Masashi Namikawa; Satoru Kakizaki; Yutaka Yata; Yuichi Yamazaki; Norio Horiguchi; Ken Sato; Hitoshi Takagi; Masatomo Mori
Posted: 02/07/2012; J Gastroenterol Hepatol. 2012;27(1):69-75. © 2012 Blackwell Publishing
Abstract and Introduction
Background and Aim: This study evaluated whether the assessment of hepatitis C virus (HCV)-RNA at 12 weeks (FW+12) post-treatment follow-up was as applicable as FW+24 to evaluate sustained virological response (SVR) using the highly sensitive real-time polymerase chain reaction (PCR) HCV assay.
Methods and Results: Two hundred and twenty-two patients with chronic hepatitis C were included in this study. Pegylated interferon (Peg-IFN) and ribavirin were administered for 24–72 weeks based on the genotype and viral load. Serum HCV-RNA was measured using real-time PCR at pretreatment, the end of treatment, FW+4, FW+8, FW+12, FW+16, FW+20 and FW+24. Two hundred patients had a virological response at the end of treatment. One hundred and forty-eight of 200 (74.0%) patients with a virological response at the end of treatment had an SVR at the FW+24. The positive predictive value (PPV) to identify patients with SVR at FW+4, FW+8, FW+12 was 87.1, 96.1, 98.0%, respectively. The viral load showed a reversion to the basal level as early as 8 weeks in relapse patients. There were only three patients who relapsed after FW+12 and all three of these patients were females with genotype Ib and a high viral load.
Conclusion: The assessment of serum HCV-RNA FW+12, using the highly sensitive real-time PCR assay, is almost as effective as FW+24 to predict SVR. However, there are false negatives in female patients with a high viral load of genotype Ib when the SVR is predicted by FW+12. The current standard with FW+24 is reasonable, but the assessment of serum HCV-RNA FW+12 may be effective in most patients.
The recommended therapy for chronic hepatitis C is a combination of pegylated interferon (Peg-IFN) and ribavirin, and the major purpose of therapy to reach the sustained virological response (SVR).[1–4] The current standard for the determination of SVR is undetectable serum hepatitis C virus (HCV)-RNA 24 weeks (FW+24) after the end of treatment and the continued viral response after FW+24.[1–4] This standard is based on the results of many previous reports that late relapse is rarely observed after FW+24.[6,7] This criterion is based on investigations using qualitative HCV-RNA assays with the lowest limits of detection of 50–100 IU/mL, to establish undetectable serum HCV-RNA at the end of therapy and after treatment.[6,7]
A highly sensitive method to detect HCV-RNA was recently developed and applied in clinical settings.[8,9] The highly sensitive methods include transcription-mediated amplification (TMA) and a real-time polymerase chain reaction (PCR) HCV assay.[8–12] Martinot-Peignoux et al. reported the efficacy of a new assay based on TMA to predict SVR at FW+12. The TMA assay has a lowest detection limit of 5–10 IU/mL. Theoretically, this highly sensitive method could detect residual serum HCV-RNA in a proportion of patients, who had been classified as having a virological response with a less sensitive assay. Therefore, the assessment of HCV-RNA FW+12, using the TMA assay, is considered to be as effective as FW+24 to predict SVR.
The real-time PCR assay is also highly sensitive to detect HCV-RNA and a popular method to assess the HCV-RNA.[9–12] Bortoletto et al. reported that TMA and the real-time PCR HCV assay have comparable sensitivity and specificity in identifying minimal residual HCV-RNA. The lower limit of quantitative detection of the real-time PCR assay is 15 IU/mL[10–12] in comparison to the lowest detection limit of 5–10 IU/mL of the TMA assay. However, even HCV-RNA signals below the quantitative limit are detected by the real-time PCR assay. Therefore, it is difficult to determine whether the TMA or the real-time HCV assays are optimal for the detection of minimal residual viremia at the end of antiviral therapy with Peg-IFN and ribavirin and in predicting relapse after withdrawal of treatment.
This study evaluated whether the assessment of HCV-RNA FW+12 by the real-time PCR HCV assay was as effective as FW+24 to evaluate SVR.
Two hundred and twenty-two patients with chronic hepatitis C treated with combination Peg-IFN and ribavirin therapy from April 2008 to March 2010 at Gunma University Hospital and its affiliated hospitals were included in this study. All patients fulfilled the following inclusion criteria: (i) more than 5 log IU/mL of HCV-RNA in the baseline serum, and (ii) an elevated serum ALT level for least 6 months before initiation of treatment. In addition, patients were excluded if they presented any of the following conditions: (i) decompensated liver disease; (ii) other causes of liver disease such as hepatitis B infection; (iii) autoimmune disorders; (iv) hemoglobin value < 11 g/dL; (v) white blood cell count < 3000/μL; (vi) thrombocytopenia < 70 000/μL; (vii) neoplastic disease; (viii) severe cardiac disease; (ix) other severe concurrent disease such as a preexisting psychiatric conditions; or (x) pregnancy or lactation. Informed consent was obtained from all patients enrolled in the study after a thorough explanation of the aims, risks and benefits of this therapy.
Peg-IFN and ribavirin were administered for 24–72 weeks according to the genotype and viral load. One hundred and seventy-five patients received Peg-IFN α2b (PegIntron, Schering Plough, Tokyo, Japan) at a dose of 1.5 μg/kg per week and ribavirin (Rebetol, Schering Plough, Tokyo, Japan) at a dose of 600–1000 mg/kg per day adjusted according to body weight (600 mg for patients weighing under 60 kg, 800 mg for those between 60 kg and 80 kg, 1000 mg for those over 80 kg) for 24–72 weeks. Forty-seven patients received Peg-IFN α2a at a dose of 180 μg/week (Pegasys, Chugai Pharmaceutical Co., Ltd, Tokyo, Japan) and weight-based ribavirin 600–1000 mg/kg per day same dosage as described above (Copegus, Chugai Pharmaceutical Co., Ltd, Tokyo, Japan). Patients infected with genotype I and all previously treated patients were treated for 48 weeks. The genotype I patients who remained positive for HCV-RNA at 12 weeks after the start of treatment, but who become negative for HCV-RNA after 13–36 weeks of treatment received an additional 24 weeks (total 72 weeks) treatment according to the 2008 Japanese guidelines for the treatment of patients with chronic hepatitis C. Patients infected with genotype II were treated for 24 weeks. The patients were followed-up for another 24 weeks after the treatment. The clinical characteristics including age, gender, body weight, and previous treatment were recorded. The aspartate aminotransferase (AST) to platelets ratio index (APRI) was calculated as the serum liver fibrosis scores according to previously published formulas: APRI = [AST (U/L)/upper limit of normal (U/L)] × 100/platelets count (109/L). The time of undetectable HCV-RNA was defined as the first time that achieved undetectable HCV-RNA during the treatment period.
The patients were included if they completed a full course of therapy. The end of treatment virological response was defined as undetectable serum HCV-RNA levels at the end of therapy. A nonresponse was defined as detectable serum HCV-RNA levels at the end of treatment. Patients with a virological end of treatment response were followed. Serum biochemistry and the HCV RNA titer were measured at pre-treatment (Pre), the end of treatment (FW+0), as well as at 4 (FW+4), 8 (FW+8), 12 (FW+12), 16 (FW+16), 20 (FW+20), and 24 (FW+24) weeks after the end of treatment. An SVR was defined as undetectable HCV-RNA in serum 24 weeks (FW+24) after the end of treatment. A virological relapse (VR) was defined as undetectable serum HCV-RNA levels at the end of treatment and detectable serum HCV-RNA at the FW+24 post treatment follow-up. Among VR patients, late VR was defined as patients with relapse occurring after FW+12. Early VR was defined as patients with relapse occurring before and/or at FW+12. The serum samples were evaluated by the real-time PCR assay (Cobas TaqMan HCV assay, Roche Diagnostics Japan, Tokyo, Japan), in which the lower limit of quantitative detection is 15 IU/mL.[10–12] HCV-RNA signals below the quantitative limit (15 IU/mL, 1.2 log IU/mL) were also detected and referred to as "low positive." HCV-RNA were measured pre-treatment and every 4 weeks after the treatment (FW+0, FW+4, FW+8, FW+12, FW+16, FW+20, FW+24) in all cases until a VR was proven. HCV-RNA assessment after the VR was optional in the cases with agreement to visit. The first time point in which viral relapse was proven was defined as VR+0, and 4 and 8 weeks after the VR+0 were defined as VR+4 and VR+8, respectively.
Single Nucleotide Polymorphisms of Interleukin-28B
The VR patient relapsed after FW+8 were genotyped for three interleukin-28B (IL28B) single nucleotide polymorphisms (SNPs), which were previously reported to be associated with therapy outcome: rs8099917, rs11881222, and rs8103142. Blood samples were genotyped using the Invader assay, as described previously.[15,16]
Continuous variables are presented as the mean ± standard deviation (SD). Fisher's exact probability test for frequency tables was used for the statistical analysis. Distributions of continuous variables were analyzed by the Mann–Whitney U-test. The positive predictive value (PPV) was defined as the probability that the patients fulfilling the criteria at each week become SVR. Results are expressed as odds ratios (ORs) with 95% confidence intervals (CIs). The comparison of continuous variables at different time points was performed using the Wilcoxon signed-rank test. A P-value < 0.05 was considered to be significant.
Clinical Characteristics and Response to Therapy
Two hundred of 222 patients demonstrated a virological response at the end of treatment. The characteristics of the 200 patients with a virological response at the end of treatment are shown in Table 1. The male : female ratio was 94:106. The mean patient age was 57.3 ± 11.1 years old (range 20–75). The mean body weight was 59.5 ± 11.5 kg. One hundred and fifty-three patients were naïve to IFN treatment, while 47 patients had received previous treatment. A schematic tree of the follow-up protocol is illustrated in Figure 1. Serum HCV-RNA outcomes during the 24 weeks post-treatment follow-up are shown in Table 2.
Figure 1. Schematic tree of the follow-up protocol.
FW+4 to FW+12 Post Treatment Follow Up
The serum HCV-RNA levels were undetectable at FW+4 in 170 (85.0%) patients of the 200 patients with a virological response at the end of treatment, and 148 of these demonstrated an SVR (PPV 87.1%, 95% CI 82.0–92.1; Table 2A). The serum HCVRNA levels were undetectable at FW+8 in 154 of 200 (77.0%) patients, and the PPV for SVR was 96.1% (95% CI 93.0–99.2). The serum HCV-RNA levels were undetectable at FW+12 in 151 of 200 (75.5%) patients; the PPV for SVR was 98.0% (95% CI 95.8–100). Therefore, 49 of 52 patients with virological relapse (VR) showed a relapse within FW+12. In the patients with genotype I, the PPV of SVR was 73.1% at FW+4, 89.1% at FW+8, 94.2 % at FW+12, respectively (Table 2B). In the patients with genotypes IIa and IIb, the PPV of SVR was 96.0% at FW+4, 100.0% at FW+8, 100.0 % at FW+12, respectively (Table 2C). In the patients with genotypes IIa and IIb, the PPV of SVR was 100.0% at and after FW+8.
FW+16 to FW+20 Post Treatment Follow Up
The serum HCV-RNA levels were undetectable at FW+16 in 150 of 200 (75.0%) patients, and the PPV for SVR was 98.7% (95% CI 96.8–100 Table 2A). The serum HCV-RNA levels were undetectable at FW+20, in 149 of 200 (74.5%) patients, and the PPV for SVR was 99.3% (95% CI 98.0–100). There was only one patient with VR at each time point. In the patients with genotype I, PPV of SVR was 96.1% at FW+16, 98.0% at FW+20, respectively (Table 2B). In the patients with genotypes IIa and IIb, PPV of SVR was 100.0% at both FW+16 and FW+20 (Table 2C).
Sustained Virological Response and Virological Relapse
One hundred and forty-eight (74%) patients were SVR at the end of the post-treatment follow-up (FW+24), and 52 (26%) patients had a VR (Table 2A). The characteristics of the patients with SVR and VR are shown in Table 1. The mean age and the ratio of female patients were significantly higher in VR patients in comparison to SVR patients. The pretreatment viral load was significantly higher in VR patients in comparison to SVR patients (P < 0.05). The ratios of genotype I and retreatment patients were significantly higher in VR patients in comparison to SVR patients (P < 0.01). The adherence of ribavirin and the time of undetectable HCV-RNA were found to be significantly worse in the VR patients in comparison to SVR patients (P < 0.01). There were no differences in the serum alanine aminotransferase, hemoglobin, platelet levels, APRI, and adherence of Peg-IFN between the SVR and VR patients.
There were six patients that showed a viral relapse after FW+8 (Table 2A). Furthermore, there were only three patients who relapsed after FW+12 (late VR). Therefore, the PPV for SVR was 98.0% at FW+12. In the patients with genotypes IIa and IIb, the PPV for SVR was 100% at FW+12. The characteristics of six patients that showed a viral relapse after FW+8 are shown in Table 3. All six of these patients were female with genotype Ib and with a high viral load. The genotypes of the IL28B SNPs were major homo type in all six of these patients. As a result, the genotypes were T/T in rs8099917, A/A in rs11881222, and T/T in rs8103142, respectively. The patient demonstrating a relapse at FW+24 (Case 6) was a 67-year-old genotype Ib patient treated for 72 weeks with the combination therapy Peg-IFN α-2a plus ribavirin. The pretreatment serum HCV-RNA load was 6.5 log IU/mL, and the liver histology showed moderate liver disease (A2/F2; Metavir scoring system). The serum HCV-RNA levels were undetectable 8 weeks after the initiation of treatment. The adherence to Peg-IFN and ribavirin therapy was 91.4% and 72.7%, respectively. A comparison of the three late VR patients (after FW+12 VR) and the other 49 early VR patients (before and/or at FW+12) is shown in Table 4. No statistical differences were found in the characteristics, including the HCV genotype, viral load, age, sex, body weight, APRI, type of Peg-IFN, adherence of Peg-IFN, adherence of ribavirin, time of undetectable HCV-RNA and previous treatment between the late VR patients and early VR patients.
Serum HCV-RNA Outcome in Relapse Patients
Fifty-two of 200 patients with an end of treatment virological response showed virological relapse during the follow-up periods. After VR was proven in these cases, some patients then had their HCV-RNA levels regularly measured every 4 weeks, but other patients did not do so. In the end, only 28 patients had their HCV-RNA levels measured every 4 weeks after VR. The first time point at which viral relapse is proven is defined as VR+0. Serum HCV-RNA levels in relapse patients at baseline, the VR+0, and 4 (VR+4), 8 (VR+8) week after the VR+0 are shown in Figure 2. Serum HCV-RNA levels were: 6.4 ± 0.7 log IU/mL at baseline, 4.3 ± 1.7 log IU/mL at VR+0, 5.5 ± 1.2 log IU/mL at VR+4, and, 5.8 ± 0.9 log IU/mL at VR+8. The viral titers of VR+0 (P < 0.01), and VR+4 (P < 0.05) were significantly smaller than those at baseline. However, there were no differences in the viral titers between baseline and VR+8. The viral load showed a reversion to the basal level at 8 weeks after VR. Forty-nine of 52 patients with VR relapsed within FW+12 and the serum HCV-RNA levels in VR patients reverted to the basal level at 8 weeks after VR. These results show that the viral load increases rapidly in patients who have relapsed; namely, reaching close to baseline levels as early as 24 weeks post-treatment.
Figure 2. Serum hepatitis C virus (HCV)-RNA outcome in 28 virological relapse (VR) patients. Baseline, pre-treatment; VR0, the first time point in which viral relapse was proved; VR+4, 4 weeks after VR+0; VR+8, 8 weeks after VR+0. **P < 0.01, *P < 0.01 in comparison to baseline.
The findings of this study demonstrated that the assessment of serum HCV-RNA 12 weeks after the end of treatment, using the highly sensitive real-time PCR assay (PPV 98.0%), is closely similar to that after 24 weeks for predicting SVR. There were only three patients who relapsed after FW+12 (late VR) and all three of these patients were female patients with a high viral load of genotype Ib. As a result, in the patients with genotypes IIa and IIb, the PPV of SVR was 100.0% at FW+12. Therefore, the assessment of serum HCV-RNA FW+12 is as applicable as FW+24 to predict SVR except in female patients with a high viral load of genotype Ib.
Martinot-Peignoux et al. reported the usefulness of a new assay based on TMA to predict SVR at the FW+12. They reported that the assessment of HCV-RNA FW+12 is as effective as that after 24 weeks to predict SVR with 99.7% PPV. The real-time PCR method, used in the current study, is also highly sensitive to detect HCV-RNA and it is commonly used in Japan. Bortoletto et al. reported that TMA and the real-time PCR HCV assay have comparable sensitivity and specificity in identifying the minimal residual serum level of HCV. This study demonstrated that the assessment of HCV-RNA by real-time PCR at FW+12 was almost as effective as that at FW+24 to evaluate SVR in 200 Japanese patients. PPV (98.0%) of FW+12 in our study was almost as relevant as those of TMA assay (99.7%). However, there were some false negative results with real-time PCR, especially in female patients demonstrating a high viral load of genotype Ib. Bortoletto et al. reported that minimal residual viremia was detectable with the real-time PCR assay in patients with genotype II and with genotype IV, but not with TMA. They noted increased sensitivity of the real-time PCR assay across all major HCV genotypes in comparison to TMA.[9,17,18] Although the current standard at FW+24 is reasonable for Japanese patients by real-time PCR, the assessment of serum HCV-RNA FW+12 may therefore be appropriate for almost all patients, except for genotype Ib female patients.
Zeuzem et al. reported similar results in patients treated with standard interferon α2a or Peg-IFN α2a by PCR (Amplicor HCV Monitor vs 2.0). Only six of 348 patients (2%) became HCV-RNA positive between FW+12 and FW+24. These results suggest that the addition of ribavirin to Peg-IFN enhances the SVR rates but does not affect the timing of relapse.[9,19] Our study also showed that the adherence of ribavirin enhances the SVR rate but it does not affect the timing of relapse. Martinot-Peignoux et al. found no differences in VR patients treated with Peg-IFN α2a and ribavirin and in patients treated with Peg-IFN α2b and ribavirin. The present study also found no differences between Peg-IFN α2a and Peg-IFN α2b. Therefore, the type of Peg-IFN does not affect the VR.
Early determination of post-treatment response status in patients can help patients make decisions and might allow relapse patients to begin alternative therapy earlier. Viral load showed a reversion to the basal level within 8 weeks after the relapse in this study. The viral load increases rapidly after relapse, nearly reaching basal levels, confirming the importance of identifying relapse patients early. Patients might benefit from early retreatment with different regimens when the viral load is still low. Indeed, some patients received additional treatment just after the appearance of VR in this study.
The baseline characteristics associated with virological relapse were age, sex, HCV genotype, viral load and past history of IFN. Patients with these characteristics showed a poor response to combination Peg-IFN and ribavirin therapy.[20–22] Therefore, genotype I female old patients with previous treatment had risk factors for VR. Three of 52 VR patients showed late VR patients (5.8%). There were no significant differences in characteristics between late VR and early VR patients. Although female patients with genotype I, and a history of receiving previous treatment tended to be included in the late VR group, it did not reach the statistical significance. This may be due to the small number of late VR patients. The adherence to Peg-IFN and ribavirin by late VR patients was 95.1 + 7.6%, and 67.8 + 9.4%, respectively. The adherence to Peg-IFN and ribavirin in early VR patients was 81.5 + 21.6%, and 60.5 + 24.8%, respectively. There were no significant differences in adherence to Peg-IFN and ribavirin between the late VR and early VR patients. A genetic polymorphism near the IL28B gene, encoding interferon-lambda-3 (IFN-lambda-3), has been reported to be strongly associated with response to treatment.[23,24] In this study, the genotypes of the IL28B SNPs were major homo type in all three late VR patients. Because IL28B SNPs predict null virological response, it seems to have no value for predicting the late VR in this study. Further study is needed to clarify the differences between late VR and early VR patients because of the small number of late VR patients.
In conclusion, testing for HCV-RNA, using the highly sensitive real-time PCR assay, at FW+12, is therefore considered to be as effective as testing at FW+24 to assess a sustained virological response in patients receiving combination Peg-IFN and ribavirin therapy. As a result, the post-treatment follow-up to identify patients with SVR or VR could be shortened to 12 weeks post-treatment. However, there was a low incidence of false negatives in female patients with a high viral load of genotype Ib for predicting the SVR by FW+12. The current standard with FW+24 is reasonable, but the assessment of serum HCV-RNA FW+12 may be also be relevant and useful for evaluating similar patients other than Ib female patients.
- Marcellin P. Hepatitis C: the clinical spectrum of the disease. J.Hepatol. 1999; 31: 9–16.
- Marcellin P, Boyer N, Gervais A et al. Long-term histological improvement and loss of detectable intrahepatic HCV RNA in patients with chronic hepatitis C and sustained response to interferon-alpha therapy. Ann. Intern. Med. 1997; 127: 875–81.
- Maylin S, Martinot-Peignoux M, Moucari M et al. Eradication of hepatitis C virus in patients successfully treated for chronic hepatitis C. Gastroenterology 2008; 135: 821–9.
- Suzuki H, Kakizaki S, Horiguchi N et al. Clinical characteristics of null responders to Peg-IFNa2b/ribavirin therapy for chronic hepatitis C. World J. Hepatol. 2010; 2: 401–5.
- Ghany MG, Strader DB, Thomas DL, Seeff LB; American Association for the Study of Liver Diseases. Diagnosis, management and treatment of hepatitis C: an update. Hepatology 2009; 49: 1335–74.
- Reichard O, Glaumann H, Fryden A, Norkrans G, Wejstal R, Weiland O. Long-term follow-up of chronic hepatitis C patients with sustained virological response to alpha-interferon. J. Hepatol. 1999; 30: 783–7.
- McHutchison JG, Poynard T, Esteban-Mur R et al. International Hepatitis Interventional Therapy Group. Hepatic HCV RNA before and after treatment with interferon alone or combined with ribavirin. Hepatology 2002; 35: 688–93.
- Martinot-Peignoux M, Stern C, Maylin S et al. Twelve weeks posttreatment follow-up is as relevant as 24 weeks to determine the sustained virologic response in patients with hepatitis C virus receiving pegylated interferon and ribavirin. Hepatology 2010; 51: 1122–6.
- Bortoletto G, Campagnolo D, Mirandola S et al. Comparable performance of TMA and Real-Time PCR in detecting minimal residual hepatitis C viraemia at the end of antiviral therapy. J. Clin.Virol. 2011; 50: 217–20.
- Sandres-Sauné K, Abravanel F, Nicot F et al. Detection and quantitation of HCV RNA using real-time PCR after automated sample processing. J. Med. Virol. 2007; 79: 1821–6.
- 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.
- Ogawa E, Furusyo N, Toyoda K et al. Excellent superiority and specificity of COBAS TaqMan HCV assay in an early viral kinetic change during pegylated interferon alpha-2b plus ribavirin treatment. BMC Gastroenterol. 2010; 10: 38.
- Kumada H, Okanoue T, Onji M et al. The Study Group for the Standardization of Treatment of Viral Hepatitis Including Cirrhosis, Ministry of Health, Labour and Welfare of Japan. Guidelines for the treatment of chronic hepatitis and cirrhosis due to hepatitis C virus infection for the fiscal year 2008 in Japan. Hepatol. Res. 2010; 40: 8–13.
- Wai CT, Greenson JK, Fontana RJ et al. A simple noninvasive index can predict both significant fibrosis and cirrhosis in patients with chronic hepatitis C. Hepatology 2003; 38: 518–26.
- Ohnishi Y, Tanaka T, Ozaki K, Yamada R, Suzuki H, Nakamura Y. A high-throughput SNP typing system for genome-wide association studies. J. Hum. Genet. 2001; 46: 471–7.
- Suzuki A, Yamada R, Chang X et al. Functional haplotypes of PADI4, encoding citrullinating enzyme peptidylarginine deiminase 4, are associated with rheumatoid arthritis. Nat. Genet. 2003; 34: 395–402.
- Sabato MF, Shiffman ML, Langley MR, Wilkinson DS, Ferreira-Gonzalez A. Comparison of performance characteristics of three Real-Time reverse transcription-PCR test systems for detection and quantification of hepatitis C virus. J. Clin. Virol. 2007; 45: 2529–36.
- Chevaliez S, Bouvier-Alias M, Pawlotsky JM. Performance of the Abbott Real-Time PCR assay using m2000sp and m2000rt for hepatitis C virus RNA quantification. J. Clin. Microbiol. 2009; 47: 1726–32.
- Zeuzem S, Heathcote EJ, Shiffman ML et al. Twelve weeks follow-up is sufficient for the determination of sustained virologic response in patients treated with interferon alpha for chronic hepatitis C. J. Hepatol. 2003; 39: 106–11.
- Sezaki H, Suzuki F, Kawamura Y et al. Poor response to pegylated interferon and ribavirin in older women infected with hepatitis C virus of genotype 1b in high viral loads. Dig. Dis. Sci. 2009; 54: 1317–24.
- Yamada G, Iino S, Okuno T et al. Virological response in patients with hepatitis C virus genotype 1b and a high viral load: impact of peginterferon-alpha-2a plus ribavirin dose reductions and host-related factors. Clin. Drug. Investig. 2008; 28: 9–16.
- Oze T, Hiramatsu N, Yakushijin T et al. Indications and limitations for aged patients with chronic hepatitis C in pegylated interferon alfa-2b plus ribavirin combination therapy. J. Hepatol. 2011; 54: 604–11.
- Ge D, Fellay J, Thompson AJ et al. Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance. Nature 2009; 461: 399–401.
- Tanaka Y, Nishida N, Sugiyama M et al. Genome-wide association of IL28B with response to pegylated interferon-alpha and ribavirin therapy for chronic hepatitis C. Nat. Genet. 2009; 41: 1105–9.