Analysis of mutations in genes associated with resistance to isoniazid (inh) in Mycobacterium tuberculosis Brazilian clinical isolates

Análise de mutações nos genes relacionados com resistência a isoniazida (INH) em isolados clínicos de Mycobacterium tuberculosis de origem brasileira

SUMMARY OF THESIS^*

Analysis of mutations in genes associated with resistance to isoniazid (inh) in Mycobacterium tuberculosis Brazilian clinical isolates

Rosilene Frenatti Cardoso

Laboratório de Bacteriologia Clínica, Universidade Estadual de Maringá, Bloco J-90 - Sala 05, 87000-000 MARINGÁ - PARANÁ, e-mail: [e-mail], [e-mail]

CARDOSO, Rosilene Fressatti - Análise de mutações nos genes relacionados com resistência a isoniazida (INH) em isolados clínicos de Mycobacterium tuberculosis de origem brasileira. São Paulo, 2003. (Tese de Doutorado - Faculdade de Ciências Famacêuticas da Universidade de São Paulo).

In the present study the mutations frequencies in the katG, kasA, inhA genes and oxyR-ahpC intergenic region of 97 resistant and 60 sensitive Mycobacterium tuberculosis clinical isolates from Brazil were investigated. The mutations screening were carried out by PCR-SSCP and confirmed by automated sequencing. The minimal inhibitory concentration for INH was done by the microplate Alamar Blue Assay (MABA). The molecular differentiation of INH resistant and sensitive isolates was carried out by Spoligotyping of the locus DR. We found 40 different Spoligotyping patterns when analyzing 97 INH resistant clinical isolates and 30 in 60 INH sensitive isolates. Our data showed no resistant isolates had a complete deletion of the katG gene. However, one resistant isolate that had a deletion at region 3' in katG gene was found and a high percentage of mutation in this gene (85.6%) mainly at codon 315 (61.9%). Twenty-five new mutations previously not described in the literature, were detected in katG gene in all resistant isolates studied and 25.8% had mutation in inhA promoter region, 5.82% had mutation in inhA structural region and 10.3% at oxyR-ahpC intergenic region where one of this is a new mutation (-48) previously not described in the literature. It was observed in kasA gene mutation in resistant and sensitive isolates. The most frequent mutation observed in kasA gene was at codon 269 that will cause alteration G269S (23.7%). Other silent mutations were detected only in INH sensitive isolates, however with a low frequency. PCR-SSCP showed good sensibility (90.7%) to detect mutations in INH resistant isolates, analyzing 5 genic regions (315 codon region of katG gene, inhA promoter and inhA structural region and oxyR-ahpC intergenic region) by PCR-SSCP. PCR-SSCP showed agreement in 100% when compared to sequencing. We can thus conclude that mutation screening by PCR-SSCP can be used to detect resistance to INH in M. tuberculosis.

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This thesis is available at the Library of the Instituto de Medicina Tropical de São Paulo

Publication Dates

• Publication in this collection
  29 Mar 2004
• Date of issue
  Feb 2004