Natural History of Patients with Recurrent Chronic Hepatitis C Virus and Occult Hepatitis B Co-Infection after Liver Transplantation

Authors


* Corresponding author: C.-K. Hui, ckhui23@gmail.com

Abstract

It is uncertain whether occult hepatitis B virus co-infection will hasten progressive liver disease in chronic hepatitis C patients after liver transplantation. This study evaluated fibrosis progression and severe fibrosis in 118 consecutive hepatitis B surface antigen-negative patients with virological and histological evidence of recurrent chronic hepatitis C infection co-infected with occult hepatitis B virus after liver transplantation. HBV DNA was detected from serum at the time of recurrent chronic hepatitis C infection by polymerase chain reaction. Each subject underwent a repeat liver biopsy 5 years post-liver transplantation. Occult hepatitis B virus co-infection was present in 41 of the 118 (34.7%) patients. At 5 years post-liver transplantation, 13 of the 41 occult hepatitis B virus co-infected patients compared with 16 of the 77 patients without occult hepatitis B virus co-infection developed fibrosis progression (31.7% vs. 20.8%, respectively, p = 0.39). Eight of 41 the occult hepatitis B virus co-infected patients compared with 13 of the 77 patients without occult hepatitis B virus co-infection had severe fibrosis (19.5% vs. 16.9%, respectively, p = 0.97). In conclusion, occult hepatitis B virus co-infection in patients with recurrent chronic hepatitis C infection was not associated with accelerated fibrosis progression or severe fibrosis after liver transplantation.

Introduction

Hepatitis B virus (HBV) infection is a major health issue globally. It is estimated that more than 350 million people worldwide and 1.25 million people in the United States are affected by HBV infection (1,2). The spectrum of HBV-related disease ranges from acute hepatitis B, asymptomatic HBV infection, chronic hepatitis to fulminant hepatitis (3,4). Chronic HBV infection is also associated with cirrhosis and hepatocellular carcinoma (HCC).

Chronic HBV infection is characterized by persistence of hepatitis surface antigen (HBsAg) and viremia (3,4). Earlier studies have shown that clearance of HBsAg in patients with HBV infection is associated with disappearance of viremia and remission of disease. However, in recent years data have indicated that a low level of HBV DNA remains detectable in serum and liver tissue in some patients after clearance of HBsAg from acute self-limited or chronic HBV infection, or even after successful anti-HBV treatment (5). Demonstration of this clinical entity has introduced the concept of 'occult' HBV infection, which is defined as the presence of HBV DNA in patients with undetectable HBsAg.

Although there have been extensive studies on occult HBV infection in immunocompetent patients, the precise prevalence of this clinical entity remains difficult to define due to several reasons (6–10). Firstly, occult HBV infection can manifest in a range of clinical scenarios, ranging from convalescence of acute self-limited hepatitis B, chronic asymptomatic carrier status to delayed HBsAg clearance complicated by cirrhosis or HCC. Secondly, most studies have been limited by small sample size, heterogeneous population groups or lack of a control group. Thirdly, although HBV DNA polymerase chain reaction (PCR) is the most sensitive test currently available for detecting HBV DNA molecules, its sensitivity is variable. Indeed, a recent study reported a high rate (10/19) of false positive HBV DNA by PCR in patients with hepatitis B core antibody (anti-HBc) positivity alone (6). Thus, a highly specific and sensitive HBV PCR amplification assay is essential for determining the precise prevalence of occult HBV infection. Finally, it has been well demonstrated that the prevalence of occult HBV infection is dependent on the time interval from HBsAg clearance to HBV DNA assessment. A longer time interval is associated with a lower prevalence of occult HBV infection. All these factors should be taken into consideration when determining the prevalence of occult HBV infection.

Occult HBV infection is frequently found in patients with chronic hepatitis C virus (HCV) infection, probably because HBV and HCV share the same route of transmission (7–10). Although liver disease has been reported to be more severe in patients with concurrent positive serology for both HBsAg (chronic hepatitis B infection) and anti-HCV than in patients with positive anti-HCV alone, the clinical relevance of occult HBV in patients with chronic HCV infection is still being assessed (11–15). With the use of sensitive real time PCR technique, an important observation in patients with occult HBV infection is the detection of a low HBV DNA level of 10–100 copies/mL in the serum (16,17). In the vast majority of patients with occult HBV infection, this low threshold level of HBV DNA (∼10–100 copies/mL) may not be sufficient to cause progressive liver disease in immunocompetent chronic HCV patients, as demonstrated in recent studies (16,18,19). On the contrary, a low-level HBV infection can reactivate in the immunocompromised individual, resulting in profound liver injury and liver failure (20–22).

Whether occult HBV infection will hasten progressive liver disease in chronic HCV patients after liver transplantation is uncertain. We, therefore, measured the HBV DNA extracted from the serum of patients undergoing liver transplantation for chronic HCV related liver complications. The goal of this study was to longitudinally compare fibrosis progression and progression to severe fibrosis on serial liver biopsies in patients with recurrent chronic HCV infection and occult HBV co-infection after liver transplantation.

Patients and Methods

A total of 231 consecutive patients undergoing orthotopic liver transplantation for chronic HCV infection related complications at the University of California, San Francisco, San Francisco, USA, from 1988 to 1996 were retrospectively identified from a computerized database and included into this study if they satisfied the following criteria: (i) HCV RNA positive after liver transplantation; (ii) patient had changes consistent with chronic hepatitis due to HCV infection according to the previously published criteria on liver biopsies within the first year after transplantation (23,24); (iii) patient and donor were HBsAg negative before liver transplantation; (iv) patient was HBsAg negative at the time recurrent chronic HCV infection after liver transplantation was diagnosed and (v) positive for anti-HCV by second generation enzyme-linked immunosorbent assay (ELISA) (Ortho Diagnostics System, Raritan, NJ, USA) before and after liver transplantation. Patients with the following were excluded from the study: (i) HBsAg co-infection (HBsAg positive) before liver transplantation; (ii) other chronic liver disease (alcoholic liver disease, hepatotoxic drugs, autoimmune chronic hepatitis, hemochromatosis); (iii) patients with an alcohol intensity of more than 20 gm/day post-liver transplantation and (iv) anti-HIV positive.

HBsAg, hepatitis B surface antibody (anti-HBs) and anti-HBc were tested with commercially available radioimmunoassay kits (Abbott Laboratories, Chicago, IL, USA). HCV genotyping was performed using INNO-LiPA (Innogenetics, Ghent, Belgium). HCV RNA was quantified by reverse-transcription PCR (Amplicor HCV Monitor Test 2.0, Roche Diagnostics, Branchburg, NJ). Donors and patients before 1993 were retrospectively tested for anti-HCV, HCV RNA and HCV genotype using stored serum samples. Anti-HBs negative patients were all vaccinated with HBV vaccine (Engerix-B, GlaxoSmithKline, Research Triangle Park, NC, USA) before liver transplantation. None of the patients receiving anti-HBc positive liver received hepatitis B immunoglobulin.

A percutaneous liver biopsy was performed in accordance with transplantation protocol and when clinically indicated. In addition to the transplantation protocol, a percutaneous liver biopsy would also be performed in patients with an elevated serum alanine aminotransaminase levels to more than 2 times the upper limit of normal (normal range 13–51 U/L). The liver biopsy was paraffin-embedded and stained with hematoxylin-eosin safran and Masson's trichrome and graded according to the METAVIR score by a staff pathologist with no prior knowledge of HBV serology or occult HBV results (25,26).

After liver transplantation, patients were followed-up every 2–4 weeks in the outpatient clinic for the first 3 months, and every 2–4 months thereafter until 5 years after liver transplantation (end of follow-up). A liver biopsy would be repeated at the end of follow-up to assess fibrosis progression. At every follow-up visit, clinical manifestations or complications were noted. Liver function tests, HBV serology and HCV RNA were routinely performed. Those who were started on anti-viral therapy for the treatment of recurrent chronic HCV infection were discontinued from the study and their last date of follow-up and liver biopsy before commencement of anti-viral was included into the analysis. Patients who developed relapsed infection due to HBV (HBsAg seroreversion) after transplantation were also discontinued from the study and the last liver biopsy before HBsAg seroreversion was detected was included into the analysis.

HBV DNA analyses

The archived sera collected at the time recurrent chronic HCV infection was diagnosed after liver transplantation was analyzed for HBV DNA. Although some of the serum samples had been stored for more than 10 years, it has been previously shown that storage of serum at −70°C can preserve the integrity of HCV RNA and HBV DNA (27–29). Our archived sera had been stored in accordance with these recommendations/guidelines.

HBV DNA was extracted from the serum by phenol/chloroform extraction as previously described (30). Guidelines to minimize the risk of contamination were closely observed (31,32). Three pairs of primers specific for Surface (S), Core and X genes of the HBV were used (Table 1) for detection of HBV DNA by PCR. Amplification was performed for 35 cycles, each consisting of denaturing for 30 s at 94°C, annealing for 45 s at 56°C and extension for 1.5 min at 72°C. The amplification products were electrophoresed on an ethidium bromide stained 1.5% agarose gel.

Table 1.  Primers and probes used for detection of HBV DNA by PCR
Primer set delegationNucleotide sequencePosition
S regionPrimer 
 [+]5′ TCT TCT TGT TGG TTC TTC TGG ACT ATC AAG 3′1791–1820
 [−]5′ CTG AAG AAA TGG CAC TAG TAA ACT GAG CCA 3′2059–2030
Probe 
 [+]5′ CGG AAA TTG CAC CTG TAT TCC CAT CCC ATC 3′1948–1977
Core region 
Core regionPrimer 
 [+]5′ TTT GGT GTC TTT CGG AGT GTG GAT TCG CAC 3′434–463
 [−]5′ AGA TTC CCG AGA TTG AGA TCT TCT GCG ACG 3′623–594
Probe 
 [+]5′ GAC CAC CAA ATG CCC CTA TCC TAT CAA CAC 3′478–507
X regionPrimer 
 [+]5′ ATG GCT GCT AGG CTG TGC TGC CAA CTG GAT 3′1522–1557
 [−]5′ CAA GAG TCC TCT TAT GTA AGA CCT TGG GCA 3′1816–1787
Probe 
 [+]5′ TGC ACT TCG CTT CAC CTC TGC ACG TCG CAT 3′1728–1757

The gel was then denatured in 0.25 N hydrochloric acid (HCL) for 15 min and denatured in 0.5 N sodium hydroxide (NaOH) for 30 min. The gel was then vacuum transferred (Model 785 Vacuum Blotter, BIO-RAD, Hercules, CA, USA) to a nylon membrane (Zeta-Probe GT, BIO-RAD) in 10 × SSC for 90 min at 5 inches Hg. The nylon membranes/filters were pre-hybridized in 5 × SSC (BIO-RAD), 0.02% SDS, 0.1% N-lauroylsarcosine and 1% blocking solution (Roche Diagnostics, Branchburg, NJ, USA) for 4 h at 55°C. After pre-hybridization, the filters were hybridized at 55°C for 16 h using three specific 30-mer oligonucleotide probes located between the amplification primers (Table 1) and 3′ end tailed with digoxigenin-dUTP (Roche Diagnostics).

The membranes were washed twice in 2 × SSC, 0.1% SDS for 15 min in room temperature and in 0.5 × SSC, 0.1% SDS at 55°C for 1 h and incubated for 30 min in 100 mL blocking solution for 30 min and in 1:10 000 anti-digoxigenin-alkaline phosphatase Fab fragments for another 30 min. The membranes were finally equilibrated in detection buffer and soaked in 1:100 CSPD and autoradiographed.

The sensitivity of our PCR assay reached 50 copies/mL as determined by serial 10-fold dilutions of cloned HBV DNA with known amount (108 copies/mL) as previously described (16). Stored serum samples collected from 1988 to 1996 from 10 anti-HCV positive and HBsAg positive patients were included as positive controls. Ten negative controls consisting of controls without nucleic acids but with the complete reaction mixture were included in the assay. In the 10 HBsAg and anti-HCV positive controls, all specimens were positive for S, Core and X genes. There was no difference in sensitivity between S, Core and X gene by PCR in these 10 HBsAg and anti-HCV positive controls although their serum was stored for a period of more than 10 years. None of the 10 negative controls were positive for S, Core or X gene PCR.

Patients were considered to be occult HBV co-infected when at least two of the three PCR assays were reactive (16,17). If only one set of primers detected HBV DNA, the result was considered negative. An investigator who was blinded to the patients' baseline characteristics, HBV serology, stage of liver biopsy and clinical outcome performed the assay and interpreted the results.

Quantification of serum HBV DNA with the TaqMan HBV test

The amount of HBV DNA in the sera of patients with occult HBV co-infection was quantified by real time PCR using upstream and downstream primers targeted to the pre-core/core region with the TaqMan HBV assay (Roche Diagnostics) in accordance with the manufacturer's instructions. The lower limit of detection of this assay was 50 copies/mL.

Study endpoint

The primary endpoint of this study was to determine whether occult HBV co-infection in patients with recurrent chronic HCV infection after liver transplantation would result in a faster rate of fibrosis progression (defined as an increase in fibrosis stage by at least 2-point) or severe fibrosis (defined as stage 3 or 4 fibrosis) on liver biopsy 5 years after liver transplantation (end of follow-up). The rate of liver fibrosis progression was also determined as described previously (26). Briefly, the fibrosis progression rate per year was calculated as the ratio between the difference in fibrosis stage (from the first and final liver biopsy) and the time interval in years between the first and final liver biopsy in years. The secondary endpoint was to determine the occurrence of relapsed infection due to HBV (defined as HBsAg reverting from negative to positive in a patient who was HBsAg negative before liver transplantation) after liver transplantation.

This study was approved by the local Institutional Review Board.

Statistical analysis

All statistical analyses were performed using the Statistical Program for Social Sciences (SPSS 11.0 for windows, SPSS Inc., Chicago, IL, USA). The Mann-Whitney U-test was used for continuous variables with skewed distribution and the chi-square with Yates' correction for continuity or Fisher's exact test for categorical variables. Cumulative probability of fibrosis progression or severe fibrosis in patients with and without occult HBV co-infection after liver transplantation at the time of analysis was calculated by Kaplan-Meier method. The cumulative probability of developing relapsed infection due to HBV in patients with and without occult HBV co-infection after liver transplantation was also measured by the Kaplan-Meier method. Continuous variables were expressed as median (range). All statistical analyses were performed on an intention-to-treat population. Statistical significance was defined as p < 0.05 (2-tailed).

Results

Out of the 231 patients, 118 (51.1%) developed recurrent chronic HCV infection in the first year after liver transplantation. Occult HBV co-infection was detected in 41 of the 118 patients (34.7%). The characteristics of the donors and patients before liver transplantation are shown in Tables 2 and 3, respectively.

Table 2.  Donors' characteristics in patients with and without occult HBV co-infection before liver transplantation
 Occult HBV positive patients (n = 41)Occult HBV negative patients (n = 77)p values
  1. HBsAg = hepatitis B surface antigen; anti-HBc = hepatitis B core antibody; anti-HBs = hepatitis B surface antibody; HCV = hepatitis C virus; HBV = hepatitis B virus.

Donor age (years)47 (34–54)39 (28–52)0.17
Donor sex (M:F)12:2926:510.62
Donor anti-HBc status 0.74
 Positive46 
 Negative3771 
Donor anti-HBs status 1.00
 Positive510 
 Negative3667 
Donor anti-HBc/anti-HBs 0.20
 status  
 +/+36 
 +/−10 
Donor anti-HCV status 0.46
 Positive01 
 Negative4176 
Table 3.  Baseline characteristic of patients undergoing liver transplantation
 Occult HBV positive patients (n = 41)Occult HBV negative patients (n = 77)p values
  1. 1Genotype before liver transplantation was available in only 74 patients.

  2. OLT = orthotopic liver transplantation; Anti-HBc = hepatitis B core antibody; Anti-HBs = hepatitis B surface antibody; HCV = hepatitis C virus; HBV = hepatitis B virus.

Age (years)47 (15–65)52 (31–65)0.22
Sex (M:F)26:1557:200.35
Patients anti-HBc status pre-OLT 0.34
 Positive2438 
 Negative1739 
Patients anti-HBs status pre-OLT 0.64
 Positive2041 
 Negative2136 
Patients anti-HBc/anti-HBs status 0.14
 +/+1935 
 +/−53 
Pre-liver transplant HBV vaccination 0.64
 Yes2136 
 No2041 
Immunosuppressive drugs 0.86
 Corticosteroid4177 
 Cyclosporin3769 
 Azathioprine3355 
 Mycophenolate mofetil1026 
 FK-50657 
Genotype pre-OLT1 0.30
 11639 
 Non-1811 
HCV RNA pre-OLT 0.42
 Detectable3149 
 Undetectable1028 
Pre-liver transplant interferon therapy 0.58
 Yes412 
 No3765 
 HCV RNA at the time of recurrent chronic HCV post-transplant, IU/mL652 000 (91 000–850 000)538 000 (45 000–850 000)0.76
Genotype at the time of recurrent HCV 0.81
 12345 
 Non-11832 
Year of liver transplantation 0.84
 1988–1989714 
 1990–19911124 
 1992–19962339 

Quantification of serum HBV DNA

Serum HBV DNA of the 41 patients with occult HBV co-infection is shown in Table 4. Serum HBV DNA was not quantifiable in 6 of the 41 patients (14.6%) with occult HBV co-infection and was detectable but below the liner range of the Roche Taqman assay in 6 of the 41 patients (14.6%) with occult HBV co-infection. The median serum HBV DNA in 29 of the 41 patients (70.7%) with quantifiable HBV DNA was 87 (range 51–644) copies/mL.

Table 4.  HBV DNA in patients with occult HBV co-infection
Occult HBV positive patient (n = 41)HBV Region by PCRSerum HBV DNA by real time PCR (copies/mL)
X regionCore regionS region
  1. BLQ = positive for HBV DNA but below limit of quantification; TND = target not detected; HBV = hepatitis B virus; += positive; −= negative.

1++51
2++65
3++53
4++TND
5++121
6++300
7++56
8++76
9++130
10++BLQ
11++87
12++97
13++112
14++BLQ
15++62
16++112
17++68
18++644
19++BLQ
20++89
21++TND
22++75
23++TND
24+++155
25++TND
26++56
27++320
28++BLQ
29++BLQ
30++67
31++58
32++60
33++76
34++110
35++190
36++TND
37++325
38++110
39++52
40++TND
41++BLQ
Median serum HBV DNA (range) in the 29 patients with quantifiable HBV DNA by Roche Taqman assay, copies/mL87 (51–644)

Clinical outcome

Changes consistent with recurrent chronic HCV infection after liver transplantation was diagnosed on liver biopsy at a median time of 5.9 (range 1.6–10.8) months after liver transplantation in those with occult HBV co-infection and 5.2 (range 2.1–11.5) months after liver transplantation in those without occult HBV co-infection (p = 0.45). The stage of fibrosis and grade of inflammation on liver biopsy at the time of recurrent chronic HCV infection are shown in Table 5. There was no significant difference between the stage of fibrosis and grade of inflammation on liver biopsy between patients with and without occult HBV co-infection at the time of recurrent chronic HCV infection post-transplantation (p = n.s.).

Table 5.  Histology of liver biopsy at the time of recurrent chronic HCV infection after liver transplantation and results of histology at the time of analysis
 Occult HBV positive patients (n = 41)Occult HBV negative patients (n = 77)p values
  1. HCV = hepatitis C virus; HBV = hepatitis B virus.

Liver biopsy at the time of recurrent chronic HCV
 Grade of inflammation 0.89
  A0–12548 
  A2–41629 
 Stage of fibrosis 0.91
  F03770 
  F147 
Size of liver biopsy, mm18 (12–22)17 (10–20)0.76
Portal tract11 (10–15)10 (10–14)0.85
Liver biopsy at 5 years after transplantation
 Grade of inflammation 0.25
  A0–11622 
  A2–42555 
 Stage of fibrosis 0.22
  F01735 
  F11023 
  F266 
  F329 
  F464 
Size of liver biopsy (mm)18 (10–24)19 (12–24)0.65
Portal tract10 (9–13)11 (9–14)0.49

Seventy-eight of the 118 patients (66.1%) had liver biopsy at the end of follow-up. Forty of the 118 patients (33.9%) patients were discontinued from the study before the end of follow-up for the following reasons: 35 patients were treated with interferon-based therapy while 5 patients developed relapsed infection due to HBV. The stage of fibrosis and grade of inflammation on the final liver biopsy for all patients are shown in Table 5.

Fibrosis progression and severe fibrosis after liver transplantation

Twenty-nine of the 118 patients (24.6%) had fibrosis progression at the end of follow-up. Thirteen of the 41 occult HBV co-infected patients compared with 16 of the 77 patients without occult HBV co-infection developed fibrosis progression (31.7% vs. 20.8%, respectively, p = 0.39) (Figure 1A). The median rate of fibrosis progression between the first and final liver biopsies in patients with occult HBV co-infection was 0.24 (range 0–2.40) METAVIR units/year compared with 0.31 (range 0–2.06) METAVIR units/year in patients without occult HBV co-infection (p = 0.40). None of the patients had regression of fibrosis stage.

Figure 1.

Graph showing cumulative survival free from (A) fibrosis progression and (B) severe fibrosis in recurrent chronic hepatitis C virus infected patients with and without occult hepatitis B virus co-infection.

Severe fibrosis occurred in 21 of the 118 patients (17.8%) at the end of follow-up. Eight of the 41 patients with occult HBV co-infection compared with 13 of the 77 patients without occult HBV co-infection progressed to severe fibrosis (19.5% vs. 16.9%, respectively, p = 0.97) (Figure 1B).

Relapsed infection due to HBV after liver transplantation

Five of the 118 recipients (4.2%) developed relapsed infection due to HBV after liver transplantation. Relapsed infection due to HBV occurred in 5 of the 41 patients (12.2%) with occult HBV co-infection compared with none of the 77 patients (0%) without occult HBV co-infection (p = 0.004) after liver transplantation (Figure 2). The clinical features of these 5 patients who developed relapsed infection due to HBV are shown in Table 6. All 5 patients were treated with 4 months of conventional interferon. None of these 5 patients had fibrosis progression on liver biopsy performed before the relapsed infection due to HBV when compared with the liver biopsy at the time recurrent HCV and occult HBV was diagnosed.

Figure 2.

Graph showing cumulative survival free from relapsed infection due to hepatitis B virus in recurrent chronic hepatitis C virus infected patients with and without occult hepatitis B virus co-infection.

Table 6.  Clinical feature of the 5 patients who developed relapsed infection due to HBV after liver transplantation
PatientPatients HBV status pre-transplantDonor HBV statusOccult HBV co-infection in patientsQuantity of HBV DNA in serum by Taqman assayTime of relapsed infection due to HBV after recurrent HCV infection diagnosed (months)Recipient HBV status at the time of last liver biopsy
HBsAgAnti-HBcAnti-HBsHBsAgAnti-HBcAnti-HBsHBsAgHBeAgAnti-HBe
  1. HBV = hepatitis B virus; HBsAg = hepatitis B surface antigen; Anti-HBc = hepatitis B core antibody; Anti-HBs = hepatitis B surface antibody; HBeAg = hepatitis B e antigen; Anti-HBe = hepatitis B e antibody; += positive; −= negative.

1+++1125.2++
2+++645.9++
3++++526.3++
4+++516.6++
5++617.8++

Interferon-based therapy for recurrent chronic HCV infection

A total of 35 patients (30.0%) were treated with 48 weeks of interferon-based therapy for recurrent chronic HCV infection; 10 patients with occult HBV co-infection and 25 patients without occult HBV co-infection. These 35 patients had histological evidence of ongoing HCV hepatitis on liver biopsy. The histology of these 35 patients on their final liver biopsy before receiving interferon-based therapy and the final histology at the end of follow-up in those who did not receive interferon-based therapy are shown in Table 7. There was no difference in the grade of inflammation or stage of fibrosis between these two groups.

Table 7.  Histology on final liver biopsy before initiation of interferon-based therapy compared with the final liver biopsy at the end of follow-up in patients without interferon-based therapy
 Interferon- based therapy (n = 35)1No interferon- based therapy (n = 83)p values
  1. 1Final liver biopsy for patients receiving interferon-based therapy is the liver biopsy, which was performed before commencement of interferon-based therapy.

Grade of inflammation on final liver biopsy0.19
 A0511 
 A1913 
 A21041 
 A3917 
 A421 
Stage of fibrosis on final liver biopsy0.19
 FO1240 
 F1924 
 F275 
 F347 
 F437 

In the 10 patients with occult HBV co-infection, 3 patients (30.0%) were treated with interferon-α (Schering-Plough, Kenilworth, NJ, USA) monotherapy, 4 patients (40.0%) with interferon-α (Schering-Plough) plus ribavirin (Schering-Plough) combination therapy and 3 patients (30.0%) with pegylated interferon-α2b (Schering-Plough) plus ribavirin (Schering-Plough) combination therapy.

In the 25 patients without occult HBV co-infection, 8 patients (32.0%) were treated with interferon-α (Schering-Plough) monotherapy, 13 patients (52.0%) with interferon-α (Schering-Plough) plus ribavirin (Schering-Plough) combination therapy and 4 patients (16.0%) with pegylated interferon-α2b (Schering-Plough) plus ribavirin (Schering-Plough) combination therapy.

The sustained virological response with interferon-based therapy in patients with and without occult HBV co-infection is shown in Table 8.

Table 8.  Patients treated with interferon-based therapy for 48 weeks and sustained virological response
 Occult HBV positive patients (n = 10)Occult HBV negative patients (n = 25)
No of patients treatedSVRNo of patients treatedSVR
  1. SVR = sustained virological response; HBV = hepatitis B virus infection.

Interferon-α monotherapy3081 (12.5%)
Interferon-α plus ribavirin therapy40134 (30.8%)
Pegylated interferon-α2b plus ribavirin therapy3041 (25.0%)

Rejection

Graft rejection occurred in 15 of the 41 patients (36.6%) with occult HBV co-infection and in 35 of the 77 patients (45.4%) without occult HBV co-infection (p = 0.35).

Interferon-based therapy in patients with and without graft rejection

Fifteen of the 50 patients (30.0%) with graft rejection compared with 20 of the 68 patients without graft rejection (29.4%) received interferon-based therapy (p = 0.48 on log rank).

Discussion

With the development of sensitive tests for HCV and HBV DNA in recent years, about 20–30% of sera and 40–50% of livers of anti-HCV positive patients with or without HCC demonstrated HBV DNA positivity (33). Studies have shown an association of HBV DNA and HCV infected patients with liver cirrhosis or HCC when compared with chronic HCV patients without liver cirrhosis or HCC (7). Occult HBV may also modify the response to interferon therapy in HCV patients (34,35). This is important in view of the controversy on the relative impact of viral, host and environmental factors on the course of HCV infection. However, the diagnosis of occult HBV infection in these studies were based on either detection of HBV DNA in the serum by one set of primers or by detection of HBV DNA in the liver with two sets of primers without taking into account the presence or absence of covalently closed circular DNA (cccDNA) in the liver (7,34,35). Without the presence of cccDNA, the detection of HBV DNA in the liver could simply represent integration of HBV DNA into the host genome rather than an intact virus (7). Thus, the diagnosis of occult HBV infection might be better reserved for those in whom HBV DNA is detected by two independent sets of primers in the sera or in those with cccDNA detected in the liver (36,37).

Recently, by using these strict criteria for diagnosing occult HBV, we and others have found that occult HBV co-infection did not seem to result in accelerated fibrosis progression or impaired the response to interferon therapy in chronic HCV-infected immunocompetent patients (17–19,38). However, whether the same observation still holds true in immunosuppressed chronic HCV patients is uncertain. We, therefore, investigated fibrosis progression in patients with recurrent chronic HCV and occult HBV co-infection after liver transplantation.

Patients with recurrent chronic HCV and occult HBV co-infection did not seem to have an accelerated fibrosis progression (Figure 1A) or accelerated progression to severe fibrosis (Figure 1B) after liver transplantation. It seems that the presence of occult HBV co-infection, with its low level of HBV viremia, does not seem to lead to an accelerated rate of progressive liver disease. The current study would suggest, that, although definitive data are lacking, the likelihood of this low level of HBV viremia resulting in progressive liver disease is low. Occult HBV infection with its low level of viremia may just be an innocent bystander. However, due to the small sample size, a definitive conclusion cannot be made even though no statistical significance in fibrosis progression or severe fibrosis was detected between those with or without occult HBV co-infection.

The rate of fibrosis progression in our study is also lower than that reported by Firpi et al. and Wali et al. (39,40). This observed difference may be accounted for by the different scoring system used in these two studies, and, the fact that Wali et al. analyzed liver biopsies performed at a median of 1.92 (range 0.24–11.48) years after liver transplantation rather than at a discreet time point as in this study. As is often the case with studies looking at fibrosis progression, one tends to assume that fibrosis progression is linear. However, this assumption may not be true.

Although we did not perform serial quantification of serum HBV DNA in patients with occult HBV infection, the median level of HBV DNA was found to be only around 87 copies/mL even though these 41 patients were on anti-rejection protocols. This low level of HBV DNA is comparable with what we have previously reported in immunocompetent bone marrow transplant donors, as well as in immunocompetent chronic HCV infected patients with occult HBV co-infection (16,17). It seems that the use of neither corticosteroids nor immunosuppressants increased the quantity of HBV DNA in occult HBV co-infected patients. Thus, it is uncertain why only 12.2% of these occult HBV co-infected patients developed relapsed infection due to HBV post-liver transplantation.

The results of this study must be interpreted with caution because of its limitations. Firstly, only 30.0% of the patients in the study were treated for recurrent chronic HCV infection. One can reasonably assume that most patients were not treated because limited data existed about the treatment outcomes of these patients at the time. Furthermore, a 'watch and wait' rationale was adopted as treatment regimens were rapidly evolving at the time. Secondly, our PCR assay of 50 copies/mL may not be sensitive enough for the diagnosis of occult HBV infection. The best option for the diagnosis of occult HBV infection may be reserved for patients with HBV DNA plus cccDNA detected on frozen liver biopsy samples. Thirdly, the use of stored serum samples for more than 10 years may have affected the amount of HBV DNA or HCV RNA quantified. Those who were excluded from the study due to undetectable HCV RNA or undetectable HBV DNA may have had recurrent chronic HCV infection and occult HBV co-infection, thus resulting in a bias. Fourthly, none of the patients receiving anti-HBc positive livers were given post-transplantation prophylaxis as our Center stopped accepting anti-HBc positive individuals as liver donors after 1993. Furthermore, the HBV DNA was only performed after the end of follow-up. Hence, were only available retrospectively. Finally, at the time, relapsed infection due to HBV was thought to be relatively benign (41), and, lamivudine was only approved by the Federal Drug Administration for treatment of chronic HBV 2 years after the last patient included in this study had undergone liver transplantation.

In conclusion, occult HBV co-infection in patients with recurrent chronic HCV infection after liver transplantation is associated with a low level of viremia. This low level of viremia does not seem to accelerate the rate of progressive liver disease. However, these patients are at a higher risk of developing relapsed infection due to HBV after liver transplantation.

Acknowledgment

Professor Sir David Todd, Professor Kar-Neng Lai and Professor Anna S Lok. CKH and GKL were supported by The Hong Kong Liver Foundation.

Ancillary