methods to identify hepatitis C virus antigen. (HCVAg) ... and recurrent hepatitis C virus (HCV) infection. ... sion with the degree of HCV replication as assessed.
Recurrent Hepatitis C Virus Infection After Liver Transplantation: Immunohistochemical Assessment of the Viral Antigen Victor Vargas,* Krzysztof Krawczynski,† Lluis Castells,* Nuria Martinez,* Juan Esteban,* Helena Allende,* Rafael Esteban,* and Jaime Guardia* Background: The value of immunohistochemical methods to identify hepatitis C virus antigen (HCVAg) in liver tissue has not been established. We have evaluated the significance of HCVAg expression in livers of patients with transplants and recurrent hepatitis C virus (HCV) infection. Methods: Forty-two liver biopsy specimens from 32 liver-transplant recipients with recurrent HCV infection were tested for HCVAg using fluorescein isothiocyanate–labeled polyclonal, polyreactive human immunoglobulin. Histologic assessment of liver and quantitation of HCV RNA in sera were carried out in specimens obtained simultaneously with biopsies. Results: HCVAg was found in 33% of the liver specimens obtained during the first month after transplantation and in all liver specimens obtained between 1 and 18 months after transplantation. Amounts of the antigen were significantly greater in specimens obtained more than 1 month after transplantation. A statistically
significant increase of the average HCV RNA level in serum was observed in samples tested after the first month after the transplantation, and some decrease in the HCV RNA level was found in those obtained between 6 and 18 months after transplantation. Larger amounts of HCVAg were observed in specimens corresponding to episodes of acute or chronic hepatitis than in those associated with minimal parenchymal evidence of rejection. Conclusions: Observations of HCVAg expression in liver biopsy specimens indicated that the presence of viral antigens in hepatocytes is a constant finding in specimens obtained 1 month or longer after transplantation. Although large amounts of HCVAg correlated with acute or chronic hepatitis, the nature of this association with the development of pathologic changes remains to be established. Copyright r 1998 by the American Association for the Study of Liver Diseases
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However, the severity of histopathologic changes, including degree of fibrosis and structural changes and development of end-stage liver disease, may vary significantly. Although concordance between liver disease and HCV replication has been observed repeatedly, the direct causative association between the viral factors and advancement of chronic hepatitis remains uncertain.8,9 Identification of HCV antigen (HCVAg) by immunohistochemical analysis enabled the localization of HCV viral proteins in the liver cells.10 The observations of HCVAg in hepatocytes have been applied to the diagnosis of HCV infection in patients and experimentally infected chimpanzees and to pathogenetic studies of HCV infection.11-13 It has also been applied to the assessment of response to antiviral therapy in HCV-infected patients with chronic hepatitis.14 Information on expression of HCVAg and especially the dynamics of virus spread and HCVAg expression in infected liver grafts is scarce.5,6 We report the results of studies of HCVAg expression in liver biopsy specimens after OLT and the correlation of this expression with the degree of HCV replication as assessed
iver graft reinfection with hepatitis C virus (HCV) is constantly observed in patients undergoing liver transplantation for end-stage chronic liver disease (CLD) related to HCV infection.1,2 Previous clinical studies have revealed that hightiter HCV viremia can be detected early after transplantation in a significant proportion of patients, and recurrence of CLD has been observed frequently.3-6 In recurrent HCV infection, histopathologic changes after orthotopic liver transplantation (OLT) appear to progress from an acute lobular hepatitis to chronic hepatitis with lymphoid aggregates in the majority of patients.7
From the *Liver Unit, Hospital General Universitari Vall d’Hebron, Universitat Auto`noma, Barcelona, Spain; and †Hepatitis Branch, Division of Viral and Rickettsial Diseases/National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA. Address reprint requests to Prof. Dr. Jaime Guardia, Servei de Medicina Interna-Hepatologia, Hospital General Universitari Vall d’Hebron, Pg Vall d’Hebron 119, 08035 Barcelona, Spain. Copyright r 1998 by the American Association for the Study of Liver Diseases 1074-3022/98/0404-0012$3.00/0
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Immunohistochemical Assessment of HCVAg
by the level of circulating HCV RNA and with the histopathologic findings.
Materials and Methods The study group included 42 biopsy specimens obtained from 32 patients who underwent OLT as a result of HCV-related end-stage CLD (24 patients with liver cirrhosis and 8 patients with cirrhosis and hepatocellular carcinoma). All patients were treated according to the same immunosuppression protocol with cyclosporine or tacrolimus plus prednisone; during acute rejection episodes, prednisone boluses or anti-OKT3 antibodies were administered. Liver biopsies were performed when clinical and biochemical data, including elevated alanine aminotransferase activity and abnormal values of alkaline phosphatase and bilirubin, suggested developing liver disease. To study HCVAg in the liver, biopsy specimens were divided into three groups according to the time elapsed from OLT. Group A included 10 post-OLT liver biopsy specimens obtained during the first 29 days after OLT (average 17.8 6 8.2 days); group B comprised 15 specimens obtained between 32 and 179 days after OLT, (average 103.3 6 51.4 days); and group C included 17 specimens obtained between 229 and 535 days (average 372.2 6 109.0 days) after OLT (Table 1). A control group included in the study consisted of 10 liver biopsy specimens obtained from 5 patients who received liver transplants because of liver disease unrelated to HCV infection (2 patients with alcoholic liver cirrhosis, 2 with fulminant hepatitis of unknown origin, and 1 with primary biliary cirrhosis). Only specimens obtained for diagnostic purposes were evaluated, and no material was taken specifically for the purposes of this study.
HCVAg and Histopathologic Analysis All of the liver biopsy specimens were obtained by using the Menghini aspiration technique. A portion of each liver biopsy specimen was snap-frozen after being embedded in OCT compound (Miles Laboratories, Elkhart, IN) and stored at 270°C. Cryostat sections 2- to 4-µm thick were fixed in chloroform for 5 minutes, air-dried, and stained with fluorescein isothiocyanate (FITC)-labeled anti-HCV reagents as described previously.11 This antibody was not reactive with liver biopsy specimens from hepatitis G virus–infected chimpanzees. The presence of HCVAg in the liver tissue was determined by direct immunofluorescence with FITC-labeled immunoglobulins obtained from serum specimens derived from patients chronically infected with HCV and with high-titer anti-HCV determined by the limiting dilution titration with a commercial enzyme immunoassay (secondgeneration HCV-EIA, Abbott Laboratories, North Chicago, IL). Absorptions with recombinant proteins expressed in Escherichia coli and yeast indicated that the
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primary HCV proteins identified by fluorescein-labeled reagents were NS3 and NS4 proteins.11 The presence and amount of HCVAg was estimated by a single observer (K.K.) blinded to the clinical diagnosis of the patient and the temporal relationship of the examined specimen to the liver transplantation. The amount of HCVAg was graded semiquantitatively and expressed as the percentage of positively stained hepatocytes: 0, no positive cells; 11, less than 5% cells with HCVAg, 21, 5% to 20% cells with HCVAg; and 31, greater than 20% HCVAg-positive cells. The intensity of immunofluorescent staining, which may reflect the amount of the viral antigen in the cytoplasm of the individual cell, was almost always concordant with the proportion of positively stained cells. The weakest fluorescence was observed in cases with the smallest number of HCVAg-positive cells, and the largest amount of the antigen in individual cells was found in cases with the largest proportion of HCVAgpositive hepatocytes (Fig. 1). A portion of liver biopsy core was fixed in buffered formalin and routinely processed for histopathologic evaluation (hematoxylin and eosin, Perl’s, and Picro Sirius red stains). The slides were evaluated by a pathologist (H.A.) without knowledge of serum HCV RNA levels, intrahepatic HCVAg, or the time at which specimens had been taken. Liver lesions were classified as acute or chronic hepatitis according to the presence of classic pathomorphologic features in lobules, limiting plates, and portal tracts. Acute cellular rejection was diagnosed if a mixed cellular infiltrate with eosinophils in the portal tract triads, bile duct damage with nonsuppurative cholangitis, and endophlebitis affecting both the portal and central veins were found. Histologic necroinflammatory and fibrosis indexes of specimens with chronic hepatitis were scored as described by Ishak et al.15 The total necroinflammatory score was the sum of individual scores for (1) periportal or periseptal interface hepatitis; (2) confluent necrosis; (3) focal lytic necrosis, apoptosis, and focal inflammation; and (4) portal inflammation.
HCV Serologic Assessment and HCV RNA Determination and Quantitation In all patients, HCV infection was determined serologically at the initial clinical diagnosis by enzyme immunoassay (Ortho HCV 3.1 ELISA, Ortho Diagnostic Systems, Nechargemund, Germany) and confirmatory assays (Chiron RIBA-HCV3, Chiron, Emeryville, CA). HCV RNA was tested by the reverse-transcriptase polymerase chain reaction (RT-PCR; Amplicor-HCV; Roche, Basel, Switzerland). All serum samples were also tested for HCV RNA level by semiquantitative PCR (HCV-Monitor-Roche, Basel, Switzerland) with an estimated dynamic range of 3.5 logs, between 103 and 106.5 HCV RNA copies/mL. The sensitivity of the technique was determined at 103 copies/mL. Samples with more than 105 HCV RNA copies/mL were diluted in normal
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Table 1. HCVAg in Liver Cells, Serum HCV RNA, and Histopathologic Diagnosis in Liver-Transplant Recipients of Three Groups of Liver Biopsy Specimens Taken at Various Times After Liver Transplantation
Patient Group A: 5-29 d post-OLT A.R.F. J.P.C. C.A.S. F.M.M. J.P.F. M.C.C. F.M.A. L.C.J. M.M.Q. M.Y.B. Group B: 32-179 d post-OLT J.A.P. A.U.A. L.J.V. J.P.F. J.N.V. J.R.R. A.M.N. M.Y.B. M.M.Q. M.V.P. M.R.T. C.L.F. R.I.A. F.L.V. R.M.M. Group C: 229-535 d post-OLT T.A.M. F.G.A. A.M.M. A.U.A. C.L.F. J.N.V. L.S.J. J.R.R. V.L.V. J.P.C. C.A.R. R.I.A. M.Y.B. F.L.V. R.M.M. J.M.C. A.Q.R.
Days After OLT
HCVAg Score by FA
Serum HCV RNA (copies Eq 3 104/mL)
5 6 15 15 15 19 22 24 28 29
0 0 0 0 3 0 1 0 2 0
32 42 46 61 63 68 70 99 124 139 147 153 158 168 179
2 1 3 3 3 3 3 3 3 3 1 3 3 3 2
170 41 886 188 125 850 1,035 130 160 405 38 480 114 1,962 80
Normal liver Acute rejection AVH AVH AVH AVH AVH & CMV AVH CAH (7, 3) CAH (5, 1) Acute rejection AVH CAH (4, 1) AVH AVH
229 232 236 268 281 289 339 347 359 380 385 416 489 504 505 534 535
3 3 3 1 3 3 3 3 3 2 3 3 1 1 1 3 1
4,147 140 104 61 160 154 40 73 56 103 550 150 26 72 30 175 31
CAH (5, 0) CAH (5, 1) CAH (6, 1) CAH (4, 1) Ischemia, sepsis Chronic rejection CAH (4, 2) CAH (9, 4) CAH (5, 1) CAH (4, 1) CAH (6, 2) CAH (5, 1) CAH (9, 2) CAH (11, 4) Acute rejection CAH (4, 1) CAH (4, 2)
,0.1 0.17 0.75 39 120 ,0.1 36 12 7 69
Histopathologic Diagnosis (NIS, F) Normal liver Acute rejection Acute rejection Normal liver AVH Normal liver Acute rejection Normal liver Normal liver Acute rejection
FA, HCVAg by immunohistochemistry (0, negative; 1, ,5% positive cells; 2, 5% to 20% positive cells; 3, .20% positive cells); NIS, necroinflammatory score; F, grading of fibrosis15; AVH, acute viral hepatitis; CMV, cytomegalovirusassociated hepatitis.
Immunohistochemical Assessment of HCVAg
Figure 1. HCVAg in the cytoplasm of hepatocytes in frozen sections identified by FITC-labeled polyclonal IgG anti-HCVAg. (A) Intensive fluorescence of massive deposits of the antigen in several cells that stained 31 (70% to 90% positive cells). Most of the liver cells contain homogeneous HCVAg in the cytoplasm; some granular deposits can also be seen (patient C.L.F., group B; original magnification 8 3 40). (B) A positive liver cell containing homogeneous and granular cytoplasmatic deposits of HCVAg (patient M.Y.B., group C; original magnification 8 3 63). human serum and retested to increase the upper sensitivity limit of the technique.
Statistical Evaluations Mean values for each group were tested for statistical significance with a nonparametric method (MannWhitney U test). Discontinuous variables were evaluated for statistical significance with a chi-squared test. Correlation studies were performed by using the Spearman test. All P values were two-sided. An a level of .05 was considered statistically significant.
Results Specimens included in this study were obtained from 23 men and 9 women ranging in age from 23 to 67 years (mean 55.36 6 8.53 years) and who
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received a liver transplant between 1992 and 1994. The results of liver HCVAg expression in the liver, serum HCV RNA levels, and histologic assessment in the 42 liver transplant biopsy specimens are shown in Table 1. In liver biopsy samples from group A, obtained in the first month after liver transplantation, HCVAg was detectable in 3 of the 10 liver biopsy samples (30%; Table 1 and Fig. 2). Serum HCV RNA levels ranged from less than 0.1 3 104 copies/mL to 120 3 104 copies/mL (mean level 28.4 6 12.5 3 104 copies/mL). In this group of specimens, liver tissue had normal histologic appearance in five samples and showed features of acute rejection in four; acute viral hepatitis was diagnosed in one specimen. In group B, among specimens obtained between 30 and 180 days after OLT, all 15 liver biopsy specimens were positive for HCVAg (Table 1 and Fig. 2). In 11 specimens (73%), the number of positive cells exceeded 20% (31), and the mean HCVAg score for all group B specimens was 2.6 6 0.19. The level of HCV RNA ranged from 38 to 1,962 3 104 copies/mL, and the mean serum HCV RNA level was 442.32 6 138.17 3 104 copies/mL. Nine specimens showed morphologic characteristics of acute viral hepatitis; chronic hepatitis was diagnosed in three; two demonstrated acute graft rejection; and in one there were no pathologic changes. Finally, in group C, which included 17 liver samples obtained more than 180 days after transplantation (229 to 535 days or almost 18 months), all specimens were positive for HCVAg, with 11 of 17 samples (65%) graded as 31 (more than 20% hepatocytes positive for HCVAg). The mean serum HCV RNA level was 357.19 6 238.68 3 104 copies/mL. Histologically, changes in 14 liver biopsy specimens were consistent with chronic hepatitis and in three specimens with nonhepatitis lesions (acute rejection, chronic rejection, ischemia/sepsis). The degree of HCVAg expression was significantly more prominent in specimens from group B or C than in those from group A (P , .001, Table 2). The highest levels of serum HCV RNA were found in serum samples from patients whose liver specimens were included in group B (Table 2). The mean HCV RNA level in this group was significantly higher than in serum samples taken simultaneously with liver transplant specimens from group A (P 5 .0002) or from group C (P 5 .041); also, the mean HCV RNA level in sera of patients included in group C was higher than those of group
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Figure 2. Amounts of HCVAg in the liver determined by the number of positive hepatocytes and histopathologic diagnosis in the 42 biopsy specimens divided into three groups according to the time intervals between liver biopsy and OLT.
A (P 5 .0016). The distribution of HCVAg was random, sometimes with a tendency to a focal pattern, when a smaller number of hepatocytes were positive. More uniform distribution of positive hepatocytes was observed in specimens with a larger number of positive cells. When specimens were classified according to their histopathologic changes, HCVAg was detectable in 8 of 15 samples (53%) with nonhepatitis histologic features (Table 1 and Fig. 2) and in only two specimens scoring 31. The mean serum HCV RNA in these cases was 50.47 6 15.71 3 104 copies/mL. All six specimens with nonhepatitis histologic features, which were obtained more than 32 days after OLT (group B), were positive for HCVAg (11 in three, 21 in one, and 31 in two specimens; Table 1 and Fig. 2). Ten specimens with acute hepatitis had a high expression of HCVAg, graded as 31 (greater than 20% positive cells) in nine and as 21 in one. Their mean serum HCV RNA level was 585.67 6 192.06 3 104 copies/mL. All 17 specimens with lesions of chronic hepatitis were positive for HCVAg. The proportion of HCVAgpositive cells in the group with chronic hepatitis
was graded as 11 in four, 21 in one, and 31 in 12 liver biopsy specimens, and the mean serum HCV RNA level in serum was 376.88 6 237.97 3 104 copies/mL. In this group, only 9 of 14 (64%) specimens studied more than 229 days after OLT had more than 20% hepatocytes positive for HCVAg, whereas all three specimens tested 124, 139, and 158 days after OLT had a very high number of positive liver cells (31, .20%). In chronic hepatitis, the level of HCVAg in specimens with a necroinflammatory score of greater than or equal to 6 was similar to that in specimens with a necroinflammatory score of less than 6 (P 5 NS). When we compared staining among specimens with acute or chronic hepatitis versus samples with nonhepatitis histologic features in each of the groups (A, B, or C), we only found statistically significant differences in group B (A, P 5 .3; B, P 5 .02; C, P 5 .9). Because the number of patients in these groups is small, we cannot definitively conclude that the amount of HCVAg correlates with the presence of hepatitis. Only when the specimens were compared irrespective of the time when the biopsy was performed was it found that the amount of HCVAg
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Immunohistochemical Assessment of HCVAg
in liver specimens with acute or chronic hepatitis was larger than in specimens with no histologic changes or with features other than viral hepatitis (P 5 .0002 and P 5 .0004, respectively; Table 3). HCVAg expression was also higher in biopsy specimens with acute hepatitis than in those specimens showing chronic hepatitis, although differences were not significant. HCV RNA levels in serum were also higher in patients with acute or chronic active hepatitis than in those with nonhepatitis histologic features (P , .001 and P , .005, respectively; Table 3). Serum HCV RNA serum levels in patients with acute hepatitis were higher than in those with chronic hepatitis (P 5 .02; Table 3). Liver HCVAg expression and HCV RNA and serum HCV RNA levels showed a significant degree of correlation (P 5 .01; r 5 .32; Fig. 3). In the control group, serum samples tested repeatedly for HCV RNA were negative, and all 10 liver biopsy specimens obtained from these patients tested negative for HCVAg.
Table 2. HCVAg in Hepatocytes and Levels of Serum HCV RNA in Three Groups of Liver Biopsy Specimens From Liver-Transplant Recipients
Group A (1 mo post-OLT)
No. of Liver Specimens
Amount of HCVAg in Each Specimen
10
01:7 11:1 21:1 31:1
28.4 6 12.5
Serum HCV RNA (mean no. copies 3 104/mL 6 SD)
Table 3. HCVAg in Hepatocytes and Levels of Serum HCV RNA in Liver Biopsy Specimens From LiverTransplant Recipients With Histopathologic Features of Nonhepatitis Changes and Acute or Chronic Hepatitis
Histopathologic Assessment
No. of Liver Specimens
Amount of HCVAg in Liver*
Nonhepatitis features†
15
01:7 11:4 21:2 31:2
50.47 6 15.71
Acute hepatitis
10
01:0 11:0 21:1 31:9
585.67 6 192.06
Chronic hepatitis
17
01:0 11:4 21:1 31:12
376.88 6 237.97
Serum HCV RNA (mean no. copies 3 104/mL 6 SD)
NOTE. Comparison of HCVAg in liver (chi-squared test for trend): nonhepatitis features v acute hepatitis, P 5 .0002; nonhepatitis features v chronic hepatitis, P 5 .0004; acute hepatitis v chronic hepatitis, P 5 NS. Comparison of serum HCV RNA levels (MannWhitney U test): nonhepatitis features v acute hepatitis, P 5 .0005; nonhepatitis features v chronic hepatitis, P 5 .0046; acute hepatitis v chronic hepatitis, P 5 .02. *HCVAg in liver from 01 to 31 scale (01: neg, 11: ,5%, 21: 5%-20%, 31: .20% positive hepatocytes). †Graft rejection (acute, n 5 7; chronic, n 5 1); ischemia sepsis, n 5 1; normal liver, n 5 6.
Discussion
B (2-6 mo post-OLT)
15
01:0 11:2 21:2 31:11
442.32 6 138.17
C (.6 mo post-OLT)
17
01:0 11:5 21:1 31:11
357.19 6 238.69
NOTE. HCVAg in liver: group A v group B, P 5 .0002; group A v group B, P 5 .0005; group B v group C, P 5 NS (chi-squared test for trend). HCV RNA serum level: group A v group B, P 5 .0002; group A v group C, P 5 .0016; group B v group C, P 5 .041 (Mann-Whitney U test). *HCVAg in liver from 01 to 31 scale (01: neg, 11: ,5%, 21: 5%-20%, 31: .20% positive hepatocytes).
HCV infection of a liver graft is a constant phenomenon observed in most HCV-positive liver-transplant recipients.1,2,5,6 HCV RNA becomes readily detectable a few weeks after transplantation, reaching levels 10- to 20-fold higher than those present before transplantation.3 In this study, we identified the presence of HCVAg in liver biopsy specimens from unselected transplant recipients, estimated the viral load in serum at the time of liver biopsy, and evaluated histopathologic changes in the liver. HCV was detected only in one third of the liver biopsy specimens obtained during the first month after transplantation. In contrast, HCVAg was found in all liver specimens obtained after the first month posttransplantation by using immunoglobulins that reacted primarily with NS3/NS4 proteins.11 Gane et
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Figure 3. Correlation between amounts of HCVAg in the liver and HCV RNA in serum (r 5 .32; P 5 .01).
al also observed HCV core and NS4 proteins by using monoclonal antibodies in 30% of patients during the first month after transplantation, but only in 60% of patients after 3 months, and, similar to our findings, in all patients after 6 months posttransplantation.6 In our studies, the amount of HCVAg estimated semiquantitatively in coded samples correlated with the level of HCV RNA in serum and suggested that an increased proportion of cells containing HCV NS3/NS4 antigens reflected a higher level of HCV replication. This observation extends previously reported data on correlation between HCV core antigen in hepatocytes and the level of HCV RNA in serum.16 These correlations have also been observed by Di Martino et al17 between liver HCV RNA and serum HCV RNA. It has been noted that HCV reinfection is concordant with biochemical and histologic evidence of acute hepatitis during the first 8 weeks after transplantation4-6 with subsequent development of chronic hepatitis and cirrhosis. In addition, a progressive severe cholestatic form of hepatitis has been observed in 10% of HCV-reinfected patients.18 Preliminary data suggest that clinical complications of cirrhosis may be evident in some patients as early as 5 years after transplantation and
HCV reinfection.9 In our study, the degree of HCVAg expression in hepatocytes in specimens with morphologic changes of acute and chronic hepatitis was significantly greater than in those without histologic features of viral hepatitis. Only in occasional specimens were large amounts of HCVAg accompanied by changes unrelated to viral hepatitis. Similar observations were reported by others who found hepatocytic HCV NS4 proteins in 45% of patients with chronic active hepatitis and in a similar proportion of HCV-reinfected patients without histologic features of viral hepatitis.5 Large amounts of HCVAg (31) were seen in 70% of liver transplant biopsy specimens with morphologic characteristics of chronic hepatitis that were observed in this study, whereas only 11% of 27 nonimmunosuppressed patients with chronic hepatitis had high expression of HCVAg as determined with the same immunohistochemical technology.14 In our studies, there was a trend toward an increased amount of hepatocellular HCVAg in those specimens with acute or chronic hepatitis lesions, but the pathogenic significance of morphologic expression of structural and nonstructural HCV proteins for the development of liver lesions remains unclear. Studies of the natural history of HCV reinfection
Immunohistochemical Assessment of HCVAg
of a grafted liver require longitudinal observations carried out under systematically designed protocols, which are difficult to introduce in clinical settings. Our results are limited to a transverse correlation of HCVAg detection in the liver, serum HCV RNA levels, and morphologic changes in the tested liver grafts obtained from multiple patients at different time points after transplantation. The consistency of correlations at the discrete posttransplantation intervals suggests a potential dynamic scenario of the viral replication profile and its morphologic consequences in liver-transplant recipients with recurrent HCV infection. Validation of our findings and correlation of the dynamics of the tested variables should be pursued in a prospective, sequential, follow-up study.
7.
8.
9.
10.
11.
Acknowledgment The authors thank Dorrie A. Carson for excellent technical assistance in preparation of immunohistochemical slides.
References 1. Wright TL, Donegan E, Hsu HH, Ferrell L, Lake JR, Kim M, et al. Recurrent and acquired hepatitis C viral infection in liver transplant recipients. Gastroenterology 1992;103:317-322. 2. Sallie R, Cohen AT, Tibbs CJ, Portmann BC, Rayner A, O’Grady JG, et al. Recurrence of hepatitis C following orthotopic liver transplantation: A polymerase chain reaction and histological study. J Hepatol 1994;21:536542. 3. Chazouilleres O, Kim M, Combs C, Ferrell L, Bachetti P, Roberts J, et al. Quantitation of hepatitis C virus RNA in liver transplant recipients. Gastroenterology 1994;106: 994-999. 4. Ferrell LD, Wright TL, Roberts J, Ascher N, Lake J. Hepatitis C viral infection in liver transplant recipients. Hepatology 1992;16:865-876. 5. Gretch DR, Bacchi CE, Corey L, dela Rosa C, Lesniewski RR, Kowdley K, et al. Persistent hepatitis C virus infection after liver transplantation: Clinical and virological features. Hepatology 1995;22:1-9. 6. Gane EJ, Naoumov NV, Qian KP, Mondelli MU, Maertens G, Portmann BC, et al. A longitudinal analysis of
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