Antiviral Therapy 14:45 54

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1 Antiviral Therapy 14:45 54 Original article Factors affecting early viral load decline of Asian chronic hepatitis C patients receiving pegylated interferon plus ribavirin therapy Ching-Sheng Hsu 1,2, Chen-Hua Liu 2,3, Chun-Jen Liu 2,3, Chi-Ling Chen 2, Ming-Yang Lai 2,3,4, Pei-Jer Chen 2,3,4, Ding-Shinn Chen 2,3 and Jia-Horng Kao 2,3,4,5 * 1 Division of Gastroenterology, Department of Internal Medicine, Buddhist Tzu Chi General Hospital, Taipei, Taiwan 2 Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine and National Taiwan University Hospital, Taipei, Taiwan 3 Department of Internal Medicine, National Taiwan University College of Medicine and National Taiwan University Hospital, Taipei, Taiwan 4 Department of Medical Research, National Taiwan University College of Medicine and National Taiwan University Hospital, Taipei, Taiwan 5 Hepatitis Research Center, National Taiwan University College of Medicine and National Taiwan University Hospital, Taipei, Taiwan *Corresponding author: kaojh@ntu.edu.tw Background: Early viral load decline following pegylated interferon-α2a and ribavirin therapy is an important predictor of the treatment responses in chronic hepatitis C (CHC) patients, thus, it is essential to evaluate the influence of host and viral factors on early viral load decline. Methods: Clinical and serial virological data were collected from 145 consecutive Asian CHC patients with pegylated interferon-α2a plus ribavirin therapy. A dose of pegylated interferon-α2a was administered at week 1 and then weekly with daily oral ribavirin for 24 or 48 weeks. Genotyping and quantification of hepatitis C virus (HCV) RNA were done using molecular methods. Results: A total of 81 patients were infected with HCV genotype 1, 61 with genotype 2 and 3 with both genotypes 1 and 2. At the end of follow-up, 110 patients attained sustained virological response (SVR). In multivariate analyses, body mass index (BMI) and genotype were related to viral load decline at day 2, baseline viral load and high-density lipoprotein (HDL) cholesterol levels were correlated with viral load decline between days 2 and 28. Genotype, baseline viral load, alanine aminotransferase (ALT) levels and BMI independently predicted rapid virological response, whereas only genotype 2, lower baseline viral load and more substantial viral load decline at day 28 predicted a higher SVR. Conclusions: HCV genotype, baseline viral load, pretreatment BMI, HDL and ALT levels have a significant effect on early viral load decline of Asian CHC patients with interferon-based therapy. Only HCV genotype, baseline viral load and viral load decline at day 28 can independently predict SVR. Introduction Hepatitis C virus (HCV) infection is the major cause of chronic liver disease and hepatocellular carcinoma worldwide [1,2]. The goal of treating chronic hepatitis C (CHC) patients is to eradicate the virus or, in a clinical term, to attain a sustained virological response (SVR; defined as undetectable serum HCV RNA level at 24 weeks after treatment cessation) [3]. However, currently approved pegylated interferon (PEG-IFN) plus ribavirin (RBV) therapy has many unpleasant side effects and is only effective in a certain proportion of CHC patients [4]. Therefore, identifying baseline and on-treatment factors predictive of SVR in CHC patients is important in terms of increasing efficacy, avoiding unnecessary side effects and saving medical costs. Several factors have been linked to the therapeutic response of CHC patients, including viral factors [5 10], host factors [11 14], metabolic factors [15 17], histological factors [18], the type of regimens used [3] and the duration of infection [19]. Among these factors, early viral kinetics following therapy has become increasingly recognized and is widely used in both clinical trials and daily practice [20]. Considering the clinical significance of early viral kinetics, it is thus reasonable to investigate the association of other predictors with viral load decline. These attempts not only provide information 2009 International Medical Press

2 C-S Hsu et al. on individualized treatment, but also elucidate the underlying mechanisms of these predictors. Although Asians are more likely to achieve an SVR to combination therapy than Caucasians with CHC [14], the influence of various host and viral factors on early viral load decline in Asian CHC patients deserves further studies [21 23]. To this end, we enrolled 145 consecutive treatmentnaive Asian CHC patients and administered a combination therapy of PEG-IFN-a2a plus RBV to evaluate the effect of various host, viral, metabolic and histological factors on early viral load decline. Methods Study population From January 2002 to December 2005, a total of 145 consecutive CHC patients were evaluated prospectively from the gastroenterological clinics of the National Taiwan University Hospital (Taipei, Taiwan) and the National Taiwan University Hospital Yun-Lin branch (Yun-Lin, Taiwan). Chronic HCV infection was defined as the positivity of both anti-hcv antibodies and serum HCV RNA for >6 months. Patients had not received prior interferon, other experimental antiviral or immunosuppressive therapy and had serum alanine aminotransferase (ALT) levels 2 the upper limit of normal on two occasions within the past 6 months. All of them were negative for hepatitis B surface antigens or HIV antibodies. None had known history or serological evidence of autoimmune liver disease, inheritable disorders (such as haemochromatosis or Wilson s disease), renal insufficiency, a history of excess alcohol intake (daily alcohol consumption >20 g) or active drug abuse. Viral parameters, biochemical, serological and anthropometric data of all the patients were recorded at enrolment. Metabolic and demographic features, and serological markers Information on sex, age, body mass index (BMI), serum fasting blood glucose, triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL) cholesterol, highdensity lipoprotein (HDL) cholesterol, albumin, aspartate aminotransferase (AST), ALT, γ-glutaryl transpeptidase (GGT) levels, complete blood count and differential count were collected. BMI was calculated as weight in kg divided by height in m 2. Baseline blood samples were collected in the morning after 12 h fasting and measured using standard laboratory techniques. Hepatitis B surface antigen and anti-hcv antibodies were assayed using commercial kits (Abbott Laboratories, North Chicago, IL, USA). Serum fasting blood glucose, albumin, TG, TC, HDL, LDL, AST, ALT and GGT were measured using an autoanalyser (Hitachi 7250, Special; Hitachi, Tokyo, Japan) according to the manufacturer s instructions. All the remnant serum samples collected from each patient were stored at -80 C until use. Virological assays: extraction, quantification and genotyping of HCV RNA Serum RNA was extracted using a commercial kit (QIAamp RNA Blood Mini Kit; Qiagen Inc., Valencia, CA, USA). Serum HCV RNA level was quantified using a LightCycler (Roche Diagnostics Applied Science, Penzberg, Germany) with the detection limit of 86 copies/ml (that is, 34 IU/ml) [24,25]. High viral load was defined as serum HCV RNA level 400,000 IU/ml. Genotyping of HCV was performed using the Light- Cycler PCR assay or reverse transcription-pcr with type-specific primers as previously described [26,27]. The detection limit of the type-specific primers genotyping method was 100 copies/ml (that is, 37 IU/ml). All samples were tested in triplicate. Histological evaluations of liver biopsy specimens Written informed consent was given. Each patient, without contraindication for liver biopsies, received an echo-guided percutaneous liver biopsy from the right hepatic lobe with a 16-gauge Temno Evolution biopsy needle (Allegiance, McGaw Park, IL, USA). The mean length of the liver specimen was 19.1 mm and the diameter was 1.4 mm. No fragmentation of the liver specimen was found. The sampling tissues were fixed with formalin, embedded with paraffin and stained with H&E and reticulin silver (Masson trichome method). The staging of hepatic fibrosis was scored using the Metavir system, which ranges from F0 to F4. Significant hepatic fibrosis and cirrhosis were defined as a fibrosis stage F2 and F4, respectively [28]. Brunt s recommendations were used to categorize hepatic steatosis and centrilobular fibrosis [29]. The classification of steatosis was grade 0 (no steatosis), grade 1 (0 33% of hepatocytes contained visible macrovesicular steatosis), grade 2 (34 66%) and grade 3 (>67%). Because portal, lobular inflammation and portal fibrosis, as well as cytological ballooning, also occur commonly in HCV-related liver disease, these findings were not used for characterization of steatohepatitis in this study. Accordingly, only centrilobular fibrosis in zone 3 of the acinus after Masson s trichome staining was denoted as steatohepatitis injury. The samples were evaluated by one experienced pathologist who was blinded to the clinical status of this study. Protocol for treatment and assessment of early viral kinetics All patients received one dose of subcutaneous 180 mg/ ml PEG-IFN-α2a at the beginning of the study (day 0). Then, 180 mg/ml PEG-IFN-α2a was International Medical Press

3 Early viral kinetics and HCV therapy administered weekly from week 1 to week 24. Oral RBV was coadministered at a daily dose of 1,000 1,200 mg from week 1 to week 24, adjusted according to body weight (1,000 mg for 75 kg and 1,200 mg for >75 kg). Although the Bureau of National Health Insurance in Taiwan (Taipei, Taiwan) only allows the reimbursement of 24 weeks of combination therapy for patients with HCV genotype 1 infection [30], they were encouraged to receive an additional 24-week treatment to week 48 in this study and all patients were followed for 24 weeks after discontinuation of treatment. When adverse effects occurred, the dosage of drugs was reduced as indicated by the product manufacturer. For the analysis of early viral kinetics, serum HCV RNA levels were quantified before the first dosing (0 h), at 4 h, 8 h and 12 h after the first dosing on day 0 and then daily for 3 consecutive days (day 1 3) in week 0, then at week 4, week 12, week 24 and every 24 weeks until the end of follow-up. Definition of treatment responses The biochemical and virological responses to therapy were on the basis of serum ALT and HCV RNA levels, respectively. Rapid virological response (RVR) was defined as undetectable serum HCV RNA level at day 28. Early virological response (EVR) was defined as undetectable serum HCV RNA levels or 2 log 10 decrease of the baseline HCV RNA levels at week 12. Complete early virological response (c-evr) was defined as undetectable serum HCV RNA at week 12 of therapy in patients who did not achieve RVR. Partial early virological response (p-evr) as 2 log 10 reduction of serum HCV RNA from baseline to week 12 of therapy in those who did not achieve RVR at week 4 and undetectable serum HCV RNA at week 12 of therapy. Sustained virological responders were defined as those having undetectable serum HCV RNA 24 weeks after discontinuation of treatment. Null response was defined as <2 log 10 decrease of the baseline HCV RNA levels after 12 weeks of treatment. Partial response was defined as 2 log 10 decrease of the baseline HCV RNA levels, but detectable HCV RNA after 24 weeks of treatment. Virological breakthrough was defined as undetectable serum HCV RNA levels at week 12, but detectable HCV RNA before discontinuation of treatment. Relapse was defined as reappearance of detectable HCV RNA levels following discontinuation of treatment [3,31]. Ethical considerations The study was performed in accordance with the principles of the Declaration of Helsinki. The study was approved by the Ethical Committee of the National Taiwan University Hospital and all patients gave informed consent before enrolment. Statistical analyses The data were presented as frequency and percentages for categorical variables and mean ±sd for continuous variables unless mentioned otherwise. Log transformation was performed for variables with significant deviation from normal distribution. Demographic, virological and histological features between CHC patients with SVR and without SVR were compared using χ 2 test or Fisher s exact probability test for categorical variables and Mann Whitney U test or Student s t-test for continuous variables. Odds ratio (OR) as well as P-values were estimated for each viral load decline using unconditional logistic regression. Stepwise estimation with forward selection logistic model was performed to examine the association between various factors and the parameters of early viral kinetics (RVR, c-evr, SVR and the viral load decline of the first 2 days and between day 28 and day 2). Day 2 results were used to account for the first phase of the biphasic decline, although this varied from patient to patient. The parameters of early viral kinetics were used as dependent variables. Genotype, baseline HCV viral load, age, sex, BMI, Metavir inflammation activity, Metavir fibrosis stage, steatosis, PEG-IFN-a2a treatment duration, RBV dosage, pretreatment white blood cell count, haemoglobin, platelet count, albumin, ferritin, a-fetoprotein, AST, ALT, GGT, fasting blood glucose, serum TC, serum TG, LDL, HDL and the decline of HCV RNA levels at 12 h, days 1, 2, 3, 28 and week 12 were used as independent variables. A P-value 0.2 was the significance level for removal from the model and a P-value <0.1 was the significance level for addition to the model. All analyses were performed using Stata statistical software (version 8.0; Stata Corp., College Station, TX, USA). All tests were two-sided and P<0.05 was considered statistically significant. Results Demographic characteristics and metabolic profiles A total of 145 CHC patients were enrolled. Demographic characteristics and metabolic profiles of the study population are shown in Table 1. BMI was available in 137 of 145 patients (94.5%). The mean BMI value was 25.9 kg/m 2 and fell in the overweight range for the Southeast Asia population. The distribution of BMI was 51 (37.2%) for <24 kg/m 2, 42 (30.7%) for kg/m 2, 26 (19.0%) for kg/m 2, 13 (9.5%) for kg/m 2 and 5 (3.6%) for >35 kg/m 2. Quantitative viral load and HCV genotype were available in all patients. There were 81 (55.9%) patients with HCV genotype 1 infection, 61 (42.1%) with genotype 2 infection and 3 (2.1%) with mixed infection of genotype 1 and 2. Antiviral Therapy

4 C-S Hsu et al. Table 1. Demographic characteristics between chronic hepatitis C patients with and without sustained virological response Characteristic SVR (n=110) (n=35) P-value Age, years 53.3 ± ± Male, n (%) 72 (65.5) 18 (51.4) Body weight, kg 66.9 ± ± Body mass index, kg/m ± ± Albumin, g/dl 4.2 ± ± Fasting blood glucose, mg/dl ± ± Triglyceride, mg/dl ± ± Total cholesterol, mg/dl ± ± Low-density lipoprotein cholesterol, mg/dl ± ± High-density lipoprotein cholesterol, mg/dl 47.9 ± ± HCV RNA, log 10 IU/ml 5.3 ± ± a Asparate aminotransferase, U/l 96.1 ± ± Alanine aminotransferase, U/l ± ± γ-glutaryl transpeptidase, U/l 62.7 ± ± White blood cell count, 1,000 cells/ml 5.0 ± ± Haemoglobin, g/dl 14.6 ± ± Platelet count, 1,000 cells/ml ± ± a-fetoprotein, ng/ml 12.6 ± ± Serum iron, mg/dl ± ± Ferritin, mg/l ± ± Genotype 2, n (%) 54 (49.1) 7 (20.0) a PEG-IFN-a2a duration ( 24 weeks/>24 weeks) 87/23 23/ Ribavirin, mg/kg 13.7 ± ± Metavir activity (A0 1/A2 3) 17/92 8/ Metavir fibrosis (F0 2/F3 4) 62/47 14/ Steatosis (<0%/ 0%) 58/39 14/ Data is shown as mean ±se unless indicated otherwise. Sustained virological response (SVR) was defined as undetectable serum hepatitis C virus (HCV) RNA 24 weeks after discontinuation of treatment. Undetectable HCV RNA level was defined as 34 IU/ml. a P<0.05 was considered as statistically significant. PEG-IFN-a2a, pegylated interferon-a2a. Histological findings All patients but one (99.3%) had histological data. Of them, 118 (82%) had a Metavir activity score >1 and 120 (83%) had a Metavir fibrosis score <4. Steatosis grading was available in 127 patients and 124 (97.6%) had steatosis grade <3. Virological response and follow-up Of 145 CHC patients, 110 received 24-week therapy, 3 dropped out, 2 were lost to follow-up and 2 did not receive a full dose or complete the therapeutic duration because of side effects (both of them had genotype 1 infection, one achieved SVR and the other one did not). The remaining 35 patients (all had genotype 1 infection) received 48-week treatment; however, 3 were lost to follow-up and 8 did not receive a full dose or complete therapeutic duration because of side effects (2 of these 8 patients did not achieve SVR). Among these patients, 111 attained RVR, 138 achieved EVR and 110 had SVR (Figure 1). Of the 111 of 143 (77.6%) patients who achieved RVR, 52 had genotype 1 infection (52/80, 65%), 56 had genotype 2 infection (56/60, 93.3%) and 3 had mixed genotype 1 and 2 infection (3/3, 100%). Two patients without RVR data were defined as non-rvr, one had genotype 1 infection and non-svr and the other had genotype 2 infection and dropped out at week 2. Serum HCV RNA data at week 12 were available in 143 patients and 138 had EVR (138/143, 96.5%). One patient (who had mixed genotype 1 and 2 infection and was classified as non-evr and non-svr) dropped out at week 12. Among those who achieved EVR, only one (genotype 1 infection) was p-evr and 28 patients were c-evr. Two genotype 1 patients who attained RVR had detectable serum HCV RNA at week 12. Among patients with undetectable serum HCV RNA at week 12, 76 had genotype 1 infection (76/81, 93.8%), 59 had genotype 2 infection (59/60, 98.3%) and 2 had mixed genotype 1 and 2 infection (2/2, 100%). Of patients with c-evr, 25 had genotype 1 infection (25/28, 89.3%) and 3 had genotype 2 infection (3/4, 75%). Serum HCV RNA data 24 weeks after discontinuation of treatment were available in 137 patients and 110 achieved SVR (110/137, 80.3%). One genotype 1 patient dropped out at week 16, 2 were lost to follow-up at week 24 (one had genotype 1 and the other International Medical Press

5 Early viral kinetics and HCV therapy Figure 1. Virological response diagram of the study 145 CHC genotype I or 2 RVR 111 RVR 32 Non-RVR 1 Lost to follow-up, 1 drop-out EVR 2 a Drop-out SVR Lost to follow-up, 1 drop-out RVR, c-evr or SVR Non-RVR, non-evr or non-svr p-evr Of 145 chronic hepatitis C (CHC) patients with pegylated interferon-a2a plus ribavirin therapy and available serial serum hepatitis C virus (HCV) RNA levels, 111 attained rapid virological response (RVR), 137 with undetectable serum HCV RNA levels, 138 attained early virological response (EVR), 28 with complete early virological response (c-evr), 1 with partial early virological response (p-evr) and 110 attained sustained virological response (SVR). Only 1 non-evr patient attained SVR, but 10 non-rvr patients achieved SVR. The white squares indicate the number of enrollers who attained virological response (RVR, c-evr or SVR). The light grey squares indicate the number of enrollers who did not attain a virological response (non-rvr, non-evr or non-svr). The dark grey squares indicate the number of enrollers who attained p-evr. a Two genotype 1 patients who attained RVR had detectable serum HCV RNA at week 12. One dropped out later and the other one was lost to follow-up at week 24. one had genotype 2), one genotype 1 patient was lost to follow-up at week 48, 2 genotype 1 patients were lost to follow-up at week 72 and all were classified as non-svr. Two genotype 1 patients who received 48-week treatment were lost to follow-up at week 48 and had viral load data at week 72. Among patients who achieved SVR, 54 had genotype 1 infection (54/76, 71.1%), 54 had genotype 2 infection (54/59, 91.5%) and 2 had mixed genotype 1 and 2 infection (2/2, 100%). Two genotype 1 patients had virological breakthrough, one had detectable HCV RNA level since week 24 and the other one at week 48. One genotype 2 patient had detectable HCV RNA levels (16,600 IU/ml) at week 24, but the viral load became undetectable after week 48. Four genotype 2 patients were defined as relapsers at week 48, three of them were non-rvr. Eleven genotype 1 patients who received 24-week treatment were defined as relapsers at week 48 and seven of them were non-rvr. Five patients received 48-week treatment had relapse at week 72 and four of them were non-rvr. Comparison between various characters and treatment response In univariate analyses, only baseline viral load and genotype were significantly different between patients with and without SVR (Table 1). Virological responses and early viral load decline were also calculated and compared between patients with and without SVR. Patients with SVR had significantly lower serum HCV RNA levels during the first 3 months of therapy than those without SVR (a difference of 0.8 log 10 IU/ml at 12 h, 0.9 log 10 IU/ml at day 1, 1.1 log 10 IU/ml at day 2, 1.1 log 10 IU/ml at day 3 and 1.5 log 10 IU/ml at day 28, but only 0.4 log 10 IU/ ml at week 12), implying better viral kinetics in those with SVR (Table 2). Predictors of early viral load decline in CHC patients In univariate analyses, several characters were associated with early viral load decline, such as age, genotype, fibrosis stage, baseline viral load, white blood cell count and platelet count (data not shown). By contrast, categorized fasting blood glucose, TG, TC and ALT levels were unrelated to viral load decline. By using multivariate analyses with stepwise approaches that include age, sex, BMI, liver fibrosis stage, baseline viral load, genotype, ALT and other clinical and demographic parameters as independent variables, genotype and BMI (adjusted OR 0.25 and 0.83, respectively) were identified as the independent predictors of viral load decline in the first 2 days (Table 3). Also, baseline viral load and HDL levels (adjusted OR 2.78 and 0.95, respectively) were identified as the independent predictors of viral load decline between days 2 and 28 (Table 3). With similar analyses, genotype, baseline viral load, ALT levels and BMI (adjusted OR 0.07, 0.41, 1.01 and 0.83, respectively) were recognized as the independent predictors of RVR (Table 4); however, none of these variables could be identified as predictors for Antiviral Therapy

6 C-S Hsu et al. c-evr or undetectable serum HCV RNA at week 12 (see Additional file). In addition, genotype and liver fibrosis stage (adjusted OR 0.27 and 0.51, respectively) were found to be independent predictors of SVR (Table 4). However, adjustment of the previous model by early viral load decline showed that only genotype, baseline viral load and viral load decline at day 28 (adjusted OR 0.27, 0.38 and 2.32, respectively) could independently predict SVR (Table 5). We further stratified the data by HCV genotype and baseline viral load (Figures 2 and 3). In genotype 1 patients, 94.4% (17/18) of patients who had low baseline viral load with RVR attained SVR. By contrast, only 33% (8/24) of patients who had high baseline viral load without RVR attained SVR. In total, 73.8% (31/42) of genotype 1 patients who received a complete course of 24-week therapy attained RVR and 65.2% (15/23) of patients who received a complete course of 48-week therapy achieved RVR (P=0.446). In addition, 91.7% (44/48) of genotype 1 patients who had RVR and undetectable serum HCV RNA level at week 12 attained SVR. Also, 37.5% (9/24) of genotype 1 patients who were non- RVR and c-evr attained SVR, but only one genotype 1 patient (who received 40 weeks of treatment) with non- RVR and detectable viral load at week 12 attained SVR. In genotype 2 patients, all patients who had low baseline viral load with RVR attained SVR, but none who had high baseline viral load without RVR achieved SVR. Table 2. Early viral titre decline and virological response in chronic hepatitis C patients with and without sustained virological response Viral titre decline and virological response SVR (n=110) (n=35) P-value Early viral titre decline a Day 0.5 HCV RNA, log 10 IU/ml 5.5 ± ±0.2 <0.001 Day 1 HCV RNA, log 10 IU/ml 4.6 ± ±0.2 <0.001 Day 2 HCV RNA, log 10 IU/ml 4.0 ± ±0.2 <0.001 Day 3 HCV RNA, log 10 IU/ml 3.8 ± ± Day 28 HCV RNA, log 10 IU/ml 1.8 ± ±0.2 <0.001 Week 12 HCV RNA, log 10 IU/ml 1.6 ± ±0.2 <0.001 Virological response RVR, n (%) 100 (90.9) 11 (31.4) <0.001 Undetectable HCV RNA at week 12, n (%) 109 (99.1) 26 (74.3) <0.001 c-evr, n 9 18 p-evr, n 0 1 Non-EVR, n 1 2 Data is shown as mean ±se unless indicated otherwise. Available hepatitis C virus (HCV) RNA, complete early virological response (c-evr) or partial early virological response (p-evr) data are listed in the table. HCV RNA level (IU/ml) is log 10 transformed for its significant deviation from normal distribution. Sustained virological response (SVR) was defined as undetectable serum HCV RNA 24 weeks after discontinuation of treatment. Rapid virological response (RVR) was defined as undetectable serum HCV RNA level at day 28. c-evr was defined as undetectable serum HCV RNA at week 12 of therapy in patients who did not achieve RVR. p-evr was defined as 2 log 10 reduction of serum HCV RNA from baseline to week 12 of therapy in those who did not achieve RVR at week 4 and undetectable serum HCV RNA at week 12 of therapy. Undetectable HCV RNA level was defined as 34 IU/ml. a The viral titre at days 0.5, 1, 2, 3 and 28, and at week 12 after pegylated interferon-a2a plus ribavirin treatment. Table 3. Multivariate analyses identifying factors associated with the early viral load decline in chronic hepatitis C patients Odds ratio (95% confidence interval) Factor First 2 days a Days 2 to 28 b Age, years 1.02 ( ) 1.00 ( ) Sex, female versus male 1.79 ( ) 0.88 ( ) Body mass index, kg/m ( ) c 0.86 ( ) HCV RNA levels, log 10 IU/ml 1.12 ( ) 2.78 ( ) c Genotype, 2 versus ( ) c 1.48 ( ) Alanine aminotransferase, U/l 1.00 ( ) 1.00 ( ) Metavir fibrosis 1.55 ( ) 1.37 ( ) γ-glutaryl transpeptidase, U/l 1.01 ( ) High-density lipoprotein cholesterol, mg/dl 0.95 ( ) c Stepwise estimation with a forward selection logistic model was used. All variables were adjusted by genotype, baseline hepatitis C virus (HCV) viral load, age, sex, body mass index, Metavir fibrosis stage and alanine aminotransferase levels. Histological data were evaluated semi-quantitatively using the Metavir scoring system. a The decline of viral load >2 log 10 in the first 2 days was used as a dependent variable and 125 patients with available complete data were included in the analyses. b The decline of viral load >1 log 10 between days 2 and 28 was used as a dependent variable and 112 patients with available complete data were included in the analysis. c P<0.05 was considered as statistically significant International Medical Press

7 Early viral kinetics and HCV therapy Discussion Studies on early viral kinetics during treatment of chronic HCV infection have greatly improved our understanding about the pathophysiology of HCV infection and thus, harboured several clinical implications. For example, knowing the influence of various factors on HCV dynamics could help individualize antiviral treatment. In this study, we found that genotype, baseline viral load, BMI, pretreatment HDL and ALT levels were independently associated with early viral load decline and that genotype, baseline viral load and the viral load decline at day 28 were independent predictors of SVR. In addition, we also found that both RVR and baseline viral load had value in predicting SVR, especially in genotype 2 patients. These data could optimize current standard of care and elucidate the underlying mechanisms of these predictors. Firstly, we found that HCV genotype was an independent predictor of early viral load decline of CHC patients during PEG-IFN-a2a plus RBV treatment, except at the interval between day 2 and day 28. Considering the proposed two-phase mathematical model of hepatitis C viral dynamics [32], this fact suggests that the different SVR rates between genotype 1 and 2 are, for the most part, caused by different viral kinetics at the first phase. Secondly, baseline viral load was found to be independently predictive of the viral load decline between days 2 and 28 as well as RVR, implying the influence of baseline viral load on therapeutic efficacy is not only a simple reflection of a lower starting viral load, but also exerts its beneficial effect on the second phase of early viral kinetics. In addition, we consistently found that HCV genotype was an important predictor of SVR [3], independent of baseline viral load and the viral load decline at day 28. Several lines of evidence have proved the association of HCV infection with lipid metabolism [33 35], it is thus reasonable to link obesity and/or lipid profiles to viral kinetics. Using similar analyses, we found that BMI, a surrogate marker of obesity and host metabolic profiles, served as an independent predictor of the viral Table 4. Multivariate analyses identifying factors associated with rapid virological response or sustained virological response in 129 chronic hepatitis C patients Odds ratio (95% confidence interval) Factor RVR SVR Age, years 0.97 ( ) 0.98 ( ) Sex, female versus male 2.36 ( ) 1.57 ( ) Body mass index, kg/m ( ) a 0.90 ( ) HCV RNA levels, log 10 IU/ml 0.41 ( ) a 0.52 ( ) Genotype, 2 versus ( ) a 0.27 ( ) a Metavir fibrosis 0.96 ( ) 0.51 ( ) a Alanine aminotransferase, U/l 1.01 ( ) a 1.00 ( ) Platelet count, 1,000 cells/ml 1.01 ( ) b Stepwise estimation with a forward selection logistic model was used. All variables were adjusted by genotype, baseline hepatitis C virus (HCV) viral load, age, sex, body mass index, Metavir fibrosis stage and alanine aminotransferase. A total of 127 patients with available complete data were included in the rapid virological response (RVR) analyses and 129 in the sustained virological response (SVR) analyses. RVR was defined as undetectable HCV RNA level at day 28, SVR was defined as undetectable serum HCV RNA 24 weeks after discontinuation of treatment and undetectable HCV RNA level was defined as 34 IU/ml. Histological data were evaluated semi-quantitatively using the Metavir scoring system. a P<0.05 was considered as statistically significant. b P= Table 5. Multivariate analyses identifying factors associated with sustained virological response in 126 chronic hepatitis C patients Factor Odds ratio 95% Confidence interval P-value Age, years Sex, female versus male Body mass index, kg/m HCV RNA levels, log 10 IU/ml Genotype, 2 versus Metavir fibrosis Alanine aminotransferase, U/l HCV RNA level on day 28, log 10 IU/ml a Stepwise estimation with a forward selection logistic model was used. All variables were adjusted by genotype, baseline hepatitis C virus (HCV) viral load, age, sex, body mass index, Metavir fibrosis stage and alanine aminotransferase. A total of 126 patients with available complete data were included in the analyses. Sustained virological response was defined as undetectable serum HCV RNA 24 weeks after discontinuation of treatment and undetectable HCV RNA level was defined as 34 IU/ml. Histological data were evaluated semi-quantitatively using the Metavir scoring system. a The decline of viral titres after 28 days of pegylated interferon-a2a plus ribavirin treatment. Antiviral Therapy

8 C-S Hsu et al. Figure 2. Virological response diagram of genotype 1 CHC patients SVR RVR CHC genotype 1 Non-EVR SVR 7 2 c-evr 22 3 HCV RNA 400,000 IU/ml HCV RNA<400,000 IU/ml 28 Non-RVR p-evr 1 0 SVR Non-EVR 2 0 According to the per-protocol analyses and after stratifying the data by hepatitis C virus (HCV) genotype and baseline viral load, 94% (17/18) of genotype 1 patients who had low baseline viral load with rapid virological response (RVR) attained sustained virological response (SVR). By contrast, only 33% (8/24) of patients who had high baseline viral load without RVR attained SVR. High viral load was defined as serum HCV RNA level 400,000 IU/ml. c-evr, complete early virological response; CHC, chronic hepatitis C; p-evr, partial early virological response. Figure 3. Virological response diagram of genotype 2 CHC patients SVR RVR CHC genotype 2 c-evr 3 0 HCV RNA 400,000 IU/ml HCV RNA<400,000 IU/ml 4 Non-RVR Non-EVR 0 1 According to the per-protocol analysis and after stratifying the data by hepatitis C virus (HCV) genotype and baseline viral load, 100% (40/40) of genotype 2 patients who had low baseline viral load with rapid virological response (RVR) attained sustained virological response (SVR) and none of genotype 2 patients without RVR could achieve SVR. High viral load was defined as serum HCV RNA level 400,000 IU/ml. c-evr, complete early virological response; CHC, chronic hepatitis C. load decline before day 2 and at day 28. Furthermore, serum HDL was also identified as a predictor of the viral load decline between days 2 and 28. Our data gave support to the hypothetic interaction between obesity and/or host metabolic profiles and HCV infection and suggested the onset of interaction might occur before day 28. Further studies are needed to confirm this interesting and important finding. Although liver histology is also a well known predictor of SVR [18], its effect on early viral kinetics remains unclear. In this study, platelet count, a surrogate marker of liver fibrosis [36], was identified as a predictor of RVR (P=0.052) and pretreatment hepatic fibrosis stage as a predictor of SVR. It has been speculated that the distorted architecture of the hepatic lobe might decrease the contact between hepatocytes and drugs, leading to the impairment of therapeutic efficacy [37]. Our results conformed to this speculation and validated the important role of liver histology in predicting therapeutic responses. In addition, serum ALT levels were recognized as a predictor of RVR. Because higher ALT levels indicate a higher degree of liver inflammation or hepatocyte necrosis, the association between higher serum ALT levels and RVR, rather than EVR or SVR, indicated a close relationship between immunological response and early viral kinetics. Finally, our model including early viral load decline showed that only HCV genotype, baseline viral load and the viral load decline at day 28 could predict SVR, independent of several known viral kinetic and clinical parameters. These lines of evidence lend support to the current concept that on-treatment virological response is the most accurate predictor of response to combination therapy. In addition, we also confirmed the usefulness of both RVR and baseline viral load in predicting SVR, especially in genotype 2 patients. All genotype 2 patients who had low baseline viral load with RVR attained SVR and those who failed to achieve RVR attained no SVR. Furthermore, our data also indicated an important role of undetectable serum HCV RNA levels at week 12 and c-evr in predicting SVR, especially for genotype 1 patients. These findings not only confirm that RVR is the key predictor of SVR [38], but also suggest a paradigm shift in our current management of genotype 1 and 2 patients. Further prospective studies are needed to address this important issue. Several limitations existed in this study. First, a testing PEG-IFN-a2a dose was used at week 1, rather than using PEG-IFN-a2a plus RBV treatment from the beginning. The first 7 days represent the viral response to PEG-IFNa2a alone and the later measurements reflect the response to PEG-IFN-a2a plus RBV treatment. In addition, the RBV dose is adjusted according to body weight and not the conventional dose for genotypes 2 and 3. This unique study design might affect viral kinetics and diminish the International Medical Press

9 Early viral kinetics and HCV therapy comparability of our results with others [39]. Second, a previous pilot study from Taiwan demonstrated a better virological response rate than the Western report (genotype 1 patients received 6 months of PEG-IFN-α2b plus RBV had an SVR up to 65.8%) [30]. In addition, data to support shorter treatment duration for genotype 2 patients were also noted [40], On the basis of these observations, the Bureau of National Health Insurance in Taiwan allows the reimbursement of 24-week rather than 48-week interferon-based treatment for HCV genotype 1 patients. This is the reason why only 43% of our genotype 1 patients received a 48-week treatment and 57% of them received a 24-week treatment. Third, the use of viral load decline is inherently limited by the value of PCR negativity, and thus the decline cannot be greater than the difference between the baseline viral load and the value of PCR negativity. However, the viral load decline could render the information other than HCV RNA negativity in monitoring on-treatment virological response. Therefore, the combination of these qualitative and quantitative data will be more informative to practicing physicians. Fourth, the duration of HCV infection, a known predictor of the therapeutic efficacy, was not included in our analyses because of possible recall bias. In conclusion, HCV genotype, baseline viral load, pretreatment ALT, HDL levels, BMI and liver fibrosis stage are significantly correlated with early viral load decline of Asian CHC patients. In the meantime, HCV genotype, baseline viral load and the viral load decline at day 28 are independent predictors of SVR. These data will certainly help clinicians optimize current therapy for CHC and elucidate the underlying mechanisms of these predictors. Acknowledgements This work was supported by grants from the National Taiwan University Hospital, the Department of Health and the National Science Council, Executive Yuan, Taiwan. Disclosure statement The authors declare no competing interests. Additional file The additional file Supplementary tables can be accessed via the Volume 14 Issue 1 contents page for Antiviral Therapy, which can be found at (by clicking on Antiviral Therapy then Journal PDFs ). References 1. Kao JH, Chen DS. Transmission of hepatitis C virus in Asia: past and present perspectives. J Gastroenterol Hepatol 2000; 15 Suppl:E91 E Chen DS. Hepatitis C virus in chronic liver disease and hepatocellular carcinoma in Taiwan. Princess Takamatsu Symp 1995; 25: Strader DB, Wright T, Thomas DL, Seeff LB. Diagnosis, management, and treatment of hepatitis C. Hepatology 2004; 39: Fried MW. Side effects of therapy of hepatitis C and their management. Hepatology 2002; 36:S237 S Davis GL, Lau JY. 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