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1.
Cells ; 8(7)2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31269695

RESUMO

Background: The aim of the study was to investigate the intra-host variability through next-generation-sequencing (NGS) of the NS5A-gene in nosocomial transmission-clusters observed in two Italian hospitals among hepatitis C virus (HCV)-genotype-1b infected patients. Methods: HCV-sequencing was performed by Sanger-sequencing (NS3 + NS5A + NS5B) and by NGS (NS5A, MiSeq-Illumina) in 15 HCV-1b infected patients [five acute with onco-hematologic-disease and 10 (4/6 acute/chronic) with ß-thalassemia]. Resistance-associated-substitutions (RAS) were analysed by Geno2pheno-algorithm. Nucleotide-sequence-variability (NSV, at 1%, 2%, 5%, 10% and 15% NGS-cutoffs) and Shannon entropy were estimated. Phylogenetic analysis was performed by Mega6-software and Bayesian-analysis. Results: Phylogenetic analysis showed five transmission-clusters: one involving four HCV-acute onco-hematologic-patients; one involving three HCV-chronic ß-thalassemia-patients and three involving both HCV-acute and chronic ß-thalassemia-patients. The NS5A-RAS Y93H was found in seven patients, distributed differently among chronic/acute patients involved in the same transmission-clusters, independently from the host-genetic IL-28-polymorphism. The intra-host NSV was higher in chronic-patients versus acute-patients, at all cutoffs analyzed (p < 0.05). Even though Shannon-entropy was higher in chronic-patients, significantly higher values were observed only in chronic ß-thalassemia-patients versus acute ß-thalassemia-patients (p = 0.01). Conclusions: In nosocomial HCV transmission-clusters, the intra-host HCV quasispecies divergence in patients with acute-infection was very low in comparison to that in chronic-infection. The NS5A-RAS Y93H was often transmitted and distributed differently within the same transmission-clusters, independently from the IL-28-polymorphism.


Assuntos
Infecção Hospitalar/virologia , Hepacivirus/genética , Hepatite C/virologia , Proteínas não Estruturais Virais/genética , Talassemia beta/terapia , Doença Aguda , Adulto , Substituição de Aminoácidos , Antivirais/farmacologia , Antivirais/uso terapêutico , Transfusão de Sangue , Doença Crônica , Infecção Hospitalar/tratamento farmacológico , Infecção Hospitalar/transmissão , Farmacorresistência Viral/genética , Feminino , Genótipo , Hepacivirus/patogenicidade , Hepatite C/tratamento farmacológico , Hepatite C/transmissão , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Interferons/genética , Interferons/imunologia , Masculino , Pessoa de Meia-Idade , Filogenia , Polimorfismo de Nucleotídeo Único , Talassemia beta/genética , Talassemia beta/imunologia
2.
Sci Rep ; 8(1): 8988, 2018 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-29895871

RESUMO

Natural resistance-associated substitutions (RASs) are reported with highly variable prevalence across different HCV genotypes (GTs). Frequency of natural RASs in a large Italian real-life cohort of patients infected with the 4 main HCV-GTs was investigated. NS3, NS5A and NS5B sequences were analysed in 1445 HCV-infected DAA-naïve patients. Sanger-sequencing was performed by home-made protocols on 464 GT1a, 585 GT1b, 92 GT2c, 199 GT3a, 16 GT4a and 99 GT4d samples. Overall, 20.7% (301/1455) of patients showed natural RASs, and the prevalence of multiclass-resistance was 7.3% (29/372 patients analysed). NS3-RASs were particularly common in GT1a and GT1b (45.2-10.8%, respectively), mainly due to 80K presence in GT1a (17%). Almost all GTs showed high prevalence of NS5A-RASs (range: 10.2-45.4%), and especially of 93H (5.1%). NS5A-RASs with fold-change >100x were detected in 6.8% GT1a (30H/R-31M-93C/H), 10.3% GT1b (31V-93H), 28.4% GT2c (28C-31M-93H), 8.5% GT3a (30K-93H), 45.5% GT4a (28M-30R-93H) and 3.8% GT4d (28V-30S-93H). Sofosbuvir RAS 282T was never detected, while the 159F and 316N RASs were found in GT1b (13.4-19.1%, respectively). Natural RASs are common in Italian patients infected with HCV-GTs 1-4. High prevalence of clinically-relevant RASs (such as Y93H) supports the appropriateness of HCV resistance-test to properly guide DAA-based therapy.


Assuntos
Farmacorresistência Viral/genética , Genótipo , Hepacivirus/genética , Hepatite C/genética , Proteínas não Estruturais Virais/genética , Adulto , Idoso , Feminino , Hepatite C/tratamento farmacológico , Hepatite C/epidemiologia , Humanos , Itália/epidemiologia , Masculino , Pessoa de Meia-Idade , Prevalência
4.
J Virol Methods ; 246: 1-7, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28408312

RESUMO

BACKGROUND & AIMS: We evaluated the performance of a rapid method to quantify HCV-RNA in the hepatic and extrahepatic compartments, by using for the first time the Abbott RealTime HCV-assay. METHODS: Non-tumoral (NT), tumoral (TT) liver samples, lymph nodes and ascitic fluid from patients undergoing orthotopic-liver-transplantation (N=18) or liver resection (N=4) were used for the HCV-RNA quantification; 5/22 patients were tested after or during direct acting antivirals (DAA) treatment. Total RNA and DNA quantification from tissue-biopsies allowed normalization of HCV-RNA concentrations in IU/µg of total RNA and IU/106 liver-cells, respectively. RESULTS: HCV-RNA was successfully quantified with high reliability in liver biopsies, lymph nodes and ascitic fluid samples. Among the 17 untreated patients, a positive and significant HCV-RNA correlation between serum and NT liver-samples was observed (Pearson: rho=0.544, p=0.024). Three DAA-treated patients were HCV-RNA "undetectable" in serum, but still "detectable" in all tested liver-tissues. Differently, only one DAA-treated patient, tested after sustained-virological-response, showed HCV-RNA "undetectability" in liver-tissue. CONCLUSIONS: HCV-RNA was successfully quantified with high reliability in liver bioptic samples and extrahepatic compartments, even when HCV-RNA was "undetectable" in serum. Abbott RealTime HCV-assay is a good diagnostic tool for HCV quantification in intra- and extra-hepatic compartments, whenever a bioptic sample is available.


Assuntos
Hepacivirus/genética , Hepacivirus/isolamento & purificação , Fígado/virologia , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Carga Viral/métodos , Idoso , Antivirais/uso terapêutico , Líquido Ascítico/virologia , Biópsia , Feminino , Hepatite C/diagnóstico , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Hepatite C Crônica/diagnóstico , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/virologia , Humanos , Fígado/patologia , Linfonodos/virologia , Masculino , Pessoa de Meia-Idade , Kit de Reagentes para Diagnóstico , Reprodutibilidade dos Testes
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