Evolutionary patterns of T cell epitopes in mycobacterium tuberculosis strains isolated in India

Background: Early diagnosis of tuberculous meningitis (TBM) is still a diagnostic challenge due to paucibacillary nature of the disease and conventional methods being quite insensitive and time consuming. We evaluated performance of three real time PCR assays targeting MPT64, IS6110 and 16s rRNA gene sequences for early diagnosis of TBM Methods & Materials: A total of 100 consecutive probable TBM patients and 50 non TBM patients were enrolled from an ongoing prospective study on tuberculous meningitis (July 2012 to Dec 2014). CSF specimens were subjected to microscopy and automated BACTEC MGIT culture. In-house Real time nested MPT64 and IS6110 PCR were standardized using H37Rv spiked CSF and evaluated for diagnostic utility along with commercially available Roche Amplicor Real time PCR assay. Results: Out of 100 clinical specimens processed, the sensitivity of smear microscopy and culture was 4% and 42% respectively. The sensitivity, specificity of Real time IS6110 PCR, Nested MPT 64 assay and Roche Amplicor Real time PCR assay was 71%, 98%; 69%, 98% and 25%, 100% respectively against probable TBM as reference standard. The Real time IS6110 PCR, Nested MPT 64 assay and Roche Amplicor Real time PCR assay could detect M. tuberculosis in only 84%, 77% and 30% of culture positive patient’s respectively Conclusion: Real time IS6110 PCR proved to be a simple and rapid method to diagnose TBM with sensitivity, specificity of 71%, 98% only. Nested MPT 64 assay though had comparable sensitivity and specificity was much more difficult due to nested design and higher risks of contamination. None of the PCR was 100% sensitive for detecting all culture positive samples suggesting that for TBM diagnosis there is no single rule out test and all the tests are contingent upon their ability to pick the target in tested volume of CSF.