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1 February 1995 | Volume 122 Issue 3 | Pages 161-168
Objective: To analyze the distribution of hepatitis C virus (HCV) genotypes among patients positive for antibody to HCV (anti-HCV) according to age, severity of liver disease, and duration of infection; to investigate the influence of HCV genotypes on response to interferon-
Design: Cross-sectional study.
Setting: 3 university hospitals and 2 research units.
Patients: 3 groups of French and Italian patients with chronic HCV infection and detectable serum HCV RNA: Group 1 included 35 patients with hepatocellular carcinoma; group 2, 71 patients with cirrhosis who did not have hepatocellular carcinoma; and group 3, 114 patients with chronic active hepatitis. 106 of the patients with chronic hepatitis or cirrhosis were treated with interferon-
Measurements: Genotyping by polymerase chain reaction with capsid-specific primers; serum HCV RNA by branched DNA (bDNA) signal amplification.
Results: Hepatitis C virus genotype 1b (II) was the most prevalent genotype (61.8%). In a univariate analysis, it was associated with older age (<40 years, 47.4%;
Conclusions: The prevalence of HCV genotypes in French and Italian patients has been changing; the prevalence of HCV type 1b (II) infection has progressively decreased, although it still accounts for most HCV-related cirrhosis and hepatocellular carcinoma. High HCV viremia levels and HCV genotype type 1b (II) are independent predictors for poor response to interferon-
For affiliations and current author addresses, see end of text.
*For a listing of collaborators, see Appendix 2.
Although each genotype was initially thought to have a distinct geographic location (HCV type I being the major genotype in the United States and Europe and types II, III, and IV being reported only in Japan), it is now clear that different genotypes are in fact distributed worldwide [16]. In this context, a major issue concerns the actual clinical effect of these different HCV genotypes.
Evidence from studies conducted in Japan and, more recently, in Europe, now suggests that the sensitivity of different HCV genotypes to interferon-
Our intent, therefore, was to study a large group of patients to elucidate the following points: whether the pattern of distribution of HCV genotypes has been changing over time; whether severity of illness (chronic hepatitis, cirrhosis, and hepatocellular carcinoma) and response to interferon-
Key terms are defined in Appendix 1.
We retrospectively analyzed 220 patients with chronic liver disease positive for antibody to HCV who were living in France and Italy. Inclusion criteria were biopsy-proven chronic hepatitis of any severity and detectable HCV RNA that allowed HCV genotyping; patients were selected from among those referred to three liver units between 1988 and 1992. Two units were located in Paris and one in Milan; all three were large reference centers with national recruitment. One hundred twenty-four patients were French, 74 were Italian, and 22 were from countries in the Mediterranean area. Patients were divided into three groups Table 1: Group 1 consisted of 35 patients with histologically confirmed hepatocellular carcinoma; group 2 consisted of 71 patients with cirrhosis and no evidence of hepatocellular carcinoma; and group 3 consisted of 114 patients with chronic hepatitis. Diagnosis of hepatitis was based on appropriate biochemical and histopathologic criteria [23]. The mode of infection was nonambiguously known for 60 patients who had had transfusions and for 27 patients who had used intravenous drugs, but no definite causes could be identified for 133 patients. The date of infection was known for 107 patients: For 78 patients, it was considered to be the date of blood transfusion or the date when intravenous drug use began; 29 patients were followed for non-A, non-B hepatitis infection for at least 10 years. ARTICLE
Hepatitis C Virus Type 1b (II) Infection in France and Italy
therapy; and to study HCV viremia levels in relation to genotypes and severity of liver disease. (3 MU subcutaneously 3 times/wk for 
6 months). 60 years, 80.4%; P = 0.001), longer duration of disease (
10 years, 40.4%; 20 years, 86.7%; P = 0.005), and cirrhosis with or without hepatocellular carcinoma (78.4% compared with 53.8% for chronic hepatitis; P < 0.001). Viremia levels did not differ between patients infected with HCV type 1b (II) and those infected with other HCV genotypes. Patients with HCV type 1b (II) responded to interferon- therapy significantly less than did patients with other HCV genotypes (P = 0.01). In a multivariate analysis, age and cirrhosis were independently associated with HCV genotype 1b (II). Genotype and HCV viremia level were independent predictors of response to interferon- therapy. therapy and should be considered in the management of patients with HCV infection.
Nucleotide sequence analyses of many strains of the hepatitis C virus (HCV) from several geographic areas have shown substantial variability among the different isolates [1-5]. The degree of variability differs markedly throughout the viral genome: High-sequence divergence is found in some nonstructural proteins and in the regions encoding for the putative envelope proteins, and the E2 region contains two hypervariable regions. In contrast, the 5' untranslated and core protein encoding domains show higher conservation among different strains. Numerous classifications have been proposed that, although unconcerted, distinguish among different HCV genotypes. A genotype is defined by the sequence similarity in both coding and noncoding parts of the viral genome: Nucleotide divergence is less than 10% within each genotype and greater than 20% between distinct genotypes [6, 7]. Genotyping can be done by sequencing HCV isolates, but this is time-consuming. Faster procedures have recently been developed, all of which are based on the polymerase chain reaction (PCR), which can be done using either type-specific or conserved primers, followed by hybridization with a specific probe [8, 9]. Additional assays have been developed using restriction fragment length polymorphism analysis in the 5' noncoding region [10, 11] or in the NS5 region [12]. Finally, Okamoto and colleagues [13] have proposed a method that uses type-specific primers located on the capsid domain and allows HCV isolates to be subgrouped into at least five types [14]. Our strategy for genotyping is based on a modification of this approach [13]. Another method has been proposed by Simmonds and colleagues [11] and is based on a phylogenetic study. Recently, a system for the nomenclature of hepatitis C viral genotypes was proposed [15]; types classified as I, II, III, IV, and V by Okamoto and colleagues are classified in this new system as 1a, 1b, 2a, 2b, and 3a, respectively. therapy might vary substantially [17, 18]. In contrast, it is still unclear whether some HCV genotypes lead to a more severe course of viral infection and are thus associated with the development of primary liver cancer. Reports from Japan addressing this question have been inconclusive; a limited study from Italy suggested that "Japanese types" of HCV (precise genotyping was not yet available) were associated with cirrhosis [19, 20]. In none of these studies, however, was sufficient attention paid to the ages of the patients or the durations of their infections; indeed, given the genetic variability of HCV, it is plausible that the relative proportion of the various HCV genomes has changed over time. Finally, whether the level of HCV viremia differs according to genotype and to severity of liver disease remains debatable. therapy differs with the HCV genotype; and whether the level of HCV viremia varies with severity of liver disease and with the HCV genotype as determined by the recently introduced branched-chain DNA (bDNA) assay [21, 22].
Methods
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Methods
Results
Discussion
Author & Article Info
References
Patients
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One hundred six patients were treated with recombinant interferon- 2b (3 MU subcutaneously 3 times/wk for 6 months). Inclusion criteria were the absence of hepatocellular carcinoma, elevated levels of alanine aminotransferase, biopsy-proven chronic hepatitis, and informed consent. Patients were divided into two groups, the first of which comprised 62 responders to interferon-. In these patients, alanine aminotransferase levels returned to normal after treatment with interferon-; 33 were considered long-term responders because alanine aminotransferase levels remained in the normal range for at least 6 months after therapy, and 29 were considered relapsers because alanine aminotransferase levels increased after cessation of treatment. The second group comprised 44 nonresponders; their alanine aminotransferase levels did not return to normal during therapy.
Studies
All serum specimens showed antibody to HCV by second-generation enzyme-linked immunosorbent assay and by RIBA2 (Ortho Diagnostic Systems, Raritan, New Jersey and Monolisa HCV, Diagnostic Pasteur, Marnes la Coquette, France) and contained HCV RNA detected by nested PCR.
Extraction of Hepatitis C Virus RNA and Synthesis of Complementary DNA
We used the method of Chomczynski and Sacchi [24] to extract RNA from plasma and serum specimens as previously described [25]. For five patients with chronic active hepatitis, RNA was also extracted from liver biopsy specimens [24].
Polymerase Chain Reaction with Universal and Type-Specific Primers
Because nested PCR considerably increases the risk for contamination, a modified procedure was developed in which both steps of nested PCR were done in a single tube [25]. In several ongoing studies, the sensitivity of this assay has been found to be similar to that of classic nested PCR. Universal and antisense-specific primers were used according to the method of Okamoto and colleagues [13, 14].
The first PCR was done for 25 cycles with universal primers. Each reaction cycle included denaturation at 94 °C for 1 minute, primer annealing at 55 °C for 1.5 minutes, and primer extension at 72 °C for 1.5 minutes. In the first amplification using external primers, complementary DNA was centrifuged for 1 minute to combine the second PCR mix with the first. The second amplification was done according to the following protocol: denaturation at 94 °C for 1 minute, primer annealing at 62 °C for 1.5 minutes, and primer extension at 72 °C for 1.5 minutes for 25 cycles, using a universal primer and one of the five type-specific primers.
The products of the second PCR were subjected to electrophoresis on a composite acrylamide-bisacrylamide gel (19%/1%), and the fragments were detected by ethidium bromide staining and ultraviolet illumination. Subtypes were determined by band position; type 1a (I), type 1b (II), type 2a (III), type 2b (IV), and type 3a (V) show bands at 49, 144, 174, 123, and 88 base pairs, respectively.
Precautions were taken to avoid carryover [26]. In addition, a negative control was inserted at the extraction step and run to the end of the test; results were considered valid if they were consistent in at least two tests done on samples derived from two independent extractions [27].
Quantitative Detection of Serum Hepatitis C Virus RNA
To quantitatively detect serum HCV RNA, a signal amplification method based on branched oligodeoxyribonucleotides (bDNA) developed by Chiron (Emeryville, California) was used according to the manufacturer's instructions [22]. These bDNAs have a unique primary segment and a set of identical secondary fragments covalently attached to the primary sequence through branched points. The primary sequence is designed to hybridize to oligonucleotide target probes bound to HCV RNA. A second set of oligonucleotide target probes mediate capture of the target nucleic acid onto a microwell. The secondary fragments of the bDNA are used to direct the binding of multiple copies of an oligonucleotide labeled with alkaline phosphatase. The latter is detected using an enzyme-triggerable dioxetane substrate, and the visible light output is recorded on a luminometer permitting quantitation of HCV RNA. The detection limit of this test is 350 000 HCV RNA equivalents per milliliter of serum. Only positive results were included in the calculation of the mean HCV viremia level. Serum specimens were available from 183 of 220 patients for quantitative detection of serum HCV RNA by bDNA.
Validation of Results
Results were validated by exchanging 20 samples between two laboratories (in Paris and Tokyo) and comparing HCV genotypes derived from serum and liver specimens from five patients; these showed the same patterns. Results were in complete agreement.
Statistical Analysis
The chi-square test or Fisher exact test was used for statistical analysis of comparison between group frequencies. When appropriate, laboratory data and titers of HCV RNA were compared using the Student t-test. When data were not normally distributed, the Wilcoxon signed-rank test and the Wilcoxon rank-sum test were used. Independent factors associated with the presence of HCV genotype 1b (II) and those associated with the response to interferon- therapy were studied using a stepwise logistic regression model. In a first model focusing on genotype 1b (II), explanatory variables were sex, type of disease (hepatocellular carcinoma, cirrhosis, chronic hepatitis), histologic findings in liver (cirrhosis or its absence), age, and bDNA assay value. Duration of disease was excluded because it was known for only 107 patients. In the second model, explanatory variables for response to interferon- therapy were sex, histologic findings in liver, age, HCV genotype, and bDNA assay value. In both models, the P value for entrance limit had to be less than 0.10. Univariate and multivariate analyses were done using BMDP statistical software (BMDP Statistical Software Inc., Los Angeles, California).
Results
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Prevalence of Hepatitis C Virus Genotypes
The results of HCV genotyping according to histopathologic liver status are given in Table 1. Infection with HCV type 1b (II) alone had the highest prevalence in all three groups of patients (61.8%). No statistically significant difference in the prevalence of infection with HCV type 1b (II) was seen among patients of different nationalities. The limited number of patients did not allow us to do a relevant statistical analysis for the other HCV genotypes. A few of our samples (26 of 220) could not be classified and, thus, may correspond to HCV types not detected by Okamoto and colleagues' method. Evidence of a mixed infection was seen in 5.4% of patients (12 of 220). Because the significance of mixed infections is currently unclear, these patients were not included for further clinicopathologic analysis despite the fact that they did not change the results. The univariate analysis is summarized in Table 2.
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Age, Duration of Disease, and Mode of Contamination
As shown in Figure 1(top), HCV type 1b (II) was present in 30.8% of patients younger than 30 years of age, 50% of patients between 30 and 40 years of age, 64% of patients between 40 and 50 years of age, 68.7% of patients between 50 and 60 years of age, and 82.3% of patients older than 60 years of age.
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We could precisely establish the date of infection in 107 of the 220 patients. Hepatitis C virus genotype 1b (II) was associated with a longer duration of disease than were other genotypes; it was seen in 40.4% of patients who had had disease for less than 10 years, 57.5% of those who had had disease for 10 to 20 years, and 86.7% of those who had had disease for 20 years or more [P = 0.005] Figure 1, top). It should be emphasized, however, that it will be important to examine whether the long-term mortality rate differs in patients infected with different HCV genotypes.
Type 1b (II) was more often seen in patients who had had transfusions (35 of 55; 63.6%) than in patients who had used intravenous drugs (7 of 23; 30.4%) (P = 0.007), but the significance of the difference did not increase after adjustment for the duration of disease. Because of the few patients with each of the non-1b (II) genotypes, the following statistical comparisons were made between patients with HCV type 1b (II) and those with non-1b (II) HCV genotypes.
Association of Genotype with Severity of Liver Disease and Response to Interferon-
Type 1b (II) alone was detected in 28 of 33 (84.8%) patients with hepatocellular carcinoma and in 52 of 69 (75.4%) patients with cirrhosis but without hepatocellular carcinoma, but it was found in only 57 of 106 (53.8%) patients with chronic hepatitis (hepatocellular carcinoma compared with cirrhosis, P > 0.05; hepatocellular carcinoma or cirrhosis compared with chronic hepatitis, P < 0.01).
Among the 106 patients treated with interferon-, 6 with mixed HCV genotypes were excluded from the analysis; thus, 100 patients were included in the study (Table 3). No difference was seen between the responders (defined as long-term responders and relapsers after interferon- withdrawal) and the nonresponders to interferon- therapy for age, sex, disease duration, mode of transmission, or mean alanine aminotransferase value.
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Furthermore, neither age nor duration of disease was associated with response to interferon-, regardless of the underlying liver disease (data not shown).
The response to interferon- was more favorable in patients infected with non-1b (II) HCV genotypes (31 of 42) than in those infected with HCV type 1b (II) (28 of 58) (P = 0.01) (Table 3).
Quantification of Hepatitis C Virus RNA
Serum HCV RNA levels were estimated by bDNA assay for the 183 patients for whom serum specimens were available.
The number of bDNA-positive samples and the mean value of the test result did not differ between patients infected with HCV type 1b (II) and those infected with other HCV genotypes (34.6 ±33.7 compared with 36.1 ±42.2 x 105 Equation genomes/mL; P > 0.05).
Among patients with a bDNA-positive test result (>350 000 Equation genomes/mL), no significant difference was seen in the mean level of HCV viremia among patients with cirrhosis and hepatocellular carcinoma (40.3 ±45.2 x 105 Equation genomes/mL; n = 15), those with cirrhosis who did not have hepatocellular carcinoma (33.8 ±37.7 x 105 Equation genomes/mL; n = 41), and those with chronic hepatitis (34.8 ±34.5 x 105 Equation genomes/mL; n = 74). There was also no difference between HCV type 1b (II) and non1b (II) genotypes in the three groups.
In patients treated with interferon-, nonresponders had a higher level of HCV viremia than did relapsers or long-term responders (39.1 ±37.4 compared with 26.5 ±24.0 x 105 Equation genomes/mL; P < 0.05). In addition, the proportion of patients whose specimens were positive for HCV RNA by PCR but negative by bDNA (for example, those with low-level HCV viremia) was significantly higher (21 of 55 compared with 6 of 40; P = 0.01) in responders than in nonresponders (Figure 2).
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In patients treated with interferon-, nonresponders infected with genotype 1b (II) had a higher level of viremia than did nonresponders infected with other types (47.4 ±39.1 [n = 26] compared with 12.2 ±6.2 x 105 Equation genomes/mL [n = 8]; P < 0.05).
Thus, a univariate analysis showed that HCV type 1b (II) was associated with older age and longer duration of chronic infection. Prevalence of HCV type 1b (II) was significantly higher in patients with cirrhosis or hepatocellular carcinoma and in those who were nonresponders to interferon-.
Multivariate Analysis
Among the variables tested (see Methods), age and presence of cirrhosis were independent factors associated with HCV type 1b (II) (Table 4).
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The response to interferon- therapy varied with the level of viremia. Those with a level less than 350 000 Equation genomes/mL were more likely to respond to therapy with interferon- than were those with levels greater than 350 000 Equation genomes/mL.
The genotype was also an independent factor for the response to treatment; patients infected with HCV type 1b (II) responded less to treatment than did patients infected with other genotypes (odds ratio, 2.8).
Discussion
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therapy. Hepatitis C Virus Genotypes and Severity of Liver Disease
Our data firmly establish the high prevalence of HCV type 1b (II) among French and Italian patients, a result that accords with some recent data [18].
Most importantly, our results show that HCV type 1b (II) is highly prevalent among patients with severe liver disease. Prevalence was 84.8% in patients with both cirrhosis and hepatocellular carcinomas, 75.4% in patients with cirrhosis who did not have hepatocellular carcinoma, and only 53.8% in patients with chronic hepatitis. In contrast, HCV infections with type 1a (I) alone, or with types 2a (III), 2b (IV) or 3a (V), were more frequently seen in patients with chronic hepatitis who did not have cirrhosis or hepatocellular carcinoma. This raises the possibility that HCV type 1b (II) exerts a specific cytopathogenic effect. Few data are available on this issue [16, 19]; moreover, the studies that produced these data had only a limited number of patients and did not include a multivariate analysis. Our results are reinforced by data that we recently obtained in studies of HCV-reinfected liver grafts; these data show that HCV type 1b (II) is associated with more severe histologic findings in liver specimens (Feray C. Personal communication). Furthermore, we recently started to test samples using a genotyping procedure based on the 5' noncoding region (LiPA, Line Probe Assay, Innogenetics, Zwijndrecht, Belgium) [9] and reached identical conclusions (unpublished data).
However, our present investigation clearly shows that age and duration of infection are major variables to be considered. Indeed, HCV type 1b (II) was highly prevalent in patients older than 40 years of age and in those with a long duration of infection. In contrast, non-1b (II) genotypes were most often identified in recently infected patients who either had had transfusions or were intravenous drug users. These findings agree with our recent observation that most patients receiving hemodialysis or renal transplants who were infected with HCV 15 to 20 years ago were infected with HCV type 1b (II) (Pol S. Personal communication). Therefore, these data do not exclude the possibility that other genotypes might also lead to severe liver disease because a long period of time is required for the development of cirrhosis and hepatocellular carcinoma. Non-1b (II) types appeared only recently; more time must elapse before this question can be answered.
However, there are only a few data about the long-term mortality rate of chronic HCV infection (51% at 18 years) [28]. Thus, it should be emphasized that we did not address whether the long-term mortality rate might differ among different genotypes, a factor of potential importance in such cross-sectional analysis. Further prospective studies should be informative.
Chronic infection by HCV is reported to be an important risk factor for hepatocellular carcinoma [29]. Whether the viral infection acts only through the associated cirrhosis or whether some strains of HCV are directly carcinogenic, however, is still unclear. The prevalence of HCV type 1b (II) was high in patients with hepatocellular carcinoma but did not differ significantly from the prevalence in patients with cirrhosis who did not have hepatocellular carcinoma. Therefore, our results support the idea that cirrhosis plays a major role in the causation of liver cancer related to HCV infection.
Hepatitis C Virus Genotypes and Quantitative Viremia
We have investigated the level of HCV viremia using the recent bDNA assay. In accordance with previous observations, we detected serum HCV RNA by bDNA in 70% of serum specimens that tested positive by PCR. Using the bDNA assay, we did not observe any differences in the level of HCV viremia in patients with chronic hepatitis, in patients with cirrhosis who did not have cancer, or in patients with hepatocellular carcinoma. Furthermore, our multivariate analysis shows that HCV viremia was not associated with the presence or absence of cirrhosis, duration of chronic infection, or age. Therefore, our results show that sustained HCV multiplication persists during the chronic course of HCV infection when cirrhosis and hepatocellular carcinoma develop; this contrasts with the frequent disappearance of hepatitis B virus replication when cirrhosis occurs in chronic hepatitis B virus carriers [30, 31].
Hepatitis C Virus Genotypes and Response to Interferon-
Several randomized controlled trials have shown that interferon- therapy effectively induces long-term remission in about 20% of patients with chronic hepatitis C [32, 33]. The basis of response or nonresponse to interferon- treatment is still unclear.
Our study confirms previous reports from Japan [17] and Europe [18] that HCV 1b (II) is an independent predictor for a poor response to interferon-. Indeed, in a multivariate analysis, infection by non-1b (II) genotypes was associated with an increased chance of response to treatment. In our study, a high level of HCV viremia was also an independent factor for a low response to interferon-. This has been previously suggested in studies from the United States [34], although genotyping was not available when these studies were done. Thus, the HCV viremia levels and the HCV genotype both act as independent prognostic factors for response to interferon-.
Genotypes 1a (I) and 3a (V) seem to be associated with a high rate of response to interferon- therapy. Although this observation needs to be further substantiated, it suggests that, as was reported in Japan for types 2a (III) and 2b (IV) [17], some genotypes might be sensitive to interferon- therapy.
Several nonexclusive hypotheses can explain both the different pathogenic effects of HCV genotypes and the different responses of these genotypes to interferon-. First, high levels of replication might contribute to resistance to interferon- and development of cirrhosis. In studies from Japan [17], HCV-RNA concentrations in patients infected with type 1b (II) were significantly higher than in patients infected with type 2a (III). This suggests that some genotypes have a greater replicative capacity than do others. The different HCV RNA concentrations among genotypes might also be attributed to the difference in susceptibility to immunologic response to the virus by the host. However, our data do not support these hypotheses. We did not find a significant difference in the level of HCV viremia between genotype II and other genotypes; nonresponders infected with type 1b (II), however, had a higher HCV RNA level than did nonresponders infected with other types.
Second, it is plausible that other properties of HCV type 1b (II) might be involved in the different courses of HCV infection. In particular, it has been established that a significant genetic variation occurs in the HCV genome during the course of infection [35-37], mostly in the hypervariable E2 envelope encoding domains, possibly as a result of the host immune selective pressure [35, 37]. In addition, the degree of variability in the aminoterminal region of the envelope E2/NS1 protein of HCV might correlate with responsiveness to interferon- [38].
It is also tempting to speculate that, as has been shown for some pestiviruses [39], differences in the properties of the HCV viral proteins might lead to different cytopathic effects; no data, however, are presently available with which to decide this issue.
Our study, although based on cross-sectional data, suggests that the prevalence of HCV genotypes in French and Italian patients has been changing, with a progressive decrease in HCV type 1b (II). This genotype is currently responsible for most HCV-related cirrhosis and hepatocellular carcinoma and shows a particular pattern of resistance to interferon-. The level of viremia and the HCV genotype are independent factors to be considered in the management of patients with HCV infection.
Appendix 1: Glossary
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Hepatitis C virus genotypes: On the basis of the variability of the HCV RNA genome, the various isolates can be schematically classified as HCV "genotypes"
Type-specific genotyping: Polymerase chain reaction done with specific primers for each genotype
Branched DNA assay: Quantitative evaluation of HCV viremia without previous amplification of the HCV RNA
Appendix 2
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Author and Article Information
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References
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