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1.
PLoS One ; 11(11): e0167093, 2016.
Article in English | MEDLINE | ID: mdl-27902737

ABSTRACT

BACKGROUND: The goal of this study was to compare the consistency of three assays for the determination of the drug resistance of Mycobacterium tuberculosis (MTB) strains with various resistance profiles isolated from the Moscow region. METHODS: A total of 144 MTB clinical isolates with a strong bias toward drug resistance were examined using Bactec MGIT 960, Sensititre MycoTB, and a microarray-based molecular assay TB-TEST to detect substitutions in the rpoB, katG, inhA, ahpC, gyrA, gyrB, rrs, eis, and embB genes that are associated with resistance to rifampin, isoniazid, fluoroquinolones, second-line injectable drugs and ethambutol. RESULTS: The average correlation for the identification of resistant and susceptible isolates using the three methods was approximately 94%. An association of mutations detected with variable resistance levels was shown. We propose a change in the breakpoint minimal inhibitory concentration for kanamycin to less than 5 µg/ml in the Sensititre MycoTB system. A pairwise comparison of the minimal inhibitory concentrations (MICs) of two different drugs revealed an increased correlation in the first-line drug group and a partial correlation in the second-line drug group, reflecting the history of the preferential simultaneous use of drugs from these groups. An increased correlation with the MICs was also observed for drugs sharing common resistance mechanisms. CONCLUSIONS: The quantitative measures of phenotypic drug resistance produced by the Sensititre MycoTB and the timely detection of mutations using the TB-TEST assay provide guidance for clinicians for the choice of the appropriate drug regimen.


Subject(s)
Antitubercular Agents/pharmacology , Drug Resistance, Bacterial/genetics , Microbial Sensitivity Tests/methods , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Oligonucleotide Array Sequence Analysis , Antitubercular Agents/therapeutic use , Genotype , Humans , Moscow , Mutation , Mycobacterium tuberculosis/isolation & purification , Mycobacterium tuberculosis/physiology , Phenotype , Tuberculosis/drug therapy
2.
BMC Infect Dis ; 13: 240, 2013 May 24.
Article in English | MEDLINE | ID: mdl-23705640

ABSTRACT

BACKGROUND: The steady rise in the spread of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) requires rapid and reliable methods to identify resistant strains. The current molecular methods to detect MTB resistance to second-line drugs either do not cover an extended spectrum of mutations to be identified or are not easily implemented in clinical laboratories. A rapid molecular technique for the detection of resistance to second-line drugs in M. tuberculosis has been developed using hybridisation analysis on microarrays. METHODS: The method allows the identification of mutations within the gyrA and gyrB genes responsible for fluoroquinolones resistance and mutations within the rrs gene and the eis promoter region associated with the resistance to injectable aminoglycosides and a cyclic peptide, capreomycin. The method was tested on 65 M. tuberculosis clinical isolates with different resistance spectra that were characterised by their resistance to ofloxacin, levofloxacin, moxifloxacin, kanamycin and capreomycin. Also, a total of 61 clinical specimens of various origin (e.g., sputum, bronchioalveolar lavage) were tested. RESULTS: The sensitivity and specificity of the method in the detection of resistance to fluoroquinolones were 98% and 100%, respectively, 97% and 94% for kanamycin, and 100% and 94% for capreomycin. The analytical sensitivity of the method was approximately 300 genome copies per assay. The diagnostic sensitivity of the assay ranging from 67% to 100%, depending on the smear grade, and the method is preferable for analysis of smear-positive specimens. CONCLUSIONS: The combined use of the developed microarray test and the previously described microarray-based test for the detection of rifampin and isoniazid resistance allows the simultaneous identification of the causative agents of MDR and XDR and the detection of their resistance profiles in a single day.


Subject(s)
Drug Resistance, Multiple, Bacterial/genetics , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Oligonucleotide Array Sequence Analysis/methods , Tuberculosis, Multidrug-Resistant/microbiology , Aminoglycosides/pharmacology , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , DNA Gyrase/genetics , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Fluoroquinolones/pharmacology , Humans , Microbial Sensitivity Tests , Mycobacterium tuberculosis/classification , Mycobacterium tuberculosis/isolation & purification , Phenotype , Sensitivity and Specificity , Sputum/microbiology , Tuberculosis, Multidrug-Resistant/diagnosis
3.
J Antimicrob Chemother ; 67(9): 2107-9, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22593564

ABSTRACT

OBJECTIVES: Kanamycin is an important second-line drug used to treat multidrug-resistant (MDR) tuberculosis (TB). Molecular analysis of the rrs gene seems to be not enough to identify every case of kanamycin resistance. In the present study we evaluated the incidence of eis mutations in kanamycin-resistant Mycobacterium tuberculosis isolates. METHODS: We analysed 70 MDR M. tuberculosis clinical isolates. All isolates were screened for rrs and eis mutations using single-strand conformation polymorphism and sequencing. Phenotypic drug susceptibility testing was performed using Bactec MGIT 960 and the absolute concentration method on Lowenstein-Jensen medium. RESULTS: eis mutations were found in 10 isolates. The most prevalent mutations were the A1401G substitution in the rrs gene and the C14T substitution in the eis promoter region. CONCLUSIONS: Our study shows that the eis promoter region is a useful molecular marker of kanamycin resistance in the Moscow region. Complex analysis of rrs and eis mutations will significantly reduce the time to diagnose kanamycin resistance in TB patients, compared with phenotypic drug resistance testing.


Subject(s)
Antigens, Bacterial/genetics , Antigens, Bacterial/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Kanamycin Resistance , Mutation , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Tuberculosis, Multidrug-Resistant/microbiology , Acetyltransferases , Humans , Microbial Sensitivity Tests , Moscow , Mutant Proteins/genetics , Mutant Proteins/metabolism , Mycobacterium tuberculosis/isolation & purification , Polymorphism, Single-Stranded Conformational , Promoter Regions, Genetic , Sequence Analysis, DNA
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