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Comparative Evaluation of CFX96TM Real Time PCR with Conventional PCR for Rapid Diagnosis of Mycobacterium tuberculosis Complex in Clinical Isolates

Jaspreet Kaur, Jaswinder Singh and Priyanka Mishra

Background: Globally, Tuberculosis (TB) still remains a major public health problem. In India, Pulmonary Tuberculosis (PTB) is the most common form of the disease; however, Extrapulmonary Tuberculosis (EPTB) comprises 10 to 20% of all TB cases. The diagnosis of EPTB cases is difficult because of paucibacillary nature and consequently is associated with low sensitivity of Zhiel- Neelson (ZN) smear and culture on Lowenstein-Jensen (LJ) media as gold standard. The present study comparatively evaluates the utility of real time quantitative PCR over nested PCR and other conventional techniques for the detection of M. tuberculosis Complex (Mtc) in clinical isolates of PTB and EPTB samples at a tertiary care centre, Shri Ram Murti Smarak Institute of Medical Sciences (SRMSIMS), Bareilly.

Methods and Findings: In total, 205 both pulmonary (24) and extrapulmonary (181) specimens were processed for ZN smear, culture on LJ media, nested PCR and real time quantitative PCR using IS6110 as a gene target. Out of 205 samples, none of the sample was found to be smearpositive and only 28 (14%) samples were found to be culture positive. The nested PCR and real time quantitative PCR positivity was observed 100% in culture positive specimens. However in majority of culture negative specimens the sensitivity was 100% in both nested PCR and real time quantitative PCR assays for EPTB and PTB specimens. The specificity was better in case of real time quatitative PCR as compared to nested PCR, 45.8% and 20% in case of nested PCR for nonrespiratory and respiratory specimens respectively. The specificity was increased upto 86.1 and 100% with real time quantitative PCR for EPTB and PTB cases respectively.

Conclusions: The combined analysis of nested PCR, real time quantitative PCR and other lab investigations can be very useful in the rapid diagnosis of M. tuberculosis in paucibacillary extrapulmonary tuberculosis samples in Indian scenario.