[Treatment of hepatitis C]. / Therapie der Hepatitis C.

Chronic hepatitis C virus (HCV) infection is the major cause of liver cirrhosis, hepatocellular carcinoma and liver transplantation in the western world. The development and approval of nine directly acting antiviral drugs in recent years has led to a dramatic improvement in therapeutic efficacy accompanied by fewer side effects. With current treatment options sustained virologic response in more than 90 % of patients can be achieved depending on HCV genotype, liver cirrhosis and prior therapies. Modern HCV treatment regimens are interferon-free and should be administered for 12-24 weeks. Shorter courses are possible in selected patients. For the treatment of HCV genotype 1 infection combinations of either the nucleotide polymerase inhibitor sofosbuvir with the protease inhibitor simeprevir or with one of the two NS5A inhibitors daclatasvir or ledipasvir on the one hand or triple DAA therapy of paritaprevir, ombitasvir and dasabuvir on the other hand are applicable. Ribavirin has still a role as an add-on in difficult to treat patients.

Proton pump inhibitor treatment is associated with the severity of liver disease and increased mortality in patients with cirrhosis.

BACKGROUND: Proton pump inhibitors (PPI) are widely used in patients with liver diseases. Within the last years, there have been concerns about the PPI use as they may promote infections in patients with cirrhosis. AIM: As there are sparse data of the prognostic relevance of PPI treatment, to perform a prospective study investigating the relation of PPI treatment and overall survival (OS) in cirrhotic individuals. METHODS: Patients with cirrhosis were enrolled and followed prospectively. The primary end point was OS. PPI treatment and additional clinical and laboratory data were assessed at the day of the study inclusion. The time until the end point death was assessed and the individual risks were calculated with Cox regression analyses. RESULTS: A total of 272 patients were included and 213 individuals (78.3%) were on PPI treatment. In multivariate logistic regression analysis, PPI treatment was associated with higher MELD scores (P = 0.027) and ascites (P = 0.039). In a multivariate Cox regression model, PPI use was an independent predictor of mortality (hazard ratio 2.330, 95% confidence interval 1.264-4.296, P = 0.007) in addition to the model of end-stage liver disease (MELD) score, hepatocellular carcinoma and hepatic decompensation. CONCLUSIONS: PPI use is an independent risk factor for mortality in patients with cirrhosis. Although a causative role for increased mortality in patients taking PPI is still missing, the prescription of PPI in cirrhotics should be considered carefully taking into account its potential adverse effects.

Soluble CD163 is an indicator of liver inflammation and fibrosis in patients chronically infected with the hepatitis B virus.

Soluble CD163 (sCD163), a marker for macrophage activation, was found to be associated with the severity of liver cirrhosis. The aim of the current study was to investigate whether serum sCD163 levels correlate with liver inflammation and fibrosis in patients with chronic hepatitis B virus (HBV) infection. In a retrospective cohort study, serum sCD163 levels were assessed by ELISA together with clinical and laboratory data in 186 patients with chronic HBV infection and 15 healthy controls. The relation between parameters for liver fibrosis and necroinflammation and sCD163 levels was analysed. Additionally, sCD163 was quantified in a subset of follow-up serum samples after initiation of antiviral treatment. sCD163 levels differed among phases of chronic HBV infection (P < 0.0001), and sCD163 concentrations were associated with inflammatory activity and fibrosis in the liver. sCD163 levels ≥ 1961 ng/l had a high specificity in the identification of subjects with substantial fibrosis (F ≥ 2). sCD163 concentrations decreased significantly after initiation of antiviral treatment. The correlation of sCD163 levels with necroinflammation and fibrosis and the sCD163 decline under treatment indicates that macrophage activation plays a role in HBV-related liver pathogenesis.

Identifying indications for percutaneous (PTC) vs. endoscopic ultrasound (EUS)- guided "rendezvous" procedure in biliary obstruction and incomplete endoscopic retrograde cholangiography (ERC).

BACKGROUND: The variety of rendezvous (RV) procedures has recently been extended by EUS- and PTCD-guided procedures as a complementary means to conventional ERCP. We have identified indication criteria and the potential of biliary PTCD-guided vs. EUS-guided RV. METHODS: Consecutive patients with bile duct obstruction who underwent RV were included. In all, ERCP alone was unable to achieve treatment success. Indication, technical success, and outcome in PTCD- vs. EUS-guided RV were retrospectively compared to identify criteria that indicate preference of RV technique. Site of obstruction, clinical scenario (stenosis with abscess vs. no abscess) and reason for previous failure of ERC were evaluated. RESULTS: In 32 patients, three different indications for RV procedures were identified: First, a one-step access to assist in failed ERCP (type 1, intra-ductal RV); second, temporary drainage for prolonged treatment of complex biliary disease (type 2, intra-ductal RV), and drainage of cholangio-abscess with re-establishing bile outflow (type 3, intra-abscess RV). Indication of PTCD- vs. EUS-guided rendezvous was competitive in type 1, but exclusive in favor of PTCD in types 2 and 3. The site of biliary obstruction indicated the anatomic location of RV procedures. CONCLUSIONS: This classification may help to define inclusion criteria for prospective studies on biliary RV procedures. Choice of therapeutic strategy depends on the anatomic location of the biliary obstruction and the type of the biliary lesion. PTCD-guided RV might improve outcome in cholangio-abscess.

Severe 25-hydroxyvitamin D deficiency identifies a poor prognosis in patients with hepatocellular carcinoma - a prospective cohort study.

BACKGROUND: Vitamin D is involved in many biological processes. The role of vitamin D in patients with hepatocellular carcinoma (HCC) remains inconclusive, although there is evolving evidence that vitamin D may modulate cancer development and progression. AIM: To evaluate serum vitamin D as prognostic parameter in HCC, we performed a prospective cohort study. METHODS: HCC patients were prospectively recruited and 25-hydroxyvitamin D3 (25(OH)D3 ) levels were determined. 25(OH)D3 levels were compared to stages of cirrhosis and HCC stages with nonparametric Kruskal-Wallis tests and Spearman correlations in 200 HCC patients. The association of the 25(OH)D3 levels and overall survival (OS) was assessed in uni- and multivariate Cox regression models. RESULTS: Two-hundred patients with HCC were included. The mean follow-up time was 322 ± 342 days with a range of 1-1508 days. Nineteen patients underwent liver transplantation and 60 patients died within the observation time. The mean serum 25(OH)D3 concentration was 17 ± 13 ng/mL with a range of 1-72 ng/mL. 25(OH)D3 serum levels negatively correlated with the stage of cirrhosis as well as with stages of HCC. Patients with severe 25(OH)D3 deficiency had the highest mortality risk (hazard ratio 2.225, 95% confidence interval 1.331-3.719, P = 0.002). Furthermore, very low 25(OH)D3 levels were associated with mortality independently from the MELD score and high alpha-Fetoprotein levels (>400 ng/mL) in a multivariate Cox regression model. CONCLUSIONS: We conclude that 25(OH)D3 deficiency is associated with advanced stages of hepatocellular carcinoma and it is a prognostic indicator for a poor outcome.

[Cytokeratin 18 as marker for non-invasive diagnosis and prognosis of acute and chronic liver diseases]. / Zytokeratin-18 als Marker zur nichtinvasiven Diagnostik und Prognose akuter und chronischer Lebererkrankungen.

INTRODUCTION: Currently liver biopsy represents the gold standard to assess severity and fibrosis grade in liver diseases. Since this laborious, costly, and invasive procedure is associated with possible complications, non-invasive methods and biomarkers, which allow for an easy, reliable, and repeatable assessment of liver disease are warranted. Cytokeratin (CK) 18 is an intermediary filament protein, expressed in hepatocytes, which is proteolytically cleaved during liver damage. The resultant CK-18 fragments are released by hepatocytes and can be detected in serum. METHODS: A selective literature search in PubMed for original publications about the detection of CK-18 cell death markers in liver diseases was undertaken. RESULTS: Assessment of CK-18 cell death biomarkers allows for the early detection of liver damage in acute and chronic liver diseases. This is even feasible when transaminases are in the normal ranges. Detection of CK-18 biomarkers can also hint at disease activity and severity. For example, patients with non-alcoholic steatohepatitis exhibit elevated serum cell-death markers compared to those with simple steatosis. Furthermore, in patients with relevant fibrosis higher CK-18 values are found as compared to those with low fibrosis. In acute liver failure, cell death biomarkers may assist decision finding for the necessity of liver transplantation. DISCUSSION: Due to promising results of various studies, CK-18 cell death markers could be applied in clinical routine soon.

Serum microRNA-122 kinetics in patients with chronic hepatitis C virus infection during antiviral therapy.

The levels of the liver-specific microRNA-122 (miR-122) circulating extracellularly in the blood have been shown to be increased upon liver damage. However, it is unknown if the levels of serum miR-122 are altered during antiviral therapy and reflect the therapeutic success. Here, we investigated miR-122 serum levels in patients with chronic hepatitis C virus (HCV) genotype 1 infection during antiviral therapy with pegylated interferon and ribavirin. Therefore, sera from 60 patients with chronic HCV infection genotype 1 showing sustained virological response (SVR), non-response or relapse to therapy obtained at baseline, 4, 12, 24 weeks, end of treatment and follow-up were analysed retrospectively for miR-122 content by quantitative real-time reverse transcription PCR. The time courses of miR-122 were correlated with HCV RNA as well as standard liver parameters. We found that while there was no relation between serum miR-122 and HCV RNA levels at baseline, the decline in HCV RNA upon beginning of the therapy closely correlated with the reduction of serum miR-122 in the three different patient groups. Moreover, the serum miR-122 level correlated well with alanine aminotransaminase, a marker of ongoing liver damage. At follow-up serum miR-122 levels remained low in SVR, but increased to baseline levels in patients not responding or showing relapse to therapy. In contrast, the serum concentration of the ubiquitously expressed miR-16 did not change during therapy. We conclude that the serum level of miR-122 well reflects the success of interferon/ribavirin therapy in patients with chronic HCV infection.

Dynamics of hepatitis B virus quasispecies heterogeneity and virologic response in patients receiving low-to-moderate genetic barrier nucleoside analogs.

We characterized the early dynamics of hepatitis B virus (HBV) quasispecies evolution during the first weeks of antiviral therapy with low-to-moderate genetic barrier antiviral drugs and associated these data with antiviral response patterns. Fifteen chronic hepatitis B patients (men, 10; mean age, 34; HBeAg positive, 6) who received lamivudine or telbivudine for at least 52 weeks were included. HBV DNA was extracted from serum, and a 910-bp fragment covering domains A-F of the reverse transcriptase region was amplified, cloned and sequenced. Parameters of quasispecies heterogeneity, genetic diversity and complexity were calculated and were correlated with complete virologic response, defined as undetectable HBV DNA at week 52. Nine patients achieved complete virologic response during the observational period. While baseline HBV DNA levels and HBeAg status were associated with virologic response, baseline quasispecies complexity and diversity of responders showed no significant difference to those of nonresponders (P > 0.05). However, at week 4, quasispecies complexity of nonresponders was significantly higher compared with that of responders on the nucleotide level (P = 0.01) and the aa level (P = 0.04). The number of synonymous substitutions per synonymous site dropped significantly in responders at week 4 (P = 0.04), while there was no difference in nonresponders. The HBV quasispecies complexity at the early stage of antiviral therapy (week 4) with the low-to-moderate genetic barrier nucleoside analogs lamivudine or telbivudine was associated with subsequent virologic response. Further studies are needed to confirm HBV quasispecies evolution as additional predictive marker for beneficial treatment outcome.

Modulation of replication efficacy of the hepatitis C virus replicon Con1 by site-directed mutagenesis of an NS4B aminoterminal basic leucine zipper.

The hepatitis C virus (HCV) nonstructural protein 4B (NS4B) is assumed to function as a membrane anchor and protein hub for the viral replication complex. The aim of the current work was to modulate HCV replication efficacy in the subgenomic Con1 replicon by mutations of specific sites within the aminoterminal-located basic leucine zipper (bZIP), a candidate motif for protein-protein interactions involving NS4B. Mutational sites and amino acid substitutes were determined by in-silico sequence analyses of the NS4B-bZIP motif in 357 isolates of HCV genotype 1b from the euHCVdB and LosAlamos database and consecutive analysis of conserved physico-chemical properties at bZIP specific positions. Mutants with predicted minor, medium or major reduction of replication efficacy were tested in the pFKI389neo/NS3-3'/ET plasmid replicon model. Four sites (L25, T29, V39 and W43) of crucial importance for bZIP-mediated protein interaction with predicted apolarity of respective amino acid positions were selected for mutational studies. Substitutes with physico-chemical properties matching the predicted requirements either well (T29A), moderately (L25W, V39W), or insufficiently (T29E, W43E) were associated with slightly improved, moderate and marked decreased replication efficacy, respectively. Spontaneous (T29G) and adaptive (A28G, E40G) mutations occurred in the T29E mutation isolate only and were associated with marked reduction of replication efficacy. The bZIP motif region of NS4B is crucial for RNA replication in the subgenomic Con1 replicon system. RNA replication efficacy can be modulated by site-directed mutagenesis at specific bZIP functional sites. New adaptive amino acid mutations were identified within the HCV NS4B protein.

New developments in HCV therapy.

About half of the patients with chronic hepatitis C are still not cured by treatment with the current standard of care, peginterferon α/ribavirin. Direct antiviral drugs may overcome the limitations of standard antiviral therapy. The most promising new agents are inhibitors of the NS3/4A protease, the NS5B polymerase and the NS5A protein. Several compounds against these targets have entered clinical evaluation. Early clinical trials have emphasized the high potential for selecting resistant Hepatitis C virus variants. Furthermore, development of several new direct antivirals was stopped because of concerns over tolerability and safety. Then, in 2010, two phase III trials with the NS3/4A protease inhibitors boceprevir (SPRINT-2) and telaprevir (ADVANCE) showed that the combination of these compounds with standard care increases sustained virologic response rates in treatment-naïve genotype 1 patients from 38-44% to 66-75%. Future goals of therapy with direct antiviral agents are to improve tolerability, shorten the duration of therapy and overcome the issue of resistance. Several studies have been initiated that combine different novel therapies, with and without interferon α/ribavirin.

Serum microRNA-122 levels in different groups of patients with chronic hepatitis B virus infection.

miR-122 is a liver-specific microRNA, which also circulates in the blood. The levels of miR-122 in serum and plasma correlate with hepatic necroinflammation in patients with hepatitis B virus (HBV) infection. Here, we investigated whether miR-122 levels correlate with surrogate markers for viral replication and translation. Furthermore, we examined whether miR-122 levels differ in the different groups of HBV-infected patients and whether miR-122 levels may be useful to identify patients with higher or lower risk for liver disease progression. Therefore, RNA was extracted from sera of therapy-naïve patients with HBV infection (n = 89) and from healthy volunteers (n = 19). The concentration of miR-122 was assessed by quantitative real-time reverse-transcription PCR. HBs antigen and HBV DNA levels were quantified as surrogate parameters for HBV replication and translation. Liver biopsies were examined according to the histological activity index and the degree of fibrosis was assessed. We found that the miR-122 serum concentration correlated with the level of ALT, HBV DNA and HBs antigen (r = 0.259, P < 0.05; r = 0.225, P < 0.05; r = 0.508, P < 0.001, respectively). The miR-122 serum levels discriminated the different patient groups infected with HBV from healthy subjects (P < 0.001), and inactive carrier patients with high (>3500 IU/mL) or low (<3500 IU/mL) levels of HBs antigen could be differentiated by the miR-122 serum concentration (P < 0.05). As serum miR-122 levels strongly correlated with HBs antigen, it might be an indicator for viral translation. Furthermore, serum miR-122 levels discriminated HBV carrier patients with high or low risk for disease progression and may, thus, be an additional marker for risk stratification.

Comparison of envelope 2 CD81 binding regions in PBMC-derived versus serum-derived hepatitis C virus isolates: higher conservation of CD81 region 2 in PBMC isolates.

UNLABELLED: The aim of the present study was to investigate the variability of hepatitis C virus (HCV) CD81 binding regions (CD81-1/2) in peripheral blood mononuclear cells (PBMC)-derived and serum-derived HCV-RNA samples. HCV-RNA was isolated from PBMC (104 cells) and serum samples from 37 patients chronically infected with HCV genotype 1a/1b (n=21/16). The hypervariable regions 1/2 (amino acid 384-410, amino acid 474-482) and regions CD81-1/2 (amino acid 474-494, amino acid 522-551) were analysed. Mutational frequency of amino acid sequences was compared between PBMC-derived and serum-derived HCV variants as well as local accumulation of mutations. Furthermore, CD81 was quantified on PBMC. Mutational frequency was not different between PBMC-derived and serum-derived HCV variants. A trend to lower mutational frequency in genotype 1a PBMC variants compared with serum-derived variants was observed in region CD81-2 (5%vs 10%). Smoothed mutational frequency analysis showed a significantly lower variability within genotype 1a CD81-2 in PBMC-derived compared to serum-derived HCV-RNA (P=0.026). CD81 expression on PBMC was not correlated with the number of mutations within the CD81 binding regions. CONCLUSION: A higher conservation was observed in region CD81-2 in PBMC-derived versus serum-derived HCV-RNA indicating selection of HCV variants on PBMC. The variability in the CD81 binding regions appeared to be independent from CD81 expression.

Prospective study of bone mineral density and metabolism in patients with chronic hepatitis C during pegylated interferon alpha and ribavirin therapy.

The importance of osteoporosis as a complication of end-stage liver disease is well known. However, significant osteopenia may occur in earlier stages of chronic hepatitis C (CHC). Furthermore, antiviral therapy may influence bone metabolism. Thirty patients with CHC genotype 1 infection and without established cirrhosis were treated with peginterferon-alfa and ribavirin. Dual-energy x-ray absorptiometry was performed at baseline, after 48 weeks of therapy, and by the end of a 24-week follow-up period. Bone mineral density (BMD), T-scores, and Z-scores were assessed. Serum C-terminal propeptide of type I collagen (CICP) and osteocalcin levels were measured. Thirteen patients had osteopenia (43%) and osteoporosis was present in four patients (13%). Antiviral therapy led to significant on-treatment increases of lumbar spine and hip BMD (P < or = 0.05) as well as T-scores (P < or = 0.05) and Z-scores (P < or = 0.01) irrespective of subsequent treatment response. Further analyses showed that in patients with sustained virological response (n = 19) most parameters remained highly above baseline values by the end of the 24-week follow-up period, while patients with virological relapse (n = 11) had decreases of BMD, T-scores and Z-scores thereafter that did not differ from baseline. Serum CICP and osteocalcin levels decreased during therapy. Osteocalcin levels remained below baseline in sustained responder, but showed an increase in relapsers by the end of the 24-week follow-up (P < or = 0.05). Osteopenia is detectable in a substantial proportion of CHC patients without established cirrhosis. Antiviral therapy leads to an on-treatment increase of BMD, which may last in those patients who achieve a sustained virological response.

Somatic hypermutation and mRNA expression levels of the BCL-6 gene in patients with hepatitis C virus-associated lymphoproliferative diseases.

Chronic hepatitis C virus (HCV) infection leads to mixed cryoglobulinaemia (MC) and B-cell non-Hodgkin lymphoma (B-NHL). Aberrant somatic hypermutation and deregulation of the oncogene BCL-6 is associated with lymphomagenesis. Recently, HCV was shown to induce BCL-6 mutations in vitro. The BCL-6 gene (area B) was cloned and sequenced from peripheral blood mononuclear cells (PBMC) of 21 chronically HCV-infected patients with or without MC and B-NHL, and six healthy controls. Mutational frequencies, genetic complexity and diversity were calculated. BCL-6 mRNA from PBMC was quantified by real-time polymerase chain reaction, and additional sustained virologic responders to antiviral therapy and HBV patients served as controls. The overall/recurrent mutational frequencies tended to be lower in MC and B-NHL patients when compared with controls (P = 0.15 and 0.06, respectively). Genetic complexity was significantly lower in MC and B-NHL patients (P = 0.025). BCL-6 mRNA concentration was decreased in all HCV patients when compared with healthy controls, sustained virologic responder and HBV patients (P = 0.005). Although HCV can induce BCL-6 mutations in vitro, lower mutational frequencies and decreased BCL-6 mRNA expression in vivo suggest no major role of aberrant somatic hypermutation in HCV-associated MC and B-NHL.

Impaired health-related quality of life in patients with chronic hepatitis C and persistently normal aminotransferase levels.

A significant impact of hepatitis C virus (HCV) infection on health-related quality of life (HRQOL) has been previously described. However, comprehensive data on the quality of life in patients with chronic hepatitis C and persistently normal aminotransferase levels (PNAL) are currently not available. One hundred fifteen patients with chronic hepatitis C (45 with persistently normal aminotransferases and 70 with elevated aminotransferases) and 50 healthy subjects were enrolled. Emotional and psychological states were assessed by Profile of Mood States (POMS) scale and HRQOL was assessed by the 'Everyday Life' questionnaire (EDLQ), a validated questionnaire related to the SF-36 Health Survey. An impairment in HRQOL was observed in patients with chronic hepatitis C showing PNAL compared with healthy subjects with significant differences for the factor scores depression and anger in the POMS scale as well as for the items body, relationship to partner, self-confidence and zest of life in the EDLQ. No differences in any questionnaire were observed between patients with chronic hepatitis C showing PNAL or elevated aminotransferase levels except of a worse mean level for factor score anger in POMS scale in patients with persistently normal aminotransferases. No association of quality of life with severity of liver disease was found. Impairment of HRQOL by chronic infection with HCV is similar in patients with PNAL and those with elevated aminotransferase levels.

Apoptotic cytokeratin 18 neoepitopes in serum of patients with chronic hepatitis C.

In patients with chronic hepatitis C, alanine aminotransferase (ALT) levels do not accurately reflect the extent of liver inflammation. The discrepancy between ALT level and liver damage could be related to the mode of cell death. In the present study, we quantified serum levels of apoptotic cytokeratin 18 (CK-18) neoepitopes that are generated by activated caspases during apoptosis. Apoptotic CK-18 neoepitopes were quantified by enzyme linked immunosorbent assay in sera from patients with chronic hepatitis C and elevated ALT levels (n = 72), patients with chronic hepatitis C and persistently normal ALT levels (n = 27) and healthy controls (n = 19). Serum CK-18 neoepitope levels were strongly correlated with ALT (r = 0.659, P < 0.0001) and the histology activity index (r = 0.374, P < 0.001). Patients with chronic hepatitis C and persistently normal ALT levels had higher apoptotic CK-18 neoepitope levels than healthy controls (P = 0.03) but lower levels than patients with chronic hepatitis C and elevated ALT levels (P < 0.001). Highest serum CK-18 neoepitope levels were observed in patients with cirrhosis (P = 0.002). Hence apoptotic CK-18 neoepitopes in serum of patients with chronic hepatitis C are associated with ALT level and histological liver damage. Serum apoptotic CK-18 neoepitope levels are elevated both in patients with chronic hepatitis C and elevated ALT levels as well as in patients with normal ALT levels indicating that also patients with chronic hepatitis C and normal ALT have an increased hepatocyte loss by apoptosis.