Automatic Identification of Individual <i>rpoB</i> Gene Mutations Responsible for Rifampin Resistance in Mycobacterium tuberculosis by Use of Melting Temperature Signatures Generated by the Xpert MTB/RIF Ultra Assay

Abstract

Molecular surveillance of rifampin-resistant <i>Mycobacterium tuberculosis</i> can help to monitor the transmission of the disease. The Xpert MTB/RIF Ultra assay detects mutations in the rifampin resistance-determining region (RRDR) of the <i>rpoB</i> gene by the use of melting temperature (<i>T_m</i> ) information from 4 <i>rpoB</i> probes which can fall in one of the 9 different assay-specified <i>T_m</i> windows. The large amount of <i>T_m</i> data generated by the assay offers the possibility of an RRDR genotyping approach more accessible than whole-genome sequencing. In this study, we developed an automated algorithm to specifically identify a wide range of mutations in the <i>rpoB</i> RRDR by utilizing the pattern of the <i>T_m</i> of the 4 probes within the 9 windows generated by the Ultra assay. The algorithm builds a RRDR mutation-specific "<i>T_m</i> signature" reference library from a set of known mutations and then identifies the RRDR genotype of an unknown sample by measuring the <i>T_m</i> distances between the test sample and the reference <i>T_m</i> values. Validated using a set of clinical isolates, the algorithm correctly identified RRDR genotypes of 93% samples with a wide range of <i>rpoB</i> single and double mutations. Our analytical approach showed a great potential for fast RRDR mutation identification and may also be used as a stand-alone method for ruling out relapse or transmission between patients. The algorithm can be further modified and optimized for higher accuracy as more Ultra data become available.