Impact of peginterferon alpha-2b and ribavirin treatment on liver tissue in patients with HCV or HCV–HIV co-infection

Journal of Infection (2007) 54, 609e616

www.elsevierhealth.com/journals/jinf

Impact of peginterferon alpha-2b and ribavirin treatment on liver tissue in patients with HCV or HCVeHIV co-infection Rui Sarmento-Castro a,*, Ana Horta a, Olga Vasconcelos a, Helena Coelho a, ˜o Seabra a, Manuela Duarte a, Josefina Mendez a, Ana Paula Tavares a, Joa ˆngela Ventura a, Leonor Chaves a, Olga Fortes a, Cristina Recalde a, A a b c ´tima Carneiro d Nuno Pires , Luciana Pinho , Nancy Dias , Fa a

Infectious Diseases Department, Hospital de Joaquim Urbano, Rua Caˆmara Pestana 348, 4369-004 Porto, Portugal Virology Department, Hospital Geral de Santo Anto´nio, Porto, Portugal c Microbiology Department, University of Porto, Porto, Portugal d Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP) and Medical Faculty of Porto/Hospital S. Joa˜o, Porto, Portugal b

Accepted 10 November 2006 Available online 27 December 2006

KEYWORDS Chronic hepatitis C; Treatment; Liver histology; Pegylated interferon alpha-2b; Ribavirin; HCV/HIV co-infection; Fibrosis; Liver biopsy

Summary Objective: To evaluate the effect of treatment with peginterferon alpha-2b and ribavirin on liver histology in patients with chronic hepatitis C (CHC) with or without HIV infection. Methods: Patients received peginterferon alpha-2b (1.5 m/kg/week during the first 4 weeks; 1.0 m/kg/week thereafter) plus ribavirin (800e1200 mg/day, adjusted for weight) for 24 (genotypes 2/3) or 48 weeks (genotypes 1/4). Paired liver biopsy specimens were obtained at baseline and at the end of follow-up. Results: 108 paired biopsy specimens were available: 67 from HCV-monoinfected and 41 from co-infected patients. At the end of follow-up, necroinflammatory activity (NIA) was significantly reduced (P < 0.001), and fibrosis scores improved by 1 point (Ishak et al criteria) in 65.7% of HCV-monoinfected patients. In co-infected patients, NIA was significantly reduced (P < 0.001), and fibrosis scores improved by 1 point in 42.5% of cases. In both groups, results were better for patients who attained sustained virological response (SVR). HCV RNA was undetectable in the second biopsy specimens of all patients who attained SVR. Conclusion: Liver fibrosis is reduced significantly after a course of therapy in patients with chronic hepatitis C. Reduction of fibrosis is more significant in patients who are monoinfected with HCV and in those who attained SVR. ª 2006 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

* Corresponding author. Tel.: þ351 22 5899550; fax: þ351 22 5106160. E-mail address: [email protected] (R. Sarmento-Castro). 0163-4453/$30 ª 2006 The British Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jinf.2006.11.010

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Introduction The introduction of highly active antiretroviral therapy (HAART) has extended the life expectancy of HIV-infected individuals. For those co-infected with hepatitis C virus (HCV) and HIV (co-infected patients), this increase in life expectancy has also increased the likelihood of developing chronic hepatitis C (CHC)dan important cause of morbidity and mortality in this patient population. End-stage liver disease is recognised as a significant cause of death in patients with HIV and, in the majority of cases, co-infection with HCV/HIV is identified as a contributing factor.1,2 The optimum approach to the treatment of hepatitis C in patients monoinfected with HCV or co-infected patients is two-pronged, coupling viral eradication with regression of liver disease. In patients monoinfected with HCV, pegylated interferon plus ribavirin is effective in realising both of these treatment goals. Sustained virological responses (SVRs) are achieved in 54e56% of all patients monoinfected with HCV,3,4 with rates reaching up to 80% in those with HCV genotype 2 or 3.3 Likewise, the rate of fibrosis progression and necroinflammatory activity (NIA) are also reduced with combination therapy.5 The risk for severe liver disease, histological cirrhosis, and decompensated liver disease has been reported to be 2- to 6-fold higher in co-infected patients than in those monoinfected with HCV.6 Furthermore, the rate of progression to severe liver disease and cirrhosis, a predictor of SVR,7 is more rapid in co-infected patients than in patients monoinfected with HCV.8 These findings may explain why treatment of CHC is less effective in reducing NIA and fibrosis in co-infected patients than in patients monoinfected with HCV and why histological response is better in patients who attain SVR.3,9 Similarly, the rates of SVR achieved in co-infected patients are inferior to those obtained in patients monoinfected with HCV.9e11 Information on histological improvement in co-infected patients treated with anti-HCV therapy is scarce and few studies have evaluated the efficacy of peginterferon alpha2b doses less than 1.5 m/kg/week in these patients. The aim of this study was to evaluate the effect of a moderate maintenance dose of peginterferon alpha-2b (1.0 m/kg/week) plus weight-based ribavirin on liver histology, in patients with CHC with or without concurrent HIV infection.

Materials and methods This study was conducted at an Infectious Diseases Department in Portugal and received the approval of the Ethics Committee and of the hospital administration board. The Portuguese Institute of Pharmacy and Drugs (INFARMED) was also notified. All patients provided written informed consent.

Patients Patients infected with HCV (confirmed by enzyme-linked immunosorbent assay [ELISA] and recombinant immunoblot assay) with or without HIV infection (diagnosed by ELISA and Western blot) were enrolled in the study between January 2002 and December 2004.

R. Sarmento-Castro et al. All patients were between 18 and 60 years of age, had detectable serum HCV RNA, had aminotransferase levels above the upper limit of normal (40 IU/ml), and showed histological evidence of chronic hepatitis, characterised by inflammatory changes in the liver parenchyma (with lymphocytic infiltrates in portal tracts), that were graded for necroinflammatory activity and fibrosis according to the system proposed by Ishak et al.12 (at least mild activity or fibrosis F1). Patients previously had not received therapy for HCV infection. Co-infected patients had no active opportunistic infections and had a CD4þ cell count >200 cells/mm3, regardless of HIV RNA level. It was required that HAART was unchanged during the 6 weeks before inclusion. Exclusion criteria included active drug abuse in the 12 months prior to study entry; excessive alcohol consumption (>40 g/day); history of liver transplantation; history or symptoms of ischaemic heart disease, autoimmune hepatitis, or decompensated cirrhosis; history of psychiatric disorders, renal impairment, uncontrolled diabetes; and coinfection with hepatitis B virus or HIV-2. Patients who had haemoglobin levels <13 g/dl (men) or <12 g/dl (women), neutrophil counts <1500 cells/mm3, or platelet counts <100,000/mm3 were also excluded. Patients with alcoholic hepatitis or other infectious, autoimmune, tumoural, or vascular diseases that affect hepatic function were also excluded. Females of childbearing potential were required to use adequate contraception.

Study design Patients were initially included in a 4-month screening period of monthly visits at which time they were evaluated for motivation toward treatment and assessed against inclusion and exclusion criteria. Patients who missed one visit without justification and those using drugs or alcohol were excluded. Liver biopsies were conducted at the end of this period for histological evaluation and quantification of HCV RNA. During this phase, patients also underwent clinical and analytical assessments, including evaluation of hepatic, renal, and thyroid function; evaluation of autoimmune phenomena; and quantification of HCV RNA (bDNA) and a-fetoprotein. Patients infected with HCV genotype 1 or 4 received treatment for 48 weeks; those infected with HCV genotype 2 or 3 were treated for 24 weeks. All patients received peginterferon alpha-2b 1.5 m/kg/week for the first 4 weeks and then 1.0 m/kg/week until the end of treatment. Ribavirin was administered according to a weight-based dosing regimen (1200 mg/day for patients weighing >75 kg; 1000 mg/ day for patients weighing >65 and 75 kg; 800 mg/day for patients weighing 65 kg). Peginterferon alpha-2b was administered weekly at the hospital. Ribavirin was dispensed in 1-week instalments and self-administered at home. Co-infected patients receiving HAART maintained their current regimens (2 nucleoside analogues combined with a non-nucleoside analogue or a protease inhibitor) throughout the study. If necessary during anti-HCV treatment, HAART was initiated in accordance with DHHS criteria in patients who had not received prior treatment.13 During treatment, patients attended regular scheduled clinic visits at weeks 1, 2, 4, 6, and 8 and monthly visits thereafter. Thyroid function (T3, T4, and thyroid-stimulating

Effect of PEG-IFN and ribavirin treatment on liver tissue hormone [TSH]) was evaluated at weeks 0, 6, 24, and 40. CD4þ cell count and HIV viral load were determined at weeks 1, 24, and 48. All patients were followed up for 24 weeks after completing their allocated treatment schedule. Final assessment (week 48 for genotype 2 or 3 patients and week 72 for genotype 1 or 4 patients) included blood testing and a second liver biopsy. Throughout the study, compliance was continually monitored by a multidisciplinary team. Immediate clinical and psychiatric support was available as required and immediate contact was made with any patient who did not attend a scheduled appointment.

Study outcomes The primary efficacy and tolerability data from this study are presented elsewhere (unpublished data; manuscript in preparation). The primary efficacy end point of the study was the evaluation of SVR, defined as undetectable HCV RNA at 6 months after the end of treatment. Biochemical response, defined as normal alanine aminotransferase (ALT) levels at the end of follow-up, was also assessed. Liver biopsy specimens were obtained before starting study medication and at the end of follow-up. Histological assessments were performed in the course of the study by one experienced pathologist blind to the clinical characteristics of the patients (e.g., HIV status, viral load, genotype) as well as to the time of biopsy (pre- or posttreatment). Histological changes were graded in accordance with the criteria of Ishak et al.12 Patients with a reduction of at least 2 points in NIA score or at least 1 point in fibrosis score were considered NIA or fibrosis responders. Detection of HCV was performed in the liver before and after treatment. Several types of tissue stains and immunocytochemical and immunohistochemical tests were used to rule out other diagnoses. Detection of HCV in the liver tissue was performed by polymerase chain reaction (PCR) and DNA enzyme immunoassay (DEIA, Roche Molecular System, Branchburg, NJ, USA). The Versant HCV RNA 3.0 Assay Kit (Bayer, Tarrytown, NY, USA; lower limit of detection <615 IU/ml) was used to quantify HCV RNA. In co-infected patients, the Versant HIV RNA 3.0 Assay Kit was used for quantification of HIV RNA (bDNA). HCV genotype was determined with use of the Roche Amplicor 2.0 amplification kit (Roche, Branchburg, NJ, USA), the inno-LIPA hybridisation technique and the Bayer Versant HCV Genotype Assay Kit.

Statistical analysis Results are expressed in absolute values and percentages. Whenever possible, the quantitative variables were evaluated using parametric tests such as the Student t-test. The dichotomous variables were analysed using contingency tables such as the chi-squared tests and the Fisher exact test. Final results were evaluated by analysing the intentto-treat population. Chi-squared test was used to identify prognosis factors for NIA and fibrosis responses. The factors identified as predictors were later analysed together using binary logistic regression. All P values recorded were bilateral. Statistical studies were performed using SPSS 11.0 for Windows (SPSS Inc., Chicago, IL, USA).

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Results In total, 133 patients were included in the analysis: 80 patients monoinfected with HCV and 53 co-infected patients. Demographic characteristics were similar between the groups. Most subjects were male and had either HCV genotype 1 or 3 (Table 1). In co-infected patients, 37 (69.8%) were receiving HAART for a mean duration of 41 months, and all were receiving zidovudine or stavudine and eight (15.1%) were also taking didanosine. Compliance with peginterferon treatment was higher than 95%. At baseline, mean leucocyte and platelet counts were significantly lower and mean values of lactate dehydrogenase and serum protein levels (albumin and globulins) were significantly higher in co-infected patients than in patients monoinfected with HCV (data not shown; all comparisons P  0.02). There were no significant differences in mean ALT (P < 0.4) and HCV RNA levels between the groups (P < 0.19).

Virological and biochemical responses Using an intent-to-treat analysis, 60 (75.0%) of 80 patients monoinfected with HCV and 19 (35.8%) of 53 co-infected patients attained SVR. Overall, sustained biochemical

Table 1

Baseline characteristics

Sex, n (%) Male Female Mean age, years Mean weight, kg Estimated duration of HCV infection, years Risk, n (%) Drug abuse Sex Transfusion Unknown HCV genotype, n (%) G1 G2 G3 G4 Anti-HBcþ, n (%) Mean ALT, IU/ml Mean HCV RNA, IU/m Mean CD4þ, cells/mm3 Mean HIV-RNA, copies/mm3 Antiretroviral therapy, n (%)

Patients monoinfected with HCV (n Z 80)

HCV/HIV co-infected patients (n Z 53)

67 (83.8) 13 (16.2) 34.9 72.8 13.0

42 (79.2) 11 (20.8) 32.6* 70.5 12.3

61 1 4 14

45 (84.9)** 8 (15.1) e e

(76.2) (1.3) (5.0) (17.5)

36 (45.0) 2 (2.5) 35 (43.8) 7 (8.7) 35/80 (43.8) 130.2 3,221,348 e e

23 (43.4) 1 (1.9) 24 (45.3) 5 (9.4) 43/51 (84.4)** 116.1 4,685,163 585 6804

e

37 (69.8)

*P < 0.05, **P < 0.001 compared with patients monoinfected with HCV.

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R. Sarmento-Castro et al.

Histological response

HCV RNA levels were measured in liver biopsy samples from 63 patients. All patients who achieved SVR (n Z 48) had undetectable hepatic HCV RNA at the end of followup; HCV RNA was detected in the remaining 15 patients who did not achieve SVR.

Patients monoinfected with HCV Paired liver biopsy samples were available for analysis for 67 of 80 patients monoinfected with HCV. Of the remaining 13 patients, four withdrew from treatment on medical advice, seven refused to undergo a second biopsy, and the biopsy samples of two were inadequate for analysis (Fig. 1). Mean NIA scores were significantly lower at the end of follow-up than at baseline (5.16 vs. 2.37; P < 0.001) (Table 2). In total, 83.6% (56 of 67) of all patients were NIA responders (Figs. 1 and 2). Of patients who attained SVR, 92.3% were NIA responders, whereas only 53.3% of those who did not attain SVR were NIA responders. Mean fibrosis scores were also significantly decreased at the end of follow-up, compared with baseline values (1.81 vs. 0.97, P < 0.001; Table 2). Overall, 65.7% (44 of 67) of all patients were fibrosis responders (Figs. 1 and 2). Of patients who attained SVR, 67.3% were also fibrosis responders, whereas only 60.0% of those who did not attain SVR were classified as fibrosis responders. At baseline, 53.7% of patients had grade F2 to F6 fibrosis; however, only 15.0% of patients remained within this category after treatment. Of three patients with cirrhosis at baseline, there was a substantial reduction in fibrosis (from grade F6 to grade F2) in one patient and a slight reduction (from grade F6 to grade F5) in another patient. The third patient did not complete treatment.

Co-infected patients Liver biopsy samples for two of 53 co-infected patients were inadequate for analysis. Of the remaining 51 patients, eight discontinued treatment, one was lost for follow-up, and one refused to undergo a second biopsy. Additionally, the liver biopsy sample from one patient was acceptable for assessment of NIA but was inadequate for analysis of fibrosis. Hence, NIA was evaluated in 41 pairs of samples and fibrosis was evaluated in 40 (Fig. 1). Before treatment 11.8% of the patients had bridging fibrosis or cirrhosis. Mean NIA scores were significantly reduced at the end of follow-up compared with baseline scores (5.24 vs. 3.22, P < 0.001; Table 2). Overall, 24 patients (58.5%) were NIA histological responders (Figs. 1 and 2). Of patients who attained SVR, 88.2% were classified as NIA responders. NIA scores improved in 37.5% of patients who did not attain SVR. Mean fibrosis scores were significantly improved at the end of treatment (1.65 vs. 1.33, P < 0.05; Table 2) and the incidence of moderate to severe fibrosis (F2eF6) was reduced at the end of follow-up compared with baseline (25% vs. 42.5%, respectively). Overall, 17 patients (42.5%) were fibrosis responders (Figs. 1 and 2). Of those patients who attained SVR, 47.0% were also fibrosis responders. Amongst non-responders, the incidence of fibrosis responders was 39.1%.

response was observed in 75.0% of patients monoinfected with HCV and in 54.5% of co-infected patients (unpublished data; manuscript in preparation).

Figure 1

Histological response in patients monoinfected with HCV and in co-infected patients.

Effect of PEG-IFN and ribavirin treatment on liver tissue Table 2

613

Histologic results of the treatment of patients monoinfected with HCV and co-infected with HCV/HIV

NIA, mean score Fibrosis, mean score Patients with moderate to severe (F2eF6) fibrosis, %

Patients monoinfected with HCV (n Z 67)

HCV/HIV co-infected patientsa (n Z 41)

Baseline

Follow-up

Baseline

Follow-up

5.16 1.81 53.7

2.37** 0.97** 14.9

5.24 1.65 42.5

3.22** 1.33* 25.0

*P < 0.05, **P < 0.001 versus baseline score. a Fibrosis was evaluated in 40 patients with evaluable biopsies at baseline and at follow-up.

All 53 co-infected patients had detectable hepatic HCV RNA before treatment. Of these, 42 patients had evaluable biopsy samples at follow-up. All patients (n Z 19) who attained SVR had undetectable hepatic HCV RNA. Among patients who did not achieve SVR, hepatic HCV RNA was either detectable (n Z 22) or inconclusive (n Z 1).

Factors related to histological response Baseline variables selected for statistical analysis were age, gender, weight, risk for HCV, haemoglobin, platelets, bilirubin, AST, ALT, alkaline phosphatase, lactate dehydrogenase, albumin, iron and ferritin levels, a-fetoprotein, NIA, fibrosis score, serum HCV RNA and HCV genotype. In co-infected patients, CD4 cell count, HIV RNA and antiretroviral regimen (PI use vs. other regimens and treated vs. untreated patients) were also included in the analysis. ANI and fibrosis responses and SVR were also evaluated in both groups. Logistic regression analysis revealed that in patients monoinfected with HCV, there was a statistically significant association between NIA response and low iron levels (<200 mg/dl), fibrosis response and SVR (odds ratio: 4.9). Fibrosis responder status was significantly associated with NIA response and pre-treatment fibrosis score (odds ratio: 2.1).

Figure 2 Patients who achieved a clinically significant improvement in necroinflammatory activity (NIA) or fibrosis at follow-up from their baseline status. Patients achieving a reduction of at least 2 points in NIA or a decrease of 1 point in fibrosis scores were considered responders.

In co-infected patients, univariate analysis showed that NIA response was significantly associated with pretreatment NIA score (P < 0.004) and SVR (P < 0.025). Patients with undetectable HIV RNA had a better response of NIA than those with detectable HIV RNA, but this difference did not reach statistical significance (P < 0.10). Improvement in fibrosis was only related with pre-treatment fibrosis score (P < 0.001). Models constructed for logistic regression analysis had no statistical significance.

Comparison of the impact of treatment Before treatment, the mean scores for NIA (monoinfected with HCV [5.16] vs. co-infected [5.24]; P < 0.79) and fibrosis (monoinfected with HCV [1.81] vs. co-infected [1.65]; P < 0.45) showed no statistically significant differences between the groups. At the end of the follow-up, the absolute difference between mean NIA values was numerically greater in patients monoinfected with HCV (2.79) than in co-infected patients (2.02). The absolute difference between mean fibrosis scores was also more pronounced in patients monoinfected with HCV (0.84) than in coinfected patients (0.32).

Discussion A significant body of evidence supports the premise that effective treatment of HCV infection significantly reduces the progression of fibrosis and inflammatory activity. A recent meta-analysis of four peginterferon alpha-2b clinical studies of patients with CHC indicated that 55% of all treated patients experience an improvement in NIA scores after treatment, with only 14% of patients experiencing worsening scores.5 In this meta-analysis, improvements in NIA ranged from 39% (in patients treated with standard interferon monotherapy for 24 weeks) to 65e73% (in patients treated with peginterferon alpha-2b plus ribavirin for 48 weeks).5 Improvement in fibrosis was less marked for patients treated with standard interferon for 24 weeks (12%) than for those treated with peginterferon alpha-2b plus ribavirin for 48 weeks (w24%). Across the 10 regimens examined in the meta-analysis, fibrosis was stabilised in 65% of all treated patients and 50% of all patients with cirrhosis before treatment experienced a ‘cirrhosis reversal’ (defined as an improvement in fibrosis score based on the post-treatment biopsy sample). Histological improvements

614 were most prominent in patients who attained SVR while receiving peginterferon alpha-2b plus high-dose ribavirin.5 The primary source of the peginterferon alpha-2b data in the meta-analysis was the study by Manns et al.3 In this study, improvement in fibrosis occurred in 21% of all patients, in 26% of those who attained SVR, and in 14% of non-responders. Ninety percent of patients who attained SVR showed an improvement in inflammation status.3 In the present series, a similar percentage of cases (92%) evolved with NIA improvement. At variance, we observed a much higher frequency of fibrosis improvement (66%) than in the study by Manns et al.3 Different methodological approaches and demographic features of the patients may account for these discrepancies. Altogether, our data further clarify the effect of peginterferon alpha-2b plus ribavirin on the improvement of liver inflammation and fibrosis. Several studies have shown that co-infection with HIV influences the progression of fibrosis; hepatic lesions are generally more advanced in co-infected patients than in patients monoinfected with HCV who have had hepatitis for a similar amount of time.14 In our series, baseline values of NIA and fibrosis were similar between the two groups of patients. However, when compared with patients monoinfected with HCV, co-infected patients were younger (P < 0.05), and had a shorter duration of exposure to HCV. In addition, co-infected patients had a mean CD4 cell count of 585 cells/mm3 and 70% were taking HAART. Taken together, all these factors may have contributed for the relatively low baseline scores of NIA and fibrosis in co-infected patients. These observations are consistent with another report that described a lower stage of fibrosis and a delayed progression of fibrosis in co-infected patients who were taking protease inhibitors and with a CD4 cell count above 200 cells/mm.3,15 Impact of PIs on fibrosis is controversial. In another study antiretroviral therapy had a negative impact on fibrosis in patients with advanced immunodeficiency and using alcohol and drugs.16 The aggressive liver disease reported in co-infected patients is associated with higher viraemia and immunological deficits, which may explain why virological, biochemical, and histological responses are generally lower in co-infected patients than in patients monoinfected with HCV. In the present study, there was no correlation between serum HCV RNA levels and CD4 cell count (in coinfected patients) and more aggressive histological lesions. Furthermore, the results achieved in patients monoinfected with HCV were better than those in co-infected patients, suggesting that other factors, such as delayed HCV RNA clearance in response to treatment,17 a higher relapse rate or poorer compliance may influence the lower response rates to anti-HCV therapy in co-infected patients. In the RIBAVIC study,9 which enrolled co-infected patients, 27% of patients receiving peginterferon alpha-2b plus ribavirin achieved SVR and, in this group, there was a mean decrease in NIA score of 1.2 points (Ishak et al. criteria). The mean reduction of NIA in patients who did not attain SVR was 0.2 points. In total, 31% of patients who attained SVR and 17% of the non-responders showed some improvement in NIA. Additionally, treatment failed to result in improvement in mean fibrosis scores, and fibrosis scores worsened in patients who did not attain SVR. Among patients who attained SVR, 11% showed a reduction in fibrosis and 9% showed worsening of fibrosis scores.9

R. Sarmento-Castro et al. In our study, we observed an improvement in NIA and fibrosis of co-infected patients after treatment. There was a mean reduction of 2.02 in NIA scores and of 0.32 in fibrosis scores. In total, 58.5% of all patients, 88.0% of patients who achieved SVR and 37.5% of non-responders, were classified as NIA responders. Globally, fibrosis improved in 42.5% of the co-infected patients. There was a reduction in fibrosis scores in 47.0% of the patients who attained SVR and in 39.1% of the non-responders. Our data, which are more encouraging than those of the RIBAVIC study, confirm the difficulties in treating coinfected patients and that generally, histological outcomes in these patients do not match those reported in patients monoinfected with HCV. Treatment had a significant influence on reduction of NIA but was less effective in the improvement of fibrosis. Noteworthy, almost 40% of nonresponders were classified as either NIA or fibrosis responders, indicating that histological improvements may not be aligned with virological response in these patients. Comparison of our results with the findings of the RIBAVIC study should be undertaken with caution, but there are several putative explanations for the discrepancies between the two studies. Our use of higher doses of ribavirin may help explain the lower rate of relapse registered in our series18 (17.5% vs. 28.6% in the RIBAVIC study). In addition, the use of moderate pegylated interferon doses and the support provided by our multidisciplinary team may also have been important factors contributing to the higher adherence and the lower dropout rate observed in our patients (16.9% vs. 39.1% in RIBAVIC). Moreover, patients included in the RIBAVIC study were older (39.5 vs. 32 years), had a longer duration of HCV infection (>16 years vs. 12.3), and had more advanced histological lesions (39% with bridging fibrosis or cirrhosis vs. 12%). A greater proportion of patients included in the RIBAVIC trial (21.6% vs. 15% in our series) were taking didanosine, a drug that in combination with pegylated interferon plus ribavirin increases the risk for hepatic decompensation.19,20 The ‘eradication’ of HCV from the liver tissue has been considered a reliable long-term marker of cure.21 HCV RNA is undetectable in the liver of most patients who attained SVR after treatment22,23 and the presence, after treatment, of HCV RNA in the liver of patients who attained SVR indicates a possible relapse.24 In a series of 400 patients who attained SVR, HCV RNA was not found in the liver of 393 patients (98%); however, it was present in the other seven patients (2%). Two of these seven patients had a relapse in the following 12 months.24 In the 105 patients for whom intrahepatic replication of HCV was evaluated, the presence or absence of hepatic HCV RNA before and after treatment was consistent with that of serum HCV RNA levels (i.e., all patients who attained SVR had undetectable HCV RNA in the liver). These findings confirm the data reported by other authors.22,23 In our co-infected patients, the rate of SVR (35.8%) was inferior to that observed in patients monoinfected with HCV. Our results were better than those obtained in two large series9,10 but inferior to those in two other trials.11,25 One explanation may be that, in HCV genotype 1 and 4 coinfected patients, the dose of peginterferon used in the present study may have been too low to achieve the desired efficacy.

Effect of PEG-IFN and ribavirin treatment on liver tissue Summing up, we think that several factors may explain the better virological and histological results obtained in our series when compared with other studies3,4,9e11: (i) some baseline ‘favourable’ features of our patients (younger, shorter duration of HCV infection, lesser aggressive hepatic lesions, lower frequency of patients infected with genotype 1; (ii) permanent support provided by our multidisciplinary team and the use of a moderate dose of pegylated interferon under direct observation (contributing to a very good adherence and a low drop-out rate); (iii) regimen therapy with administration of ribavirin at higher doses than those used in other studies (eventually contributing to a lower relapse rate). In conclusion, peginterferon alpha-2b plus ribavirin is an effective treatment choice for patients with chronic hepatitis C resulting in successful long-term virological clearance in a large number of patients coupled with reductions in hepatic fibrosis and inflammatory activity. In co-infected patients, outcomes are generally poorer than those obtained with HCV monoinfected patients. However, the degree of histological improvements observed in virological non-responders may indicate that benefits of treatment other than viral eradication may be pursued, which could ultimately reduce patient morbidity.

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Acknowledgements The authors thank Vicente Soriano for critical review of the manuscript.

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