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Abstract
BackgroundThe 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.MethodsThe 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.ResultsThe 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.ConclusionsThe 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.
Highlights
The steady rise in the spread of multidrug-resistant tuberculosis (MDR-TB) and extensively drugresistant tuberculosis (XDR-TB) requires rapid and reliable methods to identify resistant strains
Determination of minimum inhibitory concentrations (MICs) using Bactec MGIT 960 Among the 65 M. tuberculosis clinical isolates that were obtained from sputum samples, 42 (65%) were resistant to OFX, and most of the OFX-resistant strains were resistant to LVX (n = 37) and MFX (n = 40), see Table 1
There was a strong correlation between the minimum inhibitory concentrations (MICs) of both fluoroquinolones
Summary
The steady rise in the spread of multidrug-resistant tuberculosis (MDR-TB) and extensively drugresistant 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 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. 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, as early identification allows adequate. The prescription of second-line anti-TB drugs should be based on knowledge of the causative agent’s resistance and the patient’s treatment history. Including the gyrB gene as a target appears reasonable to improve the molecular detection of FQ resistance [8]
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