Keratin variants associate with progression of fibrosis during chronic hepatitis C infection

Keratins 8 and 18 (K8/K18) protect the liver from various forms of injury. Studies of liver explants from a large cohort of U.S. patients showed that K8/K18 mutations confer a risk to developing end-stage liver diseases, though which diseases are preferentially involved is unknown. We tested the hypothesis that K8/K18 variants are associated with chronic hepatitis C (CHC) and that their presence correlates with progression of fibrosis. Genomic DNA was isolated from peripheral blood of a well-characterized German cohort of 329 patients with CHC infection. Exonic regions were PCR-amplified and analyzed using denaturing high-performance liquid chromatography and DNA sequencing. Our findings showed: (1) amino acid altering keratin heterozygous variants in 24 of 329 CHC patients (7.3%) and non-coding heterozygous variants in 26 patients (7.8%), and (2) 3 new exonic K8 variants (T26R/G55A/A359T); 6 novel non-coding variants and one K18 coding variant (K18 S230T; 2 patients). The most common variants were K8 R341H (10 patients), K8 G62C (6 patients) and K8 I63V (4 patients). A novel and exclusive association of an intronic KRT8 IVS7+10delC deletion in all 10 patients with K8 R341H was observed. Notably, there was a significant association of exonic, but not of intronic K8 variants with increased fibrosis. In conclusion, previously described and novel K8 variants are present in a German population and collectively associate with progression of fibrosis in CHC infection. The unique 100% segregation of the most common K8 variant, R341H, with an intronic deletion suggests that one of these two genetic changes might lead to the other.

Targets of emerging therapies for viral hepatitis B and C

Viral hepatitis B and C, structurally two completely different viruses, commonly infect human hepatocytes and cause similar clinical manifestations. Since their discovery, IFN has been a pillar in the treatment. However, because of the different natures of the viruses, therapeutic approaches diverge and new treatment targets are tailored specifically for each virus. Herein, the authors analyse therapeutic approaches for hepatitis B virus (HBV) and hepatitis C virus (HCV) and focus on emerging concepts that are under clinical evaluation. In particular, promising viral inhibitors for HBV and HCV are reviewed and the current status of research for gene therapy for HCV is described. Immune therapy is a fast-moving field with fascinating results which include therapeutic vaccines and toll-like receptor agonists that could improve tomorrow's treatment approaches.

HCV-related advanced fibrosis/cirrhosis: randomized controlled trial of pegylated interferon alpha-2a and ribavirin

In patients with hepatitis C virus (HCV)-related advanced fibrosis/cirrhosis, 30% of sustained HCV clearance has been reported with pegylated interferon alpha-2a (PEG-IFN) alone, but the efficacy and tolerability of the PEG-IFN/ribavirin (RBV) combination remain poorly defined. A total of 124 treatment-naïve patients with biopsy proved HCV-related advanced fibrosis/cirrhosis (Ishak score F4-F6, Child-Pugh score < or =7) were randomized to 48 weeks of PEG-IFN (180 microg sc weekly) and standard dose of RBV (1000/1200 mg po daily, STD) or PEG-IFN (180 microg sc weekly) and low-dose of RBV (600/800 mg po daily, LOW). Sustained virologic response (SVR) rates with PEG-IFN/STD RBV (52%) were higher--albeit not significantly--than that with PEG-IFN/LOW RBV (38%, P = 0.153). In multivariate analysis, genotype 2/3 and a baseline platelet count > or =150 x 10(9)/L were independently associated with SVR. The likelihood of SVR was < 7% if viraemia had not declined by > or =2 log or to undetectable levels after 12 weeks. Nine adverse events in the STD RBV and 15 in the LOW RBV group were classified as severe (including two deaths); dose reductions for intolerance were required in 78% and 57% (P = 0.013), and treatment was terminated early in 23% and 27% of patients (P = n.s.). The benefit/risk ratio of treating compensated HCV-cirrhotics with STD PEG-IFN/RBV is favourable.

Interferon-inducible expression of APOBEC3 editing enzymes in human hepatocytes and inhibition of hepatitis B virus replication

Hypermutations in hepatitis B virus (HBV) DNA by APOBEC3 cytidine deaminases have been detected in vitro and in vivo, and APOBEC3G (A3G) and APOBEC3F (A3F) have been shown to inhibit the replication of HBV in vitro, but the presumably low or even absent hepatic expression of these enzymes has raised the question as to their physiological impact on HBV replication. We show that normal human liver expresses the mRNAs of APOBEC3B (A3B), APOBEC3C (A3C), A3F, and A3G. In primary human hepatocytes, interferon alpha (IFN-alpha) stimulated the expression of these cytidine deaminases up to 14-fold, and the mRNAs of A3G, A3F, and A3B reached expression levels of 10%, 3%, and 3%, respectively, relative to GAPDH mRNA abundance. On transfection, the full-length protein A3B(L) inhibited HBV replication in vitro as efficiently as A3G or A3F, whereas the truncated splice variant A3B(S) and A3C had no effect. A3B(L) and A3B(S) were detected predominantly in the nucleus of uninfected cells; however, in HBV-expressing cells both proteins were found also in the cytoplasm and were associated with HBV viral particles, similarly to A3G and A3F. Moreover, A3G, A3F, and A3B(L), but not A3B(S), induced extensive G-to-A hypermutations in a fraction of the replicated HBV genomes. In conclusion, the editing enzymes A3B(L), A3F, and most markedly A3G, which are expressed in liver and up-regulated by IFN-alpha in hepatocytes, are candidates to contribute to the noncytolytic clearance of HBV.

Hepatitis C coinfection is independently associated with decreased adherence to antiretroviral therapy in a population-based HIV cohort

OBJECTIVE: To characterize the impact of hepatitis C (HCV) serostatus on adherence to antiretroviral treatment (ART) among HIV-infected adults initiating ART. METHODS: The British Columbia HIV/AIDS Drug Treatment Program distributes, at no cost, all ART in this Canadian province. Eligible individuals used triple combination ART as their first HIV therapy and had documented HCV serology. Statistical analyses used parametric and non-parametric methods, including multivariate logistic regression. The primary outcome was > or = 95% adherence, defined as receiving > or = 95% of prescription refills during the first year of antiretroviral therapy. RESULTS: There were 1186 patients eligible for analysis, including 606 (51%) positive for HCV antibody and 580 (49%) who were negative. In adjusted analyses, adherence was independently associated with HCV seropositivity [adjusted odds ratio (AOR), 0.48; 95% confidence interval (CI), 0.23-0.97; P = 0.003], higher plasma albumin levels (AOR, 1.07; 95% CI, 1.01-1.12; P = 0.002) and male gender (AOR, 2.53; 95% CI, 1.04-6.15; P = 0.017), but not with injection drug use (IDU), age or other markers of liver injury. There was no evidence of an interaction between HCV and liver injury in adjusted analyses; comparing different strata of HCV and IDU confirmed that HCV was associated with poor adherence independent of IDU. CONCLUSIONS: HCV-coinfected individuals and those with lower albumin are less likely to be adherent to their ART.

Blood donor screening: how to decrease the risk of transfusion-transmitted hepatitis B virus?

QUESTIONS UNDER STUDY: The risk of transfusion-transmitted HBV remains significant in Switzerland, where routine screening for hepatitis B virus (HBV) in blood donations relies solely on serological hepatitis B surface antigen (HBsAg) testing. This study was designed to determine the prevalence of anti-hepatitis B core (anti-HBc) and HBV nucleic acid testing (NAT) positive donations in two different Swiss donor populations, to help in deciding whether supplemental testing may bring additional safety to blood products. METHODS: In a first population of donors, 18143 consecutive donations were screened initially for HBsAg, anti-HBc (with one EIA assay) and with HBV NAT in minipools of 24 donations. The screening repeatedly reactive anti-HBc donations were then "confirmed" with two supplemental anti-HBc assays, an anti-hepatitis B surface assay (anti-HBs) and with single donation HBV NAT. In a second population of donors, 4186 consecutive donations were screened initially with two different anti-HBc assays in addition to the mandatory HBsAg screening test. The screening repeatedly reactive donations with at least one anti-HBc assay were tested for anti-HBs. RESULTS: In the first subset of 18143 donations, 17593 (97.0%) were negative for HBsAg, anti-HBc and HBV NAT in minipools. 549 (3.0%) were HBsAg and HBV NAT negative, but repeatedly reactive for anti-HBc. Of these 549 donations, 287 could not be "confirmed" with two additional anti-HBc assays and were negative with an anti-HBs assay, as well as with single donation HBV NAT. Only 211 (1.2% of the total screened donations) were "confirmed" positive with at least one of two supplemental anti-HBc assays. One repeatedly reactive HBsAg donation, from a first-time donor, was confirmed positive for HBsAg and anti-HBc, as well as with single donation HBV NAT. In the second subset of 4186 donations, 4014 (95.9%) were screened negative for HBsAg and for anti-HBc, tested with two independent anti-HBc assays. 172 donations (4.1%) were HBsAg negative but repeatedly reactive with at least one of the two anti-HBc assays. Of these 172 samples, 86 were reactive with the first anti-HBc assay only, 13 were reactive with the second anti-HBc assay only and 73 (1.7% of the total screened donations) were "confirmed" positive with both anti-HBc assays. CONCLUSION: The prevalence of anti-HBc "confirmed" positive donations in the two Swiss blood donor populations studied was low (<2%) and we found only one HBV NAT positive (HBsAg positive) donation among more than 18000. Concerning blood product safety, an increase in the deferral rate of less than 2% of anti-HBc positive, potentially infectious donors, would in our opinion make routine anti-HBc testing of blood donations cost-effective. There is however still a need for more specific assays to avoid an unacceptably high deferral rate of "false" positive donors. In contrast, the introduction of HBV NAT in minipools gives minimal benefit due to the inadequate sensitivity of the assay. It remains to evaluate more extensively the value of individual donation NAT, alone or in addition to anti-HBc, as supplemental testing in the context of several Swiss blood donor populations.

Connective tissue growth factor, steatosis and fibrosis in patients with chronic hepatitis C

BACKGROUND/AIM: Both steatosis and insulin resistance have been linked to accelerated fibrosis in chronic hepatitis C. Connective tissue growth factor (CTGF) plays a major role in extracellular matrix production in fibrotic disorders including cirrhosis, and its expression is stimulated in vitro by insulin and glucose. We hypothesized that CTGF may link steatosis, insulin resistance and fibrosis. METHODS: We included 153 chronic hepatitis C patients enrolled in the Swiss Hepatitis C Cohort Study and for whom a liver biopsy and plasma samples were available. CTGF expression was assessed quantitatively by immunohistochemistry. In 94 patients (57 with genotypes non-3), plasma levels of glucose, insulin and leptin were also measured. CTGF synthesis was investigated by immunoblotting on LX-2 stellate cells. RESULTS: Connective tissue growth factor expression was higher in patients with steatosis (P=0.039) and in patients with fibrosis (P=0.008) than those without these features. CTGF levels were neither associated with insulinaemia or with glycaemia, nor with inflammation. By multiple regression analysis, CTGF levels were independently associated with steatosis, a past history of alcohol abuse, plasma leptin and HCV RNA levels; when only patients with genotypes non-3 were considered, CTGF levels were independently associated with a past history of alcohol abuse, plasma leptin levels and steatosis. Leptin stimulated CTGF synthesis in LX-2 cells. CONCLUSIONS: In patients with chronic hepatitis C and steatosis, CTGF may promote fibrosis independently of inflammation. CTGF may link steatosis and fibrosis via increased leptin levels.

Increased risk of hepatocellular carcinoma among patients with hepatitis C cirrhosis and diabetes mellitus

Recent studies suggest that diabetes mellitus increases the risk of developing hepatocellular carcinoma (HCC). The aim of this study is to quantify the risk of HCC among patients with both diabetes mellitus and hepatitis C in a large cohort of patients with chronic hepatitis C and advanced fibrosis. We included 541 patients of whom 85 (16%) had diabetes mellitus. The median age at inclusion was 50 years. The prevalence of diabetes mellitus was 10.5% for patients with Ishak fibrosis score 4, 12.5% for Ishak score 5, and 19.1% for Ishak score 6. Multiple logistic regression analysis showed an increased risk of diabetes mellitus for patients with an elevated body mass index (BMI) (odds ratio [OR], 1.05; 95% confidence interval [CI], 1.00-1.11; P = 0.060) and a decreased risk of diabetes mellitus for patients with higher serum albumin levels (OR, 0.81; 95% CI, 0.63-1.04; P = 0.095). During a median follow-up of 4.0 years (interquartile range, 2.0-6.7), 11 patients (13%) with diabetes mellitus versus 27 patients (5.9%) without diabetes mellitus developed HCC, the 5-year occurrence of HCC being 11.4% (95% CI, 3.0-19.8) and 5.0% (95% CI, 2.2-7.8), respectively (P = 0.013). Multivariate Cox regression analysis of patients with Ishak 6 cirrhosis showed that diabetes mellitus was independently associated with the development of HCC (hazard ratio, 3.28; 95% CI, 1.35-7.97; P = 0.009). CONCLUSION: For patients with chronic hepatitis C and advanced cirrhosis, diabetes mellitus increases the risk of developing HCC.

Hepatitis B virus infection is associated with impaired immunological recovery during antiretroviral therapy in the Swiss HIV cohort study

Hepatitis B virus (HBV) infection is a major cause of morbidity and mortality in human immunodeficiency virus (HIV)-infected patients worldwide. It is unclear whether HIV-related outcomes are affected by HBV coinfection. We compared virological suppression and immunological recovery during antiretroviral therapy (ART) of patients of different HBV serological status in the Swiss HIV Cohort Study. CD4 cell recovery during ART was significantly impaired in hepatitis B surface antigen-positive patients and in those with anti-hepatitis B core antigen alone compared with HBV-uninfected patients, despite similar virological efficacy of ART. CD4 increase in patients with resolved HBV infection was similar to that in HBV-uninfected individuals.

Hepatitis B and C in Switzerland - healthcare provider initiated testing for chronic hepatitis B and C infection

Hepatitis B and hepatitis C are contagious liver diseases caused by the hepatitis B virus (HBV) and the hepatitis C virus (HCV), respectively. In particular, chronic infection with HBV or HCV is a major public health problem throughout Europe. The majority of persons chronically infected (65%-75%) are not aware of their infection status until symptoms of advanced liver disease appear. In addition, the peak in the number of patients suffering from advanced stages of the disease, such as cirrhosis and hepatocellular carcinoma, has not yet been reached. In order to reduce the current and future morbidity and mortality associated with chronic HBV or HCV infection, the timely detection of chronically infected persons, with follow-up and case management, is crucial. However, the current screening strategies in Europe and Switzerland have to be considered as inadequate to detect the majority of chronically infected persons. Hence, we emphasise the importance of an alternative approach: the healthcare provider initiated identification of HBV or HCV infection in defined risk groups. This entails determining whether a person is not only at risk of being chronically infected, but also at risk of becoming infected with HBV or HCV and, if necessary, testing for HBV or HCV infection.

Seroprevalence of hepatitis E virus in domestic pigs and wild boars in Switzerland.

Hepatitis E is considered an emerging human viral disease in industrialized countries. Studies from Switzerland report a human seroprevalence of hepatitis E virus (HEV) of 2.6-21%, a range lower than in adjacent European countries. The aim of this study was to determine whether HEV seroprevalence in domestic pigs and wild boars is also lower in Switzerland and whether it is increasing and thus indicating that this zoonotic viral infection is emerging. Serum samples collected from 2,001 pigs in 2006 and 2011 and from 303 wild boars from 2008 to 2012 were analysed by ELISA for the presence of HEV-specific antibodies. Overall HEV seroprevalence was 58.1% in domestic pigs and 12.5% in wild boars. Prevalence in domestic pigs was significantly higher in 2006 than in 2011. In conclusion, HEV seroprevalence in domestic pigs and wild boars in Switzerland is comparable with the seroprevalence in other countries and not increasing. Therefore, prevalence of HEV in humans must be related to other factors than prevalence in pigs or wild boars.

Impact of common risk factors of fibrosis progression in chronic hepatitis C.

OBJECTIVE The natural course of chronic hepatitis C varies widely. To improve the profiling of patients at risk of developing advanced liver disease, we assessed the relative contribution of factors for liver fibrosis progression in hepatitis C. DESIGN We analysed 1461 patients with chronic hepatitis C with an estimated date of infection and at least one liver biopsy. Risk factors for accelerated fibrosis progression rate (FPR), defined as ≥0.13 Metavir fibrosis units per year, were identified by logistic regression. Examined factors included age at infection, sex, route of infection, HCV genotype, body mass index (BMI), significant alcohol drinking (≥20 g/day for ≥5 years), HIV coinfection and diabetes. In a subgroup of 575 patients, we assessed the impact of single nucleotide polymorphisms previously associated with fibrosis progression in genome-wide association studies. Results were expressed as attributable fraction (AF) of risk for accelerated FPR. RESULTS Age at infection (AF 28.7%), sex (AF 8.2%), route of infection (AF 16.5%) and HCV genotype (AF 7.9%) contributed to accelerated FPR in the Swiss Hepatitis C Cohort Study, whereas significant alcohol drinking, anti-HIV, diabetes and BMI did not. In genotyped patients, variants at rs9380516 (TULP1), rs738409 (PNPLA3), rs4374383 (MERTK) (AF 19.2%) and rs910049 (major histocompatibility complex region) significantly added to the risk of accelerated FPR. Results were replicated in three additional independent cohorts, and a meta-analysis confirmed the role of age at infection, sex, route of infection, HCV genotype, rs738409, rs4374383 and rs910049 in accelerating FPR. CONCLUSIONS Most factors accelerating liver fibrosis progression in chronic hepatitis C are unmodifiable.

Interferon-free treatment of chronic hepatitis C with faldaprevir, deleobuvir and ribavirin: SOUND-C3, a Phase 2b study.

BACKGROUND & AIMS The safety and efficacy of the interferon-free combination of faldaprevir (NS3/A4 protease inhibitor), deleobuvir (BI 207127, non-nucleoside polymerase inhibitor), and ribavirin in treatment-naïve patients chronically infected with HCV genotype-1 was explored. METHODS SOUND-C3 was a multicenter, open-label Phase 2b study. Treatment-naïve patients chronically infected with HCV genotype-1a (IL28B CC genotype only; n = 12) and genotype-1b (n = 20) were assigned to 16 weeks of treatment with faldaprevir 120 mg once daily, deleobuvir 600 mg twice daily, and weight-based ribavirin. Patients with compensated liver disease, including cirrhosis, were eligible for inclusion in this study. The primary endpoint was sustained virological response 12 weeks after completion of therapy. RESULTS Sustained virological response rates 12 weeks after completion of therapy were 17% and 95% in patients infected with HCV genotype-1a and genotype-1b respectively. All four patients with cirrhosis achieved sustained virological response 12 weeks after completion of therapy. The most frequently reported adverse events of at least moderate intensity were anaemia (16%), nausea, vomiting and fatigue (9% each). Three (9%) patients discontinued because of adverse events. CONCLUSIONS The interferon-free regimen of faldaprevir, deleobuvir and ribavirin was efficacious in patients infected with genotype-1b and generally well tolerated.