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<b>Introduction:</b> Tubercular lymphadenitis (TB-L) is the most common manifestation of extrapulmonary tuberculosis. Excisional biopsy with histopathological examination, Ziehl-Neelsen staining (ZNS) and culture and fine needle aspiration (FNA) cytology, although useful in the diagnosis of TB-L, cannot diagnose a substantial proportion of cases. We investigated the role of an in-house polymerase chain reaction (PCR) assay targeting the IS6110 gene from the FNA material in the diagnosis of the disease. <b>Materials and Methods:</b> The clinical profile of 150 patients with lymphadenopathy was noted and the fine needle aspirate was collected. After cytological processing, ZNS and culture on Lowenstein-Jensen media, mycobacterial DNA was isolated from the residual aspirate material and IS6110 gene PCR was performed. Results of cytology, ZNS, culture and IS6110 gene PCR were compared. <b>Results:</b> There were 49 confirmed patients of TB-L based on laboratory parameters (either culture isolation of <i>Mycobacterium tuberculosis</i> or any two of cytology, ZNS, PCR positive) and clinical response to therapy. Sensitivity and specificity of FNA was 89.8&#x0025; and 96&#x0025;, of ZNS was 40.8&#x0025; and 99&#x0025;, of culture was 40.8&#x0025; and 100&#x0025; and of IS6110 gene PCR test was 100&#x0025; and 92.1&#x0025;. <b>Conclusion:</b> IS6110 PCR can be considered a valuable adjunct to cytology, ZNS and culture techniques in the diagnosis of TB-L.