Enrichment of Microbial DNA in Plasma to Improve Pathogen Detection in Sepsis: A Pilot Study.
Background Diagnosis of sepsis and timely identification of pathogens in critically ill patients remains challenging. Plasma metagenomic sequencing to detect microbial cell-free DNA (mDNA) has shown promise, but low abundance of mDNA in plasma limits sensitivity and necessitates high sequencing dept...
| Publicado en: | Clinical Chemistry Vol. 71; no. 5; pp. 567 - 577 |
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| Autores principales: | , , , , , , , , , , |
| Formato: | Journal Article |
| Publicado: |
Oxford University Press / USA
May2025
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| Acceso en línea: | Ver este registro en EBSCOhost |
| fields | @attributes: recordID: 1 pdfLink: plink: https://search.ebscohost.com/login.aspx?direct=true&db=ccm&AN=185453583&site=ehost-live header: @attributes: shortDbName: ccm uiTerm: 185453583 longDbName: CINAHL Complete uiTag: AN controlInfo: bkinfo: dissinfo: jinfo: jid: 00099147 10CS jtl: Clinical Chemistry issn: 00099147 maglogo: N pubinfo: dt: May2025 vid: 71 iid: 5 pid: 622 pub: Oxford University Press / USA artinfo: ui: 185453583 10.1093/clinchem/hvaf011 185453583 ppf: 567 ppct: 10 formats: fmt: – @attributes: type: T – @attributes: type: P tig: atl: Enrichment of Microbial DNA in Plasma to Improve Pathogen Detection in Sepsis: A Pilot Study. aug: au: Dominguez, Eddie G McDonald, Bradon R Zhang, Haikun Stephens, Michelle D Dietmann, Elise C Nedden, Megan Byington, Nicole Thompson, Sydney Junak, Mary Pepperell, Caitlin S Kisat, Mehreen T affil: Department of Surgery, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI, United States sug: ab: Background Diagnosis of sepsis and timely identification of pathogens in critically ill patients remains challenging. Plasma metagenomic sequencing to detect microbial cell-free DNA (mDNA) has shown promise, but low abundance of mDNA in plasma limits sensitivity and necessitates high sequencing depth. mDNA is shorter and more fragmented than human cell-free DNA. Here, we evaluated whether combining single-stranded DNA (ssDNA) sequencing library preparation and size selection can enrich mDNA and improve pathogen detection. Methods We prospectively enrolled 48 trauma patients and collected daily blood samples during the first 10 days of intensive care unit (ICU) admission. For patients with culture-proven infections, we extracted plasma DNA, prepared double-stranded DNA (dsDNA) and ssDNA sequencing libraries, and applied size selection to exclude fragments >110 bp. Following sequencing, we performed taxonomic classification, and evaluated differences in mDNA fractions and in sensitivity for pathogen detection (compared to background noise). Results We analyzed 46 plasma samples from 5 patients who developed culture-proven infections, including 17 samples coincident with positive microbial cultures. Size-selected ssDNA libraries showed the total mDNA fraction 204-fold higher on average than conventional dsDNA libraries (P < 0.0001). However, for pathogen-specific DNA (at the genus level), the highest sensitivity was observed in size-selected dsDNA (82%), compared to dsDNA (41%), ssDNA (71%), and size-selected ssDNA (35%) library preparations. Conclusions Our results demonstrate that combining ssDNA library preparation together with fragment size selection improves mDNA yield, potentially reducing sequencing requirements. However, at the genus level, this combination also increases background noise, which limits sensitivity for pathogen detection. pubtype: Academic Journal doctype: Journal Article ougenre: Article language: English refInfo: holdings: @attributes: islocal: N |
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