Screening and characterization of mutations in isoniazid-resistant Mycobacterium tuberculosis isolates obtained in Brazil.
We investigated mutations in the genes katG, inhA (regulatory and structural regions), and kasA and the oxyR-ahpC intergenic region of 97 isoniazid (INH)-resistant and 60 INH-susceptible Mycobacterium tuberculosis isolates obtained in two states in Brazil: São Paulo and Paraná. PCR-single-strand conformational polymorphism (PCR-SSCP) was evaluated for screening mutations in regions of prevalence, including codons 315 and 463 of katG, the regulatory region and codons 16 and 94 of inhA, kasA, and the oxyR-ahpC intergenic region. DNA sequencing of PCR amplicons was performed for all isolates with altered PCR-SSCP profiles. Mutations in katG were found in 83 (85.6%) of the 97 INH-resistant isolates, including mutations in codon 315 that occurred in 60 (61.9%) of the INH-resistant isolates and 23 previously unreported katG mutations. Mutations in the inhA promoter region occurred in 25 (25.8%) of the INH-resistant isolates; 6.2% of the isolates had inhA structural gene mutations, and 10.3% had mutations in the oxyR-ahpC intergenic region (one, nucleotide -48, previously unreported). Polymorphisms in the kasA gene occurred in both INH-resistant and INH-susceptible isolates. The most frequent polymorphism encoded a G(269)A substitution. Although KatG(315) substitutions are predominant, novel mutations also appear to be responsible for INH resistance in the two states in Brazil. Since ca. 90.7% of the INH-resistant isolates had mutations identified by SSCP electrophoresis, this method may be a useful genotypic screen for INH resistance.
Department of Clincal Analysis, State University of Maringá, Paraná, Brazil.
Rosilene Fressatti
RF
Cardoso
Robert C
RC
Cooksey
Glenn P
GP
Morlock
Patricia
P
Barco
Leticia
L
Cecon
Francisco
F
Forestiero
Clarice Q F
CQ
Leite
Daisy N
DN
Sato
Maria de Lourdes
Mde L
Shikama
Elsa M
EM
Mamizuka
Rosario D C
RD
Hirata
Mario H
MH
Hirata
D000995:Antitubercular Agents / Q000494:pharmacology*; D001938:Brazil; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000235:genetics; D024881:Drug Resistance, Bacterial; D005798:Genes, Bacterial / Q000235:genetics; D007538:Isoniazid / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D009154:Mutation / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics*; D011110:Polymorphism, Genetic / Q000235:genetics; D018807:Polymorphism, Single-Stranded Conformational; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014376:Tuberculosis / Q000382:microbiology*
D000995:Antitubercular Agents; D004269:DNA, Bacterial; D007538:Isoniazid