Multiplex PCR targeting IS6110 and mpb64 Genes for Detecting Extra-Pulmonary Tuberculosis: A Cross-Sectional Study in a Tertiary Care Center, Kashmir, India

Extra-pulmonary tuberculosis (EPTB) is a significant cause of morbidity, and early diagnosis is critical for improving patient outcomes. Conventional diagnostic methods for EPTB often require improvement, highlighting the need for more rapid and sensitive diagnostic procedures. In this cross-sectional study, we aimed to evaluate the diagnostic usefulness of multiplex PCR (mPCR) using IS6110 and mpb64 as gene targets for detecting Mycobacterium tuberculosis in samples from suspected cases of EPTB. We compared the performance of mPCR with conventional methods, including Ziehl Neelsen (ZN) microscopy, culture in LJ media, and BacT/Alert system. Our study aimed to provide insight into the utility of mPCR and its different targets for diagnosing EPTB in our setting.

We conducted a cross-sectional survey of 250 non-repeat clinical samples from extrapulmonary sites to detect M. tuberculosis. Both conventional diagnostic methods, including ZN microscopy, culture in LJ media, and BacT/Alert system, and molecular methods, including multiplex PCR (mPCR) using IS6110 and mpb64 as gene targets, were performed on the samples. Of the 250 samples, results for all the diagnostic methods were available for 116 samples, which were included in the final analysis. The study population comprised 83 patients with suspected EPTB and 33 controls.

Among the 83 samples in the EPTB group, conventional diagnostic methods, including ZN microscopy, LJ culture, and BacT/Alert system, showed low positivity rates of 6.02%, 8.43%, and 15.66%, respectively. In contrast, multiplex PCR (mPCR) using IS6110 and mpb64 as gene targets showed a significantly higher positivity rate of 79.51%. The IS6110 gene was amplified in 79.51% of the samples, while mpb64 was amplified in 49.39%.

Our study demonstrates that multiplex PCR (mPCR) using IS6110 and mpb64 as gene targets is a more sensitive diagnostic method for extra-pulmonary tuberculosis (EPTB) than conventional methods. Both IS6110 and mpb64 showed high sensitivity of 100%, but mpb64 was more specific when compared with the gold standard. Our findings suggest that mPCR, particularly with the inclusion of mpb64 as the target gene, may be a valuable tool for the early and accurate diagnosis of EPTB.