{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,9]],"date-time":"2026-06-09T13:05:56Z","timestamp":1781010356671,"version":"3.54.1"},"reference-count":47,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,4,29]],"date-time":"2025-04-29T00:00:00Z","timestamp":1745884800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:sec><jats:title>Background<\/jats:title><jats:p>Plasma microbial cell-free DNA (mcfDNA) is a key biomarker for diagnosing bloodstream infections (BSIs), which contribute significantly to morbidity and mortality, particularly in patients with severe trauma, chronic illnesses, or immunosuppressive conditions. However, the baseline distribution of mcfDNA in different populations remains unclear. This study characterizes plasma mcfDNA profiles across various human populations.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>A total of 300 blood samples were collected from 10 groups: healthy individuals (Group A), patients with chronic diseases but no infections (Group B1\u2013B7), patients with mild-to-moderate infections (Group C), and patients meeting sepsis criteria (Group D). Multiplex droplet digital PCR (ddPCR) was used to detect mcfDNA from 10 common sepsis-causing bacterial species, two fungal species, and three herpesviruses (HSV-1, Epstein\u2013Barr virus [EBV], and Cytomegalovirus [CMV]).<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>Most pathogens in all groups showed low mcfDNA copy concentrations (~100 copies\/mL), forming a baseline. Group A had no pathogens exceeding this level, while Group B showed elevated <jats:italic>E. coli<\/jats:italic> and <jats:italic>S. maltophilia<\/jats:italic> (10<jats:sup>2<\/jats:sup>\u201310<jats:sup>4<\/jats:sup> copies\/mL). In Group C, 53 pathogens were detected above baseline, with EBV, CMV, and HSV-1 as the most common (copy concentrations 10<jats:sup>2<\/jats:sup>\u201310<jats:sup>4<\/jats:sup> copies\/mL). In Group D, 57 pathogens exceeded baseline, primarily EBV, <jats:italic>K. pneumoniae<\/jats:italic>, <jats:italic>A. baumannii<\/jats:italic>, <jats:italic>E. faecium<\/jats:italic>, and CMV. Although statistical analysis showed no significant differences in pathogen distribution between Groups C and D, Gram-negative bacteria were more prevalent in Group D (70% vs. 53.3%, OR\u202f=\u202f2.03), while viral pathogens were more frequently detected in Group C (93.3% vs. 76.7%, OR\u202f=\u202f0.24). The microbial profiles and mcfDNA copy concentrations in Groups C and D were similar (10<jats:sup>2<\/jats:sup>\u201310<jats:sup>4<\/jats:sup> copies\/mL), distinguishing them from Groups A and B.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion<\/jats:title><jats:p>This study provides a comprehensive characterization of mcfDNA across different health states, demonstrating the utility of ddPCR in detecting microbial infections. These findings contribute to refining infection diagnostics and improving early detection strategies for BSIs and sepsis.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fmicb.2025.1578820","type":"journal-article","created":{"date-parts":[[2025,4,29]],"date-time":"2025-04-29T05:26:16Z","timestamp":1745904376000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":3,"title":["Plasma microbial cell-free DNA characterization in different populations based on the droplet digital PCR method: a multi-cohort study"],"prefix":"10.3389","volume":"16","author":[{"given":"Juan","family":"Du","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dong","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fengliu","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xuesong","family":"Shang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jingjia","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guanhua","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jingwen","family":"Ding","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aiwu","family":"Ren","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shuang","family":"Jing","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lu","family":"Bai","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ying","family":"Liu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ye","family":"Zhao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peng","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shuyi","family":"Yang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jing","family":"Liu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xuefang","family":"Xiang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qiang","family":"Chen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yingchun","family":"Xu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiang","family":"Xia","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qiwen","family":"Yang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2025,4,29]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"1795963","DOI":"10.1080\/16549716.2020.1795963","article-title":"A twin disaster: addressing the COVID-19 pandemic and a cerebrospinal meningitis outbreak simultaneously in a low-resource country","volume":"13","author":"Adjorlolo","year":"2020","journal-title":"Glob. Health Action"},{"key":"ref2","doi-asserted-by":"publisher","first-page":"663","DOI":"10.1038\/s41564-018-0349-6","article-title":"Analytical and clinical validation of a microbial cell-free DNA sequencing test for infectious disease","volume":"4","author":"Blauwkamp","year":"2019","journal-title":"Nat. Microbiol."},{"key":"ref3","doi-asserted-by":"publisher","first-page":"899508","DOI":"10.3389\/fcimb.2022.899508","article-title":"The diagnostic value of next-generation sequencing technology in sepsis","volume":"12","author":"Cao","year":"2022","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"ref4","doi-asserted-by":"publisher","first-page":"22","DOI":"10.1016\/j.idh.2019.08.006","article-title":"Bloodstream infections in neutropenic patients with haematological malignancies","volume":"25","author":"Carvalho","year":"2020","journal-title":"Infect Dis Health"},{"key":"ref5","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/dkx324","article-title":"ECIL guidelines for the prevention, diagnosis and treatment of BK polyomavirus-associated haemorrhagic cystitis in haematopoietic stem cell transplant recipients","author":"Cesaro","year":"2017","journal-title":"J. Antimicrob. Chemother."},{"key":"ref6","doi-asserted-by":"publisher","first-page":"1678","DOI":"10.1093\/cid\/cix1095","article-title":"Detecting infections rapidly and easily for candidemia trial, part 2 (DIRECT2): a prospective, multicenter study of the T2Candida panel","volume":"66","author":"Clancy","year":"2018","journal-title":"Clin. Infect. Dis."},{"key":"ref7","doi-asserted-by":"publisher","first-page":"693","DOI":"10.1080\/17843286.2021.1944584","article-title":"A case of Flavonifractor plautii blood stream infection in a severe burn patient and a review of the literature","volume":"77","author":"Costescu Strachinaru","year":"2021","journal-title":"Acta Clin. Belg."},{"key":"ref8","doi-asserted-by":"publisher","first-page":"121","DOI":"10.1007\/s15010-021-01649-y","article-title":"The proportion, species distribution and dynamic trends of bloodstream infection cases in a tertiary hospital in China, 2010-2019","volume":"50","author":"Cui","year":"2022","journal-title":"Infection"},{"key":"ref9","first-page":"41","volume-title":"Cell-free DNA testing. Genomic medicine","author":"Decker","year":"2019"},{"key":"ref10","doi-asserted-by":"publisher","first-page":"650","DOI":"10.1038\/nrneph.2009.153","article-title":"Kidney disease: improving global outcomes","volume":"5","author":"Eckardt","year":"2009","journal-title":"Nat. Rev. Nephrol."},{"key":"ref11","doi-asserted-by":"publisher","first-page":"1285","DOI":"10.1093\/cid\/ciz406","article-title":"Clinical impact of rapid species identification from positive blood cultures with same-day phenotypic antimicrobial susceptibility testing on the management and outcome of bloodstream infections","volume":"70","author":"Ehren","year":"2019","journal-title":"Clin. Infect. Dis."},{"key":"ref12","doi-asserted-by":"publisher","first-page":"e01234","DOI":"10.1128\/JCM.01234-18","article-title":"Toward the development of a circulating free DNA-based in vitro diagnostic test for infectious diseases: a review of evidence for tuberculosis","volume":"57","author":"Fern\u00e1ndez-Carballo","year":"2019","journal-title":"J. Clin. Microbiol."},{"key":"ref13","doi-asserted-by":"publisher","first-page":"552","DOI":"10.1001\/jamaoncol.2019.4120","article-title":"Evaluation of plasma microbial cell-free DNA sequencing to predict bloodstream infection in pediatric patients with relapsed or refractory cancer","volume":"6","author":"Goggin","year":"2020","journal-title":"JAMA Oncol."},{"key":"ref14","doi-asserted-by":"publisher","first-page":"1277","DOI":"10.1097\/01.TP.0000156165.83160.09","article-title":"Polyomavirus-associated nephropathy in renal transplantation: interdisciplinary analyses and recommendations","volume":"79","author":"Hirsch","year":"2005","journal-title":"Transplantation"},{"key":"ref15","doi-asserted-by":"publisher","first-page":"210","DOI":"10.1016\/j.diagmicrobio.2018.06.009","article-title":"Liquid biopsy for infectious diseases: sequencing of cell-free plasma to detect pathogen DNA in patients with invasive fungal disease","volume":"92","author":"Hong","year":"2018","journal-title":"Diagn. Microbiol. Infect. Dis."},{"key":"ref16","doi-asserted-by":"publisher","first-page":"641202","DOI":"10.3389\/fmicb.2021.641202","article-title":"A comparison of blood pathogen detection among droplet digital PCR, metagenomic next-generation sequencing, and blood culture in critically ill patients with suspected bloodstream infections","volume":"12","author":"Hu","year":"2021","journal-title":"Front. Microbiol."},{"key":"ref17","doi-asserted-by":"publisher","first-page":"1236","DOI":"10.1097\/INF.0000000000002492","article-title":"Early-onset sepsis among preterm neonates in China, 2015 to 2018","volume":"38","author":"Jiang","year":"2019","journal-title":"Pediatr. Infect. Dis. J."},{"key":"ref18","doi-asserted-by":"publisher","first-page":"151","DOI":"10.1016\/j.cmi.2019.10.031","article-title":"Burden of bacterial bloodstream infection\u2014a brief update on epidemiology and significance of multidrug-resistant pathogens","volume":"26","author":"Kern","year":"2020","journal-title":"Clin. Microbiol. Infect."},{"key":"ref19","doi-asserted-by":"publisher","first-page":"69","DOI":"10.1016\/j.cmi.2020.03.038","article-title":"Enhanced antimicrobial stewardship based on rapid phenotypic antimicrobial susceptibility testing for bacteraemia in patients with haematological malignancies: a randomized controlled trial","volume":"27","author":"Kim","year":"2021","journal-title":"Clin. Microbiol. Infect."},{"key":"ref20","doi-asserted-by":"publisher","first-page":"4182","DOI":"10.1002\/jmv.26846","article-title":"Droplet digital PCR of viral DNA\/RNA, current progress, challenges, and future perspectives","volume":"93","author":"Kojabad","year":"2021","journal-title":"J. Med. Virol."},{"key":"ref21","doi-asserted-by":"publisher","first-page":"1057","DOI":"10.1080\/15384047.2019.1598759","article-title":"Life and death of circulating cell-free DNA","volume":"20","author":"Kustanovich","year":"2019","journal-title":"Cancer Biol. Ther."},{"key":"ref22","doi-asserted-by":"publisher","first-page":"e00419","DOI":"10.1128\/JCM.00419-20","article-title":"Assessment of the clinical utility of plasma metagenomic next-generation sequencing in a pediatric hospital population","volume":"58","author":"Lee","year":"2020","journal-title":"J. Clin. Microbiol."},{"key":"ref23","doi-asserted-by":"publisher","first-page":"45","DOI":"10.7499\/j.issn.1008-8830.2019.01.009","article-title":"Clinical value of droplet digital PCR in rapid diagnosis of invasive fungal infection in neonates","volume":"21","author":"Li","year":"2019","journal-title":"Chin J Contemp Pediatr"},{"key":"ref24","doi-asserted-by":"publisher","first-page":"2056","DOI":"10.7150\/thno.28119","article-title":"Non-invasive diagnosis of early-stage lung cancer using high-throughput targeted DNA methylation sequencing of circulating tumor DNA (ctDNA)","volume":"9","author":"Liang","year":"2019","journal-title":"Theranostics"},{"key":"ref25","doi-asserted-by":"publisher","first-page":"70","DOI":"10.4103\/2319-4170.132905","article-title":"Epstein-Barr virus reactivation in critically ill immunocompetent patients","volume":"38","author":"Libert","year":"2015","journal-title":"Biom. J."},{"key":"ref26","doi-asserted-by":"publisher","first-page":"e0137822","DOI":"10.1128\/spectrum.01378-22","article-title":"Clinical diagnostic performance of droplet digital PCR for suspected bloodstream infections","volume":"11","author":"Lin","year":"2023","journal-title":"Microbiol Spectr."},{"key":"ref27","doi-asserted-by":"publisher","first-page":"127088","DOI":"10.1016\/j.snb.2019.127088","article-title":"Self-circulating electrochemiluminescence chip for sensitive detection of circulating tumour nucleic acids in blood","volume":"301","author":"Liu","year":"2019","journal-title":"Sensors Actuators B Chem."},{"key":"ref28","doi-asserted-by":"publisher","first-page":"121","DOI":"10.1186\/s13756-021-00989-6","article-title":"Pathogens distribution and antimicrobial resistance in bloodstream infections in twenty-five neonatal intensive care units in China, 2017\u20132019","volume":"10","author":"Liu","year":"2021","journal-title":"Antimicrob. Resist. Infect. Control"},{"key":"ref29","doi-asserted-by":"publisher","first-page":"365","DOI":"10.1016\/j.arcmed.2016.08.004","article-title":"Diagnosis of sepsis with cell-free DNA by next-generation sequencing technology in ICU patients","volume":"47","author":"Long","year":"2016","journal-title":"Arch. Med. Res."},{"key":"ref30","doi-asserted-by":"publisher","first-page":"219","DOI":"10.5588\/ijtld.18.0182","article-title":"Rapid direct drug susceptibility testing of Mycobacterium tuberculosis based on culture droplet digital polymerase chain reaction","volume":"23","author":"Luo","year":"2019","journal-title":"Int. J. Tuberc. Lung Dis."},{"key":"ref31","doi-asserted-by":"publisher","first-page":"28","DOI":"10.1186\/s40635-019-0256-z","article-title":"Early herpes and TTV DNAemia in septic shock patients: a pilot study","volume":"7","author":"Mallet","year":"2019","journal-title":"Intensive Care Med. Exp."},{"key":"ref32","doi-asserted-by":"publisher","first-page":"284","DOI":"10.3390\/antibiotics11020284","article-title":"Early and late onset neonatal sepsis: epidemiology and effectiveness of empirical antibacterial therapy in a III level neonatal intensive care unit","volume":"11","author":"Mariani","year":"2022","journal-title":"Antibiotics"},{"key":"ref33","doi-asserted-by":"publisher","first-page":"63","DOI":"10.1186\/s13054-022-03948-8","article-title":"Digital PCR applications for the diagnosis and management of infection in critical care medicine","volume":"26","author":"Merino","year":"2022","journal-title":"Crit. Care"},{"key":"ref34","doi-asserted-by":"publisher","first-page":"845","DOI":"10.7326\/M18-2772","article-title":"Performance of the T2Bacteria panel for diagnosing bloodstream infections","volume":"170","author":"Nguyen","year":"2019","journal-title":"Ann. Intern. Med."},{"key":"ref35","doi-asserted-by":"publisher","first-page":"1204","DOI":"10.1093\/cid\/cix120","article-title":"Epidemiology of multiple herpes viremia in previously immunocompetent patients with septic shock","volume":"64","author":"Ong","year":"2017","journal-title":"Clin. Infect. Dis."},{"key":"ref36","doi-asserted-by":"publisher","first-page":"944","DOI":"10.1016\/j.cmi.2018.05.007","article-title":"Diagnosis of bloodstream infections from positive blood cultures and directly from blood samples: recent developments in molecular approaches","volume":"24","author":"Peker","year":"2018","journal-title":"Clin. Microbiol. Infect."},{"key":"ref37","doi-asserted-by":"publisher","first-page":"e00089","DOI":"10.1128\/CMR.00089-17","article-title":"Emerging technologies for molecular diagnosis of sepsis","volume":"31","author":"Sinha","year":"2018","journal-title":"Clin. Microbiol. Rev."},{"key":"ref38","doi-asserted-by":"crossref","DOI":"10.5772\/intechopen.80730","article-title":"Circulating cell-free DNA","volume-title":"Liquid Biopsy","author":"Sun","year":"2019"},{"key":"ref39","doi-asserted-by":"publisher","first-page":"659390","DOI":"10.3389\/fmolb.2021.659390","article-title":"Plasma microbial cell-free DNA sequencing technology for the diagnosis of sepsis in the ICU","volume":"8","author":"Wang","year":"2021","journal-title":"Front. Mol. Biosci."},{"key":"ref40","doi-asserted-by":"publisher","first-page":"378","DOI":"10.1016\/j.pt.2016.01.006","article-title":"Cell-free DNA as a diagnostic tool for human parasitic infections","volume":"32","author":"Weerakoon","year":"2016","journal-title":"Trends Parasitol."},{"key":"ref41","doi-asserted-by":"publisher","first-page":"4544","DOI":"10.7150\/thno.40532","article-title":"Tumor circulome in the liquid biopsies for cancer diagnosis and prognosis","volume":"10","author":"Wu","year":"2020","journal-title":"Theranostics"},{"key":"ref42","doi-asserted-by":"publisher","first-page":"243","DOI":"10.1186\/s13054-022-04116-8","article-title":"Clinical validation of a multiplex droplet digital PCR for diagnosing suspected bloodstream infections in ICU practice: a promising diagnostic tool","volume":"26","author":"Wu","year":"2022","journal-title":"Crit. Care"},{"key":"ref43","doi-asserted-by":"publisher","first-page":"13","DOI":"10.3389\/fcimb.2023.1228401","article-title":"Clinical characteristics of bloodstream infections in adult patients with solid tumours and a nomogram for mortality prediction: a 5-year case-controlled retrospective study in a tertiary-level hospital","volume":"13","author":"Xue","year":"2023","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"ref44","doi-asserted-by":"publisher","first-page":"665226","DOI":"10.3389\/fcimb.2021.665226","article-title":"Metagenomic next-generation sequencing of bloodstream microbial cell-free nucleic acid in children with suspected sepsis in pediatric intensive care unit","volume":"11","author":"Yan","year":"2021","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"ref45","doi-asserted-by":"publisher","first-page":"R159","DOI":"10.1186\/cc9234","article-title":"Evaluation of pathogen detection from clinical samples by real-time polymerase chain reaction using a sepsis pathogen DNA detection kit","volume":"14","author":"Yanagihara","year":"2010","journal-title":"Crit. Care"},{"key":"ref46","doi-asserted-by":"publisher","first-page":"56","DOI":"10.3389\/fcimb.2019.00056","article-title":"Evaluation of the Magicplex\u2122 sepsis real-time test for the rapid diagnosis of bloodstream infections in adults","volume":"9","author":"Zboromyrska","year":"2019","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"ref47","doi-asserted-by":"publisher","first-page":"11","DOI":"10.3389\/fcimb.2021.694093","article-title":"Emerging threat of multidrug resistant pathogens from neonatal sepsis","volume":"11","author":"Zou","year":"2021","journal-title":"Front. Cell. Infect. Microbiol."}],"container-title":["Frontiers in Microbiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2025.1578820\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,4,29]],"date-time":"2025-04-29T05:26:18Z","timestamp":1745904378000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2025.1578820\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,4,29]]},"references-count":47,"alternative-id":["10.3389\/fmicb.2025.1578820"],"URL":"https:\/\/doi.org\/10.3389\/fmicb.2025.1578820","relation":{},"ISSN":["1664-302X"],"issn-type":[{"value":"1664-302X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,4,29]]},"article-number":"1578820"}}