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Background <p>The limited genomic targeting range of current targeted next-generation sequencing (tNGS) workflows results in limited detection of pathogen-derived cell-free DNA (cfDNA), making it challenging to apply this approach to bloodstream infections (BSIs). Here, we developed an ultra-broad hybrid capture-based tNGS method to detect plasma pathogen-derived cfDNA and evaluate its suitability for the diagnosis of BSI.</p> Methods <p>This study introduced an ultra-broad hybrid capture-based tNGS method featuring an ultra-broad pathogen panel (1872 pathogens) and high-density probe coverage. To adequately evaluate its performance, we conducted retrospective tests in 208 suspected BSI patients (139 immunocompromised), comparing tNGS results with mNGS, conventional microbiological testing (CMT), and comprehensive clinical diagnoses.</p> Results <p>In pathogen detection, the concordance between ultra-broad hybrid capture-based tNGS and mNGS results was 93.75%. The diagnostic accuracy of tNGS in BSI was comparable to mNGS (76.44% vs. 75.00%) and significantly higher than CMT (45.67%, <i>p</i> &lt; 0.0001). In immunocompromised populations, the diagnostic accuracy of tNGS was similar to mNGS (77.70% vs. 76.98%). tNGS detected 92.09% (163/177) of pathogens identified by mNGS. Two of the missed pathogens were not included in the 1872 pathogens panel, and both were from the immunocompromised group.</p> Conclusions <p>Ultra-broad hybrid capture-based tNGS exhibits sensitivity and accuracy comparable to mNGS, effectively covering a relatively wide range of pathogens, and may serve as an economic screening tool for BSI in the future.</p>

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Ultra-broad hybrid capture-based targeted next-generation sequencing for sensitive plasma pathogen cfDNA detection in bloodstream infections

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年10月31日

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  • Muyun Wei,
  • Xiangzhao Ai,
  • Dejian Gu,
  • Shengyang Zhang,
  • Ke Xu,
  • Shuangshuang Li,
  • Shaowei Mao,
  • Min Li

摘要

Background

The limited genomic targeting range of current targeted next-generation sequencing (tNGS) workflows results in limited detection of pathogen-derived cell-free DNA (cfDNA), making it challenging to apply this approach to bloodstream infections (BSIs). Here, we developed an ultra-broad hybrid capture-based tNGS method to detect plasma pathogen-derived cfDNA and evaluate its suitability for the diagnosis of BSI.

Methods

This study introduced an ultra-broad hybrid capture-based tNGS method featuring an ultra-broad pathogen panel (1872 pathogens) and high-density probe coverage. To adequately evaluate its performance, we conducted retrospective tests in 208 suspected BSI patients (139 immunocompromised), comparing tNGS results with mNGS, conventional microbiological testing (CMT), and comprehensive clinical diagnoses.

Results

In pathogen detection, the concordance between ultra-broad hybrid capture-based tNGS and mNGS results was 93.75%. The diagnostic accuracy of tNGS in BSI was comparable to mNGS (76.44% vs. 75.00%) and significantly higher than CMT (45.67%, p < 0.0001). In immunocompromised populations, the diagnostic accuracy of tNGS was similar to mNGS (77.70% vs. 76.98%). tNGS detected 92.09% (163/177) of pathogens identified by mNGS. Two of the missed pathogens were not included in the 1872 pathogens panel, and both were from the immunocompromised group.

Conclusions

Ultra-broad hybrid capture-based tNGS exhibits sensitivity and accuracy comparable to mNGS, effectively covering a relatively wide range of pathogens, and may serve as an economic screening tool for BSI in the future.