n                      were compared to establish the optimal amplification system. Field validation was conducted in the Khong District, Champasak Province, Laos, during the dry season (May 2024) and wet season (August 2024) across 20 transmission sites. Method agreement between eDNA detection and malacological surveys was assessed using Cohen\u2019s kappa coefficient, and associations between\n                      <jats:italic>N. aperta<\/jats:italic>\n                      density and physicochemical variables were evaluated using Spearman\u2019s rank correlation.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      The optimized multiplex qPCR assay targeting\n                      <jats:italic>N. aperta<\/jats:italic>\n                      (16S rRNA) and\n                      <jats:italic>S. mekongi<\/jats:italic>\n                      (18S rRNA) showed high sensitivity, with limits of detection of 5 and 6 copies\/\u03bcl and limits of quantification of 40 and 46 copies\/\u03bcl, respectively. In mesocosms, both targets were detected at an infected-snail density of 1 snail\/L, with median Cq values of 35.23 for\n                      <jats:italic>N. aperta<\/jats:italic>\n                      and 26.54 for\n                      <jats:italic>S. mekongi<\/jats:italic>\n                      . During the dry-season surveys, 978\n                      <jats:italic>N. aperta<\/jats:italic>\n                      were collected, with a mean density of 46.25 snails per site; wet-season surveys were not feasible. No schistosome infections were detected by crushing microscopy. Field eDNA analysis detected\n                      <jats:italic>N. aperta<\/jats:italic>\n                      but not\n                      <jats:italic>S. mekongi<\/jats:italic>\n                      , with\n                      <jats:italic>N. aperta<\/jats:italic>\n                      eDNA positivity being 90% in the dry season and 40% in the wet season. Agreement with malacological surveys was moderate (\n                      <jats:italic>\u03ba<\/jats:italic>\n                      \u2009=\u20090.44;\n                      <jats:italic>p<\/jats:italic>\n                      \u2009=\u20090.01) and snail density was significantly associated with water temperature, pH, and dissolved oxygen.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>\n                      This multiplex eDNA assay sensitively detected\n                      <jats:italic>N. aperta<\/jats:italic>\n                      in field water samples and may help overcome seasonal limitations of malacological surveys. Although\n                      <jats:italic>S. mekongi<\/jats:italic>\n                      was detected under laboratory conditions, it was not detected in field samples, consistent with the absence of infected snails. Further validation in active-transmission sites is required before reliable field detection of\n                      <jats:italic>S. mekongi<\/jats:italic>\n                      eDNA can be assumed.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s40249-026-01466-1","type":"journal-article","created":{"date-parts":[[2026,6,4]],"date-time":"2026-06-04T07:20:33Z","timestamp":1780557633000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Development and field evaluation of a multiplex qPCR assay for environmental DNA detection of Schistosoma mekongi and its intermediate snail host Neotricula aperta in the Mekong River Basin"],"prefix":"10.1186","volume":"15","author":[{"given":"Jian","family":"He","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peter S.","family":"Andrus","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sengrloun","family":"Phonekeo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kun","family":"Yin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Leshan","family":"Xiu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shan","family":"Lv","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kun","family":"Yang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Somphou","family":"Sayasone","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiao-Nong","family":"Zhou","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,6,4]]},"reference":[{"issue":"10479","key":"1466_CR1","doi-asserted-by":"publisher","first-page":"658","DOI":"10.1016\/S0140-6736(24)02814-9","volume":"405","author":"D Buonfrate","year":"2025","unstructured":"Buonfrate D, Ferrari TCA, Adegnika AA, Stothard JR, Gobbi FG. 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Sci One Health. 2025:100132.","DOI":"10.1016\/j.soh.2025.100132"}],"container-title":["Infectious Diseases of Poverty"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/s40249-026-01466-1.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/article\/10.1186\/s40249-026-01466-1","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/s40249-026-01466-1.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,4]],"date-time":"2026-06-04T09:03:27Z","timestamp":1780563807000},"score":1,"resource":{"primary":{"URL":"https:\/\/link.springer.com\/10.1186\/s40249-026-01466-1"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6,4]]},"references-count":47,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2026,12]]}},"alternative-id":["1466"],"URL":"https:\/\/doi.org\/10.1186\/s40249-026-01466-1","relation":{},"ISSN":["2049-9957"],"issn-type":[{"value":"2049-9957","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,6,4]]},"assertion":[{"value":"20 January 2026","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"26 May 2026","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"4 June 2026","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Declarations"}},{"value":"This study was conducted in accordance with the Declaration of Helsinki and approved by the National Ethics Committee for Health Research of the Lao People\u2019s Democratic Republic (ID: 2024.32) and the Ethics Committee of the Jiangsu Institute of Parasitic Diseases (IRB00012165). Infected\n                      N. aperta\n                      specimens were obtained from the Applied Malacology Laboratory, Department of Social and Environmental Medicine, Mahidol University. Genomic DNA of other human-infecting\n                      Schistosoma\n                      species (\n                      S. japonicum\n                      ,\n                      S. haematobium\n                      ,\n                      S. mansoni\n                      ) was provided by the Jiangsu Institute of Parasitic Diseases. During field sampling, collected host snails were not returned to their habitats regardless of infection status to avoid potential reintroduction of prepatent infections.","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 no competing interests.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"65"}}