Evaluation of a 16S rRNA real-time PCR assay for detection of mycobacterium tuberculosis in comparison with GeneXpert MTB/RIF in a Vietnam hospital.

PURPOSE: Tuberculosis remains a major public health challenge in Vietnam, requiring timely and accurate diagnosis. This study aimed to evaluate the clinical performance of a simple real-time PCR assay targeting the 16S rRNA gene of Mycobacterium tuberculosis, with emphasis on its applicability in routine hospital conditions.

METHODS

A cross-sectional study was conducted at Military Hospital 103 (during 2022-2023) involving 60 newly diagnosed pulmonary TB patients (30 Acid-Fast Bacillus (AFB)-positive, 30 AFB-negative), 30 community-acquired pneumonia patients, and 30 Mycobacterium tuberculosis (MTB) plus 30 non-tuberculous mycobacteria (NTM) isolates as controls. Depending on the study group and sample availability, specimens were subjected to MGIT culture, AFB smear microscopy, GeneXpert MTB/RIF, and 16S rRNA real-time PCR according to the study protocol and routine clinical workflow. Diagnostic performance was assessed using MGIT culture as the reference standard.

RESULTS

The 16S rRNA PCR assay showed 100% sensitivity and specificity in control isolates. In clinical specimens, sensitivity and specificity were 100% and 100% in AFB-positive patients, 83.3% and 100% in AFB-negative patients, and 91.7% and 100% overall. GeneXpert MTB/RIF showed a sensitivity of 86.7% and specificity of 100% among tested samples. Notably, reduced GeneXpert sensitivity was observed in a subset of blood-contaminated specimens, whereas the 16S rRNA assay maintained consistent detection performance.

CONCLUSION

The 16S rRNA real-time PCR assay demonstrated high diagnostic accuracy across different clinical subgroups and specimen types under routine laboratory conditions. While not introducing a novel molecular target, this approach showed potential as a complementary diagnostic tool, particularly in settings where access to automated molecular platforms is limited or where specimen quality may affect test performance.