Rapid diagnosis of tuberculous meningitis: a comparative evaluation of in-house PCR assays involving three mycobacterial DNA sequences, IS6110, MPB-64 and 65 kDa antigen

Abstract

A PCR was standardized for amplifying three different mycobacterial - IS6110, MPB-64, 65 kDa DNA sequences. A comparative evaluation of the three PCR assays was carried out for the rapid diagnosis of tuberculous meningitis (TBM) using cerebrospinal fluid (CSF) specimens. While the IS6110 PCR was a single-step amplification reaction, the MPB-64 and 65 kDa antigen PCR assays were nested reactions. A total of 176 cerebrospinal fluid (CSF) samples from 176 patients were subjected to amplification of the three different mycobacterial sequences. Amongst them, 45 samples were obtained from confirmed cases of TBM (culture positive) and 56 samples were obtained from clinically suspected cases of TBM which were culture-negative. The remaining 75 CSF samples were categorized under the non-infectious and infectious illness of the central nervous system (CNS). Against a gold standard of culture, a sensitivity of 98% (NPV = 99%) and a specificity of 100% (PPV = 100%) was observed with the IS6110 PCR. Among the nested PCRs, a sensitivity of 91% (NPV = 94%) and a specificity of 91% (PPV = 85%) was observed with the MPB-64 assay, while the 65 kDa protocol had an associated sensitivity of 51% (NPV = 76%) and a specificity of 92% (PPV = 79%). These findings suggest that among the nested PCR assays, the MPB-64 PCR assay was associated with an enhanced degree of sensitivity and was comparable in terms of specificity. Our study also demonstrates that the IS6110 assay, while being a single-step PCR had the advantage of being a rapid test for the diagnosis of TBM, with increased sensitivity and enhanced specificity as compared to the nested PCR protocols.