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Background <p>Tuberculous pleurisy (TP), a predominant form of extrapulmonary tuberculosis, presents significant diagnostic challenges attributable to the paucibacillary nature of pleural effusion (PE) specimens. Cell-free <i>Mycobacterium tuberculosis</i> (MTB) DNA in PE represents a promising biomarker for TP diagnosis. This study aimed to develop and assess a novel cell-free DNA (cfDNA)-CRISPR assay targeting MTB DNA in PE supernatants.</p> Methods <p>Patients with suspected TP were prospectively enrolled at Beijing Chest Hospital. PE samples underwent centrifugation, with sediments tested by MTB/RIF Xpert (Xpert) testing and mycobacterial culture, while supernatants were analyzed using the cfDNA-CRISPR assay. Diagnostic performance was evaluated using a composite reference standard (CRS).</p> Results <p>Of 276 participants, 237 (85.9%) were included in the final analysis. Based on the CRS, cases were stratified as follows: 63 definite TP, 70probable TP, and 104 non-TP controls. The cfDNA-CRISPR assay in definite TP demonstrated superior sensitivity (81.0%) compared to mycobacterial culture (33.3%, <i>P</i> &lt; 0.001) and Xpert (42.9%, <i>P</i> &lt; 0.001). In probable TP, where both Culture and Xpert were negative, cfDNA-CRISPR maintained high sensitivity (80.0%), exceeding that of ADA testing (64.3%, <i>P</i> &lt; 0.05). Overall sensitivity of cfDNA-CRISPR for TP was 80.5%, markedly higher than Culture (15.8%) and Xpert (20.3%) (both <i>P</i> &lt; 0.001). The cfDNA-CRISPR assay exhibited a specificity of 94.2%, while both Culture and Xpert achieved 100% specificity.</p> Conclusions <p>The cfDNA-CRISPR assay based on the CRISPR-Cas13a system offers significantly improved sensitivity over conventional methods for detecting MTB in PE. It represents a promising, non-invasive diagnostic tool for enhancing TP detection in clinical practice.</p>

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An ultra-sensitive cell-free DNA-based diagnostic assay for Tuberculous pleurisy utilizing the CRISPR-Cas13a system

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年3月25日

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  • Weicong Ren,
  • Mengjie Yang,
  • You Zhou,
  • Yinghui Yang,
  • Haoran Li,
  • Yanqin Chen,
  • Shanshan Li,
  • Yu Pang

摘要

Background

Tuberculous pleurisy (TP), a predominant form of extrapulmonary tuberculosis, presents significant diagnostic challenges attributable to the paucibacillary nature of pleural effusion (PE) specimens. Cell-free Mycobacterium tuberculosis (MTB) DNA in PE represents a promising biomarker for TP diagnosis. This study aimed to develop and assess a novel cell-free DNA (cfDNA)-CRISPR assay targeting MTB DNA in PE supernatants.

Methods

Patients with suspected TP were prospectively enrolled at Beijing Chest Hospital. PE samples underwent centrifugation, with sediments tested by MTB/RIF Xpert (Xpert) testing and mycobacterial culture, while supernatants were analyzed using the cfDNA-CRISPR assay. Diagnostic performance was evaluated using a composite reference standard (CRS).

Results

Of 276 participants, 237 (85.9%) were included in the final analysis. Based on the CRS, cases were stratified as follows: 63 definite TP, 70probable TP, and 104 non-TP controls. The cfDNA-CRISPR assay in definite TP demonstrated superior sensitivity (81.0%) compared to mycobacterial culture (33.3%, P < 0.001) and Xpert (42.9%, P < 0.001). In probable TP, where both Culture and Xpert were negative, cfDNA-CRISPR maintained high sensitivity (80.0%), exceeding that of ADA testing (64.3%, P < 0.05). Overall sensitivity of cfDNA-CRISPR for TP was 80.5%, markedly higher than Culture (15.8%) and Xpert (20.3%) (both P < 0.001). The cfDNA-CRISPR assay exhibited a specificity of 94.2%, while both Culture and Xpert achieved 100% specificity.

Conclusions

The cfDNA-CRISPR assay based on the CRISPR-Cas13a system offers significantly improved sensitivity over conventional methods for detecting MTB in PE. It represents a promising, non-invasive diagnostic tool for enhancing TP detection in clinical practice.