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Dual hepatitis B virus and hepatitis C virus infection: To treat or not to treat, and how? EDITORIAL
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PETR HUSA
Department of Infectious Diseases, Teaching Hospital Brno, Czech Republic
Journal of Gastroenterology and Hepatology
May 2005
See original study article below
Hepatitis B virus (HBV) and hepatitis C virus (HCV) share several modes of transmission; not surprisingly, coinfection by the two viruses is not uncommon, particularly in areas where the two viruses are endemic and among subjects with a high risk of parenteral infection. Seroprevalence studies have shown that approximately 3-18% of patients with chronic HBV infection are also infected with HCV. 1-7 In addition, HBV/HCV coinfection is of clinical relevance because it is associated with severe chronic liver diseases poorly sensitive to interferon (IFN) treatment and with a high risk of hepatocellular carcinoma (HCC) development. 8-11 The virologic profiles of HBV and HCV and their interplay in coinfection are still largely undefined because most studies of the natural and post-therapeutic course of chronic hepatitis C have, among the main exclusion criteria, coexisting HBV infection and vice versa. These patients may show different virologic profiles: they may be either hepatitis B e antigen (HBeAg) or anti-HBe positive; they may have low HBV-DNA levels with high levels of HCV-RNA, and vice versa low or high levels for both viruses. 12,13 However, all these data come from cross-sectional studies and, consequently, we do not know whether the levels of viremia of the two viruses represent a constant feature or only temporary changes in complex kinetics evolving with respect to diagnosis and therapy in patients with HBV/HCV coinfection. 14
In most patients, HBV replication is suppressed while HCV replication remains active. However, the opposite has also been observed. In an Italian multicenter study, 837 hepatitis B surface antigen (HBsAg) positive patients were evaluated. Anti-HCV antibodies were present in 59 cases (7%). Independent predictors of dual infection were age >42 years, history of intravenous drug use, blood transfusion and residence in the south of the country. Of 36 HBV/HCV coinfected patients, 16 (44%) had only HBV-DNA in serum, five (14%) had both HBV-DNA and HCV-RNA, nine (25%) had HCV-RNA alone and six (17%) tested negative for both. In this series of unselected HBsAg positive patients, 89% of all the cases were HBeAg negative/anti-HBe positive and 100% of the patients with dual HBV/HCV infection were positive for anti-HBe. This virologic profile reflects the profound modifications in the epidemiological and clinical profile of HBV infection in southern Europe, and other developed countries that have emerged in the last decade. A spontaneous downtrend in the infection rate coupled with the vaccination campaign against HBV have caused a progressive reduction in newly acquired HBV infections. As a consequence, the proportion of chronic HBeAg positive infections has rapidly decreased over time, while in the aging cohort of HBV carriers, HBeAg-negative mutants are increasingly selected. 7
Persons with dual HBV and HCV infection are difficult-to-treat patients. They respond poorly to IFN alpha monotherapy. 8,15-17 IFN alpha monotherapy was more effective in clearing HBV infection than HCV infection 16 and when investigators used higher doses of IFN alpha (9 million units [MU] instead of 6 MU three times weekly). 17
In a pilot study by Liu et al., 21 HBV-DNA and HCV-RNA positive patients were treated for 24 weeks with a combination of ribavirin (1200 mg daily) and IFN alpha-2a (6 MU thrice weekly for 12 weeks, followed by 3 MU thrice weekly for the subsequent 12 weeks). They reported that combination therapy could achieve a similar HCV clearance rate in dual infection cases, comparable with HCV infection alone. 18
In this issue of the Journal, Hung et al. 19 add strong evidence to this notion. They treated 36 patients, all HBsAg, anti-HCV and HCV RNA positive with 18 of these HBV-DNA positive by Amplicor (Cobas Amplicor Monitor, Roche Diagnostics, Branchburg, NJ, USA), with a combination of IFN alpha-2b and ribavirin. Thirteen patients received 3 MU and 23 patients received 5 MU IFN thrice weekly for 24 weeks. Ribavirin was given at a daily dose of 800-1200 mg according to the weight of the patients. The sustained virologic response (SVR) was evaluated 48 weeks after discontinuation of the treatment. Another 72 patients with HCV infection alone served as controls. The study demonstrated that combination therapy induced a similar response against HCV in patients with dual HBV/HCV infection or HCV single infection (69%vs 71% of SVR). There was no significant difference in sustained HCV clearance between the 3 MU group and the 5 MU group (85%vs 61%).
At the end of 48 weeks follow-up, only two (11%) of 18 pretreatment viremic patients had negative serum HBV-DNA (< 200 copies/mL). This shows that such therapy was not sufficient to obtain satisfactory efficacy in clearing HBV viremia.
The bad news from this study is the reappearance of HBV-DNA in the serum of two (13%), six (40%) and eight (53%) of 15 patients without pretreatment HBV viremia at the end of treatment, and at the 24 and 48 weeks of follow-up, respectively. All these eight patients had negative HCV-RNA in serum at the end of treatment. The authors found the reappearance of both HBV-DNA and HCV-RNA in two patients at post-treatment week 48. The remaining six patients with reactivation of HBV infection achieved sustained clearance of HCV replication. IFN alpha therapy did not induce a severe HBV reactivation following suppression of HCV during and after the treatment course. The results of this study are in accordance with a previous report that HBV reactivation might develop following a decrease in HCV replication with IFN alpha therapy in a patient with positive HCV-RNA, anti-HBe and low serum HBV level. 20
In conclusion, combination therapy with IFN alpha and ribavirin can be efficient for sustained clearance of HCV replication in a significant proportion of patients with dual HBV/HCV infection, and the HCV clearance rate in these patients is comparable with those infected with HCV alone. The influence of this treatment on HBV replication is lower. The danger and significance of reactivation of HBV infection after a decrease of HCV replication needs to be evaluated in longitudinal studies. At present, majority of patients with chronic hepatitis C are treated with a combination of pegylated IFN alpha and ribavirin. This combination is more efficient and better tolerated in patients with chronic HCV infection alone. It is very probable that it will be the same in patients with HBV/HCV coinfection but it is necessary to confirm this hypothesis in clinical trials.
References
1 Fattovich G, Tagger A, Brollo L et al. Hepatitis C virus infection in chronic hepatitis B carriers. J. Infect. Dis. 1991; 163: 400-2.
2 Pontisso P, Ruvoletto MG, Fattovich G et al. Clinical and virological profiles in patients with multiple hepatitis infections. Gastroenterology 1993; 105: 1529-33.
3 Sato S, Shigetoshi F, Tanaka M et al. Coinfection of hepatitis C virus in patients with chronic hepatitis B infection. J. Hepatol. 1994; 21: 159-66.
4 Crespo J, Lozano JL, de la Cruz F et al. Prevalence and significance of hepatitis C viremia in chronic active hepatitis B. Am. J. Gastroenterol. 1994; 89: 1147-51.
5 Zarski J-P, Bohn B, Bastie A et al. Characteristics of patients with dual infection by hepatitis B and C viruses. J. Hepatol. 1998; 28: 27-33.
6 Buti M, Costa X, Valdes A et al. Study of hepatitis B virus replication and infection by other hepatitis viruses in patients with chronic hepatitis B virus infection. Gastroenterologia Y. Hepatologia 2002; 25: 295-8 (in Spanish).
7 Gaeta GB, Stornaiuolo G, Precone DF et al. Epidemiological and clinical burden of chronic hepatitis B virus/hepatitis C virus infection. A multicenter Italian study. J. Hepatol. 2003; 39: 1036-41.
8 Weltman MD, Brotodihardjo A, Crewe EB et al. Coinfection with hepatitis B and C or B, C and delta viruses results in severe chronic liver disease and respond poorly to interferon-alpha treatment. J. Viral Hepatol. 1995; 2: 39-45.
9 Shiratori Y, Shiina S, Zhang PY et al. Does dual infection by hepatitis B and C viruses play an important role in the pathogenesis of hepatocellular carcinoma in Japan? Cancer 1997; 80: 2060-7.
10 Mazzella G, Saracco G, Festi D et al. Long-term results with interferon therapy in chronic type B hepatitis: a prospective randomized trial. Am. J. Gastroenterol. 1999; 94: 2246-50.
11 Chiaramonte M, Stroffolini T, Vian A et al. Rate of incidence of hepatocellular carcinoma in patients with compensated viral cirrhosis. Cancer 1999; 85: 2132-7.
12 Sagnelli E, Coppola N, Scolastico C et al. Virologic and clinical expressions of reciprocal inhibitory effect of hepatitis B, C, and delta viruses in patients with chronic hepatitis. Hepatology 2000; 32: 1106-10.
13 Squadrito G, Orlando ME, Pollicino T et al. Virological profiles in patients with chronic hepatitis C and overt or occult HBV infection. Am. J. Gastroenterol. 2002; 97: 1518-23.
14 Raimondo G, Pollicino T, Squadrito G. Clinical virology of hepatitis B virus infection. J. Hepatol. 2003; 39 (Suppl. 1): S26-S30.
15 Liaw XF, Chien RN, Lin SM et al. Response of patients with dual hepatitis B virus and C virus infection to interferon therapy. J. Interf. Cytok. Res. 1997; 17: 449-52.
16 Guptan RC, Thakur V, Raina V, Sarin SK. Alpha-interferon therapy in chronic hepatitis due to active dual infection with hepatitis B and C viruses. J. Gastroenterol. Hepatol. 1999; 14: 893-8.
17 Villa E, Grottola A, Buttafoco P et al. High doses of alpha-interferon are required in chronic hepatitis due to coinfection with hepatitis B virus and hepatitis C virus: long term results of a prospective randomized trial. Am. J. Gastroenterol. 2001; 96: 2973-7.
18 Liu CJ, Chen PJ, Lai MY et al. Ribavirin and interferon is effective for hepatitis C virus clearance in hepatitis B and C dually infected patients. Hepatology 2003; 37: 568-76.
19 Hung CH, Lee CM, Lu SN et al. Combination therapy with interferon-alpha and ribavirin in patients with dual hepatitis B and hepatitis C virus infection. J. Gastroenterol. Hepatol. 2005; 20: 727-32.
20 Villa E, Grottola A, Trande P et al. Reactivation of hepatitis B virus infection induced by interferon (IFN) in HBsAg-positive, anti-HCV-positive patients. Lancet 1993; 341: 1413.
Combination therapy with interferon-alpha and ribavirin in patients with dual hepatitis B and hepatitis C virus infection
Journal of Gastroenterology and Hepatology
May 2005
CHAO-HUNG HUNG*, CHUAN-MO LEE*, SHENG-NAN LU*, JING-HOUNG WANG*, HUNG-DA TUNG*, CHIEN-HUNG CHEN* and CHI-SIN CHANGCHIEN*
*Division of Hepatogastroenterology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan
ABSTRACT
Background: Patients with dual hepatitis B virus (HBV) and hepatitis C virus (HCV) infection have responded poorly to interferon (IFN) monotherapy. The purpose of the present paper was to assess the effect of combined IFN-alpha and ribavirin therapy in patients infected with both hepatitis B and C.
Methods: Thirty-six patients received 3 or 5 MU IFN-alpha-2b thrice weekly and oral ribavirin (800-1200 mg/day) for 24 weeks. All patients had positive hepatitis B surface antigen, antibody to HCV, and HCV-RNA. Before treatment, one patient had positive hepatitis B e antigen. Eighteen patients had positive HBV-DNA tested by Amplicor (Cobas Amplicor Monitor, Roche Diagnostics, Branchburg, NJ, USA), with a mean HBV-DNA level of 3.1 ± 0.9 log copies/mL. Another 72 patients with HCV infection alone served as controls.
Results: Adverse events led to withdrawal in three patients receiving 5 MU IFN. Based on an intent-to-treat analysis, the biochemical response and serum HCV clearance rate at the end of 48 weeks follow up was similar in patients with dual infection and HCV infection alone (56% vs 72%; and 69% vs 71%, respectively). There was no significant difference in sustained HCV clearance rate between the 3-MU group (n = 13) and the 5-MU group (n = 23; 85% vs 61%). At the end of 48 weeks follow up, two (11%) of 18 pretreatment viremic patients had negative serum HBV-DNA (<200 copies/mL), while eight of those without pretreatment viremia had reoccurrence of HBV-DNA.
Conclusions: Combination therapy with IFN-alpha and ribavirin was effective in achieving sustained HCV clearance in patients with dual HBV and HCV infection, comparable to those with hepatitis C infection alone. Combination therapy using 3 MU IFN-alpha seemed as effective as 5 MU, and was well tolerated in the study population. However, large-scale control trials are necessary to clarify these findings.
INTRODUCTION
Chronic infection with hepatitis B virus (HBV) and hepatitis C virus (HCV) accounts for a substantial proportion of liver disease worldwide. Because both agents share the same transmission routes, dual infection may occur and seems not uncommon, particularly in areas where the two viruses are endemic and among people of high risk for parenteral infections. 1-5 Seroprevalence studies have shown that approximately 10-20% of patients with chronic HBV infection are also infected by HCV. 3-7 It has been reported that patients with dual HBV and HCV infection have more severe outcomes than those with single viral infection. Dual infection tends to aggravate the severity and progression of chronic liver disease and has a much higher relative risk for the development of hepatocellular carcinoma. 8-12
At present, interferon (IFN) is the antiviral agent approved for the treatment of chronic HBV or HCV infection. Interferon-alpha monotherapy produces a loss of HBV-DNA and hepatitis B e antigen (HBeAg) in 30-40% of chronic HBV patients and a sustained virological response in <20% of chronic hepatitis C cases. 13,14 The recent introduction of IFN plus ribavirin combination therapy has been shown to achieve double the sustained virological response rate of IFN therapy alone and is considered to be the most effective treatment for chronic HCV infection. 15,16 In contrast, a pilot study showed that combination therapy with IFN-alpha and ribavirin is efficacious to treat viremic anti-HBe-positive patients who have failed previous IFN therapy. 17 According to a number of reports, it appears that HBV and HCV co-infection are resistant to IFN monotherapy. 9,18-21 Recently, Liu et al. treated 21 patients infected with both hepatitis B and C with 24 weeks of combination therapy using ribavirin and 6 MU IFN-alpha2a thrice weekly for 12 weeks, followed by 3 MU IFN-alpha2a thrice weekly for the subsequent 12 weeks. They reported that combination therapy could achieve a similar HCV clearance rate in dual infection cases, comparable with HCV infection alone. 22 Nevertheless, studies on combination therapy for dual HBV and HCV infection are limited and an optimal IFN dose needs to be defined. Therefore, we conducted the present study.
AUTHOR DISCUSSION
In this present study we investigated the efficacy of combination therapy with IFN-alpha-2b and ribavirin in patients with HBV and HCV dual infection. We demonstrated that 69% of the patients had sustained HCV-RNA clearance, and 11% had cleared serum HBV-DNA (<200 copies/mL) at the end of 48 weeks follow up. The sustained biochemical response rate was 56%, which was more closely related to HCV clearance rate. In addition, our study demonstrated that combination therapy induced a similar response against HCV in patients with dual HBV-HCV infection or sole HCV infection. The response rate was higher than that seen in the previous studies using IFN monotherapy. 9,18-21 However, the present data are in accordance with a recent study using combination therapy, suggesting that the addition of ribavirin greatly enhances HCV-RNA clearance in patients with dual HBV and HCV infection. 22 Although there were no factors with statistical significance contributing to sustained HCV clearance in patients with dual infection, the sustained responders had a lower ratio of genotype 1b (40% vs 64%) than non-responders. The predictors of sustained response were not significantly different between patients with dual infection and sole HCV infection, implying that HBV co-infection did not interfere with the response to combination therapy against HCV.
In the present study patients received 3 or 5 MU of IFN-alpha2b thrice weekly in combination with ribavirin for 24 weeks. Although this was not a randomized controlled trial, it seemed that 3 MU of IFN-alpha2b was as effective as 5 MU; and was well tolerated in the study population. However, it was not possible to reach a firm conclusion because of small sample size, therefore large-scale control trials are necessary to confirm this point. The present sustained HCV clearance rate was higher than that of the other studies using combination therapy, but was similar to the data we previously reported. 23 The main reason is probably the low percentage of HCV genotype 1b in the present patients.
In contrast, in the present study serum HBV-DNA was positive in 50% of the patients before treatment. At the end of treatment HBV-DNA clearance was obtained in five patients (28%), and in two patients (11%) at the end of 48 weeks follow up. Eight of the patients without pretreatment viremia had reoccurrence of HBV-DNA, thus indicating that such therapy was not sufficient to obtain satisfactory efficacy in clearing hepatitis B viremia.
It is worth noting that the majority of the present patients were anti-HBe positive and had a lower serum HBV titer (median approx. 103 copies/mL). Because HBV infection in endemic areas such as Taiwan is usually acquired perinatally or at early childhood, it is likely that the present patients with dual HBV and HCV infection had chronic HBV infection before they were superinfected with HCV. 25 In such cases, HCV may replace HBV as the cause of the continuing chronic hepatitis. 26 A previous report has demonstrated that HBV reactivation might develop following a decrease in HCV replication with IFN therapy in a patient with positive HCV-RNA, anti-HBe, and low serum HBV titer. 27 Villa et al. therefore claimed that high doses of IFN (9 MU thrice weekly for 6 months) were recommended in inhibiting HBV and HCV replication at the same time when treating such patients dually infected with hepatitis B and C. 21 However, in the present study the combined ribavirin and 3- or 5-MU of IFN therapy did not seem to induce a severe HBV reactivation following suppression of HCV during and after the treatment course. The HBV changes were not related to the appearance of hepatitis C viremia in the anti-HBe-positive patients. In addition, the initial HBeAg-positive patient encountered a transient HBV surge and a small rise in ALT levels following HCV-RNA clearance after the 16th week of treatment, which reverted to normal levels at the end of therapy. Thereafter the patient achieved sustained HCV clearance, and the HBV titer did not rebound at the end of follow up.
In summary, the present study indicates that in patients with dual HBV and HCV infection, combination therapy of IFN with ribavirin could achieve a sustained HCV clearance rate comparable with those infected with HCV alone. The HBV-DNA clearance in HBV-HCV dual infection seemed relatively low in the treatment but the biochemical response was more closely related to HCV clearance.
RESULTS
Of the 36 patients, 33 completed the treatment course and were followed up for at least 48 weeks. Three patients in the 5-MU group withdrew because of intolerant influenza-like symptoms in two, and neurological side-effects in one. Forty-seven percent were of HCV genotype-1b. One patient was found positive for HBeAg and negative for anti-HBe, and the remaining 35 patients were found to be negative for HBeAg and positive for anti-HBe. The serum HBV-DNA was positive in 18 patients (50%), assayed using quantitative PCR assay (COBAS Amplicor HBV Monitor). Pathology of the liver biopsy specimens revealed that three of the patients (9%) had evidence of cirrhosis before treatment.
As shown in Table 1, patients with dual HBV-HCV infection and sole HCV infection were similar regarding age, gender, ratio of HCV genotype-1b, HCV viral load, initial ALT levels, histological activity index (HAI) scores, and IFN dosage.
Based on an intent-to-treat analysis, the responses to combination therapy during and after the end of treatment in patients with dual HBV-HCV infection and sole HCV infection are shown in Table 2. At the end of the 24 weeks of therapy, the biochemical response of patients with dual infection was lower than that of those with single HCV infection (67% vs 92%, P = 0.002). However, the ALT normalization rate was similar in both groups at 24-week follow up (61% vs 72%, P = 0.276), and at 48-week follow up (56% vs 72%, P = 0.089). There was no significant difference in the HCV clearance rate between these two groups at the end of treatment (92% vs 94%, P = 0.684) and at the end of 48 weeks follow up (69% vs 71%, P = 1.0). Eighteen patients had positive pretreatment HBV-DNA in serum, which became negative in five patients (28%) at the end of treatment, and in two patients (11%) at the end of 48 weeks follow up. None of the patients lost serum HBsAg.
Table 3 shows the factors contributing to sustained HCV clearance in patients with dual HBV and HCV infection and with sole HCV infection. There was no significant difference in the sustained HCV clearance rate between the 3-MU group (n = 13) and the 5-MU group (n = 23; 85% vs 61%). In the group with sole HCV infection, the predictors of sustained HCV clearance were non-genotype 1b (P = 0.002) and a low pretreatment viral load (P = 0.007). Multiple logistic regression analysis revealed that genotype non-1b (odds ratio [OR]: 6.56; 95% confidence interval [CI]: 1.94-22.2, P = 0.002) and log HCV-RNA (OR: 2.12; 95%CI: 1.15-3.91, P = 0.016) were significant predictors. However, in the group with dual infection there was no factor significantly predicting the response to sustained HCV clearance.
Table 4 shows the profiles of hepatitis B virus loads and hepatitis C viremia in 33 dual-infection patients receiving a complete course of combination therapy. Of the 15 patients with negative pretreatment HBV-DNA, two (13%), six (40%), and eight (53%) had reoccurrence of hepatitis B viremia at the end of treatment, and at 24- and 48-week follow up, respectively. The HBV changes did not correlate with the appearance of hepatitis C viremia. The negativity of HBV-DNA at the end of 48 weeks follow up was associated with the absence of pretreatment HBV-DNA (47% vs 11%, P = 0.047).
In the present study only one patient had initially positive HBeAg. This patient experienced HBV reactivation and a mildly elevated ALT after HCV clearance from the 16th week of therapy. The ALT reverted to a normal level at the end of therapy, and HCV-RNA was still negative at the end of 48 weeks follow up, accompanied with a lowering titer of HBeAg and without a rebound of HBV titer.
Patients
Between April 1999 and December 2001, 36 consecutive patients (22 male, 14 female) with chronic dual HBV and HCV infection, aged 48.8 ± 12.6 years (range 19-64 years), were enrolled in the present study. All patients had positive hepatitis B surface antigen (HBsAg), antibody to HCV, detectable HCV-RNA (Amplicor, Roche Diagnostics, Branchburg, NJ, USA), and had elevated alanine aminotransferase (ALT) values at least two times the normal upper limit on three occasions within 6 months before enrollment. Exclusion criteria included human immunodeficiency virus infection, alcohol abuse, autoimmune hepatitis, other causes of liver disease, and major contraindications to IFN or ribavirin therapy. The patients underwent liver biopsies within 3 months before the start of therapy, except for one patient with a contraindication (thrombocytopenia). The histological grading and staging of chronic liver diseases were based on a modified Knodell histology index, reflecting the degree of hepatic inflammation and fibrosis, respectively.
All patients gave informed consent prior to treatment. They were treated with a 24-week course of 3 MU (n = 13) or 5 MU (n = 23) of IFN-alpha2b (Intron-A, Schering-Plough, Kenilworth, NJ, USA) subcutaneously thrice weekly and with oral ribavirin daily (Rebetol, Schering-Plough, Auxerre, France). The dosage of IFN-alpha2b was not randomized and was given by the clinical doctor. Ribavirin was given at a total daily dose of 1000 mg for patients who weighed <=75 kg and 1200 mg for patients who weighed >75 kg. The dosage of ribavirin was modified according to the drop of hemoglobin, ranging from 800 to 1200 mg every day. All patients were observed every 1-2 weeks for the first 4 weeks and every 4 weeks thereafter during treatment. The protocol was the same for the patients with single HCV infection as in our previous studies. 23,24 The ALT levels were followed monthly until 6 months after the end of therapy, and were measured every 3 months thereafter. Serum HBV-DNA and HCV-RNA were assessed at enrollment, at the end of treatment, and at weeks 24 and 48 after treatment. Sustained virological response was defined as the absence of HBV-DNA or HCV-RNA in serum at week 48 after treatment. During the same period (April 1999-December 2001), control patients with sole HCV infection were recruited with a ratio of 1:2 from our database at Kaohsiung Chang Gung Memorial Hospital. They were matched with 36 dual-infection patients according to sex, genotype (1b or non-1b), IFN dosage (3 or 5 MU), age (±5 years), viral load (>=2 x 106 or <2 x 106 copies/mL), initial ALT level (>=100 or <100 IU/L), and fibrosis score (0-2 or 3-4).
Laboratory investigations
Hepatitis B virus markers, including HBsAg, HBeAg, antibody to HBeAg (anti-HBe), and antibodies to hepatitis D virus, were assayed using commercially available enzyme immunoassay kits (Abbott Laboratories, North Chicago, USA). Serum HBV-DNA was quantified with a sensitive polymerase chain reaction (PCR) assay (COBAS Amplicor HBV Monitor, Roche Diagnostics) with a detectable limit at 200 copies/mL.
Anti-HCV antibody was assessed using third-generation ELISA (Ax SYM HCV 3.0, Abbott Laboratories, Chicago, IL, USA). Qualitative detection of HCV-RNA was performed by a standardized qualitative reverse transcription-polymerase chain reaction (RT-PCR) assay (Amplicor, Roche Diagnostics), using biotinylated primers for the 5' non-coding region. The lowest detection limit of this assay was 100 copies/mL. Serum HCV-RNA levels were determined by a branched-DNA (b-DNA) signal amplification assay (VERSANT HCV-RNA 3.0. Assay, Bayer Diagnostics, Emeryville, CA, USA). This assay was a sandwich nucleic-acid hybridization procedure with a detectable limit at 3400 copies/mL. Genotyping of HCV was done by reverse hybridization assay (Inno-LiPA HCV II; Innogenetics NV, Gent, Belgium) in the HCV-Amplicor products.
Statistical analysis
Quantitative variables were expressed as mean ± SD. Mann-Whitney U-test was used to compare continuous variables between the two groups. Differences between dichotomous variables were evaluated with c2 analysis or Fisher's exact test depending on the size of the sample. Multiple logistic regression analysis was used to identify the independent factors that might influence the response to sustained HCV clearance. P < 0.05 was considered statistically significant.
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