Potential conflict of interest: Nothing to report.
This study was supported in part by grants from the Department of Health, National Science Council (100-2325-B-002-052 and 101-2325-B-002-073, Resource Center, National Research Program for Biopharmaceuticals), Executive Yuan, Taiwan; and National Taiwan University, Taiwan.
Results of this study were presented at the 22nd Conference of the Asian Pacific Association for the Study of the Liver, Taipei, Taiwan, February 16-19, 2012, and at the 47th Annual Meeting of the European Association for the Study of the Liver, Barcelona, Spain, April 18-22, 2012.
Patients dually infected with hepatitis C virus (HCV)/hepatitis B virus (HBV) have a higher risk of developing advanced liver disease or hepatocellular carcinoma compared with monoinfected patients. Yet, there is a similar rate of sustained virologic response (SVR) after peginterferon alfa-2a and ribavirin combination therapy in these patients compared with HCV-monoinfected patients and a high hepatitis B surface antigen (HBsAg) seroclearance rate. The durability of hepatitis C and B clearance in coinfected patients was investigated in a 5-year follow-up study. Patients with active HCV genotype 1, both HBV-coinfected (n = 97) and HBV-monoinfected (n = 110), underwent 48-week combination therapy with peginterferon alfa-2a plus ribavirin. In patients with active HCV genotype 2 or 3, both HBV-coinfected (n = 64) and monoinfected (n = 50) patients underwent 24-week combination therapy. A total of 295 (91.9%) patients completed treatment and 24 weeks posttreatment follow-up; 264 (89.5%) patients agreed to receive additional follow-up for up to 5 years after the end of treatment. After a median follow-up of 4.6 ± 1.0 years, six of the 232 patients achieving SVR developed HCV RNA reappearance, including five HCV genotype 1/HBV-coinfected patients and one HCV genotype 2/3-monoinfected patient. Subgenomic analysis of the HCV core gene indicated that five patients developed delayed recurrence of HCV infection. Overall, the cumulative recurrence rate of HCV infection was 2.3% (0.4%/year; 95% confidence interval [CI], 0.9%-5.5%). The cumulative HBsAg seroclearance rate was 30.0% (95% CI, 21.5%-42.0%); with 33.1% (95% CI, 21.8%-50.1%) in the 48-week combination therapy group and 24.3% (95% CI, 13.7%-42.9%) in the 24-week therapy group. Conclusion: Peginterferon alfa-2a and ribavirin therapy provides good HCV SVR durability and a high accumulative HBsAg seroclearance rate in patients who are coinfected with HCV and HBV. (HEPATOLOGY 2013;)
In areas where hepatitis B virus (HBV) or hepatitis C virus (HCV) infection is endemic, a substantial number of patients are infected with both viruses.1-3 Those dually infected with HCV and HBV have been reported to carry a significantly higher risk of developing advanced liver diseases and hepatocellular carcinoma (HCC) than those with either infection alone.3-7 Consequently, this group of patients needs to be treated more actively. In patients with HCV genotype 1 infection, the rate of sustained virologic response (SVR) at 24 weeks (SVR24) after the end of combination therapy with peginterferon alfa-2a and ribavirin was 72.2% in coinfected patients versus 77.3% in monoinfected patients; for patients with HCV genotype 2/3 infection, the SVR24 values were 82.8% and 84.0%, respectively.8 These results suggest that combination therapy is equally effective in patients with HCV monoinfection and in those with chronic HCV/HBV coinfection. In addition, posttreatment hepatitis B surface antigen (HBsAg) seroclearance was observed in 11.2% of 161 coinfected patients.8, 9 It is noteworthy that serum HBV DNA eventually appeared in 36.3% of the 77 coinfected patients with undetectable pretreatment levels of HBV DNA.
Previous studies have suggested that hepatitis C may relapse in 0.9% to 10% of simple chronic hepatitis C patients who initially obtained SVR24 after the end of treatment.10-12 They thus concluded that in patients with chronic hepatitis C who have no detectable serum HCV RNA 24 weeks after interferon therapy, long-term sustained biochemical and virologic response is anticipated. However, whether HCV SVR24 could be maintained in patients with chronic hepatitis B and C coinfection has not been reported.
For the treatment of chronic hepatitis B, the virologic and serologic responses may also not be durable.13, 14 Furthermore, previous studies suggest that therapeutic efficacy might not be seen during the treatment period but rather occur during the prolonged follow-up period in patients receiving immunomodulatory therapy such as interferon.15 Therefore, it is important to clarify the long-term treatment outcome in this dually infected population.
The goals of this study were to assess whether HCV SVR24 is sustained, to assess the long-term HBV virologic and serologic responses, and to clarify whether any incremental response occurs during 5-year post-treatment follow-up.
anti-HCV, antibody to hepatitis C virus; BCP, basal core promoter; CI, confidence interval; HBV, hepatitis B virus; HBsAg, hepatitis B surface antigen; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; LTFU, long-term follow-up; SVR, sustained virologic response; SVR24, sustained virologic response at 24 weeks.
Patients and Methods
All 321 patients enrolled in the original multicenter study (ClinicalTrials.gov registry no. NCT00361179) using peginterferon alfa-2a (Pegasys; F. Hoffman-La Roche Ltd., Basel, Switzerland) plus ribavirin (Robatrol; F. Hoffman-La Roche Ltd., Basel, Switzerland) for the treatment of HCV/HBV-coinfected patients with active hepatitis C (study group) versus HCV-monoinfected patients with active hepatitis C but seronegative for HBsAg (control group), were eligible for inclusion in this long-term follow-up (LTFU) study.8 The inclusion and exclusion criteria have been reported.8 In short, the study group (either sex, age ≥18 years) comprised HCV/HBV-coinfected patients with active hepatitis C and hepatitis B e antigen–negative chronic HBV infection. Coinfection was defined by seropositivity for HBsAg and antibodies to HCV (anti-HCV) for more than 6 months together with a serum HCV RNA level of ≥200 IU/mL. The control group consisted of patients mono-infected with HCV who fulfilled the same eligibility criteria except that they were seronegative for HBsAg.
Patients with HCV genotype 1 infection received 48 weeks of combination therapy with peginterferon alfa-2a 180 μg weekly plus daily ribavirin. Those with HCV genotype 2/3 infection were treated for 24 weeks. Ribavirin was dosed according to HCV genotype and body weight: 800 mg/day for HCV genotype 2/3; for genotype 1, 1,000 mg/day for patients whose body weight was <75 kg and 1,200 mg/day for patients whose body weight was ≥75 kg.
For the LTFU study, patients were periodically evaluated at each participating center. The investigator assessed clinical signs and symptoms of liver disease, the development of HCC, liver transplantation, mortality, and administration of any antiviral therapy for chronic hepatitis B or C after the initial study. Follow-up time was calculated from the end of the original combination treatment to the last visit in the LTFU study.
Assessment of Efficacy.
Primary efficacy of the original trial was HCV SVR at 24 weeks posttreatment (HCV SVR24), defined as HCV RNA undetectable using a commercial quantitative real-time polymerase chain reaction assay (COBAS TaqMan HCV Test version 2.0, Roche Diagnostics GmbH, Mannheim, Germany; lower detection of limit: 25 IU/mL) both at end of treatment and at 24 weeks after end of treatment.
The primary outcome for the follow-up study was sustainability of HCV SVR during LTFU (HCV SVR-LTFU) in patients with HCV SVR24. Patients who were retreated for chronic hepatitis C after the initial study were considered nonresponders (patients with HCV RNA detectable at end of treatment) or relapsers (patients with HCV RNA undetectable at end of treatment but detectable at posttreatment follow-up).
Secondary outcomes included HBV serologic and virologic responses. HBsAg seroclearance was defined as undetectable serum HBsAg level (ARCHITECT HBsAg; Abbott Diagnostics Division, Wiesbaden, Germany [sensitivity: 0.05 IU/mL]) at last visit. HBV DNA reappearance was defined by any serum HBV DNA ≥200 IU/mL during treatment or follow-up in patients with baseline serum HBV DNA <200 IU/mL. HBV virologic response was defined by serum HBV DNA <200 IU/mL at last visit in those patients with baseline serum HBV DNA ≥200 IU/mL.
Visit Schedules and Laboratory Testing During Long-Term Follow-Up.
Patients were followed for up to 5 years after the end of the treatment period, including 24 weeks posttreatment follow-up in the original study and an additional 4.5 years follow-up in this study. Clinical assessments were performed at 24 weeks and at years 1, 2, 3, 4, and 5 during the posttreatment follow-up period. At each visit, blood cell counts, liver function test, serum HCV RNA level, and abdominal ultrasonography were performed for all patients. Serum HBsAg level and HBV DNA level were also determined at these scheduled visits in coinfected patients. Any intervening or significant clinical events related to chronic hepatitis C or B were documented.
Hepatitis Virus Markers and Virologic Assays.
Pretreatment HBsAg and anti-HCV were tested with commercial kits at each study site. Antibody against hepatitis D virus was screened with a commercial kit in a central laboratory (Hepatitis Research Center, National Taiwan University Hospital). Serum HBsAg level at each visit of the follow-up study was also measured in the central laboratory using a standard quantitative Chemiluminescent Microparticle Immunoassay (ARCHITECT HBsAg; Abbott Diagnostics Division).
Quantification of Serum HBV DNA and HCV RNA.
Serum HCV RNA level and HBV DNA level were determined in a central laboratory (Hepatitis Research Center, National Taiwan University Hospital) via commercial real-time polymerase chain reaction assays (COBAS TaqMan HCV Test version 2.0 and HBV Test [lower detection of limit: 6 IU/mL], Roche Diagnostics, respectively).
The follow-up protocol was approved by the Institutional Review Board at each medical center. The study was conducted according to the 1975 Declaration of Helsinki and Good Clinical Practice. Patients were enrolled in the LTFU study after they gave written informed consent.
All categorical and continuous variables were analyzed by chi-square test or Fisher's exact test, and Student t test with equal or unequal variance, respectively, whenever appropriate. The person-years were calculated from the start of combination therapy to the dates of death, the dates of initiation of further antiviral therapy (for HCV or for HBV) during follow-up, the dates of lost to follow-up, or the dates of completing last follow-up, whichever came first. The HCV SVR-LTFU rate was calculated as the number of patients with HCV SVR-LTFU divided by the number of person-years accumulated by those who obtained HCV SVR24. The annual and cumulative rates of HBsAg seroclearance was calculated from the start of therapy in those coinfected patients who completed combination treatment. HBV DNA reappearance rate was calculated as the number of patients with any serum HBV DNA ≥200 IU/mL during the treatment and the follow-up period divided by the number of patients with baseline serum HBV DNA <200 IU/mL. HBV virologic response rate was calculated as the number of patients with serum HBV DNA <200 IU/mL at last visit divided by the number of patients with baseline serum HBV DNA ≥200 IU/mL. For patients with undetectable serum HBV DNA and HBsAg levels, the lower limit of detection (15 IU/mL for HBV DNA and 0.05 IU/mL for HBsAg) were assigned for statistical analysis. The Kaplan-Meier method was used to calculate the cumulative incidence of HBsAg seroclearance, and a log-rank test was conducted to test the statistical significance of the difference in HBsAg seroclearance rates between HCV genotype 1 versus genotype 2/3. Key event rates including delayed HCV recurrence and HBsAg seroclearance were also calculated with 95% confidence interval (CI). A P value of 0.05 was considered statistically significant (all two-sided). All analyses were performed using Stata statistical software (version 12.1; Stata Corp., College Station, Texas).
Characteristics of Participants.
Of the 321 patients participating in our previous multicenter clinical trial, 295 (91.9%) patients completed the treatment and 24 weeks posttreatment follow-up. Thereafter, 264 (89.5%) patients of the 295 patients agreed to receive extended follow-up, including 232 patients who obtained HCV SVR24 and 32 patients without HCV SVR24. The remaining 31 patients did not participate in the LTFU study due to unwillingness (n = 22), loss to follow-up (n = 6), and travel abroad (n = 3). The LTFU study overview is shown in Fig. 1.
Baseline characteristics of patients in the initial study (n = 321) and those who were included in the LTFU study (n = 264) were comparable in terms of demographics and virologic characteristics (Table 1). Patients were followed for a mean of 4.6 ± 1.0 years (range, 1-5 years) after the end of treatment.
Table 1. Baseline Patient Characteristics
Original Cohort (n = 321)
LTFU Subcohort (n = 264)
Abbreviations: ND, not determined; SVR-6m, sustained virologic response at 6 months after end of treatment.
No. of patients
HCV SVR-6m, %
Male sex, %
Age, years, mean ± SD
48.7 ± 11.7
51.1 ± 10.4
50.7 ± 11.5
50.2 ± 9.7
HCV RNA, IU/mL, median, range
2.59 ×106 (103-2.62 ×109)
3.06 ×106 (1,970-5.85 ×108)
2.66 ×106 (103-2.20 ×109)
3.44 ×106 (2,390-5.85 ×108)
HBV DNA level, IU/mL, median (range)
236 (0-1.02 ×106)
0 (0-1.02 ×106)
HBV genotype, B/C/mixed/ND
HBV precore mutation, no/yes/ND
HBV basal core promoter mutation, no/yes/ND
Stage of fibrosis (Metavir), mean ± SD
1.7 ± 1.2
2.3 ± 1.1
1.6 ± 1.2
2.2 ± 1.0
ALT, IU/L, mean ± SD
125 ± 82
121 ± 84
131 ± 84
123 ± 87
Posttreatment follow-up, years, mean ± SD
4.6 ± 1.1
4.6 ± 1.0
Durability of HCV Response and Retreatment of HCV.
During extended posttreatment follow-up, only six of the 232 patients with HCV SVR24 developed a delayed HCV RNA reappearance, including five HCV genotype 1/HBV-coinfected patients and one HCV genotype 2/3-monoinfected patient. The time of RNA reappearance from the end of treatment was 1 year in three patients, 3 years in one patient, and 4 years in two patients. An elevated serum alanine aminotransferase level was noted in one patient at the time of reappearance (Table 2). To clarify the possible origin of the reappeared HCV during follow-up in these six patients, we performed subgenomic analysis of the HCV core gene (∼420 base pairs) using paired serum samples obtained before treatment and at the time of HCV reappearance. We found that the similarity of the HCV subgenomic sequence was >98% in five (83.3%) patients but was only 74% in the remaining one patient (Table 2), indicating potential recurrence of HCV infection in five patients. Overall, the cumulative recurrence rate of HCV infection was 2.3% (average, 0.4%/year; 95% CI, 0.94%-5.47%). Four of five patients recurred with the same subgenotype (1b), and one recurred with a different genotype.
Table 2. Clinical and Virologic Characteristics of Six Patients With Delayed HCV Reappearance During LTFU
At HCV RNA Reappearance
HCV Subgenome Similarity
Time to Relapse, Years
HCV Titer, IU/mL
HBV Titer, IU/mL
Precore/ BCP Sequence
HCV titer, IU/mL
HBV Titer, IU/mL
Serum ALT Level, IU/L
Core (420 pb), %
Abbreviation: ND, not determined.
Of the five patients with possible HCV recurrence posttreatment, we further characterized their clinical, virologic, and treatment features. We found that none of the patients received immunosuppressive therapy, none had risk behavior for reinfection of HCV, and one had seroclearance of HBsAg at the time point of HCV recurrence. During the treatment course, one patient had transient reduction of peginterferon dosage (adherence rate, 96%) and another two patients had reduction of ribavirin dosage (adherence rate, 99% and 72%, respectively). Older age at baseline and serum HBV DNA ≥200 IU/mL at end of treatment correlated significantly with the development of HCV recurrence on univariate analysis (Table 3). In addition, we provided the profiles of HCV and HBV markers in these five patients (Fig. 2).
Table 3. Factors Correlated With HCV Recurrence in All Coinfected and Monoinfected Patients: Univariate Analysis
In total, 38 of this followed cohort had a relapse of HCV activity (including 32 cases at 6 months posttreatment and six cases with delayed reappearance). Ten (26%) of the 38 patients received anti-HCV retreatment, and two patients obtained HCV SVR.
HBV Serologic Response, Virologic Response, and Reappearance.
Overall, 45 patients developed HBsAg seroclearance after the start of peginterferon-based therapy, which was sustained in 40 patients and was only transient in five patients. The cumulative rate of sustained HBsAg seroclearance during the 5-year after treatment follow-up duration was 30.0% (95% CI, 21.5%-42.0%), yielding an average annual seroclearance rate of 5.0% when counting person-years from the start of the treatment. Anti-HBs developed in 15 (37.5%) of the 40 patients with sustained HBsAg seroclearance. A subgroup analysis revealed that for HCV genotype 1 coinfected patients who received 48-week treatment, the cumulative HBsAg seroclearance rate was 33.1% (95% CI, 21.8%-50.1%) (average annual rate, 5.5%) (Fig. 3). For HCV genotype 2/3 coinfected patients who received 24-week treatment, the cumulative rate of sustained HBsAg seroclearance was 24.3% (95% CI, 13.7%-42.9%) (average annual rate, 4.0%) (Fig. 3). However, the difference in HBsAg seroclearance rates did not reach statistical significance in two groups (P = 0.273). Among baseline variables, lower pretreatment serum HBV DNA and HBsAg level were correlated significantly with sustained HBsAg seroclearance during follow-up (P < 0.05) (Table 4). Analysis of end-of-treatment parameters also revealed that only low HBsAg level correlated with the seroclearance of HBsAg (Table 4).
Table 4. Factors Correlated With HBsAg Seroclearance During Treatment or Follow-Up
HBsAg Seroclearance (n = 40)
HBsAg Persistence (n = 110)
Abbreviation: ND, not determined.
Fisher's exact test.
Patients were excluded from analysis if mutation status was not determined.
Before treatment, serum HBV DNA was ≥200 IU/mL in 62 (45.7%) of the 138 coinfected patients. At last visit, HBV virologic response was obtained in 33 (53.2%) patients. Baseline hepatitis B viral load did not correlate with HBsAg seroclearance. After treatment, serum HBsAg seroclearance occurred in 17 (51.5%) of the 33 patients with HBV virologic response but none of the remaining 29 patients without HBV virologic response.
Of 76 patients with pretreatment serum HBV DNA <200 IU/mL, reappearance of HBV DNA was found in 47 (61.8%) patients, either during the course of treatment (n = 18 [38.3%]) or during post-treatment follow-up (n = 29 [61.7%]). Reappearance was transient in 21 (44.7%) of the 47 patients, intermittent in 12 (25.5%), and sustained in 14 (29.8%). None of the recurrent hepatitis B replication was associated with hepatitis flare indicated by an elevation of serum alanine aminotransferase level >80 IU/L, and none of our patients received anti-HBV therapy for hepatitis B reactivation. Serum HBsAg seroclearance was found in 18 (62.1%) of the 29 patients without hepatitis B reappearance. In contrast, among the 47 patients developing hepatitis B reappearance, HBsAg seroclearance occurred in nine (19.1%) patients.
Effect of HBV Genotype, Precore Mutation, and Basal Core Promoter Mutation on Treatment Outcomes.
Recent studies have identified the role of HBV genotype and precore/basal core promoter (BCP) mutations as predictors for HBsAg seroclearance. We thus examined the value of HBV genotype, and precore/BCP mutation in determining the treatment outcomes among coinfected patients. Of 138 patients coinfected with HCV and HBV, HBV genotype, precore, and BCP sequence status could be successfully determined in 70, 60, and 38 patients, respectively. A precore mutant was present in 52 patients, and a BCP mutant was present in 24 patients. We found that HBV genotype (B versus C) and the presence of precore or BCP mutant versus wild-type did not correlate with HBsAg seroclearance (Table 4).
Development of HCC.
Nine patients developed HCC during the study period. At baseline, eight (88.9%) of the nine patients had HCV/HBV coinfection, and only one (11.1%) had HCV monoinfection. Five (55.6%) patients had cirrhosis, three (33.3%) had stage 2 fibrosis, and one (11.1%) had stage 1 fibrosis. After treatment, seven of the nine patients obtained HCV SVR-LTFU, seven had biochemical remission, and three developed seroclearance of HBsAg. The median time from end of treatment to diagnosis of HCC was 3 years (range, 1-5 years).
Our previous study in Taiwanese patients demonstrated that, using peginterferon and ribavirin, a sustained HCV clearance rate of 72% was achieved in the difficult-to-treat patients coinfected with HCV genotype 1 and HBV at 24 weeks after end of treatment. This LTFU study supported that the virologic response was durable in 97% of the coinfected patients who obtained HCV SVR24. The results indicated that HCV SVR-LTFU rates would be similar in coinfected patients versus in HCV-monoinfected patients.
Recent studies have suggested that SVR in HCV-monoinfected patients after peginterferon plus ribavirin combination therapy is durable in 99% of patients.10 Our posttreatment LTFU study consistently revealed that HCV SVR was also durable in coinfected patients. Subgenomic analysis of the HCV core gene further demonstrated that five out of six patients with HCV RNA reappearance may have delayed HCV recurrence, all being HBV-coinfected patients. Because subgenotype 1b is common in our population, in the absence of quasispecies analysis, it is difficult to ascertain that the subgenotype 1b cases are recurrences versus de novo reinfection. Whatever the underlying mechanism, for the treatment of chronic HCV in patients coinfected with HBV, prolonged follow-up after the end of treatment would be needed to confirm the sustained seroclearance of HCV RNA.
In patients with HCV monoinfection, successful anti-HCV therapy has markedly decreased the incidence of HCC and liver-cause mortality.16 In addition to the cure of HCV infection in the short term, determining whether anti-HCV therapy with peginterferon plus ribavirin could decrease the incidence of HCC and improve overall survival in HCV/HBV-coinfected patients will require further long-term follow-up studies.
During the treatment of HCV/HBV coinfection, virologic response of HBV to peginterferon and the possible reappearance of HBV after the control of HCV are two major clinical issues that need to be addressed. We found that HBV virologic response was obtained in 53% of coinfected patients with pretreatment hepatitis B viremia after LTFU. Intriguingly, posttreatment HBsAg clearance was noted in 5% of coinfected patients annually, a finding that is consistent with the results of our previous pilot study.17 This figure is far beyond the previously reported spontaneous or treatment-induced HBsAg clearance of 0% to 3% annually.18-22 On the other hand, as much as 62% of the 76 coinfected patients whose pretreatment serum HBV DNA was undetectable had a reappearance of HBV. Nevertheless, the reappearance of HBV did not result in clinically evident hepatitis, and none of the patients received another course of antiviral therapy due to HBV reactivation. The significance of HBV reappearance after effective treatment of HCV in patients with chronic HCV/HBV coinfection requires further study.23
In conclusion, combination therapy of peginterferon alfa-2a and ribavirin appears to be just as effective and durable for the treatment of HBsAg-positive patients chronically infected with active chronic HCV as it is in patients with HCV monoinfection. Annually, ∼5% of coinfected patients developed HBsAg seroclearance posttreatment. Notably, this group of patients may still develop HCC even after achieving HBsAg seroclearance, thus they should be kept under regular surveillance even after SVR.
The following investigators also participated in this study: Ming-Yang Lai (National Taiwan University Hospital), Chia-Yen Dai (Kaohsiung Medical University Hospital), Zu-Yau Lin (Kaohsiung Medical University Hospital), Jing-Houng Wang (Chang Gung Memorial Hospital-Kaohsiung), Hung-Da Tung (Chi Mei Medical Center), Tsung-Hui Hu (Chang Gung Memorial Hospital-Kaohsiung), Chien-Hung Chen (Chang Gung Memorial Hospital-Kaohsiung), and Chao-Hung Hung (Chang Gung Memorial Hospital-Kaohsiung). We thank Roche Diagnostics Ltd, Taiwan, for supplying COBAS TaqMan HCV Test version 2.0 and HBV test kits.