Detection of Mycobacterium tuberculosis resistance mutations to rifampin and isoniazid by real-time PCR.
OBJECTIVE
The objective of our study was to evaluate the use of a real-time polymerase chain reaction (PCR)-based technique for the prediction of phenotypic resistance of Mycobacterium tuberculosis.


METHODS
We tested 67 M tuberculosis strains (26 drug resistant and 41 drug susceptible) using a method recommended for the LightCycler platform. The susceptibility testing was performed by the absolute concentration method. For rifampin resistance, two regions of the rpoB gene were targeted, while for identification of isoniazid resistance, we searched for mutations in katG and inhA genes.


RESULTS
The sensitivity and specificity of this method for rapid detection of mutations for isoniazid resistance were 96% (95% CI: 88% to 100%) and 95% (95% CI: 89% to 100%), respectively. For detection of rifampin resistance, the sensitivity and specificity were 92% (95% CI: 81% to 100%) and 74% (95% CI: 61% to 87%), respectively. The main isoniazid resistance mechanism identified in our isolates is related to changes in the katG gene that encodes catalase. We found that for rifampin resistance the concordance between the predicted and observed phenotype was less than satisfactory.


CONCLUSIONS
Using this method, the best accuracy for genotyping compared with phenotypic resistance testing was obtained for detecting isoniazid resistance mutations. Although real-time PCR assay may be a valuable diagnostic tool, it is not yet completely satisfactory for detection of drug resistance mutations in M tuberculosis.
Hristea
A
A
Prof Dr Matei Bals National Institute for Infectious Diseases, Str Calistrat Grozovici, Nr 1, Sector 2, 021105 Bucharest, Romania.
Otelea
D
D
Paraschiv
S
S
Macri
A
A
Baicus
C
C
Moldovan
O
O
Tinischi
M
M
Arama
V
V
Streinu-Cercel
A
A
D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics; D002374:Catalase / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D024881:Drug Resistance, Bacterial; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests / Q000379:methods; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics; D010088:Oxidoreductases / Q000235:genetics; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012293:Rifampin / Q000494:pharmacology*; D012680:Sensitivity and Specificity
D000995:Antitubercular Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D010088:Oxidoreductases; D002374:Catalase; C480353:katG protein, Mycobacterium tuberculosis; C085257:InhA protein, Mycobacterium; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit; D007538:Isoniazid; D012293:Rifampin