Detection of viable Mycobacterium tuberculosis in ocular fluids using mRNA-based multiplex polymerase chain reaction.

ABSTRACT

PURPOSE: Intra ocular tuberculosis (IOTB) can affect nearly any part of the eye and delayed diagnosis can result in permanent loss of vision. mRNA, with the ability to detect viable bacilli just like culture, could serve as an important tool to differentiate whether ocular manifestations of IOTB are due to active infection or hypersensitivity response to dead mycobacteria. METHODS: A total of 19 patients with suspected IOTB and 20 controls (10 patients with active inflammation but no evidence of IOTB and 10 patients with no intraocular inflammation) were included. Vitreous fluid samples were collected from all patients and subjected to DNA and mRNA extraction followed by multiplex polymerase chain reaction (MPCR) using three specific targets for M. tuberculosis, i.e. IS6110, MPB64 and protein b. All samples were also subjected to Gene Xpert MTB/RIF assay. RESULTS: Among the 19 patients in group 1, the highest test positivity was noted for DNA-MPCR (73.7%) followed by mRNA-MPCR (42.1%) and GeneXpert MTB/RIF assay (10.5%). All the three tests were negative in the 20 control samples demonstrating a 100% specificity. The negative predictive value was highest for DNA-MPCR (80%), followed by mRNA-PCR (64.5%) and Xpert MTB/RIF assay (48.8%). All patients with any one test positive were treated with anti-tubercular therapy (ATT) and there was good clinical response noted in all. CONCLUSION: mRNA-based MPCR can be used as a marker for detecting viable M. tuberculosis in IOTB cases.