Annals of clinical microbiology and antimicrobials · 2026
An ultra-sensitive cell-free DNA-based diagnostic assay for Tuberculous pleurisy utilizing the CRISPR-Cas13a system.
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Ren Weicong, Yang Mengjie, Zhou You, Yang Yinghui, Li Haoran, Chen Yanqin, Li Shanshan, Pang Yu
Аннотация
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.
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.