Comparative Analysis of mpb-64 and Nested PCR targeting IS6110 for the Detection of Extra Pulmonary Tuberculosis

Abstract

Introduction: Extra Pulmonary Tuberculosis can be seen in more than half of the patients with concurrent AIDS and tuberculosis. A high index of suspicion is necessary for timely diagnosis and prompt initiation of therapy. PCR has allowed great progress to be made in the rapid and accurate diagnosis of infections due to organisms for which culture is too insensitive and paubacillary nature of the pathogens in the specimens. Aim: Evaluation of the efficacy of dual targets; mpb-64 and Nested PCR targeting IS6110 for the detection of Extra Pulmonary Tuberculosis. Place and Duration of Study: Done at Molecular Research Laboratory, Biochemistry Department, SGRRIMand HS, Dehradun, India, in between the duration, 2012-2013. Methodology: A total of ninety clinical specimens, which includes; 14 pus, 32 CSF, 4 ascetic fluid, 6 biopsy, 10 blood, 3 urine, 6 endometrial tissues, 6 pleural fluids, 1 endometrial curetting, and 2 synovial fluid, collected from the various departments of SMI Hospital, Patel Nagar, Dehradun. Specimens were further processed for ZN staining, PCR utilizing; mpb-64 and nested PCR (N-PCR) using IS6110 as a target for the detection of extra pulmonary tuberculosis cases. Results: Out of 90 clinical specimens processed, 29 (32.2%) came N-PCR positive and 23 (25.5%) are mpb64 TB PCR positive. When analyzed comparatively for both the molecular targets, it was seen that 20 samples came positive by both the targets i.e. IS6110 as well as mpb64 gene, but 9 specimens alone came positive only by IS6110 NTPCR, and the same came negative for mpb64 gene. 3 specimens showed positive result status for mpb64 gene by conventional assay and at the same time they were negative for IS6110 gene. Conclusion: An essential element in the management of extra pulmonary tuberculosis is the availability of rapid, sensitive and specific identification of the causative agent. The laboratory diagnosis is largely based on direct microscopy and culture for mycobacterium. Direct microscopic examination is used to detect acid-fast bacilli (AFB) and has very low sensitivity. The gold standard for isolation of mycobacterium is not only time-consuming but also has low sensitivity in case of extra pulmonary tuberculosis. TB PCR diagnostics kits must be prepared, targeting mpb-64 as well as IS6110 as a targets to avoid false results.