- Email: [email protected]
Safe use of livers from donors with positive hepatitis B core antibody
Safe Use of Livers From Donors With Positive Hepatitis B Core Antibody Cosme Manzarbeitia, David J. Reich, Jorge A. Ortiz, Kenneth D. Rothstein, Victor R. Araya, and Santiago J. Munoz Thirty-five patients received liver transplants using liver donors who had positive test results for the hepatitis B core antibody (HBcAb). In the same time frame, 195 patients received HBcAb-negative liver donors. Mean follow up for patients receiving HBcAb-positive donors was 25 months. All patients receiving HBcAb-positive donors were monitored for recurrence of hepatitis B (HBV) with HBV DNA assays. There was no de novo HBV in recipients of HBcAb-negative grafts. In the group of patients receiving HBcAb-positive donors, 4 of 35 patients died within 3 months after transplant with no evidence of HBV recurrence at time of death. Four patients were transplanted for HBV-related disease and were postoperatively placed on lamivudine and hepatitis B immune globulin (HBIG). HBV recurrence was seen in one of these patients. Of the remaining 27 patients, three of three mismatched patients (HBcAb-positive donor to HBcAbnegative recipient) developed de novo HBV (100%). Of 24 matched patients (HBcAb-positive donor to HBcAbpositive recipient), only two (7%) developed recurrent HBV allograft reinfection. All de novo and recurrent HBV infections were successfully managed with HBIG and lamivudine therapy. Survival for this subgroup of patients receiving HBcAb-positive donors for non-HBV–related liver disease was 100%. We conclude that the judicious use of HBcAb-positive donors is reasonably safe and associated with low morbidity and mortality, with the appropriate follow-up protocols. Additionally, lamivudine use can be reserved for those cases with de novo or recurrent HBV in the liver allograft, or, selectively, as prophylaxis in those recipients patients who are naı¨ve to HBV and receive an HBcAb-positive donor. (Liver Transpl 2002;8: 556-561.)
A
s the field of transplantation has evolved, organ procurement has expanded to try to meet the everincreasing demands. However, the need for cadaveric organs and tissues far exceeds the current supply and many patients still die before a suitable organ is found. Hepatic transplantation, as a therapy for end-stage liver
From the Center for Liver Diseases and Liver Transplant Program, Albert Einstein Medical Center, Philadelphia, PA. Address reprint requests to Cosme Manzarbeitia, MD, Albert Einstein Medical Center, 5401 Old York Rd, Klein #509, Philadelphia, PA 19141. Telephone: 215-456-4985; FAX: 215-456-8058; E-mail: [email protected] Copyright © 2002 by the American Association for the Study of Liver Diseases 1527-6465/02/0806-0058$35.00/0 doi:10.1053/jlts.2002.33451
556
disease, has grown exponentially since cyclosporine immunosupression was introduced in 1981.1,2 Because of the severe shortage of organ donors, the transplant community has turned to the use of the so-called extended donors. Among this extended pool are those liver donors with hepatitis B core antibody (HBcAb)– positive serology but negative hepatitis B surface antigen (HBsAg), which can comprise up to 4% of all liver donors3; however, it is unclear whether these organs should be used for transplant.4,5 There have been documented instances of de novo occurrence of hepatitis B (HBV) infection in recipients of these organs.3,5,6-11 The reported rates of de novo HBV in HBcAb-negative recipients receiving an HBcAb-positive donor liver vary from 30%6-8 to up to 80%3,9,20 when no antiviral prophylaxis is used after surgery. With this in mind, we reviewed our own experience with the use of donors with HBcAb-positive serology. Our aim was to evaluate the safety of these donors for use in hepatic transplantation as it relates to potential transmission or reactivation of HBV.
Materials and Methods From June, 1995 through September, 2000, 254 liver transplants (OLTX) were performed in 230 patients at our institution. Thirty-five patients (15.2 %) received transplants from donors that tested positive for HBcAb, and 195 others received transplants from HBcAb-negative donors. All donors in our program were HBsAg-negative. The characteristics of the donors can be seen in Table 1. Informed consent was obtained from prospective recipients before using these donors. Of these 35 patients, 10 (28.6 %) were high-priority (United Network for Organ Sharing [UNOS] 1 or 2A), and the rest were medium- to low-priority (UNOS 2, 2B, and 3). Clinical and laboratory data were prospectively collected in our database, and a postoperative screening protocol was initiated for these patients. Mean follow-up for these patients was 25.6 months (range, 3 to 65 months). During this period, any evidence of allograft dysfunction was worked up to include, among others, testing for HBsAg and HBV DNA. In addition to their routine postoperative follow-up testing, these patients were monitored for recurrence of HBV with determination of HBV DNA by branched-DNA assay (normal ⬍ 0.7 ⫻ 106 Eq/mL, or ⬍ 200 pg/mL, Quest Diagnostics Inc, San Juan Capistrano, CA) every 3 months, whenever there was allograft
Liver Transplantation, Vol 8, No 6 (June), 2002: pp 556-561
557
Liver Donors With Hepatitis B Core Antibody
Table 1. HBcAb-Positive Donor Demographics
Age (yr) Sex HBsAb status
Range 15-77 Male 19 Positive 18
Mean 51 Female 16 Negative 5
Median 53
SD 16
episodes were managed with corticosteroid recycle and, in 5 patients, conversion to tacrolimus. In three patients, mycophenolate was added to the regimen. OKT3 was required in only one patient. Disease Recurrence
Not Done 11
dysfunction, or both. If HBV DNA turned positive, a full serum HBV profile was performed. Lamivudine prophylaxis was not used except in those patients who were positive for HBV before OLTX. This was reserved for therapy and administered only after HBV DNA seroconversion, with or without allograft dysfunction, and irrespective of HBsAg positivity. In instances of allograft dysfunction, biopsy procedures were performed to evaluate for rejection, disease recurrence, or active hepatitis.
Results Survival For all 35 patients receiving HBcAb-positive donors, the one-year actuarial survival rate was 88.6%. Four deaths occurred within 3 months after transplant, without evidence of HBV recurrence at time of death or at autopsy. If we exclude these four patients, overall survival for the subgroup of patients receiving HBcAbpositive donors was 100%, with a mean follow-up of 25 months. This subgroup includes 25 patients who have follow-up periods of more than a year. Immunosuppression and Rejection In the 31 patients surviving more than 3 months, immunosuppression consisted of cyclosporine A and corticosteroids, with or without either azathioprine or mycophenolate in 12 patients (38.7%), and tacrolimus and corticosteroids in the other 19 (61.3 %) patients. There were 13 episodes of allograft rejection documented by biopsy in these 31 patients (42%). Rejection
Of the 31 surviving patients receiving HBcAb-positive grafts, 4 were transplanted for HBV-related liver disease: 1 for HBsAg-positive fulminant hepatic failure and 3 for HBV cirrhosis. All of these patients were placed on Lamivudine and HBV immune globulin (HBIG) after surgery. In this group, HBV recurrence was seen in one patient who received a transplant for HBV cirrhosis, with a resistant HBV strain that ultimately led to graft failure and death over 4 years after transplantation. In the 27 patients transplanted for non-HBV–related liver disease, development of de novo HBV in patients who were previously negative for HBcAb and who received HBcAb-positive grafts was universal (3 of 3, or 100%, unmatched group). All of these patients received transplants in extremis. Lamivudine was not used as prophylaxis in any of these patients, as it was not available yet for the first 2 patients and, in the third instance, the recipient had a positive test result for HBV surface antibody (HBsAb). However, of the first 2 patients, one received prophylactic HBIG at doses of 10,000 IU intravenously (IV) every month thereafter for 6 months, aiming at quantitative titers of ⱖ 1/100. These patients developed detectable HBV DNA at 24, 17, and 9 months after transplantation; one of these patients also developed allograft dysfunction with clinical hepatitis. Management consisted of institution of lamivudine therapy with or without concomitant HBIG. All of these patients are alive and well without allograft dysfunction at 47, 63, and 19 months after transplantation, respectively (Table 2). All patients were rendered HBsAg-negative by lamivudine therapy, although one patient (mismatch group) has experienced a late viral breakthrough (lamivudine resistance)
Table 2. HBcAb-Negative Patients Receiving HBcAb-Positive Donor Livers Pretransplant Serologies Patient HBcAb HBsAb HBsAg 1 2 3
Neg Neg Neg
Neg Neg Pos
Posttransplant Mo After Allograft HBV DNA⫹ OLT Dysfunction? Biopsy Therapy Prophylaxis?
Neg Neg Neg
Abbreviations: Neg, negative; Pos, positive.
Yes Yes Yes
17 24 9
Yes No No
Yes No No
Yes Yes Yes
Outcome
Yes (HBIG) Alive at 63 mo No Alive at 47 mo No Alive at 19 mo
558
Manzarbeitia et al
Table 3. HBcAb-Positive Patients Receiving HBcAb-Positive Donor Livers Pretransplant Serologies Patient HBcAb HBsAb HBsAg 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos
Pos Pos Neg Pos Pos Neg Pos Neg Neg Neg Pos Pos Neg Neg Pos Neg Pos Pos Neg Pos Neg Pos Pos Neg
Posttransplant Mo After Allograft HBV DNA OLT Dysfunction? Biopsy Therapy Prophylaxis?
Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg
No No No No No No No Pos No No No No No No No No No No No No Pos No No No
— — — — — — — 18 — — — — — — — — — — — — 6 — — —
No No No No No No No No No No No No No No No No No No No No Yes No No No
No No No No No No No No No No No No No No No No No No No No Yes No No No
No No No No No No No Yes No No No No No No No No No No No No Yes No No No
No No No No No No No No No No No No No No No No No No No No No No No No
Outcome Alive UnrelatedDeath Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive
Abbreviations: Pos, positive; Neg, negative.
with active viral replication, normal allograft function, and mild chronic hepatitis on liver biopsy. Twenty-four patients receiving HBcAb-positive donors were already positive for HBcAb in their pretransplant serologies (matched group), with or without HBsAb positivity (see Table 3). In this group, only two patients (7%) developed HBV recurrence in the allograft at 6 and 18 months after transplant, respectively. The first patient developed allograft dysfunction and histologic hepatitis, whereas the other one had normal allograft function and was identified only by the monitoring protocol outlined above. Both patients were HBsAb-negative, so they were placed on both HBIG and lamivudine. Hepatitis resolved in the first patient, and HBV DNA test results became negative in the second patient. The rest of the matched patients remain free of HBV disease at a mean follow up period of 25 months. Surveillance continues in this group. Within this match group, 24 patients have follow-up periods of more than 1 year. In this subgroup, only one patient was noted to have HBV recurrence. Of these 24, 13 also were HBsAb-positive.
Influence of Recipient HBsAb Of the 24 matched patients, the rate of recurrent HBV infection was different depending on the HBsAb status of the recipient (Table 3). For those patients with a positive HBsAb, the rate of HBV infection was 0% (0 of 13), whereas for those with negative HBsAb it was 18% (2/11). Hence, it is our preference to match both antibodies, surface and core, at time of allocation, if possible.
Discussion High-risk factors for transmission of HBV infection through organ transplantation include the presence of HBsAg, HB-precore antigen, HBV DNA, and immunoglobulin M (IgM) HBcAb in donor serum, indicating active viral replication.12-14 However, for donors with positive test results for IgG HBcAb, transmissibility depends largely on the organ transplanted,8,15 with the highest incidence in liver transplantation.16 About 4% of liver donors are positive for HBcAb.3 For recip-
559
Liver Donors With Hepatitis B Core Antibody
Table 4. HBV Recurrence Rates in Recipients of HBcAb-Positive Liver Donors Recipient Status Author Wachs et al,8 1995 Dodson et al,3 1997 Dickson et al,9 1997 Douglas et al,6 1997 Current series Overall
n
HBsAb⫹ HBcAb⫺
HBsAb⫺ or ⫹ HBcAb⫹
HBsAb⫺ HBcAb⫺
6 47 26 9 27 115
— 0/7 (0%) 1/3 (33%) 0/1 (0%) 1/1 (100%) 2/12 (17%)
0/1 (0%) 2/15 (13%) 0/2 (0%) — 2/24 (7%) 4/42 (9.5%)
3/5 (60%) 18/25 (72%) 18/21 (86%) 3/8 (37%) 2/2 (100%) 44/61 (72%)
Average Follow-up (mo) 15 — — — 25
NOTE. Includes only patients with full screening after transplant.
ients of livers bearing this marker, HBV has been reported to occur anywhere from 30% to 50%6-8 to up to 78%.9 This is concerning, because in the absence of therapy, HBV infection can progress to severe allograft dysfunction, graft loss, and even patient death.8 Retransplantation also is fraught with disastrous results in this setting. In a recent multicenter study,17 only 5% (1 of 20 patients) survived more than 6 months after retransplantation. Altogether, these concerns can ultimately result in exclusion of potential liver donors.4 HBV infection in liver transplant recipients is manifested by allograft dysfunction, conversion to HBsAg positivity from HBsAg negativity, and positive HBV DNA in serum. Clinically, HBV infection in the liver transplant recipient can be classified as de novo or recurrent, depending on whether there is preexisting evidence of exposure to the HBV virus before transplant. Histologically, HBV infection presents with allograft hepatitis, which can progress to fibrosing cholestatic hepatitis and cirrhosis. De novo HBV disease also can follow an aggressive course,8 but it is often mild.6,9,16 Our results seem to indicate that patients at greatest risk for de novo occurrence of HBV show negative results for HBcAb in their serum, reflecting a lack of an immune response to HBV. Use of HBcAb-positive donors in this population should be avoided when possible; however, if the transplant proceeds in such a mismatched patient, our results would suggest the use of prophylactic lamivudine in view of the universal recurrence of disease under these conditions. The results of pooled series can be seen in Table 4. Wachs et al8 found a high rate of HBV recurrence and do not advocate using HBcAb-positive donors for liver transplantation. In a recent report, Dodson et al3 concluded that the risk of HBV transmission by HBcAbpositive donors is related to the previous immunologic status of the recipient. In their experience, the presence
of HBsAb positivity conferred full protection against HBV reinfection after transplantation of HBcAb-positive organs. Others have not confirmed this finding.9 In a recent report from Spain, Prieto et al11 reported on 30 patients receiving HBC-positive donor livers. In this series, none of the recipients who were either HBcAbpositive or HBsAb-positive developed de novo HBV, including 3 patients who were only positive for HBcAb. In another report by Douglas et al,6 only 1 patient with HBsAb positivity received a core-positive organ, and no HBV recurrence was seen in this patient. However, Dickson et al9 reported an HBV recurrence rate of 33% in this subgroup. In our series, the only patient who was HBsAb-positive and HBcAb-negative before transplant had recurrence of HBV. This patient had low titers of HBsAb, and this may have played a role in the development of HBV infection, although this sample is too small to draw conclusions. Although in our patients we did not routinely measure HBsAb titers, this may, at least theoretically, explain some of the differences. However, the well-known fact that cirrhotics frequently fail to develop detectable HBsAb levels after full vaccination18 makes even this debatable. Based on our experience, we feel that selectively using donors with a positive HBcAb can lead to reasonable outcomes. Paramount in the success of this program is the institution of a match-and-monitor policy, in which an attempt is made to match the surface and core antibodies of donor and recipient at the time of organ allocation. This is based on the assumption that previous exposure to HBV infection, manifested by the presence of antibodies, may exert a protective effect on HBV recurrence, as suggested by Dodson et al3 as well as experimental data.19 In fact, our own experience shows a lower recurrence level (0% versus 18%) in those matched recipients with a positive HBsAb result. Serologic testing routinely used by organ procurement
560
Manzarbeitia et al
organizations includes (among others) HBcAb, HBsAg, and, on occasion, HBsAb. Once these test results are known, we attempt to match the recipient’s HBsAb, HBcAb, or both to that of the donor. Informed consent also is obtained from the prospective recipient, and the follow-up surveillance program is thoroughly explained. No perioperative HBV prophylaxis is used in view of the low recurrence rate and good response to available therapy, unless the recipient is HBcAb- and HBsAb-negative. Markowitz et al20 advocates combination lamivudine/HBIG therapy starting in the perioperative period for those patients with active viral replication in the preoperative period, as evidenced by HBV DNA positivity. This includes those patients whose transplant indication is chronic HBV cirrhosis or fulminant HBV with active viral replication. Other antiviral therapies that have been used in an attempt to prevent HBV recurrence after OLTX include sequential prostaglandin E,21 and gancyclovir/famcyclovir.22 However, the results are inferior when compared with primary lamivudine therapy in preventing the need for retransplantation. Dodson et al10 also have proposed using combination HBIG and lamivudine prophylaxis in high-risk patients only. More recently, Chung et al23 have made the same recommendation for all recipients of HBCpositive donors, regardless of risk. We have not routinely used lamivudine and HBIG prophylaxis in the postoperative period, except in those patients with preexisting HBsAg positivity. However, in view of our current findings, we plan to use prophylactic lamivudine for those exceptional cases in which we are forced to use an HBcAb-positive donor liver into an HBcAb/ HBsAb-negative recipient. Prophylaxis with combinations of HBV hyperimmune globulin,24 gancyclovir, and lamivudine25-27 may prevent or delay infection in patients receiving a transplant for HBV-related liver disease. One of our concerns with using routine prophylaxis is the emergence of resistant HBV strains,28,29 because of a specific mutation in the HBV RNA– dependent DNA polymerase at the YMDD locus.30 Therefore, we reserve this as a therapeutic approach whenever we identify HBV recurrence in these recipients through our monitoring program. However, we have used combination HBIG and lamivudine for therapy in our patients with either de novo or recurrent HBV, because we believe that aggressive combination therapy is justified in these immunosupressed patients who can have serious graft-threatening forms of HBV reinfection.
Conclusions With appropriate matching and follow-up protocols, the impact of allograft HBV infection in liver transplant recipients of HBcAb-positive donors can be minimized. In this context, using HBcAb-positive donors is safe and associated with low morbidity and mortality. In fact, 15 % of donors used by our program are positive for HBcAb. Lamivudine should be used as prophylaxis in HBcAb-negative recipients of HBcAb-positive donors. In those patients who are HBsAb-positive, the routine use of lamivudine needs further clarification. Therapy with lamivudine and HBIG should be reserved for recurrent HBV in recipients of HBcAbpositive donors. Careful surveillance also will enable early detection of resistant strains, which may require additional therapy. In our experience, the judicious use of these organs is safe and can increase organ availability for certain recipients.
References 1. UNOS 1996 Annual Report, The US Scientific Registry of Transplant Recipients and the Organ Procurement and Transplantation Network Data as of September 7, 1996. Richmond, VA, UNOS and the US Department of Health and Human Services, 1996. 2. UNOS official Waiting List Statistics, July 30, 1997. Available at: www.unos.org. Accessed: January 15, 2001. 3. Dodson SF, Issa S, Araya V, Gayowski T, Pinna A, Eghtesad B, et al. Infectivity of hepatic allografts with antibodies to hepatitis B virus. Transplantation 1997;64:1582-1584. 4. Turner DPJ, Zuckerman M, Alexander GJM, Waite J, Wreghitt T. Risk of inappropriate exclusion of organ donors by introduction of hepatitis B core antibody testing. Transplantation 1997; 63:775-778. 5. Radomski JS, Moritz MJ, Armenti VT, Munoz SJ. Hepatitis B transmission from a liver donor who tested negative for hepatitis B surface antigen and positive for hepatitis B core antibody. Liver Transpl Surg 1996;2:130-131. 6. Douglas DD, Rakela J, Wright TL, Krom RAF, Wiesner RH. The clincial course of transplantation-associated De Novo hepatitis B infection in the liver transplant recipient. Liver Transpl Surg 1997;63:105-111. 7. Kadian M, Hawkins K, Schwartz M, Miller C. use of hepatits B core antibody-positive multiorgan donors. J Transplant Coord 1994; 4:57-60. 8. Wachs ME, Amend WJ, Ascher NL, Bretan PN, Emond J, Lake JR, et al. The risk of transmission of hepatitis B from HBsAg (-), HBcAb (⫹), HBIgM (-) organ donors. Transplantation 1995; 59:230-234. 9. Dickson RC, Everhart JE, Lake JR, Wei Y, Seaberg EC, Wiesner RH, et al. Transmission of hepatitis B by transplantation of livers from donors positive for antibody to hepatitis B core antigen. Gastroenterology 1997; 113:1668-1674. 10. Dodson SF, Bonham CA, Geller DA, Cacciarelli TV, Rakela J, Fung JJ. Prevention of de novo hepatitis B infection in recipients of hepatic allografts from anti-HBc positive donors. Transplantation 1999;68:1058-1061.
Liver Donors With Hepatitis B Core Antibody
11. Prieto M, Gomez MD, Berenguer M, Cordoba J, Rayon JM, Pastor M, et al. De novo hepatitis B after liver transplantation from hepatitis B core antibody-positive donors in an area with high prevalence of anti-HBc positivity in the donor population. Liver Transpl 2001;7:51-58. 12. Chan M, Chang WK. Renal transplantation from HBsAg positive donors to HBsAg negative recipients. Br J Med 1988; 297: 522-523. 13. Lutwick LI, Sywassink JM, Corry RJ, Shorey JW. The transmission of hepatitis B by renal transplantation. Clin Nephrol 1983; 19:317-319. 14. Wolf JL, Perkins H, Schreeder MT, Vincenti F. The transplanted kidney as a source of hepatitis B infection. Ann Intern Med 1979;91:412-413. 15. Madayag RM, Johnson LB, Bartlett ST, Schweitzer EJ, Constantine NT, McCarter RJ Jr, et al. Use of renal allografts from donors positive for hepatitis B core antibody confers minimal risk for subsequent development of clinical hepatitis B virus disease. Transplantation 1997;64:1781-1786. 16. Chazouilleres O, Mamish D, Kim M, Carey K, Ferrell L, Roberts JP, et al. “Occult” hepatitis B virus as a source of infection in liver transplant recipients. Lancet 1994;343:142-146. 17. Crippin J, Foster B, Carlen S, Borcich A, Bodenheimer H. Retransplantation in hepatits B: A multicenter experience. Transplantation 1994;57:823-826. 18. Van Thiel D, El-Ashmawy L, Love K, Gavaler JS, Starel TE. Response to hepatitis B vaccination by liver transplant candidates. Dig Dis Sci 1992;37:1245-1249. 19. Iwarson S, Tabor, E, Thomas HC, Snoy P, Gerety RJ. Protection against hepatitis B virus infection by immunization with hepatitis B core antigen. Gastroenterology 1985;88:763-767. 20. Markowitz J, Martin P, Conrad A, Markmann JF, Seu P, Yersiz H, et al. Prophylaxis against hepatitis B recurrence following liver transplantation using combination lamivudine and hepatitis B immune globulin. Hepatology 1998;28:585-589. 21. Boker KH, Ringe B, Kruger M, Pichlmayr R, Manns MP. Prostaglandin E plus famcyclovir: A new concept for the treatment of severe hepatitis B after liver transplantation. Transplantation 1994;57:1706-1708.
561
22. McCaughan G, Angus P, Bowden S, Shaw T, Breschkin A, Sheil R, Locarnini S. Retransplantation for precore mutant-related chronic hepatitis B infection: Prolonged survival in a patient receiving sequential gancyclovir/famcyclovir therapy. Liver Transpl Surg, 2:472-474,1996. 23. Chung RT, Feng S, Delmonico FL. Approach to the management of allograft recipients following the detection of hepatitis B virus in the prospective organ donor. Am J Transpl 2001;1:185191. 24. Kiyasu PK, Ishitani MB, McGory RW, Gaffey MJ, Dickson RC, Caldwell SH, et al. Prevention of hepatitis B recurrence after a second liver transplant: The role of maintenance polyclonal HBIG therpay. Transplantation 1994;58:954-955. 25. Ben-Ari Z, Shmueli D, Mor E, Shapira Z, Tur-Kaspa R. Beneficial effect of lamivudine in recurrent hepatitis B after liver transplantation. Transplantation 1997;63:393-396. 26. Terrault NA, Zhou S, Combs C, Hahn JA, Lake JR, Roberts JP, et al. Prophylaxis in liver transplant recipients using a fixed dosing schedule of hepatitis B immunoglobulin. Hepatology 1996; 24:1327-1333. 27. Yoshida EM, Wolber RA, Mahmood WA, Anderson FH, Scudamore CH , Erb SR. Attempted resolution of acute recurrent hepatitis B in a transplanted liver allograft by the administration of gancyclovir. Transplantation 1994;58:956958. 28. Bartholomew MM, Jansen RW, Jeffers LJ, Reddy KR, Johnson LC, Bunzendahl H, et al. Hepatitis B virus resistance to lamivudine giver for recurrent infection after orthotopic liver transplantation. Lancet 1997;349:20-22. 29. Bain VG, Kneteman NM, Ma MM, Gutfreund K, Shapiro JA, Fischer K, et al. Efficacy of lamivudine in chronic hepatitis B patients with active viralreplication and decompensated cirrhosis undergoing liver transplantation. Transplantation 1996;62: 1456-1462. 30. Tipples GA, Ma MM, Fischer KP, Bain VG, Kneteman NM, Tyrrell DL. Mutation in HBV RNA– dependent DNA polymerase confers resistance to lamivudine in vivo. Hepatology 1996; 24:714-717
A
s the field of transplantation has evolved, organ procurement has expanded to try to meet the everincreasing demands. However, the need for cadaveric organs and tissues far exceeds the current supply and many patients still die before a suitable organ is found. Hepatic transplantation, as a therapy for end-stage liver
From the Center for Liver Diseases and Liver Transplant Program, Albert Einstein Medical Center, Philadelphia, PA. Address reprint requests to Cosme Manzarbeitia, MD, Albert Einstein Medical Center, 5401 Old York Rd, Klein #509, Philadelphia, PA 19141. Telephone: 215-456-4985; FAX: 215-456-8058; E-mail: [email protected] Copyright © 2002 by the American Association for the Study of Liver Diseases 1527-6465/02/0806-0058$35.00/0 doi:10.1053/jlts.2002.33451
556
disease, has grown exponentially since cyclosporine immunosupression was introduced in 1981.1,2 Because of the severe shortage of organ donors, the transplant community has turned to the use of the so-called extended donors. Among this extended pool are those liver donors with hepatitis B core antibody (HBcAb)– positive serology but negative hepatitis B surface antigen (HBsAg), which can comprise up to 4% of all liver donors3; however, it is unclear whether these organs should be used for transplant.4,5 There have been documented instances of de novo occurrence of hepatitis B (HBV) infection in recipients of these organs.3,5,6-11 The reported rates of de novo HBV in HBcAb-negative recipients receiving an HBcAb-positive donor liver vary from 30%6-8 to up to 80%3,9,20 when no antiviral prophylaxis is used after surgery. With this in mind, we reviewed our own experience with the use of donors with HBcAb-positive serology. Our aim was to evaluate the safety of these donors for use in hepatic transplantation as it relates to potential transmission or reactivation of HBV.
Materials and Methods From June, 1995 through September, 2000, 254 liver transplants (OLTX) were performed in 230 patients at our institution. Thirty-five patients (15.2 %) received transplants from donors that tested positive for HBcAb, and 195 others received transplants from HBcAb-negative donors. All donors in our program were HBsAg-negative. The characteristics of the donors can be seen in Table 1. Informed consent was obtained from prospective recipients before using these donors. Of these 35 patients, 10 (28.6 %) were high-priority (United Network for Organ Sharing [UNOS] 1 or 2A), and the rest were medium- to low-priority (UNOS 2, 2B, and 3). Clinical and laboratory data were prospectively collected in our database, and a postoperative screening protocol was initiated for these patients. Mean follow-up for these patients was 25.6 months (range, 3 to 65 months). During this period, any evidence of allograft dysfunction was worked up to include, among others, testing for HBsAg and HBV DNA. In addition to their routine postoperative follow-up testing, these patients were monitored for recurrence of HBV with determination of HBV DNA by branched-DNA assay (normal ⬍ 0.7 ⫻ 106 Eq/mL, or ⬍ 200 pg/mL, Quest Diagnostics Inc, San Juan Capistrano, CA) every 3 months, whenever there was allograft
Liver Transplantation, Vol 8, No 6 (June), 2002: pp 556-561
557
Liver Donors With Hepatitis B Core Antibody
Table 1. HBcAb-Positive Donor Demographics
Age (yr) Sex HBsAb status
Range 15-77 Male 19 Positive 18
Mean 51 Female 16 Negative 5
Median 53
SD 16
episodes were managed with corticosteroid recycle and, in 5 patients, conversion to tacrolimus. In three patients, mycophenolate was added to the regimen. OKT3 was required in only one patient. Disease Recurrence
Not Done 11
dysfunction, or both. If HBV DNA turned positive, a full serum HBV profile was performed. Lamivudine prophylaxis was not used except in those patients who were positive for HBV before OLTX. This was reserved for therapy and administered only after HBV DNA seroconversion, with or without allograft dysfunction, and irrespective of HBsAg positivity. In instances of allograft dysfunction, biopsy procedures were performed to evaluate for rejection, disease recurrence, or active hepatitis.
Results Survival For all 35 patients receiving HBcAb-positive donors, the one-year actuarial survival rate was 88.6%. Four deaths occurred within 3 months after transplant, without evidence of HBV recurrence at time of death or at autopsy. If we exclude these four patients, overall survival for the subgroup of patients receiving HBcAbpositive donors was 100%, with a mean follow-up of 25 months. This subgroup includes 25 patients who have follow-up periods of more than a year. Immunosuppression and Rejection In the 31 patients surviving more than 3 months, immunosuppression consisted of cyclosporine A and corticosteroids, with or without either azathioprine or mycophenolate in 12 patients (38.7%), and tacrolimus and corticosteroids in the other 19 (61.3 %) patients. There were 13 episodes of allograft rejection documented by biopsy in these 31 patients (42%). Rejection
Of the 31 surviving patients receiving HBcAb-positive grafts, 4 were transplanted for HBV-related liver disease: 1 for HBsAg-positive fulminant hepatic failure and 3 for HBV cirrhosis. All of these patients were placed on Lamivudine and HBV immune globulin (HBIG) after surgery. In this group, HBV recurrence was seen in one patient who received a transplant for HBV cirrhosis, with a resistant HBV strain that ultimately led to graft failure and death over 4 years after transplantation. In the 27 patients transplanted for non-HBV–related liver disease, development of de novo HBV in patients who were previously negative for HBcAb and who received HBcAb-positive grafts was universal (3 of 3, or 100%, unmatched group). All of these patients received transplants in extremis. Lamivudine was not used as prophylaxis in any of these patients, as it was not available yet for the first 2 patients and, in the third instance, the recipient had a positive test result for HBV surface antibody (HBsAb). However, of the first 2 patients, one received prophylactic HBIG at doses of 10,000 IU intravenously (IV) every month thereafter for 6 months, aiming at quantitative titers of ⱖ 1/100. These patients developed detectable HBV DNA at 24, 17, and 9 months after transplantation; one of these patients also developed allograft dysfunction with clinical hepatitis. Management consisted of institution of lamivudine therapy with or without concomitant HBIG. All of these patients are alive and well without allograft dysfunction at 47, 63, and 19 months after transplantation, respectively (Table 2). All patients were rendered HBsAg-negative by lamivudine therapy, although one patient (mismatch group) has experienced a late viral breakthrough (lamivudine resistance)
Table 2. HBcAb-Negative Patients Receiving HBcAb-Positive Donor Livers Pretransplant Serologies Patient HBcAb HBsAb HBsAg 1 2 3
Neg Neg Neg
Neg Neg Pos
Posttransplant Mo After Allograft HBV DNA⫹ OLT Dysfunction? Biopsy Therapy Prophylaxis?
Neg Neg Neg
Abbreviations: Neg, negative; Pos, positive.
Yes Yes Yes
17 24 9
Yes No No
Yes No No
Yes Yes Yes
Outcome
Yes (HBIG) Alive at 63 mo No Alive at 47 mo No Alive at 19 mo
558
Manzarbeitia et al
Table 3. HBcAb-Positive Patients Receiving HBcAb-Positive Donor Livers Pretransplant Serologies Patient HBcAb HBsAb HBsAg 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos
Pos Pos Neg Pos Pos Neg Pos Neg Neg Neg Pos Pos Neg Neg Pos Neg Pos Pos Neg Pos Neg Pos Pos Neg
Posttransplant Mo After Allograft HBV DNA OLT Dysfunction? Biopsy Therapy Prophylaxis?
Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg
No No No No No No No Pos No No No No No No No No No No No No Pos No No No
— — — — — — — 18 — — — — — — — — — — — — 6 — — —
No No No No No No No No No No No No No No No No No No No No Yes No No No
No No No No No No No No No No No No No No No No No No No No Yes No No No
No No No No No No No Yes No No No No No No No No No No No No Yes No No No
No No No No No No No No No No No No No No No No No No No No No No No No
Outcome Alive UnrelatedDeath Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive
Abbreviations: Pos, positive; Neg, negative.
with active viral replication, normal allograft function, and mild chronic hepatitis on liver biopsy. Twenty-four patients receiving HBcAb-positive donors were already positive for HBcAb in their pretransplant serologies (matched group), with or without HBsAb positivity (see Table 3). In this group, only two patients (7%) developed HBV recurrence in the allograft at 6 and 18 months after transplant, respectively. The first patient developed allograft dysfunction and histologic hepatitis, whereas the other one had normal allograft function and was identified only by the monitoring protocol outlined above. Both patients were HBsAb-negative, so they were placed on both HBIG and lamivudine. Hepatitis resolved in the first patient, and HBV DNA test results became negative in the second patient. The rest of the matched patients remain free of HBV disease at a mean follow up period of 25 months. Surveillance continues in this group. Within this match group, 24 patients have follow-up periods of more than 1 year. In this subgroup, only one patient was noted to have HBV recurrence. Of these 24, 13 also were HBsAb-positive.
Influence of Recipient HBsAb Of the 24 matched patients, the rate of recurrent HBV infection was different depending on the HBsAb status of the recipient (Table 3). For those patients with a positive HBsAb, the rate of HBV infection was 0% (0 of 13), whereas for those with negative HBsAb it was 18% (2/11). Hence, it is our preference to match both antibodies, surface and core, at time of allocation, if possible.
Discussion High-risk factors for transmission of HBV infection through organ transplantation include the presence of HBsAg, HB-precore antigen, HBV DNA, and immunoglobulin M (IgM) HBcAb in donor serum, indicating active viral replication.12-14 However, for donors with positive test results for IgG HBcAb, transmissibility depends largely on the organ transplanted,8,15 with the highest incidence in liver transplantation.16 About 4% of liver donors are positive for HBcAb.3 For recip-
559
Liver Donors With Hepatitis B Core Antibody
Table 4. HBV Recurrence Rates in Recipients of HBcAb-Positive Liver Donors Recipient Status Author Wachs et al,8 1995 Dodson et al,3 1997 Dickson et al,9 1997 Douglas et al,6 1997 Current series Overall
n
HBsAb⫹ HBcAb⫺
HBsAb⫺ or ⫹ HBcAb⫹
HBsAb⫺ HBcAb⫺
6 47 26 9 27 115
— 0/7 (0%) 1/3 (33%) 0/1 (0%) 1/1 (100%) 2/12 (17%)
0/1 (0%) 2/15 (13%) 0/2 (0%) — 2/24 (7%) 4/42 (9.5%)
3/5 (60%) 18/25 (72%) 18/21 (86%) 3/8 (37%) 2/2 (100%) 44/61 (72%)
Average Follow-up (mo) 15 — — — 25
NOTE. Includes only patients with full screening after transplant.
ients of livers bearing this marker, HBV has been reported to occur anywhere from 30% to 50%6-8 to up to 78%.9 This is concerning, because in the absence of therapy, HBV infection can progress to severe allograft dysfunction, graft loss, and even patient death.8 Retransplantation also is fraught with disastrous results in this setting. In a recent multicenter study,17 only 5% (1 of 20 patients) survived more than 6 months after retransplantation. Altogether, these concerns can ultimately result in exclusion of potential liver donors.4 HBV infection in liver transplant recipients is manifested by allograft dysfunction, conversion to HBsAg positivity from HBsAg negativity, and positive HBV DNA in serum. Clinically, HBV infection in the liver transplant recipient can be classified as de novo or recurrent, depending on whether there is preexisting evidence of exposure to the HBV virus before transplant. Histologically, HBV infection presents with allograft hepatitis, which can progress to fibrosing cholestatic hepatitis and cirrhosis. De novo HBV disease also can follow an aggressive course,8 but it is often mild.6,9,16 Our results seem to indicate that patients at greatest risk for de novo occurrence of HBV show negative results for HBcAb in their serum, reflecting a lack of an immune response to HBV. Use of HBcAb-positive donors in this population should be avoided when possible; however, if the transplant proceeds in such a mismatched patient, our results would suggest the use of prophylactic lamivudine in view of the universal recurrence of disease under these conditions. The results of pooled series can be seen in Table 4. Wachs et al8 found a high rate of HBV recurrence and do not advocate using HBcAb-positive donors for liver transplantation. In a recent report, Dodson et al3 concluded that the risk of HBV transmission by HBcAbpositive donors is related to the previous immunologic status of the recipient. In their experience, the presence
of HBsAb positivity conferred full protection against HBV reinfection after transplantation of HBcAb-positive organs. Others have not confirmed this finding.9 In a recent report from Spain, Prieto et al11 reported on 30 patients receiving HBC-positive donor livers. In this series, none of the recipients who were either HBcAbpositive or HBsAb-positive developed de novo HBV, including 3 patients who were only positive for HBcAb. In another report by Douglas et al,6 only 1 patient with HBsAb positivity received a core-positive organ, and no HBV recurrence was seen in this patient. However, Dickson et al9 reported an HBV recurrence rate of 33% in this subgroup. In our series, the only patient who was HBsAb-positive and HBcAb-negative before transplant had recurrence of HBV. This patient had low titers of HBsAb, and this may have played a role in the development of HBV infection, although this sample is too small to draw conclusions. Although in our patients we did not routinely measure HBsAb titers, this may, at least theoretically, explain some of the differences. However, the well-known fact that cirrhotics frequently fail to develop detectable HBsAb levels after full vaccination18 makes even this debatable. Based on our experience, we feel that selectively using donors with a positive HBcAb can lead to reasonable outcomes. Paramount in the success of this program is the institution of a match-and-monitor policy, in which an attempt is made to match the surface and core antibodies of donor and recipient at the time of organ allocation. This is based on the assumption that previous exposure to HBV infection, manifested by the presence of antibodies, may exert a protective effect on HBV recurrence, as suggested by Dodson et al3 as well as experimental data.19 In fact, our own experience shows a lower recurrence level (0% versus 18%) in those matched recipients with a positive HBsAb result. Serologic testing routinely used by organ procurement
560
Manzarbeitia et al
organizations includes (among others) HBcAb, HBsAg, and, on occasion, HBsAb. Once these test results are known, we attempt to match the recipient’s HBsAb, HBcAb, or both to that of the donor. Informed consent also is obtained from the prospective recipient, and the follow-up surveillance program is thoroughly explained. No perioperative HBV prophylaxis is used in view of the low recurrence rate and good response to available therapy, unless the recipient is HBcAb- and HBsAb-negative. Markowitz et al20 advocates combination lamivudine/HBIG therapy starting in the perioperative period for those patients with active viral replication in the preoperative period, as evidenced by HBV DNA positivity. This includes those patients whose transplant indication is chronic HBV cirrhosis or fulminant HBV with active viral replication. Other antiviral therapies that have been used in an attempt to prevent HBV recurrence after OLTX include sequential prostaglandin E,21 and gancyclovir/famcyclovir.22 However, the results are inferior when compared with primary lamivudine therapy in preventing the need for retransplantation. Dodson et al10 also have proposed using combination HBIG and lamivudine prophylaxis in high-risk patients only. More recently, Chung et al23 have made the same recommendation for all recipients of HBCpositive donors, regardless of risk. We have not routinely used lamivudine and HBIG prophylaxis in the postoperative period, except in those patients with preexisting HBsAg positivity. However, in view of our current findings, we plan to use prophylactic lamivudine for those exceptional cases in which we are forced to use an HBcAb-positive donor liver into an HBcAb/ HBsAb-negative recipient. Prophylaxis with combinations of HBV hyperimmune globulin,24 gancyclovir, and lamivudine25-27 may prevent or delay infection in patients receiving a transplant for HBV-related liver disease. One of our concerns with using routine prophylaxis is the emergence of resistant HBV strains,28,29 because of a specific mutation in the HBV RNA– dependent DNA polymerase at the YMDD locus.30 Therefore, we reserve this as a therapeutic approach whenever we identify HBV recurrence in these recipients through our monitoring program. However, we have used combination HBIG and lamivudine for therapy in our patients with either de novo or recurrent HBV, because we believe that aggressive combination therapy is justified in these immunosupressed patients who can have serious graft-threatening forms of HBV reinfection.
Conclusions With appropriate matching and follow-up protocols, the impact of allograft HBV infection in liver transplant recipients of HBcAb-positive donors can be minimized. In this context, using HBcAb-positive donors is safe and associated with low morbidity and mortality. In fact, 15 % of donors used by our program are positive for HBcAb. Lamivudine should be used as prophylaxis in HBcAb-negative recipients of HBcAb-positive donors. In those patients who are HBsAb-positive, the routine use of lamivudine needs further clarification. Therapy with lamivudine and HBIG should be reserved for recurrent HBV in recipients of HBcAbpositive donors. Careful surveillance also will enable early detection of resistant strains, which may require additional therapy. In our experience, the judicious use of these organs is safe and can increase organ availability for certain recipients.
References 1. UNOS 1996 Annual Report, The US Scientific Registry of Transplant Recipients and the Organ Procurement and Transplantation Network Data as of September 7, 1996. Richmond, VA, UNOS and the US Department of Health and Human Services, 1996. 2. UNOS official Waiting List Statistics, July 30, 1997. Available at: www.unos.org. Accessed: January 15, 2001. 3. Dodson SF, Issa S, Araya V, Gayowski T, Pinna A, Eghtesad B, et al. Infectivity of hepatic allografts with antibodies to hepatitis B virus. Transplantation 1997;64:1582-1584. 4. Turner DPJ, Zuckerman M, Alexander GJM, Waite J, Wreghitt T. Risk of inappropriate exclusion of organ donors by introduction of hepatitis B core antibody testing. Transplantation 1997; 63:775-778. 5. Radomski JS, Moritz MJ, Armenti VT, Munoz SJ. Hepatitis B transmission from a liver donor who tested negative for hepatitis B surface antigen and positive for hepatitis B core antibody. Liver Transpl Surg 1996;2:130-131. 6. Douglas DD, Rakela J, Wright TL, Krom RAF, Wiesner RH. The clincial course of transplantation-associated De Novo hepatitis B infection in the liver transplant recipient. Liver Transpl Surg 1997;63:105-111. 7. Kadian M, Hawkins K, Schwartz M, Miller C. use of hepatits B core antibody-positive multiorgan donors. J Transplant Coord 1994; 4:57-60. 8. Wachs ME, Amend WJ, Ascher NL, Bretan PN, Emond J, Lake JR, et al. The risk of transmission of hepatitis B from HBsAg (-), HBcAb (⫹), HBIgM (-) organ donors. Transplantation 1995; 59:230-234. 9. Dickson RC, Everhart JE, Lake JR, Wei Y, Seaberg EC, Wiesner RH, et al. Transmission of hepatitis B by transplantation of livers from donors positive for antibody to hepatitis B core antigen. Gastroenterology 1997; 113:1668-1674. 10. Dodson SF, Bonham CA, Geller DA, Cacciarelli TV, Rakela J, Fung JJ. Prevention of de novo hepatitis B infection in recipients of hepatic allografts from anti-HBc positive donors. Transplantation 1999;68:1058-1061.
Liver Donors With Hepatitis B Core Antibody
11. Prieto M, Gomez MD, Berenguer M, Cordoba J, Rayon JM, Pastor M, et al. De novo hepatitis B after liver transplantation from hepatitis B core antibody-positive donors in an area with high prevalence of anti-HBc positivity in the donor population. Liver Transpl 2001;7:51-58. 12. Chan M, Chang WK. Renal transplantation from HBsAg positive donors to HBsAg negative recipients. Br J Med 1988; 297: 522-523. 13. Lutwick LI, Sywassink JM, Corry RJ, Shorey JW. The transmission of hepatitis B by renal transplantation. Clin Nephrol 1983; 19:317-319. 14. Wolf JL, Perkins H, Schreeder MT, Vincenti F. The transplanted kidney as a source of hepatitis B infection. Ann Intern Med 1979;91:412-413. 15. Madayag RM, Johnson LB, Bartlett ST, Schweitzer EJ, Constantine NT, McCarter RJ Jr, et al. Use of renal allografts from donors positive for hepatitis B core antibody confers minimal risk for subsequent development of clinical hepatitis B virus disease. Transplantation 1997;64:1781-1786. 16. Chazouilleres O, Mamish D, Kim M, Carey K, Ferrell L, Roberts JP, et al. “Occult” hepatitis B virus as a source of infection in liver transplant recipients. Lancet 1994;343:142-146. 17. Crippin J, Foster B, Carlen S, Borcich A, Bodenheimer H. Retransplantation in hepatits B: A multicenter experience. Transplantation 1994;57:823-826. 18. Van Thiel D, El-Ashmawy L, Love K, Gavaler JS, Starel TE. Response to hepatitis B vaccination by liver transplant candidates. Dig Dis Sci 1992;37:1245-1249. 19. Iwarson S, Tabor, E, Thomas HC, Snoy P, Gerety RJ. Protection against hepatitis B virus infection by immunization with hepatitis B core antigen. Gastroenterology 1985;88:763-767. 20. Markowitz J, Martin P, Conrad A, Markmann JF, Seu P, Yersiz H, et al. Prophylaxis against hepatitis B recurrence following liver transplantation using combination lamivudine and hepatitis B immune globulin. Hepatology 1998;28:585-589. 21. Boker KH, Ringe B, Kruger M, Pichlmayr R, Manns MP. Prostaglandin E plus famcyclovir: A new concept for the treatment of severe hepatitis B after liver transplantation. Transplantation 1994;57:1706-1708.
561
22. McCaughan G, Angus P, Bowden S, Shaw T, Breschkin A, Sheil R, Locarnini S. Retransplantation for precore mutant-related chronic hepatitis B infection: Prolonged survival in a patient receiving sequential gancyclovir/famcyclovir therapy. Liver Transpl Surg, 2:472-474,1996. 23. Chung RT, Feng S, Delmonico FL. Approach to the management of allograft recipients following the detection of hepatitis B virus in the prospective organ donor. Am J Transpl 2001;1:185191. 24. Kiyasu PK, Ishitani MB, McGory RW, Gaffey MJ, Dickson RC, Caldwell SH, et al. Prevention of hepatitis B recurrence after a second liver transplant: The role of maintenance polyclonal HBIG therpay. Transplantation 1994;58:954-955. 25. Ben-Ari Z, Shmueli D, Mor E, Shapira Z, Tur-Kaspa R. Beneficial effect of lamivudine in recurrent hepatitis B after liver transplantation. Transplantation 1997;63:393-396. 26. Terrault NA, Zhou S, Combs C, Hahn JA, Lake JR, Roberts JP, et al. Prophylaxis in liver transplant recipients using a fixed dosing schedule of hepatitis B immunoglobulin. Hepatology 1996; 24:1327-1333. 27. Yoshida EM, Wolber RA, Mahmood WA, Anderson FH, Scudamore CH , Erb SR. Attempted resolution of acute recurrent hepatitis B in a transplanted liver allograft by the administration of gancyclovir. Transplantation 1994;58:956958. 28. Bartholomew MM, Jansen RW, Jeffers LJ, Reddy KR, Johnson LC, Bunzendahl H, et al. Hepatitis B virus resistance to lamivudine giver for recurrent infection after orthotopic liver transplantation. Lancet 1997;349:20-22. 29. Bain VG, Kneteman NM, Ma MM, Gutfreund K, Shapiro JA, Fischer K, et al. Efficacy of lamivudine in chronic hepatitis B patients with active viralreplication and decompensated cirrhosis undergoing liver transplantation. Transplantation 1996;62: 1456-1462. 30. Tipples GA, Ma MM, Fischer KP, Bain VG, Kneteman NM, Tyrrell DL. Mutation in HBV RNA– dependent DNA polymerase confers resistance to lamivudine in vivo. Hepatology 1996; 24:714-717