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Keywords:

  • Genetic evolution;
  • hepatitis C virus;
  • transplantation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

In patients with hepatitis C virus (HCV)-related cirrhosis, infection recurrence is universal after liver transplantation (LT). The relevance of host and virus-related factors on the outcome of hepatitis C recurrence is poorly understood. This study analyzed the relationship between the genetic evolution of the Non-Structural (NS)3 protease and NS5B polymerase regions of HCV and the severity of hepatitis C recurrence. Thirty-three patients were classified as having mild (n = 16) or severe recurrence (n = 17), according to the degree of fibrosis in liver biopsies obtained 1 year after transplantation. Viral load and consensus sequences of the NS3 and NS5B domains were determined in a pre-LT and in four post-LT sequential serum samples. At week 12 after LT, viremia was significantly higher in patients with severe recurrence. NS3 and NS5b regions evolved independently after LT. The genetic evolution of NS3 domain was not related to the severity of the recurrence. However, the diversification in the NS5B region later than 12 weeks after LT was greater in patients with mild than in those with severe recurrence, suggesting a stronger immune pressure in the first group. These observations highlight the complex interplay between viral evolution and clinical outcomes in the LT setting.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Advanced liver disease as a consequence of chronic hepatitis C virus (HCV) infection is a major indication for liver transplantation (LT). Regretfully, HCV infection of the graft occurs universally after LT and causes chronic hepatitis in most patients, although the severity of liver damage is variable. After HCV infection, recurrence liver disease remains mild for a long time in some patients whereas liver cirrhosis rapidly develops in others (1,2). A recent study by our group showed that 30% of patients transplanted for HCV-related liver disease already had cirrhosis 5 years after LT (3).

The diseased liver, which is the main source of HCV, is removed during LT leading to a substantial reduction in the viral population. HCV infection of the liver graft takes place during the first days, or even the first hours, after transplantation (4). Infection is caused mainly by viruses remaining in the circulation, although viral particles produced in non-hepatic reservoirs may contribute as well (4,5). Emerging HCV quasispecies rapidly adapt to the graft, and the viral variants that most efficiently replicate become predominant. In addition, new mutants with increased fitness for the graft may emerge and compete with pre-LT variants. Nevertheless, HCV quasiespecies become more homogeneous after LT (6).

A number of variables have been associated with a greater severity of liver disease after LT, including high pretransplantation viral load, old donor age, transplantation from a living donor, presence of steatosis in the graft and more aggressive immunosupressive therapy (2,3,7–9). The influence of virological factors, such as the HCV genetic sequence or the degree of amino acids (Aa) diversification, remains controversial. Several studies have analyzed the role of genetic heterogeneity in the envelope region E2 of HCV on the outcome of the hepatitis C recurrence, with differing results (10–14). One study showed that HCV core sequences isolated from patients with similar degree of liver damage showed a close genetic distance (GD) (15), but these findings have not been confirmed by other authors (16).

The aim of our study was to investigate if the genetic evolution of HCV during the early phase after LT varied in individuals with different disease outcomes. We focused on the Non-Structural (NS)3 protease and NS5B polymerase regions of the HCV genome, which are essential for HCV replication and are major targets for the development of novel therapeutic agents (17–20). Analysis of the early genetic changes in the NS3 and NS5B regions after LT might improve our understanding of HCV adaptation in the graft and its influence on the severity of liver disease recurrence.

Patients and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Patients

Patients who underwent LT for HCV-related cirrhosis from March 2000 to August 2003 were considered for study. Only patients infected with genotype 1b in whom a liver biopsy performed 1 year after LT was available were considered for inclusion. Exclusion criteria were: infection with the hepatitis B virus or the human immunodeficiency virus, fibrosing cholestatic hepatitis or antiviral treatment during the first year after LT.

Serum samples were obtained immediately before LT and 1, 4, 12 and 48 weeks after LT. Follow-up liver biopsies were obtained 3 and 12 months after LT, but only 12-month biopsies were considered for the purpose of this study. Histological assessment was performed by a single pathologist using the Scheuer Fibrosis score (21). Mild disease recurrence was defined by the absence of fibrosis or fibrosis restricted to the portal tract (F0-F1), whereas fibrosis beyond the portal tract, bridging fibrosis or cirrhosis (F2-F4) defined severe disease recurrence. This classification was based on several published articles showing that significant fibrosis or increased portal pressure (measured as hepatic venous pressure gradient [HVPG]) 1 year after LT identifies with high-accuracy patients at risk of severe hepatitis C recurrence (22,23). All patients gave their informed consent and the Ethics Committee of our institution approved the study.

HCV genotyping and viral load determination

HCV genotype was determined by restriction fragment length polymorphism of the 5' non-coding region of HCV genome, as previously described (4). HCV-RNA levels in serum samples were measured by RT-PCR with a commercially available assay (Cobas Amplicor HCV Monitor vs.2.0, Roche Diagnostics, Branchburg, NJ), following the manufacturer's instructions. Samples were diluted 1/10 to prevent underestimation of values in the upper range of the assay (500 000 IU/mL) (24) and retested without dilution if negative. Samples with HCV-RNA concentration exceeding 5 ×106 IU/mL were diluted 1/100 and retested.

RNA extraction and nested-PCR

Total RNA was extracted from 140 μL of serum (QIAmp Viral RNA Mini kit, Qiagen, Hilden, Germany), following the manufacturer's instructions. The NS3 protease (nucleotides 3405–4083 according the HCV-J prototype (25) and NS5B regions (nucleotides 8252–9208 according to HCV-J prototype, including part of motif A, motifs B–E and the thumb domain) (26,27) were retrotranscripted and amplified by nested PCR as previously described (28). Two sets of primers were designed for NS3 amplification: NS3 external primers: 5′-CGGCGTGTGGGGACATCATCT-3′ (Forward) and 5′-AGGTGGCAACGGACGGGTTCAGGA-3′ (Reverse); NS3 internal primers: 5′-CTTGCGCCTATCACGGCCTATT-3′ (Forward) and 5′-CACCTTGTACCCTTGGGCTGCAT-3′ (Reverse). Cycling conditions for the NS3 outer PCR were: 94°C for 5 min; 35 cycles of 94°C for 30 s; 55°C for 30 s; 72°C for 1 min; and a final extension at 72°C for 10 min, and for the nested NS3 PCR were: 94°C for 5 min; 35 cycles of 94°C for 30 s; 60°C for 30 s; 72°C for 1 min; and 72°C for 10 min. For NS5B amplification external primers were: 5′-CGCCTTATCGTATTCCCAGA-3′ (Forward) and 5′-GCACGAGACAGGCTGTGATA-3′ (Reverse) and NS5B internal primers were: 5′-CTGCTTTGACTCAACGGTCA-3′ (forward) and 5′-CCGGGATTGGAGTGAGTTTA-3′ (reverse). Cycling conditions for both NS5B PCRs were: 94°C for 5 min; 35 cycles of 94°C for 30 s; 56° for 1 min; 72°C for 2 min; and a final extension at 72°C for 10 min.

DNA sequencing

PCR products were purified with the QIAquick PCR purification kit (QIAGEN). 5–20 ng of purified DNA were bi-directionally sequenced using the set of primers of the corresponding internal PCR and the ABI Prism Big Dye Terminator Ready reaction kit, v3.1 (Applied Biosystems, Foster City, CA). Two additional primers were designed for NS5B region sequencing: Pol7 (5′-TGATGTTGCCTAGCCAGGAGTTGA-3′) (reverse) and Pol9 (5′-ACTCAACTCCTGGCTAGGCAACAT-3′) (forward). Sequences were verified and strands were assembled using the Sequencher 4,6 program (Gene Codes Corporation, Ann Arbor, MI).

Sequence comparison and phylogenetic analyses

Nucleotide and Aa consensus sequences obtained from each patient were compared with the sequence obtained just before LT and to the previous post-LT sequences using the MEGA program (29). The rate of accumulation of mutations was calculated as the number of Aa mutations fixed per total number of Aa analyzed per unit of time (year).

The intersample genetic distance (GD) for each patient was estimated by pairwise comparison using the p-distance method in the MEGA package. The average number of synonymous nucleotide substitutions per synonymous site (dS) and the average number of non-synonymous nucleotide substitutions per non-synonymous site (dN) were estimated with the Nei Gojobori Method and the Jukes Cantor correction using the MEGA package.

Phylogenetic trees were constructed using the PHYLIP package (Phylogeny Inference Package, version 3.5 c, Felsenstein, J. 1993). Bootstrap values were determined from 1000 bootstrap resamplings of the original data. The final graphic output was created with the TREEVIEW program.

Nucleotide sequences

HCV sequences have been submitted to Genebank under accession numbers EF608622 to EF608774 for NS3 region and EF608775 to EF608925 for NS5B region.

Statistical analysis

Quantitative variables are expressed as mean and standard deviation. Differences between groups were analyzed using a non-parametric test (Mann-Whitney for unpaired groups and Wilcoxon for paired groups). A value of p < 0.05 was considered significant. Statistical calculations were performed with the SPSS v11.0 package.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Patient characteristics

Thirty-three patients were included in the study. The main features of the patients are summarized in Table 1. We did not find significant differences regarding age, sex, pretransplantation viral load, type of LT, donor age, presence of hepatocellular carcinoma pre-LT and type of immunosuppression between patients with mild or severe recurrence.

Table 1.  Features of patients undergoing liver transplantation according to the severity of hepatitis C recurrence
 Severe (n = 17)Mild (n = 16)p-Value
  1. 1Values are expressed as median (range).

  2. n. s.= not significant; LDLT = living donor LT; DDLT = deceased donor LT; FK = tacrolimus; CyA = cyclosporine A.

Recipient age (years)159 (34–70)63 (52–72)n. s.
Gender (male/female)10/79/7n. s.
Pretransplant viral load (log10 IU/mL)15.71 (4.79–6.54)5.48 (4.19–6.32)n. s.
Hepatocellular carcinoma9 (53%)7 (44%)n. s.
Type of transplantation (LDLT/DDLT)4/133/13n. s.
Donor age (years)144 (15–74)37 (15–79)n. s.
Immunosuppression (FK/CyA)6/119/7n. s.

HCV infection recurred after LT in all patients. One week after LT, HCV-RNA was detected in 29 out of 32 (91%) patients. Histological evaluation of liver biopsies obtained 1 year after LT revealed absence of (F0) (n = 8) or mild (F1) (n = 8) fibrosis in 16 patients (48.5%) (mild recurrence). Biopsies from the remaining individuals exhibited fibrosis beyond the portal tract (F2) (n = 5) or bridging fibrosis (F3) (n = 12) and hepatitis recurrence was considered severe (n = 17) (51.5%). This histological classification was strongly supported by the follow-up of these patients: in individuals classified as having mild recurrence (F0-F1) 1 year after LT, liver fibrosis remained stable (F0 or F1) in 12 out of 15 (80%) patients within 3 years after transplantation. In the remaining three patients, liver fibrosis progressed to F2 stage 2 or 3 years after transplantation. In this group, one patient underwent retransplantation due to biliary complications. In the group of patients classified as severe recurrence (n = 17), disease progressed to F3 or F4 and caused clinical descompensation (ascitis) in 10 patients (59%) within 3 years following transplantation. In the remaining patients, fibrosis remained stable or progressed to F3 (except in one individual who achieved sustained viral clearance after antiviral therapy).

Viral load was determined in serum samples obtained immediately before and 1, 4, 12 and 48 weeks after LT. HCV-RNA serum concentration increased rapidly after LT and at week 4 after LT HCV-RNA levels were significantly higher than pretransplant levels (p = 0.001), as previously described (4). Figure 1 shows the kinetics of viral load according to recurrence severity. HCV-RNA levels measured at week 12 were significantly higher in patients with severe recurrence (6.86 ± 0.52 log IU/mL) than in those with mild recurrence (6.31 ± 0.65 log IU/mL) (p = 0.023). Viral load at this time point was significantly higher in patients with bridging fibrosis (F3) 1 year after LT (6.98 ± 0.53 log IU/mL) than in the remaining patients (F0-F2) (6.37 ± 0.60 log IU/mL) (p = 0.009).

image

Figure 1. HCV kinetics after LT according to the severity of the hepatitis C recurrence. Patients were grouped as having mild (n = 16) or severe (n = 17) recurrence. Viral load is shown as mean ± standard error in a logarithmic scale. *p < 0.05 vs. severe recurrence.

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NS5B genetic evolution and hepatitis recurrence

The genetic evolution of the NS5B region after LT was assessed by comparing the viral sequences from consecutive samples obtained after LT with the sequence obtained immediately before LT. As depicted in Figure 2A, the mean intersample GD throughout the first year after LT were greater in patients who developed mild recurrence. At week 12 and 1 year after LT, GD values were significantly higher in patients with mild recurrence compared to those with severe recurrence: 1.8 × 10−3 versus 0.41 × 10−3 (p = 0.049) and 2.3 × 10−3 vs. 1.0 × 10−3 (p = 0.024), respectively. As expected, similar differences were found concerning the emergence of Aa mutations over time. Serial Aa sequences of each patient were compared with all the preceding sequences and the results are represented in the Table 2. Patients with mild recurrence presented a trend to a higher number of Aa changes per Aa position at all time points than those with severe recurrence. Based on the regression analysis of sequential samples obtained over the first year after LT, the rate of fixation of Aa mutations in the NS5B region was 1.52 × 10−3 mutations/site/year for mild recurrence and 0.51 × 10−3 mutations/site/year for severe recurrence.

image

Figure 2. Genetic distance in NS5b and NS3 protease regions according to the severity of the hepatitis recurrence. Genetic distances at each time point are referred to the sequences in viral isolates obtained immediately before transplantation and are depicted as mean + standard error. *p < 0.05 vs. severe recurrence.

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Table 2.  Number of Aa changes accumulated in the NS5b region at different time points according to the severity of hepatitis C recurrence
 W1W4W12W48
  1. Aa consensus sequences obtained from each patient were compared to the sequence obtained just before LT (Pre-LT) and to all the previous post-LT sequences (W1, W4, W12, W48: Weeks after liver transplantation). Values are expressed as mean (range).

  2. 1 Differences were not significant compared to severe recurrence.

PreMild10.50 (0–2)0.47 (0–1)0.73 (0–8)0.80 (0–2)
 Severe0.33 (0–2)0.20 (0–2)0.24 (0–1)0.62 (0–3)
W1Mild1 0.50 (0–3)1.00 (0–7)0.85 (0–4)
 Severe 0.31 (0–2)0.40 (0–3)0.67 (0–4)
W4Mild1 0.93 (0–7)0.73 (0–2)
 Severe 0.27 (0–2)0.46 (0–2)
W12Mild1 1.00 (0–6)
 Severe 0.46 (0–2)

NS5B genetic evolution and viral replication

The relationship between the accumulation of mutations in NS5B and the efficiency of viral replication in the graft was also analyzed. Samples were grouped according to the fixation or absence of mutations in the NS5B region at different time points after LT and the mean viral load in each group was then compared (Figure 3). No significant differences were observed at any time point studied.

image

Figure 3. Relationship between the emergence of Aa changes in the NS5B region and viral load. At the different time points, samples were grouped according to the absence or the presence of Aa mutations within the NS5B region compared to the sequence obtained immediatly before transplantation. Viral load is represented as a box plot (the top and the bottom of the boxes being the 25th and 75th percentiles ± standard error). Solid lines indicate the median values. No significant differences were observed between both groups.

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NS3 protease genetic evolution and hepatitis recurrence

The intersample GD measured in the NS3 region rose in patients undergoing severe recurrence during the first year after LT (Figure 2B). After LT, though, no significant differences at any time point were detected compared to patients with mild recurrence (Figure 2B). Similarly, the emergence of Aa substitutions in NS3 protease tended to be more frequent in patients with severe recurrence (Table 3), resulting in an increased rate of fixation of Aa changes during the first year after LT in patients developing severe recurrence compared to those with mild recurrence (6.01 × 10−3 vs. 0.87 × 10−3 mutations/site/year).

Table 3.  Number of Aa changes accumulated in the NS3 region at different time points according to the severity of hepatitis C recurrence
 W1W4W12W48
  1. Aa consensus sequences obtained from each patient were compared to the sequence obtained just before LT (Pre-LT) and to all the previous post-LT sequences (W1, W4, W12, W48: Weeks after liver transplantation). Values are expressed as mean (range).

  2. 1Differences were not significant compared to severe recurrence.

Pre-LTMild10.31 (0–1)0.13 (0–1)0.31 (0–1)0.37 (0–2)
 Severe0.60 (0–3)0.50 (0–2)0.88 (0–4)1.15 (0–5)
W1Mild1 0.31 (0–1)0.15 (0–1)0.46 (0–2)
 Severe 0.15 (0–2)0.27 (0–2)0.50 (0–3)
W4Mild1 0.19 (0–1)0.40 (0–2)
 Severe 0.21 (0 – 1)0.58 (0–3)
W12Mild1 0.44 (0–1)
 Severe 1.38 (0–8)

No differences were observed in the ratio of dS/dN between the two groups of patients at any point time (data not shown). In addition, no differences in the fixation on mutations in relevant NS3 T-cell epitopes (30,31) were detected according to the severity of fibrosis (data not shown). Emergence of mutations (Aa and nucleotides) in the NS3 protease region was not related to viral load (data not shown).

Phylogenetic analysis of the the NS3 protease and NS5B regions

Phylogenetic analyses of the NS3 protease and NS5b regions were carried out using the Neighbor-joining method from a Kimura two-parameter distance matrix. Phylogenic trees were constructed with all consecutive sequences from each patient (data not shown). All serial samples from the same patient were closely related, indicating that HCV infection with a new strain did not occur in any case. The NS3 and NS5B phylogenetic trees constructed with samples obtained before LT are represented in Figure 4. Sequence clustering in both trees had a low statistical support since only three nodes in NS3 and two nodes in NS5b showed bootstrap support values higher than 50%. Moreover, the supported nodes were different in both trees. Two sequences were related with a bootstrap value of 92.1% in the NS3 tree. However, the severity of the recurrence was different in both samples. Therefore, no relationship was observed between the genetic similarity of the NS3 and NS5b sequences and the severity of hepatitis recurrence or viral replication. Similar data were obtained when the trees were constructed with the sequences obtained after LT (data not shown).

image

Figure 4. Phylogenetic trees of the NS3 protease (A) and NS5B (B) regions from samples obtained before LT. Trees were constructed by the neighbor-joining algorithm from a Kimura two-parameter distance matrix. Bootstrap values >50% of 1000 replicates are shown in the corresponding node. Boxes identify patients with mild recurrence (M) and circles patients with severe recurrence (S).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Recurrent HCV infection after LT is an important clinical challenge for transplantation programs worldwide. Disease progression is accelerated in immunosuppressed patients (2,32) and, subsequently, graft and patient survival are significantly worse in patients undergoing LT for HCV-related cirrhosis than in those transplanted for other causes (11,33). Nevertheless, disease progression is highly variable and while some patients develop cirrhosis within a few years, the disease remains mild and does not progress in others. The mechanisms that determine these outcomes are not well known, although a complex interplay between host and viral factors is probably involved.

In this study, we analyzed the relationship between several virological factors and the severity of HCV infection recurrence, including pre- and posttransplant viral load, as well as the genetic evolution of two important functional regions of the virus (NS3 and NS5B). NS3 has two domains that code for the viral serin-protease (189 N-terminal Aa) and helicase (442 C-terminal Aa). The NS3 protease functions as a heterodimer complexed with the NS4 A protein and cleaves all downstream viral polyprotein junctions (NS3/4 A, NS4 A/B, NS4B/5 A and NS5 A/5B) (34,35). NS5B is an RNA-dependent RNA polymerase (RdRp) and is responsible for replicating the entire HCV genome. Using the subgenomic HCV RNA replicon system (36), several single and combined mutations within the NS3 and NS5B genes that notably affect viral replication have been described (37,38). However, the ‘in vivo’ prevalence and significance of such mutations have not been firmly established (39). In addition to their role in viral replication, the NS3 protease and NS5b domains contain immunologically relevant T-cell epitopes and escape mutations in these regions have been involved in HCV persistence (30,40,41).

Our results reveal that there is an inverse correlation between the diversification of the NS5b region and the severity of liver damage during the first year after LT. Patients with mild recurrence presented higher intersample GD and, a trend towards a greater number of Aa fixations, as compared with patients undergoing severe recurrence. Although multiple variables (such as host-related factors and the genetic back-ground of the graft) may influence the severity of the recurrence, previous studies have shown that severe hepatitis recurrence after LT occurred in patients with impaired HCV-specific T-cell responses, whereas the histological and clinical outcomes were better in those with efficient T-cell response (42,43). Therefore, the increased degree of genetic diversification of the NS5b region in patients with mild recurrence might be a consequence of a more prominent immunological pressure in this group of patients. These results are in keeping with a previous study in which the degree of diversification of the hypervariable region-1 (HVR-1) of the HCV-E2 domain was inversely correlated with the severity of hepatitis recurrence (10). HVR-1 contains major neutralizing epitopes and is also involved in the generation of escape mutants.

The differences in the NS5B GD between these two groups of patients increased over time and became statistically significant at weeks 12 and 48 after LT. The low-grade genetic evolution of NS5B observed in the very early phase after LT might be related to the weak immune pressure against HCV (44,45) resulting from the immunosuppressive therapy, which is usually stronger during the initial posttransplantation period. However, these findings may also be a result of the particular evolutionary nature of RNA viruses, in which the overall genetic diversity remains low during the phase of maximum fitness increase, but rises once fitness becomes asymptotic (46).

At week 12 after LT, patients with mild recurrence showed a more elevated genetic evolution of the NS5B region along with lower HCV-RNA concentrations, whereas a significant proportion of patients with severe histological recurrence presented high levels of viremia along with little genetic evolution of NS5B (data not shown). The possible influence of Aa mutations in NS5B function ‘in vivo’ is a matter of controversy. Some authors have reported that the presence of mutations in NS5B were associated with a decreased viral load in non-transplanted patients (27,47), but these findings have not been confirmed by others (39,48). In fact, the lower viral load in these patients might reflect the better control of HCV by the immune system.

Analysis of the genetic evolution of the NS3 protease did not show a significant association between changes in this region and the severity of hepatitis recurrence, although the intersample GD and the emergence of mutations tended to be higher in patients who developed more severe liver damage. These findings are in keeping with previous reports indicating that NS3 sequences are highly preserved after LT and that the emergence of mutations in T-cell epitopes of the NS3 protein were not related to the different outcome of liver disease recurrence (49,50). Most likely, the pattern of genetic evolution is diverse and has different implications in different domains of the HCV genome.

Our study has several limitations. First, the sample size is small and further studies including a higher number of patients are needed to elucidate the role of NS5b evolution on the outcome of HCV disease recurrence and on the replication efficiency of HCV. In addition, analysis of the NS3 protease and NS5b regions of HCV has been performed by direct sequencing, which does not take into account possible changes in the quasiespecies composition.

In summary, we analyzed the possible association of evolutionary changes in two different regions of HCV with the severity of hepatitis recurrence after LT. The genetic evolution in NS5B was greater in patients with mild disease recurrence compared to those with severe recurrence. This might reflect a stronger immunological pressure in the first group of patients. On the contrary, we did not observe a well-defined relationship between the genetic changes in NS3 and the severity of hepatitis C recurrence. The divergent results obtained in these two regions emphasize the difficulties in establishing clear associations between HCV genetic evolution and clinical outcomes. The high number of variables that modulate HCV genetic evolution and the fact that HCV disease recurrence cannot be easily categorized (since it is a continuous spectrum ranging from a normal liver to very severe disease) may explain, at least in part, our findings.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This work was supported in part through a grant from Fondo de Investigación Sanitaria (FIS 03–0217) and the European Surveillance Network for Vigilance against Viral Resistance (VIRGIL). Santseharay Ramírez was granted by IDIBAPS.

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  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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