en Diagnostic/screening methods and protocols Diagnostic/screening methods and protocols Original article Original article <b><span style="font-size:10.0pt"><span style="line-height:107%"><span new="" roman="" style="font-family:" times="">Introduction</span></span></span></b><span style="font-size:10.0pt"><span style="line-height:107%"><span new="" roman="" style="font-family:" times="">: 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 <i>IS6110</i> and <i>mpb64</i> as gene targets for detecting <i>Mycobacterium tuberculosis</i> 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. <b>Methods</b>: We conducted a cross-sectional survey of 250 non-repeat clinical samples from extrapulmonary sites to detect <i>M. tuberculosis</i>. Both conventional diagnostic methods, including ZN microscopy, culture in LJ media, and BacT/Alert system, and molecular methods, including multiplex PCR (mPCR) using <i>IS6110</i> and <i>mpb64</i> 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. <b>Results</b>: 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 <i>IS6110</i> and <i>mpb64</i> as gene targets showed a significantly higher positivity rate of 79.51%. The <i>IS6110</i> gene was amplified in 79.51% of the samples, while <i>mpb64</i> was amplified in 49.39%. <b>Conclusion</b>: Our study demonstrates that multiplex PCR (mPCR) using <i>IS6110</i> and <i>mpb64</i> as gene targets is a more sensitive diagnostic method for extra-pulmonary tuberculosis (EPTB) than conventional methods. Both <i>IS6110</i> and <i>mpb64</i> showed high sensitivity of 100%, but <i>mpb64</i> was more specific when compared with the gold standard. Our findings suggest that mPCR, particularly with the inclusion of <i>mpb64</i> as the target gene, may be a valuable tool for the early and accurate diagnosis of EPTB.</span></span></span>&nbsp; EPTB, mPCR, mpb64, IS6110, Tuberculosis (TB), Diagnostic accuracy, Sensitivity, Specificity 78 85 http://jommid.pasteur.ac.ir/browse.php?a_code=A-10-438-1&slc_lang=en&sid=1 Dekyong Angmo angmodekyong1@gmail.com 0009-0006-2919-5413 No Department of Microbiology, Government Medical College, Srinagar. Jammu and Kashmir, India Gulnaz Bashir drgulnazbashir@hotmail.com 0000-0002-8048-0854 Yes Department of Microbiology, Sher-i-Kashmir Institute of Medical Sciences, Soura, Srinagar, Jammu and Kashmir, India Abiroo Jan abroo_naqash@yahoo.com 0000-0002-3150-2906 No Department of Microbiology, Government Medical College, Anantnag, Jammu and Kashmir, India Mushtaq A. Khan drmushtaqkhan@hotmail.com 0000-0002-1257-6002 No Department of Gastroenterology, Sher-i-Kashmir Institute of Medical Sciences, Soura, Srinagar, Jammu and Kashmir, India Syed Besina Yasin besina.yasin@skims.ac.in 0000-0001-9886-1113 No Department of Pathology, Sher-i-Kashmir Institute of Medical Sciences, Soura, Srinagar, Jammu and Kashmir, India