Evaluation of clinical usefulness of a commercial multiplex PCR for diagnosing mycobacterial infections: a multicenter study

Rapid and accurate differentiation between Mycobacterium tuberculosis complex (MTBC) and clinically relevant nontuberculous mycobacteria (NTM) is essential for appropriate patient management but remains challenging with conventional diagnostic methods. We evaluated the clinical performance of a commercial multiplex real-time PCR assay for the direct detection of MTBC, Mycobacterium avium complex (MAC), and Mycobacterium abscessus complex (MABSC) in respiratory specimens. In this prospective multicenter study, 845 respiratory samples from patients with suspected mycobacterial infection were collected across 15 microbiology laboratories in the Madrid region (Spain). PCR results were compared with mycobacterial culture as the reference standard and with acid-fast bacilli (AFB) staining. Overall, 301 samples (35.6%) were culture-positive for mycobacteria. The multiplex PCR detected mycobacteria in 245 specimens, identifying MTBC ( n = 147), MAC ( n = 77), and MABSC ( n = 21). For MTBC, sensitivity and specificity were 90.3% and 98.5%, respectively, increasing to 96.3% sensitivity in AFB smear-positive specimens. Sensitivity for MAC was 61.0% overall and 76.9% in AFB-positive samples, while MABSC sensitivity was 72.0%; specificity exceeded 99% for all targets. Culture identified additional NTM species not included in the PCR panel, underscoring the continued importance of culture for comprehensive diagnosis. In conclusion, this multiplex real-time PCR assay provides rapid and highly specific detection of MTBC and major NTM complexes directly from respiratory samples, with high sensitivity in smear-positive specimens. Its use may facilitate earlier differentiation between tuberculosis and NTM disease, supporting timely and appropriate clinical management. IMPORTANCE This prospective multicenter study evaluates a commercial multiplex real-time PCR assay for the direct detection of Mycobacterium tuberculosis complex and important nontuberculous mycobacteria in respiratory samples. The inclusion of a large number of specimens ( n = 845) collected across multiple hospitals reflects real-world diagnostic practice and strengthens the clinical relevance of the findings, which demonstrate high specificity and excellent sensitivity for tuberculosis, particularly in smear-positive specimens, supporting the value of rapid molecular diagnostics for timely patient management.