Multiplex PCR targeting IS۶۱۱۰ and mpb۶۴ Genes for Detecting Extra-Pulmonary Tuberculosis: A Cross-Sectional Study in a Tertiary Care Center, Kashmir, India

Introduction: 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 IS۶۱۱۰ and mpb۶۴ 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. Methods: We conducted a cross-sectional survey of ۲۵۰ 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 IS۶۱۱۰ and mpb۶۴ as gene targets, were performed on the samples. Of the ۲۵۰ samples, results for all the diagnostic methods were available for ۱۱۶ samples, which were included in the final analysis. The study population comprised ۸۳ patients with suspected EPTB and ۳۳ controls. Results: Among the ۸۳ samples in the EPTB group, conventional diagnostic methods, including ZN microscopy, LJ culture, and BacT/Alert system, showed low positivity rates of ۶.۰۲%, ۸.۴۳%, and ۱۵.۶۶%, respectively. In contrast, multiplex PCR (mPCR) using IS۶۱۱۰ and mpb۶۴ as gene targets showed a significantly higher positivity rate of ۷۹.۵۱%. The IS۶۱۱۰ gene was amplified in ۷۹.۵۱% of the samples, while mpb۶۴ was amplified in ۴۹.۳۹%. Conclusion: Our study demonstrates that multiplex PCR (mPCR) using IS۶۱۱۰ and mpb۶۴ as gene targets is a more sensitive diagnostic method for extra-pulmonary tuberculosis (EPTB) than conventional methods. Both IS۶۱۱۰ and mpb۶۴ showed high sensitivity of ۱۰۰%, but mpb۶۴ was more specific when compared with the gold standard. Our findings suggest that mPCR, particularly with the inclusion of mpb۶۴ as the target gene, may be a valuable tool for the early and accurate diagnosis of EPTB.