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

Abstract

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 (Tm) information from 4 rpoB probes which can fall in one of the 9 different assay-specified Tm windows. The large amount of Tm 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 Tm of the 4 probes within the 9 windows generated by the Ultra assay. The algorithm builds a RRDR mutation-specific “Tm signature” reference library from a set of known mutations and then identifies the RRDR genotype of an unknown sample by measuring the Tm distances between the test sample and the reference Tm 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.

Xpert MTB/XDR: a 10-Color Reflex Assay Suitable for Point-of-Care Settings To Detect Isoniazid, Fluoroquinolone, and Second-Line-Injectable-Drug Resistance Directly from Mycobacterium tuberculosis-Positive Sputum

“…Several iterative probe designs were tested based on the probes present in our prototype cartridge and a combination of three overlapping probes were chosen to generate a series of Tm signatures, which not only individually identified the three different mutations and differentiated them from other QRDR mutations, but also generated the same WT Tm values for the codon 95 polymorphism. We successfully used this Tm signature principle to identify these mutations by placing the mutant Tm values in carefully chosen, specific WT and mutant Tm windows for each probe, which underscores the previously described capacity for SMB probe tiling to accurately identify DNA sequences (40). The Xpert MTB/XDR assay targeted at least two regions in the M. tuberculosis genome which contained mutations spread over relatively long stretches, which would be very difficult for a single probe to query.…”

Genomic Analysis Identifies Mutations Concerning Drug-Resistance and Beijing Genotype in Multidrug-Resistant Mycobacterium tuberculosis Isolated From China

“…Compared to the molecular assays of DST, such as line-probe arrays, PCR methods based on TaqMan probes, or melting curves (Boehme et al, 2010;Chen et al, 2014;Zhao et al, 2015b;World Health Organization, 2016;Makhado et al, 2018;Cao et al, 2019), WGS displays better performance to predict drug resistance according to the limited known mutations as well as a big catalog of mutations in various genes. However, among the mutations found by WGS, synonymous mutations, mutations of lineage markers, and some well-known nonsynonymous polymorphisms (e.g., katG R463L, gyrA S95T) (Feuerriegel et al, 2014;Zhao et al, 2014b), which are universally acknowledged to be unrelated with drug resistance, must be excluded before predicting resistance.…”

Comparison of Analytical Sensitivity (Limit of Detection) of Xpert MTB/RIF and Xpert MTB/RIF Ultra for Non-Sputum Specimens

“…The Ultra test primers amplify the same core region of the rpoB gene as the US-IVD test, but with four probes. Mutations in the RRDR are captured by a shift in the melting temperature (Tm) of at least one of the four probes away from the Tm expected to occur in the presence of wild-type [ 11 ]. In addition, the primers in the Ultra test amplify portions of the multi-copy insertion elements IS1081 and IS6110 , which enhance the detection of MTB [ 12 ].…”