title: Drug resistance in Mycobacterium tuberculosis clinical isolates from Brazil: phenotypic and genotypic methods.
abstract: We determined the susceptibility profile of 80 Mycobacterium tuberculosis (MTB) clinical isolates from Brazil against isoniazid (INH) and rifampicin (RIF) drugs by two phenotypic methods (Resazurin Microtiter Assay - REMA and BACTEC™ MGIT™ Mycobacterial Detection System). DNA polymorphisms were also determined by PCR-SSCP in isolates resistant to INH and RIF. BACTEC™ MGIT™ 960 detected 22 susceptible isolates to INH and RIF, 48 MDR isolates (resistant at least to INH and RIF) and nine mono-resistant isolates (eight to INH and one to RIF). REMA performance was determined by Receiver Operating Characteristic curve, whose assay was validated utilizing as reference the BACTEC™ MGIT™ 960 system. ROC curve showed cut-off values of 0.0625μg/mL and 0.125μg/mL, for INH and RIF, respectively. REMA-INH demonstrated sensitivity and specificity of 100% while REMA-RIF showed sensitivity of 97.2% and specificity of 100%. PCR-SSCP detected DNA polymorphisms in 87.5% and 75.5% of isolates classified as INH-resistant and RIF-resistant, respectively. One discordant sample found to RIF (resistant by BACTEC™ MGIT™ 960 and susceptible by REMA) showed no mutation by PCR-SSCP. In conclusion, our studies demonstrated that the combination of phenotypic method REMA, which allowed rapid detection of MDR-MTB with higher levels of sensitivity and specificity, with the genotypic method PCR-SSCP, which demonstrated high accuracy in the search of polymorphisms in the resistance genes, proved to be a useful strategy to study MDR-MTB clinical isolates from national reference center located in São Paulo city.
authors: School of Pharmaceutical Sciences, Biological Sciences Department, Paulista State University, Rodovia Araraquara-Jaú km 1, 14800-901 Araraquara, SP, Brazil. marcelo_miyatabr@yahoo.com.br
authors: Marcelo
authors: M
authors: Miyata
authors: Fernando Rogério
authors: FR
authors: Pavan
authors: Daisy Nakamura
authors: DN
authors: Sato
authors: Leonardo Biancolino
authors: LB
authors: Marino
authors: Mario Hiroyuki
authors: MH
authors: Hirata
authors: Rosilene Fressati
authors: RF
authors: Cardoso
authors: Fernando Augusto Fiúza
authors: FA
authors: de Melo
authors: Cleslei Fernando
authors: CF
authors: Zanelli
authors: Clarice Queico Fujimura
authors: CQ
authors: Leite
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology; D001938:Brazil; D024901:Drug Resistance, Multiple, Bacterial; D005798:Genes, Bacterial; D019450:Genes, MDR; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D058889:Molecular Typing; D009169:Mycobacterium tuberculosis / Q000145:classification* / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D015203:Reproducibility of Results; D012293:Rifampin / Q000494:pharmacology; D012680:Sensitivity and Specificity; D018088:Tuberculosis, Multidrug-Resistant / Q000175:diagnosis / Q000188:drug therapy / Q000382:microbiology*
chemical_list: D000995:Antitubercular Agents; D007538:Isoniazid; D012293:Rifampin