Comparison of polymerase chain reaction single-strand conformation polymorphism with DNA sequencing to detect drug resistance of mycobacterium tuberculosis isolates

Objectives: Advancements in molecular technology increased our understanding of genetic mechanism of drug resistance. Nowadays, the chance of rapid detection of resistant Mycobacterium tuberculosis (M. tuberculosis) strains is increased. In the present study, we aimed to investigate the sensitivity and specificity of PCR-SSCP for detecting susceptible and resistant strains of M. tuberculosis compared with DNA sequencing.

To calculate the sensitivity and specificity of PCR-SSCP assay to detect drug resistance in M. tuberculosis, respiratory samples were collected from suspected patients referred to Mycobacteriology Research Center (Masieh Daneshvary Hosptial) since 2002. Susceptibility testing against first line drugs was performed on 74 culturepositive specimens. Consequently, PCR-SSCP and DNA sequencing were performed on katG, inhA, ahpC and rpoB genes.

Results: Drug-susceptibility testing by the proportional method in selected samples revealed 16 MDR (21.6%), 23 mono-drug resistant (31%) and 35 susceptible strains (47.3%). In comparison with DNA sequencing as a gold standard for molecular methods, the sensitivity of PCR-SSCP assay for detecting of mutation in 315 codon of katG gene was 94.74% (CI=73.97%-99.87%) with 100% (CI=93.51%-100%) specificity. In contrast, the sensitivity and specificity of this assay in detecting of rpoB gene were 70.8 (CI=48.91%-87.38%) and 88 (CI=75.69%-95.47%), respectively.

Conclusion: PCR-SSCP in combination with DNA sequencing can be used as screening method to detect MDR-TB and mono-drug resistant cases.