“Preliminary study of Mycobacterium tuberculosis clinical isolates resistant to isoniazid and rifampicin by MAS-PCR”.

IMPERIALE BELÉN; MORCILLO NORA; CATALDI ANGEL

Plovdiv

Congreso; 29 Congreso Anual de la Sociedad Europea de Micobacteriología (ESM); 2008

Sociedad Europea de Micobacteriología (ESM)

Introduction: Timely detection of drug resistance (DR) and multidrug-resistance to anti-tuberculosis agents is important in the effective management of tuberculosis (TB) cases. Purpose: The aim of this study was to detect point mutations conferring isoniazid (INH) and rifampicin (RIF) resistance in clinical isolates from patients with TB. Methods: Drug susceptibility testing (DST) to first-line drugs were performed by indirect proportion method on Löwenstein Jensen (ILJ) and MGIT 960. A total of 30 laboratory strains plus the reference strain H37Rv were subcultured on LJ and included in the study. A multiplex allele-specific polymerase chain reaction (MAS-PCR) involving the genes rpoB, katG, and the mabA-inhA promoter region was used to detect the point mutations presumably related to the DR profile of the isolates. Results: Seventy nine DR clinical isolates were recovered from LJ and MGIT 960 media: 71 isolates were resistant to INH (INH-R) and 42 to RIF (RIF-R). Using MAS-PCR we found point mutations in 55 (77.5%) of the INH-R strains: 36 (50.7%) with mutation in codon 315 of katG; 19 with a mutation in the promoter region (-15) of inhA (27.0%); and 16 (22.5%) without mutations detected. Thirty four (81.0%) of the RIF-R strains showed mutations in the rpoB gene studied codons. Both drug-susceptible and DR laboratory strains were correctly identified by MAS-PCR system. In clinical isolates 22 (52.4%) mutations were found in codon 531, 12 (28.6%) in 526, and no mutations appeared in codon 516 of rpoB gene. Conclusions: The MAS-PCR used in this preliminary study allowed us to detect mutations conferring resistance to INH and RIF from pure cultures, within a working day and with a low cost. Therefore it could be a useful tool for a rapid detection of drug-resistance at clinical level and for describing frequent point mutations in our geographical area.