Background:Extrapulmonary tuberculosis (EPTB) constitutes about 15% to 20% of all cases of tuberculosis (TB). The confirmation of EPTB has always been a challenge to laboratory personnel. We aim to evaluate the diagnostic potential of immunostaining with anti-MPT64 in various EPTB specimens.Materials and Methods:We studied a total of 51 TB cases and 38 non-TB control specimens comprising of fine-needle aspirates and formalin-fixed biopsies. These were investigated using a combination of the Ziehl-Neelsen method, the Lowenstein-Jensen culture, immunostaining with anti-MPT64 and anti-BCG, and nested-polymerase chain reaction (PCR) for IS6110. Results of all the tests were compared using nested-PCR as the gold standard.Results:Diagnostic validation of immunostaining for anti-MPT64 was performed using nested-PCR as the gold standard. The overall sensitivity, specificity, and positive and negative predictive values for immunostaining with anti-MPT64 were 100%, 97%, 97%, and 100%, respectively.Conclusions:Immunostaining using anti-MPT64 is a rapid and sensitive method for establishing an early and specific diagnosis of Mycobacterium tuberculosis infection. The technique is simple to be incorporated into routine pathology laboratories.