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Antimicrobial Agents and Chemotherapy, September 2004, p. 3373-3381, Vol. 48, No. 9
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.9.3373-3381.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Screening and Characterization of Mutations in Isoniazid-Resistant Mycobacterium tuberculosis Isolates Obtained in Brazil

Department of Clinical Analysis, State University of Maringá, Paraná,¹ Department of Biologic Sciences, Paulista State University, Paulista,⁴ Institute Adolfo Lutz, Ribeirão Preto,⁵ Institute Adolfo Lutz, Sorocaba,⁶ University of São Paulo, São Paulo, Brazil,² Division of AIDS, STD, and TB Laboratory Research, National Center for HIV, STD, and TB Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia³

Received 12 April 2004/ Returned for modification 20 May 2004/ Accepted 24 May 2004

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 G₂₆₉A substitution. Although KatG₃₁₅ 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.

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