Preliminary results of Multidrug-resistant M. tuberculosis by MAS-PCR

IMPERIALE BELÉN; MORCILLO NORA

Instituto Karolinska, Estocolmo, Suecia

Workshop; Primera reunión del proyecto “Development of a two-approach plate system for the fast and simultaneous detection of MDR and XDR M. tuberculosis” (FAST-XDR-DETECT).; 2008

Instituto Karolinska

Purpose: The aim of this study was to detect point mutations conferring isoniazid (INH) and rifampicin (RIF) resistance in clinical isolates from TB patients, by multiplex allele e specific PCR (MAS-PCR) system. Materials and Methods: In the study were included 35 RIF-coded strains, and 111 clinical isolates: 42 MDR, 7 RIF-R, 38 INH-R and 24 MTS. The reference strain H37Rv were used as a control in all the used methods. The cultures were obtained by Lowenstein-Jensen (LJ) and MGIT 960. Drug susceptibility testing to first line drugs were performed using indirect LJ and MGIT 960. A MAS-PCR involving the genes: katG (315), inhA (-15) and rpoB (516, 526, 531) were used to detect point mutations presumably related to the drug-resistant profile of the isolates. Results: Using MAS-PCR we could find point mutations presented in 62 (77.5%) of the INH-R strains: 41 (51.3%) with mutation in codon 315 of katG, 21 (26.2%) with mutations in the promoter region ( -15) of inhA and 18 (22.5%) strains without mutations for these studied genes. Fourty (81.6%) of the RIF-R strains showed mutation in these studied codons of the hot spot region of the rpoB gene. Both the drug-susceptible and drug-resistant coded strains were correctly identified by the MAS-PCR system. In clinical isolates no mutations appeared in codon 516 of the rpoB gene. Thirteen mutations (26.6%) occurred in codon 526 and 27 (55.1%) in codon 531 of rpoB gene. Conclusions: The MAS-PCR system 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 as well as to describe frequent point mutations in our geographical area.