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We present a human immunodeficiency virus-infected patient with severe decompensated hepatitis C virus-related cirrhosis awaiting liver transplantation (LT) who received a 24-week course of interferon/ribavirin-free antiviral treatment with sofosbuvir and daclatasvir on a compassionate basis. Rapid viral suppression was associated with progressive improvement of his liver function tests. The patient achieved a sustained virological response and concomitant clinical improvement, which prompted removal from the LT list 12 weeks after the end of treatment.

Objectives This study aimed to characterize the chronically infected general hepatitis C virus (HCV) population in Barcelona using a highly sensitive subtyping method that can identify the 67 recognized HCV subtypes and diagnose mixed infection by various genotypes/subtypes in a single individual. The resulting information has implications for selecting optimal direct-acting antiviral (DAA) treatment for each patient and establishing public healthcare policies in our setting. Methods Consecutive HCV patients (treatment-naive or interferon-based failures) attending Vall d'Hebron Hospital outpatient clinics from February 2015 to May 2016 ( N =1473) were included in the study. Patient samples were characterized using HCV subtyping by next-generation ultra-deep pyrosequencing. Results The following genotypes (G) were found: G1 (1126/1473 (76.4%)), G4 (145/1473 (9.8%)), G3 (135/1473 (9.2%)), G2 (51/1473 (3.5%)), and G5 (1/1473 (0.1%)). Twenty-two subtypes were seen: 1b (790/1473 (53.6%)), 1a (332/1473 (22.5%)), 3a (133/1473 (9.0%)), 4d (105/1473 (7.1%)), 4a (29/1473 (2.0%)), and 2c (25/1473 (1.7%)), with 16 low-prevalence subtypes accounting for the remaining 3.0% (44/1473). There was a worrisome 1.0% (15/1473) of mixed infections. G2 (51/1473 (3.5%)) showed a high level of heterogeneity. Analyses by age groups showed a predominance of G1b over G1a (428/506 (84.6%) vs. 24/506 (4.7%)) in patients born before 1950 ( N =506/1473), and similar percentages of these subtypes in those born between 1951 and 1975 ( N =834/1473) (315/834, 37.8% vs. 266/834, 31.9%) and after 1976 ( N =133/1473) (47/133, 35.3% vs. 42/133, 31.6%). Conclusions Subtype distribution showed a higher level of heterogeneity than was expected, particularly for G2. Prevalence of mixed infections was around 1%. HCV subtype distribution related to patient age group suggested that patients born from 1936 to 1975 in our setting should undergo screening for the infection. Next-generation sequencing enabled better classification of candidates for DAA-based treatment.

SUMMARY. Epidemiological, viral and host factors are associated with the outcome of hepatitis C virus (HCV) infection, and strong host immune responses against HCV favour viral clearance. Recently, genome-wide association studies have shown a strong correlation between single-nucleotide polymorphisms (SNPs) near the interleukin-28B (IL28B) gene and spontaneous or treatment-induced HCV clearance. We have investigated whether protective IL28B genetic variants are associated with HCV-specific T-cell responses among Spanish blood donors. The rs12979860 IL28B haplotype was determined in 69 anti-HCV-positive blood donors (21 HCV RNA negative and 48 HCV RNA positive) and 30 seronegative donors. In all cases, HCV-specific CD4 + T-cell responses to HCV recombinant proteins (core, NS3 and NS3 helicase) were assessed by ex vivo interferon-c ELISpot assay. The rs12979860-CC genotype was highly overrepresented in donors with spontaneous HCV clearance when compared to those with chronic infection (76.2% vs 29.2%, P < 0.001; odds ratio, 7.77; 95% confidence interval, 2.4–25.3, P < 0.001). HCV-specific CD4 + T-cell responses were detected in 16 (76.2%) spontaneous resolvers especially towards nonstructural proteins, but with no correlation with IL28B genotype. Chronic individuals had a significantly lower overall T-cell response again irrespective of IL28B genotype. When spontaneous resolvers and chronic individuals were stratified according to their IL28B genotype, significantly stronger T-cell responses were only observed among those with non-CC haplotypes. Although the protective rs12979860 IL28B CC genotype is associated with spontaneous HCV clearance, stronger CD4 + T-cell responses towards NS3 were only evident among those with non-CC haplotypes.

The aims of the study were to verify the longterm effect of time on viral clearance in hepatitis C virus (HCV) patients and to find out factors possibly associated with disease progression. A total of 1641 patients recruited from eight European centres in 1996-1.997 were re-analysed 5-7 years after inclusion. The occurrence of decompensated cirrhosis, hepatocellular carcinoma (HCC) and liver transplantation was analysed in relation to different host and viral factors. Ninety-three per cent of the HCV patients who had cleared the virus (spontaneously or after antiviral therapy) remained HCV-RNA-negative during follow up and may be considered as 'cured'. Among patients who were sustained responders at inclusion, 2.3% developed liver complications during follow up, and 31% of non-responders did. Advanced age at infection and presence of the human leucocyte antigen (HLA) DRBI*1201-3 allele were possibly associated with a higher rate of progression to decompen- sated cirrhosis or HCC. Decompensated cirrhosis might be further associated with male gender, non-response to previous therapy, and lack of FILA DRBI*1301 allele, whereas HCC seems to be associated with the presence of the HLA DQ02 allele. Long-term follow up of HCV patients indicates that virological response persists over time and is associated with a very low incidence of liver complications. Advanced age at inclusion. advanced age at infection, viral genotype 1, non-response to previous therapy and possibly some specific HLA alleles are factors independently associated with a faster rate of progression towards liver complications. The large proportion of patients lost to follow up stresses the need for a strengthened and optimized management of HCV patients. (Less)

A new quantitative marker of HCV viremia based on the detection of the core antigen of the virus has recently become commercially available in Europe. The usefulness of this test was examined for the management of patients treated with pegylated interferon/ribavirin. One hundred twenty-eight pegylated interferon/ribavirin treated patients were studied. Serum samples were available at baseline, week 4 and week 12 time-points, respectively. Core antigen was quantified using the trak-C assay (Ortho Clinical Diagnostics, Raritan, NJ). For all genotypes at week 4, the positive and negative predictive values of HCV core antigen were 81.4 and 92.9%, respectively, while at week 12 they were 67.9 and 100%, respectively. These predictive values varied substantially according to viral genotype. Among patients with a negative core antigen level (

The aim of this study was to determine whether specific sequences of the phosphorylation homology domain (PePHD) region could be correlated with differences in response to antiviral therapy in patients infected with hepatitis C virus subtypes 1b, 2c, 3a and 4c/d. We included 43 patients (22 sustained responders and 21 nonresponders or relapsers) in the study, who were classified according to early viral decline during the first weeks of antiviral treatment and response at end of follow up. Type of mutations, mutation frequency, genetic diversity and phylogenetic relationships were compared at the PePHD and flanking regions. Phylogenetic trees showed that each sequence clustered together with those of the same subtype. Sequences from subtypes 1b and 4c/d resembled more closely the phosphorylation sites of protein kinase R and eIF2 alpha than sequences from genotypes 2c and 3a, the latter with higher response rates to interferon-alpha (IFN alpha) treatment. However, within specific subtypes, no separate clusters of responders and nonresponders were observed either at the beginning or at the end of follow up. We were not able to find any particular sequence or mutation in the PePHD region or in any other subregion of the fragment studied that allowed prediction of treatment response.

Hepatitis C is now recognized as the most common infection causing chronic liver disease in the European population. Our aim was to assess the prevalence of the antibody to hepatitis C virus (HCV), and the incidence of HCV seroconversion in the general population and the main risk groups, namely intravenous drug users, haemodialysis and transfused patients, in seven countries of the European Union, by carrying out a critical analysis of the literature. Data sources used were the Medline database and a manual search using the key words: hepatitis C, prevalence, incidence, transmission, risk factors and epidemiology. Articles published between January 1990 and March 1997 were reviewed. Articles were reviewed according to a critical analysis method regarding title, type of article, study design, period and population, tests, results and their consistency with data. The tests performed were mainly second- or third-generation serological tests. The average prevalence rate in blood donors was 1%, with a north-south gradient ranging from 0.04% to 2%. Prevalence varied from 20% to 30% in haemodialysis patients. The incidence in transfused patients was less than 1% after 1991. The prevalence in intravenous drug users was about 80%. Multicentre studies conducted in larger samples are needed to obtain more accurate and reliable results, in particular. However, the epidemiological studies available allowed us to assess the magnitude of HCV infection in Europe.

Analysing significant numbers of cDNA clones of the hepatitis C virus (HCV) from single isolates provides unquestionable proof that the viral genome cannot be defined by a single sequence, but rather by a population of variant sequences closely related to one another. This way of organizing the genetic information is referred to as quasispecies. Throughout HCV infection, the number and composition of the variants in the viral population keeps changing owing to environmental influences, resulting in a virus that is constantly redefining itself both genetically and phenotypically. Therefore, the virus has often been investigated in population terms. Many clinical studies have tried to unravel, through the parameters that characterize the HCV quasispecies, prognostic markers of the disease and its response to treatment. Other investigations have focused on discovering how the virus and host interact during chronic infection. The consensus sequence, the rate of fixation of mutations and the complexity of the viral population are useful parameters for describing the viral population behaviour and its interaction with the host. In addition to sequencing, several other methods, based on electrophoretic mobility, have been used to study these parameters, such as temperature gradient-gel electrophoresis, single-strand conformation polymorphism and gel-shift analysis. The viral region examined, the source of clinical specimen, as well as the methodology employed, will be decisive in interpreting the information obtained.

Summary The success of current antiviral treatment for hepatitis C virus (HCV) recurrence in liver transplant (LT) recipients remains limited. We aimed at evaluating the value of IL28B genotype and early viral kinetics to predict response to standard treatment in the transplant setting. We retrospectively evaluated 104 LT recipients treated for HCV genotype 1 recurrence between 2001 and 2010. Baseline variables, including IL28B genotype, and early viral kinetics were compared among patients who did or did not achieve a sustained virological response (SVR). Logistic regression analyses of candidate variables were conducted to generate a reliable predictive model based on the minimum set of variables. Twenty-nine (28%) achieved an SVR. On multivariate analysis, the magnitude of HCV RNA decline at 4 weeks (OR: 3.74, 95% CI: 1.64–9.39; P = 0.003) and treatment compliance (OR: 35.27, 95% CI: 3.35–365.54; P = 0.003) were the only independent predictors of SVR. Favourable recipient IL28B genotype significantly correlates with virological response at week 4 (OR 3.23; 95% CI, 1.12–9.15; P = 0.03). By logistic regression analysis, a model including donor age, recipient rs12979860 genotype and viral load at 4 weeks showed the best predictive value for SVR with an area under the receiver operating curve of 0.861. Favourable recipient IL28B genotype strongly correlates with the viral response at week 4 which is the strongest predictor of response. The combination of recipient IL28B genotype and donor age with the week 4 response reliably estimates the probability of SVR early on-treatment and may facilitate therapeutic strategies incorporating new antiviral agents.

In order to study the prevalence of hepatitis E virus (HEV) infection in developed countries, IgG and IgM anti-HEV were determined in serum samples from 382 patients with acute viral hepatitis (244 hepatitis A, 48 hepatitis B and/or D, and 90 non-A, non-B, non-C hepatitis), 76 healthy subjects, 55 hemophiliacs and 50 patients on hemodialysis. IgG anti-HEV antibodies were detected and confirmed by a synthetic peptide-based EIA in 5 (5.6%) non-A, non-B, non-C acute hepatitis, in 3 (6.5%) B and D acute hepatitis, in 10 (4%) acute A hepatitis, in 3 (5.5%) of 54 healthy adults in none of the hemophiliacs and in 3 (6%) patients on hemodialysis. IgM anti-HEV antibodies were only detected in two cases of acute hepatitis B and/or D. Analysis of serial serum samples demonstrated IgG anti-HEV seroconversion in 3 of the 18 confirmed cases; one of them was also positive for IgM anti-HEV. All 3 acute anti-HEV-positive hepatitis cases occurred in adults, were community-acquired (two of them were intravenous drug addicts) and had a self-limited course. These results demonstrate that HEV is a minor cause of acute hepatitis in Spain. A similar low rate of IgG anti-HEV antibodies was detected in patients with different diseases, suggesting that HEV has a very low epidemiological impact. An apparent association of HEV infection with hepatitis B and D suggests a possible parenteral transmission of a mainly enteral pathogen.

Hepatitis viruses comprise a group of very diverse pathogens that primarily infect the liver, but belong to very different virus families with very different replication strategies (hepatitis A virus (HAV) Picornaviridae, hepatitis B virus (HBV) Hepadnaviridae, hepatitis C virus (HCV) Flaviviridae, hepatitis delta virus (HDV) genus Deltavirus, not assigned to a family yet, and hepatitis E virus (HEV) Hepeviridae). All of them have in common a high genome plasticity, and have received special attention because of their worldwide distribution in human population, infecting hundreds of million people, causing either acute and/or chronic infections that in many cases lead to liver cirrhosis and hepatocellular carcinoma, which is one of the leading causes of death worldwide. The huge number of infected people all over the world is the best proof of how different replication and transmission strategies, with the common factor of variability, may succeed in terms of viral persistence.

Treatment of chronic hepatitis C in humanimmunodeficiency virus (HIV)-infected patients is associatedwith low response rates and high incidence of side effects.One hundred twenty-one hepatitis C virus (HCV)–HIV-co-infected patients were randomized to receive interferonalpha-2b (3 MU thrice weekly; n ¼ 61) or peginterferonalpha-2b (1.5 lg/kg/week; n ¼ 60), plus ribavirin (800 mgdaily), for 24 (genotype 2 or 3) or 48 weeks (genotype 1 or4). We assessed early virological response at 4, 8 and12 weeks to predict sustained virological response (SVR).Safety assessment included frequent blood lactate measure-ment and relative quantitation of mitochondrial DNA(mtDNA) content in peripheral blood mononuclear cells. Inintention-to-treat analysis, the SVR rate was higher in thepeginterferon group (55% vs 26%; P ¼ 0.002). The differ-ence for HCV genotypes 1 and 4 was 45% vs 14% (P ¼0.009) and 50% vs 27% (P ¼ 0.387), respectively, and forgenotype 2 or 3, 71% vs 43% (P ¼ 0.12) Viral response at 4,8 and 12 weeks of treatment was highly predictive of SVR.Among genotype 3 patients, 17 of 20 (85%) whose HCVRNA was already undetectable at 4 weeks had an SVR after24 weeks of treatment. Hyperlactataemia occurred in 22patients and was clinically significant in six, two of whomdied. mtDNA decreased significantly 4–12 weeks after thestart of treatment in patients developing clinically significanthyperlactataemia. Peginterferon alpha-2b plus ribavirin wasmore effective than interferon alpha-2b plus ribavirin inHIV-coinfected patients. Frequent monitoring of virologicalresponse may be very helpful to optimize treatment compli-ance, to tailor treatment duration and to minimize sideeffects.Keywords: hyperlactataemia and liver failure, mitochondrialDNA by real-time PCR, mitochondrial toxicity, treatment ofchronic hepatitis C.

We have investigated the value of early hepatitis C virus (HCV) RNA decline (DeltaHCV RNA) to predict response to combination therapy in 66 chronic hepatitis C patients treated with IFN-alpha2b (3 MU thrice weekly) and ribavirin (800 mg daily) for 12 months [25 sustained responders (SR) and 41 nonresponders or relapsers (NR)]. Serum HCV RNA was retrospectively measured in samples obtained at baseline and 4, 8 and 12 weeks after treatment onset, using a commercially available quantitative RT-PCR assay. At 4 weeks, serum HCV RNA had decreased a mean of 2.6 +/- 0.8 logs among SR as compared with only 0.5 +/- 0.8 logs in NR (P0.001), and was already undetectable (600 IU/mL) in 12 (48%) of the SR but in none of the NR. At 8 weeks, HCV RNA was undetectable in 21 SR and in 2 NR and mean DeltaHCV RNA were 4.2 +/- 1.3 and 0.8 +/- 1.0 logs, respectively (P0.001). At week 12 all SR had undetectable HCV RNA as compared with only five NR (P0.001). Stepwise logistic regression analysis identified DeltaHCV RNA at 12 weeks as the strongest predictor of sustained response. Receiver operating characteristic (ROC) curves of DeltaHCV RNA for sustained response prediction identified sensitivity peaks with 100% negative predictive value corresponding to DeltaHCV RNA1 log at 4 weeks,2 logs at 8 weeks and3 logs at 12 weeks. Our results show that early changes in the HCV RNA level may reliably identify patients having no chance of a sustained virological response during the first 3 months of combination therapy, thus providing an excellent tool for optimizing antiviral treatment of chronic hepatitis C.

To compare three quantitative assays measuring viral load in patients with chronic hepatitis C and to determine their value in predicting response to interferon (IFN) therapy, we analysed serum from 896 patients from eight European Centres using QUANTIPLEXtrade mark bDNA, MONITOR AMPLICORtrade mark and SUPERQUANTtrade mark assays. Analyses were performed on the same sample. Viral genotype was assessed using INNO-LiPA HCV II kits. Intercentre variations were observed that were related to the handling of specimens not processed and stored within 6 h of blood sampling. Among sera with optimal handling, a stronger correlation was observed between bDNA and SUPERQUANT (0.806) than between bDNA and MONITOR (0.677) and between MONITOR and SUPERQUANT (0.632). These discrepancies were greatest with genotype 2 (bDNA/SUPERQUANT= 0.772; bDNA/MONITOR=0. 456; SUPERQUANT/MONITOR= 0.299). This correlation was influenced by viraemia level and was better at lower viral loads. The proportion of sera with undetectable viral load was 15% with bDNA, 9.7% with MONITOR and 7.7% with SUPERQUANT. For the three measurements, the best cut-offs of sustained response to IFN treatment were located at their detection threshold. Among patients with viral load below the detection level, a sustained response was observed in 35% tested with bDNA, 38% with MONITOR and 80% with SUPERQUANT. Hence a stronger correlation was observed between bDNA and SUPERQUANT than between either of these assays and MONITOR. SUPERQUANT was the most sensitive assay and this greater sensitivity was associated with a better predictive value of treatment response.

We have studied the distribution of viral sequences from the 5' noncoding region and from a fragment of the E2/NS2 region of the hepatitis C virus (HCV) genome in samples obtained before and after liver transplantation in two patients with HCV cirrhosis. The population of viral sequences in both regions were established by sequencing cloned PCR products. In both cases, the complexity of the viral quasispecies decreased after transplantation, although the consensus nucleotide and amino acid sequences remained unchanged. It is suggested that both positive and negative selection and random sampling events contribute substantially in shaping the genetic composition of HCV quasispecies and that recurrence of HCV infection may occur under equilibrium conditions.

1324 QUASISPECIES HOMOGENIZATION AND IL-28 GENOTYPE (RS12979860 AND RS8099917) MAY FAVOUR SELF-RESOLVING INFECTION AFTER HCV BOTTLENECKING TRANSMISSION M. Cubero, J.I. Esteban, D. Garcia-Cehic, F. Rodriguez-Frias, I. Ortega, E. Domingo, S. Sauleda, M. Bes, A. Sanchez, R. Esteban, J. Guardia, J. Quer. Liver Unit-Department of Internal Medicine, Institut de Recerca (IRVH), Universitat Autonoma Barcelona (UAB), Barcelona, Ciber de Enfermedades Hepaticas y Digestivas (CIBEREHD), Instituto de Salud Carlos III, Madrid, Biochemistry Department, Hospital Universitari Vall d’Hebron, Institut de Recerca (IRVH), Universitat Autonoma Barcelona (UAB), Unitat Estadistica Bioinformatica, Institut de Recerca (IRVH), Hospital Universitari Vall d’Hebron, Universitat Autonoma Barcelona (UAB), Barcelona, Biologia Molecular, Centro de Biologia Molecular Severo Ochoa, CSIC-Universidad Autonoma de Madrid, Madrid, Banc de Sang i Teixits, Hospital Universitari Vall d’Hebron, Barcelona, Spain E-mail: mcubero@ir.vhebron.net

Of 30,231 donors tested, 368 (1.2%) were anti-HCV positive. Of these, 254 have been evaluated, with the following results: only 25% have a history of parenteral risk, seroprevalence increases with age and approximately 80% of those that are anti-HCV positive in our population are probably infected with HCV. In addition, an unexpectedly large number of these persons have chronic and/or severe liver disease and will require combined diagnostic approaches for accurate evaluation.

Sequencing of multiple recombinant clones generated from polymerase chain reaction-amplified products demonstrated that the degree of heterogeneity of two well-conserved regions of the hepatitis C virus (HCV) genome within individual plasma samples from a single patient was consistent with a quasispecies structure of HCV genomic RNA. About half of circulating RNA molecules were identical, while the remaining consisted of a spectrum of mutants differing from each other in one to four nucleotides. Mutant sequence diversity ranged from silent mutations to appearance of in-frame stop codons and included both conservative and nonconservative amino acid substitutions. From the relative proportion of essentially defective sequences, we estimated that most circulating particles should contain defective genomes. These observations might have important implications in the physiopathology of HCV infection and underline the need for a population-based approach when one is analyzing HCV genomes.

We have analyzed the population of hepatitis C virus (HCV) sequences in paired liver and serum samples from four patients with chronic hepatitis C. Sequences from three different biopsy specimens from a liver explant from one patient were compared with each other and with the circulating sequences. Our results demonstrate that the circulating quasispecies does not necessarily reflect the viral population replicating in the liver and that this is not due to a macroscopic anatomic compartmentalization of HCV replication. This finding has important implications for the pathogenesis and natural history of chronic HCV infection.