Clinical Evaluation of a Tandem qPCR-CRISPR Assay for Multidrug-Resistant or Rifampicin-Resistant Tuberculosis: A Prospective Diagnostic Accuracy Study

CRISPR-based assays have shown the potential to enhance the diagnosis of tuberculosis (TB), but evidence remains limited owing to scarce independent validation. We prospectively evaluated a tandem qPCR-CRISPR assay detecting Mycobacterium tuberculosis (Mtb) and rifampicin resistance. Respiratory specimens for smear microscopy, mycobacterial culture, Xpert MTB/RIF Ultra (Xpert) and the investigational assay were collected from suspected TB patients. Phenotypic drug susceptibility testing (pDST) was carried out on culture-positive cases. Diagnostic performance was evaluated against the microbiological reference standard (MRS) and composite reference standard (CRS). The predictive performance for drug resistance was assessed using pDST and Xpert as reference standards. A total of 179 adults were enrolled, including 84 confirmed TB, 18 clinical TB, and 77 non-TB cases. The tandem qPCR-CRISPR assay showed 84.5% (95% CI 75.0–91.5%) sensitivity and 95.8% (95% CI 89.6–98.8%) specificity against MRS, and 70.6% (95% CI 60.6–79.0%) sensitivity and 96.1% (95% CI 88.3–99.0%) specificity against CRS. For drug resistance, the assay achieved a sensitivity of 77.5% (95% CI 61.1–88.6%) and a specificity of 90.9% (95% CI 77.4–97.0%). Sensitivity was higher in sputum than in bronchoalveolar lavage fluid. The tandem qPCR-CRISPR assay showed suboptimal performance for detecting TB and MDR/RR-TB and requires further optimization before clinical application.