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Background <p>Invasive pulmonary fungal infections (IPFIs) pose significant diagnostic challenges, particularly in immunocompromised patients. Accurate and timely diagnosis is crucial to improve outcomes. While metagenomic next-generation sequencing (mNGS) is widely utilized, it is expensive and affected by host DNA interference. Targeted next-generation sequencing (tNGS) offers a cost-effective and efficient alternative for fungal pathogen detection.</p> Methods <p>We developed the Fi-tNGS assay, a targeted next-generation sequencing method, specifically designed to detect 64 fungal species. Analytical performance was validated by assessing its limit of detection (LoD), reproducibility, and resistance to host DNA interference. Subsequently, a prospective clinical study was conducted, enrolling 104 patients with suspected IPFIs. Clinical diagnostic performance was evaluated by comparing Fi-tNGS, mNGS, and conventional microbial culture against a comprehensive diagnostic standard.</p> Results <p>Fi-tNGS detected 109 pathogens, compared to 110 for mNGS and 77 for culture. The sensitivity and specificity of tNGS were 89.7% and 94.2%, respectively, outperforming culture (65.8% sensitivity, 100% specificity). Combining culture with tNGS or mNGS significantly improved sensitivity to 94.8% and 94.0%, respectively. These findings demonstrate the added diagnostic value of NGS methods for IPFIs.</p> Conclusions <p>tNGS provides accurate and efficient fungal pathogen detection, with sensitivity comparable to mNGS and superior to culture. Its cost-effectiveness and shorter turnaround time highlight its potential as a practical tool for the rapid and precise diagnosis of IPFIs in clinical settings.</p>

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An innovative fungal-specific targeted next-generation sequencing method: analytical performance and a single-center prospective clinical study

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年7月24日

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  • Ruixuan Wang,
  • Shitong Cheng,
  • Wei Du,
  • Fengshuo Sun,
  • Yang Gao,
  • Zheming Zhu,
  • Chuan He,
  • Jingjing Chen,
  • Sufei Tian,
  • Yunzhuo Chu,
  • Gang Zhou,
  • Hui Miao,
  • Lifeng Li,
  • Xiaolong Dong,
  • Xiaoxu Han

摘要

Background

Invasive pulmonary fungal infections (IPFIs) pose significant diagnostic challenges, particularly in immunocompromised patients. Accurate and timely diagnosis is crucial to improve outcomes. While metagenomic next-generation sequencing (mNGS) is widely utilized, it is expensive and affected by host DNA interference. Targeted next-generation sequencing (tNGS) offers a cost-effective and efficient alternative for fungal pathogen detection.

Methods

We developed the Fi-tNGS assay, a targeted next-generation sequencing method, specifically designed to detect 64 fungal species. Analytical performance was validated by assessing its limit of detection (LoD), reproducibility, and resistance to host DNA interference. Subsequently, a prospective clinical study was conducted, enrolling 104 patients with suspected IPFIs. Clinical diagnostic performance was evaluated by comparing Fi-tNGS, mNGS, and conventional microbial culture against a comprehensive diagnostic standard.

Results

Fi-tNGS detected 109 pathogens, compared to 110 for mNGS and 77 for culture. The sensitivity and specificity of tNGS were 89.7% and 94.2%, respectively, outperforming culture (65.8% sensitivity, 100% specificity). Combining culture with tNGS or mNGS significantly improved sensitivity to 94.8% and 94.0%, respectively. These findings demonstrate the added diagnostic value of NGS methods for IPFIs.

Conclusions

tNGS provides accurate and efficient fungal pathogen detection, with sensitivity comparable to mNGS and superior to culture. Its cost-effectiveness and shorter turnaround time highlight its potential as a practical tool for the rapid and precise diagnosis of IPFIs in clinical settings.