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Here, we present a fast, cost-effective mcfDNA multiplex workflow to complement or improve the sensitivity of microbiological diagnostics for systemic and focal infections which can be challenged by prior antibiotic treatment or non-culturable pathogens. In an explorative study of 18 patients, mcfDNA sequencing matched the clinical diagnosis in 16 cases (89%), was detected up to 16\u00a0days after starting targeted antibiotic therapy and identified\n                    <jats:italic>Staphylococcus aureus<\/jats:italic>\n                    in two cases of culture-negative endocarditis. By providing an open-access protocol, we aim to promote broader accessibility and globally available application.\n                  <\/jats:p>","DOI":"10.1186\/s13073-026-01700-3","type":"journal-article","created":{"date-parts":[[2026,6,19]],"date-time":"2026-06-19T06:05:51Z","timestamp":1781849151000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Microbial cell-free DNA for rapid pathogen identification in clinical diagnostics: a proof of concept study"],"prefix":"10.1186","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8813-5388","authenticated-orcid":false,"given":"Micha","family":"Banz","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stefan","family":"Hagel","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sebastian","family":"Freiburger","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Frederik","family":"Huhn","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Merlin","family":"Krause","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stefan","family":"Gl\u00f6ckner","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bettina","family":"L\u00f6ffler","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dennis","family":"Nurjadi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Uthayakumar","family":"Muthukumarasamy","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mateusz","family":"Jundzill","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Achim J.","family":"Kaasch","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mathias W.","family":"Pletz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christian","family":"Brandt","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,6,19]]},"reference":[{"key":"1700_CR1","doi-asserted-by":"publisher","unstructured":"Scheer CS, Fuchs C, Gr\u00fcndling M, Vollmer M, Bast J, Bohnert JA, Zimmermann K, Hahnenkamp K, Rehberg S, and Kuhn SO. 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Written informed consent was obtained from all participants.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"Not applicable. All presented data are de-identified and anonymized.","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":"88"}}