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Molecular surveillance of rifampin-resistant Mycobacterium tuberculosis 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 rpoB gene by the use of melting temperature ( T m  ) information from 4 rpoB probes which can fall in one of the 9 different assay-specified T m  windows. The large amount of T m  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 rpoB RRDR by utilizing the pattern of the T m  of the 4 probes within the 9 windows generated by the Ultra assay. The algorithm builds a RRDR mutation-specific " T m  signature" reference library from a set of known mutations and then identifies the RRDR genotype of an unknown sample by measuring the T m  distances between the test sample and the reference T m  values. Validated using a set of clinical isolates, the algorithm correctly identified RRDR genotypes of 93% samples with a wide range of rpoB 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.

journal of clinical microbiology

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

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