RESUMO
OBJECTIVES: Genetic variability in NS5A is associated with different levels of resistance to the currently licensed NS5A inhibitors. The aim of this study was to detect NS5A inhibitor resistance associated substitutions (RASs) in hepatitis C virus (HCV) genotype 1 (GT1) patients who are naive to direct-acting HCV antivirals. METHODS: Amplification, Sanger sequencing and phylogenetic analysis of the HCV NS5A region were performed on plasma obtained from 122 consecutive patients with HCV chronic infection attending four different clinics in Italy. RESULTS: NS5A inhibitor RASs were detected in 14/61 (23.0%) HCV GT1b and 3/61 (4.9%) HCV GT1a infected patients (p 0.007). The pan-genotypic RAS Y93H was detected in 1 (1.6%) GT1a and 4 (6.6%) GT1b patients. GT1a sequences clustered into two different clades with RASs detected in 1/34 (2.9%) clade I and 2/27 (7.4%) clade II sequences. CONCLUSIONS: Although the impact of naturally occurring NS5A RASs might be limited with upcoming pan-genotypic treatment regimens, this information is still useful to map naturally occurring HCV variants in different geographic areas in the context of current HCV therapy.
Assuntos
Farmacorresistência Viral , Genótipo , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Hepatite C Crônica/virologia , Mutação de Sentido Incorreto , Proteínas não Estruturais Virais/genética , Feminino , Frequência do Gene , Humanos , Itália , Masculino , Pessoa de Meia-Idade , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
To evaluate the impact of highly active antiretroviral therapy (HAART) on the course of hepatitis C (HCV) infection, we studied the biological and virological characteristics of 23 HCV/HIV-coinfected HAART-naive patients. The HCV genotype, HCV and HIV viral loads, serum alanine aminotransferase, CD4+ and CD8+ cell/mm3 were determined at baseline, 1 month, 6 months and 12 months after initiation of HAART. Results were analyzed both in terms of total population and of HCV genotype. The study of the total population suggests that this therapy did not determine a significant alteration of HCV viremia and levels of ALT, while a significant decrease in HIV viremia (-1.7log10 at one year from the start of HAART) and increase in CD4+ counts was observed (P < 0.005). The biological and virological parameters of HCV/HIV coinfection differed according to the HCV genotype. In particular, only genotype 4 showed a significant inverse correlation between HCV and HIV viral loads.
Assuntos
Terapia Antirretroviral de Alta Atividade , Infecções por HIV/complicações , Hepacivirus/classificação , Hepacivirus/genética , Hepatite C/complicações , Adulto , Alanina Transaminase/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Genótipo , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/fisiologia , Hepacivirus/isolamento & purificação , Hepatite C/tratamento farmacológico , Hepatite C/imunologia , Hepatite C/virologia , Humanos , Estudos Longitudinais , Contagem de Linfócitos , Masculino , RNA Viral/sangue , Carga ViralRESUMO
It has been postulated that host factors, such as the human leucocyte antigen (HLA) system, may play a predominant role in the pathogenesis of HCV-related extra-hepatic manifestations. This study was performed to investigate the role of HLA- DR and DQ alleles in a group of Italian patients, with HCV infection and associated extrahepatic manifestations and to test whether an association between HCV genotype, HLA locus and clinical or serological manifestations can be demonstrated. Thirty unrelated patients affected by HCV infection with extra-hepatic manifestations were consecutively included in the study. One hundred and sixty-three HCV patients without extrahepatic manifestations were tested as controls for the prevalence of HCV genotypes, and 283 healthy donors were used as controls for HLA class II alleles distribution. HCV-RNA was quantified by an reverse transcription-PCR. HLA class II alleles typing was performed using a standard microlymphocytotoxicity assay on B lymphocyte purified. HCV 2c genotype was found in 53.3% compared to 18.4% of controls (p=0.00001; OR=5.1). Cryoglobulins were detected in 72.7% DR6+ patients and in 31.6% DR6- patients (p=0.05; OR=3.21). Rheumatoid factor was found in 90.9% of DR6+ patients and in 42.1% DR6- patients (p=0.018; OR 13.7). Only two DR5+ patients (20%) had cryoglobulinemia, while 6 patients (30%) in the DR5- group had cryoglobulinemia (p=0.02; OR=0.07). Associations were found between DR7 and ANA (OR=1.74) and between DQ2 and ANA (OR=1.97). According to our findings HLA-DR6 might play an important role in developing extra-hepatic manifestations and genotype 2c could be considered as a risk factor for their onset.
Assuntos
Alelos , Genes MHC da Classe II , Genótipo , Hepacivirus/genética , Hepatite C/genética , Hepatite C/virologia , Idoso , Linfócitos B/metabolismo , Crioglobulinemia/metabolismo , Crioglobulinas/metabolismo , Feminino , Antígenos HLA-DQ/metabolismo , Antígeno HLA-DR6/metabolismo , Hepacivirus/metabolismo , Hepatite C/complicações , Humanos , Fígado/metabolismo , Masculino , Pessoa de Meia-Idade , Razão de Chances , RNA/química , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de RiscoRESUMO
A multiplex polymerase chain reaction (PCR) was applied to clinical samples for simultaneous detection of hepatitis C virus (HCV) and GBV-C/HGV genome. With both RNA viruses, the amplification was performed with primers of the 5' UTR region starting from the single viral RNA reverse transcripted (cDNA) with random hexanucleotide primer mix. GBV-C/HGV RNA was detected in plasma sample of seven out of 50 transfused patients (14%). The multiplex PCR demonstrated a sensitivity up to 7.8 x 10(2) copies/ml respectively for GBV-C/HGV and HCV RNA in plasma samples of 5/50 patients with GBV-C/HGV/HCV co-infection and in patients with HCV (27/50) or GBV-C/HGV infection alone (2/50).
Assuntos
Transfusão de Sangue , Flaviviridae , Hepatite C/diagnóstico , Hepatite Viral Humana/diagnóstico , Reação em Cadeia da Polimerase , Flaviviridae/genética , Flaviviridae/isolamento & purificação , Genoma Viral , Hepacivirus/genética , Hepacivirus/isolamento & purificação , Hepatite C/complicações , Hepatite Viral Humana/complicações , Humanos , RNA Viral/sangue , Sensibilidade e Especificidade , Transcrição GênicaRESUMO
The water protein spin-lattice relaxation time (T1) was measured in suspensions of human peripheral blood mononuclear cells (PBMC), uninfected or infected with type 1 herpes simplex virus, human cytomegalovirus and rubella virus. In the infected samples, T1 enhancements, which linearly depend on virus concentration, were observed. This T1 increase can be related to the early changes induced by the virus adsorption of the cells, not always confirmed by virus-induced cytopathic effect (CPE) in cocultures of infected PBMC and other sensitive cells. Compared with other conventional virological techniques, this NMR method seems to be rapid and sensitive. The NMR response was reproducible and specific, since neutralization of the viral infection by homologous antisera consistently matched the neutralization of the virus-induced NMR effects. These observations suggest that fast and sensitive 1H-NMR relaxation techniques can be implemented in virological diagnosis directly on pathological materials.
Assuntos
Infecções por Citomegalovirus/diagnóstico , Herpes Simples/diagnóstico , Leucócitos Mononucleares/microbiologia , Espectroscopia de Ressonância Magnética , Rubéola (Sarampo Alemão)/diagnóstico , Anticorpos Antivirais/imunologia , Linhagem Celular , Citomegalovirus/imunologia , Citomegalovirus/fisiologia , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 1/fisiologia , Humanos , Testes de Neutralização , Vírus da Rubéola/imunologia , Vírus da Rubéola/fisiologia , Sensibilidade e EspecificidadeRESUMO
We have mapped continuous epitopes, for positions 591-673 of the human cytomegalovirus 58-kDa glycoprotein using overlapping synthetic peptides and human sera. This region contains a fragment previously described as including the dominant site for induction of human-cytomegalovirus antibodies. Since the selected sequence is highly conserved among herpes viruses, we have considered the possible presence of antigenic cross-reactivity, particularly with the Epstein-Barr virus. Several peptides in the studied region were antigenic and two main continuous epitopes have been identified. Serological cross-reactions observed with Epstein-Barr virus are discussed, focusing on the possible implications of structural features and sequence similarity between human-cytomegalovirus and Epstein-Barr-virus glycoproteins.
Assuntos
Antígenos Virais/imunologia , Citomegalovirus/química , Herpesvirus Humano 4/química , Oligopeptídeos/imunologia , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Anticorpos Antivirais , Antígenos Virais/química , Reações Cruzadas , Citomegalovirus/imunologia , Ensaio de Imunoadsorção Enzimática , Epitopos , Humanos , Dados de Sequência Molecular , Oligopeptídeos/química , Alinhamento de Sequência , Proteínas do Envelope Viral/químicaRESUMO
A 72-year-old man developed chronic sensory neuropathy (CSN) during chronic hepatitis C (HCV) infection. Neurological symptoms began one year after acute HCV hepatitis and slowly worsened over three years. No conventional cause for CSN was found. Circulating antinervous tissue antibodies (including anti-Hu) and inflammatory infiltrates in sural nerve biopsy specimens were absent. However, the presence of anti-HCV antibody and HCV-RNA in cerebrospinal fluid indicated that HCV had reached the intrathecal compartment, suggesting the direct viral involvement in the pathogenesis of CSN.
Assuntos
Hepacivirus/patogenicidade , Hepatite C/complicações , Hepatite Crônica/complicações , Doenças do Sistema Nervoso/virologia , Idoso , Anticorpos Antivirais/líquido cefalorraquidiano , Hepacivirus/genética , Hepacivirus/imunologia , Humanos , Técnicas Imunoenzimáticas , Masculino , Doenças do Sistema Nervoso/líquido cefalorraquidiano , Neurônios Aferentes , Reação em Cadeia da Polimerase , RNA Viral/líquido cefalorraquidianoRESUMO
In a prospective study of 33 infants born to hepatitis C virus (HCV)-positive human immunodeficiency virus-negative mothers the vertical transmission of HCV occurred in 6.8%. The evolution of HCV infection in two babies was studied from birth up to 5 or 6 years of age, and the sequencing of the hypervariable region (HVR) of the putative envelope-encoding E2 region of the HCV genome was performed. The HVR1 sequence variability and the different serological profiles during follow-up could reflect the differences in HCV transmission routes, HCV genotypes, and clinical evolution of infection.
Assuntos
Regiões Determinantes de Complementaridade/genética , Hepacivirus/genética , Hepatite C/genética , Hepatite C/transmissão , Transmissão Vertical de Doenças Infecciosas , Complicações Infecciosas na Gravidez/virologia , Sequência de Aminoácidos , Primers do DNA , Evolução Molecular , Feminino , Seguimentos , Soronegatividade para HIV , Hepatite C/embriologia , Humanos , Recém-Nascido , Gravidez , Fatores de Tempo , Viremia/diagnósticoRESUMO
Muscle biopsy tissue from a patient with chronic hepatitis, who was hepatitis C virus (HCV) positive and showed slight weakness of the right arm and leg associated with increased serum creatine kinase levels, was studied using immunocytochemical and polymerase chain reaction (PCR) techniques. Muscle biopsy showed changes compatible with an inflammatory myopathy. Immunohistochemical studies included the use of monoclonal antibodies against human T lymphocytes, macrophages, immunoglobulins, major histocompatibility complex class I molecules (MHC-I), and the neoantigens of the terminal C5b-9 complement membrane attack complex (MAC). In addition to confirming the potential importance of cytotoxic T cells and MHC-I antigen expression in inducing muscle pathology, we demonstrated MAC deposition and the presence of HCV-RNA in the muscle of our patient, suggesting that direct involvement of the virus leading to complement activation might be important in inducing muscle damage.
Assuntos
Hepatite C Crônica/patologia , Miosite/patologia , Adulto , Complexo de Ataque à Membrana do Sistema Complemento/análise , Antígenos de Histocompatibilidade Classe I/análise , Humanos , Masculino , Microscopia Eletrônica , Músculo Esquelético/patologia , Músculo Esquelético/ultraestrutura , Reação em Cadeia da Polimerase , RNA Mensageiro/análiseRESUMO
To evaluate the concordance between viremia and antibody testing in hepatitis C virus (HCV) diagnosis, 682 serum or plasma samples collected from patients with known or suspected HCV infection were tested. An overall concordance of 77% between serological and PCR results was found, 5% was RNA positive/antibody negative and 18% antibody positive/RNA negative. The relationship between HCV infection, risk group and clinical diagnosis was studied in 116 patients: the presence of anti-HCV antibody without viremia was shown in 72.7% of asymptomatic subjects and 17.6% of chronic hepatitis subjects without interferon treatment. However, the detection of HCV-RNA in peripheral blood mononuclear cells (PBMC) in four out of 38 plasma viremia-negative HCV-seropositive subjects (10.5%), showed that HCV-RNA could persist in PBMC and could begin the viral replication again at different times. The detection of HCV-RNA in PBMC in anti-HCV-positive subjects without viremia could reduce false-negative results of HCV-RNA testing by RT-PCR in serum or plasma.
Assuntos
Hepacivirus/isolamento & purificação , Anticorpos Anti-Hepatite C/sangue , Hepatite C/diagnóstico , RNA Viral/sangue , Viremia/diagnóstico , Viremia/virologia , Primers do DNA , Reações Falso-Negativas , Hepacivirus/genética , Hepacivirus/imunologia , Hepatite C/imunologia , Humanos , Leucócitos Mononucleares/imunologia , Reação em Cadeia da Polimerase/métodos , Valor Preditivo dos Testes , DNA Polimerase Dirigida por RNA , RiscoRESUMO
A new restriction fragment length polymorphism (RFLP) analysis has been developed for hepatitis C virus (HCV) typing in the viral 5' non-coding region and contiguous core region. These genomic sequences were chosen for the relative nucleotide homology among different genotypes and for the presence of polymorphic sites. By employing two endonucleases (AccI and MboI) and, in some instances, a third one (EcoRII), we can unambiguously and reproducibly distinguish between genotypes and subtypes 1a, 1b, 1c, 2a, 2c, 2b, 3a, 3b, 4a, 5a and 6a. The method was applied for diagnosing two Italian groups of HCV-infected individuals reflecting a randomly collected population and a group of intravenous drug users. The accuracy of this method has been validated by comparison with INNOLiPA and by sequencing. Our approach represents an improvement over previous RFLP methods, since typing is accurate and simpler.