Nucleosome-targeted host DNA depletion enables automated plasma metagenomic sequencing for sensitive detection of bloodstream pathogens

BACKGROUND: Bloodstream infections (BSIs) are leading causes of sepsis-related mortality. Although metagenomic next-generation sequencing (mNGS) enables culture-independent pathogen detection, its clinical utility in plasma is limited by the overwhelming abundance of host cell-free DNA (cfDNA) and labor-intensive manual workflows. METHODS: A plasma host DNA depletion mNGS (HD-mNGS) assay was developed which integrated nucleosome-targeted host DNA depletion with automated DNA extraction and library preparation. Analytical performance was evaluated through limit of detection, linearity, precision, and contamination control. Clinical performance was assessed in a cohort of 107 patients with suspected BSI and benchmarked against blood culture (BC), conventional microbiological testing (CMT), and standard mNGS without host depletion, using a composite clinical reference standard. RESULTS: = 0.915-0.989). Furthermore, the automated workflow maintained strong quantitative correlation with manual protocols while significantly reducing common skin and environmental contaminants by 71.7% and 83.7%, respectively. In a cohort of 107 patients, HD-mNGS demonstrates improved diagnostic performance for BSI, achieving a significantly higher pathogen detection rate (64.49%) and clinical positive percent agreement (PPA: 95.24%) than standard mNGS, BC, and CMT (P < 0.001). Crucially, HD-mNGS demonstrates enhanced performance in detecting rare, fastidious, and intracellular pathogens (such as Mycobacterium tuberculosis and Rickettsia) that yield extremely low concentrations of circulating DNA, overcoming the limitations of traditional methods while maintaining high overall diagnostic total percent agreement (TPA: 88.79%). CONCLUSIONS: Nucleosome-targeted host DNA depletion integrated with a fully automated mNGS platform significantly enhances microbial detection in plasma and provides a scalable approach for standardized BSI diagnostics.