Abstract
BackgroundAccurate 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.MethodsA 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.ResultsAmong 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.ConclusionsFour 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.
Highlights
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 (MDRTB)
Accurate drug susceptibility testing (DST) of M. tuberculosis in clinical specimens and culture isolates to first-line drugs is crucial for rapid diagnosis of Multidrug-resistant tuberculosis (MDR-TB) for proper patient management, for limiting further transmission of MDR-TB and development of Extensively drug-resistant tuberculosis (XDR-TB) [2, 5]
Phenotypic DST data by Mycobacteria growth indicator tube (MGIT) 960 system Phenotypic DST by MGIT 960 system showed that 64 isolates were fully susceptible to all four (RIF, INH, SM and EMB) anti-TB drugs, 15 isolates were resistant to SM only, 51 isolates were resistant to INH only while the remaining isolates were resistant to more than one drug
Summary
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 (MDRTB). Widespread occurrence of drug-resistant tuberculosis (TB) and multidrug-resistant (MDR)-TB (infection with Mycobacterium tuberculosis strain resistant at least to rifampicin, RIF and isoniazid, INH; the two most effective first-line anti-TB drugs) is a serious threat to TB control success worldwide. Accurate drug susceptibility testing (DST) of M. tuberculosis in clinical specimens and culture isolates to first-line drugs is crucial for rapid diagnosis of MDR-TB for proper patient management, for limiting further transmission of MDR-TB and development of XDR-TB [2, 5]. Rapid liquid culture-based phenotypic DST methods are considered as the gold standard by WHO for identifying resistance to RIF, INH and other first-line drugs, these methods still require 1– 2 weeks to report results [5, 6]. Molecular DST methods rapidly detect genetic mutations associated with drug resistance [2, 7]
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