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‘Easy to treat’ genotypes were not created equal: Can rapid virological response (RVR) level the playing field?

Open AccessPublished:February 21, 2011DOI:https://doi.org/10.1016/j.jhep.2011.02.004
      Genotypes 2 and 3 (G2/G3) of hepatitis C virus have been lumped together as ‘easy to treat’. As a result, guidelines recommend 24 weeks of peginterferon/ribavirin for both. However, a closer look at trials shows that these genotypes are not the same, with G2 infection proving more responsive to peginterferon. The data supporting this conclusion are presented along with possible explanations for the differences observed. Ultimately, decisions must be made about therapy. Rapid virological response (RVR) may be the best parameter predicting successful antiviral therapy. For patients with G2 infection who achieve an RVR, shortened courses of therapy are effective. In contrast, for G3 patients without an RVR, there may be benefit to extending therapy to 48 weeks; however, this requires confirmation in prospective studies. Using RVR to guide therapy may level the playing field between these ‘easy to treat’ genotypes.

      Abbreviations:

      pegIFN (peginterferon), ribavirin (RBV), G (genotype (G1-4)), SVR (sustained virological response), HCV (hepatitis C virus), RVR/EVR (rapid/early virological response), ALT (alanine aminotransferase), OR/95%CI (odds ratio/95% confidence interval), CHC (chronic hepatitis C), ISG (interferon-stimulated genes), Jak/STAT (Janus kinase transducers and activators of transcription), SNP (single nucleotide polymorphisms), IL28B (interleukin 28b), SREBPc (sterol regulatory element-binding protein), MTP (microsomal triglyceride transfer)

      Keywords

      Introduction

      The current standard of care for patients with chronic hepatitis C consists of pegylated interferon alpha (pegIFN) and ribavirin (RBV), with duration of therapy and dosing of RBV varying according to the genotype of the virus [
      • Ghany M.G.
      • Strader D.B.
      • Thomas D.L.
      • Seeff L.B.
      American association for the study of liver diseases. Diagnosis, management, and treatment of hepatitis C: an update.
      ,
      • Antaki N.
      • Craxi A.
      • Kamal S.
      • et al.
      The neglected hepatitis C virus genotypes 4, 5 and 6: an international consensus report.
      ]. In patients with genotypes 2 and 3 (G2/G3) the recommended therapy is a 24-week course with a reduced dose of RBV, achieving a sustained virological response (SVR) in 75–80% of cases. This rate of successful therapy highly contrasts with the 45–55% reported for genotype 1 (G1), despite higher doses of therapy given for 48 weeks. The striking difference seemed to justify the grouping together of G2 and G3 in clinical trials and scientific reports and the early registration trials seemed to validate this approach [
      • Hadziyannis S.J.
      • Sette H.
      • Morgan T.R.
      • et al.
      Peginterferon-alpha2a and ribavirin combination therapy in chronic hepatitis C: a randomized study of treatment duration and ribavirin dose.
      ]. However, a closer look at published trials suggests a clinically meaningful difference in response to treatment between G2 and G3 and raises the question of whether it is in fact appropriate to lump them together. Additionally, close inspection of the data suggests that it may be appropriate to have different therapeutic approaches for these different genotypes. Viral kinetics might be a useful tool for tailoring the therapy to improve efficacy.

      Predictors of viral eradication

      Several viral and host factors have been shown to affect the response to antiviral therapy (Fig. 1). Although most experience derives from trials including predominantly patients with G1, many aspects also apply to G2 and G3. The role played by each factor in a given patient is almost impossible to dissect, but great enthusiasm has recently been paid to viral kinetics. Given that early viral kinetics are themselves a measure of treatment response, it is not surprising that they predict ultimate treatment outcome – response predicts response. As such, viral kinetics can and should form the basis for most therapeutic decisions.
      Figure thumbnail gr1
      Fig. 1Factors associated with response to treatment in chronic hepatitis C infection. Genetic factors such as IL28B genotype and hepatic gene expression profiles are important host determinants of response. Metabolic factors such as obesity, steatosis, insulin resistance, and type 2 diabetes mellitus also play an important role.
      Ferenci et al. first showed that the rapidity of HCV RNA decline was of utmost importance for reaching an SVR, and a therapeutic advantage was observed in patients achieving an RVR (87%) when compared to those with a >2 log10 IU/ml decrease but detectable levels at 4 weeks of treatment (52%) [
      • Ferenci P.
      • Fried M.W.
      • Shiffman M.L.
      • et al.
      Predicting sustained virological responses in chronic hepatitis C patients treated with peginterferon alfa-2a (40 kDa)/ribavirin.
      ]. More recent studies including patients with G1 infection have confirmed that the greater the viral suppression during the first 4 weeks of therapy, the greater the likelihood of reaching an SVR. In the Virahep-C study, Hoofnagle et al. found that early viral kinetics were very useful, showing an increasing rate of SVR for every 1 log10 decrease in the hepatitis C virus (HCV) RNA level at 4 weeks into therapy (Table 1) [
      • Hoofnagle J.H.
      • Wahed A.S.
      • Brown R.S.
      • Howell C.D.
      • Belle S.H.
      Virahep-C study group. Early changes in hepatitis C virus (HCV) levels in response to peginterferon and ribavirin treatment in patients with chronic HCV genotype 1 infection.
      ].
      Table 1Relationship between the decrease in viral load after four weeks of antiviral therapy and the rate of sustained virological response in the Virahep-C study.
      Both the early virological response at 12 weeks of therapy (EVR) and RVR have proven useful to tailor treatment in G1 infection, however, for slightly different purposes [
      • Sánchez-Tapias J.M.
      • Diago M.
      • Escartín 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.
      ,
      • 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.
      ,
      • Ferenci P.
      • Laferl H.
      • Scherzer T.M.
      • et al.
      Peginterferon alfa-2a and ribavirin for 24 weeks in hepatitis C type 1 and 4 patients with rapid virological response.
      ]. EVR has been used to define the minimum necessary response for patients to ultimately achieve an SVR. In other words, it has very good negative predictive value and thus is used to define treatment stopping rules. In contrast, RVR is used for its positive predictive value; achievement of RVR is highly predictive of SVR. RVR is a useful predictor in G1 and may even allow for shortening of therapy to 24 weeks, but unfortunately it is a rare event, occurring in only 15–30% of patients. In contrast, RVR is achieved in 60–80% of patients with G2 and G3 infection. With the shorter duration of treatment, the high rate of RVR and the strong correlation with subsequent SVR, RVR may be the most useful viral kinetic parameter for tailoring the duration of antiviral therapy in patients with G2 and G3 infection.

      Clinical trials with genotypes 2 and 3 assessing the RVR

      Large clinical trials assessing the efficacy of antiviral treatment arbitrarily combined G2 and G3 patients, based on their under-representation and higher rate of SVR when compared with G1 [
      • Hadziyannis S.J.
      • Sette H.
      • Morgan T.R.
      • et al.
      Peginterferon-alpha2a and ribavirin combination therapy in chronic hepatitis C: a randomized study of treatment duration and ribavirin dose.
      ,
      • McHutchison J.G.
      • Gordon S.C.
      • Schiff E.R.
      • et al.
      Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. Hepatitis interventional therapy group.
      ,
      • Poynard T.
      • Marcellin P.
      • Lee S.S.
      • et al.
      Randomised trial of interferon alpha2b plus ribavirin for 48 weeks or for 24 weeks versus interferon alpha2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis C virus. International hepatitis interventional therapy group (IHIT).
      ,
      • Manns M.P.
      • McHutchison J.G.
      • Gordon S.C.
      • et al.
      Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial.
      ,
      • Fried M.W.
      • Shiffman M.L.
      • Reddy K.R.
      • et al.
      Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection.
      ]. This has led to the false notion that G2 and G3 have a similar rate of response to pegIFN and RBV, but a careful examination of the evidence would suggest otherwise.
      Most studies, in which comparison is available, have found at least a trend toward improved outcomes in patients with G2 compared to G3, although not surprisingly the differences in rates of SVR are small between genotypes if patients achieve an RVR. Larger differences emerge when comparing patients with slower responses i.e. those who do not achieve an RVR. Because no study has directly assessed the issue of G2 vs G3 as its primary endpoint, any conclusions based on genotype must be teased out of studies assessing other factors.
      A meta-analysis compared the results from trials between G2 and G3. Summarizing the experience from 8 studies including 2275 patients (not all randomized trials), patients with G2 had a significantly higher SVR rate (74%) when compared to G3 (69%), with a weighted difference of 8.7% (95% CI = 5.1–12.3) and a pooled OR: 1.49 (95% CI = 1.23–1.80). Furthermore, in patients with a high viral load (mostly defined as >600,000 IU/ml) the respective SVR rates were 75% and 58%, and the weighted difference rose to 24.9% (12.8–37) with an OR: 2.36 (95% CI = 1.80–3.09). Confirming the importance of viral titer in G3, they found that even in patients receiving the standard 24-week regimen who achieved an RVR, G3 patients with a high baseline viral load experienced a lower rate of SVR (81%) compared to those with G2 (94%). The weighted difference was 12.6% (95% CI = 1.3–23.9), with an OR: 2.94 (95% CI = 0.99–8.72). The difference between genotypes was again more striking in patients who did not achieve an RVR, with a pooled SVR rate for G2 of 62%, compared to 46% for G3, for a weighted difference of 17.8% (95% CI = 8.7–27) and an OR: 2.06 (95% CI = 1.4–3.02) [
      • Andriulli A.
      • Mangia A.
      • Iacobellis A.
      • Ippolito A.
      • Leandro G.
      • Zeuzem S.
      Meta-analysis: the outcome of anti-viral therapy in HCV genotype 2 and genotype 3 infected patients with chronic hepatitis.
      ].
      Examining data from landmark studies, differences between G2 and G3 beyond absolute rates of SVR with standard therapy emerge. The ACCELERATE study was a large (n = 1469) multinational trial designed to assess if a shortened course of therapy (16 weeks) was non-inferior to the standard duration (24 weeks). As shown in Fig. 2 the SVR rate was different between the groups receiving 24 and 16 weeks only in G2 patients. Notably, however, the absolute difference in response at 24 weeks between G2 and G3 was 9%. Multivariable analysis revealed that G2 infection was independently associated with SVR along with 24 weeks of treatment duration, low viral load (≤800,000 IU/ml), age ≤45 years, weight ≤80 kg, high ALT (>3 times the upper limit of normal) and absence of bridging fibrosis/cirrhosis. Importantly, however, in a post hoc analysis they found that patients achieving an RVR had a higher rate of SVR, independent of HCV genotype or treatment duration [
      • Shiffman M.L.
      • Suter F.
      • Bacon B.R.
      • et al.
      Peginterferon alfa-2a and ribavirin for 16 or 24 weeks in HCV genotype 2 or 3.
      ]. The NORDynamIC trial compared 24 vs 12 weeks of treatment in 382 G2/3 patients. In this study, 24 weeks of therapy was superior, both for G2 and G3 and the absolute difference between G2 and G3 treated for 24 weeks was only 4%. However, patients with G2 infection were significantly older, perhaps explaining the reduced difference between G2 and G3 when compared to ACCELERATE (Fig. 2). Multivariable analysis identified RVR and age <40 years as predictors of SVR. The presence of bridging fibrosis/cirrhosis had a major impact in decreasing the SVR rate, even in patients under the 24-week regimen: 84% if non-significant fibrosis, 76% in bridging fibrosis, 57% in cirrhosis [
      • Lagging M.
      • Langeland N.
      • Pedersen C.
      • et al.
      Randomized comparison of 12 or 24 weeks of peginterferon alpha-2a and ribavirin in chronic hepatitis C virus genotype 2/3 infection.
      ]. The studies, both using 800 mg of RBV, showed that shorter courses of therapy were not equivalent to the 24 week standard and G2 patients fared at least somewhat better than those with G3. As expected, RVR was a strong predictor of subsequent SVR, regardless of genotype or treatment duration.
      Figure thumbnail gr2
      Fig. 2Bar chart showing the sustained virological response rates in the ACCELERATE (Shiffman et al.) and NORDynamIC (Lagging et al.) trials, which focused on patients with G2/G3 infection. The results are shown according to genotype and duration of treatment p value <0.01.
      To directly assess whether RVR can be used to determine the length of therapy, randomized, open-label studies have assessed the possibility of giving shortened courses of therapy to patients who achieve an RVR. Fig. 3 shows the SVR rates in two of these trials, and contrasts their results with the post hoc analysis of the ACCELERATE study according to RVR. Mangia et al. randomized 283 patients to receive pegIFN-α2b 1.0 μg/kg and weight-based RBV 1000–1200 mg to a standard 24-week regimen (n = 70), or to a variable one where subjects were assigned to be treated for 12 weeks if achieving an RVR (n = 133) or 24 weeks if not (n = 80). Their overall rate of RVR was 63%. As shown in Fig. 3, among patients achieving an RVR there was a small difference in the SVR rate favoring G2 in the shortened therapy groups (10%), but the numbers were too small for a fair comparison between the 24-week treatment arms. In patients not achieving an RVR (24-week regimen), there was a larger difference in the SVR rate between G2 and G3 (31%), although again, the number of patients in the G3 group (n = 9) was quite small [
      • Mangia A.
      • Santoro R.
      • Minerva N.
      • et al.
      Peginterferon alfa-2b and ribavirin for 12 vs. 24 weeks in HCV genotype 2 or 3.
      ]. Dalgard et al. treated 428 patients with pegIFN-α2b 1.5 μg/kg and weight-based RBV 800–1400 mg, and randomized 298 achieving RVR (RVR rate 71%, 302/428) to a shortened course of 14 vs 24 weeks of therapy. The overall rates of SVR in the intention-to-treat analysis for G2 and G3 were 70% and 66%, respectively. Among patients with an RVR, the differences by genotype were small comparing patients treated for the same duration (Fig. 3). In contrast, in patients not achieving an RVR but treated with a full 24-week course there was a notable difference of 19% in the rate of SVR between G2 and G3 [
      • Dalgard O.
      • Bjøro K.
      • Ring-Larsen H.
      • et al.
      Pegylated interferon alfa and ribavirin for 14 versus 24 weeks in patients with hepatitis C virus genotype 2 or 3 and rapid virological response.
      ]. These data all show a similar trend; rates of SVR are higher in G2 than G3 but for patients achieving an RVR, the differences are less striking.
      Figure thumbnail gr3
      Fig. 3The sustained virological response rates (SVR) obtained in randomized controlled trials (Mangia et al. and Dalgard et al.) comparing 24-week and short courses of pegIFN and RBV according to rapid virological response (RVR) are shown. The colored bars show the response rates in patients who achieved RVR. The different colors show the different genotypes and the duration of therapy, short vs standard 24 weeks. The white bars at the bottom show the rates of SVR in those who did not achieve RVR. The results are divided by genotype but notably, all non-RVR patients were treated for 24 weeks. The results from the ACCELERATE trial (Shiffman et al.), in which treatment duration was not dependent on RVR, are depicted in the same way for comparison.
      A second meta-analysis looked specifically at duration of therapy in G2/3. Slavenburg et al. found that pooling of data from 5 trials (including 3 unpublished studies) with a total of 785 patients, who were randomized after the achievement of an RVR, failed to demonstrate any difference in SVR between standard- and short-duration regimens (83% and 82%, respectively; RR: 1.00, 95% CI: 0.92–1.09). However, for this analysis, patients with G2/3 were combined. A subanalysis of patients with G3 alone showed a trend toward superiority of 24 weeks of therapy, while for patients with G2, despite relatively small numbers, there was no difference between standard and shortened therapy [
      • Slavenburg S.
      • Weggelaar I.
      • van Oijen M.G.
      • Drenth J.P.
      Optimal length of antiviral therapy in patients with hepatitis C virus genotypes 2 and 3: a meta-analysis.
      ].
      Taken together, all of the above evidence points out that a regimen of 24 weeks of therapy is appropriate for the majority of patients with chronic hepatitis C (CHC) caused by G2/G3. Shortened courses may be offered to G2 patients achieving an RVR, irrespective of the viral load, as it is a cost-effective approach [
      • Dalgard O.
      • Bjøro K.
      • Ring-Larsen H.
      • et al.
      Pegylated interferon alfa and ribavirin for 14 versus 24 weeks in patients with hepatitis C virus genotype 2 or 3 and rapid virological response.
      ] and retreatment of relapsers for 24 weeks is not compromised [
      • Mangia A.
      • Minerva N.
      • Bacca D.
      • Cozzolongo R.
      • Agostinacchio E.
      • Sogari F.
      • et al.
      Determinants of relapse after a short (12 weeks) course of antiviral therapy and re-treatment efficacy of a prolonged course in patients with chronic hepatitis C virus genotype 2 or 3 infection.
      ]. However, the data suggest it may not be prudent to shorten therapy in patients bearing other factors associated with failure to achieve SVR (i.e. age >45 years, ≥F3 fibrosis). Treatment for G3 is less straightforward. Viral load seems to be more relevant in G3 than G2, as it affects both the rate of RVR [
      • Mangia A.
      • Bandiera F.
      • Montalto G.
      • Mottola L.
      • Piazzolla V.
      • Minerva N.
      • et al.
      Individualized treatment with combination of peg-interferon alpha 2b and ribavirin in patients infected with HCV genotype 3.
      ] and increases the chance of relapse [
      • Zeuzem S.
      • Hultcrantz R.
      • Bourliere M.
      • et al.
      Peginterferon alfa-2b plus ribavirin for treatment of chronic hepatitis C in previously untreated patients infected with HCV genotypes 2 or 3.
      ]. Patients who achieve RVR do well with a standard 24-week course of therapy and some data suggest that treatment can be shortened in such individuals. For those who do not achieve RVR, particularly if starting with a high viral load, rates of SVR approach those for G1 and thus it seems prudent not to consider this subgroup of G3 as ‘easy to treat’. Whether these patients may benefit from an extended course of 48 weeks, just as with G1 infection, remains an open question.

      Extended or intensive courses of therapy in genotypes 2 and 3

      The need to treat G2 and G3 patients for 48 weeks (extended therapy) or with weight-based RBV (intensive therapy) was shown to be unnecessary in the registration study of pegIFN-α2a by Hadziyannis et al. The authors randomized 492 patients to any of four arms including pegIFN-α2a 180 mg/week: with 800 mg/day of RBV for 24 or 48 weeks, and with 1000–1200 mg/day (weight-based) for 24 or 48 weeks. There was no difference in the rate of SVR among the four groups (Fig. 4). Stratification according to the presence of low or high viral load (cut-off value: 600,000 IU/ml), or to the presence/absence of bridging fibrosis and cirrhosis also failed to identify any differences. Thus, this landmark study showed no benefit from an extended or intensive therapy in those infected with G2/G3 [
      • Hadziyannis S.J.
      • Sette H.
      • Morgan T.R.
      • et al.
      Peginterferon-alpha2a and ribavirin combination therapy in chronic hepatitis C: a randomized study of treatment duration and ribavirin dose.
      ]. However, in an interesting abstract from the same authors, the same data were re-analyzed for patients without an RVR, and it was found that extended/intensified therapy with weight-based RBV for 48 weeks offered a 9% gain in SVR over the standard 24 week, low-dose ribavirin regimen (Fig. 4). This was accomplished by a remarkable lowering of the relapse rate: ∼25% for the 24-week arms vs 13% and 4% in the 48-week arms with 800 mg and weight-based RBV, respectively [
      • Willems W.B.
      • Hadziyannis H.S.J.
      • Morgan M.T.R.
      • et al.
      Should treatment with peginterferon plus ribavirin be intensified in patients with HCV genotype 2/3 without a rapid virological response?.
      ]. Unfortunately, given that the trial achieved a high rate of RVR (75%), the number of patients included in this analysis was small (<40 patients per group) and the differentiation of G3 from G2 patients was not possible. Nevertheless, the 76% rate of SVR observed in non-RVR patients with extended and intensive therapy contrasts with the 39–53% observed in the ACCELERATE trial in the 24-week, 800 mg of RBV regimen. Data from the NORDynamIC trial also suggested a benefit to higher RBV dosing. They found a gradient effect with higher plasma concentration of RBV being associated with higher rates of SVR, and levels >2 mg/L at day 29 of therapy as an independent predictor of SVR [

      Pedersen C, Alsiö A, Lagging M, et al. Ribavirin plasma concentration is a predictor of sustained virological response in patients treated for chronic hepatitis C virus genotype 2/3 infection. J Viral Hepat 2010. Article first published online: 1 APR 2010/doi:10.1111/j.1365-2893.2010.01303.x.

      ]. A post-hoc analysis combining data from two clinical trials from Scandinavia and Italy (using weight-based RBV) showed that a RBV dose ≥15 mg/kg during the first 4 weeks of antiviral therapy was associated with a higher rate of RVR, and this was particularly evident in patients with G3 infection. Moreover, the maintenance of the full dose throughout treatment was an independent predictor of SVR in patients with shortened therapy (12 or 14 weeks) [
      • Mangia A.
      • Dalgard O.
      • Minerva N.
      • Verbaan H.
      • Bacca D.
      • Ring-Larsen H.
      • et al.
      Ribavirin dosage in patients with HCV genotypes 2 and 3 who completed short therapy with peg-interferon alpha-2b and ribavirin.
      ]. Collectively these data make a case for using weight-based ribavirin, particularly if shortened therapy is to be considered.
      Figure thumbnail gr4
      Fig. 4Results from the pegIFN-α2a registration trial (Hadziyannis et al.) demonstrating the lack of difference in sustained virological response (SVR) according to length of therapy (between 24-week and 48-week) and RBV dose (standard vs weight-based), in genotype 2 and genotype 3 patients. Reanalysis of the same data from Willems et al. demonstrates the superior SVR rate from the extended/intensive regimen (48-week and weight-based RBV) in those who failed to achieve a rapid virological response.
      A recent clinical trial from Mangia et al. examined the length of therapy according to achievement of RVR. Patients with G3 infection were randomized to a standard 24-week regimen (pegIFN-α2b 1.5 μg/kg and weight-based RBV 1000–1200 mg) or to a variable duration therapy of 12 or 36 weeks according to the presence or absence of RVR, respectively. In patients with an RVR, a difference in SVR could not be demonstrated between patients receiving the standard (82%) or 12-week (83%) regimens, although a non-significant higher rate of relapse was observed in patients who received the shorter course (10% and 15%, respectively). When considering patients without an RVR, extended therapy (36-week) yielded a 10% increase in SVR (62% vs 52%) when compared to the standard 24 weeks, although this did not reach statistical significance. Notably, this difference was not related to a decrease in the relapse rate (36-week: 20%, 24-week: 18%). Baseline viral load did not affect the rate of SVR in patients with an RVR, but unfortunately this was not reported for those without an RVR [
      • Mangia A.
      • Bandiera F.
      • Montalto G.
      • Mottola L.
      • Piazzolla V.
      • Minerva N.
      • et al.
      Individualized treatment with combination of peg-interferon alpha 2b and ribavirin in patients infected with HCV genotype 3.
      ]. Although this well-conducted study sheds light into the role of RVR for tailoring the length of treatment in G3 patients, their non-inferiority design is only appropriate for RVR patients (12- vs 24-week comparison), but not for the non-RVR group (24- vs 36-week comparison), thereby, limiting the interpretation of these exciting results. Of note, the SVR rate in non-RVR was similar to that observed in G1 patients, and a clinically meaningful increase to 62% was observed by prolonging therapy by 12 weeks. The question remains as to whether a 48-week regimen in patients with known risk factors for therapeutic failure can decrease the relapse rate and further increase SVR.
      The proposal to extend and intensify treatment (48 weeks with weight-based RBV) in patients with G3 infection who do not achieve RVR needs to be confirmed by prospective studies. These would need to stratify by at least two of the major adverse factors for an SVR, fibrosis stage, and baseline viral load [
      • Hadziyannis S.J.
      • Sette H.
      • Morgan T.R.
      • et al.
      Peginterferon-alpha2a and ribavirin combination therapy in chronic hepatitis C: a randomized study of treatment duration and ribavirin dose.
      ,
      • Shiffman M.L.
      • Suter F.
      • Bacon B.R.
      • et al.
      Peginterferon alfa-2a and ribavirin for 16 or 24 weeks in HCV genotype 2 or 3.
      ], as they can significantly influence the outcome of antiviral therapy. This was shown when re-analyzing the experience of a Canadian multi-centered and non-randomized study in which the rate of SVR in G3 patients with cirrhosis was 17%, in comparison to >60% in the absence of cirrhosis [
      • Powis J.
      • Peltekian K.M.
      • Lee S.S.
      • et al.
      Exploring differences in response to treatment with peginterferon alpha 2a (40 kDa) and ribavirin in chronic hepatitis C between genotypes 2 and 3.
      ]. The ongoing EXACT-R(3) is a randomized clinical trial exploring the benefit of an extended regimen (48- vs 24-week) of pegIFN-α2b, using weight-based RBV, with proper stratification for fibrosis and baseline viral load. The study will also clarify other predictive factors of response, such as ethnicity and metabolic factors. The results of this trial are eagerly awaited to clarify the management of G3 HCV, particularly given that the new direct-acting antivirals appear to have minimal activity in this population.

      Why is G3 more difficult to treat than G2?

      Genotype is a marker of interferon responsiveness

      The exogenous administration of alpha interferon enhances the innate and adaptive immune response against the virus, mainly by boosting the induction of interferon-stimulated genes (ISG) through activation of the Janus kinase transducers and activators of transcription (Jak/STAT) pathway. ISG promote antiviral, antiproliferative, and immunoregulatory signals in the host cell that can potentially lead to eradication of HCV [
      • Chung R.T.
      • Gale M.
      • Polyak S.J.
      • Lemon S.M.
      • Liang T.J.
      • Hoofnagle J.H.
      Mechanisms of action of interferon and ribavirin in chronic hepatitis c: summary of a workshop.
      ,
      • Feld J.J.
      • Hoofnagle J.H.
      Mechanism of action of interferon and ribavirin in treatment of hepatitis C.
      ]. However, intracellular pathways and their effector mechanisms need to be very finely tuned for the therapy to be effective.
      Microarray analysis from pre-treatment liver biopsies has shown that that non-responders to pegIFN-based therapy have increased basal expression of hepatic ISG prior to treatment [
      • Chen L.
      • Borozan I.
      • Feld J.
      • et al.
      Hepatic gene expression discriminates responders and nonresponders in treatment of chronic hepatitis C viral infection.
      ,
      • Asselah T.
      • Bièche I.
      • Sabbagh A.
      • et al.
      Gene expression and hepatitis C virus infection.
      ]. Near maximal ISG expression prior to therapy prevents further induction with pegIFN treatment [
      • Feld J.J.
      • Nanda S.
      • Huang Y.
      • et al.
      Hepatic gene expression during treatment with peginterferon and ribavirin: identifying molecular pathways for treatment response.
      ,
      • Sarasin-Filipowicz M.
      • Oakeley E.J.
      • Duong F.H.
      • et al.
      Interferon signaling and treatment outcome in chronic hepatitis C.
      ]. A recent study from Chen et al. showed that ISG pre-activation is a stronger predictor of outcome than viral genotype. Patients with G1 infection are more likely to have the non-responder phenotype (ISG pre-activation) than those with G2/G3, however, the relatively uncommon G2/G3 non-responders have the identical gene expression signature as non-responders with G1 [
      • Chen L.
      • Borozan I.
      • Sun J.
      • et al.
      Cell-type specific gene expression signature in liver underlies response to interferon therapy in chronic hepatitis C infection.
      ]. Why G1 HCV more commonly promotes ISG pre-activation is not yet understood but likely underlies the genotypic difference in treatment response.
      Careful analyses of very early viral kinetics from clinical trials have shed light on the mechanisms of action of interferon alpha and the differential effects seen in different HCV genotypes. Changes in HCV RNA levels occur in a biphasic pattern with a steep decrease in the first 24–48 h after the initial dose of interferon, with a more gradual reduction thereafter. The initial drop is thought to be related to inhibition of viral replication and clearance of free circulating virus; whereas the slower second phase represents the rate of clearance of infected hepatocytes [
      • Neumann A.U.
      • Lam N.P.
      • Dahari H.
      • et al.
      Hepatitis C viral dynamics in vivo and the antiviral efficacy of interferon-alpha therapy.
      ]. Non-responders to pegIFN show little or no decrease in viral load from the start of therapy, suggesting an intrinsic resistance to the antiviral activity of interferon alpha. The decreased response observed in the first and second phases of viral kinetics, continues with lack of RVR and culminates in failure to eradicate the virus.
      The gene expression pattern typically induced by each viral genotype correlates well with the responsiveness to interferon. Comparison of kinetics during interferon monotherapy showed that G2 patients have a faster first and second phase decline than those with G1 infection [
      • Neumann A.U.
      • Lam N.P.
      • Dahari H.
      • et al.
      Differences in viral dynamics between genotypes 1 and 2 of hepatitis C virus.
      ]. Similar results were later reproduced with pegIFN treatment with G3 infection, showing an intermediate susceptibility between G2 and G1 [
      • Zeuzem S.
      • Herrmann E.
      • Lee J.H.
      • et al.
      Viral kinetics in patients with chronic hepatitis C treated with standard or peginterferon alpha2a.
      ]. Rather than expressing true interferon resistance, it seems that HCV genotypes are more or less likely to promote the ISG pre-activation phenotype (G1>G4>G3>>G2), which ultimately prevents hepatocytes from responding to pegIFN treatment leading to poor early viral kinetics and ultimately to failure to clear virus.
      The recent identification of single nucleotide polymorphisms (SNP) upstream of the IL28B gene that are associated with response to pegIFN-based therapy is a landmark observation. Multiple groups have independently shown that patients with the favorable SNP have at least a 2.5-fold increase in the rate of SVR. The closest gene to the identified SNP is known as IL28B or interferon lambda 3 (IFN-λ3), a type 3 interferon that signals via the JAK-STAT pathway to induce ISG but uses a different receptor than interferon-alpha or beta [
      • O’Brien T.R.
      Interferon-alfa, interferon-lambda and hepatitis C.
      ]. The SNP was also shown to be significantly more prevalent in patients who spontaneously clear HCV [
      • Thomas D.L.
      • Thio C.L.
      • Martin M.P.
      • et al.
      Genetic variation in IL28B and spontaneous clearance of hepatitis C virus.
      ]. The IL28B genotype could at least in part explain the pre-activation of ISG in non-responders, as those with the unfavorable IL28B genotype show increased levels of intra-hepatic ISG [
      • Honda M.
      • Sakai A.
      • Yamashita T.
      • Nakamoto Y.
      • Mizukoshi E.
      • Sakai Y.
      • et al.
      Hepatic ISG expression is associated with genetic variation in interleukin 28B and the outcome of IFN therapy for chronic hepatitis C.
      ,
      • Urban T.J.
      • Thompson A.J.
      • Bradrick S.S.
      • Fellay J.
      • Schuppan D.
      • Cronin K.D.
      • et al.
      IL28B genotype is associated with differential expression of intrahepatic interferon-stimulated genes in patients with chronic hepatitis C.
      ].
      To date most of the data on the IL28B SNP come from patients with G1 infection [
      • Ge D.
      • Fellay J.
      • Thompson A.J.
      • et al.
      Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance.
      ,
      • Thompson A.J.
      • Muir A.J.
      • Sulkowski M.S.
      • et al.
      IL28B polymorphism improves viral kinetics and is the strongest pre-treatment predictor of SVR in HCV-1 patients.
      ]. Mangia et al. studied this polymorphism in G2/G3 patients from their randomized clinical trial comparing short and 24-week therapy according to RVR. A positive effect on SVR was observed in patients bearing the favorable IL28B allele, although it was attenuated when compared to G1. This related to the high RVR rate (63%), as the effect was only seen in patients not achieving an RVR. Surprisingly, in the non-RVR group patients, the CT variant showed an intermediate effect between CC and TT (SVR: 67%, 87% and 29%, respectively). Although their reduced sample of G3 patients limited interpretation of the data, the prognostic usefulness of IL28B was higher for G3 (OR: 5.5, 95% CI = 1.2–25) than for G2 (OR: 2.3, 95% CI = 1.2–4.5) [
      • Mangia A.
      • Thompson A.J.
      • Santoro R.
      • Piazzolla V.
      • Tillmann H.L.
      • Patel K.
      • et al.
      An IL28B polymorphism determines treatment response of hepatitis C virus genotype 2 or 3 patients who do not achieve a rapid virologic response.
      ].
      It may be that certain viral genotypes are inherently more likely to induce ISG pre-activation and the likelihood of this occurrence is modified by the presence of the IL28B SNP. Once present, ISG pre-activation is the strongest predictor of ultimate treatment outcome, regardless of the viral genotype or the SNP [
      • Dill M.T.
      • Duong F.H.T.
      • Vogt J.E.
      • et al.
      IFN stimulated gene expression in the liver is a better predictor of treatment response in chronic hepatitis C than the IL28B genotype.
      ,
      • Feld J.J.
      • Chen L.
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      • Patullo V.
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      • et al.
      Hepatic gene expression profile is a better predictor of treatment outcome in HCV infection than IL28B genotype.
      ]. Because liver biopsy is invasive and gene expression analysis is not widely available, using ISG pre-activation as a predictor of response is rather impractical and other pre and on-treatment markers have been examined, but will need further validation before becoming clinically useful. Until better markers are validated, RVR remains the best predictor.

      Do insulin resistance and steatosis contribute to poor outcome in genotype 3?

      The metabolic syndrome clusters disease traits related to insulin resistance (i.e. hyperglycemia/hyperinsulinemia, adiposity, dyslipidemia, arterial hypertension), to predict an increased risk of cardiovascular morbidity and type-2 diabetes mellitus [
      • Aguilar-Salinas C.A.
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      ,
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      ]. Obesity [
      • Chen T.
      • Jia H.
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      • Tian H.
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      ], insulin resistance [
      • Shaheen M.
      • Echeverry D.
      • Oblad M.G.
      • Montoya M.I.
      • Teklehaimanot S.
      • Hepatitis Akhtar.A.J.
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      Metabolic syndrome, and inflammatory markers: results from the third national health and nutrition examination survey [NHANES III].
      ], impaired fasting glucose, and diabetes mellitus [
      • Mehta S.H.
      • Brancati F.L.
      • Strathdee S.A.
      • et al.
      Hepatitis C virus infection and incident type 2 diabetes.
      ,
      • Wang C.S.
      • Wang S.T.
      • Yao W.J.
      • Chang T.T.
      • Chou P.
      Hepatitis C virus infection and the development of type 2 diabetes in a community-based longitudinal study.
      ,
      • Huang J.F.
      • Yu M.L.
      • Dai C.Y.
      • et al.
      Reappraisal of the characteristics of glucose abnormalities in patients with chronic hepatitis C infection.
      ,
      • Lecube A.
      • Hernández C.
      • Genescà J.
      • Esteban J.I.
      • Jardí R.
      • Simó R.
      High prevalence of glucose abnormalities in patients with hepatitis C virus infection: a multivariate analysis considering the liver injury.
      ] have been strongly associated with chronic HCV infection, and their presence predicts decreased likelihood for an SVR with pegIFN and RBV therapy [
      • Bressler B.L.
      • Guindi M.
      • Tomlinson G.
      • Heathcote J.
      High body mass index is an independent risk factor for nonresponse to antiviral treatment in chronic hepatitis C.
      ,
      • Romero-Gómez M.
      • Del Mar Viloria M.
      • Andrade R.J.
      • et al.
      Insulin resistance impairs sustained response rate to peginterferon plus ribavirin in chronic hepatitis C patients.
      ,
      • Conjeevaram H.S.
      • Kleiner D.E.
      • Everhart J.E.
      • et al.
      Race, insulin resistance and hepatic steatosis in chronic hepatitis C.
      ,
      • Romero-Gómez M.
      • Fernández-Rodríguez C.M.
      • Andrade R.J.
      • et al.
      Effect of sustained virological response to treatment on the incidence of abnormal glucose values in chronic hepatitis C.
      ,
      • Elgouhari H.M.
      • Zein C.O.
      • Hanouneh I.
      • Feldstein A.E.
      • Zein N.N.
      Diabetes mellitus is associated with impaired response to antiviral therapy in chronic hepatitis C infection.
      ,
      • Dai C.Y.
      • Huang J.F.
      • Hsieh M.Y.
      • et al.
      Insulin resistance predicts response to peginterferon-alpha/ribavirin combination therapy in chronic hepatitis C patients.
      ,
      • Grasso A.
      • Malfatti F.
      • De Leo P.
      • et al.
      Insulin resistance predicts rapid virological response in non-diabetic, non-cirrhotic genotype 1 HCV patients treated with peginterferon alpha-2b plus ribavirin.
      ]. Further, viral eradication is followed by an improvement in insulin resistance [
      • Delgado-Borrego A.
      • Jordan S.H.
      • Negre B.
      • et al.
      Reduction of insulin resistance with effective clearance of hepatitis C infection: results from the HALT-C trial.
      ], and a decreased risk for future type 2 diabetes mellitus [
      • Arase Y.
      • Suzuki F.
      • Suzuki Y.
      • et al.
      Sustained virological response reduces incidence of onset of type 2 diabetes in chronic hepatitis C.
      ]. Although, most evidence comes from trials assessing G1 patients, obesity (BMI ≥30 kg/m2) and insulin resistance have also been shown to impair the rate of SVR in G2/G3 infection [
      • Mangia A.
      • Minerva N.
      • Bacca D.
      • Cozzolongo R.
      • Agostinacchio E.
      • Sogari F.
      • et al.
      Determinants of relapse after a short (12 weeks) course of antiviral therapy and re-treatment efficacy of a prolonged course in patients with chronic hepatitis C virus genotype 2 or 3 infection.
      ,
      • Poustchi H.
      • Negro F.
      • Hui J.
      • et al.
      Insulin resistance and response to therapy in patients infected with chronic hepatitis C virus genotypes 2 and 3.
      ].
      Liver steatosis is now recognized as an additional feature of the metabolic syndrome. It is commonly found in patients with CHC [
      • Younossi Z.M.
      • McCullough A.J.
      Metabolic syndrome, non-alcoholic fatty liver disease and hepatitis C virus: impact on disease progression and treatment response.
      ], but seems to be more prevalent in patients with G3 infection [
      • Hayman A.V.
      • Sofair A.N.
      • Manos M.M.
      • et al.
      Prevalence and predictors of hepatic steatosis in adults with newly diagnosed chronic liver disease due to hepatitis C.
      ]. Importantly, the presence of steatosis, independent of other metabolic risk factors, has also been associated with treatment non-response [
      • Grasso A.
      • Malfatti F.
      • De Leo P.
      • et al.
      Insulin resistance predicts rapid virological response in non-diabetic, non-cirrhotic genotype 1 HCV patients treated with peginterferon alpha-2b plus ribavirin.
      ,
      • Poynard T.
      • Ratziu V.
      • McHutchison J.
      • et al.
      Effect of treatment with peginterferon or interferon alfa-2b and ribavirin on steatosis in patients infected with hepatitis C.
      ,
      • Cammà C.
      • Bruno S.
      • Di Marco V.
      • et al.
      Insulin resistance is associated with steatosis in nondiabetic patients with genotype 1 chronic hepatitis C.
      ,
      • McHutchison J.G.
      • Lawitz E.J.
      • Shiffman M.L.
      • et al.
      Peginterferon alfa-2b or alfa-2a with ribavirin for treatment of hepatitis C infection.
      ]. In the ACCELERATE trial, liver steatosis was associated with a decreased rate of SVR (60% vs 75%, in the 24-week arm), and was also reported to negatively impact treatment outcome in the NORDynamIC study [
      • Shiffman M.L.
      • Suter F.
      • Bacon B.R.
      • et al.
      Peginterferon alfa-2a and ribavirin for 16 or 24 weeks in HCV genotype 2 or 3.
      ,
      • Lagging M.
      • Langeland N.
      • Pedersen C.
      • et al.
      Randomized comparison of 12 or 24 weeks of peginterferon alpha-2a and ribavirin in chronic hepatitis C virus genotype 2/3 infection.
      ]. In an open-label study including G2/G3 CHC, Zeuzem et al. found that steatosis affected the rate of SVR mostly in G3 patients [
      • Zeuzem S.
      • Hultcrantz R.
      • Bourliere M.
      • et al.
      Peginterferon alfa-2b plus ribavirin for treatment of chronic hepatitis C in previously untreated patients infected with HCV genotypes 2 or 3.
      ]. It has also been associated with accelerated fibrosis in G3 patients [
      • Westin J.
      • Nordlinder H.
      • Lagging M.
      • Norkrans G.
      • Wejstål R.
      Steatosis accelerates fibrosis development over time in hepatitis C virus genotype 3 infected patients.
      ]. Notably, steatosis in G3 infection disappears entirely after viral eradication, suggesting that it is induced directly by the virus [
      • Poynard T.
      • Ratziu V.
      • McHutchison J.
      • et al.
      Effect of treatment with peginterferon or interferon alfa-2b and ribavirin on steatosis in patients infected with hepatitis C.
      ].
      Traditionally, steatosis in G3 has been attributed directly to the virus itself, whereas in non-G3 it is thought to be caused by host metabolic factors [
      • Adinolfi L.E.
      • Durante-Mangoni E.
      • Zampino R.
      • Ruggiero G.
      Review article: hepatitis C virus-associated steatosis–pathogenic mechanisms and clinical implications.
      ]. Such a strict dichotomy may be too simplistic. Unlike type 2 diabetes mellitus and the metabolic syndrome, in HCV infection the association between liver steatosis and insulin resistance is not straightforward for any genotype. Experimental and clinical research have shown that independent of viral genotype, the HCV lifecycle interacts directly with the glucose and lipid machinery of the cell (i.e. insulin receptor substrate, apolipoproteins, sterol regulatory element-binding protein or SREBP1c, microsomal triglyceride transfer protein or MTP, peroxisome proliferator-activated receptors) [
      • McHutchison J.G.
      • Lawitz E.J.
      • Shiffman M.L.
      • et al.
      Peginterferon alfa-2b or alfa-2a with ribavirin for treatment of hepatitis C infection.
      ,
      • Westin J.
      • Nordlinder H.
      • Lagging M.
      • Norkrans G.
      • Wejstål R.
      Steatosis accelerates fibrosis development over time in hepatitis C virus genotype 3 infected patients.
      ,
      • Adinolfi L.E.
      • Durante-Mangoni E.
      • Zampino R.
      • Ruggiero G.
      Review article: hepatitis C virus-associated steatosis–pathogenic mechanisms and clinical implications.
      ,
      • Kawaguchi T.
      • Ide T.
      • Taniguchi E.
      • et al.
      Clearance of HCV improves insulin resistance, beta-cell function, and hepatic expression of insulin receptor substrate 1 and 2.
      ,
      • Perrault M.
      • Pécheur E.I.
      The hepatitis C virus and its hepatic environment: a toxic but finely tuned partnership.
      ,
      • Benga W.J.
      • Krieger S.E.
      • Dimitrova M.
      • et al.
      Apolipoprotein E interacts with hepatitis C virus nonstructural protein 5A and determines assembly of infectious particles.
      ,
      • Lerat H.
      • Kammoun H.L.
      • Hainault I.
      • et al.
      Hepatitis C virus proteins induce lipogenesis and defective triglyceride secretion in transgenic mice.
      ,
      • Dharancy S.
      • Malapel M.
      • Perlemuter G.
      • et al.
      Impaired expression of the peroxisome proliferator-activated receptor alpha during hepatitis C virus infection.
      ,
      • Pazienza V.
      • Clément S.
      • Pugnale P.
      • et al.
      The hepatitis C virus core protein of genotypes 3a and 1b downregulates insulin receptor substrate 1 through genotype-specific mechanisms.
      ] that may ultimately cause either insulin resistance or liver steatosis alone (i.e. independently of insulin resistance). Studies with the euglycemic-hyperinsulinemic clamp have shown both hepatic and particularly systemic insulin resistance in patients with CHC (G1 and G3) without any metabolic risk factors and irrespective of the presence of hepatic steatosis [
      • Vanni E.
      • Abate M.L.
      • Gentilcore E.
      • et al.
      Sites and mechanisms of insulin resistance in nonobese, nondiabetic patients with chronic hepatitis C.
      ,
      • Milner K.L.
      • van der Poorten D.
      • Trenell M.
      • et al.
      Chronic hepatitis C is associated with peripheral rather than hepatic insulin resistance.
      ]. Thus, it is possible that steatosis in HCV infection is more related to a viral-induced defect in the synthesis and intracellular transport of triglycerides (induction of SREBP1c and decrease in MTP, respectively) [
      • Lerat H.
      • Kammoun H.L.
      • Hainault I.
      • et al.
      Hepatitis C virus proteins induce lipogenesis and defective triglyceride secretion in transgenic mice.
      ], than to insulin resistance. The mechanisms for these effects may even differ between genotypes [
      • Pazienza V.
      • Clément S.
      • Pugnale P.
      • et al.
      The hepatitis C virus core protein of genotypes 3a and 1b downregulates insulin receptor substrate 1 through genotype-specific mechanisms.
      ], however, because of the myriad of complex host–viral metabolic interactions, this may be difficult to tease apart.
      The steatogenic nature of G3 may partially account for the poorer responses observed with G3 than G2, although in patients with risk factors for the metabolic syndrome, insulin resistance may also play a major role. As both conditions finally affect the response to pegIFN and RBV, they may be considered before initiating therapy. Whether insulin sensitizers may be useful to improve response in some G3 patients remains an open question. Clarifying how metabolic abnormalities impair the interferon response and why G3 is steatogenic will hopefully shed some light on the differences in response between the ‘favorable’ genotypes.

      Conclusions

      The grouping of G2 and G3 under the label of ‘easy to treat’ genotypes was an unfortunate consequence of their under-representation in the large registration clinical trials that led to the current standard of care. A careful examination of data from many trials shows fairly clearly that patients infected with G3 do not respond as well as those with G2 infection to pegIFN-based therapy. Patients with G3 infection sometimes behave more like those with G1 infection, whereas G2 is almost universally a truly ‘easy to treat’ genotype. What underlies this difference is less clear. The different genotypes may be intrinsically more or less sensitive to the effects of interferon, or alternatively, and seemingly more likely, the different genotypes may differentially affect the ability of the host liver cells to respond to exogenous interferon, an effect that may be modulated by the host genotype (IL28B SNP). Why certain genotypes are more likely to induce ISG pre-activation and/or hepatic steatogenesis, both of which impair interferon responses, is not well understood but may explain the differences in treatment outcome across genotypes.
      Fortunately, early viral responses, and in particular the achievement of an RVR, are very helpful in predicting the ultimate outcome of therapy. For patients who achieve an RVR, the consideration of shortened therapy (16 weeks) may be reasonable, even though SVR rates are likely still slightly higher with 24 weeks of therapy. However, the presence of poor prognostic factors, such as advanced fibrosis, obesity, increased age, and probably insulin resistance and liver steatosis in the specific case of G3, may discourage a shortened course of therapy. Whether extending therapy (48 weeks) in patients who do not achieve an RVR would be beneficial, particularly in those with G3 and/or poor prognostic factors will have to be formally assessed in clinical trials, but seems like an attractive option. New data suggest a prognostic role for IL28B polymorphisms mostly in G3 patients not achieving an RVR, and these could also be considered for improved tailoring of therapy (Fig. 5). Although the initial trials found low-dose RBV (800 mg) to be adequate in G2/G3, most subsequent data suggest that weight-based RBV is more appropriate. Weight-based dosing is particularly important if a shortened course of therapy is to be considered. Although we are on the cusp of a new era in HCV therapy, the first generation of direct acting antivirals have limited or no activity against G3 infection. As such, pegIFN and RBV will remain the standard of care for G3 infection for the foreseeable future. As a result, researchers will need to focus on better understanding the mechanisms impairing interferon-responses in G3 infection and clinicians will have to tailor current therapy to optimize treatment outcomes.
      Figure thumbnail gr5
      Fig. 5Algorithm for addressing length of therapy in genotype 2 (G2) and genotype 3 (G3) infection, according to rapid virological response (RVR). Broken arrows show proposed strategies drawn from available evidence, but for which randomized clinical trials are lacking or currently ongoing. Whether the presence of IR/steatosis should be considered within these negative parameters remains in question. In patients without an RVR it is likely that extending therapy to 48 weeks may increase the rate of sustained virological response, and trials are ongoing. This may be especially true for patients with unfavorable IL28B genotypes (CT and especially TT variants). BMI, body mass index; F, fibrosis; VL, viral load; IR, insulin resistance.

      Conflict of interest

      The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

      Acknowledgment

      Andrés Duarte-Rojo receives funding for a Clinical Fellowship from the Instituto de Ciencia y Tecnología del Distrito Federal, ICyTDF (México), and The American College of Gastroenterology, ACG: International Training Grant 2009 (USA).

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