RESUMO
INNO-LiPA Mycobacteria (LiPA; Innogenetics, Zwijnaarde, Belgium) is a kit for the simultaneous detection and identification of Mycobacterium species in culture and identifies the Mycobacterium tuberculosis complex, the M. avium complex (MAC), and the following Mycobacterium species: M. kansasii, M. avium, M. intracellulare, M. scrofulaceum, M. gordonae, M. xenopi, and the M. chelonae-M. abscessus complex. The assay, which targets the 16S-23S rRNA spacer region, was evaluated on 157 mycobacterial strains that had been identified by conventional techniques and PCR-restriction enzyme analysis of the hsp65 gene (PRA). Forty-seven reference strains consisting of 37 different species and 110 human clinical isolates were submitted to the test, and all were hybridized with the Mycobacterium genus probe (MYC) on the LiPA strip (100% sensitivity). Ninety-four isolates hybridized to their corresponding species- or complex-specific probes; only one isolate phenotypically identified as M. gordonae did not react with its specific probe (99.4% accuracy). Thirty-seven MAC strains were phenotypically identified to the complex level and to the species level by LiPA as M. avium (n = 18) or M. intracellulare (n = 7) or as belonging to the M. avium-M. intracellulare-M. scrofulaceum complex (n = 12). Of the last 12 strains, 10 had M. avium PRA patterns and 2 had M. intracellulare PRA patterns. Three isolates that had been identified as a single species by conventional identification were proven to be mixed cultures by the LiPA assay. The whole procedure can be performed in 1 working day, starting with the supernatant of a small amount of bacterial mass that had been treated by freezing and then boiling.
Assuntos
Proteínas de Bactérias , DNA Espaçador Ribossômico/genética , Infecções por Mycobacterium/microbiologia , Mycobacterium/classificação , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Kit de Reagentes para Diagnóstico , Chaperonina 60 , Chaperoninas/genética , Sondas de DNA , Humanos , Mycobacterium/genética , Hibridização de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Especificidade da Espécie , Fatores de TempoRESUMO
Since the introduction of antiviral compounds such as lamivudine and famciclovir in the treatment schedules of patients with chronic hepatitis B virus (HBV) infection, the accumulation of a variety of mutations in the HBV polymerase gene has been observed. The selection of these mutations is generally considered the cause of viral nonresponsiveness and treatment failure. Therefore, the detection of these mutations is of clinical importance. Previously genotyped HBV strains isolated from treated and untreated patients were amplified with primers specific for the HBV polymerase region from amino acids 465 to 562. Amplified products were cloned into plasmid vectors. The clones were used as reference strains. A set of 38 highly specific oligonucleotide probes covering three different codon positions, L528M, M552V/I, and V/L/M555I, were selected. These probes were applied as 19 different lines on a membrane strip. The strips were then hybridized with PCR fragments from the reference panel, revealing the amino acids at the three codon positions simultaneously for each clone. PCR products generated from two patients infected with HBV genotypes A and C, respectively, and treated with nucleoside analogs were analyzed on these strips. A gradual increase in genetic HBV polymerase complexity was observed in follow-up samples compared to that in pretreatment samples. Additional analysis of HBV polymerase DNA fragments in recombinant plasmid clones demonstrated the existence of (i) clones with double mutations, (ii) clones with single mutations at either codon 528, 552, or 555, and (iii) the simultaneous occurrence of two or more viral populations within one sample. This line probe assay detected the complex quasispecies nature of HBV and provided some insight into the dynamics of resistance mutations.
Assuntos
Antivirais/farmacologia , DNA Polimerase Dirigida por DNA/genética , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/genética , Hepatite B/virologia , Sequência de Aminoácidos , Antivirais/uso terapêutico , Sequência de Bases , DNA Viral/genética , DNA Polimerase Dirigida por DNA/química , Resistência Microbiana a Medicamentos/genética , Hepatite B/tratamento farmacológico , Vírus da Hepatite B/enzimologia , Humanos , Dados de Sequência Molecular , Mutação , Sondas de Oligonucleotídeos , Plasmídeos/genética , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de DNARESUMO
Fulminant and severe viral hepatitis are frequently associated with mutant hepatitis B virus (HBV) strains. In this study, the genetic background of a viral strain causing severe subfulminant outcome in heart-transplanted patients was studied and compared with viral hepatitis B strains that were not linked to severe liver disease in the same setting. A total of 46 patients infected nosocomially with HBV genotype A were studied. Five different viral strains were detected, infecting 3, 9, 5, 24, and 5 patients, respectively. Only one viral strain was found to be associated with the subfulminant outcome and 3 patient deaths as a consequence of severe liver disease. The remaining 43 patients with posttransplantation HBV infection did not show this fatal outcome. Instead, symptoms of hepatitis were generally mild or clinically undiagnosed. Comparison of this virus genome with the four other strains showed an accumulation of mutations in the basic core promoter, a region that influences viral replication, but also in hepatitis B X protein (HBX) (7 mutant motifs), core (10 mutant motifs), the preS1 region (5 mutant motifs), and the HBpolymerase open reading frame (17 motifs). Some of these variations, such as those in the core region, were located on the tip of the protruding spike of the viral capsid (codons 60 to 90), also known in part as an important HLA class II-restricted epitope region. These mutations might therefore influence the immune-mediated response. The viral strain causing subfulminant hepatitis was, in addition, the only strain with a preCore stop codon mutation and, thus, hepatitis B e antigen (HBeAg) expression was never observed. The combination of these specific viral factors is thought to be responsible for the fatal outcome in these immune-suppressed heart-transplant recipients.
Assuntos
Infecção Hospitalar/transmissão , Transplante de Coração , Encefalopatia Hepática/etiologia , Vírus da Hepatite B/genética , Hepatite B/transmissão , Complicações Pós-Operatórias , Sequência de Bases , Estudos de Casos e Controles , Sequência Consenso , Infecção Hospitalar/fisiopatologia , DNA Viral/sangue , DNA Viral/genética , Evolução Fatal , Genótipo , Hepatite B/fisiopatologia , Anticorpos Anti-Hepatite B/sangue , Antígenos de Superfície da Hepatite B/sangue , Vírus da Hepatite B/isolamento & purificação , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação , Reação em Cadeia da Polimerase , Estudos Retrospectivos , Alinhamento de SequênciaRESUMO
A panel of samples, previously typed by serology, was retyped using a line probe assay. One sample from a Brazilian Caucasian individual was serologically typed as B52/B39, but showed an aberrant HLA-B pattern on the diagnostic strip and was typed as B*52012/B*39new. Further analysis by allele-specific amplification and subsequent sequencing of exons 2 and 3 revealed a G(B*3908)-to-T nucleotide substitution at position 467 (codon 156) resulting in an Arg (B*3908)-to-Leu substitution. Furthermore, the sequence revealed a silent mutation at position 174 (codon 58): a G(B*3908)-to-A nucleotide switch. The sequence has been sent to the EMBL databank and the HLA Nomenclature Committee, and the allele was named B*3913.
