Mycobacterium tuberculosis resistant to isoniazid and rifampicin detection by MAS-PCR system

B IMPERIALE; A. CATALDI; MORCILLO N

Florida

Jornada; Jornadas Interdisciplinarias y actualización en Trasplante pulmonar en Fibrosis Quistica; 2010

Hospital Dr. Antonio A. Cetrángolo

Objective: to detect point mutations in M. tuberculosis clinical isolates resistant to isoniazid (INH-R) and rifampicin (RIF-R). Methods: 168 isolates were recovered from solid and liquid media: 54 INH-R, 12 RIF-R, 64 multidrug-resistant and 38 fully drug susceptible (MTS) strains. Drug susceptibility testing was performed by indirect proportion method and MGIT 960. A multiplex allele-specific polymerase chain reaction (MAS-PCR) involving the genes rpoβ, katG, and the mabA-inhA promoter region, was used to detect point mutations related to the drug-resistant profile of the isolates. Results: Using MAS-PCR we found point mutations in 94 (79.6%) of INH-R strains: 57 (48.3%) mutated in katG codon 315; 34 (28.8%) with mutation in -15 of inhA, 3 (2.5%) with double mutation, and 24 (20.3%) without mutations in these genes. Fifty eight (76.3%) RIF-R strains showed mutation in the rpoβ gene. No mutations appeared in codon 516; 15 (19.7%) occurred in codon 526 and 43 (56.6%) in 531 of the rpoβ gene; 18 (23.7%) didn´t show mutations in these regions. MTS isolates were correctly identified. Sequencing of these genes was performed on resistant isolates. Correlation values between MAS-PCR and sequencing were 100.0% for 315 katG, 84.0 % for -15 inhA and 94.0% for rpoβ. Conclusions: MAS-PCR is a rapid and a low cost method, useful to detect mutations conferring INH-R and RIF-R from cultures. It could be used for a rapid detection of drug-resistance at clinical level and for describing point mutations in different geographical areas.