End-stage liver disease secondary to hepatitis C virus (HCV) is the most common indication for liver transplantation (LT) in developed countries. HCV recurrence after transplantation is almost universal and most patients will develop chronic hepatitis on the graft during follow-up.1 The course of HCV graft disease is accelerated in transplant recipients compared with immunocompetent patients.2 In liver transplant recipients, chronic HCV infection leads to cirrhosis in up to 30% of patients at 5 yr after LT,3 and many of these patients experience their first clinical decompensation within 1 yr after diagnosis.4 Analysis of the United Network for Organ Sharing and European Liver Transplant Registry database demonstrates a lower survival rate of LT patients with recurrent hepatitis C than in patients transplanted for other indications.5, 6 Indeed, the main cause of death in these HCV transplant recipients was allograft failure due to HCV recurrence.
Thus, the outcome of recurrent hepatitis C in LT is a matter of increasing concern. In this context, it appears to be important to consider antiviral therapy with the aim of delaying the progression of liver injury and thus preventing graft failure. Theoretically, HCV infection in LT may also be managed either in the immediate posttransplantation period, with the aim of preventing the development of chronic hepatitis in the graft, or prior to transplantation, to prevent graft infection, but the scarce available information seems to discourage these strategies.7
The aim of this study was to evaluate the efficacy and safety of treatment with pegylated interferon alpha 2 (PEG-IFN) and ribavirin in LT patients with established HCV recurrence.
Since November 2001, consecutive HCV-transplanted patients with recurrent HCV infection were prospectively evaluated as candidates to receive antiviral therapy with PEG-IFN and ribavirin. Patients were eligible for treatment when they fulfilled the following criteria: 1) they were at least 18 yr of age; 2) had abnormal liver function tests; 3) had a positive serum HCV-ribonucleic acid (RNA); and 4) they had evidence of chronic hepatitis on graft biopsy (fibrosis index ≥ F2 or F1 with necroinflammatory index ≥3). Written informed consent was obtained from each patient, and the study protocol followed the ethical guidelines of the Declaration of Helsinki. Patients excluded from treatment included those with ductopenic or acute rejection, concurrent biliary or vascular complications, persistently normal alanine aminotransferase (ALT) values, hemoglobin levels less than 10 gm/dL, absolute neutrophil count less than 1500/μL, platelet count less than 65000/μL or renal failure (creatinine clearance below 35 mL/minute) or who denied to give the informed consent. In addition, patients with severe depression, renal transplant or a history of ischemic cardiopathy within the previous 12 months were excluded. Participants also had to be IFN naive following LT and to accept not to drink alcohol. Patients were receiving cyclosporine or tacrolimus ± steroids as immunosuppressive therapy. In those patients treated with mycophenolate mofetil, this drug was discontinued. For the purpose of this study, only patients who completed the treatment and had at least 6 months of follow-up after the end of therapy were included in the analysis.
All patients were treated with PEG-IFN (Peg-Intron; Schering-Plough, Kenilworth, NJ, USA) and ribavirin (Rebetol; Schering-Plough). The treatment period was 48 weeks. PEG-IFN was initiated at 1.5 μg/kg/week and was reduced to 1.0 μg/kg/week when neutrophils dropped below 500/μL or platelets below 35,000/μL. If neutrophils or platelets persisted below these limits, PEG-IFN was discontinued. Ribavirin was initially given at a dose of 600 mg/day for patients with a body weight less than 70 kg and 800 mg/day for patients with a body weight greater than 70 kg. The dose of ribavirin was increased 200 mg after 4 weeks of treatment if hemoglobin level was above 12 gm/dL. When hemoglobin dropped below 10.5 gm/dL, darbepoetin alpha (Aranesp; Amgen SA, Barcelona, Spain) was initiated at a dose of 30-100 μg once weekly to maintain hemoglobin above 11 gm/dL. Ribavirin was discontinued when hemoglobin level dropped below 9 gm/dL despite darbepoetin alpha administration. To evaluate the influence of tolerance on the outcome of the patients, we have considered a complete antiviral treatment when the patient was able to receive more than 80% of the proposed dose of antiviral therapy during more than 80% of the time for which the treatment was scheduled.
Evaluation and Follow-Up
Clinical history, physical examination, and hematological and biochemical studies were performed at baseline, every 2 weeks for the first 6 weeks and then every 4 weeks during the treatment period, and 4, 12, 24 and 48 weeks after completion of treatment. In addition, these evaluations were checked more frequently when clinically indicated.
HCV-RNA and Genotype Determination
Qualitative HCV-RNA was determined by a polymerase chain reaction-based assay (Amplicor HCV 2.0; Roche Diagnostics, Branchburg, NJ, USA; for detection of HCV-RNA levels as low as 50 UI/mL). HCV-RNA was detected in serum at baseline and then every 12 weeks. The HCV-RNA viral load was determined at baseline by a quantitative assay (Cobas Amplicor HCV Monitor 2.0; Roche Diagnostics; for detection of HCV RNA levels as low as 600 UI/mL). Genotyping was performed using a reverse hybridization assay (Innolipa; Innogenetics, Rungis, France) after amplification by polymerase chain reaction.
Liver biopsy specimens were obtained before treatment and at the end of follow-up. Liver biopsy was also performed when a biochemical abnormality was detected during treatment. The slides were stained with hematoxylin-eosin and trichrome. Histological recurrence of HCV hepatitis was based on the presence of lobular and portal inflammation lacking diagnostic features of rejection. Inflammatory activity and fibrosis were scored according to the Scheuer scoring system.8
Diagnosis of recurrent cholestatic hepatitis C was diagnosed according to the criteria of the International Liver Transplantation Society Consensus definition9 and were as follows: 1) longer than 1 month posttransplantation (usually <6 months); 2) serum bilirubin level greater than 6 mg/dL; 3) serum alkaline phosphatase and γ-glutamyltransferase levels greater than 5 times the upper limits of normal; 4) characteristic histological state with ballooning degeneration of hepatocytes predominantly in the perivenular zone (not necrosis or fallout), paucity of inflammation in portal tract and interface, and variable degrees of cholangiolar proliferation without bile duct loss; 5) very high serum HCV RNA levels (in our case we have used a cutoff of >2,5 × 106 UI/mL); and 6) absence of surgical biliary complications (normal cholangiogram) and absence of evidence for hepatic artery thrombosis.
The primary endpoint was the achievement of a sustained virological response (SVR), as defined by a negative serum HCV-RNA test at the end of the follow-up period (at least 24 weeks after completion of therapy). Secondary endpoints included biochemical response (normalization of serum ALT levels at the end of treatment), end-of-treatment virological response (undetectable HCV-RNA at the end of therapy) and the improvement of the Scheuer index in a liver biopsy at the end of the follow-up. In addition, early virological response (EVR) was defined as HCV-RNA negativity after 12 weeks of treatment.
Results were reported in an intention-to-treat analysis. Quantitative variables were expressed as mean ± standard deviation. Categorical variables were analyzed by the χ2 test or Fisher's exact test as appropriate. For quantitative variables, group differences were analyzed using Student's t test. P values less than 0.05 were considered to be significant.
From November 2001 to April 2004, 88 liver transplant recipients with hepatitis C recurrence were screened as potential candidates to combined ribavirin and pegylated interferon treatment. A total of 23 patients did not qualify to receive antiviral therapy; 10 patients did not show fibrosis (F0 in the Scheuer index) or had mild fibrosis (F1 in the Scheuer index) with a mild necroinflammatory index in the liver biopsy; 3 patients were discarded due to the presence of ductopenic rejection or biliary complications, 3 due to anemia and/or leukopenia; and the remaining 7 patients did not give their consent to receive antiviral treatment. Additionally, 18 patients are still receiving antiviral therapy or have a follow-up period shorter than 6 months and were excluded from the analysis. Therefore, 47 patients were finally included in the current analysis. Baseline characteristics of the 47 patients are shown in Table 1. The median age of the participants was 52 yr; 28 (60%) were men, and all patients were Caucasian. The mean time for initiation of antiviral therapy after transplantation was 32 months (range: 3-149 months). Ten (21%) patients had cholestatic hepatitis and 8 (17%) patients had a severe (F3 or F4) fibrosis score at baseline liver biopsy. Among these 47 patients, 44 were infected by genotype 1 and only 3 were infected by a non-1 genotype (2 with genotype 2 and 1 with genotype 3). The mean HCV RNA load was 3,519,482 UI/mL (range: 31,000-200 × 106 UI/mL). Serum HCV RNA levels were greater than 850,000 UI/mL in 64% of patients. Patients with cholestatic hepatitis C had a mean HCV RNA value of 15,2 × 106 UI/mL (range: 4.5 × 106 to 200 × 106 UI/mL). Three patients had a history of antiviral treatment with interferon and ribavirin long before transplantation. Immunosuppressive treatment included tacrolimus in 35 patients and cyclosporine in 12 patients. Two patients were transplanted from a living donor.
Table 1. Baseline Characteristics of Patients With Post-LT HCV Recurrence (n = 47)
52.2 ± 9.2
Time since transplantation (month)
32 ± 25
201 ± 151
327 ± 368
Alkaline phosphatase (IU/L)
368 ± 282
3.7 ± 5.7
2.6 ± 0.8
2.7 ± 0.7
1.6 ± 0.9
Fibrosis score ≥ 3
HCV genotype 1
Serum HCV RNA > 850,000 UI/mL
Response to Therapy
A total of 33 of the 47 patients (70%) had normalized serum ALT levels and 17 (36%) tested negative for serum HCV RNA at the end of combination therapy. Eleven patients (23%) achieved SVR. After 12 weeks of treatment, 21 of 47 patients (45%) presented EVR and 11 of them (52%) had SVR. No patient with an HCV-RNA-positive test after 12 weeks of treatment achieved SVR. A total of 37 patients had completed the 48-week therapy with PEG-IFN and ribavirin, with 10 additional recipients in whom antiviral therapy was discontinued due to drug-related side effects. Among those who had completed therapy, biochemical response was observed in 33 (89%) patients, virological response at the end of treatment in 17 (46%) patients, and SVR in 11 (30%) patients. The 3 patients who were treated with interferon alpha and ribavirin before transplantation were nonresponders both before and after surgery.
The outcome of the patients with cholestatic hepatitis C is shown in Figure 1. Overall, 2 of 10 (20%) patients achieved SVR. Five patients achieved biochemical response at the end of the 48-week antiviral therapy, but remained HCV-RNA-positive. Three of them developed a severe cholestatic hepatitis when antiviral therapy was stopped; 1 of them died due to graft failure, and in the remaining 2 recipients who are still alive the reintroduction of antiviral therapy resulted in a normalization of ALT levels. Two patients with severe cholestatic hepatitis C, in whom liver function declined during antiviral therapy, died due to graft failure. The remaining patient with severe cholestatic hepatitis achieved EVR but he suffered acute rejection after 4 months of antiviral therapy and this was discontinued.
Predictors of Response
On an intention-to-treat basis, several baseline characteristics were examined to determine whether they could predict SVR (Table 2). Low basal serum γ-glutamyl transpeptidase levels and low HCV-RNA load were associated with a higher probability for SVR (P < 0.05). Additionally, SVR was more probable among patients who completed antiviral therapy, as defined previously (P < 0.05), and in those who had achieved an EVR (P < 0.01).
Table 2. Analysis of Prognostic Factors Associated With a Sustained Virological Response (SVR)
SVR (n = 11)
Non-SVR (n = 36)
The patient was able to receive more than 80% of the proposed dose of antiviral therapy during more than 80% of the time for which the treatment was scheduled.
Paired liver biopsies at baseline and at the end of combination treatment were available in 16 out of 37 patients who did not discontinue antiviral therapy, 7 patients who had SVR and 9 nonresponder patients (Fig. 2). The mean inflammatory portal activity in the SVR group decreased from 2.3 ± 0.7 to 1.3 ± 0.7 (P = 0.038). Similarly, the mean inflammatory lobular score decreased from 2.8 ± 0.7 to 0.8 ± 0.9 (P = 0.0016). No statistically significant change was appreciated in the mean fibrosis score (1.5 ± 1.1 to 1.16 ± 1.0 P = 0.52) in patients with SVR. However, no patient who achieved SVR showed a worsening of the fibrosis score; actually, the fibrosis score improved in 5 and remained unchanged in 2 patients. On the contrary, there were no significant changes neither in the mean activity index nor in the fibrosis score in nonresponder patients. At the end of therapy, fibrosis had progressed in 4 of these patients, remained unchanged in another 4 and improved only in 1 (F3 to F2).
Tolerability and Adverse Effects
The most frequent side effects of antiviral therapy were asthenia, fever, flu-like symptoms, headache, myalgia, depression and hematological side effects (Table 3). Although 17 (36%) patients developed depression, no patient was withdrawn from the protocol because this of side effect. There were no cirrhotic decompensations during therapy. Anemia occurred in 36 (77%) patients. A total of 17 of them (36%) required darbepoetin administration and 15 (32%) ribavirin dose reductions. In 8 (17%) patients, blood transfusion was needed. Overall, ribavirin was definitely discontinued in 5 patients. The dose of the PEG-IFN was decreased to a dose of 1.0 μg/kg/week in 6 (13%) patients due to neutropenia.
Table 3. Side Effects During Treatment
Anemia (Hb < 10.5 gm/dL)
Neutropenia (neutrophils < 1,000/μL)
Thrombocytopenia (platelets < 90,000/μL)
Treatment was prematurely discontinued in 10 (21%) patients (Table 4). The most common reason for treatment discontinuation was noncompliance in 4 patients due to fatigue within the first 4 weeks of therapy. One patient with severe cholestatic recurrence and EVR suffered acute rejection after 4 months of treatment and antiviral therapy was interrupted. Another patient developed ductopenic rejection after 4 months of treatment. These 2 patients had receiving cyclosporine as immunosuppressive therapy. Overall, only 20 of 47 (42.5%) patients received a complete antiviral therapy as previously defined.
Table 4. Reasons for Therapy Discontinuation
Reason for discontinuation
n = 10
Severe cholestatic hepatitis
Cardiac insufficiency (anemia)
Severe thrombocytopenia and neutropenia
Mortality During the Study
Eight out of 47 patients died during the study. Three of 10 (30%) recipients with severe cholestatic recurrence died due to liver failure (2 of them in the first 2 months after initially antiviral therapy and 1 at the end of the treatment). Five of the 37 (13%) recipients with recurrent non-cholestatic hepatitis died due to chronic rejection (2), hepatocarcinoma recurrence (1), massive esophageal variceal bleeding (1), and progressive liver failure due to hepatitis (1).
The management of hepatitis C in the post-LT setting is a matter of increasing concern. Due to the accelerated natural history of recurrent hepatitis C virus infection after LT, most authors recommend offering antiviral therapy at least to patients with established recurrent hepatitis C, although its use in patients with mild or absent fibrosis is controversial.10, 11 However, a standard treatment for recurrent hepatitis C after LT has not been established yet, but based on the previous experience in the nontransplant setting, pegylated interferon plus ribavirin combination therapy seems to be the most attractive alternative.12, 13
In our study, combination therapy achieved a SVR in 23% of patients, end of treatment virological response in 36%, and a normalization of serum ALT levels in 70% of patients during treatment. Moreover, among patients who did not discontinue antiviral therapy, biochemical response was observed in 33 (89%) patients, end of treatment virological response in 17 (46%) patients, and SVR in 11 (30%) patients.
Our results in terms of clinical and virological outcomes are in the lower range to those previously reported results, which ranged from 21 to 45%.14–17 Most reports on the efficacy of antiviral therapy in the treatment of recurrent hepatitis C differ widely in important factors such as viral genotype, antiviral dose, timing of the introduction of antiviral therapy, immunosuppressive therapy, etc., which may significantly influence the final outcome. In the current study, 93% of the patients were infected by the 1b genotype, which presents the worst response to antiviral therapy. Additionally, we only initiated treatment when a clear evidence of chronic hepatitis was found in a graft biopsy. The exact timing to initiate HCV-antiviral therapy after LT is controversial. The experience in the nontransplant setting indicates that treatment is more effective when it is administered early. However, we have not found differences in the outcome when antiviral therapy was administered during or after the first year after transplantation. Moreover, some authors have suggested a longer duration of antiviral therapy in transplant recipients to decrease the rate of virological relapse.11
The most important drawback of antiviral therapy for the treatment of recurrent hepatitis C in liver transplant recipients is drug-related toxicity. In fact, 1 in every 5 patients in our study discontinued medication due to drug-related side effects and only 20 of 47 patients were able to receive more than 80% of the scheduled antiviral therapy. Even though we administered darbepoetin alpha in 1 of 3 of the patients, anemia was severe enough to require transfusion in 17% of patients and discontinuation of ribavirin therapy in 11%; in an additional 32% of patients, ribavirin dose needed to be reduced. Neutropenia was also a frequent event (53%), although it was the cause of treatment discontinuation in only 1 case. Discontinuation or dose reductions are important factors for reducing the likelihood of a favorable outcome. In our experience, the rate of SVR was significantly higher in patients who completed therapy, defined as patients who were able to receive more than 80% of the proposed dose of antiviral therapy during more than 80% of the time for which the treatment was scheduled. The frequency of side effects necessitating cessation of therapy has been reported to range between 20% and 44%.14–17 Dumortier et al.14 reported a low rate of withdrawals (20%) and a high SVR rate (45%) using a progressive increase in the PEG-IFN and ribavirin doses, which we have partially used in the case of ribavirin.
Due to the relatively small number of paired liver biopsies included in our study, we should be cautious about the interpretation of the histological outcome of our population. However, it is important to note that SVR was associated with a significant improvement of the necroinflammatory activity score in the liver biopsy. Although the mean fibrosis score did not significantly change in 7 patients with SVR, the fibrosis score improved in 5 and remained unchanged in the remaining 2 patients. It is possible that the long-term effect of viral clearance may result in the regression of the histological lesions, including liver fibrosis. In the study by Bizollon et al.,18 93% of patients who achieved SVR showed a marked histological improvement at 3 yr after the end of combination therapy with standard interferon alpha plus ribavirin. Abdelmalek et al.19 showed that both the grade of inflammation and the fibrosis score improved by 3-5 yr after antiviral treatment in those patients with a SVR.
IFN-alpha has immunostimulatory properties, resulting in a theoretically enhanced risk of graft rejection. An early trial suggested that interferon treatment may be associated with an increased risk of acute and chronic rejection.20 However, more recent studies have not confirmed that the rejection risk is increased in the liver transplant recipients who have been treated with standard interferon and ribavirin.21–27 Likewise, in 1 randomized controlled study, the risk of acute and chronic rejection was no higher in those patients who received 48 weeks of interferon alpha-2b plus ribavirin than in the untreated controls.28 In the report of Chalasani et al.,29 the incidence of acute rejection was similar in patients treated with PEG-IFN monotherapy and in untreated groups whether the therapy was administered in the immediate posttransplantation period or delayed to the demonstration of chronic hepatitis in the graft. In our study, only 1 patient developed acute cellular rejection (2%) and 2 developed ductopenic rejection (4%). Similar results have been reported by others.15–17 However, Dumortier et al.14 reported a particularly high acute rejection rate (25%) in a series of 20 patients. In this study, liver biopsies were performed every 6 months when ALT levels were within the normal ranges and every 3 months when ALT levels were higher than the upper normal limit. In all cases, acute rejection was mild and could be treated only by increasing the immunosuppressive regimen. Interestingly, in our study all cases of rejection developed in patients who were receiving cyclosporine but not tacrolimus as immunosuppressive therapy. Nevertheless, due to the risk of developing chronic rejection, antiviral therapy should probably be reserved to those patients in whom disease progression is well documented and, in the absence of new data, recipients with mild disease (F0-F1 in liver biopsy) should not receive treatment.
Cholestatic hepatitis C is an aggressive form of recurrent HCV disease in the setting of LT. It is a predictor of long-term liver allograft injury that, in 5% of patients, may lead to a severe and rapidly progressive liver damage with graft dysfunction when left untreated.30, 31 In our study, 20% of patients with cholestatic hepatitis C achieved SVR. One patient with a pretreatment bilirubin level greater than 18 mg/dL achieved an EVR, although treatment was stopped because he developed acute allograft rejection at the fourth month of treatment. The other 2 patients with similar levels of bilirubin suffered a worse outcome and died because of liver failure. The other 5 patients achieved biochemical response but not virological response. Severe recurrence was seen at the end of treatment in 3 patients: 1 died because of hepatic failure and 2 normalized ALT levels again after reintroduction of the antiviral therapy. On the basis of these results, we propose a prolonged antiviral therapy in cholestatic hepatitis C LT recipients if they do not achieve a virological response during treatment. A definition of the dose and total duration of maintenance therapy in these patients is yet to be established.
Due to the toxicity of HCV-antiviral therapy in LT recipients, a definition of factors associated with a better/worse outcome is urgently needed. In our experience a negative serum HCV-RNA test after 12 weeks of treatment was associated with a more favorable outcome. Actually, as occurs in the non-transplant setting, no patient with a positive HCV-RNA test at the week 12 of therapy achieved SVR. Similarly, a low HCV-RNA viral load before therapy was associated with a higher likelihood of healing, although an isolate patient with a high viral load (150 × 106 UI/mL) achieved SVR.
As previously pointed out, most authors have associated cholestatic hepatitis of the graft with a poorer outcome. However, 2 of 10 patients with recurrent cholestatic hepatitis responded to our treatment, although, likely due to the small sample size, differences in the SVR of recipients with cholestatic or noncholestatic hepatitis were not significant. Moreover, a lower level of baseline γ-glutamyltransferase was significantly associated with SVR and the baseline level of alkaline phosphatase nearly reaches statistical significance, suggesting that the presence of a cholestatic profile is associated with a worse response to treatment.
In summary, combination therapy of PEG-IFN alpha 2b with ribavirin may clear HCV infection and improves histological damage in some LT recipients. Tolerability is the most important drawback of this therapy, often provoking therapy discontinuation or dose-reduction, which are associated with a lower probability of healing. Given the low efficacy and poor tolerability of current antiviral therapy, treatment should probably be reserved to those individuals in whom disease progression is well documented. A better definition of factors linked to a favorable outcome and strategies directed to ameliorate treatment toxicity may improve current results of the antiviral therapy for HCV infection in the posttransplantation setting.