Liver International ISSN 1478-3223
LIVER TRANSPLANTATION
Occult hepatitis B virus infection predicts de novo hepatitis B infection in patients with alcoholic cirrhosis after liver transplantation Man Xie1,2,*, Wei Rao3,4,*, Tao Yang3,4, Yonglin Deng3,4, Hong Zheng3,4, Cheng Pan3,4, Yihe Liu3,4, Zhongyang Shen3,4 and Jidong Jia1,2 1 2 3 4
Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing, 100050, China Beijing Key Laboratory of Translational Medicine in Liver Cirrhosis, Beijing, 100050, China Organ Transplantation Department, Tianjin First Central Hospital, Tianjin, 300192, China Tianjin Key Laboratory of Organ Transplantation, Tianjin, 300192, China
Keywords alcohol – cirrhosis – de novo HBV infection – hepatitis B virus infection – orthotopic liver transplantation Abbreviations ADV, adefovir dipivoxil; AN, abnormal; antiHBc, hepatitis B core antibody; anti-HBs, hepatitis B surface antibody; BW, body weight; ETV, entecavir; HCC, hepatocellular carcinoma; LdT, telbivudine; LT, liver transplantation; MDF, Maddrey’s discriminant function; MELD score, model of end-stage liver disease score; N, normal; NUCs, nucleos(t)ide analogues; OBI, occult hepatitis B infection; TAC, tacrolimus. Correspondence Jidong Jia, MD, PhD. Liver Research Center, Beijing Friendship Hospital, Capital Medical University, 95 Yong-an Road, Xi-Cheng District, Beijing 100050, China Tel: (86 10) 6313 9816 Fax: (8610) 83165944 e-mail: [email protected]
Abstract Background & Aims: Occult hepatitis B virus infection (OBI) in patients undergoing liver transplantation (LT) is a suspected source of de novo hepatitis B virus (HBV) infection after LT. This study aimed to investigate the prevalence of OBI in liver transplant recipients with alcoholic cirrhosis and demonstrate the association between OBI and de novo HBV infection after LT in these patients. Methods: Forty-three patients with alcoholic cirrhosis who were negative for HBsAg before LT were recruited in this retrospective study. DNA was extracted from paraffin-embedded native liver tissues and quantified for HBV DNA by real-time PCR. Correlation between de novo HBV infection after LT (positive HBsAg and/or detectable HBV DNA in serum) and detection of intrahepatic HBV DNA before LT was analysed. Results: Detectable HBV DNA in the explanted liver was found in 41.9% (18/43) of the patients and was thus defined as OBI, which was correlated with the presence of serum hepatitis B core antibody (P = 0.008). De novo HBV infection occurred in 18.6% (8/43) of the recipients at a median of 10 months after LT. The rate of de novo HBV infection was 38.9% (7/18) in patients with OBI, compared with 4% (1/25) in patients without OBI (P = 0.004). Furthermore, de novo HBV infection was inversely correlated with the presence of hepatitis B surface antibody in recipients with OBI (P = 0.026). Conclusion: With a prevalence of 41.9% in liver transplant recipients with alcoholic cirrhosis, OBI in the native liver can predict de novo HBV infection after LT.
Received 14 October 2013 Accepted 17 April 2014 DOI:10.1111/liv.12567 Liver Int. 2015; 35: 897–904
It is well known that the recurrence rate of hepatitis B virus (HBV) infection is almost 80% in HBV-related liver transplant recipients without antiviral prophylaxis treatment (1, 2). Currently, combinations of hepatitis B immunoglobulin (HBIG) and nucleos(t)ide analogues (NUCs), with lamivudine being the most commonly used NUCs, are considered the standard of care for prophylaxis against HBV recurrence after liver transplantation (LT) and have been shown to be effective (3).
*These authors contributed equally to this work.
Liver International (2015) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Besides HBV recurrence, de novo HBV infection is another form of HBV infection after LT. There are several possible routes of de novo HBV infection after LT, including hepatitis B core antibody (anti-HBc)-positive allografts (4), reactivation of occult HBV infection (OBI) in anti-HBc-positive recipients (5), transfusion and contact with hospital personnel (6). The past decade saw great interest in OBI, which was mainly related to the growing, widely debated evidence of OBI’s clinical impact. In general, the impact of OBI is thought to lie in several different clinical contexts, including the possible transmission of the infection
897
OBI predicts de novo HBV infection
(mainly through transfusion and LT), the risk of reactivation, and contribution to liver disease progression and development of hepatocellular carcinoma (HCC) (7–9). In fact, most studies at present concerning the clinical significance of OBI in the area of LT have mainly investigated the correlation between anti-HBc-positive donors and de novo HBV infection (10, 11). The few studies examined the correlation of the recipient’s pre-operative OBI and de novo HBV infection have found OBI in the explanted liver to be a source of de novo HBV infection after LT (5, 12–16). However, the majority of those studies were carried out in patients with hepatitis C virus (HCV)-related end-stage liver disease; data for other aetiologies in HBV-endemic areas were extremely limited. In this study, we investigated the prevalence of OBI among the liver transplant recipients with alcoholic cirrhosis and analysed the association between OBI in explanted native livers and de novo HBV infection after LT. Patients and methods Patients enrolled
Xie et al.
three patients received cyclosporine-based and one patient received sirolimus-based immunosuppressive regimen, together with corticosteroids in the first 6 months. The trough level of TAC was maintained in the range of 8–15 ng/ml during the first month after transplantation and in the range of 5–10 ng/ml thereafter. Cyclosporine dosage was calculated to obtain serum trough levels in the range of 350–500 lg/ml during the first month after transplantation and in the range of 200–350 lg/ml thereafter. The concentration of sirolimus was maintained in the range of 10–15 ng/ml during the first 3 months after transplantation and from 5 to 10 ng/ml thereafter. Corticosteroids tapering was completed within 6 months after transplantation in all patients. The conduct of our study was approved by the Ethical Affairs committee of Tianjin First Central Hospital and adhered to the tenets of the Declaration of Helsinki. Written informed consent was obtained from the patients or guardians. Quantification of intrahepatic HBV DNA
Between June 2008 and June 2012, all charts of patients undergoing LT for alcoholic cirrhosis at Tianjin First Center Hospital were retrospectively reviewed. Inclusion criteria included negative hepatitis B surface antigen (HBsAg), serum HBV DNA and hepatitis C antibody (anti-HCV) before LT and a history (longer than 10 years) of chronic alcohol intake of over 80 g per day. Exclusion criteria included lost follow-up, incomplete medical data, re-transplantation, death within 3 months of LT and donor anti-HBc positivity. All clinical data were followed up as of June 2013. The demographic data of all enrolled patients were reviewed, including age, gender, date of liver transplant, HBV serological markers in both recipients and donors before and after transplantation [including HBsAg, hepatitis B surface antibody (anti-HBs), hepatitis B e antigen (HBeAg), hepatitis B e antigen antibody (antiHBe) and anti-HBc], serum HBV DNA before and after transplantation (a value less than 100 IU/ml was considered negative; 500 IU/ml was the lowest limit of detection before 2010), model of end-stage liver disease (MELD) score and Maddrey’s discriminant function (MDF) score before transplantation, blood transfusion, level of immunosuppressant, dosage of steroids, biochemical profile and antiviral treatment after diagnosis of de novo HBV infection, and follow-up time. After transplantation, recipients were routinely tested for HBV serological and virological markers – every 6 months for the first year and every year thereafter – and observed for abnormal liver function.
Samples of recipient liver tissues were tested for occult HBV DNA according to previously reported techniques (17, 18). Briefly, DNA was extracted from freshly cut sections of formalin-fixed, paraffin-embedded explanted liver tissues, with a thickness of 5 lm and an area of almost 100 mm2, using the QIAamp DNA FFPE Tissue kit (cat. no. 56404; Qiagen, Hilden, Germany) according to the manufacturer’s protocol. An external standard curve constructed with 10-fold serial dilutions of a previously quantified plasmid carrying the HBV genome was used for quantification. The sensitivity of the assay was 100 copies/ml (dynamic range from 102 to 107 copies/ml). Real-time PCR was performed to detect the S gene and C gene. Only the case in which HBV DNA was detected using the both sets of primers was considered positive for the HBV infection. All primers and probes were synthesized by Applied Biosystems (Foster City, CA, USA) Intrahepatic HBV DNA was quantified for tissues from 10 HBsAg-positive recipients as positive controls and for 10 liver specimens from HBV-uninfected patients (HBsAg and anti-HBc negative with undetectable HBV DNA in serum by PCR) as negative controls. All specimens were run in duplicate, and negative controls and positive controls were included in each run. To quantify cell numbers, a set of primers and a probe for the human b-actin reference control DNA segment were used for real-time PCR analysis. The amount of HBV DNA in liver tissues was expressed as copies per cell.
Immunosuppression
Diagnosis of OBI
All patients were maintained on an immunosuppressive regimen that was tacrolimus (TAC)-based, except that
Occult hepatitis B virus (HBV) infection (OBI) can be defined as the long-lasting persistence of hepatitis B
898
Liver International (2015) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Xie et al.
viral genomes in the liver tissue (and in some cases also in the serum) of individuals negative for the HBV surface antigen (HBsAg) (19, 20). De novo HBV infection is defined as the detection of positive HBsAg and/or detectable serum HBV DNA in previously HBsAg-negative recipients after LT. (10) Antiviral druginduced resistance was identified by virological breakthrough after treatment with NUCs.
OBI predicts de novo HBV infection
Alcoholic cirrhosis patients admitted for LT (n = 67) Died before LT (n = 2) Patients transplanted (n = 65) Lost follow up or incomplete data (n = 11)
Died in 3 months after LT or Re-transplantation (n = 9)
Anti-HBc positive donor (n = 2) Patients analyzed (n = 43)
Statistical analysis
Data are presented as mean ± standard deviation (SD) or as median and range, as appropriate. Independent t tests or Mann–Whitney rank sum tests were used for continuous variables. Categorical variables were compared using the chi-square test. Kaplan–Meier method was used for actuarial recurrence analysis. All statistical analyses were performed using SPSS version 17.0 (SPSS, Inc., Chicago, IL, USA). A P-value less than 0.05 was considered statistically significant. Results Demographics and baseline characteristics
Between June 2008 and June 2012, 67 patients were admitted to our centre for liver transplantation because of alcoholic cirrhosis-related complications. Sixty-five of the patients were survivors before surgery and were recruited into our study. Among these patients, four lacked adequate clinical data, seven were lost to followup, three received a re-transplant, six died because of fatal complications unrelated to HBV within 3 months after LT, and two had anti-HBc-positive donors, resulting in these individuals being excluded from the study, leaving 43 eligible patients in total. A flow chart summary of the patients enrolled is presented in Fig. 1. The average age of the patients was 51.1 ± 8.8 years, and 95.3% (41/43) of them were male. The average model of end-stage liver disease (MELD) score and Maddrey’s discriminant function (MDF) before LT were 18 ± 7 and 42 ± 29 respectively. Before LT, 81% (35/ 43) of the recipients were anti-HBc positive, while 63% (27/43) were anti-HBs positive. Prevalence and characteristics of OBI in LT recipients with alcoholic cirrhosis
Of the total 43 patients analysed, intrahepatic HBV DNA was detectable in 18 (41.9%) explanted livers with a median HBV DNA level of 0.12 copies per cell (range, 0.01–271.45 copies per cell); the corresponding patients were assigned to the OBI group. The remaining 25 patients were assigned to the OBI-free group (Table 1). The median intrahepatic HBV DNA level of the positive control group was 172.9 copies per cell (range, 20.0– 1444.64 copies per cell), which was significantly higher than that of the alcohol-related recipients (P < 0.01). Liver International (2015) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Explanted liver HBV DNA positive (n = 18)
Explanted liver HBV DNA negative (n = 25)
De novo hepatitis B infection (n = 7)
De novo hepatitis B infection (n = 1)
Fig. 1. Flow chart of patients enrolment. Table 1. Demographics and clinical characteristics of patients with and without OBI Characteristics of recipients
OBI-free group (n = 25)
OBI group (n = 18)
P-value
Age (year) Gender (F/M) MELD score MDF score Anti-HBs (pos/neg) Anti-HBc (pos/neg) Anti-HBs/Anti-HBc (pos/neg) HCC De novo HBV infection Follow-up (month) median (range)
51.2 ± 9.5 1/24 17 ± 6 37.2 ± 21.6 14/11 17/8 11/14
51.1 ± 8.2 1/17 20 ± 9 49.1 ± 35.6 13/5 18/0 13/5
0.49 0.81 0.15 0.09 0.28 0.008 0.065
3/21 1/25 22 (6–60)
2/16 7/18 24 (5–51)
0.928 0.004 0.834
Bold values denote statistical significance.
None of the patients in the negative control group was found to be HBV DNA positive. Several additional clinical factors that may affect the prevalence of OBI in alcohol-related liver transplant recipients were analysed, including age, gender, MELD score, MDF, HCC status, and presence of anti-HBs, anti-HBc, or both (anti-HBs/anti-HBc). No significant difference was observed between the OBI group and the OBI-free group in terms of age, gender, MELD score, MDF, HCC status, presence of anti-HBs and presence of anti-HBs/anti-HBc, but the proportion of patients positive for anti-HBc was significantly higher in the OBI group than in the OBI-free group (P = 0.008) (Table 1). Correlation of pre-operation OBI and de novo HBV infection after LT
In our study, the incidence of de novo HBV infection was 18.6% (8/43) in a median 23 months (range, 5–60 months) after LT. Seven of the patients who developed de novo HBV infection belonged to the OBI group
899
OBI predicts de novo HBV infection
Xie et al.
statistically significant difference was observed between the two groups in gender (P = 0.412) and age (P = 0.273). Risk factors that may have affected de novo HBV infection were analysed, including MELD score, MDF, presence of anti-HBs, presence of anti-HBc, total steroid dosage after surgery, trough level of immunosuppressant, amount of blood transfusion and level of intrahepatic HBV DNA. No significant difference was observed between the two groups except in the presence of anti-HBs (P = 0.026) (Table 2). Characteristics of patients with de novo HBV infection
Fig. 2. Actuarial recurrence analyses of occult hepatitis B infection (OBI) group and OBI-free group.
(n = 18), compared with one in the OBI-free group (n = 25). The prevalence of de novo HBV infection in patients with OBI was significantly higher than that in patients without OBI (7/18 vs. 1/25, P = 0.004) (Table 1). Actuarial recurrence analysis of OBI group and OBI-free group was presented in Fig. 2. Risk factors of de novo HBV infection among alcoholrelated liver transplant recipients with OBI
Patients with OBI, all of whom were anti-HBc positive before LT, were divided into a de novo HBV group (n = 7) and a de novo HBV–free group (n = 11) according to their status of HBV infection after LT. No
The clinical characteristics of the eight patients with de novo HBV infection are presented in Table 3. Before LT, positive anti-HBc was observed in all eight patients, and three of them were anti-HBs positive. When de novo HBV infection was diagnosed, which occurred at a median of 10 months after LT (range, 4–20 months), all eight patients were positive for serum HBsAg and HBV DNA, and abnormal liver enzymes were observed in five of them. All eight patients received antiviral treatment with NUCs. Entecavir (ETV), telbivudine (LdT) and adefovir dipivoxil (ADV) were prescribed to five, two and one patients respectively. HBV resistance mutations were observed in two patients: one was ETV-treated and the other was LdT-treated previously. ADV was added for the ETV-treated patient, and ETV was prescribed with LdT discontinued for the LdT-treated patient. A strongly antiviral suppression effect was observed after drug adjustment. No fatal complication such as liver failure was observed. All eight grafts were surviving at the end of follow-up (median, 32 months; Table 3). Two of the eight patients had liver biopsy before antiviral treatment and showed HBsAg-positive hepatocytes with a lobular hepatitis feature.
Table 2. Comparison of patients with and without de novo HBV after LT among patients with OBI Characteristics Age (year) Gender (F/M) Pre-op MELD score Pre-op MDF score Anti-HBs (pos/neg) Anti-HBc (pos/neg) RBC transfusion (ml) Median (Range) Plasma transfusion (ml) Median (Range) Intrahepatic HBV-DNA (copies/cell) Median (Range) Trough level of TAC (ng/ml) Steroid during LT/BW (mg/kg) Period of follow-up (month) Median (Range)
De novo HBV-free group (n = 11)
De novo HBV group (n = 7)
P-value
50.1 ± 7.8 1/10 22 ± 9 53 ± 38 10/1 11/0 3200 (1200–6400)
52.6 ± 9.3 0/7 17 ± 9. 44 ± 34 3/4 7/0 2200 (1600–5600)
0.273 0.412 0.157 0.306 0.026 1.000 0.716
2000 (1800–4800)
2000 (1000–3200)
0.673
0.14 (0.01–271.43)
0.02 (0.01–65.23)
1.000
6.6 ± 1.9 42 ± 6 16 (5–51)
6.4 ± 1.6* 40 ± 6 29 (6–43)
0.410 0.289 0.163
Bold values denote statistical significance. *One patient received sirolimus-based immunosuppressive regimen.
900
Liver International (2015) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
1.14E+02 4.56E+02 2.16E+02
LIVER TRANSPLANTATION
Occult hepatitis B virus infection predicts de novo hepatitis B infection in patients with alcoholic cirrhosis after liver transplantation Man Xie1,2,*, Wei Rao3,4,*, Tao Yang3,4, Yonglin Deng3,4, Hong Zheng3,4, Cheng Pan3,4, Yihe Liu3,4, Zhongyang Shen3,4 and Jidong Jia1,2 1 2 3 4
Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing, 100050, China Beijing Key Laboratory of Translational Medicine in Liver Cirrhosis, Beijing, 100050, China Organ Transplantation Department, Tianjin First Central Hospital, Tianjin, 300192, China Tianjin Key Laboratory of Organ Transplantation, Tianjin, 300192, China
Keywords alcohol – cirrhosis – de novo HBV infection – hepatitis B virus infection – orthotopic liver transplantation Abbreviations ADV, adefovir dipivoxil; AN, abnormal; antiHBc, hepatitis B core antibody; anti-HBs, hepatitis B surface antibody; BW, body weight; ETV, entecavir; HCC, hepatocellular carcinoma; LdT, telbivudine; LT, liver transplantation; MDF, Maddrey’s discriminant function; MELD score, model of end-stage liver disease score; N, normal; NUCs, nucleos(t)ide analogues; OBI, occult hepatitis B infection; TAC, tacrolimus. Correspondence Jidong Jia, MD, PhD. Liver Research Center, Beijing Friendship Hospital, Capital Medical University, 95 Yong-an Road, Xi-Cheng District, Beijing 100050, China Tel: (86 10) 6313 9816 Fax: (8610) 83165944 e-mail: [email protected]
Abstract Background & Aims: Occult hepatitis B virus infection (OBI) in patients undergoing liver transplantation (LT) is a suspected source of de novo hepatitis B virus (HBV) infection after LT. This study aimed to investigate the prevalence of OBI in liver transplant recipients with alcoholic cirrhosis and demonstrate the association between OBI and de novo HBV infection after LT in these patients. Methods: Forty-three patients with alcoholic cirrhosis who were negative for HBsAg before LT were recruited in this retrospective study. DNA was extracted from paraffin-embedded native liver tissues and quantified for HBV DNA by real-time PCR. Correlation between de novo HBV infection after LT (positive HBsAg and/or detectable HBV DNA in serum) and detection of intrahepatic HBV DNA before LT was analysed. Results: Detectable HBV DNA in the explanted liver was found in 41.9% (18/43) of the patients and was thus defined as OBI, which was correlated with the presence of serum hepatitis B core antibody (P = 0.008). De novo HBV infection occurred in 18.6% (8/43) of the recipients at a median of 10 months after LT. The rate of de novo HBV infection was 38.9% (7/18) in patients with OBI, compared with 4% (1/25) in patients without OBI (P = 0.004). Furthermore, de novo HBV infection was inversely correlated with the presence of hepatitis B surface antibody in recipients with OBI (P = 0.026). Conclusion: With a prevalence of 41.9% in liver transplant recipients with alcoholic cirrhosis, OBI in the native liver can predict de novo HBV infection after LT.
Received 14 October 2013 Accepted 17 April 2014 DOI:10.1111/liv.12567 Liver Int. 2015; 35: 897–904
It is well known that the recurrence rate of hepatitis B virus (HBV) infection is almost 80% in HBV-related liver transplant recipients without antiviral prophylaxis treatment (1, 2). Currently, combinations of hepatitis B immunoglobulin (HBIG) and nucleos(t)ide analogues (NUCs), with lamivudine being the most commonly used NUCs, are considered the standard of care for prophylaxis against HBV recurrence after liver transplantation (LT) and have been shown to be effective (3).
*These authors contributed equally to this work.
Liver International (2015) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Besides HBV recurrence, de novo HBV infection is another form of HBV infection after LT. There are several possible routes of de novo HBV infection after LT, including hepatitis B core antibody (anti-HBc)-positive allografts (4), reactivation of occult HBV infection (OBI) in anti-HBc-positive recipients (5), transfusion and contact with hospital personnel (6). The past decade saw great interest in OBI, which was mainly related to the growing, widely debated evidence of OBI’s clinical impact. In general, the impact of OBI is thought to lie in several different clinical contexts, including the possible transmission of the infection
897
OBI predicts de novo HBV infection
(mainly through transfusion and LT), the risk of reactivation, and contribution to liver disease progression and development of hepatocellular carcinoma (HCC) (7–9). In fact, most studies at present concerning the clinical significance of OBI in the area of LT have mainly investigated the correlation between anti-HBc-positive donors and de novo HBV infection (10, 11). The few studies examined the correlation of the recipient’s pre-operative OBI and de novo HBV infection have found OBI in the explanted liver to be a source of de novo HBV infection after LT (5, 12–16). However, the majority of those studies were carried out in patients with hepatitis C virus (HCV)-related end-stage liver disease; data for other aetiologies in HBV-endemic areas were extremely limited. In this study, we investigated the prevalence of OBI among the liver transplant recipients with alcoholic cirrhosis and analysed the association between OBI in explanted native livers and de novo HBV infection after LT. Patients and methods Patients enrolled
Xie et al.
three patients received cyclosporine-based and one patient received sirolimus-based immunosuppressive regimen, together with corticosteroids in the first 6 months. The trough level of TAC was maintained in the range of 8–15 ng/ml during the first month after transplantation and in the range of 5–10 ng/ml thereafter. Cyclosporine dosage was calculated to obtain serum trough levels in the range of 350–500 lg/ml during the first month after transplantation and in the range of 200–350 lg/ml thereafter. The concentration of sirolimus was maintained in the range of 10–15 ng/ml during the first 3 months after transplantation and from 5 to 10 ng/ml thereafter. Corticosteroids tapering was completed within 6 months after transplantation in all patients. The conduct of our study was approved by the Ethical Affairs committee of Tianjin First Central Hospital and adhered to the tenets of the Declaration of Helsinki. Written informed consent was obtained from the patients or guardians. Quantification of intrahepatic HBV DNA
Between June 2008 and June 2012, all charts of patients undergoing LT for alcoholic cirrhosis at Tianjin First Center Hospital were retrospectively reviewed. Inclusion criteria included negative hepatitis B surface antigen (HBsAg), serum HBV DNA and hepatitis C antibody (anti-HCV) before LT and a history (longer than 10 years) of chronic alcohol intake of over 80 g per day. Exclusion criteria included lost follow-up, incomplete medical data, re-transplantation, death within 3 months of LT and donor anti-HBc positivity. All clinical data were followed up as of June 2013. The demographic data of all enrolled patients were reviewed, including age, gender, date of liver transplant, HBV serological markers in both recipients and donors before and after transplantation [including HBsAg, hepatitis B surface antibody (anti-HBs), hepatitis B e antigen (HBeAg), hepatitis B e antigen antibody (antiHBe) and anti-HBc], serum HBV DNA before and after transplantation (a value less than 100 IU/ml was considered negative; 500 IU/ml was the lowest limit of detection before 2010), model of end-stage liver disease (MELD) score and Maddrey’s discriminant function (MDF) score before transplantation, blood transfusion, level of immunosuppressant, dosage of steroids, biochemical profile and antiviral treatment after diagnosis of de novo HBV infection, and follow-up time. After transplantation, recipients were routinely tested for HBV serological and virological markers – every 6 months for the first year and every year thereafter – and observed for abnormal liver function.
Samples of recipient liver tissues were tested for occult HBV DNA according to previously reported techniques (17, 18). Briefly, DNA was extracted from freshly cut sections of formalin-fixed, paraffin-embedded explanted liver tissues, with a thickness of 5 lm and an area of almost 100 mm2, using the QIAamp DNA FFPE Tissue kit (cat. no. 56404; Qiagen, Hilden, Germany) according to the manufacturer’s protocol. An external standard curve constructed with 10-fold serial dilutions of a previously quantified plasmid carrying the HBV genome was used for quantification. The sensitivity of the assay was 100 copies/ml (dynamic range from 102 to 107 copies/ml). Real-time PCR was performed to detect the S gene and C gene. Only the case in which HBV DNA was detected using the both sets of primers was considered positive for the HBV infection. All primers and probes were synthesized by Applied Biosystems (Foster City, CA, USA) Intrahepatic HBV DNA was quantified for tissues from 10 HBsAg-positive recipients as positive controls and for 10 liver specimens from HBV-uninfected patients (HBsAg and anti-HBc negative with undetectable HBV DNA in serum by PCR) as negative controls. All specimens were run in duplicate, and negative controls and positive controls were included in each run. To quantify cell numbers, a set of primers and a probe for the human b-actin reference control DNA segment were used for real-time PCR analysis. The amount of HBV DNA in liver tissues was expressed as copies per cell.
Immunosuppression
Diagnosis of OBI
All patients were maintained on an immunosuppressive regimen that was tacrolimus (TAC)-based, except that
Occult hepatitis B virus (HBV) infection (OBI) can be defined as the long-lasting persistence of hepatitis B
898
Liver International (2015) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Xie et al.
viral genomes in the liver tissue (and in some cases also in the serum) of individuals negative for the HBV surface antigen (HBsAg) (19, 20). De novo HBV infection is defined as the detection of positive HBsAg and/or detectable serum HBV DNA in previously HBsAg-negative recipients after LT. (10) Antiviral druginduced resistance was identified by virological breakthrough after treatment with NUCs.
OBI predicts de novo HBV infection
Alcoholic cirrhosis patients admitted for LT (n = 67) Died before LT (n = 2) Patients transplanted (n = 65) Lost follow up or incomplete data (n = 11)
Died in 3 months after LT or Re-transplantation (n = 9)
Anti-HBc positive donor (n = 2) Patients analyzed (n = 43)
Statistical analysis
Data are presented as mean ± standard deviation (SD) or as median and range, as appropriate. Independent t tests or Mann–Whitney rank sum tests were used for continuous variables. Categorical variables were compared using the chi-square test. Kaplan–Meier method was used for actuarial recurrence analysis. All statistical analyses were performed using SPSS version 17.0 (SPSS, Inc., Chicago, IL, USA). A P-value less than 0.05 was considered statistically significant. Results Demographics and baseline characteristics
Between June 2008 and June 2012, 67 patients were admitted to our centre for liver transplantation because of alcoholic cirrhosis-related complications. Sixty-five of the patients were survivors before surgery and were recruited into our study. Among these patients, four lacked adequate clinical data, seven were lost to followup, three received a re-transplant, six died because of fatal complications unrelated to HBV within 3 months after LT, and two had anti-HBc-positive donors, resulting in these individuals being excluded from the study, leaving 43 eligible patients in total. A flow chart summary of the patients enrolled is presented in Fig. 1. The average age of the patients was 51.1 ± 8.8 years, and 95.3% (41/43) of them were male. The average model of end-stage liver disease (MELD) score and Maddrey’s discriminant function (MDF) before LT were 18 ± 7 and 42 ± 29 respectively. Before LT, 81% (35/ 43) of the recipients were anti-HBc positive, while 63% (27/43) were anti-HBs positive. Prevalence and characteristics of OBI in LT recipients with alcoholic cirrhosis
Of the total 43 patients analysed, intrahepatic HBV DNA was detectable in 18 (41.9%) explanted livers with a median HBV DNA level of 0.12 copies per cell (range, 0.01–271.45 copies per cell); the corresponding patients were assigned to the OBI group. The remaining 25 patients were assigned to the OBI-free group (Table 1). The median intrahepatic HBV DNA level of the positive control group was 172.9 copies per cell (range, 20.0– 1444.64 copies per cell), which was significantly higher than that of the alcohol-related recipients (P < 0.01). Liver International (2015) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Explanted liver HBV DNA positive (n = 18)
Explanted liver HBV DNA negative (n = 25)
De novo hepatitis B infection (n = 7)
De novo hepatitis B infection (n = 1)
Fig. 1. Flow chart of patients enrolment. Table 1. Demographics and clinical characteristics of patients with and without OBI Characteristics of recipients
OBI-free group (n = 25)
OBI group (n = 18)
P-value
Age (year) Gender (F/M) MELD score MDF score Anti-HBs (pos/neg) Anti-HBc (pos/neg) Anti-HBs/Anti-HBc (pos/neg) HCC De novo HBV infection Follow-up (month) median (range)
51.2 ± 9.5 1/24 17 ± 6 37.2 ± 21.6 14/11 17/8 11/14
51.1 ± 8.2 1/17 20 ± 9 49.1 ± 35.6 13/5 18/0 13/5
0.49 0.81 0.15 0.09 0.28 0.008 0.065
3/21 1/25 22 (6–60)
2/16 7/18 24 (5–51)
0.928 0.004 0.834
Bold values denote statistical significance.
None of the patients in the negative control group was found to be HBV DNA positive. Several additional clinical factors that may affect the prevalence of OBI in alcohol-related liver transplant recipients were analysed, including age, gender, MELD score, MDF, HCC status, and presence of anti-HBs, anti-HBc, or both (anti-HBs/anti-HBc). No significant difference was observed between the OBI group and the OBI-free group in terms of age, gender, MELD score, MDF, HCC status, presence of anti-HBs and presence of anti-HBs/anti-HBc, but the proportion of patients positive for anti-HBc was significantly higher in the OBI group than in the OBI-free group (P = 0.008) (Table 1). Correlation of pre-operation OBI and de novo HBV infection after LT
In our study, the incidence of de novo HBV infection was 18.6% (8/43) in a median 23 months (range, 5–60 months) after LT. Seven of the patients who developed de novo HBV infection belonged to the OBI group
899
OBI predicts de novo HBV infection
Xie et al.
statistically significant difference was observed between the two groups in gender (P = 0.412) and age (P = 0.273). Risk factors that may have affected de novo HBV infection were analysed, including MELD score, MDF, presence of anti-HBs, presence of anti-HBc, total steroid dosage after surgery, trough level of immunosuppressant, amount of blood transfusion and level of intrahepatic HBV DNA. No significant difference was observed between the two groups except in the presence of anti-HBs (P = 0.026) (Table 2). Characteristics of patients with de novo HBV infection
Fig. 2. Actuarial recurrence analyses of occult hepatitis B infection (OBI) group and OBI-free group.
(n = 18), compared with one in the OBI-free group (n = 25). The prevalence of de novo HBV infection in patients with OBI was significantly higher than that in patients without OBI (7/18 vs. 1/25, P = 0.004) (Table 1). Actuarial recurrence analysis of OBI group and OBI-free group was presented in Fig. 2. Risk factors of de novo HBV infection among alcoholrelated liver transplant recipients with OBI
Patients with OBI, all of whom were anti-HBc positive before LT, were divided into a de novo HBV group (n = 7) and a de novo HBV–free group (n = 11) according to their status of HBV infection after LT. No
The clinical characteristics of the eight patients with de novo HBV infection are presented in Table 3. Before LT, positive anti-HBc was observed in all eight patients, and three of them were anti-HBs positive. When de novo HBV infection was diagnosed, which occurred at a median of 10 months after LT (range, 4–20 months), all eight patients were positive for serum HBsAg and HBV DNA, and abnormal liver enzymes were observed in five of them. All eight patients received antiviral treatment with NUCs. Entecavir (ETV), telbivudine (LdT) and adefovir dipivoxil (ADV) were prescribed to five, two and one patients respectively. HBV resistance mutations were observed in two patients: one was ETV-treated and the other was LdT-treated previously. ADV was added for the ETV-treated patient, and ETV was prescribed with LdT discontinued for the LdT-treated patient. A strongly antiviral suppression effect was observed after drug adjustment. No fatal complication such as liver failure was observed. All eight grafts were surviving at the end of follow-up (median, 32 months; Table 3). Two of the eight patients had liver biopsy before antiviral treatment and showed HBsAg-positive hepatocytes with a lobular hepatitis feature.
Table 2. Comparison of patients with and without de novo HBV after LT among patients with OBI Characteristics Age (year) Gender (F/M) Pre-op MELD score Pre-op MDF score Anti-HBs (pos/neg) Anti-HBc (pos/neg) RBC transfusion (ml) Median (Range) Plasma transfusion (ml) Median (Range) Intrahepatic HBV-DNA (copies/cell) Median (Range) Trough level of TAC (ng/ml) Steroid during LT/BW (mg/kg) Period of follow-up (month) Median (Range)
De novo HBV-free group (n = 11)
De novo HBV group (n = 7)
P-value
50.1 ± 7.8 1/10 22 ± 9 53 ± 38 10/1 11/0 3200 (1200–6400)
52.6 ± 9.3 0/7 17 ± 9. 44 ± 34 3/4 7/0 2200 (1600–5600)
0.273 0.412 0.157 0.306 0.026 1.000 0.716
2000 (1800–4800)
2000 (1000–3200)
0.673
0.14 (0.01–271.43)
0.02 (0.01–65.23)
1.000
6.6 ± 1.9 42 ± 6 16 (5–51)
6.4 ± 1.6* 40 ± 6 29 (6–43)
0.410 0.289 0.163
Bold values denote statistical significance. *One patient received sirolimus-based immunosuppressive regimen.
900
Liver International (2015) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
1.14E+02 4.56E+02 2.16E+02
