Profiling of human and microbial cell-free DNA reflects early host–pathogen interactions in sepsis

Objectives: In sepsis, circulating cell-free DNA (cfDNA) originates from host cells (damage-associated molecular patterns, DAMPs) and pathogens (pathogen-associated molecular patterns, PAMPs), contributing to immune activation and offering potential as both a biomarker and a therapeutic target. While DAMPs are thought to predominate in early sepsis, this study aimed to quantify their relative abundance and assess their correlation with inflammatory markers compared to PAMPs. Methods: In this prospective observational study, blood samples of 18 ICU patients were collected within 24 hours of sepsis diagnosis. Plasma cfDNA was analyzed via qPCR (targeting human LINE-1) and iSEP-SEQ nanopore sequencing. Human and microbial cfDNA were quantified, and method correlation was assessed using Kendall's tau-b correlation. Associations with inflammatory biomarkers were tested using Spearman correlation analysis and group comparisons between human and non-human reads were analyzed with Pearson correlation analysis. The study received ethical approval from the Landesärztekammer Rheinland-Pfalz (Approval Number: 2020-15535). Results: cfDNA was predominantly of human origin, comprising 99.86% of classified reads, with microbial cfDNA accounting for only 0.077% (p < 0.001). qPCR-based cfDNA concentrations strongly correlated with human read counts from sequencing (τ = 0.712; p < 0.001). Human cfDNA levels were significantly associated with LDH, WBC, and CRP. Microbial cfDNA, although low in abundance, correlated with WBC, CRP, and D-dimer. Conclusions: In early sepsis, human cfDNA is markedly more abundant than microbial cfDNA. However, both exhibit strong correlations with inflammatory and tissue injury markers. These findings support a model of PAMP-triggered and DAMP-driven inflammation and identify human cfDNA as a promising biomarker and potential therapeutic target. Clinical Trial Registration: https://drks.de/search/de/trial/DRKS00025222/details, identifier DRKS-ID: 00025222.