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was used to check the number of cells subjected to PCR. ... histological score for fibrosis stage and activity.30. An acquired HBV infection ... The Mann-Whitney test was used to com- ...... Lee KW, Lee DS, Lee HH, Kim SJ, Joh JW, Seo JM, et al.
LIVER TRANSPLANTATION 16:885-894, 2010

ORIGINAL ARTICLE

Escape Hepatitis B Virus Mutations in Recipients of Antibody to Hepatitis B Core Antigen–Positive Liver Grafts Receiving Hepatitis B Immunoglobulins Bruno Roche,1,4,5 Anne-Marie Roque-Afonso,2,4,5 Mylene Sebagh,3,4,5 Valerie Delvart,1 Jean Charles Duclos-Vallee,1,4,5 Denis Castaing,1,4,5 and Didier Samuel1,4,5 1 Centre Hepato-Biliaire; 2Laboratoire de Virologie; 3Laboratoire Anatomie Pathologique, AP-HP Hopital Paul Brousse, Villejuif, France; 4Unite´ 785, INSERM, Villejuif, France; and 5Unite´ Mixte de Recherche en Sante´ 785, Universite´ Paris-Sud, Villejuif, France

A variety of prophylactic strategies are used to prevent the risk of hepatitis B virus (HBV) transmission from antibody to hepatitis B core antigen (anti-HBc)–positive donors. The mechanisms underlying the failure of HBV immunoglobulin monoprophylaxis have been poorly evaluated. Seventy-seven anti-HBc–positive grafts were used in 21 hepatitis B surface antigen (HBsAg)–positive recipients and 56 HBsAg-negative recipients. HBsAg-positive recipients received prophylaxis comprising hepatitis B immunoglobulins (HBIG) and antiviral agents, 45 HBsAg-negative recipients received a modified HBIG regimen, and 11 HBsAg-negative recipients received no prophylaxis. Both donors and recipients were screened for HBsAg, antibody to HBsAg (anti-HBs) and anti-HBc in their sera and for HBV DNA in both their sera and liver. S gene mutations were investigated after HBV reinfection. HBV infection occurred in 15 HBsAg-negative recipients (19.4%) at a median interval of 16 months (range ¼ 6-67 months) post-transplant and in none of the HBsAg-positive recipients. HBV infections were observed in 31.6% of HBV-naive recipients and 7.7% of HBV-immune recipients receiving HBIG prophylaxis versus 100% of HBV-naive recipients (P ¼ 0.0068) and 33% of HBV-immune recipients (P ¼ 0.08) with no such prophylaxis. S gene mutations were identified in 9 recipients. In conclusion, priority should be given to using anti-HBc positive grafts for HBsAg-positive or HBV-immune recipients. Our study has confirmed the high risk of HBV transmission to naive recipients. HBIG monoprophylaxis was associated with a significant risk of de novo HBV infection and HBV escape mutations. In these patients, we therefore recommend prophylaxis with lamivudine or new nucleos(t)ides analogues. The potential benefits of HBIG prophylaxis combined with antiviral drugs require further evaluations. Long-term prophylaxis is needed because of the long interval of de novo HBV infection post-transplant in some patients. Liver Transpl 16:885-894, 2010. V 2010 AASLD. C

Received January 13, 2010; accepted April 5, 2010. Liver grafts from hepatitis B surface antigen (HBsAg)– negative and antibody to hepatitis B core antigen (anti-HBc)–positive donors are used to increase the pool of grafts available. In Europe, the prevalence of anti-HBc–positive grafts may reach 10% in some regions.1-3 A recently published multivariate analysis

of the United Network for Organ Sharing database has shown that the use of anti-HBc–positive grafts is not an independent determinant of graft or patient survival.4 However, these grafts are associated with a risk of hepatitis B virus (HBV) reactivation after transplantation.5-8 In the absence of any prophylaxis, the

Abbreviations: ADV, adefovir; anti-HBc, antibody to hepatitis B core antigen; anti-HBe, antibody to hepatitis B e antigen; antiHBs, antibody to hepatitis B surface antigen; CI, confidence interval; ETV, entecavir; FTC, emtricitabine; HBIG, hepatitis B immunoglobulin; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HCV, hepatitis C virus; HDV, hepatitis delta virus; LAM, lamivudine; N, negative; NA, not available; NASH, nonalcoholic steatohepatitis; OR, odds ratio; P, positive; PBC, primary biliary cirrhosis; PCR, polymerase chain reaction; PSC, primary sclerosing cholangitis; TDF, tenofovir; WT, wild type. Address reprint requests to Didier Samuel, MD, PhD, Centre He´pato-Biliaire, Hoˆpital Paul Brousse, 12-14 Avenue Paul Vaillant Couturier, Villejuif, France 94804. Telephone: 33 01 45 58 33 31; FAX: 33 01 45 59 38 57; E-mail: [email protected] DOI 10.1002/lt.22084 Published online in Wiley InterScience (www.interscience.wiley.com). LIVER TRANSPLANTATION.DOI 10.1002/lt. Published on behalf of the American Association for the Study of Liver Diseases

C 2010 American Association for the Study of Liver Diseases. V

2006 2005 2004 2003 2002 2001 2000 1999 1998 1997 Year

TABLE 1. Frequency of Anti-HBc–Positive Donors

Liver transplantation (n) 86 86 71 96 82 100 105 101 112 108 947 Anti-HBc–positive 7 (8.1%) 7 (8.1%) 5 (7%) 8 (8.3%) 10 (12.2%) 9 (9%) 8 (7.6%) 7 (6.9%) 7 (6.2%) 9 (8.3%) 77 (8.1%) donors [n (%)] Donor age in years 42 (28-56) 49 (32-58) 47 (23-62) 42.5 (21-60) 51 (33-70) 55 (24-75) 53 (23-68) 49 (18-75) 61 (41-77) 67 (46-79) 51 (18-79) [median (range)]

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All

886 ROCHE ET AL.

probability of HBV infection depends on the HBV serological status of the recipient: this probability is lowest in antibody to hepatitis B surface antigen (anti-HBs)–positive and anti-HBc–positive recipients (0%-5%) and intermediate in anti-HBc–positive or anti-HBs–positive recipients (10%-18%), whereas the rate of de novo infection in naive recipients can reach 70% to 80%.1,2,5-10 A variety of prophylactic strategies have been used in small patient series: hepatitis B immunoglobulins (HBIG), lamivudine (LAM), pretransplant or posttransplant vaccination, and combination prophylaxis.2,10-20 However, prophylactic regimens remain empirical, and there is no standardized approach.21 The continuous use of HBIG in the context of liver transplantation for hepatitis B–infected patients has been shown to enable a dramatic reduction in the incidence of recurrent HBV infection.22 Since 1998 in our institution, recipients of anti-HBc– positive grafts have been given HBIG without additional antiviral drugs in order to prevent de novo HBV infection. We previously reported encouraging results with this strategy in a cohort of 22 patients who were also included in the present study.2 Some unanswered questions remain regarding the efficacy of HBIG versus antiviral agents, the limitations of using HBIG and particularly the risk of HBIG-induced viral mutations, and the potential additional benefit of using combination prophylaxis. Several case reports of HBV mutants with amino acid changes in the ‘‘a’’ determinant of the S gene have been reported in recipients receiving HBIG prophylaxis or vaccination.23-27 The aims of the present study were to report our experience with HBIG monoprophylaxis over a period of 10 years in 77 consecutive recipients of anti-HBc–positive grafts, to assess the incidence and risk factors associated with de novo HBV infection, and to explore the risk of HBV mutations induced by HBIG prophylaxis.

PATIENTS AND METHODS Patients Between January 1997 and December 2006, 77 of 947 patients (8.1%) received grafts from HBsAg-negative, anti-HBc–positive donors (Table 1). The median donor age was 51 years (range ¼ 18-79 years), and it had increased in recent years. Five of these 77 patients underwent combined liver and kidney transplantation, and 12 received partial grafts (8 living donor liver transplants, 3 right split liver grafts, and 1 left split liver graft). These grafts were transplanted into 21 HBsAg-positive recipients and 56 HBsAg-negative recipients (Tables 2-4). The HBsAg-positive recipients underwent transplantation for HBV-related cirrhosis (n ¼ 15) or B-delta– related cirrhosis (n ¼ 6), and hepatocellular carcinoma was present in 5 cases (Table 2). At the time of transplantation, all these patients were HBV DNA– negative as revealed by real-time polymerase chain

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reaction (PCR) testing. Ten patients received antiviral therapy prior to transplantation: 8 received LAM, 1 received adefovir (ADV), and 1 received LAM and ADV. After transplantation, 11 recipients received HBIG (Laboratoire Franc¸ais de Biotechnologie, Les Ulis, France) as long-term monoprophylaxis, and 10 received combination prophylaxis using HBIG and pretransplant antiviral therapy. The HBIG prophylactic regimen consisted of 10,000 IU of HBIGs in a daily intravenous infusion for 7 days after transplantation and then whenever the anti-HBs titer fell to less than 200 to 500 IU/mL (ie, 6-12 infusions per year). HBsAg-negative recipients underwent transplantation for hepatitis C virus (HCV) cirrhosis (n ¼ 20), alcoholic cirrhosis (n ¼ 13), amyloid neuropathy (n ¼ 10), and other reasons (n ¼ 13; Tables 3 and 4). Before July 1998, 6 recipients received no HBIG prophylaxis, but after that time, 45 of 50 HBsAg-negative recipients were given a modified HBIG prophylactic protocol whatever their pretransplant HBV serological status was: an infusion of 5000 IU of HBIG during the perioperative period and thereafter to maintain the anti-HBs titer above 100 IU/mL (ie, 2-4 infusions per year). Five patients received no HBIG prophylaxis because the donor anti-HBc positivity was unknown

TABLE 2. Outcomes of Anti-HBc–Positive Grafts in HBsAg-Positive Recipients HBsAg-Positive Recipients (n ¼ 21) Diagnosis (n) Recipient pretransplant HBV status HBV prophylaxis (n)

Follow-up in months [median (range)] De novo HBV infection

HBV cirrhosis (15) HDV cirrhosis (6) HBsAg-positive HBIG (11) LAM/HBIG (8) ADV/HBIG (1) LAM/ADV/HBIG (1) 59 (3-124) 0%

at the time of transplantation. Trained transplant coordinators assessed patient compliance by monitoring the results of anti-HBs titers and HBIG infusions. Twenty-one HBV-naive recipients received a course of HBV vaccinations (40 lg per dose via the intramuscular route administered at 1, 30, and 60 days; Engerix B, SmithKline Beecham, France) during the pretransplant assessment. Immunosuppression was ensured with a combination of corticosteroids, cyclosporine or tacrolimus, and azathioprine or mycophenolate mofetil. The dosage was adjusted as a function of therapeutic drug levels, underlying disease, and renal function. The corticosteroid dosage followed a routine protocol, and it was withdrawn after a period based on the underlying disease.

Virological Testing and Histology The donor HBV virological status was systematically reexamined by our laboratory. Sera were tested with commercial enzyme immunoassays for HBsAg (DiaSorin, Sallugia, Italy), anti-HBs (DiaSorin), and anti-HBc (Biorad, Marnes-la-Coquette, France), and HBV DNA was detected retrospectively with real-time PCR with a lower limit of detection of 15 IU/mL (Cobas TaqMan HBV, Roche Diagnostics, Meylan, France). Total HBV DNA and covalently closed circular DNA were detected on paraffinembedded prereperfusion liver biopsy specimens with real-time PCR on a LightCycler system, as previously described.28 The real-time amplification of b-globin genes was used to check the number of cells subjected to PCR. Prior to transplantation, recipients were tested for HBsAg, anti-HBs, anti-HBc, and HBV DNA. HBV DNA was determined from 1997 to 2005 with the Cobas HBV Monitor assay (Roche Diagnostics) and after 2005 with the Cobas TaqMan HBV test, which presents an excellent correlation with the Cobas HBV Monitor assay.29 After transplantation, recipients were routinely screened at least once a month for anti-HBs and every 4 months for HBsAg and HBV DNA. Liver biopsy was performed before reperfusion, routinely after transplantation (in years 1, 2, and 5 and then every 5 years), and if liver test abnormalities occurred or HBsAg emerged. Liver biopsy samples

TABLE 3. Frequency of De Novo HBV Infection in HBsAg-Negative Recipients Without HBV Prophylaxis as a Function of the Recipient Pretransplant HBV Status HBsAg-Negative Recipients (n ¼ 11) HCV cirrhosis (3), alcoholic cirrhosis (3), amyloid neuropathy (2), cholestatic liver Diagnosis (n) Recipient pretransplant HBV status (n) HBV prophylaxis Follow-up in months [median (range)] De novo HBV infection [n (%)]

disease (1), hepatic metastasis of neuroendocrine tumor (1), and fulminant hepatitis (1) anti-HBc-negative,

anti-HBs-positive,

anti-HBs-negative,

anti-HBs-positive,

anti-HBs-negative

anti-HBc-negative

anti-HBc-positive

anti-HBc-positive

(5)

(3)

(2)

(1)

None 94 (29-130)

None 129 (33-129)

None 54 (33-75)

None 35

5 (100%)

1 (33%)

1 (50%)

0

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TABLE 4. Frequency of De Novo HBV Infection in HBsAg-Negative Recipients Receiving HBIG Prophylaxis as a Function of the Recipient Pretransplant HBV Status HBsAg-Negative Recipients (n ¼ 45) HCV cirrhosis (17), alcoholic cirrhosis (10), other cirrhosis: cholestatic liver disease (4), Diagnosis (n)

Recipient pretransplant HBV status (n) HBV prophylaxis Follow-up in months [median (range)] De novo HBV infection [n (%)]

NASH (1),hemochromatosis (1), amyloid neuropathy (8), and fulminant hepatitis (4) anti-HBc-negative, anti-HBs-negative

anti-HBs-positive, anti-HBc-negative

anti-HBs-negative, anti-HBc-positive

anti-HBs-positive, anti-HBc-positive

(19)

(14)

(4)

(8)

HBIG 66 (1-118)

HBIG 72 (1-113)

HBIG 23 (3-118)

HBIG 56 (8-114)

6 (31.6%)*

1 (7%)

0

1 (12.5%)*

*Two of these patients were HBV DNA–positive and HBsAg-negative.

were staged and graded according to the METAVIR histological score for fibrosis stage and activity.30 An acquired HBV infection was defined as a positive test for HBsAg and/or HBV DNA. In the case of HBsAg positivity, sera were tested for HBeAg/antibody to hepatitis B e antigen (anti-HBe) and HBV DNA. Mutations affecting surface and reverse-transcriptase genes were investigated via the sequencing of a fragment encompassing nucleotides 366 to 1015 of the HBV genome.31

Statistical Analysis Statistical analyses were performed with StatView statistical software (Abacus Concepts, Inc., Berkeley, CA). Continuous variables were expressed as medians and ranges, and categorical variables were expressed as proportions. The Mann-Whitney test was used to compare continuous variables, and v2 and Fisher’s exact tests were used to compare categorical variables. A P value of 10 IU/mL in 52 of 76 tested donors (68%). Serum HBV DNA was negative in the 73 donors tested. There was testing for HBV DNA and the b-globin gene in 56 prereperfusion liver graft biopsy samples. b-Globin was undetectable in 5 samples because of insufficient amounts of liver material. Total HBV DNA was detectable in 20 of the 51 remaining samples (39%). The histological assessment of prereperfusion liver graft biopsy samples did not reveal any evidence of acute or chronic hepatitis.

Outcome of HBsAg-Positive Recipients When HBIG was used with or without antiviral prophylaxis, no HBV infection occurred in the 21 HBsAg-positive recipients throughout a median follow-up period of

59 months (range ¼ 3-124 months; Table 2). Four patients died during the follow-up period; these events were related to a recurrence of hepatocellular carcinoma (month 3), amyloid cardiopathy (month 15), pulmonary disease (month 116), and posttransplant lymphoma (month 77). One patient underwent repeat transplantation for chronic rejection at month 10.

Outcome of HBsAg-Negative Recipients Recipients Without HBIG Prophylaxis Eleven HBsAg-negative recipients were not given HBIG prophylaxis. At the time of transplantation, 5 were HBV-naive, 3 were anti-HBs–positive (anti-HBs titer > 100 IU/mL in 2 patients and anti-HBs titer ¼ 21 IU/ mL in 1 patient), 2 were anti-HBc–positive, and 1 was anti-HBc–positive and anti-HBs–positive (Table 3). HBV infection developed in the 5 naive recipients (100%) after a median posttransplant interval of 12 months (range ¼ 7-67 months) and in 2 of 6 HBVimmune recipients (33%): at month 6 in an anti-HBs– positive and anti-HBc–negative recipient (pretransplant anti-HBs titer ¼ 21 IU/mL) and at month 9 in a patient who was anti-HBc–positive and anti-HBs–negative (Table 3). These 7 patients showed evidence of anti-HBc seroconversion at a median of 10 months (range ¼ 3-67 months) post-transplant. One anti-HBc–negative recipient showed evidence of anti-HBc seroconversion at month 3 post-transplant without other serological HBV markers of infection. Anti-HBc positivity persisted throughout follow-up. Two patients died during follow-up because of a lung tumor (months 36 and 75). One patient underwent repeat transplantation because of portal vein thrombosis and biliary complications in month 22.

Recipients Receiving HBIG Prophylaxis A modified HBIG protocol was administered to 45 HBsAg-negative recipients. At the time of transplantation, 19 were HBV-naive, 14 were anti-HBs–positive, and 12 were anti-HBc–positive (n ¼ 4) or anti-HBc– positive and anti-HBs–positive (n ¼ 8; Table 4).

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TABLE 5. Predictive Factors Associated with De Novo HBV Infection in HBsAg-Negative Recipients

Anti-HBs–positive donor [n (%)] HBV DNA–positive donor liver [n (%)] Anti-HBs–negative recipient [n (%)] Anti-HBc–negative recipient [n (%)] HBV-naive recipient [n (%)]

HBIG prophylaxis [n (%)]

De Novo HBV

No HBV

Univariate

Infection

Infection

Analysis

Multivariate

(n ¼ 15)

(n ¼ 41)

P Value

Analysis

12 (85.7%)

28 (68.3%)

0.20

NS

6 (42.9%)

8 (29.6%)

0.39

NS

12 (80%)

18 (43.9%)

0.016

NS

13 (86.7%)

28 (68.3%)

0.16

NS

11 (73.3%)

13 (31.7%)

0.0053

8 (53.3%)

37 (90.2%)

0.0021

OR ¼ 9.4, CI ¼ 1.8-49.3, P ¼ 0.0079 OR ¼ 13.6, CI ¼ 2.2-83, P ¼ 0.0048

Among the anti-HBs–positive recipients, anti-HBs titers were between 10 and 100 IU/mL in 9 patients (median ¼ 53 IU/mL, range ¼ 18-78 IU/mL) and >100 IU/mL in 5 patients. Five of 21 patients who were vaccinated prior to transplantation (24%) became anti-HBs–positive before transplantation; their anti-HBs titers at the time of surgery were 31, 41, 54, 61, and >100 IU/mL, respectively. De novo HBV infection developed in 6 of 19 naive recipients (31.6%) after a median interval of 20.5 months (range ¼ 14-57 months) post-transplant and in 2 of 26 HBV-immune recipients (7.7%): in month 56 in an anti-HBs–positive and anti-HBc–negative recipient (pretransplant anti-HBs titer ¼ 61 IU/mL) and in month 20 in an anti-HBc–positive and anti-HBs– positive recipient (Table 4). Seven of the 8 recipients with de novo HBV infection showed evidence of antiHBc seroconversion at a median of 22 months (range ¼ 3-59 months) post-transplant. Eight of 25 antiHBc–negative recipients (32%) showed evidence of anti-HBc seroconversion at a median of 6 months (range ¼ 3-24 months) post-transplant without other serological HBV markers of infection. Anti-HBc positivity persisted throughout the follow-up period. Fourteen patients died during follow-up (median period elapsing ¼ 12 months, range ¼ 1-114 months) from multiorgan failure (n ¼ 6), HCV recurrence (n ¼ 4), heart failure (n ¼ 3), or gastric cancer (n ¼ 1). Four patients underwent repeat transplantation because of chronic rejection (month 35), HCV recurrence (months 38 and 103), or arterial thrombosis (month 19).

Risk Factors for De Novo HBV Graft Infection The median follow-up of the 77 recipients after transplantation was 65 months (range ¼ 1-130 months). Fifteen of 77 patients (19.4%) developed de novo HBV infection post-transplant after a median interval of 16 months (range ¼ 6-67 months): none of the HBsAg-

positive recipients and 15 of the 56 HBsAg-negative recipients (26.7%). Among the HBsAg-negative recipients, HBV infection occurred in 8 of 45 patients with prophylaxis (17.7%) versus 7 of 11 recipients without prophylaxis (63.6%; P ¼ 0.0021). The mean time to the onset of HBV infection was 30.8 months (618.4 months) in patients with HBIG prophylaxis and 18.1 months (621.7 months) in patients without HBIG prophylaxis (P ¼ 0.24). De novo HBV infection occurred in 11 of 24 HBV-naive recipients (45.8%) versus 4 of 32 HBVimmune recipients (12.5%; P ¼ 0.0053). HBV infection developed in 6 of 19 HBV-naive recipients with prophylaxis (31.6%) versus all naive recipients without prophylaxis (P ¼ 0.0063) and in 2 of 26 HBV-immune recipients with prophylaxis (7.7%) versus 2 of 6 without prophylaxis (33%; P ¼ 0.08). HBV DNA was detectable in 14 prereperfusion liver biopsy samples, undetectable in 27, and unavailable in 15. De novo HBV infection occurred in 6 of 14 recipients with detectable HBV DNA in the liver graft (42.8%) and in 8 of 27 with undetectable HBV DNA (57.1%; P ¼ 0.39). In multivariate analysis, factors associated with HBV infection in HBsAg-negative recipients were HBV-naive recipient status [odds ratio (OR) ¼ 9.4, 95% confidence interval (CI) ¼ 1.8-49.3, P ¼ 0.0079] and an absence of HBIG prophylaxis (OR ¼ 13.6, 95% CI ¼ 2.2-83, P ¼ 0.0048; Table 5).

Viral Characterization of De Novo Infection At the time of HBV infection, HBV DNA was detectable in sera and liver tissue from 15 patients; 13 were positive for HBsAg, and 10 were positive for HBeAg (Table 6). HBsAg-negative de novo infection developed in 2 patients. Patient 15 was anti-HBs–positive and antiHBc–positive before transplantation for amyloid neuropathy. HBIG were infused to maintain the anti-HBs titer above 100 IU/mL. Twenty months after transplantation, HBV DNA was detected at levels of 53 and

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TABLE 6. Outcomes of HBV Graft Infection Pretransplant HBV Status of Recipient

HBV Infection Pretransplant HBV

anti-HBs Patient 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Diagnosis (IU/mL) anti-HBc Prophylaxis

Positive Test for

Positive Test

Graft HBV DNA for HBsAg HBV DNA (months)

HBV

HBs

(months) HBeAg Genotype Sequence

Budd-Chiari Alcoholic cirrhosis Alcoholic cirrhosis HCV cirrhosis

N N

N N

0 0

P P

10 16

4 20

P P

D D

WT WT

N

N

0

P

7

19

P

D

WT

N

N

0

N

67

67

P

D

Amyloid neuropathy Alcoholic cirrhosis PSC Alcoholic cirrhosis PBC HCV cirrhosis NASH Alcoholic cirrhosis Alcoholic cirrhosis PBC Amyloid neuropathy

N

N

0

N

12

12

N

A

P120S D144E WT

N

P

0

N

9

10

P

A

Q129R

21 N

N N

0 HBIG

N P

6 17

12 17

P N

B A

G145R D144A

N N N N

N N N N

HBIG HBIG HBIG HBIG

N NA N P

15 44 14 57

17 44 No 59

P N N P

D A A E

G145R D144E WT G145R

N

N

HBIG

P

24

24

P

A

G145R

61 >100

N P

HBIG HBIG

N N

56 20

59 No

P N

D NA

G145R NA

36 IU/mL in a control test. The patient was treated with entecavir (ETV), and HBIG were withdrawn. HBV DNA levels became negative. This patient died 4 months later from heart failure. Patient 11 was HBVnaive prior to transplantation for nonalcoholic steatohepatitis (NASH). He received HBIG to maintain his anti-HBs titer above 100 IU/mL. HBV DNA was detected at a level of 94 IU/mL 14 months after transplantation and at a level of 1306 IU/mL at 16 months. The patient was treated with LAM, and HBIG were withdrawn. HBV DNA levels remained negative after 18 months of follow-up. Viral sequencing was possible in 14 of 15 patients with de novo infections, including 1 HBsAg-negative patient. HBV DNA levels were insufficient to allow sequencing in 1 patient. The HBV genotype was A in 6 patients, D in 6 patients, B in 1 patient, and E in 1 patient (Table 6). A wild-type (WT) surface gene was detected in 5 recipients: 4 patients who received no HBIG prophylaxis and 1 who received HBIG (Table 6). S gene mutations were identified in the remaining 9 recipients: sG145R in 5, sD144A in 1, sD144E in 1, sD144E and sP120S in 1, and sQ129R in 1. The last patient had received no HBIG prophylaxis. Six of the 8 patients with sG145 or sD144 mutations received HBIG prophylaxis, and patient 7 received a course of vaccination prior to

transplantation (Table 6). At the time of de novo HBV infection, no resistance mutations were observed in the overlapping polymerase gene.

Outcome of De Novo HBV Graft Infection At the time of HBV infection, aminotransferase levels were normal or moderately elevated. Liver biopsy samples revealed mild lobular inflammation. First-line antiviral therapy comprised LAM in 10 patients, LAM and ADV in 4 patients, and ETV in 1 patient (Table 7). Antiviral therapy was modified in 5 patients because of LAM resistance mutations (L180M and M204V). S gene mutations previously observed in 3 of these 5 patients were still present at the time of viral breakthrough. Antiviral therapy was modified in 2 patients due to a suboptimal virological response [the patients were switched from LAM and ADV to emtricitabine (FTC) and tenofovir (TDF)]. At the last follow-up assessment, HBV DNA was positive in 3 of 15 patients. During antiviral therapy, 5 of 10 patients presented with anti-HBe seroconversion, 6 presented with HBsAg negativization, and 3 presented with anti-HBs seroconversion. Liver biopsy samples at the end of follow-up (median interval after an HBsAg-positive test ¼ 4.7 years,

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TABLE 7. Virological and Histological Outcomes of HBV Graft Infection Follow-Up

Patient

Antiviral Therapy

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

LAM þ ADV LAM LAM þ ADV LAM þ ADV FTC þ TDF LAM þ ADV FTC þ TDF LAM þ ADV LAM LAM þ ADV LAM LAM LAM LAM þ ADV LAM þ ADV LAM þ ADV ETV

Fibrosis

After HBV

Serum HBV DNA

anti-HBe Seroconversion

HBsAg/ anti-HBs

Stage on Last Biopsy*

Infection (months)

N P N P

No No No No

P/N P/N P/N P/N

F0 F0 F1 F0

126 18 120 31

N

—

N/P

F1

19

P N N N N N N N N N

No Yes — Yes — — Yes Yes Yes —

P/N N/N P/N N/P N/P — N/N P/N N/N —

F1 F0 F0 F0 F1 F0 F1 F1 F1 F0

60 30 104 84 52 20 21 60 20 6

*According to METAVIR.

range ¼ 1-10 years), available for all patients, revealed stage 1 fibrosis in 7 patients and no fibrosis in 8 patients.

DISCUSSION One strategy for expanding the donor pool is to use grafts from donors who display evidence of prior exposure to HBV. The percentage of anti-HBc–positive donors has been estimated to range from 5.5% in the United States and 10% in certain parts of Europe (8% in our series) to 50% or more in Asia.1-3,13 We report our experience with 77 consecutive recipients of antiHBc–positive grafts. In the absence of prophylaxis, HBV infection can be reactivated in the graft at a frequency that is linked to the HBV serological status of the recipient: this frequency is lowest in HBV-immune recipients (0%-18%) and highest in HBV-naive recipients (70%-80%).1,2,5-10 Our findings confirmed that patients who underwent transplantation for non– HBV-related disease were at a significant risk of HBV reactivation in the absence of prophylaxis: this was constant in HBV-naive recipients (100%) and intermediate in HBV-immune recipients (33%). With the HBIG monoprophylaxis protocol, the risk decreased significantly to 31.6% in naive recipients (P ¼ 0.0063). The risk decreased to 7.7% in HBV-immune recipients (P ¼ 0.08), but the difference was not statistically significant, probably because of the small number of patients. In multivariate analysis, the factors associated with de novo HBV infection in HBsAg-negative recipients were HBV-naive recipient status and an absence of HBIG prophylaxis. S gene mutations were identified in 9 recipients with de novo HBV graft infec-

tion. Six of them received HBIG prophylaxis, and 1 received HBV vaccination prior to transplantation. With a prophylactic regimen comprising HBIG with or without antiviral therapy as a function of the pretransplant HBV viral load, no HBV infection occurred in the 21 HBsAg-positive recipients. Combination prophylaxis, which was highly effective in preventing a recurrence of HBV, was also able to prevent HBV reactivation in the HBsAg-positive recipients in this study. This suggests that anti-HBc–positive grafts should be directed preferentially toward HBsAg-positive recipients, who should then be given appropriate prophylaxis against a recurrence of HBV. In theory, after transplantation of an anti-HBc–positive graft, the risk of HBV infection is expected to be higher in HBsAg-positive recipients receiving HBIG monoprophylaxis than in HBsAg-negative recipients using the same prophylaxis because of a presumably greater viral load in the former group. However, the site of HBV replication and the mechanisms of HBV reinfection could be different in these 2 groups of patients. In patients who undergo transplantation for non– HBV-related disease, a variety of prophylactic strategies have been used in small, heterogeneous, singlecenter cohorts in order to prevent the risk of HBV reactivation; these include HBIG, LAM, pretransplant or posttransplant vaccination, and combination prophylaxis.2,10-20 However, prophylactic regimens remain empirical, and there is no standardized approach. A recent survey reported on HBV prevention strategies for anti-HBc–positive donors in 52 liver transplant programs.21 All physicians used nucleoside analogue therapy, which mainly involved LAM (65%), usually for an indefinite period (81%). Sixty-one

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percent of these physicians applied combination therapy including HBIG, generally for a period of 1 year. The continuous use of high-dose HBIG in liver transplantation for hepatitis B–infected patients has been shown to dramatically reduce the incidence of recurrent HBV infection.22 During a previous study, we also showed that long-term HBIG prophylaxis or postvaccine anti-HBs antibodies could control HBV replication in liver grafts from anti-HBc–positive donors without the addition of antiviral drugs.2 Among 16 recipients receiving HBIG and 2 vaccinated patients, only 1 patient became HBsAg-positive.2 One reason for the use of HBIG is their ability to neutralize HBsAg-coated virions and inhibit the release of virions from infected cells. When the present study was initiated, the use of LAM prophylaxis was limited by the risk of drug-induced viral mutations. This prompted us to administer HBIG monotherapy but under a less stringent HBIG protocol because we assumed that there were no circulating particles or at least fewer circulating particles in comparison with HBsAg-positive recipients. In HBsAg-negative recipients under HBIG monoprophylaxis, de novo HBV infection occurred in 6 of 19 naive recipients (31.6%) versus 2 of 26 HBVimmune recipients (7.7%; 1 anti-HBs–positive recipient with an anti-HBs titer of 61 IU/mL and 1 anti-HBs– positive and anti-HBc–positive recipient). These results underline the greatest risk of HBV reactivation in naive recipients. The long-term detection of anti-HBc without other serological HBV markers (HBV DNA or anti-HBc immunoglobulin M) in 9 of 26 anti-HBc–negative recipients (34.6%) indicated an immunological response to the hepatitis B core antigen and suggested that low levels of HBV antigens had been transmitted. The administration of HBIG in these patients probably prevented overt HBV infection. Indeed, it has been shown that HBIG can be internalized by hepatocytes and interact with HBsAg in the cells and thus prevent HBsAg secretion.32 Regular anti-HBc testing after transplantation may provide the first signs of a risk of HBV reactivation in the liver graft. During this study, HBV mutations in the ‘‘a’’ determinant of the S gene commonly associated with HBIG or vaccine escape (sD144 and sG145) were identified in 8 recipients. One of them had received a course of vaccinations prior to transplantation, and 6 received HBIG after transplantation. The emergence of these mutations is suggestive of insufficient viral suppression by active or passive anti-HBs. Several case reports of HBV mutants causing amino acid changes in the ‘‘a’’ determinant of the S gene have been reported after transplantation from anti-HBc–positive donors in recipients who had been vaccinated or received HBIG prophylaxis.12,23-27 These surface gene mutants can escape the immune response against HBV and cause infection. This failure may be dependent on the mechanism of action of HBIG, which have no effect on viral replication. It appears that HBIG are unable to clear HBV infection, as shown by detectable HBV DNA levels in the serum, liver, or peripheral blood mononuclear cells for up to 10 years in patients

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who underwent liver transplantation for HBV-related disease without HBV recurrence and were treated with long-term HBIG.33 In HBsAg-negative recipients of anti-HBc–positive grafts, there are few or no circulating HBsAg-coated virions to be neutralized by HBIG. Because of the overlapping reading frames between s gene sequence and HBV polymerase, patients with surface gene mutant infections could develop HBV polymerase mutations more frequently. Three of 5 patients with LAM resistance mutations had surface gene mutant HBV infections in our study. The other limitations to using HBIG are their parenteral administration and high cost. The failures observed in the 2 HBV-immune recipients during the present study may have been linked to a low pretransplant anti-HBs titer in 1 patient (ie, 61 IU/mL). A recent pediatric study suggested that a pretransplant anti-HBs titer > 200 IU/mL might be sufficient to prevent HBV infection in the recipients of anti-HBc–positive grafts.34 The evolution of posttransplant anti-HBs levels over time may also be important, and it could be necessary to maintain anti-HBs titers > 100 IU/mL.26 The use of vaccination boosters after transplantation should be evaluated in this setting. We can confirm that pretransplant HBV vaccination designed to supply endogenous anti-HBs is unlikely to be successful in the majority of patients.35 Five of 21 patients who were vaccinated before transplantation (24%) became anti-HBs– positive before transplantation but with low anti-HBs titers in the majority of them: 31, 41, 54, 61, and >100 IU/mL. Pretransplant vaccination alone does not appear to be an effective strategy because de novo HBV infection developed in 10% of successfully vaccinated recipients without any posttransplant prophylaxis.2,10,13,25 Posttransplant vaccination alone is ineffective.36 The efficacy of new immunogenic HBV vaccines before or after transplantation for obtaining high anti-HBs titers should be evaluated.37 In studies of LAM monoprophylaxis, HBV infection was observed in 2.6% of recipients: 4% of HBVimmune recipients and 3.4% of HBV-naı¨ve recipients.10 However, it should be emphasized that the number of reported studies is still small, and the follow-up is short. The development of a YMDD mutant after prophylaxis with LAM has also been described.9,14 Compliance with antiviral therapy should be closely evaluated because some patients developed HBV infection after LAM discontinuation. HBV infection was observed in 2.8% of the recipients using HBIG and LAM combination prophylaxis, the most widely used approach.9,12,13,16,17 Given the low probability of de novo HBV infection with LAM, the benefit of HBIG and LAM combination prophylaxis over monoprophylaxis with LAM is unclear from the present literature and deserves new studies. The specific situation of HBV-naive recipients may plead in favor of the use of combination prophylaxis. Despite the absence of current data on the use of more potent antiviral agents (ETV and TDF) in this setting, it is expected that they could be more effective than LAM with a lower risk of resistance mutations.

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HBV molecular testing of the serum or liver with the techniques now available does not provide a definitive measurement of the infectious risk of anti-HBc–positive donors. During the present study, no donor serum samples were positive for HBV DNA, and this confirmed the very low HBV viral load in the sera.17 HBV DNA was detected at a high frequency in liver grafts (39%), as previously reported.11,17,38 Nevertheless, HBV DNA positivity in the liver graft was not predictive of the infectious risk because HBV DNA was positive in 7 of 14 grafts in patients who developed de novo infection and in 14 of 37 grafts in patients who did not (this difference was not significant). However, the HBV detection rate may have been underestimated because of the low viral load, insufficient sensitivity of the PCR assay, or the focal distribution of HBV infection. In conclusion, the use of liver grafts from anti-HBc– positive donors is a relevant strategy for expanding the liver graft pool in an era of graft shortages. These grafts may be suitable for HBsAg-positive recipients because they are going to receive combination HBV prophylaxis. In our study, no case of HBV infection occurred in this group. For recipients with high antiHBs titers and anti-HBc, the risk of de novo infection is very low, and HBV prophylaxis could be unnecessary. However, anti-HBs titers should be closely monitored after transplantation. For anti-HBs–positive or anti-HBc–positive recipients, the risk of de novo infection is intermediate, and monoprophylaxis with LAM or new antiviral drugs appears to offer satisfactory protection. The use of anti-HBc–positive grafts in naive recipients should be considered with care. HBIG monoprophylaxis seems to provide a far from optimum solution because there is a high risk of the occurrence of de novo HBV infection (31.6% in our study) and HBV mutations. Despite a low response rate, these patients should be immunized against HBV before transplantation. After surgery, they should receive nucleos(t)ides analogues with or without HBIG. This combination prophylaxis is currently employed by our center. Long-term prophylaxis is needed because of the long interval of de novo HBV infection post-transplant in some patients. Further studies are necessary to define the utility and modalities of HBIG in combination with antiviral prophylaxis. The use of new-generation antiviral agents is certainly promising but requires further investigation.

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