| Summary: | 博士 === 高雄醫學大學 === 醫學研究所 === 96 === Taiwan is a hepatitis B endemic area. In addition, the chronic hepatitis C virus (HCV) infection becomes another important agent responsible for hepatic diseases. The prevalence of CHC differs allover the world. The average anti-HCV positive rate is 1-3% in Taiwan which seems similar to that allover the world. However, some HCV hyperendemic areas in southern Taiwan have been elucidated. Hence the epidemiologic study in southern Taiwan is mandatory for the further prevention and treatment of CHC. Interferon-α (IFN-α) is the effective anti-HCV agent which has become the standard agent for treatment of acute or chronic HCV infection. With the oral ribavirin, combined therapy with IFN-α and ribavirin have significantly improved the efficacy of anti-HCV therapy than IFN-α monotherapy. The the combination therapy with IFN-α and ribavirin has been accepted as the recommendation to treat CHC. In addition to the hepatotropic viruses of hepatitis A-E virus, some viruses have been proposed to be associated with liver diseases. The post-transfusion hepatitis have implicated other agents might be responsible for the liver damage. The TT virus (TTV) also was found from patients with unknown hepatitis. The initial primers designed for TTV detection vy Okamoto’s have been found to be less sensitive than the primers by Takahashi et al. in 1998。More recently the SEN Virus (SENV) was isolated. The phylogenetic tree study showed that there are eight strains (A-H) for SENV. Reported have shown the two strains (SENV-D and SENV-H) were possibly associated with post-transfusion non A-E hepatitis. The impacts of the concurrent infection of other viruses on the clinical characteristics and the effect of interferon-based therapy need further studies. To accurately predict the therapeutic response is very important in the treatment for CHC. The pretreatment predictors for sustain viral responses (SVR) after anti-HCV therapy are fundamental for the development of individualized therapy. The predictors included virological and host factors. Cytokines are important mediators in the human immune response, The tumor necrosis factor-α, TNF-α and interferon-γ (IFN-γ）have also been shown to be associated with degree of fibrosis and the response to IFN-α. By the way, some cytokine gene polymorphisms which might influence the amount of cytokine (expression phenotype) have been shown. For example, the TNF-α promoter -308 (G and A) and -238 (G and A) and intron 1 gene +874 (T and A) of interferon-γheve been shown to be associated with cytokines level. Whether these genetic polymorphisms will play a role on the response to anti-HCV therapy deserved further studies. The present study included three sections aimed to investigate the epidemiology of HCV and the response of CHC patients in southern Taiwan to high-dose based therapy. The impacts of coinfection of hepatitis B virus, TTV, SENV-D and SENV-H on the HCV response to high dose IFN-α–based treatment were also investigated. The role of gene polymorphisms on the response to therapy is elucidated to determine the possible host predictors which will be helpful for the development of individualized therapy for HCV.
Section I: Prevalence of chronic hepatitis C virus infection in Kaohsiung area. Total 11239 subjects were enrolled and tested for alanine aminotransferase (ALT), hepatitis B surface antigen (HBsAg) and HCV antibody (anti-HCV) by commercially available enzyme-linked immunosorbent assay kit (Abbott Laboratories, Chicago, IL, USA). For those who were positive for anti-HCV, HCV RNA was tested by polymerase chain reaction assay (Cobas Amplicor Hepatitis C Virus Test, version 2.0; Roche Diagnostics, Branchburg, NJ; detection limit: 50 IU/mL) and the HCV genotypes were determined by type-specific primers by Okamoto et al. The prevalence of HBsAg and anti-HCVwere 13.7% and 6.3%, respectively, and eighty-four (0.7%) subjects were positive for both HBsAg and anti-HCV. Six hundred and forty-two anti-HCV-positive subjects were tested for HCV RNA and 478 (74.5%) of them were positive for HCV RNA. The distribution of HCV genotype 1b was 43.0%. The HCV RNA-positive subjects had significantly higher mean ALT level (64.2±60.5 vs. 27.8±28.3 IU/L, P <0.001) and proportion of abnormal ALT level (90.3%vs.9.7%, P<0.001) then those who were HCV RNA-negative. In stepwise logistic regression analysis, positive HBsAg was the only independent factor significantly associated with negative HCV RNA in anti-HCV subjects (Odds ratio: 0.348; 95% confidence intervals: 0.211-0.574, P<0.001).
Section II: Concurrent infection of other viruses and the therapeutic responses to interferon α- based therapy in CHC patient. Total 102 HCV RNA-positive CHC patients were enrolled in TTV study and tested for TTV DNA (using PCR primers derived from 5’non-coding region and open reading frame 2), alanine aminotransferase (ALT), genotype and RNA levels of HCV. The regimen of therapy was IFN-α 6 MU, thrice a week for 24 weeks with or without the extended therapy with IFN-α 3 MU, thrice a week for 12 weeks [total dose 432 (35 patients) and 540 MU (67patietns)]。The prevalence of TTV DNA was 51.0%. Based on multivariate regression analyses, SVR were significantly associated with pretreatment HCV RNA levels (Odds ratio, OR:0.247, 95% confidence interval, CI:0.110-0.556), HCV genotype (OR: 0.367; 95% CI: 0.137-0.980) and pretreatment levels of ALT (OR:1.014; 95% CI:1.005-1.022) but not TTV viremia. Total 164 naive CHC patients were enrolled in the SENV-D study. Sixty-one (37.2%) patients were positive for SENV-D DNA and had higher mean age than those who were negative (50.7±10.6 years vs 46.6±11.6 years, P = 0.026). There are 156 patients receiving combination therapy with IFN 6 MU plus ribavirin for 24 wk. The rate of sustained viral response (SVR) for HCV was 67.3% (105/156). By multivariate analyses, HCV genotype non-1b, younger age and lower pretreatment HCV RNA levels were significantly associated with HCV SVR (odds ratio (OR)/95% confidence interval (CI): 12.098/0.02-0.19, 0.936/0.890-0.998, and 3.131/1.080-9.077, respectively). A total of 151naïve CHC patients were enrolled in this SENV-H study and 29 (19.2%) were positive for SENV-H DNA. After combination therapy, the rate of SVR of HCV was 66.9% (101/151). By multivariate analyses, the significant factors associated with HCV SVR after combination therapy were HCV genotype non-1b, younger age and pretreatment HCV RNA levels less than 200,000 IU/mL (OR/95% CI: 0.06/0.020-0.190, 0.94/0.90-0.99 and 0.33/0.11-0.98, respectively). Total 42 (29 naïve and 13 relapsers from IFN-α monotherapy) patients with dual HBV/HCV infection and 84 age-, sex- and previous treatment-matched patients with chronic HCV infection alone were enrolled. The rate of HCV sustained virological response (SVR) was comparable among IFN-naive and IFN-relapsed HCV/HBV-coinfected patients and IFN-naive and IFNrelapsed HCV-monoinfected patients (69.0%, 69.2%, 67.2% and 57.7%, respectively; intention-to-treat analysis). For 42 dual HBV/HCV infected patients, HCV genotype 1b was significantly associated with non-response. For all 126 patietns, HCV genotype 1b [Odds ratio (OR): 0.043; 95% confidence interval (CI): 0.013–0.169], high pretreatment HCV RNA levels (OR: 0.551; 95% CI: 0.308–0.986) and more severe liver fibrosis (OR: 0.654; 95% CI: 0.427–1.002) were significantly associated with a lower HCV SVR by multivariate analyses.
Section III: High dose interferon-α and ribavirin combination therapy for CHC: Association between cytokine gene polymorphism and response. The present study aimed to investigate the distribution of polymorphic sequence in the tumor necrosis factor (TNF)-α promoter at position -308 and -238 in Taiwanese chronic hepatitis C (CHC) patients and the impacts of these polymorphism on the response to combination therapy with high dose interferon-α (IFN-α) and ribavirin. Total 141 CHC patients undergoing combination therapy with high dose IFN-α (6MU thrice weekly) and ribavirin (1000-1200mg per day according to the body weight; 1,000mg for <75 kg，1,200mg for >75 kg) for 24 weeks were enrolled for thestudy of polymorphic sequence in the tumor necrosis factor (TNF)-α promoter at position -308 and -238. The distribution of TNF-α promoter polymorphism at position -308 was: TNF308.1(G)/ TNF308.1: TNF308.1/ TNF308.2(A): TNF308.2/ TNF308.2 = 108 (76.6%): 30 (21.3%): 3 (2.1%)，The frequency of TNF308.1 (low level of TNF-α) 及TNF308.2 (high level of TNF-α) was 87.2% and 12.8%, respectively. Two (1.4%) of the 141 CHC patients TNF238.1/ TNF238.2 and all the others had TNF238.1/ TNF238.1. One hundred (70.9%) patients achieved SVR。Multivarite analyses showed that the HCV genotype (Odds ratio, OR: 10.464; 95% conficence interval, CI: 3.916-27.961)、pretreatment HCV RNA level(OR: 0.396; 95% CI:0.214-0.733)、without TNF308.2 (OR:3.648; 95% CI:1.273-10.455) and higher necroinflammatory activity (OR: 1.284; 95% CI:1.034-1.594) were factors associated with SVR to combination therapy with high dose IFN-α and ribavirin. Of 63 patients with genotype 1b infection who received combination therapy, the independent factors predicting HCV SVR, based on multivariate regression analyses, were pretreatment HCV RNA levels lower than 200,000IU/ml and TNF308.2 allele non-carrier (OR/ 95% C.I.: 22.610 /3.844-132.987 and 5.53 /1.031-29.651, respectively). We further stratified the patients to groups by HCV 1b or non-1b genotype infection or with high (>=200,000IU/ml) or low (<200,000IU/ml) pretreatment HCV RNA levels. The proportion of patients achieving SVR was significantly lower among 43 patients with genotype 1b infection and high pretreatment HCV RNA levels (defined as “difficult-to-treat” patients) than the others. When considering -308 TNF alleles for these 43 difficult-to-treat patients, 8 of them were TNF308.2 carriers, and none of these 8 patients achieved SVR after therapy. The proportion of patients achieving SVR was significantly lower than the 40% (14/35) of the other patients without TNF308.2 allele (P=0.029). Total 302 histologically-proved CHC patients were enrolled in the study of polymorphic sequence at position +874 in the IFN- γ gene. The distribution of genotypes for IFN–gamma at position +874 were T/T: 12 (4.0%), T/A: 71 (23.5%) and A/A: 219 (72.5%). The frequency of the T allele was 15.7% Multivariate analyses showed that patietns with T allele (Odds ratio, OR: 2.15，95% confidence interval, CI: 1.128-5.622) and higher age (OR: 1.065，95% CI:1.128-5.622) are independent factors associated with cirrhosis. Total 150 histologically-proved CHC patients undergoing combination therapy with high dose IFN-α (6MU thrice weekly) and ribavirin (1000-1200mg per day according to the body weight; 1,000mg for <75 kg，1,200mg for >75 kg) for 24 weeks. 70.7% (106/150) of patients achieved (SVR). Based on multivariate regression analyses, the independent factors predicting HCV SVR after combination therapy were HCV genotype non-1b (P<0.001) and low pretreatment HCV RNA levels (P=0.041) (odds ratios/ 95% C.I.: 10.150/ 4.023-25.609 and 0.581/ 0.345-0.979, respectively).
Conclusions: The prevalence of anti-HCV and HBsAg was 6.3% and 3.7% among adults aged 40 to 65 years in southern Taiwan. HCV viremia was associated with a higher ALT level and status of HBsAg which implicated the viral interference between these two virused. TTV viremia is highly prevalent among Taiwanese CHC patients (51%) and related to increased ages. Neither severity of liver disease nor replication and genotype distribution of HCV was affected by concurrent TTV infection. The prevalences of SENV-D and SENV-H coinfection were 37.2% and 19.2%, respectively in CHC patient in southern Taiwan. Coexistent SENV-D infection is associated with higher ages and coexistent SENV-H infection is associated with HCV genotype 1b infection. Viremia of both viruses was not associated with severity of liver disease in CHC patients. The inheritance of the IFN–gamma polymorphism at the position +847 is associated with cirrhosis in patients with CHC. The high dose IFN-α therapy achieved 41.2% of SVR rate which was associated with pretreatment HCV RNA, ALT levels and HCV genotype, and TTV viremia did not influence the HCV response. Around two-thirds of CHC patients achieved SVR to combination therapy with high dose interferon-α (IFN-α) (6 MU thrice a week) and ribavirin (1000-1200mg per day) for 24 weeks. HCV genotype, pretreatment HCV RNA levels and age are predictive factors for HCV SVR and CHB, SENV-D or SENV-H coinfection and inheritance of the IFN-γ polymorphism at the position +847 do not affect the HCV response.
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