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

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 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.