Occurrence of disputed rpoB mutations among Mycobacterium tuberculosis isolates phenotypically susceptible to rifampicin in a country with a low incidence of multidrug-resistant tuberculosis.

BACKGROUND: Accurate drug susceptibility testing (DST) of Mycobacterium tuberculosis in clinical specimens and culture isolates to first-line drugs is crucial for diagnosis and management of multidrug-resistant tuberculosis (MDR-TB). Resistance of M. tuberculosis to rifampicin is mainly due to mutations in hot-spot region of rpoB gene (HSR-rpoB). The prevalence of disputed (generally missed by rapid phenotypic DST methods) rpoB mutations, which mainly include L511P, D516Y, H526N, H526L, H526S, and L533P in HSR-rpoB and I572F in cluster II region of rpoB gene, is largely unknown. This study determined the occurrence of all disputed mutations in HSR-rpoB and at rpoB codon 572 in M. tuberculosis strains phenotypically susceptible to rifampicin in Kuwait. METHODS: A total of 242 M. tuberculosis isolates phenotypically susceptible to rifampicin were used. The DST against first-line drugs was performed by Mycobacteria growth indicator tube (MGIT) 960 system. Mutations in HSR-rpoB (and katG codon 315 and inhA-regulatory region for isoniazid resistance) were detected by GenoType MDBDRplus assay. The I572F mutation in cluster II region of rpoB was detected by developing a multiplex allele-specific (MAS)-PCR assay. Results were confirmed by PCR-sequencing of respective loci. Molecular detection of resistance for ethambutol and pyrazinamide and fingerprinting by spoligotyping were also performed for isolates with an rpoB mutation. RESULTS: Among 242 rifampicin-susceptible isolates, 0 of 130 pansusceptible/monodrug-resistant isolates but 4 of 112 polydrug-resistant isolates contained a disputed rpoB mutation. All 4 isolates were also resistant to isoniazid and molecular screening identified additional resistance to pyrazinamide and ethambutol in one isolate each. In final analysis, 2 of 4 isolates were resistant to all 4 first-line drugs. Spoligotyping showed that the isolates belonged to different M. tuberculosis lineages. CONCLUSIONS: Four of 242 (1.7%) rifampicin-susceptible M. tuberculosis isolates contained a disputed rpoB mutation including 2 isolates resistant to all four first-line drugs. The occurrence of a disputed rpoB mutation in polydrug-resistant M. tuberculosis isolates resistant at least to isoniazid (MDR-TB) suggests that polydrug-resistant strains should be checked for genotypic rifampicin resistance for optimal patient management since the failure/relapse rates are nearly same in isolates with a canonical or disputed rpoB mutation.

Discordance between Xpert MTB/RIF assay and Bactec MGIT 960 Culture System for detection of rifampin-resistant Mycobacterium tuberculosis isolates in a country with a low tuberculosis (TB) incidence.

Among 452 samples that were positive by the Xpert MTB/RIF (Xpert) assay and MGIT 960 system (MGIT), 440 and 10 Mycobacterium tuberculosis samples were detected as rifampin susceptible and rifampin resistant, respectively. Two isolates that were rifampin susceptible by the MGIT system were rifampin resistant by the Xpert assay. rpoB sequencing identified a silent (CTG521TTG) mutation in one isolate and a missense (GAC516TAC) mutation in another. The detection of rifampin resistance is imperfect with both the Xpert assay and MGIT system. Any discordant rifampin resistance results should be confirmed by sequencing of the rpoB gene.

Discordance across Phenotypic and Molecular Methods for Drug Susceptibility Testing of Drug-Resistant Mycobacterium tuberculosis Isolates in a Low TB Incidence Country.

With increasing incidence of multidrug-resistant tuberculosis (MDR-TB), accurate drug susceptibility testing (DST) of Mycobacterium tuberculosis to first-line drugs has become crucial for proper patient management. We evaluated concordance of DST results for 70 M. tuberculosis isolates across two phenotypic and two molecular methods: BACTEC 460TB, MGIT 960 system, GenoType MTBDRplus and DNA sequencing of gene segments most commonly implicated in conferring resistance to anti-TB drugs. Most (84%) M. tuberculosis isolates were multidrug-resistant. Twenty-four isolates yielded discrepant DST results. For rifampicin, isoniazid and streptomycin, 96%, 97% and 93% of isolates, respectively, were susceptible or resistant by all four methods, whereas for ethambutol, this agreement was observed for only 76% of isolates (P<0.05 for rifampicin or isoniazid or streptomycin versus ethambutol). Occurrence of rare mutations in three isolates that confer low-level resistance caused lower agreement for rifampicin among the four methods (kappa coefficient (κ) range, 0.84 to 0.95). For isoniazid, there was perfect agreement among phenotypic methods and molecular methods (κ, 1.00) but lower agreement between phenotypic and molecular methods. Three isolates were detected as polydrug-resistant by MGIT 960 system but as multidrug-resistant by DNA sequence-based method. The agreement was higher for streptomycin among the two phenotypic methods (κ, 0.97) while targeted sequencing yielded lower agreement (κ range, 0.86 to 0.89). The discrepancy for ethambutol resulted largely due to lower concordance of MGIT 960 results (κ range, 0.53 to 0.64). The MGIT 960 system is an accurate method for DST of M. tuberculosis against isoniazid and streptomycin while the results of rifampicin susceptibility should be complemented with DNA sequencing-based method when the suspicion for resistance is high. The possibility of false susceptibility to ethambutol with MGIT 960 system suggests that molecular or other phenotypic methods may be more useful when accurate ethambutol susceptibility results are warranted.