Low-Level Rifampin Resistance and rpoB Mutations in Mycobacterium tuberculosis: an Analysis of Whole-Genome Sequencing and Drug Susceptibility Test Data in New York

Abstract

Rapid and reliable detection of rifampin (RIF) resistance is critical for the diagnosis and treatment of drug-resistant and multi-drug resistant (MDR) tuberculosis. Discordant RIF phenotype/genotype susceptibility results remain a challenge due to the presence of rpoB mutations which do not confer high levels of RIF resistance as have been exhibited in strains with mutations such as Ser450Leu. These strains, termed low-level RIF resistant, exhibit elevated RIF minimum inhibitory concentrations (MICs) compared to fully susceptible strains, however remain phenotypically susceptible by mycobacteria growth indicator tube (MGIT) testing and have been associated with poor patient outcomes. Here we assess RIF resistance prediction by whole-genome sequencing (WGS) among a set of 1779 prospectively tested strains by both prevalence of rpoB gene mutation and phenotype as part of routine clinical testing during a 21/2-year period. During this time, 139 strains were found to have nonsynonymous rpoB mutations, 53 of which were associated with RIF resistance, including both low-level and high-level resistance. Resistance to RIF (1.0 μg/mL in MGIT) was identified in 43 (81.1%) isolates. The remaining 10 (18.9%) strains were susceptible by MGIT, however were confirmed to be low-level RIF resistant by MIC testing. Full rpoB gene sequencing overcame the limitations of critical concentration phenotyping, probe-based genotyping, and partial-gene sequencing methods. Universal clinical WGS with concurrent phenotypic testing provided a more complete understanding of the prevalence and type of rpoB mutations and their association with RIF resistance in New York.

On the Consequences of Poorly Defined Breakpoints for Rifampin Susceptibility Testing of Mycobacterium tuberculosis Complex

“…Importantly, some RRDR mutations might be excluded from the expert rule, should conclusive evidence emerge that these do not confer elevated MICs and/or are not clinically significant. The MIC data from Shea et al are in line with an earlier report from Haiti by Ocheretina et al that suggested that rpoB T427A may be one of these exceptions (6,35). However, given that this mutation appears to have evolved independently in lineages 3 and 4, which is typically a signal of selection, additional MIC testing is needed using a method for which a welldefined ECV exists and the quality control range of the laboratory strain H37Rv is not truncated to ensure high-quality testing (17).…”

“…In this issue of the Journal of Clinical Microbiology, Shea et al add to this picture by providing evidence that the CLSI-endorsed RIF CC for the Sensititre MYCOTB broth microdilution plate by Thermo Fisher, which is not FDA approved but is CE-IVD marked in the European Union, is likely set too high (6,17). Specifically, their findings are in line with an earlier study by Torrea et al and indicate that the CC needs to be lowered to the tentative ECV of 0.5mg/ml, following a more detailed review of additional data sets (25).…”

“…1). This applies to the MYCOTB plate and all WHO-endorsed media, including LJ (i.e., it is a misconception that LJ is a reliable confirmatory test for borderline resistance mutations) (6,25). It should be noted, however, that even when using the new WHO CCs, the rate of misclassification of these borderline resistance mutations as susceptible will likely remain higher for MGIT than for LJ and 7H10, which could be due to one of two reasons.…”

“…Nevertheless, only 61% of bacteriologically confirmed pulmonary TB cases were tested for RIF resistance in 2019 compared to the target of 100% set by the World Health Organization (WHO) in the END TB Strategy (3). More fundamentally, the molecular understanding of resistance provided crucial evidence that RIF resistance had not been defined rigorously on the phenotypic level for more than half a century, complicating routine diagnostics and patient treatment (6,7).…”

rpoB gene mutations in rifampin-resistant Mycobacterium tuberculosis isolates from rural areas of Zhejiang, China

“…Universal clinical whole-genome sequencing could overcome the limitations of critical concentration phenotyping and partial gene sequencing and provide a more complete understanding of the prevalence and type of rpoB mutations and their association with RIF R . 27 Second, we focused only on MTB isolates of RIF R using LJ medium and the DST proportional method ; therefore, we were unable to associate treatment outcomes with various mutation types. Finally, all isolates were isolated from culture-positive sputum specimens from patients with suspected TB, and we did not include patients without isolates available for drug resistance profiles and rpoB mutations in our analyses.…”