Special Issue: Proceedings of the 4th Paris Hepatitis Conference. The publication of this supplement was supported by an unrestricted educational grant from F. Hoffmann-Laroche Ltd.
Volume 31, Issue Supplement s1, pages 3–12, January 2011
Philipp de Leuw, Christoph Sarrazin, Stefan Zeuzem
Article first published online: 4 JAN 2011
© 2011 John Wiley & Sons A/S
Medizinische Klinik I, Klinikum der Johann-Wolfgang Goethe-Universität, Frankfurt am Main, Germany
* Correspondence: Correspondence Prof. Dr. med. Stefan Zeuzem, Medizinische Klinik 1, Klinikum der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany Tel:+49 69 6301 4544 Fax:+49 69 6301 87676 e-mail: email@example.com
hepatitis C virus; interferon-α; molecular biology; real-time PCR; ribavirin; serological tests
Approximately 180 million individuals are chronically infected with hepatitis C, which is strongly associated with the development of cirrhosis, end-stage liver disease and hepatocellular carcinoma. Several virological tools (anti-HCV antibody assays, measurement of HCV-RNA, HCV-genotyping) are useful in management of hepatitis C infected patients. The primary goal of antiviral therapy in chronic hepatitis C is a sustained virological response (SVR). The HCV genotype should be determined in every patient considered for antiviral therapy because the currently recommended treatment duration and ribavirin doses differ among HCV genotypes. Exact subtyping might gain increased importance for future therapies with direct-acting antiviral agents (DAA) because of differences of antiviral activities and barriers to resistance among HCV subtypes. Monitoring HCV RNA by a highly sensitive assay (LOD≤15 IU/ml) is the basis for management of response guided therapy of chronic hepatitis C with pegylated IFN plus ribavirin. Rules for early discontinuation of antiviral therapy in non-responders and determination of optimal treatment durations in virologic responders have been developed for application of individualized treatment strategies.
b.w., body weight; CAM, COBAS® Amplicor HCV Monitor 2.0; CAP/CTM, COBAS® Ampliprep/COBAS®-TaqMan®; CIA, chemiluminescence immunoassay; EIA, enzyme immunoassay; HCV, hepatitis C virus; LOD, limit of detection; NA, not applicable; NPV, negative predictive value; PPV, positive predictive value; RT-PCR, reverse transcription-polymerase chain reaction; RVR, rapid virological response; SVR, sustained virological response; VD, viral decline; VL, viral load.
Approximately 180 million individuals (i.e. 2.5% of the world population) are estimated to have chronic hepatitis C virus (HCV) infection, with the highest prevalence reported in Egypt and the lowest in Finland (1, 2). HCV belongs to the Flaviviridae family and is a small 55 nm virus with a lipid envelope and a single-stranded RNA viral genome with approximately 9600 nucleotides (3, 4). The positive-strand RNA genome includes a 5′-non-coding region with an internal ribosome entry site, an open reading frame that encodes structural (core, envelope 1, 2, p7) and non-structural (NS2, NS3, NS4A, NS4B, NS5A, NS5B) proteins and a 3′-non-coding region. The internal ribosome entry site causes the translation of a polyprotein precursor, which is processed into mature structural and non-structural proteins (5). HCV is mainly transmitted via various parenteral routes such as blood and blood product transfusions, (intravenous) drug abuse, contaminated medical equipment, tattoos, as well as sexually or perinatally. Acute HCV infection usually occurs 2–12 weeks after exposure. Patients with symptomatic acute hepatitis C develop chronic infection in 48–75% of the cases while asymptomatic courses result in chronic infection in most cases (85–90%) (6–8). Chronic hepatitis C is strongly associated with the development of cirrhosis, end-stage liver disease and hepatocellular carcinoma (HCC). Antiviral therapy can prevent these complications (9, 10). The current standard of care includes the administration of PEG-IFN-α/ribavirin combination therapy. In HCV genotype 2- or 3-infected patients, sustained virological response (SVR) rates of ∼80% (11) are achieved while in genotype 1-infected patients, viral eradication is achieved in only 40–50% (12–14).
Anti-hepatitis C virus antibody detection
The most common screening test for HCV is an immunoassay [enzyme immunoassay (EIA), microparticle EIA, chemiluminescence immunoassay (CIA)] that detects anti-HCV antibodies. These assays have many advantages for diagnosis: they are easy to use, automation is simple, have a low variability and are inexpensive.
Nowadays, the detection of antibodies directed against various HCV epitopes in plasma or serum is based on the use of third-generation EIAs. These assays detect antibodies to recombinant antigens from core (c22) and non-structural proteins 3 (c33), 4 (c100, c200) and 5. The specificity and sensitivity of third-generation immunoassays in patients with chronic liver disease were found to be >98 and >97% respectively (15–17). The mean time to seroconversion is shortened by 2–3 weeks with third-generation compared with second-generation assays with the detection of HCV-specific antibodies approximately 4–6 weeks after infection (18). Anti-HCV-IgM detection cannot discriminate between acute and chronic hepatitis C because some chronically infected patients produce anti-HCV IgM intermittently and not all patients respond to acute HCV infection by producing anti-HCV IgM. Anti-HCV antibodies may become undetectable via commercial assays in some patients many years after successful treatment.
Detection and quantification of hepatitis C virus RNA
There are a number of different commercial assays approved by the FDA and EMEA for the determination of HCV RNA (19–24).
Cobas® Amplicor HCV version 2.0 (Roche Molecular Diagnostics, Pleasonton, CA, USA) based on a standard reverse transcription-polymerase chain reaction (RT-PCR) is available for the qualitative measurement of HCV RNA. The lowest detection limit is 50 IU/ml whatever the HCV genotype (19).
The versant HCV qualitative assay (Siemens Healthcare Diagnostics, Deerfield, IL, USA) based on transcription-mediated amplification is also a highly sensitive test for a qualitative HCV RNA measurement with the lower detection limit of 5–10 IU/ml whatever the HCV genotype (25).
The versant HCV quantitative Test (Siemens Healthcare Diagnostics) is a quantitative HCV RNA assay based on signal amplification by branched DNA (bDNA). The bDNA assay version 3.0 is standardized for IU, and the assay has been reported to be linear over its entire dynamic range from the lower detection limit of 615 IU/ml to 8 million IU/ml whatever the HCV genotype (22).
The COBAS® Amplicor HCV Monitor 2.0 (CAM, Roche Molecular Diagnostics) is a standard RT-PCR-based assay with a linear detection range of 500–500 000 IU/ml, whatever the HCV genotype. For higher HCV RNA concentrations, predilution of the original sample is required (21).
Currently, two real-time PCR assays are available: the COBAS® Ampliprep/Cobas® TaqMan® assay (CAP/CTM, Roche Molecular Diagnostics) and the real-time HCV assay (also named AccuGene® HCV, Abbott Molecular Inc., Des Plaines, IL, USA). These assays have the advantage of having a broad dynamic range of amplification, thus improving the limits of detection (LOD) to ≤10 IU/ml, and linear quantification up to 107–108 IU/ml (26, 27). Four different modes of results are possible with the Abbott HCV assay: (a) undetectable (below the LOD of the assay), (b) possible detection but below 12 IU/ml, (c) positive above 12 IU/ml with an exact HCV RNA concentration or (d) positive above 8.0 log10 IU/ml (which represents the upper limit of quantification).
The results of the Roche CAP/CTM are reported in four stages: (a) undetectable (below the detection limit), (b) positive but unquantifiable (<15 IU/ml), (c) detectable and quantifiable (>15 IU/ml), reported as an exact HCV RNA concentration in IU/ml above 30–40 IU/ml, or (d) detectable, quantifiable but above the upper limit (>6.9 × 107 IU/ml) (28, 29).
It has also been shown that the results of assays can vary significantly with different HCV genotypes despite IU standardization. Generally, the HCV RNA levels in genotype 1 samples measured with the Roche CAP/CTM are higher than those obtained by the Abbott real-time HCV assay and the Siemens bDNA assay (approximately 0.5 log10 IU/ml), while the HCV RNA concentrations in samples with genotype 4 are lower (26, 29–31).
The HCV genotype can be determined by direct sequencing from subgenomic regions such as core/E1 or NS5B. The most conserved regions of the HCV genome are the 5′-untranslated region and a nearly invariant 98 nucleotide RNA element (X-tail) within the 3′-untranslated region (32, 33). The most variable region of the HCV genome is the hypervariable region of E2 (34, 35). For everyday use, there are few commercial kits that use direct sequence analysis of the 5′-untranslated region (Trugene® 5′NC HCV Genotyping Kit, Siemens Healthcare Diagnostics) or reverse hybridization with genotype-specific probes complementary to the 5′-untranslated and the core region (INNO-LiPA HCV II, Siemens Healthcare Diagnostics) (36–39).
The determination of HCV subtypes has no clinical relevance for the current standard of care with pegylated IFN/ribavirin therapy, while different treatment durations based on viral kinetics are recommended based on different HCV genotypes.
Novel, direct-acting antiviral agents (DAA), also called ‘specifically targeted antiviral therapy for hepatitis C’, are currently under clinical development and are substantially improving the SVR rates in genotype 1 patients (40). Subtype determination may become important in future clinical practice mainly because of resistance profiles for these DAA agents according to HCV genotype and subtype. The correct identification of HCV subtypes 1a and 1b has been reported in >96% of cases for second-generation INNO-LiPA assays (41).
Clinical application of virological tools
Acute hepatitis C
The diagnosis of acute HCV infection can be difficult. Although the most reliable approach is proof of seroconversion to HCV antibodies in a previously seronegative individual (42), this is rarely possible in clinical practice. The absence of detectable HCV antibodies in the acute phase does not exclude acute hepatitis C, because the appearance of antibodies can be delayed in as many as 30% of patients at the onset of symptoms (43). An impaired ability to develop antibodies is especially seen in immunocompromised patients. HCV RNA is typically detected in a seronegative patient, followed by the development of HCV antibodies several days or weeks later. When patients are positive for both anti-HCV antibodies and HCV RNA at the initial presentation, it can be difficult to discriminate between acute and acute exacerbated chronic hepatitis C. In these cases, it may be helpful to monitor liver enzymes and to evaluate the risks of HCV transmission. The value of IgM antibodies in the diagnosis of acute infection is considered to be low, because they are detected in both acute and chronic infection (44). Although measurable HCV RNA serum concentrations emerge within the first days after infection, HCV RNA can fluctuate during acute hepatitis C. Therefore, HCV RNA tests must be performed again several weeks later in all negatively tested patients suspected of acute hepatitis C.
Chronic hepatitis C
The primary goal of antiviral therapy in chronic hepatitis C is a SVR, defined as undetectable serum HCV RNA by a sensitive molecular assay 24 weeks after the end of therapy. Recently, it has been suggested that 12 weeks post-treatment follow-up is as relevant as 24 weeks to determine the SVR in patients with HCV receiving PEG-IFN-α and ribavirin (45).
With the current standard therapy of 24–48 weeks of treatment with PEG-IFN-α and ribavirin, the SVR rates are still unsatisfactory and only reach about 40–50% (12–14). In recent years, treatment regimens have been individualized in an attempt to improve the treatment response with the identification of several viral- and host-related factors that affect response to antiviral therapy. Monitoring HCV RNA was found to be a key parameter in the management of response-guided therapy of chronic hepatitis C with PEG-IFN plus ribavirin.
The treatment recommendations of the current German/Austrian/Swiss guidelines are described below (46).
If HCV RNA has decreased by <2 log10 by week 12 compared with the concentrations before the initiation of therapy, the probability of an SVR is minimal (0–3%) and discontinuation of therapy is recommended (47–49). An absolute HCV RNA concentration of >30 000 IU/ml at week 12 has been suggested (50, 51) as an alternative to the <2 log decline stopping rule (Fig. 1).
Figure 1. Treatment algorithm for genotype 1-infected patients (46). 1. Sensitive HCV RNA assays (limit of detection 12–15 or 50 IU/ml) at weeks 4, 12 and 24 may determine the treatment duration. 2. Limit for baseline viral load: pegylated interferon-α2b 600 000 IU/ml; pegylated interferon-α2a 800 000 IU/ml. Shortening the treatment is not recommended in patients with advanced fibrosis/cirrhosis, insulin resistance, metabolic syndrome or hepatic steatosis. There are no data for patients with normal transaminases at baseline. cEVR, complete early virological response; HCV, hepatitis C virus; RVR, rapid virological response.
Treatment can also be discontinued in patients with detectable HCV RNA (≥50 IU/ml) after 24 weeks of therapy. Once again, the chances of an SVR in these patients are minimal (1–3%) (12, 48, 52).
Because of the higher sensitivity of the current most extensively used real-time PCR-based HCV RNA assays (LOD≤10 IU/ml) and with the extension of therapy to up to 72 weeks, the negative predictive value (NPV) of the 2 log rule and the stopping rule at week 24 based on HCV RNA detectability must be re-evaluated. In the INDIV-2 study, for example, patients with initial HCV RNA negativity by a highly sensitive assay at week 30 were treated for 72 weeks and achieved SVR rates of 50% (53).
Low-dose monotherapy with PEG-IFN-α in patients who fail to respond to a full course of antiviral therapy cannot be recommended. Three independent studies did not show a significant improvement in histological and/or clinical courses in these patients. Whether certain sub-groups (i.e. patients with portal hypertension) can benefit from low-dose PEG-IFN monotherapy remains to be determined (54–56).
Treatment duration for genotype 1(4)-infected patients
Twenty-four weeks of treatment is recommended for genotype 1 patients with a low baseline viral load (VL) (<600 000–800 000 IU/ml) who achieve a rapid virological response (RVR; HCV RNA<50 IU/ml at week 4 of treatment). There was no significant difference in the SVR rates when patients with RVR and low VL were treated for 24 weeks or 48 weeks (57–59) (Fig. 1). There are few data on a reduced treatment duration in patients with advanced fibrosis, steatosis and insulin resistance, and thus shortened treatment should not be considered for these groups (60–62).
Various studies have investigated the benefit of extending treatment in genotype 1-infected slow responders [patients with detectable (≥50 IU/ml) HCV RNA levels at week 12, but undetectable HCV RNA at week 24] (50, 52, 63–66) (Table 1). Analysis of three different European studies showed significantly higher SVR and lower relapse rates in the 72-week compared with the 48-week treatment group (52) (Fig. 1). A trend towards lower relapse rates in slow responders treated for 72 weeks compared with 48 weeks was reported in a recent prospective study, which investigated extending treatment (SUCCESS). However, because of a uniform trend in all studies and the significant improvement found in the meta-analysis of three European studies, 72 weeks of treatment can still be considered in slow responders who tolerate therapy (66).
Patients who attain a complete early virological response (cEVR; undetectable HCV RNA at week 12 using an assay with a lower limit of quantification cut-off of 50 IU/ml) should be treated for 48 weeks (50, 52, 64, 65) (Fig. 1). Currently, baseline VL is only used to shorten the treatment duration in rapid responders. However, the determination of low and high baseline VL may also be useful in patients with cEVR. Higher SVR rates were reported in patients with high baseline viraemia and cEVR if therapy was prolonged to 60–72 weeks (53, 64).
Treatment duration for genotype 2/3-infected patients
Various studies have investigated the reduction of treatment to 12–16 weeks. Overall, reducing the treatment to <24 weeks increases the number of relapsers. However, many HCV genotype 2/3 patients may be treated for 12–16 weeks if certain preconditions are fulfilled, especially an RVR because only patients with RVR at week 4 had high SVR rates after 16, 14 or even 12 weeks of treatment (67–72) (Table 2). Nevertheless, in the ACCELERATE study, patients who received PEG-IFN-α2a (180 μg/week) plus a fixed ribavirin dose of 800 mg per day achieved significantly lower SVR rates with a shorter treatment (79 vs 85%) (67).
In addition to RVR, the specific HCV genotype and the baseline VL are associated with a virological response. HCV genotype 2 patients respond better to pegylated IFN and ribavirin than those infected with genotype 3 (61). Furthermore, patients with a low baseline VL (<400 000 IU/ml) had significantly higher SVR rates than those with high HCV RNA concentrations at baseline (>400 000–800 000 IU/ml) (67, 69). Genotype 2/3-infected patients with a baseline VL≤400 000–800 000 IU/ml and RVR can be considered for a shorter treatment. Ribavirin dosing appears to be an important factor in defining the treatment outcome: shorter treatment durations have mainly been effective in studies with a weight-based ribavirin regimen, whereas studies with a fixed ribavirin dose of 800 mg per day have generally resulted in a significant decrease in SVR with a shorter treatment. However, reducing the duration of treatment is not recommended in patients with advanced liver fibrosis or those with low alanine transaminase values at baseline (67, 70, 74). Genotype 2/3-infected patients who do not achieve RVR showed low SVR rates (45–55%)(67, 72, 75). Whether patients without an RVR should be treated for longer than 24 weeks is based on retrospective studies. These data show that genotype 2/3-infected patients without an RVR who receive 48 weeks of PEG-IFN-α2a plus ribavirin 1000/1200 mg/day have higher SVR rates than those receiving 24 weeks of PEG-IFN-α2a plus ribavirin 800 mg/day (76 vs 67%; relapse: 4 vs 26%) (76). Prospective studies have begun to investigate extending the treatment to 36 or 48 weeks in non-RVR patients (Fig. 2).
Figure 2. Treatment algorithm for genotype 2/3-infected patients (46). 1. Sensitive HCV RNA assays (limit of detection 12–15 or 50 IU/ml) at weeks 4 and 12 may determine the treatment duration. 2. Shorter treatment regimens are not approved. Shortening the treatment is not recommended in patients with advanced fibrosis/cirrhosis. Negative predictive factors such as hepatic steatosis and low alanine transaminase values at baseline should be considered. There are no data for patients with normal transaminases at baseline. 3. Treatment can be discontinued in all patients with detectable HCV RNA (≥12–15 IU/ml) after 24 weeks of therapy. 4. The treatment duration (36, 48 or 72 weeks) for slow responders is not established. Prospective studies have been initiated to investigate treatment extension. HCV, hepatitis C virus.
Does the use of highly sensitive assays affect recent recommendations for a response-guided therapy?
Recommendations for the duration of treatment and early discontinuation were established using HCV RNA assays with a detection limit of ≤50 IU/ml. Numerous studies have shown that patients with a low baseline VL (<400 000–800 000 IU/ml) and a RVR, defined by undetectable HCV RNA at week 4, are appropriate candidates for shorter treatment regimens (12–16 and 24 weeks in genotype 2/3- and 1-infected patients respectively).
As mentioned above, the currently used real-time PCR-based CAP–CTM test has a detection limit of ≤15 IU/ml. Sarrazin and colleagues re-analysed frozen serum samples with CAP–CTM from patients with chronic hepatitis C enrolled in two large, randomized studies. The RVR rates were highly concordant for the CAM, with a LOD of 50 IU/ml, and the CAP–CTM. Although a significantly smaller number of samples had undetectable HCV RNA with the CAP–CTM, there was no difference in the SVR rates after shorter therapy in patients with an RVR<50 IU/ml, an RVR<15 IU/ml and undetectable RVR (82, 83 and 83% for 24 weeks for genotype 1 and 95, 95 and 94% for 16 weeks genotype 2/3) (77). Treatment regimens can therefore be shortened to 16/24 weeks on the basis of an RVR with HCV RNA concentrations <15 IU/ml by the CAP–CTM.
Genotype 1-infected patients with residual viraemia at week 12 (<15 IU/ml, but detectable by CAP–CTM) have also been shown to have a high relapse rate (55%). This group may benefit from prolonged treatment (72 weeks). Furthermore, low viraemia (between 15 and 50 IU/ml) at week 12 in genotype 1-infected patients was associated with even higher relapse rates (75%) (77).
Is the time point a relevant predictor of sustained virological response?
Up-to-date virological response profiles at weeks 4 and 12 provide a robust framework for predicting SVR in patients with genotype 1 infection. Neumann and colleagues investigated the positive predictive value (PPV) and NPV of an EVR at weeks 2 and 4 during treatment with IFN-α2b or pegylated IFN-α2a in treatment-naïve patients infected with genotype 1. All patients with undetectable HCV RNA at week 2 achieved an SVR (PPV: 100%). Patients with a rapid initial virological response at week 2, defined as an HCV RNA decline >2 log10IU/ml, had a high PPV for SVR of 88–97%. A VL>6 log10 IU/ml at week 2 has a high NPV (82–100%) for achieving an SVR. The combination of VL and viral decline (VD) at week 4 had the best NPV. A VL>5.5 log10 IU/ml and VD<2 log10 IU/ml had a 100% NPV in all treatment arms (4–13% of all patients; specificity: 12–29%) (78).
Despite an improvement in anti-HCV therapy in the last few years, the treatment of chronic hepatitis C is still challenging and must be improved. Many viral-related factors have been evaluated in association with the virological response to PEG-IFN-α and ribavirin-based therapy. It is currently agreed that the algorithms for treatment duration and early discontinuation can be applied based on highly sensitive HCV RNA assays (LOD≤15 IU/ml) (77, 79).
One of the most important patient predictors of a reduced SVR rate is advanced fibrosis and cirrhosis. Other patient-related factors negatively influencing the treatment outcome are ethnicity, male gender, older age, higher body weight, liver steatosis, elevated pretreatment serum γ-glutamyltransferase levels and the recently identified polymorphism upstream from IL-28B (rs12979860) (80–82).
Several independent genome-wide association studies have reported the presence of single nucleotide polymorphisms in the IL28B region to be associated with response to treatment (83, 84). The exact mechanisms underlying this association between IL28B polymorphism and response to treatment are unknown (85).
The gene IL-28B on chromosome 19 codes for IFN-λ-3. The protein product is one of the three members of the recently described type 3 IFN family (86). In Caucasians infected with genotype 1, the CC IL-28B type was associated with improved early viral kinetics and a greater likelihood of RVR (28 vs 5 vs 5% for CC, CT, TT respectively) and cEVR (87 vs 38 vs 28%) and SVR (69 vs 33 vs 37%) (82). Recently, it was shown that the IL28B polymorphism (rs12979860) also determines the treatment response in genotype 2- or 3-infected patients who do not achieve RVR (87).
In the future, DAA such as the protease inhibitors telaprevir and boceprevir in addition to PEG-IFN-α and ribavirin will improve the SVR rates in treatment-naïve genotype 1 patients as well as in genotype 1-infected non-responders and relapsers to standard therapy. Clinical trials with the NS3/4A protease inhibitor telaprevir show that the treatment duration can be shortened to 24 weeks in more than 60% of patients (40). The results from on-treatment HCV RNA measurements must be analysed and response-guided algorithms must be established based on the correlation of baseline VL with RVR and SVR. Telaprevir will be approved for a 24-week course in patients with undetectable HCV RNA at weeks 4 and 12 (extended RVR). For all other patients, 48 weeks of treatment is recommended. Treatment algorithms for boceprevir will be different. After a 4-week lead-in phase of PEG-IFN-α plus ribavirin, boceprevir will be added for either 24 or 48 weeks. Individuals achieving an RVR at week 8 will be treated for a total of 28 weeks, while patients showing a clearance of the virus between weeks 8 and 12 of treatment will continue for a total of 48 weeks.
Conflicts of interest
C. Sarrazin has served as a clinical investigator, consultant and/or member of speakers' bureau for Abbott, Roche and Siemens. S. Zeuzem has served as a clinical investigator, consultant and/or member of speakers' bureau for Abbott, Achillion, Anadys, BMS, Gilead, Merck, Novartis, Pfizer, Roche, Tibotec and Vertex. P. de Leuw has no conflicts to declare.
1 Lavanchy D. The global burden of hepatitis C. Liver Int 2009; 29 (Suppl. 1): 74–81.
2 Esteban JI, Sauleda S, Quer J. The changing epidemiology of hepatitis C virus infection in Europe. J Hepatol 2008; 48: 148–62.
3 Houghton M, Weiner A, Han J, et al. Molecular biology of the hepatitis C viruses: implications for diagnosis, development and control of viral disease. Hepatology 1991; 14: 381–8.
4 Wakita T, Pietschmann T, Kato T, et al. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 2005; 11: 791–6.
5 Moradpour D, Penin F, Rice CM. Replication of hepatitis C virus. Nat Rev Microbiol 2007; 5: 453–63.
6 Villano SA, Vlahov D, Nelson KE, et al. Persistence of viremia and the importance of long-term follow-up after acute hepatitis C infection. Hepatology 1999; 29: 908–14.
7 Gerlach JT, Diepolder HM, Zachoval R, et al. Acute hepatitis C: high rate of both spontaneous and treatment-induced viral clearance. Gastroenterology 2003; 125: 80–8.
8 Orland JR, Wright TL, Cooper S. Acute hepatitis C. Hepatology 2001; 33: 321–7.
9 Veldt BJ, Heathcote EJ, Wedemeyer H, et al. Sustained virologic response and clinical outcomes in patients with chronic hepatitis C and advanced fibrosis. Ann Intern Med 2007; 147: 677–84.
10 Morgan TR, Ghany MG, Kim HY, et al. Outcome of sustained virological responders with histologically advanced chronic hepatitis C. Hepatology 2010; 52: 833–44.
11 Zeuzem S, Berg T, Moeller B, et al. Expert opinion on the treatment of patients with chronic hepatitis C. J Viral Hepat 2009; 16: 75–90.
12 Manns MP, McHutchison JG, Gordon SC, et al. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial. Lancet. 2001; 358: 958–65.
13 Fried MW, Shiffman ML, Reddy KR, et al. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med 2002; 347: 975–82.
14 McHutchison JG, Lawitz EJ, IDEAL Study Team et al. Peginterferon alfa-2b or alfa-2a with ribavirin for treatment of hepatitis C infection. N Engl J Med 2009; 361: 580–93.
15 Colin C, Lanoir D, HEPATITIS Group et al. Sensitivity and specificity of third-generation hepatitis C virus antibody detection assays: an analysis of the literature. J Viral Hepat 2001; 8: 87–95.
16 Sookoian S, Castaño G. Evaluation of a third generation anti-HCV assay in predicting viremia in patients with positive HCV antibodies. Ann Hepatol 2002; 1: 179–82.
17 Alborino F, Burighel A, Tiller FW, van Helden J, et al. Multicenter evaluation of a fully automated third-generation anti-HCV antibody screening test with excellent sensitivity and specificity. Med Microbiol Immunol 24 September 2010 [Epub ahead of print] DOI: DOI: 10.1007/s00430-010-0171-0.
18 Uyttendaele S, Claeys H, Mertens W, et al. Evaluation of third-generation screening and confirmatory assays for HCV antibodies. Vox Sang 1994; 66: 122–9.
19 Nolte FS, Fried MW, Shiffman ML, et al. Prospective multicenter clinical evaluation of AMPLICOR and COBAS AMPLICOR hepatitis C virus tests. J Clin Microbiol 2001; 39: 4005–12.
20 Lee SC, Antony A, Lee N, et al. Improved version 2.0 qualitative and quantitative AMPLICOR reverse transcription-PCR tests for hepatitis C virus RNA: calibration to international units, enhanced genotype reactivity, and performance characteristics. J Clin Microbiol 2000; 38: 4171–9.
21 Gerken G, Rothaar T, Rumi MG, et al. Performance of the COBAS AMPLICOR HCV MONITOR test, version 2.0, an automated reverse transcription-PCR quantitative system for hepatitis C virus load determination. J Clin Microbiol 2000; 38: 2210–4.
22 Ross RS, Viazov S, Sarr S, et al. Quantitation of hepatitis C virus RNA by third generation branched DNA-based signal amplification assay. J Virol Methods 2002; 101: 159–68.
23 Sarrazin C, Teuber G, Kokka R, et al. Detection of residual hepatitis C virus RNA by transcription-mediated amplification in patients with complete virologic response according to polymerase chain reaction-based assays. Hepatology 2000; 32: 818–23.
24 Leckie G, Schneider G, Abravaya K, et al. Performance attributes of the LCx HCV RNA quantitative assay. J Virol Methods 2004; 115: 207–15.
25 Hendricks DA, Friesenhahn M, Tanimoto L, et al. Multicenter evaluation of the VERSANT HCV RNA qualitative assay for detection of hepatitis C virus RNA. J Clin Microbiol 2003; 41: 651–6.
26 Vermehren J, Kau A, Gärtner BC, et al. Differences between two real-time PCR-based hepatitis C virus (HCV) assays (RealTime HCV and Cobas AmpliPrep/Cobas TaqMan) and one signal amplification assay (Versant HCV RNA 3.0) for RNA detection and quantification. J Clin Microbiol 2008; 46: 388–91.
27 Schutten M, Fries E, Burghoorn-Maas C, et al. Evaluation of the analytical performance of the new Abbott RealTime RT-PCRs for the quantitative detection of HCV and HIV-1 RNA. J Clin Virol 2007; 40: 99–104.
28 Sarrazin C, Dragan A, Gärtner BC, et al. Evaluation of an automated, highly sensitive, real-time PCR-based assay (COBAS Ampliprep/COBAS TaqMan) for quantification of HCV RNA. J Clin Virol 2008; 43: 162–8.
29 Sizmann D, Boeck C, Boelter J, et al. Fully automated quantification of hepatitis C virus (HCV) RNA in human plasma and human serum by the COBAS AmpliPrep/COBAS TaqMan system. J Clin Virol 2007; 38: 326–33.
30 Sarrazin C, Gärtner BC, Sizmann D, et al. Comparison of conventional PCR with real-time PCR and branched DNA-based assays for hepatitis C virus RNA quantification and clinical significance for genotypes 1 to 5. J Clin Microbiol 2006; 44: 729–37.
31 Chevaliez S, Bouvier-Alias M, Brillet R, et al. Overestimation and underestimation of hepatitis C virus RNA levels in a widely used real-time polymerase chain reaction-based method. Hepatology 2007; 46: 22–31.
32 Kolykhalov AA, Feinstone SM, Rice CM. Identification of a highly conserved sequence element at the 3′ terminus of hepatitis C virus genome RNA. J Virol 1996; 70: 3363–71.
33 Tanaka T, Kato N, Cho MJ, et al. Structure of the 3′ terminus of the hepatitis C virus genome. J Virol 1996; 70: 3307–12.
34 Simmonds P. Genetic diversity and evolution of hepatitis C virus – 15 years on. J Gen Virol 2004; 85: 3173–88.
35 Simmonds P, Bukh J, Combet C, et al. Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes. Hepatology 2005; 42: 962–73.
36 Germer JJ, Vandenameele JN, Mitchell PS, et al. Automated sample preparation for the TRUGENE HIV-1 genotyping kit using the MagNA pure LC instrument. Diagn Microbiol Infect Dis 2004; 49: 59–61.
37 Stuyver L, Wyseur A, van Arnhem W, et al. Second-generation line probe assay for hepatitis C virus genotyping. J Clin Microbiol 1996; 34: 2259–66.
38 Stuyver L, Wyseur A, van Arnhem W, et al. Hepatitis C virus genotyping by means of 5′-UR/core line probe assays and molecular analysis of untypeable samples. Virus Res 1995; 38: 137–57.
39 Zheng X, Pang M, Chan A, et al. Direct comparison of hepatitis C virus genotypes tested by INNO-LiPA HCV II and TRUGENE HCV genotyping methods. J Clin Virol 2003; 28: 214–6.
40 Lange CM, Sarrazin C, Zeuzem S. Review article: specifically targeted anti-viral therapy for hepatitis C − a new era in therapy. Aliment Pharmacol Ther 2010; 32: 14–28.
41 Chevaliez S, Bouvier-Alias M, Brillet R, et al. Hepatitis C virus (HCV) genotype 1 subtype identification in new HCV drug development and future clinical practice. PLoS One 2009; 4: e8209.
42 Maheshwari A, Ray S, Thuluvath PJ. Acute hepatitis C. Lancet 2008; 372: 321–32.
43 Farci P, Alter HJ, Wong D, et al. A long-term study of hepatitis C virus replication in non-A, non-B hepatitis. N Engl J Med 1991; 325: 98–104.
44 Quiroga JA, Campillo ML, Catillo I, et al. IgM antibody to hepatitis C virus in acute and chronic hepatitis C. Hepatology 1991; 14: 38–43.
45 Martinot-Peignoux M, Stern C, Ripault M-P, et al. Twelve weeks post-treatment follow-up is as relevant as 24 weeks to determine the SVR in patients with HCV receiving PEG-IFN and ribavirin. Hepatology 2010; 51: 1122–6.
46 Sarrazin C, Berg T, Ross RS, et al. Prophylaxis, diagnosis and therapy of hepatitis c virus (HCV) infection: the German guidelines on the management of HCV infection. Z Gastroenterol 2010; 48: 289–351.
47 Fried MW, Shiffman ML, Reddy KR, et al. Peginterferon alpha-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med 2002; 347: 975–82.
48 Davis GL, Wong JB, McHutchison JG, et al. Early virologic response to treatment with peginterferon alpha-2b plus ribavirin in patients with chronic hepatitis C. Hepatology 2003; 38: 645–52.
49 Ferenci P, Fried MW, Shiffman ML, et al. Predicting sustained virological responses in chronic hepatitis C patients treated with peginterferon alpha-2a (40 KD)/ribavirin. J Hepatol 2005; 43: 425–33.
50 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-alpha-2a plus ribavirin. Gastroenterology 2006; 130: 1086–97.
51 Berg T, Sarrazin C, Herrmann E, et al. Prediction of treatment outcome in patients with chronic hepatitis C: significance of baseline parameters and viral dynamics during therapy. Hepatology 2003; 37: 600–9.
52 Sánchez-Tapias JM, Diago M, TeraViC-4 Study Group 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: 451–60.
53 Sarrazin C, Schwendy S, Möller B, et al. COMPLETELY Individualized treatment durations (24, 30, 36, 42, 48, 60 OR 72 Weeks) with peginterferon-alfa-2B and ribavirin in hcv genotype 1-infected patients (INDIV-2 STUDY). J Hepatol 2010; 52 (Suppl. 1): S25–6.
54 Afdhal N, Levine R, Brown R, et al. Colchicine versus PEG-Interferon alpha 2b long term therapy: results of the 4 year copilot trial. J Hepatol 2008; 48(Suppl. 2): S4.
55 Di Bisceglie AM, Shiffman ML, Everson GT, et al. HALT-C Trial Investigators. Prolonged therapy of advanced chronic hepatitis C with low dose peginterferon. N Engl J Med 2008; 359: 2429–41.
56 Bruix J, Poynard T, Colombo M, et al. Pegintron maintenance therapy in cirrhotic (Metavir F4) HCV patients, who failed to respond to interferon/ribavirin (IR) therapy: final results of the EPIC3 cirrhosis maintenance trial. J Hepatol 2009; 50: A49.
57 Zeuzem S, Buti M, Ferenci P, et al. Efficacy of 24 weeks treatment with peginterferon alpha-2b plus ribavirin in patients with chronic hepatitis C infected with genotype 1 and low pretreatment viremia. J Hepatol 2006; 44: 97–103.
58 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: 954–60.
59 Ferenci P, Laferl H, Scherzer TM, et al. Peginterferon alpha-2a and ribavirin for 24 weeks in hepatitis C type 1 and 4 patients with rapid virological response. Gastroenterology 2008; 135: 451–8.
60 Hadziyannis SJ, Sette Jr H, Morgan TR, et al. Peginterferon-alpha2a and ribavirin combination therapy in chronic hepatitis C: a randomized study of treatment duration and ribavirin dose. Ann Intern Med 2004; 140: 346–55.
61 Nasta P, Gatti F, Puoti M, et al. Insulin resistance impairs rapid virologic response in HIV/hepatitis C virus coinfected patients on peginterferon-alpha-2a. AIDS 2008; 22: 857–61.
62 Kau A, Vermehren J, Sarrazin C. Treatment predictors of a sustained virologic response in hepatitis B and C. J Hepatol 2008; 49: 634–51.
63 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: 1688–94.
64 Mangia A, Minerva N, Bacca D, et al. Individualized treatment duration for hepatitis C genotype 1 patients: A randomized controlled trial. Hepatology 2008; 47: 43–50.
65 Ferenci P, Laferl H, Scherzer TM, et al. Customizing treatment with peginterferon alpha-2a (40KD) plus ribavirin in patients with HCV genotype 1 or 4 infection: interimresults of a prospective randomized trial. Hepatology 2006; 44: 336A.
66 Buti M, Lurie Y, SUCCESS Study Investigators et al. Randomized trial of peginterferon alfa-2b and ribavirin for 48 or 72 weeks in patients with hepatitis C virus genotype 1 and slow virologic response. Hepatology 2010; 52: 1201–7.
67 Shiffman ML, Suter F, Bacon BR, et al. Peginterferon alpha-2a and ribavirin for 16 or 24 weeks in HCV genotype 2 or 3. N Engl J Med 2007; 357: 124–3.
68 Dalgard O, Bjoro K, Hellum KB, et al. Treatment with pegylated interferon and ribavarin in HCV infection with genotype 2 or 3 for 14weeks: a pilot study. Hepatology 2004; 40: 1260–5.
69 von Wagner M, Huber M, Berg T, et al. Peginterferon-alpha-2a (40KD) and ribavirin for 16 or 24 weeks in patients with genotype 2 or 3 chronic hepatitis C. Gastroenterology 2005; 129: 522–7.
70 Mangia A, Santoro R, Minerva N, et al. Peginterferon alpha-2b and ribavirin for 12 vs. 24 weeks in HCV genotype 2 or 3. N Engl J Med 2005; 352: 2609–17.
71 Yu ML, Dai CY, Huang JF, et al. A randomised study of peginterferon and ribavirin for 16 versus 24 weeks in patients with genotype 2 chronic hepatitis C. Gut 2007; 56: 553–9.
72 Dalgard O, Bjoro K, Ring-Larsen H, et al. Pegylated interferon alpha and ribavirin for 14 versus 24 weeks in patients with hepatitis C virus genotype 2 or 3 and rapid virological response. Hepatology 2008; 47: 35–42.
73 Diago M, Shiffman ML, Bronowicki JP, et al. Identifying hepatitis C virus genotype 2/3 patients who can receive a 16-week abbreviated course of peginterferon alfa-2a (40KD) plus ribavirin. Hepatology 2010; 51: 1897–903.
74 Andriulli A, Dalgard O, Bjoro K, et al. Short-term treatment duration for HCV-2 and HCV-3 infected patients. Dig Liver Dis 2006; 38: 741–8.
75 Mangia A, Ricci GL, Persico M, et al. A randomized controlled trial of pegylated interferon alpha-2a (40 KD) or interferon alpha-2a plus ribavirin and amantadine v. interferon alpha-2a and ribavirin in treatment-naive patients with chronic hepatitis C. J Viral Hepat 2005; 12: 292–9.
76 Willems B, Hadziyannis SJ, Morgan TR, et al. Should treatment with peginterferon plus ribavirin be intesified in patients with genotype 2/3 without a rapid virological response? # 8. J Hepatol 2007; 46(Suppl. 1): S6.
77 Sarrazin C, Shiffman ML, Hadziyannis SJ, et al. Definition of rapid virologic response with a highly sensitive real-time PCR-based HCV RNA assay in peginterferon alfa-2a plus ribavirin response-guided therapy. J Hepatol 2010; 52: 832–8.
78 Neumann AU, Pianko S, Zeuzem S, et al. Positive and negative prediction of sustained virologic response at weeks 2 and 4 of treatment with albinterferon alfa-2b or peginterferon alfa-2a in treatment-naïve patients with genotype 1, chronic hepatitis C. J Hepatol 2009; 51: 21–8.
79 Martinot-Peignoux M, Maylin S, Moucari R, et al. Virological response at 4 weeks to predict outcome of hepatitis C treatment with pegylated interferon and ribavirin. Antivir Ther 2009; 14: 501–11.
80 Mihm U, Herrmann E, Sarrazin C, Zeuzem S. Review article: predicting response in hepatitis C virus therapy. Aliment Pharmacol Ther 2006; 23: 1043–54.
81 Ge D, Fellay J, Thompson AJ, et al. Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance. Nature 2009; 461: 399–401.
82 Thompson AJ, Muir AJ, Sulkowski MS, et al. Interleukin-28B Polymorphism Improves Viral Kinetics and Is the Strongest Pretreatment Predictor of Sustained Virologic Response in Genotype 1 Hepatitis C Virus. Gastroenterology 2010; 139: 120–9.
83 Suppiah V, Moldovan M, Ahlenstiel G, et al. IL28B is associated with response to chronic hepatitis C interferonalpha and ribavirin therapy. Nat Genet 2009; 41: 1100–4.
84 Tanaka Y, Nishida N, Sugiyama M, et al. Genome-wide association of IL28B with response to pegylated interferonalpha and ribavirin therapy for chronic hepatitis C. Nat Genet 2009; 41: 1105–9.
85 Asselah T. Genetic polymorphism and response to treatment in chronic hepatitis C: the future of personalized medicine. J Hepatol 2010; 52: 452–4.
86 Kotenko SV, Gallagher G, Baurin VV, et al. IFN-lambdas mediate antiviral protection through a distinct class II cytokine receptor complex. Nat Immunol 2003; 4: 69–77.
87 Mangia A, Thompson AJ, Santoro R, 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. Gastroenterology 2010; 139: 821–7.