Michele Gigou

European collaborative study on factors influencing outcome after liver transplantation for hepatitis C

BACKGROUND & AIMS Liver transplantation for hepatitis C virus (HCV)-related liver disease is characterized by frequent graft infection by HCV. The prognosis and risk factors for morbidity and mortality in this condition were determined. METHODS A retrospective study of 652 consecutive anti-HCV-positive patients undergoing liver transplantation between 1984 and 1995 in 15 European centers was conducted; 102 patients coinfected with hepatitis B virus (HBV) received immunoglobulin prophylaxis for antibody to hepatitis B surface antigen. RESULTS Overall, 5-year survival was 72%. Five-year actuarial rates of hepatitis and cirrhosis were 80% and 10%. Genotypes 1b, 1a, and 2 were detected in 214 (80%), 24 (9%), and 24 (9%) of 268 patients analyzed. The only discriminant factor for patient or graft survival was hepatocellular carcinoma as primary indication. Independent risk factors for recurrent hepatitis included the absence of HBV coinfection before transplantation (relative risk [RR], 1.7; 95% confidence interval [CI], 1.2-2.6; P = 0.005), genotype 1b (RR, 2; 95% CI, 1.3-2.9; P = 0.01), and age > 49 years (RR, 1.4; 95% CI, 1.1-1.8; P = 0.01). CONCLUSIONS The results of transplantation for HCV-related disease are compromised by a significant risk of cirrhosis, although 5-year survival is satisfactory. Genotype 1b, age, and absence of pretransplantation coinfection by HBV are risk factors for recurrent HCV.

Compartmentalization of Hepatitis C Virus Genotypes between Plasma and Peripheral Blood Mononuclear Cells

ABSTRACT Differences in hepatitis C virus (HCV) variants of the highly conserved 5′ untranslated region (UTR) have been observed between plasma and peripheral blood mononuclear cells (PBMC). The prevalence and the mechanisms of this compartmentalization are unknown. Plasma and PBMC HCV variants were compared by single-strand conformation polymorphism (SSCP) and by cloning or by genotyping with a line probe assay (LiPA) in 116 chronically infected patients, including 44 liver transplant recipients. SSCP patterns differed between compartments in 43/109 analyzable patients (39%). Differences were significantly more frequent in patients with transplants (21/38 [55%] versus 22/71 [31%]; P < 0.01) and in those who acquired HCV through multiple transfusions before 1991 (15/20; 75%) or through drug injection (16/31; 52%) than in those infected through an unknown route (7/29; 24%) or through a single transfusion (5/29; 17%; P < 0.001). Cloning of the 5′ UTR, LiPA analysis, and nonstructural region 5B sequencing revealed different genotypes in the two compartments from 10 patients (9%). In nine patients, the genotype detected in PBMC was not detected in plasma and was weak or undetectable in the liver in three cases. This genotypic compartmentalization persisted for years in three patients and after liver transplantation in two. The present study shows that a significant proportion of HCV-infected subjects harbor in their PBMC highly divergent variants which were likely acquired through superinfections.

Incidence of Hepatitis C in Patients Receiving Different Preparations of Hepatitis B Immunoglobulins after Liver Transplantation

Cirrhosis related to hepatitis C virus (HCV) is becoming the most common indication for liver transplantation; it now accounts for 25% of elective procedures in the United States. Infection with HCV always persists after liver transplantation and often leads to chronic hepatitis. Cirrhosis occurs in up to 10% of HCV-infected patients during the first 5 years after transplantation, and long-term graft survival will no doubt be compromised in this population. In addition, many patients who underwent transplantation before the introduction of routine blood and organ donor screening for antibody to HCV (anti-HCV) acquired HCV via transplantation [1, 2]. Polyclonal immunoglobulins against hepatitis B surface antigen (HBIGs) can prevent the recurrence of hepatitis B virus (HBV) infection and have drastically improved the outcome of transplantation in transplant recipients who are positive for hepatitis B surface antigen (HBsAg) [3]. However, polyclonal immunoglobulins enriched in anti-HCV are not yet available to prevent recurrent hepatitis C after liver transplantation. Until recently [4], there was no formal evidence of humoral neutralization of HCV. Furthermore, neutralizing antibodies to HCV seem to be strain-specific, and transmission of HCV through polyclonal immunoglobulins has been described [5, 6]. Liver transplantation in HBsAg-positive patients who receive long-term prophylaxis with HBIG provides an opportunity to study the influence of polyclonal immunoglobulins on HCV infection. Indeed, before March 1990 in France, donors positive for anti-HCV were not detected, and HBIG obtained from large numbers of donors probably contained anti-HCV and antibody to HBV [7]. To analyze the influence of both HBIG and the date on which HCV screening tests were introduced in France (March 1990), we examined the frequency of HCV infection in patients who had transplantation before and after this date according to HCV infection status before transplantation and HBIG administration after transplantation. Methods Five hundred thirty-eight patients who underwent transplantation between December 1984 and March 1994 were initially considered for our study. This group comprised all patients who received transplants for HBV- or HCV-related cirrhosis and all patients who received transplants for nonviral cirrhosis before March 1990. Blood-product exposure was assessed in every case as the total number of units of packed red blood cells, frozen plasma, and platelets transfused during transplantation and during the first year after transplantation and as the number of blood donors (that is, donors of packed red blood cells, fresh frozen plasma, and platelets). As of October 1989, 41 patients had undergone transfusion with pooled clotting factors (Centre National Transfusion Sanguine, Paris, France) to minimize perioperative bleeding. Patients positive for HBsAg were prescribed long-term HBIG therapy (Laboratoire Francais de Fractionnement et de Biotechnologie, Les Ulis, France), as described elsewhere [8]. Briefly, these patients received 10 000 IU of HBIG during the anhepatic phase and 10 000 IU every day during the first 6 days after surgery. The titer of antibody to HBsAg (anti-HBsAg) was then assessed weekly; 10 000 IU of HBIG was administered when the titer was less than 100 IU/L. Immunosuppression In 401 patients, basic immunosuppression was induced by a combination of cyclosporine (Sandoz, Bales, Switzerland), steroids, and azathioprine. Twenty-seven patients received a combination of steroids and tacrolimus (Fujisawa, Osaka, Japan) without azathioprine [9]. Forty-three patients had initially been enrolled in a prospective multicenter trial [10] of antilymphocyte antithymocyte globulin (Biomerieux, Lyon, France), given for renal impairment (n = 15), or Orthoclone OKT3 (Ortho, Raritan, New Jersey) (n = 28), given for primary prophylaxis of rejection. All episodes of rejection were first treated with one to three boluses of steroids and then with Orthoclone OKT3 if necessary. Follow-up All patients were seen at our outpatient clinic by the same team of hepatologists or surgeons at least monthly during the first year and every 2 to 6 months thereafter. Liver graft biopsies were performed when liver test results were abnormal, and all specimen slides were interpreted by the same pathologist. Protocol biopsies were done routinely 1, 2, and 5 years after transplantation in 93%, 88%, and 90% of survivors, respectively. These survivors had the same liver graft at these follow-up examinations. All patients underwent at least one liver biopsy more than 6 months after transplantation. Lobular hepatitis was defined as hepatocyte necrosis and portal and lobular infiltration by mononuclear cells. Serum samples have been stored at 30C from 1984 to the present in the same location. Screening of Organs, Blood, and Donors for Polyclonal Hepatitis B Immunoglobulin In March 1990, serologic screening for HCV with a first-generation commercial kit became obligatory in France for all blood and liver donors, although it was initiated in most French blood banks as early as December 1989. All batches of HBIG prepared from the plasma of patients not tested for HCV had to be destroyed in 1991. In March 1990, however, our blood bank destroyed all batches of HBIG harvested before December 1989 (the start of effective screening in France). Therefore, all 182 patients who had transplantation after March 1990 received grafts, blood products, and HBIG from donors who were negative for anti-HCV. Markers of Hepatitis B Virus Until 1993, all HBsAg-positive patients were tested for serum HBV DNA before liver transplantation by using the spot-test technique [11]; after 1993, testing was done with the Hybrid capture system (Digen Sharp Signal System, Beltsville, Maryland). Patients receiving HBIG underwent weekly titration of anti-HBsAg during the first 2 years; after that point, titration was done at least bimonthly. If anti-HBsAg titers were low (<100 IU/L), routine testing for HBsAg was done. Markers of Hepatitis C Virus Antibodies to HCV were confirmed retrospectively in all serum samples obtained before transplantation by using a recombinant immunoblot assay (RIBA-2 and then RIBA-3, Test System, Chiron, Emeryville, California). At the end of 1991, serologic tests for HCV were done yearly in every case. In addition, serum samples harvested during the first month after transplantation from 28 patients who were negative for anti-HCV and were receiving HBIG (15 underwent transplantation before 1990 and 13 underwent transplantation after this date) were tested retrospectively with both enzyme-linked immunosorbent assay (HCV Elisa 3.0, Orthodiagnostic system) and a line immunoassay (InnoLia HCV Ab III, Innogenetics, Gent, Belgium). These assays are more sensitive than the recombinant immunoblot assay. All serum samples obtained before and after transplantation were tested for HCV RNA. Serum samples were obtained a mean of 6 weeks (range, 0 to 6 months) before liver transplantation and a mean of 12 months (range, 10 to 16 months) after liver transplantation. In patients receiving HBIG, serum samples were obtained a few hours before perfusion of HBIG. A commercial polymerase chain reaction assay (Amplicor kit, Produits Roche, Basel, Switzerland) was used to detect HCV RNA, according to the manufacturers instructions. Samples with negative Amplicor signals were retested in a one-tube nested polymerase chain reaction procedure [12]. The HCV genotypes were determined by using a line probe assay (InnoLipa, Innogenetics) according to the manufacturers instructions. In all amplification procedures, decontamination was based on classic recommendations [13] and on the routine use of uracil-N-glycosylase (AmpErase, Perkin-Elmer, Norwalk, Connecticut). Statistical Analysis We used BMDP statistical software (University of California Press, Berkeley, California). Analysis was done separately on the incidence of HCV viremia 1 year after transplantation and on the actuarial rate of HCV-related hepatitis. The latter was defined as a combination of histologically proven hepatitis and serum positivity for HCV RNA. Infection with HCV was defined as acquired if the patient was negative for both anti-HCV and HCV RNA before transplantation; infection was defined as recurrent if at least one of these two markers was detected. Histologic follow-up began on the date of the first liver transplantation and continued until the last available biopsy of the first liver graft. Presence of HCV viremia at 1 year and actuarial rates of hepatitis due to HCV were analyzed according to the following variables: age; sex; pretransplantation markers of HCV (serologic results and HCV RNA), HBV (HBsAg), and hepatitis delta virus (antibody to hepatitis delta virus); use of HBIG; immunoprophylaxis of rejection (antilymphocyte serum or Orthoclone OKT3); year of transplantation; amount and number of donors of each perfused blood product (red blood cells, platelets, frozen plasma, and pooled clotting factors); numbers of steroid boluses during the first year after transplantation; and steroid-resistant rejection. Incidence of viremia 1 year after transplantation was analyzed by using the chi-square test and logistic regression (maximum likelihood). Analysis of actuarial rates of histologically defined hepatitis C was based on the log-rank test and Cox model. All data are presented with 95% CIs. Role of Funding Source The institutions that supported this study had no direct role in gathering, analyzing, or interpreting the data and played no role in the decision to publish the study findings. Results Of the 538 patients initially eligible for the study, 64 died during the first year. Death was caused by sepsis (n = 33), cancer recurrence (n = 6), cardiovascular complications (n = 10), perioperative complications (n = 4), de novo cancer (n = 5), transplant rejection (n = 5), or unexplained graft failure (n = 1).

Influence of CD4 Cell Counts on the Genetic Heterogeneity of Hepatitis C Virus in Patients Coinfected with Human Immunodeficiency Virus

To study the effects of reduced CD4 T cell activity on hepatitis C virus (HCV) genetic heterogeneity, HCV quasi-species complexity and diversification over time were analyzed for 56 human immunodeficiency virus-coinfected patients. Patients were selected retrospectively from the French Seroconverter Cohort (SEROCO) and the French Hemophilia Cohort (HEMOCO) for having stable CD4 cell counts for 3 years. HCV complexity was assessed by single-strand conformation polymorphism analysis of the envelope-coding region (HVR) and the core region at 2 time points 3 years apart. Increased HVR complexity was associated with higher CD4 cell count and HCV genotype 1 infection. Qualitative variation of HVR and core region was not related to CD4 cell count and depended on the initial complexity. Complexity of both regions remained unchanged over 3 years. Among these HCV-HIV-coinfected patients with stable CD4 cell counts, viral genotype and CD4 cell count may have influenced HVR complexity before their inclusion in the study but were not involved in HVR diversification during the 3-year follow-up period.

Long-term ganciclovir therapy for hepatitis B virus infection after liver transplantation

BACKGROUND/AIMS Hepatitis B virus (HBV) disease on a liver graft is associated with florid viral replication and graft failure. The aim of this study performed between 1992 and 1995 was to investigate the safety and efficacy of long-term intravenous ganciclovir for HBV infection in liver transplant recipients. METHODS Twelve patients with HBV re-infection and four with de novo HBV infection were studied. HBV DNA was positive in all (median titer: 437.5 pg/ml) and HBeAg was positive in seven. Intravenous ganciclovir was started after a median of 14.5 months from HBsAg positivation and continued for a median of 10 months. RESULTS A complete response with HBV DNA negativation was seen in ten cases, a partial response with a decrease of more than 50% of initial HBV DNA levels in four and no response in two. Overall tolerance was good. Among the ten complete responders, two seroconverted for both HBsAg and HBeAg and one for HBsAg alone. Among these ten patients, three were re-transplanted for liver failure: two of them are alive; three had a viral breakthrough during treatment; and four remained HBV DNA negative: two are alive and two died. Partial responders and nonresponders were treated with other antiviral agents and three were re-transplanted, two of them are alive. Overall 12 out of 16 patients (75%) survived with a median follow up of 46 months. CONCLUSIONS Long-term intravenous ganciclovir can persistently inhibit HBV DNA replication in liver transplant recipients and is well tolerated. Further evaluation should be encouraged, especially for HBV recurrence after first-line treatments.