Comparison of Xpert MTB/RIF Assay and GenoType MTBDRplus DNA Probes for Detection of Mutations Associated with Rifampicin Resistance in Mycobacterium tuberculosis

Abstract

BackgroundGeneXpert MTB/RIF (Xpert) and Genotype MTBDRplus (DRplus) are two World Health Organization (WHO) endorsed probe based molecular drug susceptibility testing (DST) methods for rapid diagnosis of drug resistant tuberculosis. Both methods target the same 81 bp Rifampicin Resistance Determining Region (RRDR) of bacterial RNA polymerase β subunit (rpoB) for detection of Rifampicin (RIF) resistance associated mutations using DNA probes. So there is a correspondence of the probes of each other and expected similarity of probe binding.MethodsWe analyzed 92 sputum specimens by Xpert, DRplus and LJ proportion method (LJ-DST). We compared molecular DSTs with gold standard LJ-DST. We wanted to see the agreement level of two molecular methods for detection of RIF resistance associated mutations. The 81bp RRDR region of rpoB gene of discrepant cases between the two molecular methods was sequenced by Sanger sequencing.ResultsThe agreement of Xpert and DRplus with LJ-DST for detection of RIF susceptibility was found to be 93.5% and 92.4%, respectively. We also found 92.4% overall agreement of two molecular methods for the detection of RIF susceptibility. A total of 84 out of 92 samples (91.3%) had agreement on the molecular locus of RRDR mutation by DRplus and Xpert. Sanger sequencing of 81bp RRDR revealed that Xpert probes detected seven of eight discrepant cases correctly and DRplus was erroneous in all the eight cases.ConclusionAlthough the overall concordance with LJ-DST was similar for both Xpert and DRplus assay, Xpert demonstrated more accuracy in the detection of RIF susceptibility for discrepant isolates compared with DRplus. This observation would be helpful for the improvement of probe based detection of drug resistance associated mutations especially rpoB mutation in M. tuberculosis.

Comprehensive Whole-Genome Sequencing and Reporting of Drug Resistance Profiles on Clinical Cases of Mycobacterium tuberculosis in New York State

“…Commonly utilized molecular tests for drug resistance detection include the Xpert MTB/RIF (Cepheid, Sunnyvale, CA, USA) and the MTBDRplus (Hain Lifescience, GmbH, Nehren, Germany) assays. Limitations of the Xpert MTB/RIF and the MTBDRplus include detecting mutations only in the RRDR and yielding occasional false rifampin-resistant results due to silent rpoB mutations (31)(32)(33). Isoniazid resistance can arise from a number of mutations across multiple genes, resulting in imperfect sensitivities for targeted molecular assays, such as the MTBDRplus (34) and katG or combined katG-inhA pyrosequencing (28,35).…”

“…Paired-end 250-bp DNA sequencing was carried out using the Illumina MiSeq platform following Nextera XT library prep with a 15-cycle PCR indexing step (33). Sequencing runs were either composed fully of MTBC (15 to 17 samples) or of MTBC samples and other bacterial samples.…”

The diagnostic utility of line probe assays for multidrug-resistant tuberculosis

“…This is possibly because of certain mutations that the LPA, in contrast to the Xpert MTB/Rif assay, does not encompass. 20 DNA sequencing studies demonstrate that more than 95% of rifampicin-resistant M. tuberculosis strains have a mutation within the 81-bp hot-spot region (codons 507-533) of the RNA polymerase B subunit (rpoB) gene. While the Xpert MTB/Rif detects mutations in all but codon 530, the line probe assay fails to detect mutations from codon 527-530.…”

Clinical Performances of Pure TB-Lamp Kit for <i>M. tuberculosis</i> Complex Detection in Sputum Samples

“…Considering emergence of TB drug resistance, although utility of TB-Lamp assay for TB diagnosis is clearly established, it is unable to detect drug-resistant patterns notably surrogate marker of multi-drug resistance [17] [18] [19].…”