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Background. In recent years, there has been an alarming increase in cases of multidrug-resistant tuberculosis (MDR-TB), and also extensively drug-resistant tuberculosis (XDR-TB). The emergence of these forms of tuberculosis (TB) worldwide has alerted scientists to the need for improved diagnostic tests that would enable rapid identification and decision-making on patient management. Over the past 15 years, in particular, new technologies have been introduced that enabled much improved methods of detection of anti-TB drug susceptibility, notably at the molecular level. One of the unfortunate consequences of these advances, however, is the fact that different drug sensitivity test (DST) assays might not always agree in the description of drug resistance status of isolates. This is also true for the detection of resistance to the key anti-TB drug, rifampicin (RIF). Specifically, phenotypic resistance as detected by culture-based methods, often differs from genotypic resistance detected by molecular methods. In this study, we describe the degree to which several currently used assays differ in their description of RIF-susceptibility in a sample of Mycobacterium tuberculosis (M. tuberculosis) isolates with discordant phenotypic and genotypic results. We also searched for mutations on the rpoB gene by Sanger sequencing and by next-generation whole-genome sequencing (NGS) in a subset of these isolates, in an attempt to distinguish between mutations that have clinical relevance, and those that do not. Materials and Methods. A convenient sample of 89 M. tuberculosis isolates were selected from routine sputum specimen submissions to the NHLS Tshwane Academic Hospital TB Laboratory at the University of Pretoria. Current commercially available phenotypic and genotypic methods were used to describe RIF-susceptibility in the study sample. Of the 89 isolates, 34 showed discordance in RIF DST results between GeneXpert® MTB/Rif (Cepheid, California, USA) (Xpert resistant) and BACTECTM MGITTM 960 (BD, Sparks MD, USA) (MGIT susceptible). Other isolates showed concordance on DST (resistant = 21; susceptible = 31). One isolate had missing MGIT results for RIF and Xpert failed to detect resistance in another isolate, confirmed as resistant by MGIT and Hain MDRTBplus (HainLifescience GmbH, Nehren, Germany) line probe assay (LPA). Whole genome sequencing was performed on 40 randomly selected isolates. In addition, microplate alamar blue assay (MABA) was performed on 77 of the 89 isolates to determine the relationship between RIF minimum inhibitory concentration (MIC) of the isolates and detection of resistance by other assays. Results. On Sanger sequencing, the most frequent rpoB gene mutations which conferred resistance to RIF occurred in codons 531, 516, and 526 (41%, 29% and 11% respectively) and 1 isolate had a novel mutation (S601T) outside the rpoB rifampicin resistance determining region (RRDR). The most frequently identified mutations in discordant isolates were (L511P and D516Y), 40% and 40%, respectively. Ten isolates susceptible to RIF on both Xpert and MGIT revealed mutations outside the RRDR, and in rpoC and efflux pump genes (Rv 1145, Rv 1146 and Rv0933). Twelve of 22 (54%) isolates resistant to RIF on MGIT had RIF MICs greater than 1 ?g/ml, whereas three had RIF MICs below the critical concentration used by MGIT. Twenty-two of 27 (81%) isolates with discordant results had RIF MICs below 1 ?g/ml. Twelve of 19 (63%) patients who failed RIF-based TB therapy had no rpoB mutations (based on currently used molecular-tests) in the hotspot region. Conclusions. Our data support the recent reports of other investigators that the expression of efflux pump mutations in mmpL 13a and pstB genes, and alterations in rpoC and gyrA may have a synergy with other mutations in the rpoB gene within or outside the hotspot region. These associations have previously been shown to result in low levels of RIF resistance. We conclude that NGS of all isolates that show discrepancies in DST between MGIT and Xpert or LPA be routinely performed in order to better inform treatment regimens for TB suspects.