Although liver transplantation (LT) is the only promising method to cure HBV-related end-stage hepatopathy, it has promised not too much up to now because the reinfection rate is as high as 70%-80%, with a 1-year survival rate of 68% and a 3-year survival rate of 44%.[1] Since the late 1990s, liver transplantation has been expanding throughout China with more than 500 new recipients each year, of whom over 80% are HBV-related and over 50% have active HBV replication.[2] This kind of risk is becoming more and more important while recipients and allografts have a longer survival period. It has been reported that the reinfection of liver allograft after liver transplantation could be prevented and treated successfully through the administration of lamivudine plus hepatitis B immunoglobulin (HBIG),[3-9] but similar studies are not reported in China. From February 1999 to December 2001, we systematically used lamivudine on 25 out of 50 liver transplantation patients to prevent the reinfection of HBV and conducted a non-randomized control study with a follow-up of 6-36 months.

Methods

Patients

In 50 recipients who had LT from February 1999 to December 2001, 41 (82%) were due to end-stage hepatopathy with HBsAg (hepatitis B surface antigen) positive. 22 (54%) of these patients had active HBV DNA replication or/and positive HBeAg, but 7 of them died soon after surgery because of causes irrelevant to HBV reinfection, and they were excluded. Other 19 patients had only positive HBsAg or positive HBV markers on their native livers detected by immunohistochemistry with inactive HBV DNA replication. Of the 19 patients, 8 had primary liver cancer and 1 died soon after surgery from pulmonary infection, so 10 patients were also excluded. At last a total of 25 patients in our study were followed up for 6-36 months (Table 1). The recipients included were those with preoperative decompensated hepatic disease showing HBsAg positive in serum or/and HBV DNA active or/and HBcAg immunohistochemically positive in their liver tissue. Those with anti-HCV positive or cytomegalovirus IgG/IgM positive or HBcAb positive were excluded from this study. Those who needed long time anti-virus therapy were not included either because the activeness of HBV was disturbed. Informed consent was obtained from all patients in this study and the study was approved by the ethics committee of the hospital.

Methods

The 25 patients were divided non-randomized into two groups of active HBV DNA replication group (15 patients with HBV DNA positive or/and HBeAg positive) and inactive HBV DNA replication group (10 patients with HBsAg positive but HBV DNA/HBeAg negative). Of the latter group, 3 patients were not given lamivudine because of its unavailability in China before 2000, and they acted unintentionally as negative controls. The administration of lamivudine was as follows: 100 mg po qd for as early as possible before operation and 100 mg po qd in two years after operation. The dynamic alteration of HBV markers in liver tissue and serum was observed and the 1 and 2-year survival rates were documented. Successful prevention and treatment was defined as the following items:[1,9-12] clinical clearance, immunohistochemically negative HBV markers in serum and liver tissue; negative HBV DNA in serum assessed by fluorescent quantitative method; negative HBV DNA in liver tissue determined by hybridization in situ; effective antibodies alone present in serum; HBV clearance and HBsAb rising up to 10 IU/L[12] and lasting for more than 2 years spontaneously or after vaccination. Prevention and treatment failed when HBsAg positive in serum, or HBV DNA positive revealed by fluorescent quantitative method, or HBV markers positive in liver tissue could be considered HBV reinfection. These indexes plus elevated levels of bilirubin or transaminase and clinical manifestations could be considered HB recurrence. HBeAg negative and HBV DNA positive could be explained as failure of lamivudine prophylaxis.

Enzyme-linked immunoassay method (Abbott Laboratories, North Chicago, IL, USA) was used to detect HBsAg, HBeAg and corresponding antibodies. HBV DNA fluorescent quantitative analysis was made by the Hepatitis Research Lab of this hospital, with a lower value of 0.05 pg/ml (10³copy/ml of HBV DNA by PCR). HBsAg and HBcAg in liver tissue were detected by SLAB method (from BioGenex, San Romon, CA 94583, USA). HBV DNA in liver tissue was measured by hybridization in situ method labelled by digoxin (probe label box bought from Behringer Mannuheim GmBH, Germany), and digoxin antibody purchased from Roche Diagnostics Corperation, Indianapolis, USA. Biopsy specimens were taken regularly from the native host liver, the allograft before implantation, the transplanted allograft before closing the abdomen, and 1, 4, 12, 24, 48, 72, 96 weeks or whenever biopsy was needed respectively after transplantation. Informed consent was obtained from the patients and their families for each needle biopsy, and the coupled samples were judged by two pathologists who knew nothing about the patients and their clinical data. The criteria for judgement included the degree of liver lesions and the staining of HBsAg and HBcAg by peroxidase.

Statistical analysis

All data were represented as means?/SPAN>standard deviation; the long-term survival rate was analyzed using the Kaplan-Meier method.

Results

Dynamic alteration of HBV markers in recipients with active HBV replication

The average HBV DNA was 9.2?/SPAN>10⁶copy/ml in the 15 patients with active HBV replication at the beginning. After being given lamivudine for 2 weeks before operation, 12 of them (80%) showed HBV DNA in their serum. Only those patients who had had preoperative lamivudine administration less than 1 week were HBV DNA positive shown by fluorescent quantitative method. All the patients in this group continued using lamivudine after surgery. HBsAg and HBeAg remained negative in the serum, and the corresponding antibodies appeared as early as 1 week after operation. Nine patients had HBsAb, 13 had HBcAb and 11 had HBeAb; all these antibodies disappeared gradually within 6 months. In patients who were HBsAb positive 1 week after operation, the average amount of antibodies was more than 164 IU/L, which was considered to be related to the blood and blood products administered. The HBV DNA quantity in these patients remained undetectable using fluorescent quantitative method. The bilirubin and ALT levels in serum increased moderately in 2 weeks, decreased after 3 weeks, and normalized 3 months after operation. Needle biopsy of the liver 1-4 weeks after surgery found that only 2 patients had mild positive reaction of HBsAg, 1 patient was HBcAg positive, and all HBV markers described as above became negative in 3 months. All patients remained HBV DNA negative after digoxin-marked HBV DNA hybridization in situ (Table 2). Follow-up for 12-44 weeks showed that HBV markers disappeared in serum and liver tissue in 10 patients, with an average disappearance time of 23.7?/SPAN>13.05 weeks and 4 weeks respectively. The clinical clearance was irrelevant to the HBV DNA level in serum before operation.

One patient with clinical clearance at 1 year showed HBsAb 764 IU/L in the first week postoperatively, 84.5 IU/L at the 24th week, and less than 2 IU/L at 1 year. At the end of 2 years, HBsAg became positive, HBV DNA fluorescent-quantitatively positive and HBeAg negative with no corresponding elevation of bilirubin and transaminase. This phenomenon could actually be YMDD variation (1/22, 4.6%). Unfortunately, another patient died from HB recurrence at 2 years; at 1 year the patient showed good outcome of clinical clearance in this group.

Dynamic alteration of HBV markers in recipients with inactive HBV replication

The results of using lamivudine in 7 recipients with inactive HBV replication were similar to those in patients with active HBV replication. HBsAg disappeared immediately after operation, and the corresponding antibodies could be detected within 1 week postoperatively (HBsAb 4/7, HBcAb 6/7, HBeAb 2/7). These antibodies lasted for about 24 weeks, but HBcAb and HBeAb lasted longer than HBsAb. The HBV markers disappeared in 1-2 years in 3 patients (Table 3). HBV remained immunohistochemically negative in liver tissues for 6 months. Although HBcAg and HBsAg became positive in liver tissues in two patients after half a year, no change was observed in biochemical indexes and HBV DNA remained negative after hybridization in situ (Table 3).

Dynamic alteration of HBV markers in 3 controls

In the 3 patients who were not given lamivudine, their HBsAg and HBcAg disappeared immediately after operation and the antibodies emerged within 1 week and existed for half a year. HBsAg in liver tissues was positive at the 12th week, and HBsAg and HBcAg were detected in half a year. Eight-12 months after operation, HBV DNA in serum became positive and the levels of bilirubin and transaminase elevated. HBV DNA was positive after hybridization in situ at the same time. In 1 patient dead liver tissue showed the characteristics of fibrosing-cholestatic hepatitis. In 2 patients who were treated comprehensively including the use of lamivudine, HBV DNA became negative with decreased levels of bilirubin and transaminase (Table 4).Follow-up revealed that 1 patient in the control group died from HB recurrence (fibrosing-cholestatic hepatitis), from the cerebral haemorrhage irrelevant to HB recurrence, and still 1 from HB recurrence because of allograft HBcAb positive. According to the mentioned definition, the 24-144-week clinical clearance of HBV in the 22 patients in the two groups was at least 59% (13/22), HBV reinfection was 9% (2/22), HB recurrence and YMDD variation were 4.5% (1/22) respectively, and one patient died from HB recurrence. The clinical clearance in the control group was 0 (0/3), HBV allograft reinfection and HB recurrence were 100% (3/3); one (33%) died in the first year and the death rate was 33% (1/3).

Discussion

In the 1990s, Samuell et al[1] reported that HB recurrence rate in patients with active HBV replication after LT was 90%-100%. HBIG was once used to prevent such recurrence reducing the rate to 33%, but still not satisfactory for HBV DNA positive patients.[1,9] Hence patients with active HBV replication were strongly contraindicated for LT. Greillier in 1996 reported 10 LT patients with HBV DNA positive who were given lamivudine. In the following 18-90 weeks, HBsAg in 9 patients became negative at the 24th week, HBV DNA in 1 patient remained positive and recurred at the 72nd week. From that time on, lamivudine has been used widely to prevent HB recurrence in LT recipients for HB related hepatopathy, and this kind of disease is indicated again for LT.[13-18] Because of many HB patients in China and other Asian countries the improvement of this remedy is of special significance.[19]

In our study, 80% (12/15) recipients with HBV DNA positive showed decreased HBV DNA in serum to an undetectable level when lamivudine was given 2 weeks before surgery. Administration of lamivudine less than 7 days could hardly make HBV DNA negative. This effect is seemingly better than that in HBV active replicating patients receiving no liver transplantation.[20] Using lamivudine decreased HBV particles in blood circulation before LT as many as 97%, thus decreased the chance for virus to reenter the allograft. Our study also confirmed the advice that the recipients with active HBV replication should use lamivudine at least for 2 weeks ahead of surgery to prevent HB recurrence.[2] The markers in serum in patients who had received lamivudine prophylaxis regime dropped sharply, HBsAg and HBeAg disappeared after surgery, and the corresponding antibodies emerged within 1 week (HBsAb 9/15, HBeAb 11/15, HBcAb 13/15) and existed for 24 weeks. Most importantly, HBsAb showed an emergence rate of over 60%. In another study we suggested that it could possibly be introduced during transfusion of blood and blood products after operation.[21,22] In a week, its concentration in serum could be higher than 160 IU/L, recognized as an effective protective level.[1,13] Subsequently, HBsAb began to decrease significantly within 6 months. Needle liver biopsy and HBV DNA hybridization in situ showed no reappearance of HBV markers, indicating that allograft is well protected. A small volume of HBsAg and HBcAg was shown immunohistochemically in liver tissues in the 1st-4th weeks, but they soon became negative. If experimental error could be excluded, the following auto-clearance mechanisms of recipients might be involved: promptly or rapid decrease of HBV DNA particles and inhibition of HBV replication after lamivudine administration; elimination of the host nest of HBV in the native liver; dilution of viruses in serum after intraoperatively transfusion of a large volume of blood and fluid; HBsAb neutralization of HBV viruses in over 60% patients who receive HBsAb passively and unconsciously. The stage was called security period under lamivudine coverage (may conjoined with HBIG) after LT operation. HB recurrence was not observed in patients of this stage and HBV was cleared clinically in over 59% patients. In contrast, other reports showed that HB recurrence was within half a year.[1,9]

Twelve-44 weeks after operation in this study, HBV markers in serum and liver tissues became negative in 10 of 15 (66.7%) recipients within an average of 24 weeks. Clinical clearance was obtained but no complete elimination of HBV was indicated. Because the replicating mechanism of HBV remained unclear and the observation time was short, more time was needed for further evaluation.

In our study one patient (4.5%) had YMDD variation after lamivudine administration for 2 years. We consider that despite the use of both lamivudine and HBIG, HB recurrence may indicate the risk of virus variation. In case of clinical clearance, it might be necessary to stimulate active immunity in recipients after HBV vaccination to protect the hosts from HBV infection.[12]

It was reported that the 1-year recurrence rate in patients with HB related hepatopathy after LT was 70%-80%, and the actual 2-year survival rate was 54%.[1] HBV could be activated again under immunosuppressive state in recipients with inactive replicative HB related hepatopathy (HBsAg+, HBV DNA-).[14] Three patients in our control group were not given lamivudine promptly, resulting in HBV DNA positive at the 8th month, HBsAg and HBeAg in serum positive at the 10th month, and HBsAg and HBcAg in liver tissue positive at the 12th month. On the contrary, no one had HB recurrence in 7 recipients who received lamivudine, and 3 of them had clinical clearance. Further, no recurrence was observed in a year. This result showed that lamivudine prophylaxis is reasonable and necessary to prevent HBV allograft reinfection and HB recurrence even in patients with inactive HBV replication after LT.[23-28]

In our study, 22 patients had one HB recurrence on average after follow-up for one year except for YMDD variation in one patient (4.5%), which was less than 14% reported by Lai with 1 and 2-year survival rates of more than 83%. In our control group, the rates of HBV allograft reinfection and HB recurrence were 100%, and the mortality was more than 33%. This study has demonstrated that lamivudine is used satisfactorily to prevent HBV allograft reinfection and HB recurrence after LT, but the clinical significance of HBsAb emergence demands further study.[7]

Competing interest

No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

References

1 Samuel D, Bismuth H, Marcellin P, Benhamou JP. Liver transplantation in the management of chronic viral hepatitis in: Zuckerman AJ, Thomas HC, eds. Viral Hepatitis, 2nd Edition. Churchill Livingstone: Harcourt Asia; 2001:237-244.

2 Lu SC, Yan LN, Li B, Ma YK, Liu C, Wen TF, et al. Dynamic alteration of HBV markers on active HBV replicative recipients after liver transplantation: a preliminary report. Hepatobiliary Pancreat Dis Int 2003;2:196-201.

3 Seehofer D, Rayes N, Neuman U, Neuhaus R, N?/SPAN>ller AR, Tullius SG, et al. Preoperative antiviral treatment and postoperative prophylaxis in HBV-DNA positive patients undergoing liver transplantation. Transplantation 2001;72:1381-1385.

4 Lee KW, Lee SK, Joh JW, Kim SJ, Park JH, Chan SE, et al. Comparison of the efficacy in preventing of hepatitis B virus recurrence after liver transplantation between HBIG and lamivudine. Transplant Proc 2001;33:3643-3644.

5 Rosenau J, Tillmann HL, Hahr MJ, Trautwein C, Boeker KH, Nashan B, et al. Successful hepatitis B reinfection prophylaxis with lamivudine and hepatitis B immune globulin in patients with positive HBV-DNA at time of liver transplantation. Transplant Proc 2001;33:3637-3638.

6 Marzan A, Salizzoni M, Debernardi-Venon W, Smedile A, Franchello A, Ciancio A, et al. Prevention of hepatitis B virus recurrence after liver transplantation in cirrhotic patients treated with lamivudine and passive immunoprophylaxis. J Hepatol 2001;34:903-910.

7 Rosenau J, Bahr UJ, Tillmann HL, Trautwein C, Klempnauer J, Uanns MP, et al. Lamivudine and low-dose hepatitis B immune globulin for prophylaxis of hepatitis B reinfection after liver transplantation: possible role of mutations in the YMDD motif prior to transplantation as a risk factor for reinfection. J Hepatol 2001;34:895-902.

8 Fontana RJ, Hann HW, Wright T, Everson G, Baker A, Schiff ER, et al. Lamivudine compassionate use study group. A multicenter study of lamivudine treatment in 33 patients with hepatitis B after liver transplantation. Liver Transplant 2001;7:504-510.

9 Han SH, Ofman J, Holt C, King K, Kunder G, Chen P, et al. An efficacy and cost-effectiveness analysis of combination hepatitis B immune globulin and lamivudine to prevent recurrent hepatitis B after orthotopic liver transplantation compared with hepatitis B immune globulin monotherapy. Liver Transplant 2000;6:741-748.

10 Seehofer D, Rayes N, Steinmuller T, Muller AR, Settmacher U, Neuhaus R, et al. Occurrence and clinical outcome of lamivudine-resistant hepatitis B infection after liver transplantation. Liver Transplant 2001;7:976-982.

11 Segovia R, Sanchez-Fueyo A, Rimola A, Grande L, Bruguera M, Costa J, et al. Evidence of serious graft damage induced by de novo hepatitis B virus infection after liver transplantation. Liver Transplant 2001;7:106-112.

12 Sanchez-Fueyo A, Rimola A, Grande L, Costa J, Mos A, Navasa M, et al. Hepatitis B immunoglobulin discontinuation followed by hepatitis B virus vaccination: A new strategy in the prophylaxis of hepatitis B virus recurrence after liver transplantation. Hepatology 2000; 31:496-500.

13 Steinmuller T, Seehofer D, Rayes N, Muller AR, Settmacher U, Jonas S, et al. Increasing applicability of liver transplantation for patients with hepatitis B-related liver disease. Hepatology 2002;35:1528-1535.

14 Naoumov NV, Lopes AR, Burra P, Caccamo L, Temmolo RM, de Man RA, et al. Randomized trial of lamivudine versus hepatitis B immunoglobulin for long-term prophylaxis of hepatitis B recurrence after liver transplantation. J Hepatol 2001;34:888-894.

15 Ben-Ari Z, Mor E, Shapira Z, Tur-Kaspa R. Long-term experience with lamivudine therapy for hepatitis B virus infection after liver transplantation. Liver Transplant 2001;7:113-117.

16 Perrillo RP, Wright T, Rakala J, Levy G, Schiff E, Gish R, et al. A multicenter United States-Canadian trial to assess lamivudine monotherapy before and after liver transplantation for chronic hepatitis B. Hepatology 2001;33:424-432.

17 Macedo G, Maia JC, Gomes A, Beleza M, Teixeira AS, Ribeiro A. Lamivudine: a new strategy to suppress HBV replication in the pretransplant setting. Transplant Proc 2002;32:2642.

18 Yao FY, Terrault NA, Freise C, Maslow L, Bass NM. Lamivudine treatment is beneficial in patients with severely decompensated cirrhosis and actively replicating hepatitis B infection awaiting liver transplantation: a comparative study using a matched, untreated cohort. Hepatology 2001;34:411-416.

19 Lo MC, Cheung ST, Lai CL, Liu CL, Ng IO, Yuan MF, et al. Liver transplantation in Asian patients with chronic hepatitis B using lamivudine prophylaxis. Ann Surg 2001;233:276-281.

20 Lai CL, Chien RN, Leung NWY, Chang TT, Guan R, Tai DI, et al. A one-year trial of lamivudine for chronic hepatitis B. N Engl J Med 1998;339:61-68.

21 Lu SC, Yan LN, Li B, Liu Y, Wen TF, Zhao JC, et al. The analysis for the cause of anti-HBs emergence after liver transplantation for HB-related liver disease (In press).

22 Lu SC, Yan LN, Li B, Wen TF, Cheng NS, Zhao JC, et al. The etiology and management of early postoperative hyperbilirubinemia after liver transplantation. Chin J Organ Transplant 2003;24:73-75.

23 Zhu JP, Zhang TL, Li L, Yuan J, Song SB, Xiu DR, et al. Prevention and treatment of hepatitis B recurrence after liver transplantation. Hepatobiliary Pancreat Dis Int 2003;2:500-503.

24 Sponseller CA, Bacon BR, Di Bisceglie AM. Clinical improvement in patients with decompensated liver disease caused by hepatitis B after treatment with lamivudine. Liver Transplant 2000;6:715-720.

25 Kitay-Cohen Y, Ben-Ari Z, Tur-Kaspa R, Fainguelernt H, Lishner M. Extension of transplantation free time by lamivudine in patients with hepatitis B-induced decompensated cirrhosis. Transplantation 2000;69:2382-2383.

26 Barcena R, Dominguez-Antonaya M, Lopez-Sanroman A, Martinez-Turnes A, Urman J, del Campo S, et al. Lamivudine therapy of hepatitis B virus-related liver disease: cirrhosis, posttransplantation recurrence, and de novo infection. Transplant Proc 1999;31:2457-2458.

27 Ben-Ari Z, Pappo O, Zemel R, Mor E, Tur-Kaspa R. Association of lamivudine resistance in recurrent hepatitis B after liver transplantation with advanced hepatic fibrosis. Transplantation 1999;68:232-236.

28 So SK, Esquivel CO, Imperial JC, Garcia G, Monge H, Keeffe EB. Does Asian race affect hepatitis B virus recurrence or survival following liver transplantation for hepatitis B cirrhosis? J Gastroenterol Hepatol 1999;14:S48-52.

Received September 16, 2003

Accepted after revision November 3, 2003