title: Genomic mutations in the katG, inhA and aphC genes are useful for the prediction of isoniazid resistance in Mycobacterium tuberculosis isolates from Kwazulu Natal, South Africa.
abstract: Genotypic analysis of isoniazid (INH) resistance in 79 isolates of M. tuberculosis (MTB) was undertaken by PCR-single strand conformation polymorphism (SSCP), Msp1 restriction enzyme analysis and sequence analysis of specific regions of three genes (part of the coding sequence of katG, and promoter regions of the inhA operon and ahpC) in order to determine the particular allelic variants within these genes. The epidemiologic relatedness was determined using IS6110 and polymorphic G-C region (PGRS (MTB484(1)) based restriction fragment length polymorphism (RFLP). Mutations in katG, inhA locus and ahpC were identified in 77/79, 19/79 and 10/79 isolates respectively. The ability of PCR-SSCP to detect mutations associated with INH resistance in katG, inhA and ahpC genes was 100% (CI 91.2-99.7%), 98.7% (CI 74.0-99.9%), and 100% (CI 69.2-100%) respectively. Specificity was 100%. All isolates with mutations in the 209 bp fragment of the MTB katG gene containing the Ser315Thr codon were positive by PCR-RFLP using Msp1 enzyme restriction analysis. Sixteen of 19 isolates with alterations on the 3' end of the ribosome binding site upstream of mabA in inhA locus simultaneously harbored Ser315Thr mutations in KatG. In 9/10 isolates, mutations in the ahpC promoter region were located in the 105 bp oxyR-ahpC intergenic region. None of 17 INH drug susceptible isolates harbored mutations in any of the three genetic regions, although the katG1 allele (Arg 463 Leu) was present in one isolate. Characterization by IS6110/PGRS(MTB484(1))RFLP analysis revealed that a number of drug resistant clones are widespread in the community. We conclude that the frequency of the Ser315Thr katG mutation in the local strain population makes the PCR-RFLP MTB katG assay a reliable, rapid and useful method for detecting INH resistance.
authors.affiliation: Department of Virology, University of Natal, Faculty of Medicine, Durban, South Africa. kiepiela@med.und.ac.za
authors.forename: P
authors.initials: P
authors.lastname: Kiepiela
authors.forename: K S
authors.initials: KS
authors.lastname: Bishop
authors.forename: A N
authors.initials: AN
authors.lastname: Smith
authors.forename: L
authors.initials: L
authors.lastname: Roux
authors.forename: D F
authors.initials: DF
authors.lastname: York
mesh_terms: D000483:Alleles; D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins; D004252:DNA Mutational Analysis; D004352:Drug Resistance, Microbial / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology*; D009154:Mutation / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics*; D010088:Oxidoreductases / Q000235:genetics; D010544:Peroxidases / Q000235:genetics; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D011237:Predictive Value of Tests; D011401:Promoter Regions, Genetic / Q000235:genetics; D012680:Sensitivity and Specificity; D013019:South Africa
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D010088:Oxidoreductases; D010544:Peroxidases; C076136:catalase HPI; C085257:InhA protein, Mycobacterium; D007538:Isoniazid