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The HEP-NET B/C co-infection trial: A prospective multicenter study to investigate the efficacy of pegylated interferon-β2b and ribavirin in patients with HBV/HCV co-infection
  Journal of Hepatology
May 22, 2008
Articles in Press
Andrej Potthoff1, Heiner Wedemeyer1, Wulf O. Boecher2, Thomas Berg3, Stefan Zeuzem4, Joachim Arnold5, Ulrich Spengler6, Kurt Gruengreiff7, Thomas Kaeser8, Marcus Schuchmann2, Alexandra Bergk3, Nicole Forestier4, Katja Deterding1, Michael P. Manns1, Christian Trautwein19, for the Hep-Net B/C Co-infection Study Group
1 Department of Gastroenterology, Hepatology and Endocrinology, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30623 Hannover, Germany
2 I. Medizinische Klinik und Poliklinik, Johannes Gutenberg Universitat, Langenbeckstr. 1, 55131 Mainz, Germany
3 Department of Gastroenterology and Hepatology, Universitatsklinik Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, 13353 Berlin, Germany
4 Department of Gastroenterology, Hepatology, Nutritial Medicine and Pneumology, Klinikum der Johann-Wolfgang Goethe-Universitat, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany
5 II Medizinische Klinik, Diakoniekrankenhaus Rotenburg (Wumme), Elise-Averdieck-Strasse 17, 27356 Rotenburg (Wumme), Germany
6 Medizinische Klinik und Poliklinik I, Universitatsklinikum Bonn, Sigmund-Freud-Straβe 25, 53127 Bonn, Germany
7 Gastroenterologische Praxis, Heydeckstr. 9, 39104 Magdeburg, Germany
8 Gastroenterologische Praxis, Marktplatz 14, 72250 Freudenstadt, Germany
9 Medizinische Klinik III, Universitatsklinikum Aachen, Pauwelstrasse 30, 52074 Aachen, Germany
Corrected Proof
Infections with the hepatitis B virus (HBV) and hepatitis C virus (HCV) either alone or in combination are major causes of chronic liver disease in the Western world. Patients co-infected with HBV and HCV show faster fibrosis progression and frequently present with more severe liver disease [1], [2], [3]. In addition, co-infected patients have a markedly increased risk of developing hepatocellular carcinoma (HCC) [4], [5] People diagnosed with hepatitis B and hepatitis C co-infection have a markedly increased risk of liver-related death and overall mortality compared with the standard population [6].
Several reports suggested that HBV and HCV replication may interact in co-infected patients. A dominant role of HCV has been suggested by some investigators [7], [8], while other groups reported a reciprocal interference or even a dominant effect of HBV [9], [10]. Subsequently, severe HBV reactivations have been described in single patients after successful HCV treatment in dual HBV and HCV infected patients [11], [12], [13]. Currently, there is no established standard treatment for HBV/HCV co-infected patients. In general, treatment guidelines recommend applying the same criteria to patients either mono-infected or dually HBV/HCV infected [14]. Interferon alpha (IFN-_) is the most studied agent in treating co-infected patients because of its known activity against HBV and HCV. In most studies IFN monotherapy resulted in a similar rate of sustained biochemical response compared to patients with chronic HCV infection alone [15]. Villa et al. conducted a prospective randomized trial showing some efficacy, if higher doses of standard interferon alpha were used [16]. The addition of lamivudine to IFN-_ may be effective in co-infected patients with chronic HCV and active HBV replication [17]. A study on 21 HBV/HCV co-infected patients demonstrated the effectiveness of combined IFN-_2b and ribavirin in this cohort with an HCV response of 43% in co-infected patients as compared to an HCV-SVR of 60% in HCV mono-infected controls [18]. In 2005, Hung et al. reported a higher rate of HCV-SVR using IFN-_2b plus ribavirin (69%). Interestingly, 53% of patients with negative HBV-DNA at baseline had reactivation of HBV-DNA at end of treatment [19]. Another study using a higher dose of IFN-_2b (6MU three times weekly for 24 weeks) and ribavirin (1000-1200mg daily for 24 weeks) led to comparable rates of SVR in HCV co-infected (69.0%) and mono-infected (67.2%) patients (IFN-naive) [20].
At present, no studies have been published using PEG-IFN in co-infected patients, although PEG-IFN-_2b plus ribavirin combination therapy is considered the most effective treatment option for chronic HCV infection [21]. In chronic hepatitis B PEG-IFN has been successfully applied in about one third of patients [22], [23], [24]. For HBV/HCV co-infected patients two case reports have been published using combination therapy of PEG-IFN-a2b and ribavirin [25], [26]. The first case resulted in a HBeAg seroconversion at the end of treatment, while the second case showed not only a sustained HCV-RNA response but also HBs-seroconversion after an individualized course of PEG-IFN-a2b, ribavirin and additional active HBV vaccination.
Based on these data, we initiated a prospective, controlled multicenter study in the German Hep-Net to evaluate the efficacy of 48 weeks of PEG-IFN-_2b in combination with ribavirin in HBV/HCV co-infected patients.

The efficacy of pegylated interferon alpha and ribavirin in HBV/HCV co-infected patients is unknown.
Nineteen patients with chronic HBV/HCV co-infection (HBsAg and HCV-RNA positive; 10 HCV-genotype 1; 9 HCV-genotype 2 or 3) were included in this prospective multicenter pilot study. Baseline HBV-DNA was negative in 13 individuals. All patients received weight-adjusted PEG-IFN-_2b and ribavirin for 48 weeks.
In the intent-to-treat analysis, a biochemical and an HCV-RNA response were observed in 12 and 14 patients, respectively (63% and 74%). At the end of the treatment as well as at the end of the follow-up the HCV-RNA response was 93% (14/15) in patients adherent to therapy (86% in genotype 1 and 100% in genotypes 2 and 3 infection). Two of the five initially HBV-DNA positive patients with follow-up available were HBV-DNA negative at follow-up week 24. In contrast, HBV-DNA became detectable after the clearance of HCV in four initially HBV-DNA negative patients.
Combination therapy with PEG-IFN-a2b and ribavirin is highly effective in inducing a virological response concerning HCV in patients with HBV/HCV co-infection. However, HBV replication may increase after the clearance of HCV and thus close monitoring for both the viruses is recommended even in patients with initially undetectable HBV-DNA.
Associate Editor: J.G. McHutchison
Antiviral therapy was initiated in 19 patients (14 males, and 5 females). Baseline demographics are shown in Table 1. Four of the 19 patients had a history of drug abuse, while 4 patients acquired HBV/HCV co-infection after blood transfusion. In the remaining 11 patients the acquisition mode was unknown. Six of the 19 patients had a history of hepatitis A infection in the past, indicated by HAV-IgM negative and HAV-IgG positive. Grading of liver biopsies inflammation revealed grade 1 in 8 patients and grade 2 in the remaining 11 patients. Staging of liver biopsies resulted in portal fibrosis in 9/19 (47%). In seven of 19 patients an incomplete porto-portal fibrosis was observed (stage 2). Pre-cirrhotic changes were found in 1 patient (stage 3) and 1 patient had cirrhosis (stage 4). One patient had no fibrosis, but was enrolled in the study due to high elevated serum aminotransferases. Biochemical and virological characteristics are shown in Table 1, Table 2.
Fifteen of 19 patients (79%) completed the treatment course and were followed up for at least 24 weeks after end of therapy. In 4 patients antiviral treatment was not completed. One patient had a serious adverse event (psychosis, treatment week 36), and 3 patients were lost during follow-up (treatment weeks 12 and 24 in two cases).
HCV-RNA response
Based on the "intent to treat" analysis 10 of 19 (53%) patients showed an early virological response (EVR) with undetectable HCV-RNA at treatment week 12. At treatment week 24 14/19 patients were negative for HCV-RNA and no patient experienced a relapse during further treatment and follow-up. Therefore, the overall virological response to HCV was 74% (Fig. 1). One patient (#11) infected with HCV genotype 1 did not respond to PEG-IFN-a2b and RBV and was still positive for HCV-RNA at FU24 (Nonresponse). This patient was the one with the highest baseline HBV-DNA of 1.2_107IU/ml. All patients infected with genotypes 2 and 3 who completed treatment experienced a virological response concerning HCV. Considering only the 15 patients who completed treatment, we found an HCV-RNA response of 93% (genotype 1 86% and genotype 2 or 3 100%).
HBV-DNA response
All patients remained HBsAg-positive throughout treatment and follow-up. The HBe antigen status remained unchanged in all patients. HBV-DNA became negative in two of the six HBV-DNA positive patients (patients #12 and #13), both individuals were infected with HBV-genotype D. Two patients (#8 and #11) remained HBV-DNA positive and two were lost to follow-up. Four of the 13 patients (31%) with undectectable pre-treatment HBV-DNA had an HBV viremia at the end of follow-up. In three of them HBV-DNA was low with 0.4_103IU/ml (#6), 0.6_103IU/ml (#5) and 1.2_103IU/ml (#9), respectively. One initially HBV-DNA negative patient (#4) showed a high HBV viremia at FU week 24 with 5.0_106IU/ml. The complete virological profile is shown in Table 2.
Biochemical outcome
ALT levels correlated with the virological course of HCV clearance in most patients. The mean ALT of 120IU/ml (53-395) normalized to a mean of 36IU/mL (18-107) at treatment week 12. Based on the intent-to-treat analysis ALT values at the end of follow-up were normal in 12 of 19 patients (mean 31U/mL). At the end of treatment liver function - as determined by albumin levels, prothrombin time and bilirubin levels - was within normal ranges and comparable to baseline levels prior to treatment in all 15 patients. None of the four initially HBV-DNA negative patients who became HBV-DNA positive after clearance of hepatitis C experienced an ALT flare, as defined by an increase of at least three times compared to the lowest level after initial response was observed.
Adverse events (AEs) were recorded for all patients during treatment and follow-up. Nearly all the AEs recorded were attributed to treatment. Most of the adverse events occurred during the first 12 weeks of treatment. The symptoms described by patients were very similar to the side-effects known to be associated with combination therapy of PEG-IFN-_2b and ribavirin. Serious adverse events (SAEs) occurred in 3 patients. One patient developed psychosis hence treatment was withdrawn. One patient developed lobular pneumonia with consecutive hospitalization. Another patient had a traffic accident with several injuries. Antiviral treatment was discontinued for 6 weeks until discharge and complete recovery of pneumonia in 1 patient, while treatment was discontinued for 3 weeks in the other patient. However, both patients still remained HCV-RNA negative at the end of follow-up.
In the present study, we investigated the efficacy of combination therapy with PEG-IFN-_2b and ribavirin in patients with HBV/HCV co-infection. We demonstrated that virological response concerning HCV clearance occured in 74% of the patients (14/19). Only one of the patients completing therapy did not respond with negativity for HCV-RNA at the end of treatment and 24 weeks after follow-up. Two of 6 patients (33%) with pre-treatment HBV viremia also cleared serum HBV-DNA and thus responded for both viruses at follow-up week 24. Thus, our study suggests that combination therapy with PEG-IFN-_2b and ribavirin is at least as effective in patients with HBV/HCV co-infection as in individuals infected with HCV alone, which is in line with most previous studies using conventional interferon alpha in combination with ribavirin [11], [15], [16], [18], [19], [20], [29].
Interestingly, response rates in adherent patients were even higher than in most previous reports on HCV monoinfection [21]. This might be partially explained by lower HCV-viremias in the context of HBV co-infection, lower baseline HCV-RNA levels were found in our trial as only four of the 19 patients had an HCV-RNA above 106IU/ml while 5 patients showed HCV-RNA levels below 100,000IU/ml.
Both HCV-genotype 1 and HCV genotype non-1-infected patients attained excellent response rates. These results may indicate that shorter treatments might also be promising in HBV/HCV co-infected patients which needs to be evaluated in future trials.
In the present study, pre-treatment serum HBV-DNA was positive in 26% (6/19) of patients. At the end of antiviral therapy HBV-DNA clearance was achieved in two of the 6 patients. Unexpectedly, these 2 patients were infected with HBV-genotype D. In previous trials of PEG-IFN-_ in hepatitis B, HBV-genotype D showed the lowest response rate [24], [30]. Larger studies are needed to address the role of HBV genotypes in HBV/HCV co-infected patients.
Several studies reported that IFN treatment in HBV/HCV co-infected patients carries the risk of a severe hepatitis flare if the suppressive effect of one virus is removed, thereby allowing the other virus to replicate [7], [31], [32], [33]. Post-treatment HBV viremia was detected in four out of 13 patients who were HBV-DNA negative before treatment. Fortunately, three of them had rather low levels of HBV-DNA (<2000IU/mL) and thus no antiviral therapy for HBV was indicated in accordance with consensus guidelines [14]. Importantly, the HBV rebound was not accompanied by ALT flares. However, 1 patient developed rather high HBV-DNA levels and thus antiviral therapy with an HBV polymerase inhibitor should be considered.
Although virological response rates were excellent in terms of HCV clearance, it would be interesting to investigate possible factors leading to treatment failure in the context of HBV/HCV co-infection. HBV viremia might be such a factor. In line with this hypothesis would be the observation that the only HCV non-responder in this trial had the highest HBV-viral load of all patients. In line with high HBV viremia, HCV-RNA was rather low confirming reciprocal inhibition of replication between HBV and HCV [2], [34]. This patient did not clear HCV despite his low HCV viral load suggesting a possible inhibitory effect of HBV on IFN response. HBV may impair dendritic-cell function [35] and is also associated with increased regulatory T cell (Treg) frequency [36]. Thus, it is tempting to speculate that HBV has contributed to suppression of HCV-specific T cell responses.
The frequency of serious adverse events (SAEs) was comparable to trials in HCV mono-infected patients and thus our study suggests that there are no specific safety concerns in treating HBV/HCV co-infected patients with PEG-IFN-_ and ribavirin [37], [38].
However, our study has some limitations. First, the number of individuals included was rather limited. Secondly, only few patients were viremic for HBV-DNA and two of those patients were lost to follow-up. Thirdly, in 3 patients virological measurements were performed by a different assay. Unfortunately, stored serum samples were lost for the 3 patients and thus, only data of the local assay were available for these 3 patients. Fourthly, nucleos(t)ides against HBV were not explored in our trial. In addition, patients with HBV dominant co-infection (HBV-DNA positive/HCV-RNA negative) were excluded from this trial. Thus, future studies have to address the role of interferon-alpha in patients with HBV dominance. Moreover, no control group was included in this trial. Moreover, we have also to consider that dominance patterns in HBV/HCV co-infection may change over time. Therefore, some of the observed fluctuations in HBV viremia may not necessarily be a consequence of treatment but rather reflect the natural course of disease.
Despite these limitations, this first prospective study in HBV/HCV co-infected study using PEG-IFN plus RBV strongly suggests that this regimen at present should be the treatment of choice in patients with dominant HCV replication. In patients with dominant HBV disease other options especially nucleos(t)ide analogue alone or in combination with interferons should be tested.

Between June 2003 and November 2005 20 HBV/HCV co-infected patients were recruited in eight centers in Germany. One patient cleared HBsAg spontaneously between the screening and baseline visit, thus 19 treatment-naive patients were enrolled in the study.
All patients were positive for hepatitis B surface antigen (HBsAg), antibodies to HCV (anti-HCV), HCV-RNA and had elevated alanine aminotransferase (ALT) levels on two occasions in the 4 weeks before enrollment. Exclusion criteria were evidence for hepatitis delta virus (HDV) superinfection, human immunodeficiency virus (HIV) infection, alcohol abuse, autoimmune hepatitis, other causes of liver disease, decompensated liver cirrhosis, previous IFN treatment and contraindications to IFN-_ or ribavirin therapy. All patients gave a written informed consent prior to treatment. Virological response concerning HCV was defined as HCV-RNA negativity at the end of treatment and 24 weeks after end of treatment. For hepatitis B, a virological response was defined as HBV-DNA levels below 300copies/ml or HBsAg seroconversion. All patients who received at least one injection of PEG-IFN-_2b were included in the "intent-to-treat" analysis (n=19). Only patients having completed follow-up week 24 (FU24) were considered for the "per protocol" analysis.
Study medication and visit schedule
All patients (HCV-genotype 1 and HCV-genotype non-1) were treated with a 48-week course of weight-adjusted PEG-IFN-_2b (1.5_g/kg PegIntron, Schering-Plough, Kenilworth, NJ, USA) subcutaneously once weekly and with oral ribavirin daily (Rebetol, Schering-Plough, Kenilworth, NJ, USA). Ribavirin was given at a daily dose of 1000mg for patients up to 75kg of weight and 1200mg for patients who weighed more than 75kg. The dosage of ribavirin was modified based on the recommendations given in the package insert.
All patients were monitored by the investigator at treatment weeks 0, 2, 4, 8, and 12. Thereafter, patients were monitored every 6 weeks during treatment and at follow-up weeks 4, 12, and 24. The treatment visits included measurements of ALT and AST levels, blood counts, liver function tests, pregnancy test and thyroid function test. Serum HBV-DNA and HCV-RNA were assessed at enrollment, at treatment weeks 12, 24, and 48, and during the follow-up phase at weeks 12 and 24.
Liver biopsy
The patients underwent liver biopsy during the 6 months prior to the start of antiviral therapy. In 1 patient pre-treatment liver biopsy was performed 9 months before study enrollment. Histological grading and staging of chronic liver diseases were performed by the local pathologists based on the Desmet scoring system reflecting the degree of hepatic inflammation and fibrosis, respectively [27].
Hepatitis virus and biochemical markers
Hepatitis B virus markers, including HBsAg, HBeAg, antibodies to HBeAg (anti-HBe), and antibodies to hepatitis D virus (anti-HDV) were assayed using commercially available enzyme immunoassay kits (Abbott Laboratories, North Chicago, USA). Serum HBV-DNA was quantified by real-time PCR (Cobas Amplicor, Roche Diagnostics, Mannheim, Germany) and results are given in IU/mL. In 3 patients a bDNA test was used (Versant HBV DNA 3.0, Bayer Diagnostics, Germany). The HBV-genotypes of samples with detectable HBV-DNA were determined using a line probe assay (INNO-LiPA HBV Genotyping, Innogenetics NV, Ghent, Belgium), as described previously [24]. HCV-RNA quantification was performed using Cobas Amplicor (Roche Diagnostics, Germany). In 3 patients a bDNA test was used (Versant HCV-RNA 3.0 Bayer Diagnostics, limit of detection >615IU/ml). Genotyping of HCV was done according to the classification of Simmonds et al. [28] by reverse hybridization assay (Inno-LiPA HCV II; Innogenetics NV, Gent, Belgium) in the HCV-Amplicor products. HBV-DNA and HBV genotyping of HBV viremic patients were reassessed as necessary in our local laboratory.
Biochemical liver function tests were performed by standard laboratory tests. ALT was measured at 37°C and the upper limit was 45U/l for men and 35U/l for women.
The effects of antiviral therapy on course and outcome of chronic HBV/HCV co-infection were assessed by determination of (i) HBV replication (as indicated by qualitative serum HBV-DNA levels), a complete response to HBV infection was defined as HBV-DNA levels below 60IU/ml (300copies/ml) 24 weeks after end of treatment (ii) HCV replication, a complete response to HCV infection was defined as a HCV-RNA PCR (Cobas amplicor) 24 weeks after end of treatment (iii) biochemical responses (serum ALT activities) and (iv) safety parameters.
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