Relationship of NAT2, CYP2E1 and GSTM1/GSTT1 polymorphisms with mild elevation of liver enzymes in Brazilian individuals under anti-tuberculosis drug therapy.

The relationship of NAT2, CYP2E1 and GSTM1/GSTT1 polymorphisms with mild elevation of liver biomarkers was investigated in individuals under anti-tuberculosis drug therapy. Tuberculosis outpatients (18-70 y) with (n=59) and without (n=40) mild increase of liver enzymes (MILE) at two-month treatment were selected. Blood samples were obtained for DNA extraction and evaluation of serum markers of liver function. NAT2, CYP2E1 and GSTM1/GSTT1 polymorphisms were detected by DNA sequencing, PCR-RFLP, and PCR multiplex. Frequency of NAT2*5/*5 genotype was higher in MILE than in non-MILE group (p=0.04). Patients carrying NAT2*5/*5 genotype had increased susceptibility to MILE (OR: 9.00, 95CI: 1.46-55.48, p=0.018). CYP2E1*5B allele (*1A/*5B plus *5B/*5B genotypes) carriers had a trend for reduced risk for MILE (OR: 0.34, 95CI: 0.11-1.03, p=0.056) that was confirmed by lower levels of liver markers than CYP2E1*1A/*1A carriers after treatment (p<0.05). Moreover, increased post-treatment ALT, AST and total bilirubin were associated with GSTM1*1/GSTT1*1 genotypes (p<0.05). Patients taking CYP2E1 inhibitors had increased susceptibility to MILE (OR: 7.39, 95CI: 1.93-28.29, p=0.003), which was independent of the studied polymorphisms. These results are suggestive that NAT2, CYP2E1 and GSTM1/GSTT1 polymorphisms and concomitant use of CYP2E1 inhibitors contribute to the susceptibility to mild alterations in liver enzymes in patients under anti-tuberculosis drug therapy.

Transcriptional responses of host peripheral blood cells to tuberculosis infection.

Host responses following exposure to Mycobacterium tuberculosis (TB) are complex and can significantly affect clinical outcome. These responses, which are largely mediated by complex immune mechanisms involving peripheral blood cells (PBCs) such as T-lymphocytes, NK cells and monocyte-derived macrophages, have not been fully characterized. We hypothesize that different clinical outcome following TB exposure will be uniquely reflected in host gene expression profiles, and expression profiling of PBCs can be used to discriminate between different TB infectious outcomes. In this study, microarray analysis was performed on PBCs from three TB groups (BCG-vaccinated, latent TB infection, and active TB infection) and a control healthy group. Supervised learning algorithms were used to identify signature genomic responses that differentiate among group samples. Gene Set Enrichment Analysis was used to determine sets of genes that were co-regulated. Multivariate permutation analysis (p < 0.01) gave 645 genes differentially expressed among the four groups, with both distinct and common patterns of gene expression observed for each group. A 127-probeset, representing 77 known genes, capable of accurately classifying samples into their respective groups was identified. In addition, 13 insulin-sensitive genes were found to be differentially regulated in all three TB infected groups, underscoring the functional association between insulin signaling pathway and TB infection.

Screening and characterization of mutations in isoniazid-resistant Mycobacterium tuberculosis isolates obtained in Brazil.

We investigated mutations in the genes katG, inhA (regulatory and structural regions), and kasA and the oxyR-ahpC intergenic region of 97 isoniazid (INH)-resistant and 60 INH-susceptible Mycobacterium tuberculosis isolates obtained in two states in Brazil: São Paulo and Paraná. PCR-single-strand conformational polymorphism (PCR-SSCP) was evaluated for screening mutations in regions of prevalence, including codons 315 and 463 of katG, the regulatory region and codons 16 and 94 of inhA, kasA, and the oxyR-ahpC intergenic region. DNA sequencing of PCR amplicons was performed for all isolates with altered PCR-SSCP profiles. Mutations in katG were found in 83 (85.6%) of the 97 INH-resistant isolates, including mutations in codon 315 that occurred in 60 (61.9%) of the INH-resistant isolates and 23 previously unreported katG mutations. Mutations in the inhA promoter region occurred in 25 (25.8%) of the INH-resistant isolates; 6.2% of the isolates had inhA structural gene mutations, and 10.3% had mutations in the oxyR-ahpC intergenic region (one, nucleotide -48, previously unreported). Polymorphisms in the kasA gene occurred in both INH-resistant and INH-susceptible isolates. The most frequent polymorphism encoded a G(269)A substitution. Although KatG(315) substitutions are predominant, novel mutations also appear to be responsible for INH resistance in the two states in Brazil. Since ca. 90.7% of the INH-resistant isolates had mutations identified by SSCP electrophoresis, this method may be a useful genotypic screen for INH resistance.

Mutações no gene nat de isolados de Mycobacterium tuberculosis: efeito na atividade enzimática e no perfil de resistência à isoniazida / Mutation in the nat gene of Mycobacterium tuberculosis strains: effect on the enzymatic activity and on the isoniazid-resistance profile

Como entre 25%-50% dos isolados de Mycobacterium tuberculosis resistentes à INH não apresentam mutações nos genes katG, inhA, ahpC e kasA que possam justificar sua resistência, foi proposta a influência de mutações específicas no gene nat nos mecanismos de resistência e atividade da NAT. Todos os isolados obtidos (n=125) foram identificados e caracterizados através da amplificação pela PCR da IS6110 e por MIRU-VNTR, respectivamente. A determinação da concentração inibitória mínima (CIM) foi realizada pelo método REMA. Após triagem de mutações nos genes caracteristicamente envolvidos com resistência pela PCR-SSCP, seguida de seqüenciamento de DNA, foram selecionados 45 isolados para o estudo de mutações específicas (pela PCR e sequenciamento) e expressão gênica do mRNA do gene nat através da RT-PCR em tempo real. Confirmou-se que mutação no gene katG é a mais correlacionada com a resistência à INH, pois 68,4% das cepas resistentes apresentaram mutação neste gene. Mutações na região promotora do gene inhA, na região intergênica oxyR-ahpC e no gene kasA foram encontradas em 8,8%, 5,6% e 21,6% dos isolados, respectivamente...