Monitoring tuberculosis treatment response using molecular techniques

Abstract

This study looks to monitor tuberculosis treatment response using molecular techniques. An assay for the detection and enumeration of Mycobacterium tuberculosis already existed in the form of the Mycobacterial Load Assay (MBL). This utilised reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) to detect M. tuberculosis 16S ribosomal RNA (rRNA) and thereby quantify bacillary loads in patient sputa. By measuring labile RNA the assay was thought to only measure viable bacilli.

However, unpublished data from the Gillespie lab suggested that 16S rRNA remains stable even once cell are non-viable. Two new markers were sought to represent the viable bacilli: M. tuberculosis precursor 16S rRNA (pre-16S rRNA) and transfer- messenger RNA (tm-RNA).

Both of these novel biomarkers were chosen because of their complex secondary structures giving them increased stability allowing for their detection in adverse environments.

Development and of a multiplex assay using the same messenger RNA (mRNA) phyB internal control utilised by the MBL assay, then the fielding testing of these multiplex assays was performed with the subsequent failure of the phyB control.

The internal control was then redesigned to be more robust and reproducible utilising a novel M. marinum control. The new multiplex assays with 16S rRNA, pre-16S rRNA and tm-RNA with the M. marinum internal control were then tested on 520 patient samples to demonstrate that the assays could be used for monitoring treatment response.