M. Lindh, B. Arnholm, P. Björkman, K. Hellstrand, M. Lagging, S. Nilsson, T. Wahlberg, E. Wallmark, O. Weiland, R. Wejstal, J. Westin, A. Widel, G. Norkrans
J Viral Hepat. 2013;20(4):e82-e89.
Abstract and Introduction
The treatment durations for hepatitis C are guided by the analysis of hepatitis C virus (HCV) RNA in blood at certain time points. This multicentre, randomized open label trial evaluated the utility and performance of individualized treatment durations guided by viral decline rates in 103 patients with HCV genotype 1 infection. Pegylated interferon 2a and ribavirin were given as standard of care (SOC) for 24, 48 or 72 weeks or as dynamic treatment (DT) for 24–72 weeks. The DT duration was based on the time point when log HCV RNA would reach 0 log copies/mL, as estimated by the second-phase decline. The rate of sustained virologic response was 63% for SOC and 54% for DT, but this difference was not significant in multiple regression analysis taking predictive factors such as interleukin-28B genotypes, age and baseline viremia into account (P = 0.45). The mean required treatment time per cured patient was 51 weeks for DT as compared with 58 weeks for SOC (P = 0.22) when given per protocol (n = 95) and was significantly shorter (42 vs 51 weeks) among patients who achieved undetectable HCV RNA (P = 0.01). We conclude that DT was feasible and increased efficiency. The estimated time point for 0 log viral copies/mL is a new and quantitative response variable, which may be used as a complement to the qualitative variable rapid virologic response. The outcome parameter treatment weeks per cured patient could become a useful tool for comparing treatment efficiency also in the era of directly acting antivirals.
Peg-IFN and ribavirin treatment given 48 weeks cures approximately 50% of patients with chronic hepatitis C genotype 1. Response-guided therapy directed by the time point for hepatitis C virus (HCV) RNA negativity may improve treatment efficiency. Thus, shorter treatment may reduce cost and side effects for patients with rapid response,[2, 3] and extended treatment may increase cure rate for slow responders.[4–6] However, the strategy to guide treatment length on the basis of test negativity has some disadvantages. Firstly, it requires sampling at fixed time points, that is, after 4, 12 and 24 weeks. Secondly, qualitative measurements (undetectable HCV RNA) may be influenced by the sensitivity of the HCV RNA assay. Thirdly, in responding patients, viremia levels will during a significant time span be close to the HCV RNA assay's detection limit where the reproducibility is poor, and accordingly the decision regarding treatment duration may be guided by unreliable test results. Fourthly, the stepwise increase in treatment duration (24, 48 or 72 weeks) will cause some patients to be treated longer than necessary.
We previously performed a study of viral kinetics in patients given 48 weeks of treatment. Based on our findings, we proposed an algorithm for calculating treatment durations from the time point when the HCV RNA decline is predicted to reach 0 log copies/mL. This time point can be assessed from when a regression line for the second phase of viral decline crosses the x-axis (time). The aim of the present study was to evaluate the performance of this algorithm in a randomized trial with the main outcome measures sustained virologic response (SVR) and a treatment weeks per cured patient.
We intended to include 100 patients, a sample size that was predicted to be sufficient for identifying a difference in treatment efficacy in terms of treatment weeks per cured patient, but not for non-inferiority analysis concerning SVR. Patients aged ≥18 years with chronic HCV genotype 1 infection were recruited at six infectious diseases clinics in Sweden between November 2007 and March 2009. Exclusion criteria comprised signs of decompensated cirrhosis, autoimmune disease, severe psychiatric disease, concurrent drug abuse or infection with hepatitis B virus or HIV. All patients were treated with a combination of pegylated interferon alfa-2a 180 μg once weekly and oral ribavirin 1000 or 1200 mg daily, depending on if weight at inclusion was below or above 75 kg. The median time from screening to start of treatment was 4.0 weeks, and 90% started within 9.2 weeks.
As depicted in Fig. 1, premature treatment terminations occurred in three patients in the standard of care (SOC) treatment group (one after 2 weeks because of fatigue and irritability, one after 11 weeks because of injection drug use, one after 1 week after consent withdrawal) and in three patients in the dynamic treatment (DT) group (one after 5 weeks because of fatigue and mood changes, one after 11 weeks because of severe depression and one after 20 weeks because of allergic reaction). One patient in the SOC group received erythropoietin because of severe anaemia. One patient in the SOC group was lost to follow-up (left for a long travel abroad). This patient and the two patients who discontinued treatment already after ≤2 weeks (all in the SOC group) were excluded in the analyses. Thus, 103 patients remained (39 in Gothenburg, 21 in Malmö, 20 in Stockholm, 11 in Borås, 6 in Lund and 6 in Skövde), and 86 of these patients (83.5%) received at least 80% of Peg-IFN and 80% of ribavirin for at least 80% of the planned treatment.
Flow chart for inclusion, treatment and follow-up in the TTG1 trial.
The patients were allocated to SOC or DT by randomization with stratification in blocks of four for age (above or below 45 years), fibrosis (mild-moderate or severe, see below) and baseline HCV RNA levels (above or below 5.8 log IU/mL). The computer-generated randomization table was kept at the organizing study centre, and after inclusion of a patient, the allocated treatment procedure was sent by fax to the study nurse at the infectious diseases clinic. Patients randomized to SOC were treated for 24, 48 or 72 weeks depending on response: 24 weeks if HCV RNA was negative at week 4, 48 weeks if negative at week 12, and 72 weeks if HCV RNA was detectable but had declined by at least 2 log10 units at week 12; it was discontinued if HCV RNA had not declined by 2 log10 at week 12 or was detectable at week 24.
Patients randomized to DT were given peg-IFN-2a and ribavirin at the same doses but with treatment durations that were individualized and dynamic. If the viral decline was slower than 0.3 log/week (based of levels at day 14, 21 and 28), treatment should be stopped after 5 weeks. Patients with declines steeper than 0.3 log/day continued treatment, and the final duration was calculated after additional testing of HCV RNA at day 49, using an algorithm that takes into account the HCV RNA level at day 21 and the second-phase slope including values from day 14, 21, 28 and 49 (Fig. 2a). This algorithm was developed on the basis of previous findings, and the decision to give a minimum duration of 24 weeks: Duration = 24 weeks plus twice the predicted time it takes from day 21 until the level reaches 0 log copies/mL (i.e. −0.5 log IU/mL), or D = 24 + 2 × [(log HCV RNA day 21 + 0.5)/log decline per week]. If the calculated required duration was above 72 weeks, treatment was stopped after 8 weeks, else it was individualized to a calculated duration between 24 and 72 weeks.
(a) Treatment durations were calculated on the basis of the time point (arrow) when a regression line for the second-phase slope (dotted) would cross the x-axis, that is, when hepatitis C virus RNA would be ≈0 log copies/mL (−0.5 log IU/mL). (b) Durations of treatment that were given to patients achieving or not achieving sustained virologic response by standard or dynamic treatment.
HCV RNA Quantification HCV RNA levels were determined by the fully automated COBAS Ampliprep/COBAS TaqMan HCV Test version 1.0 (Roche Diagnostics, Branchburg, NJ, USA). Analyses required by the protocol were performed during treatment; later additional testing on samples frozen at −70 °C was performed to achieve for all patients HCV RNA levels at baseline, 7, 14, 21, 28 days, and 7, 12, and 24 weeks of treatment, at end of treatment, and 24 weeks after end of treatment.
Classification of Response Sustained virologic response was defined as negative HCV RNA results by COBAS TaqMan 24 and 26 weeks after end of treatment. Patients who did not achieve HCV RNA negativity were classified as having non-SVR. Reappearance of HCV RNA during treatment was defined as breakthrough, while reappearance of detectable HCV RNA after end of treatment was defined as relapse.
Fibrosis Classification Pretreatment liver biopsies were performed in 76 cases and were scored for fibrosis as described by Ludwig-Batts (0–4; n = 51) or Ishak (0–6; n = 25). Liver elasticity was tested by Fibroscan as an alternative to biopsy in 11 cases. The fibrosis was classified on the basis of biopsy stage or Fibroscan as mild-moderate (Ludwig-Batts 0–2, Ishak 0–3, Fibroscan ≤10 kPa), or severe fibrosis (Ludwig-Batts 3–4, Ishak 4–6, Fibroscan >10 kPa). Fibrosis could not be classified in 16 cases because neither biopsy nor Fibroscan was performed (randomization was based on APRI score <1.0 or ≥1.0 in these cases).
IL28B Genotyping Genetic polymorphisms within or upstream of the interleukin-28B (IL28B) gene were investigated by TaqMan real-time PCR as described previously.
The study was approved by the regional ethical review board, and a written informed consent was obtained from each patient.
Clinical Trial Registration
The study was registered at the National Institutes of Health trial registry with ClinicalTrials.gov identifier NCT00910975.
The predefined primary outcome measures were the number of treatment weeks utilized per cured patient, SVR and relapse rates. The former parameter measures treatment efficiency and was assessed by dividing the treatment duration for a patient with the SVR rate for the subset of patients to which that patient belongs (i.e. the treatment regimen alone or restricted to patients treated per protocol or achieving on-treatment negativity of HCV RNA).
Fisher's exact and chi-square tests were used for comparisons between groups. Continuous values were compared by Mann–Whitney U-test. Logistic regression analysis was performed to evaluate a possible correlation between SVR (dependent variable) and baseline variables (age, gender, BMI, liver fibrosis, HCV RNA, IL28B genotype and ALT) and treatment regimen. The softwares Statview (Statview 5.0, SAS Institute, Inc., Cary, NC, USA) and SPSS Statistics 19 (SPSS; IBM, New York, NY, USA) were used for statistical analysis.
Baseline characteristics are described in : the median age was 48.1 years, median BMI was 25.6, 40.4% were females, and 17.4% had severe fibrosis. The treatment groups were similar as regards response predictors, except for IL28B genotypes, a predictor that was identified after the start of the study. Thus, the favourable CC genotype at rs12979860 was more common in the SOC group (30% vs 22%), the unfavourable GG genotype at rs8099917 was more common in the DT group (11% vs 0%), and accordingly, patients receiving SOC were more often HCV RNA negative at week 4 (rapid virologic response, RVR; 20% vs 11%) and at week 12 (complete early virologic response; 58% vs 52%) as compared with patients in the DT group.
Table 1. Baseline status of 103 patients receiving tailored or SOC treatment
| ||Total||DT (n = 54)||SOC (n = 49)||P value|
|Age, median (range)||48.1||48.0 (23.1–70.1)||48.7 (24.3–70.3)||0.94|
|BMI, median (range)||25.6||25.6 (18.6–38.7)||25.5 (17.5–38.2)||0.35|
|HCV RNA, median IU/mL (range)||6.20||6.15 (5.15–6.91)||6.37 (4.76–7.22)||0.42|
|ALT/ULN, median (range)||1.91||1.60 (0.54–7.64)||1.17 0.34–8.93)||0.29|
|APRI score, median (range)||0.55||0.63 (0.20–7.19)||0.49 (0.19–2.82)||0.10|
|Fibrosis class (mild-moderate/severe)||72/16||36/9||36/7||0.78|
|IP-10, median pg/mL (range)||385||441 (111–1500)||368 (69–1500)||0.25|
P value is given as a measure of random unbalance between treatment groups.
DT, dynamic treatment (24–72 weeks); SOC, standard of care (24 48 or 72 weeks); HCV, hepatitis C virus.
The overall SVR rate was 58% and associated with well-known response predictors such as age, baseline HCV RNA level, fibrosis, IP-10 levels and IL28B genotype (). The SVR rate was 63% in the SOC group as compared with 54% in the DT group (P = 0.42). Patients with non-SVR were further classified as described in Table S1. In line with the less favourable baseline factors, the number of patients who never reached undetectable HCV RNA (non-responders) or who experienced breakthrough during treatment was higher in the DT group (18/54; 33%) than in the SOC group (11/49; 22%).
Table 2. Outcome in relation to baseline status and treatment regimen
| ||Total||SVR (n = 60)||Non-SVR (n = 43)||P value|
|HCV RNA, median IU/mL||6.20||6.04||6.39||0.0007b|
|APRI score, median||0.55||0.49||0.63||0.09b|
|IP-10, median pg/mL||385||325||574||0.0002b|
|Fibrosis class (mild-moderate/severe)||72/16||44/7||28/9||0.26b|
DT, dynamic treatment (24–72 weeks); SOC, standard of care (24, 48 or 72 weeks); HCV, hepatitis C virus; SVR, sustained virologic response.
aFisher's exact test.
cMantel-Haenszel linear-by-linear association.
Treatment durations were shorter in the DT as compared with the SOC group (mean, 30.5 vs 39.3 weeks), both for patients with and without SVR (Fig. 2b). The length of administered treatment per cured patient was shorter in the DT as compared with the SOC group, but not with statistical significance (, mean, 57 vs 62 weeks, P = 0.38). However, this difference was significant when comparing only the patients who were treated per protocol and were relapsers or achieved SVR (mean, 42 vs 51 weeks, P = 0.01), a comparison justified because the treatment regimen (i.e. duration) should in theory have impact only on these subgroups.
Table 3. Treatment efficiency
| ||SVR rate (%)||P value||Treatment weeks per cured patient (mean)||P value|
|All patients (n = 103)||54||63||0.42||57||62||0.38|
|Per protocola (n = 95)||55||67||0.52||51||58||0.22|
|Per protocol and achieving negative HCV RNA (n = 66)||91||91||1.00||42||51||0.01|
DT, dynamic treatment (24–72 weeks); SOC, standard of care (24, 48 or 72 weeks); HCV, hepatitis C virus; SVR, sustained virologic response.
a Including patients achieving SVR even if they did not fulfil per protocol criteria.
To quantify the potential gain in terms of shorter duration, we also compared the treatment duration for each patient by the DT algorithm with duration by application of SOC rules (setting SOC durations for all patients in the DT group who discontinued after 5 or 8 weeks to 13 weeks despite the fact that some of them might not have discontinued until 25th week). By this comparison (illustrated by Figure S1), the mean difference in treatment duration was 9.46 weeks (31.3 by the DT algorithm, 40.8 weeks by SOC, P < 0.0001).
Ten patients experienced relapse, five in the DT and five in the SOC group. The relapsers in the DT group had received shorter treatment (30, 33, 40, 62, 72 weeks) as compared with those in the SOC group (48, 48, 48, 72 and 72 weeks). In four cases, dose reductions may have contributed to relapse: two in the DT group (treated for 33 and 72 weeks) and two in the SOC group (treated for 48 and 72 weeks). Eight of the 10 relapsers were older than 45 years, and nine had baseline HCV RNA above 5.8 log IU/mL (P = 0.10 and P = 0.08 when compared with age and baseline HCV RNA in patients who achieved SVR).
Figure 3 shows the viral declines during the first 12 weeks of treatment. Patients achieving SVR had steeper declines than those who did not, but a considerable variation was seen within the SVR group, corresponding to a broad range of required treatment durations. Table S2 describes first week reduction in HCV RNA and second-phase decline rates. The decline rates based on HCV RNA week 2, 3, 4 and 7 were >0.30 in patients achieving SVR, and slower than 0.30 log IU/week in non-responders. Thirteen patients had undetectable HCV RNA at week 4 (RVR; five in the DT, eight in the SOC group), all of them achieved SVR.
Viral declines in patients achieving (a and c) or not achieving sustained virologic response (SVR) (b and d). Green lines, SVR; blue, relapse; red, non-response; orange, breakthrough. Dotted lines, not treated per protocol.
Impact of IL28B
The impact of the rs12979860 SNP on response kinetics is shown in Figure S2, demonstrating significantly steeper decline in patients with the CC genotype. Because the difference in IL28B genotype distribution between treatment groups could have influenced outcome, a multiple regression analysis was performed, including treatment regimen as an independent variable. As shown in Table S3, rs12979860, baseline levels of HCV RNA and IP-10, and age were independent predictors of SVR, whereas treatment regimen was not.
This study demonstrates that individualized dynamic tailoring of treatment with peg-IFN and ribavirin for chronic hepatitis C is feasible and efficient, resulting in reduction (almost 20%) in treatment duration and medication required to cure a patient, and accordingly reduced side effects and costs. These are goals for all response-guided treatment regimens, but tailoring determined by our algorithm has advantages compared with fixed treatment durations. It allows greater flexibility as regards dates of sampling, results in a dynamic rather than a stepwise increase in treatment duration for patients with slower viral response. It is also less dependent on HCV RNA assay sensitivity, but a WHO standard needs to be included in the assay to calibrate calculation of the zero-log time point, and the model requires one additional sampling time point as compared with the standard protocol.
Models for individualized treatment have been previously proposed or tested. A clinical trial applying a novel algorithm recently showed that treatment given for 24, 30, 36, 42, 48, 60 or 72 weeks as guided by baseline viral load and the time point when HCV RNA was undetectable by a highly sensitive assay (TMA, limit of detection 5 IU/mL) was noninferior to SOC treatment given for 48 weeks. In that study, individualized treatment resulted in 55% SVR, as compared with 54% in our study, whereas mean treatment duration for patients achieving SVR was 48 weeks, as compared with 38 weeks by our regimen. Baseline factors were similar or somewhat more favourable for their patients as compared with our (mean age, 43.1 vs 45.8, baseline HCV RNA 5.6 vs 6.2 log IU/mL, IL28B CC genotype 32% vs 27%), but the relapse rate was higher for their patients given individualized treatment (27% vs 15% in our study). These observations suggest that the efficiency of our tailoring algorithm is similar as, or possibly even higher than for the algorithm presented by Sarrazin et al. However, our study was not dimensioned to show noninferiority as compared with standard treatment.
In addition to a dynamic assignment of treatment durations, a potential advantage with our algorithm is an earlier stopping rule, applied after 5 or 8 weeks rather than after 13 weeks in a standard regimen. Our stopping rule implies that treatment should be discontinued after 5 weeks if the second-phase decline is slower than 0.3 log IU/mL per week, or at week 8 if required treatment duration is longer than 72 weeks. These rules were supported by the observations that, in the SOC group, all non-responders had declines slower, and all patients achieving SVR had declines faster than 0.3 log IU/mL per week and that all patient with SVR in the SOC group had estimated duration shorter than 72 weeks.
This study was not sized for non-inferiority analysis, and comparison of SVR rates was difficult due to unexpected skewed distributions of IL28B variation, with favourable SNP genotypes being more common among patients receiving SOC. Thus, the SVR rate was higher in patients given SOC (63% vs 54%), but in multivariate analysis taking IL28B variation as well as other baseline factors into account, no significant association between treatment regimen and outcome was seen.
The recent introduction of new treatment modalities, including protease inhibitors, limits the utility of the tailored treatment described here, but the findings may become useful also in the new era of directly acting antiviral (DAA) treatment. Firstly, DT durations calculated on the basis of the time point when HCV RNA is predicted to reach 0 log copies/mL may be applicable and useful for improving outcome and efficiency also for regimens including DAA drugs. However, because the decline of HCV RNA is more rapid when DAAs are used, the time points for sampling need to be earlier during treatment. The algorithm also needs to be modified, and this requires new studies analysing how treatment outcomes are related to early viral kinetics.
Secondly, treatment with only peg-IFN and ribavirin may remain an option for patients with favourable response predictors, but also due to financial reasons. Patients for whom this could be an alternative are those with low baseline viral load, age below 45 years, favourable IL28B genotype and moderate fibrosis, that is, factors predicting a high chance to achieve SVR also with shorter treatment duration with only peg-IFN and ribavirin. In such cases, assessment of HCV RNA decline rate, as described here, and application of our algorithm should be useful for confirming a high probability of achieving SVR with short treatment duration. In our study, 32 patients (31%) had a CT genotype at rs12979860 and baseline HCV RNA below 1 million IU/mL, or a CC genotype and HCV RNA below 2 million IU/mL, and 14 (44%) of these patients had estimated required treatment durations ≤30 weeks, that is, not substantially longer recommended for regimens including protease inhibitors. For this subgroup (≈30% of all patients), it might be reasonable – in particular if treatment is considered for patients with fibrosis stadium 1 or 2 – to start with peg-IFN and ribavirin alone and add a protease inhibitor after 4 weeks only when HCV RNA declines predict that treatment durations longer than 28–30 weeks are required to achieve SVR.
In summary, three main observations were made in this study. Firstly, the time point for 0 log HCV RNA copies/mL was a valid quantitative viral kinetics variable that can be utilized as a complement to the qualitative variable RVR. Secondly, individualized DT based on this variable was feasible and similarly effective as SOC treatment durations. Such dynamic tailoring has advantages over traditional response-guided treatment and may be useful in the next era of treatment, for optimizing therapy with DAA and for selecting patients for which peg-IFN and ribavirin alone may be sufficient. Thirdly, we introduce treatment weeks per cured patient as a measure of treatment efficiency. This parameter should be useful as a complement to SVR when new treatment regimens are compared and optimized.
Ghany MG, Strader DB, Thomas DL, Seeff LB. Diagnosis, management, and treatment of hepatitis C: an update. Hepatology 2009; 49: 1335–1374.
Jensen DM, Morgan TR, Marcellin P et al. Early identification of HCV genotype 1 patients responding to 24 weeks peginterferon alpha-2a (40 kd)/ribavirin therapy. Hepatology 2006; 43: 954–960.
Zeuzem S, Buti M, Ferenci P et al. Efficacy of 24 weeks treatment with peginterferon alfa-2b plus ribavirin in patients with chronic hepatitis C infected with genotype 1 and low pretreatment viremia. J Hepatol 2006; 44: 97–103.
Berg T, von Wagner M, Nasser S et al. Extended treatment duration for hepatitis C virus type 1: comparing 48 versus 72 weeks of peginterferon- alfa-2a plus ribavirin. Gastroenterology 2006; 130: 1086–1097.
Pearlman BL, Ehleben C, Saifee S. Treatment extension to 72 weeks of peginterferon and ribavirin in hepatitis c genotype 1-infected slow responders. Hepatology 2007; 46: 1688–1694.
Sanchez-Tapias JM, Diago M, Escartin P et al. Peginterferon-alfa2a plus ribavirin for 48 versus 72 weeks in patients with detectable hepatitis C virus RNA at week 4 of treatment. Gastroenterology 2006; 131: 451–460.
Ogawa E, Furusyo N, Toyoda K et al. Excellent superiority and specificity of COBAS TaqMan HCV assay in an early viral kinetic change during pegylated interferon alpha-2b plus ribavirin treatment. BMC Gastroenterol 2010; 10: 38.
Germer JJ, Harmsen WS, Mandrekar JN, Mitchell PS, Yao JD. Evaluation of the COBAS TaqMan HCV test with automated sample processing using the MagNA pure LC instrument. J Clin Microbiol 2005; 43: 293–298.
Lindh M, Alestig E, Arnholm B et al. Response prediction and treatment tailoring for chronic hepatitis C virus genotype 1 infection. J Clin Microbiol 2007; 45: 2439–2445.
Dahari H, Shudo E, Cotler SJ, Layden TJ, Perelson AS. Modelling hepatitis C virus kinetics: the relationship between the infected cell loss rate and the final slope of viral decay. Antivir Ther 2009; 14: 459–464.
Ge D, Fellay J, Thompson AJ et al. Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance. Nature 2009; 461: 399–401.
Lindh M, Lagging M, Farkkila M et al. Interleukin 28B gene variation at rs12979860 determines early viral kinetics during treatment in patients carrying genotypes 2 or 3 of hepatitis C virus. J Infect Dis 2011; 203: 1748–1752.
Drusano GL, Preston SL. A 48-week duration of therapy with pegylated interferon alpha 2b plus ribavirin may be too short to maximize longterm response among patients infected with genotype-1 hepatitis C virus. J Infect Dis 2004; 189: 964–970.
Berg T, Weich V, Teuber G et al. Individualized treatment strategy according to early viral kinetics in hepatitis C virus type 1-infected patients. Hepatology 2009; 50: 369–377.
Sarrazin C, Schwendy S, Moller B et al. Improved Responses to Pegylated Interferon Alfa-2b and Ribavirin by Individualizing Treatment for 24–72 Weeks. Gastroenterology 2011; 141: 1656–1664.
Adiwijaya BS, Hare B, Caron PR et al. Rapid decrease of wild-type hepatitis C virus on telaprevir treatment. Antivir Ther 2009; 14: 591–595.
Fried MW, Hadziyannis SJ, Shiffman ML, Messinger D, Zeuzem S. Rapid virological response is the most important predictor of sustained virological response across genotypes in patients with chronic hepatitis C virus infection. J Hepatol 2011; 55: 69–75.
We thank Ulrika Nillroth and Ann-Sofi Lallerstedt at Roche Pharma for their assistance, and the staff at Clinical Virology in Gothenburg for dedicated work in this project.
This work was supported by grants from Västra Götalandsregionen [VGFOUREG-85861], ALF-LUA [#146611], and Roche Pharma. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
J Viral Hepat. 2013;20(4):e82-e89. © 2013 Blackwell Publishing