RESUMO
BACKGROUND: Hepatitis E virus (HEV) is an increasing cause of acute viral hepatitis in developed countries. Serological testing alone may fail to diagnose acute infection, especially in immunocompromised patients, which justifies the use of molecular assays for diagnosis. Few studies have compared accuracy of HEV RNA detection assays. OBJECTIVES: The performances of five real-time PCR procedures for HEV RNA detection were compared. STUDY DESIGN: First, RNA quantification of hepatitis E diluted standards of 3a and 3b genotypes were performed. Secondly, forty-seven clinical samples of patients with known acute HEV infection were tested using five hepatitis E RNA detection methods of assigned letters A, B, C, D and E. RESULTS: Standards of HEV 3a genotype were detected in 100% of replicates with 2500 UI/ml of sensitivity by using A, B and C assays. Standards of HEV 3b genotype were more accurately detected with a sensitivity of 25 UI/ml in 100% of replicates using C assay and were detected in 100% of replicates with 2500 UI/ml of sensitivity by using A, B and E assays. Overall, B assay detected all of 250 UI/ml dilution and occasionally the 25 UI/ml dilution on both subtypes. The detection rates of clinical samples were 100%, 100% 97%, 97% and 83% for the respective A, B, C, D and E assay. Assays A and B were well correlated, independently of the subtype. However, discrepancies were observed when these techniques were compared to C, D and E assays according to the different subtypes. CONCLUSION: A and B assays appear reliable for HEV RNA detection. These assays target the ORF2/3 overlapping region, described as more conserved than ORF2.
Assuntos
Vírus da Hepatite E/isolamento & purificação , Hepatite E/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Virologia/métodos , Hepatite E/virologia , Vírus da Hepatite E/genética , Humanos , RNA Viral/genética , Sensibilidade e EspecificidadeRESUMO
Among hepatitis E virus (HEV) infections diagnosed in 2011 by the French Reference Centre for HEV, 9 were due to genotype 4, which until recently was limited to Asia. Sequences from autochthonous cases formed a single cluster very similar to Belgian swine sequences. Clinical presentation differed from genotype 3 infections.
Assuntos
Vírus da Hepatite E/classificação , Vírus da Hepatite E/genética , Hepatite E/epidemiologia , Hepatite E/patologia , Adulto , Idoso , Análise por Conglomerados , Feminino , França/epidemiologia , Genótipo , Hepatite E/virologia , Vírus da Hepatite E/isolamento & purificação , Humanos , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Dados de Sequência Molecular , Filogenia , RNA Viral/genética , Análise de Sequência de DNARESUMO
Mutations in the internal ribosome entry site (IRES) of hepatitis A virus (HAV) have been associated with enhanced in vitro replication and viral attenuation in animal models. To address the possible role of IRES variability in clinical presentation, IRES sequences were obtained from HAV isolates associated with benign (n = 8) or severe (n = 4) hepatitis. IRES activity was assessed using a bicistronic dual-luciferase expression system in adenocarcinoma (HeLa) and hepatoma (HuH7) cell lines. Activity was higher in HuH7 than in HeLa cells, except for an infrequently isolated genotype IIA strain. Though globally low, significant variation in IRES-dependent translation efficiency was observed between field isolates, reflecting the low but significant genetic variability of this region (94.2% +/- 0.5% nucleotide identity). No mutation was exclusive of benign or severe hepatitis, and variations in IRES activity were not associated with a clinical phenotype, indirectly supporting the preponderance of host factors in determining the clinical presentation.
Assuntos
Regiões 5' não Traduzidas/genética , Vírus da Hepatite A/genética , Vírus da Hepatite A/patogenicidade , Hepatite A/virologia , RNA Viral/genética , Doença Aguda , Adolescente , Adulto , Sequência de Bases , Linhagem Celular , Criança , Primers do DNA/genética , DNA Viral/genética , França , Variação Genética , Genótipo , Células HeLa , Vírus da Hepatite A/isolamento & purificação , Vírus da Hepatite A/fisiologia , Humanos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Conformação de Ácido Nucleico , Filogenia , Biossíntese de Proteínas , RNA Viral/química , Virulência/genética , Adulto JovemRESUMO
Investigation of hepatitis A virus (HAV) outbreaks often implies nucleotide sequence analysis. As an alternative method for the identification of related strains, single strand conformation polymorphism method (SSCP) was compared to sequence analysis. Twenty-three strains from sporadic and outbreak cases were studied retrospectively. SSCP, sequence identity and phylogenetic analyses were conducted on a 267 bp fragment of the VP1-2A variable region. The results of SSCP pattern comparison and sequence identity were highly correlated (r = 0.92, P < 0.001). If SSCP showed similar patterns, the VP1-2A fragments had a high and significant probability to have a sequence identity over 99.6%. Results were concordant for outbreak strains. The only discordant result concerned a cluster of three sporadic cases evidenced by phylogenetic analysis while SSCP showed similar patterns for only two of these three cases. A prospective SSCP analysis of a recent HAV outbreak confirmed the reliability of this technique. SSCP may thus provide a rapid and cost-effective tool for preliminary investigation of HAV outbreaks, before undertaking exhaustive nucleotide sequence analysis.
