Diagnostic Value of Cross-priming Amplification Combined With CRISPR-Cas12b in Detecting Cell-free DNA in Tuberculous Pleural Effusion

Abstract

Background Diagnosis of tuberculous pleural effusion (TPE) remains challenging. Studies have shown that detecting cell-free Mycobacterium tuberculosis DNA (cf-TB) in pleural effusion can improve TPE diagnosis. This study aimed to evaluate the diagnostic value of our recently developed Cross-Priming Amplification (CPA) combined with CRISPR-Cas12b (TB One-Pot) assay in detecting cf-TB for TPE. Methods Pleural effusion samples were collected from suspected TPE inpatients at Hangzhou Red Cross Hospital. After centrifugation, the precipitate was used for culture, Xpert, and pleural effusion cytological testing, while the supernatant was used for biochemical and cf-TB assays (including TB One-Pot and the quantitative PCR method (cf-TB-PCR)). Diagnostic performance was assessed based on a comprehensive reference standard (CRS). Results A total of 115 patients were included, comprising 88 CRS-diagnosed TPE cases and 27 non-TPE cases. The sensitivity of TB One-Pot in detecting pleural cf-TB for diagnosing TPE was 64.8%, with an area under the curve (AUC) of 0.805, significantly superior to culture and Xpert (P <0.05). Compared with cf-TB-PCR (sensitivity: 53.4%, AUC: 0.767) and ADA (sensitivity: 52.3%, AUC: 0.761), TB One-Pot demonstrated slightly higher sensitivity and AUC, but the differences were not statistically significant (P >0.05). The specificity of TB One-Pot was 96.3%, while the specificity of other tests was 100%, with no statistically significant differences (P > 0.05). Conclusions cf-TB provides direct evidence of the etiology of TPE. TB One-Pot for detecting cf-TB in diagnosing TPE outperforms existing TB laboratory tests and may represent a more effective approach for TPE diagnosis in resource-limited settings.