The introduction of highly active antiretroviral therapy (HAART) has dramatically improved the survival of patients infected with human immunodeficiency virus (HIV) . End-stage liver disease (ESLD) has thus become the main cause of death among HIV patients coinfected with hepatitis C (HCV) or hepatitis B (HBV) viruses. HIV coinfection accelerates the course of liver disease and increases the mortality rate . Data available in the literature on HBV/HIV coinfection have suggested that coinfected patients are at risk from rapidly progressing liver disease and hepatocellular carcinoma (HCC), particularly in the context of a decreased CD4 cell count [3–5]. However, the outcome of liver transplantation in HIV-coinfected patients has mostly been reported in HIV/HCV coinfected patients in whom the recurrence of HCV infection and mitochondrial toxicity on the liver graft constituted the two principal problems. Small reports have analyzed the outcome of liver transplantation in HBV/HIV-coinfected patients [6–10]. They all revealed excellent results, with median survival at 1 year of 100% without a recurrence of HBV on the liver graft [6–10]. It still remains necessary to assess some important parameters, including long-term follow-up, mitochondrial toxicity potentially induced by HAART and possible persistence of HBV-DNA replication after liver transplantation. These are addressed in the present study in a prospective manner with respect to all HIV/HBV-coinfected patients undergoing liver transplantation in our center.
Patients and methods
Between December 1999 and June 2007, 75 consecutive HIV-infected patients underwent a liver transplantation in our center. Thirteen of them were HIV/HBV-coinfected patients (11 men, two women), with a mean age of 46.5 years (range 30–57 years). The patients' clinical and laboratory data are summarized in Table 1. Patient 4 had additional hepatitis delta virus (HDV) infection, patients 7 and 12 had additional HCV infection and patients 6, 8, 9 and 13 suffered from both HDV and HCV infections in addition to HIV/HBV.
HBV carriage during the pretransplantation period was documented by positive results for hepatitis B surface antigen (HBsAg) (EtiMak4 ELISA; DiaSorin, Salluggia, Italy). Patients were screened for hepatitis B e antigen (HBeAg) and anti-HBe antibodies (BioMérieux, Marcy l'Etoile, France) and antidelta antibodies (Diasorin) prior to liver transplantation. Circulating HBV-DNA levels were quantified by PCR using the Cobas Taqman HBV assay, with a lowest detection limit of 12 IU/ml (Roche Diagnostics, Meylan, France). All patients had an undetectable HBV viral load at the time of liver transplantation. HDV replication was assessed using quantitative real time-PCR, as described elsewhere, with a lowest detection limit of 1000 copies/ml . HCV serology tests were performed using the Innotest HCV assay (Innogenetics, Ghent, Belgium). HCV-RNA was quantified using the Abbott real time HCV assay (Abbott Diagnostics, Lake County, Illinois, USA), with a lowest detection limit of 12 IU/ml. HCV genotype was determined by direct sequencing of the NS5B region, as described elsewhere .
All patients had HIV infection that was under control, with a CD4+ cell count more than 100/μl, no previous AIDS events or opportunistic infections, and a HIV plasma viral load less than 40 copies/ml at the time of their registration on the liver transplantation waiting list (<40 copies/ml, with Cobas Taqman HIV assay, Roche Diagnostics). Treatment regimens were not standardized. Antiretroviral therapy could include any of the 14 drugs available, namely zidovudine (ZDV), lamivudine (3TC), abacavir (ABC), tenofovir (TDF), nevirapine (NVP), efavirenz (EFV), ritonavir (RTV), saquinavir (SQV), indinavir (IDV), fosamprenavir (fAPV), nelfinavir (NFV), lopinavir (LPV/r), enfurtivide (ENF) and atazanavir (ATV). In each patient, the dosage of antiretroviral agents was adapted as a function of residual plasma concentrations of protease inhibitors or nonnucleoside reverse transcriptase inhibitors (NNRTI), in order to ensure that plasma concentrations remained at therapeutic levels and plasma viral load less than 40 copies/ml. All therapeutic regimens included at least one drug active against HBV. Resistance to lamivudine was revealed by sequence analysis of HIV and HBV in patient 3.
The indications for liver transplantation were decompensated cirrhosis (n = 10) [mean model for end-stage liver disease (MELD) score 16.7, range 7–35] and HCC (n = 3). A 5 cm HCC nodule was found in liver segment IV of patient 2 4 months before liver transplantation, for which he underwent chemoembolization 2 months before liver transplantation. Patient 7, who had a history of additional alcohol abuse, had a 4 cm HCC nodule in liver segment 4, 10 months before liver transplantation, for which he did not receive treatment. Patient 11 had a 2 cm HCC nodule in segment 5, which was treated 2 months before liver transplantation by percutaneous radiofrequency. Patients 2 and 5 underwent renal transplantation because of terminal renal failure at the same time as liver transplantation.
Liver transplantation and immunosuppressive therapy
Liver grafts were obtained from cadaveric, living donors or domino. In the latter case, they came from patients transplanted for familial amyloidotic polyneuropathy (domino program) (n = 2) or a relation (n = 1). The median age of donors was 44 years (range 25–84 years) (Table 2).
After transplantation, all patients received 100 mg fluconazole for antifungal prophylaxis and 600 mg cotrimoxazole as prophylaxis against Pneumocystis carinii. Ganciclovir 10 mg/kg per day was given for 3 months after liver transplantation as prophylaxis against cytomegalovirus. Primary immunosuppression was based on tacrolimus (n = 2), tacrolimus and mycophenolate mofetil (MMF) (n = 4), cyclosporine A (n = 3) and cyclosporine A and MMF (n = 4). In all patients, corticosteroids were initiated at a dose of 5 mg/kg per day, and then reduced to 0.3 mg/kg per day at the end of the first week, tapered to 10–15 mg a day until the third month and gradually withdrawn during the next 3 months except for the two patients who underwent a liver and renal transplantation. MMF was given in patients with renal insufficiency or as additional immunosuppression for those experiencing acute rejection. Physical examinations and liver biochemistry tests were performed daily during the first 3 weeks of the postoperative period, three times a week during the next month, then once a week for 3 months and once a month thereafter. Therapy, blood draws and follow-up were all part of standard of care.
Prophylaxis against hepatitis B virus recurrence and antiretroviral therapy after liver transplantation
All patients received long-term passive anti-HBs immunoprophylaxis after liver transplantation, as described in . Briefly, 10 000 IU of anti-HBs Ig (HBIG) was given during the anhepatic phase of liver transplantation, then daily for the first 6 days after surgery. Subsequently and throughout follow-up, 10 000 IU of HBIG was given intravenously whenever circulating anti-HBs antibody levels fell below 500 IU/l. All patients received combined anti-HBV prophylaxis with HBIG and lamivudine (n = 3) or lamivudine and TDF (n = 10). Antiviral drugs were reintroduced after the 14th day after liver transplantation when liver function was stable. Antiretroviral therapy was similar to that administered prior to liver transplantation.
Serum HBsAg levels were determined every 3 months after liver transplantation. Anti-HBs antibody titers were tested weekly during the first month after transplantation. Thereafter, anti-HBs were tested 1 month after the last HBIG administration and then every 2 weeks. After the first year, patients exhibiting stable levels underwent screening for anti-HBs antibody titers every 2–3 months, on an individual basis. HBV-DNA, HDV-RNA, HCV-RNA and HIV-RNA were determined every 3–4 months after liver transplantation.
Quantification of hepatitis B virus cccDNA and total DNA in liver tissue
Total HBV-DNA and cccDNA levels were assessed on frozen sections of the native liver and on the 1-year protocol biopsy using real-time PCR, as previously described . Betaglobin gene amplification was used to estimate the number of cells submitted to PCR. The estimated cell number in reaction tubes ranged from 50 to 1330, depending on the liver specimen. As the sensitivity of the total HBV-DNA assay is less than 10 copies/reaction, the lowest detection limit for total HBV-DNA in this specific series ranged from 0.2 to 0.007 copies/cell.
Monitoring of liver disease
Graft liver biopsies were planned for all patients at day 0 before perfusion, at 6 months after liver transplantation and then once a year after liver transplantation. Additional liver biopsies were performed when liver function test results were abnormal. One portion of each liver biopsy was fixed in AFA and paraffin embedded for histology (Picrosirius and Perls). Acute or chronic rejection or both was diagnosed according to the Banff classification [15,16]. Activity and fibrosis scores were assessed using the METAVIR scoring system . Immunohistochemistry with the following polyclonal antibodies and dilutions was performed on 5 mm paraffin sections from all biopsies: anti-HBs 1/2000 (DAKO SA, Trappes, France), anti-HBc 1/2000 (DAKO SA), anti-PreS1 (kindly provided by Dr M.A. Petit, Inserm U271, Lyon, France) and fluorescein isothiocyanate (FITC) human antidelta 1/320 (kindly provided by Prof. Rizzetto, Turin, Italy).
Assessment of mitochondrial toxicity
One portion of each liver biopsy was frozen immediately in liquid nitrogen and stored at −80° until it was used to perform molecular analyses of mitochondrial toxicity, as described in . Briefly, spectrophotometric analysis of respiratory complexes I, II and IV was performed on homogenate from frozen liver biopsy specimens; the activity of citrate synthase, a Krebs cycle enzyme, was determined as a control for the amount of mitochondria in the tissue. Quantifications of mitochondrial DNA (mtDNA) were performed using real time PCR amplification on a light cycler instrument (Roche Diagnostics) and Power SYBR green mix (Applied Biosystems, Foster City, California, USA), the results being expressed as the number of mtDNA copies/10 pg of nuclear DNA, which roughly represents the DNA mass of a normal diploid human cell.
Survival rates of patients and grafts
Patients were followed for a mean duration of 32 ± 5.2 months after liver transplantation (range 10–63 months) (Table 2). Overall cumulative patient and graft survival reached 100%.
Acute rejection was observed in patients 1, 4 and 7, at 6 weeks, 10 days and 10 days after liver transplantation, respectively. The Banff score was 5 for patients 1 and 7, but was not calculated for patient 4, whose acute rejection was very mild. The last biopsy was performed 7–60 months after liver transplantation (Table 2). Fibrosis and activity scores, assessed in 10 out of 13 patients for fibrosis and 11 out of 13 patients for activity, showed no fibrosis (seven patients), F1 (two patients) and F2 (one patient), no activity (seven patients), A1 (two patients) and A2 (two patients).
Hepatitis B virus infection
HBV-DNA was less than 12 IU/ml in serum samples from all 13 patients at liver transplantation and remained undetectable during the follow-up period. HBsAgs became undetectable within 2 days of liver transplantation. Positive HBeAg results in patients 1 and 3 became negative within 2 days of liver transplantation. In the native liver, total HBV-DNA was detectable in patients 2, 3, 6 and 12 (Table 1) with viral loads ranging from 0.14 to 0.83 copies/cell, whereas cccDNA was quantifiable in the native liver of patient 3 at 0.14 copies/cell. One year after liver transplantation, total HBV-DNA and cccDNA were analyzed in the grafted liver of nine patients, including three with detectable HBV-DNA in their native livers. The results were negative in all nine specimens.
Hepatitis delta virus infection
HDV-RNA was detectable before liver transplantation in three of the five patients with positive antidelta serology, with viral loads ranging from 4.07 to 6.62 log copies/ml (Table 1). After liver transplantation, HDV-RNA remained negative, with a maximum follow-up of 38 months.
Hepatitis C virus infection
HCV-RNA was detectable before liver transplantation in three of the six patients with a positive anti-HCV serology, with viral loads ranging from 4.42 to 5.58 log IU/ml (Table 1). HCV recurrence was only observed in these three patients. Of them, patient 8 was treated with PEG-interferon and ribavirin from month 9 after liver transplantation. After a 12-month course of treatment, a sustained virological response was achieved.
Plasma HIV-RNA remained negative in all patients during follow-up. No viral breakthrough was observed. CD4 cell counts were maintained within the range of 80–900 cells/μl. No septic complication, opportunistic infection or lymphoproliferative disorder occurred.
Mitochondrial toxicity analysis
Steatosis was searched for in liver biopsies from all patients following liver transplantation. It was only observed in patient 7 in case of whom it was microvesicular and involved 5% of hepatocytes at 7 months after liver transplantation and 10% of hepatocytes at 14 months after liver transplantation.
Analysis of different mitochondrial activities and quantification of the liver mtDNA content were possible in liver biopsies from seven patients, either once during progression (patients 1, 2, 3 and 5) or several times in successive biopsy specimens (Table 3). mtDNA copy number in patients' liver (median value 2959, 5–95th percentile: 1403–5797) was lower than those in control livers (median value 5041, 5–95th percentile: 2252–6980), P = 0.054. However, they were still within the normal range with the sole exception of the M6 biopsy specimen from patient 4, which fell clearly below the 5th percentile of control values.
The activities of both respiratory complex I and respiratory complex IV are dependent on several mtDNA-encoded subunits. Both were increased in HIV/HBV liver samples with a median value (5th–95th percentile range) of 34 (24–46) nmol/l per min per mg protein for respiratory complex I activity versus 26 (10–44) in control liver specimens (P = 0.037) and of 97 (44–128) nmol/l per min per mg protein for respiratory complex IV activity versus 54 (34–88) in control liver specimens (P = 0.002).
The two other mitochondrial activities, respiratory complex II and citrate synthase, are only dependent on nuclear DNA-encoded proteins. Neither differed significantly between HIV/HBV infected and control liver specimens. In HIV/HBV liver samples, the median value (5th–95th percentile range) of respiratory complex II activity was 111 (82–53) nmol/l per min per mg protein versus 106 (54–196) in control liver specimens (P = 0.460) and 41 (17–109) nmol/l per min per mg protein for citrate synthase versus 47 (22–130) in control liver specimens (P = 0.689).
Several liver transplantation programs have been developed for HIV-infected patients, and we and others have demonstrated that liver transplantation is feasible in this setting. Few reports have addressed the outcome of liver transplantation in HIV/HBV-coinfected patients. This study revealed 100% graft and patient survival after a mean follow-up of 32 months (for a maximum follow-up of 63 months), thus demonstrating the appropriateness of liver transplantation in this subgroup of patients.
In recent studies of liver transplantation outcome in HIV/HCV-coinfected patients, we demonstrated that the two major problems encountered were mitochondrial toxicity and a recurrence of HCV infection on the liver graft [18,19]. Mitochondrial toxicity was mainly due to nucleoside analogs such as didanosine and stavudine. Avoidance of these drugs in the present series of patients was accompanied by better preservation of the liver mtDNA content, which fell only once and transiently below the 5th percentile of control values. The observed increase in mitochondrial activities with mtDNA-encoded subunits demonstrated the presence of a mitochondrial response, most probably elicited by the treatment. The absence of steatosis and the preservation of liver function showed the appropriateness of that response with respect to liver physiology.
The second issue was the severity of HCV recurrence. During the present study, three HBV/HCV-coinfected patients experienced HCV recurrence with F1–F2 fibrosis scores 2 years after liver transplantation. As in other patients' series, such as those reported by Terrault et al., all our HBV/HIV-coinfected patients were HBsAg negative and had HBV-DNA less than 12 IU/ml. Of course, all our patients were receiving immunoprophylaxis in combination with HBIG and antiviral therapy, which has now demonstrated its efficacy in several studies. Our group demonstrated that the actuarial risk of HBV recurrence under combined prophylaxis was 11.8% at 5 years in HIV-negative individuals . In a recent study, our group also demonstrated that HCC during the pre-liver transplantation period, a viral load at transplantation of at least 100 000 copies/ml and HBIG monoprophylaxis were independent risk factors of HBV recurrence in cirrhotic patients . In this latter study, the overall actuarial rates of HBV-DNA recurrence in serum at 1, 2, 5 and 8 years were 6.3, 10.9, 17.3 and 17.3%, respectively . The best option for the prevention of recurrent HBV infection in HBV-DNA-positive patients seems to be a combination of pretransplantation and posttransplantation antiviral therapy with long-term HBIG administration. As the nucleoside analogue lamivudine has both anti-HIV and anti-HBV activity, many HIV-infected patients with HBV coinfection have received prolonged lamivudine therapy as a component of antiretroviral therapy (ART). In HIV/HBV-coinfected patients, the incidence of lamivudine-resistant HBV is approximately 50% after 2 years of therapy and approaches nearly 100% after 4 years; it has been found to be present in 67% of patients referred for liver transplantation . The presence of HBV resistance to lamivudine at the time of transplantation constitutes a potential risk for HBV recurrence after transplantation . None of our patients, however, acquired lamivudine resistance, and all had HBV viral load less than 12 IU/ml prior to liver transplantation. Indeed, most of them (11 out of 13 patients) were treated with a combination of 3TC and TDF, with the exception of patients 7 and 11 who were suffering from active HCV infection and received anti-HBV therapy with 3TC only. Despite a HBV viral load of less than 12 IU/ml at liver transplantation, total HBV-DNA was detected in the native liver of four patients, though at the low titers to be expected in patients with efficiently suppressed viral replication . Interestingly, for the first time in a group of HIV-infected patients, we demonstrated that HBV replication was perfectly controlled after transplantation because of the absence of detectable total and cccDNA in the liver graft. The replication of hepatitis viruses, and interactions between them, may differ in HIV-immunosuppressed patients, in whom higher viremia and a loss of the reciprocal inhibitory effect found in immunocompetent individuals may occur.
As for immunosuppressive status after liver transplantation, the doses of immunosuppressant drugs were adjusted to maintain target blood levels as pharmacological interactions between calcineurin inhibitors and ART regimens have been documented. Indeed, calcineurin inhibitors and most antiretroviral drugs (particularly nonnucleoside inhibitors of reverse transcriptase and protease inhibitors) are metabolized via the cytochrome P450 3A enzymatic pathway. This requires the monitoring of tacrolimus and cyclosporine A blood levels in order to prevent their accumulation and toxicity, especially when introducing or discontinuing a protease inhibitor. This point is of crucial importance for the prevention of an immunosuppressive status, which might affect a severe recurrence of HBV or HCV infection or both on the liver graft.
Interestingly, no mitochondrial toxicity was observed in the seven patients analyzed. Microvesicular steatosis was absent in all but one patient (patient 7) in whom it was very mild. Furthermore, these seven patients had normal mtDNA levels and normal or increased activities of the respiratory chain. We postulate that this benefit may have been secondary to the avoidance of nucleoside analogs such as didanosine or stavudine, which can be very deleterious to the liver graft .
Our series thus demonstrate excellent results in terms of survival and the control of HBV replication following liver transplantation in HIV/HBV-coinfected patients.
This work received support from the Agence Nationale de Recherches sur le Sida et les Hépatites Virales (ANRS-HC08 program). F.M. received financial support from SIDACTION.
The authors thank René Adam and Daniel Azoulay, senior surgeons, Catherine Guettier, Head of the Pathology Department, Elisabeth Dussaix, Head of the Virology Unit, Daniel Vittecoq, Head of the Infectious Disease Department, Philippe Ichai and Faouzi Saliba, senior physicians of the intensive care unit, for their active involvement into the HIV transplantation program, and Nicolas Moniaux of Inserm Unit U785, Claire Mony, Frédérique Blandin and Victoria Hawken for their technical help.
1. Weber R, Sabin CA, Friis-Moller N, Reiss P, El-Sadr WM, Kirk O, et al
. Liver-related deaths in persons infected with the human immunodeficiency virus
: the D:A:D study. Arch Intern Med 2006; 166:1632–1641.
2. Marcellin P, Pequignot F, Delarocque-Astagneau E, Zarski JP, Ganne N, Hillon P, et al
. Mortality related to chronic hepatitis B and chronic hepatitis C in France: evidence for the role of HIV coinfection
and alcohol consumption. J Hepatol 2008; 48:200–207.
3. Di Martino V, Thevenot T, Colin JF, Boyer N, Martinot M, Degos F, et al
. Influence of HIV infection on the response to interferon therapy and the long-term outcome of chronic hepatitis B. Gastroenterology 2002; 123:1812–1822.
4. Bonacini M, Louie S, Bzowej N, Wohl AR. Survival in patients with HIV infection and viral hepatitis B or C: a cohort study. AIDS 2004; 18:2039–2045.
5. Brau N, Fox RK, Xiao P, Marks K, Naqvi Z, Taylor LE, et al
. Presentation and outcome of hepatocellular carcinoma in HIV-infected patients: a U.S.-Canadian multicenter study. J Hepatol 2007; 47:527–537.
6. Schreibman I, Gaynor JJ, Jayaweera D, Pyrsopoulos N, Weppler D, Tzakis A, et al
. Outcomes after orthotopic liver transplantation
in 15 HIV-infected patients. Transplantation 2007; 84:697–705.
7. Roland ME, Barin B, Carlson L, Frassetto LA, Terrault NA, Hirose R, et al
. HIV-infected liver and kidney transplant recipients: 1- and 3-year outcomes. Am J Transplant 2008; 8:355–365.
8. Duclos-Vallee JC, Feray C, Sebagh M, Teicher E, Roque-Afonso AM, Roche B, et al
. Liver transplantation
in HIV-HCV and HIV-HBV coinfected patients: a large experience in a single centre. J Hepatol 2006; 44:S8.
9. Norris S, Taylor C, Muiesan P, Portmann BC, Knisely AS, Bowles M, et al
. Outcomes of liver transplantation
in HIV-infected individuals: the impact of HCV and HBV infection. Liver Transpl 2004; 10:1271–1278.
10. Fung J, Eghtesad B, Patel-Tom K, DeVera M, Chapman H, Ragni M. Liver transplantation
in patients with HIV infection. Liver Transpl 2004; 10:S39–53.
11. Le Gal F, Gordien E, Affolabi D, Hanslik T, Alloui C, Deny P, Gault E. Quantification of hepatitis delta virus RNA in serum by consensus real-time PCR indicates different patterns of virological response to interferon therapy in chronically infected patients. J Clin Microbiol 2005; 43:2363–2369.
12. Morice Y, Cantaloube JF, Beaucourt S, Barbotte L, De Gendt S, Goncales FL, et al
. Molecular epidemiology of hepatitis C virus
subtype 3a in injecting drug users. J Med Virol 2006; 78:1296–1303.
13. Roche B, Feray C, Gigou M, Roque-Afonso AM, Arulnaden JL, Delvart V, et al
. HBV DNA persistence 10 years after liver transplantation
despite successful anti-HBS passive immunoprophylaxis. Hepatology 2003; 38:86–95.
14. Werle-Lapostolle B, Bowden S, Locarnini S, Wursthorn K, Petersen J, Lau G, et al
. Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy. Gastroenterology 2004; 126:1750–1758.
15. Banff schema for grading liver allograft rejection: an international consensus document.Hepatology
16. Demetris A, Adams D, Bellamy C, Blakolmer K, Clouston A, Dhillon AP, et al
. Update of the International Banff Schema for Liver Allograft Rejection: working recommendations for the histopathologic staging and reporting of chronic rejection. An International Panel. Hepatology 2000; 31:792–799.
17. Bedossa P, Poynard T. An algorithm for the grading of activity in chronic hepatitis C. The METAVIR Cooperative Study Group. Hepatology 1996; 24:289–293.
18. Duclos-Vallee JC, Vittecoq D, Teicher E, Feray C, Roque-Afonso AM, Lombes A, et al
. Hepatitis C virus
viral recurrence and liver mitochondrial damage after liver transplantation
in HIV-HCV co-infected patients. J Hepatol 2005; 42:341–349.
19. Duclos-Vallee JC, Feray C, Sebagh M, Teicher E, Roque-Afonso AM, Roche B, et al
. Survival and recurrence of hepatitis C after liver transplantation
in patients coinfected with human immunodeficiency virus
and hepatitis C virus
. Hepatology 2008; 47:407–417.
20. Terrault NA, Carter JT, Carlson L, Roland ME, Stock PG. Outcome of patients with hepatitis B virus
and human immunodeficiency virus
infections referred for liver transplantation
. Liver Transpl 2006; 12:801–807.
21. Faria LC, Gigou M, Roque-Afonso AM, Sebagh M, Roche B, Fallot G, et al
. Hepatocellular carcinoma is associated with an increased risk of hepatitis B virus
recurrence after liver transplantation
. Gastroenterology 2008; 134:1890–1899, quiz 2155.