Novel multiplex allele-specific PCR assays for the detection of resistance to second-line drugs in Mycobacterium tuberculosis.
OBJECTIVE
The use of rapid molecular assays for the detection of resistance to second-line drugs would significantly decrease the time delay in diagnosing drug-resistant tuberculosis (TB) that is associated with conventional phenotypic drug susceptibility testing. In this study, multiplex allele-specific (MAS)-PCR assays designed to detect the GyrA D94G and rrs A1401G mutations were evaluated for detection of ofloxacin and kanamycin resistance.


METHODS
GyrA D94G and rrs A1401G MAS-PCR assays were carried out on 288 Mycobacterium tuberculosis isolates. Phenotypic drug susceptibility testing of ofloxacin and kanamycin was performed on selected multidrug-resistant TB isolates using the indirect proportions method.


RESULTS
MAS-PCR assays detected GyrA D94G and rrs A1401G mutations in phenotypically resistant isolates with clinical sensitivities of 54.5% (6 of 11) and 90.0% (9 of 10), respectively, and specificities of 100% were obtained for both assays. A GyrA A90V mutation was identified in 4 of 11 (36.4%) ofloxacin-resistant isolates that did not carry a D94G substitution.


CONCLUSIONS
Rapid genotypic assays designed to detect GyrA D94G and A90V mutations and rrs A1401G mutations could detect up to 90.0% of extensively drug-resistant (XDR)-TB in the Western Cape region. The use of these assays in the clinical setting would significantly reduce the time to diagnosis of XDR-TB, enabling the administration of appropriate treatment regimens at the outset of therapy.
Evans
Joanna
J
Division of Medical Microbiology, Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Cape Town, South Africa.
Segal
Heidi
H
D000483:Alleles; D000995:Antitubercular Agents / Q000494:pharmacology*; D027081:DNA Gyrase / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D024881:Drug Resistance, Bacterial; D054908:Extensively Drug-Resistant Tuberculosis / Q000175:diagnosis; D005798:Genes, Bacterial; D020459:Genes, rRNA / Q000235:genetics; D005838:Genotype; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*
D000995:Antitubercular Agents; D004269:DNA, Bacterial; D027081:DNA Gyrase