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Background <p>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.</p> Methods <p>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.</p> Results <p>Nucleosome depletion markedly reduced host DNA background by an average of 66-fold, consequently enriching microbial reads by approximately 46.73-fold. The automated HD-mNGS assay exhibited robust analytical sensitivity, with limits of detection (LoD) ranging from 9.1 to 38 genome equivalents (GE) /mL for bacteria and fungi, and from 283 to 321 GE/mL for viruses and excellent linearity across tested concentrations (R<sup>2</sup> = 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 (<i>P &lt; 0.001</i>). Crucially, HD-mNGS demonstrates enhanced performance in detecting rare, fastidious, and intracellular pathogens (such as <i>Mycobacterium tuberculosis</i> and <i>Rickettsia</i>) 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%).</p> Conclusions <p>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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年7月9日

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  • Shaohua Zhan,
  • Yafeng Zheng,
  • Tongyao Wu,
  • Xin Hou,
  • Jing Li,
  • Sisi Ma,
  • Wei Gai,
  • Ning Shen,
  • Jiajia Zheng

摘要

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

Nucleosome depletion markedly reduced host DNA background by an average of 66-fold, consequently enriching microbial reads by approximately 46.73-fold. The automated HD-mNGS assay exhibited robust analytical sensitivity, with limits of detection (LoD) ranging from 9.1 to 38 genome equivalents (GE) /mL for bacteria and fungi, and from 283 to 321 GE/mL for viruses and excellent linearity across tested concentrations (R2 = 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.