Cirrhosis caused by hepatitis C virus (HCV) infection is the leading indication for liver transplantation1, 2 and HCV viremia persists in up to 95% of patients posttransplantation.3 By 5 yr posttransplantation, HCV-induced hepatitis and cirrhosis occur in approximately 80% and 10% of patients, respectively.4, 5 Herpesvirus reactivation is common after liver transplantation. Cytomegalovirus (CMV) infection and disease is especially common in CMV donor seropositive/recipient seronegative transplant recipients. Other herpesviruses, including human herpesviruses (HHV)-6 and HHV-7, also commonly reactivate after transplantation and coinfections with these viruses may manifest as febrile syndromes.6
The interaction between herpesviruses and HCV has been proposed to be clinically important in HCV-infected liver transplant recipients.7–10 CMV in particular may have immunomodulatory effects, and lead to cytokine dysregulation, resulting in a number of indirect effects such as an increased risk of opportunistic infection, posttransplantation lymphoproliferative disease (PTLD), and acute and chronic graft injury.11 CMV7 and HHV-67, 9 have also been associated with more severe forms of HCV recurrence. Although fewer data are available, other immunomodulatory effects have also been proposed for other β-herpesviruses, including HHV-6 and HHV-7.11
It is less clear whether HCV can promote herpes viral infection. Although viral interactions are presumed to be bidirectional, a clear immunomodulatory role for HCV has not been demonstrated. Some studies have demonstrated that opportunistic infections are more common in HCV-infected patients than liver transplant patients without HCV.12 Patients with HCV infection have been shown to be more susceptible to infections that are associated with defects in cell-mediated immunity such as CMV and fungal infections.13 In addition, HCV has been identified as a risk factor for the development of PTLD,14, 15 which is usually causally related to Epstein-Barr virus (EBV) infection.
The primary objective of this study was to assess whether herpesvirus reactivation occurs more commonly in HCV-infected compared with HCV-uninfected patients in a large cohort of well-characterized liver transplant recipients. To assess whether HCV promotes herpesvirus replication, we compared the incidence and the degree of viral replication over time for several herpesviruses, including CMV, HHV-6, HHV-7, HHV-8, VZV, and EBV in liver transplant patients with and without HCV infection.
A total of 177 CMV donor positive/recipient negative (D+/R−) liver transplant recipients were included in the study. This cohort was part of a larger study that evaluated CMV prophylaxis in 364 donor positive/recipient negative transplant recipients. The parent study was a randomized, multicenter trial assessing the efficacy and safety of valganciclovir (Valcyte; F Hoffmann-La Roche, Basel, Switzerland) and oral ganciclovir (Cymevene/Cytovene; F Hoffman-La Roche) in preventing CMV disease in solid organ transplant recipients.16 Inclusion and exclusion criteria have been described previously.16 Briefly, in the parent study, eligible patients were ≥13 yr of age receiving a first liver, heart, kidney, or pancreas allograft with a CMV serostatus of D+/R−. The study was approved by the Independent Ethics Committees/Institutional Review Boards of participating centers and was conducted in accordance with the declaration of Helsinki. A priori substudies were built into the parent study, including an assessment of herpesvirus reactivation at regular intervals. Patients signed a separate consent form for participation in all substudies. HCV-positive patients were identified based on pretransplantation serology using a current generation enzyme-linked immunosorbent assay test. Pretransplantation testing for HCV was preformed at each local center at an accredited laboratory.
Antivirals and Immunosuppression
For CMV prophylaxis all patients received either valganciclovir 900 mg once daily or oral ganciclovir 1,000 mg 3 times a day, adjusted for renal function (randomized in a 2:1 fashion), for up to 100 days after transplantation. Immunosuppression therapy was administered to patients according to the standard practice at the participating center. All patients were followed up to 12 months posttransplantation. Therapeutic practices for HCV were as per center-specific protocols. However, any form of interferon therapy was prohibited during the first 100 days posttransplantation.
Blood specimens for viral testing were gathered prospectively. Plasma and whole blood were stored at −80°C until further testing. Blood samples were taken at baseline and on days 14, 42, 70, and 100, and at months 4, 5, 6, 8, and 12 posttransplantation. CMV, EBV, VZV, HHV-6, HHV-7, and HHV-8 viral loads were assessed by quantitative polymerase chain reaction assays. For CMV, viral loads were performed on plasma samples using the Cobas Amplicor Monitor Test (Roche Diagnostic Systems, Branchburg, NJ). The lower limit of detection for this assay was 400 copies/mL. For all other herpesviruses, in-house real-time polymerase chain reaction assays based on the Lightcycler format were performed on stored whole blood samples using methods previously described.17 The lower limit of detection for these assays was ∼5 copies/mL of whole blood. CMV viral load testing was done in all patients while other herpesviruses were tested for in patients who provided consent for these specific tests. HCV viral load testing was performed on a subset of plasma samples using the Roche assay (Roche Diagnostic Systems) according to manufacturer's instructions (lower limit of detection 600 copies/mL).
In HCV-positive patients, HCV viral load testing was performed at baseline (between days 0-10), day 28, day 56, day 100, and 6 and 12 months after transplantation.
Herpesviruses, and in particular CMV, have been shown to have immunomodulatory effects. Some previous studies have suggested more rapid progression of HCV in liver transplant recipients coinfected with CMV or HHV-6.7–10 Since viral interactions may be bidirectional, we evaluated whether HCV infection promoted herpesvirus replication using sensitive quantitative polymerase chain reaction assays. In this study of 177 liver transplant patients, there was no evidence for an immunomodulatory role for HCV with respect to herpesvirus reactivation. The incidence of CMV infection and disease was not significantly different between HCV-positive and HCV-negative patients. Similarly, the incidence of EBV, HHV-6, and HHV-7 were not higher in HCV patients. In fact, most trends favored less reactivation in the HCV group. The only significant finding was a lower incidence of HHV-7 infection in the HCV-positive patients. Therefore, our results suggest that chronic HCV infection does not appear to promote herpesvirus reactivation or replication. It should be emphasized that this is a select subgroup of CMV D+/R− patients, all of whom received prolonged antiviral prophylaxis. It is possible that the results may not be generalizable to CMV R+ patients.
Most previous studies have focused on the alternative hypothesis, which is whether herpesvirus infections promote HCV replication and thereby result in accelerated progression of liver disease due to HCV. Rosen et al.10 evaluated 43 HCV-positive liver transplant recipients, of whom 8 had CMV viremia. Patients with CMV viremia had a higher mean total Knodell score at last follow-up and a higher incidence of cirrhosis. Razonable et al.8 investigated the effect of β-herpesviruses in 92 HCV-infected liver transplant recipients. CMV infection and disease, but not HHV-6 infection, were independently associated with allograft failure and mortality. Patients with CMV disease had a higher fibrosis stage and had a trend toward a higher hepatitis activity index and HCV load at 16 weeks after liver transplantation.8 Interestingly, while both HHV-6 and CMV infection were common in this cohort, HHV-7 was not detected in the plasma of the patients.8 This is similar to our finding of a lower rate of HHV-7 infection in the HCV-positive group vs. the HCV-negative group. In another study,7 no correlation was observed between HHV-6, CMV, and HCV viral loads in 66 liver transplant recipients, although more severe degrees of fibrosis and hepatitis were seen in patients with CMV disease and HHV-6 infection. Unlike the current trial, the above trials did not use controlled antiviral prophylaxis. In this study, HCV viral load did not differ between those with and without CMV disease; however, this study was not designed to assess the effects of herpesviruses on HCV replication, and only a small subset of patients could be assessed. It would be interesting to investigate the effects of herpesviruses on HCV viral load and replication in a study using controlled antiviral prophylaxis.
The mechanisms behind these interactions are unclear. Viral reactivation may merely be a marker of a more profound immunosuppressed state promoting both HCV and herpesvirus replication. Alternatively, more specific interactions may exist. CMV-induced immunosuppression may lead to poor immunologic control of HCV. Also, both CMV and HHV-6 result in cytokine dysregulation and upregulation of tumor necrosis factor-alpha production, which may be important in the pathogenesis of HCV.18 Finally, cross-reactive immunologic responses may also lead to more severe forms of allograft injury.
Fewer studies have analyzed whether HCV infection has an immunomodulatory effect and can promote herpesvirus replication. Although immunomodulatory effects have been long suspected, there are limited clinical data for this. In vitro, HCV proteins (e.g., soluble core protein) can be demonstrated to cause dose-dependent suppression of T-cell proliferation, and can potentiate suppression by cyclosporine.19 In a multivariate analysis of risk for PTLD in 16 of 480 liver transplant recipients, HCV infection was found to be an independent predictor of PTLD (RR = 8.7; 95% confidence interval = 1-78.3).20 Since the majority of PTLDs are related to EBV, one might expect a higher incidence of EBV viremia in transplant patients with HCV if an interaction existed. In the current study, although the incidence of EBV viremia was high in the HCV-positive group, no difference was observed compared with the HCV-negative group. Singh et al.12 analyzed infectious complications in 100 liver transplant recipients. There was a higher incidence of fungal infections (18% vs. 6%) and CMV disease (32% vs. 9%) in patients with recurrent HCV infection than in HCV-negative patients. This is in contrast to the findings in our study; however, we did not specifically compare those with recurrent HCV disease with those without recurrent disease.
Our study had several limitations. First, this was a substudy of a larger CMV prophylaxis study and thus may have suffered from selection bias. However, the virological substudies were built into the protocol a priori and thus selection of patients was performed according to the randomization process. Second, it is possible that antiviral prophylaxis may have modified or blunted any potential interactions. Specifically, the antiviral prophylaxis while directed against CMV, may have had effects on EBV and HHV-6 viral loads. It is possible that more difference in viral loads may have been observed if no prophylaxis was given or if a purely preemptive strategy for CMV was used. Third, the protocol liver biopsies were not performed and thus the effect of herpesviruses on allograft cirrhosis and recurrent hepatitis could not be assessed. Fourth, the duration of follow-up was only 1 yr, and thus we could not assess the impact of herpesvirus reactivation on long-term HCV-induced liver disease. However, the primary purpose of this study was to assess if HCV infection promoted herpesvirus replication. Finally, there may have been subtle differences in immunosuppression between HCV-positive vs. HCV-negative patients that were not picked up by the current analysis, and that may have affected the results. The strengths of the study include the relatively large prospectively followed samples used and the use of sensitive molecular diagnostics at regular intervals posttransplantation to determine herpesvirus reactivation.
In conclusion, using sensitive molecular diagnostic assays, we observed that liver transplant recipients with HCV do not have a higher incidence or degree of herpesvirus reactivation. These data do not support an in vivo immunomodulatory role for HCV. Future studies should include specific measures of cellular immune function to further define the potential immunomodulatory effects of viral infection.
VALGANCICLOVIR STUDY GROUP
There follows a complete list of the members of the Valganciclovir Solid Organ Transplant Study Group in alphabetical order by country:
In Australia: Josie Eris, Royal Prince Alfred Hospital, Camperdown, NSW; Anne Keogh, St. Vincent's Hospital, Darlinghurst, NSW; Tim Mathew, Queen Elizabeth Hospital, Woodville, SA; Geoff McCaughan, Royal Prince Alfred Hospital, Camperdown, NSW; Kathy Nicholls, Royal Melbourne Hospital, Parkville, VIC; Simone Strasser, Royal Prince Alfred Hospital, Camperdown, NSW.
In Canada: Atul Humar, Toronto General Hospital, Toronto, ON; Richard Lalonde, Montreal Chest Institute, Montreal, QC; Paul Marotta, London Health Sciences Center University Campus, London, ON; Jutta Preiksaitis, University of Alberta Hospital, Edmonton, AB; Eric Yoshida, Vancouver Hospital and Health Sciences Center, Vancouver, BC.
In France: Iradj Gandjbakch, Pitie-Salpetriere Hospital, Paris; Yvon Lebranchu, Bretonneau Hospital, Tours; Christophe Legendre, Saint-Louis Hospital, Paris; Faouzi Saliba, Hopital Paul Brousse, Villejuif.
In Ireland: Oscar Traynor, St. Vincents University Public Hospital, Dublin.
In Italy: Paolo Angeli, Azienda Ospedaliera Di Padova, Padova; Francesco Menichetti, Ospedale Cisanello, Pisa.
In New Zealand: Ed Gane, Auckland Hospital, Auckland.
In the United Kingdom: Ali Bakran, Royal Liverpool University Hospital, Liverpool; John Forsythe, Edinburgh Royal Infirmary, Edinburgh; Nigel Heaton, Kings College Hospital, London; Peter Lodge, St. James Hospital, Leeds; Derek Manas, Freeman Hospital, Newcastle Upon Tyne; Peter Morris, Churchill Hospital, Oxford; Jayan Parameshwar, Papworth Hospital, Papworth Everard; Nizar Yonan, Wythenshawe Hospital, Manchester.
In the United States: Barbara Alexander, Duke University Medical Center, Durham, NC; Emily Blumberg, Hospital of the University of Pennsylvania, Philadelphia, PA; Daniel C. Brennan, Barnes Jewish Hospital, St. Louis, MO; Robert Brown, Columbia Presbyterian Medical Center, New York, NY; Ronald W. Busuttil, UCLA School of Medicine, Los Angeles, CA; Ken Chavin, Medical University South Carolina, Charleston, SC; David Conti, Albany Medical Center, Albany, NY; Angelo DeMattos, Oregon Health Sciences University, Portland, OR; Ed Dominguez, University of Nebraska Medical Center, Omaha, NE; Howard J. Eisen, Temple University School of Medicine, Philadelphia, PA; Dan Fishbein, University of Washington, Seattle, WA; Thomas Fishbein, Mt. Sinai Medical Center, New York, NY; Robert Fisher, Medical College of Virginia Hospital, Richmond, VA; Richard Freeman, New England Medical Center, Boston, MA; Chris Freise, University of California San Francisco, San Francisco, CA; Marquis Hart, UC San Diego Medical Center, San Diego, CA; Thomas Heffron, Emory University, Atlanta, GA; Ray E. Hershberger, Oregon Health Sciences University, Portland, OR; Richard J. Howard, University of Florida, Gainesville, FL; Sandra A. Kemmerly, Alton Ochsner Medical Institution, New Orleans, LA; Richard Knight, Mt. Sinai Medical Center, New York, NY; Bernard Kubak, UCLA School of Medicine, Los Angeles, CA; Shimon Kusne, University of Pittsburgh, Pittsburgh, PA; Steven Mawhorter, Cleveland Clinical Foundation, Cleveland, OH; Martin Mullen, Loyola University Medical Center, Maywood, IL; Carlos Paya, Mayo Clinic, Rochester, MN; Mark Pescovitz, Indiana University Medical Center, Indianapolis, IN; John Pirsch, University of Wisconsin Medical School, Madison, WI; Timothy L. Pruett, University of Virginia Health Systems, Charlottesville, VA; Jeffrey Punch, University of Michigan Medical Center, Ann Arbor, MI; John Rabkin, Oregon Health Sciences University, Portland, OR; Robert Rubin, Massachusetts General Hospital, Boston, MA; John Scandling, Stanford University Medical Center, Palo Alto, CA; Michael Shapiro, Hackensack University Medical Center, Hackensack, NJ; Randi Silibovsky, Albert Einstein Medical Center, Philadelphia, PA; Kenneth Washburn, University of Texas Health Service Center, San Antonio, TX; Sam Weinstein, LifeLink Transplant Institute, Tampa, FL.