Comparison of Xpert MTB/RIF Assay and the Targeted Next-Generation Sequencing for the Diagnosis of Pleural Tuberculosis: A Prospective Comparative Diagnostic Accuracy Study

Peng Wen,1,* Shang Xue Wang,1,* Xiao Man Ma,1 Hong Yan Chen,1 Zeng Xia Ma,1 Yun Zeng Zhang,2 Min Wei1 1Department of Respiratory Medicine, Public Health Clinical Center Affiliated to Shandong University, Jinan, People’s Republic of China; 2Department of Thoracic Surgery, Public Health Clinical Center Affiliated to Shandong University, Jinan, People’s Republic of China*These authors contributed equally to this workCorrespondence: Min Wei, Department of Respiratory Medicine, Public Health Clinical Center Affiliated to Shandong University, 2999# Gangxing West Road, Jinan, 250100, People’s Republic of China, Email 13793131591@163.com Yun Zeng Zhang, Department of Thoracic Surgery, Public Health Clinical Center Affiliated to Shandong University, 2999# Gangxing West Road, Jinan, 250100, People’s Republic of China, Email Zhangyz1981@163.comPurpose: To evaluate the diagnostic performance of targeted next-generation sequencing (tNGS), a multiplex PCR–based and hybrid-capture sequencing platform targeting 198 respiratory pathogens, versus Xpert MTB/RIF in diagnosing pleural tuberculosis (TB) across diverse specimen types, given the limited sensitivity of existing assays in paucibacillary disease.Patients and Methods: Consecutive patients with suspected pleural TB were prospectively enrolled. Final diagnosis was established using a composite reference standard incorporating microbiological, histopathological, and clinical criteria. Paired sputum, pleural fluid, and pleural biopsy specimens underwent tNGS, Xpert MTB/RIF, and mycobacterial culture. Diagnostic performance metrics, including sensitivity and specificity, were calculated. A predefined paired comparison (n=105) between tNGS and Xpert MTB/RIF was performed, with agreement assessed using Cohen’s kappa.Results: In the overall cohort (N = 142), tNGS demonstrated higher sensitivity than Xpert MTB/RIF and mycobacterial culture across specimen types when evaluated against the composite reference standard, while maintaining 100% specificity in the non-TB group (n = 8). In sputum samples, tNGS achieved a sensitivity of 72.73%, significantly outperforming Xpert MTB/RIF (21.43%) and MTB culture (16.30%). In pleural fluid, tNGS sensitivity (43.64%) exceeded that of Xpert MTB/RIF (30.49%) and MTB culture (24.14%), comparable to the performance of ADA ≥ 40 U/L (46.22%). The paired analysis confirmed that tNGS significantly outperformed Xpert MTB/RIF in overall diagnostic yield (p < 0.01), detecting 26 positive cases missed by Xpert. Among patients with culture-negative pleural tuberculosis, tNGS detected 24/60 (40.0%) cases compared with 6/60 (10.0%) by Xpert MTB/RIF.Conclusion: tNGS provides incremental diagnostic value over conventional molecular and culture-based assays in pleural tuberculosis, particularly in paucibacillary and culture-negative disease. It increases microbiological confirmation in culture- and Xpert-negative cases and is best positioned as a complementary second-line assay within a tiered diagnostic framework, despite no assessment of downstream clinical impact.Keywords: targeted next-generation sequencing, pleural tuberculosis, Xpert MTB/RIF, diagnosis