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Abstract

  1. Top of page
  2. Abstract
  3. Case 1
  4. Case 2
  5. Discussion
  6. References

Fibrosing cholestatic hepatitis (FCH) is a peculiar variant of hepatitis B virus (HBV) infection in immunocompromised patients characterized by rapid viral replication. Posttransplant patients receiving lamivudine for prophylaxis or treatment of HBV infection may develop drug resistance due to viral mutants, but FCH is rare because escape mutants are usually replication deficient. We report the development of FCH due to lamivudine-resistant HBV mutants in 2 patients at 12 and 13 months after liver transplantation. Rapidly progressive graft failure, accompanied by an escalating HBV DNA level, developed within weeks of onset. Analysis of gene sequence variation by polymerase chain reaction (PCR) and direct sequencing showed that both had a core promoter variant A1762T/G1764A and 1 had a concomitant precore stop codon G1896A variant in prelamivudine and postrecurrence serum samples. Comparison of the HBV polymerase gene in the 2 serum samples revealed a single mutation with methionine-to-isoleucine substitution at codon 552 (M552I) in both patients. “Add-in” treatment with adefovir dipivoxil resulted in a more than 2 to 3 log10 reduction in HBV DNA level within 2 weeks and retransplantation was performed with adefovir dipivoxil and hepatitis B immunoglobulin (HBIG) prophylaxis. Both patients were alive at 15 months and 48 months after retransplantation, with normal graft function and serum negative for HBsAg and HBV DNA by quantitative PCR (< 200 copies/mL). The current report demonstrates that recurrent graft infection by precore/core promoter variant with lamivudine-resistant escape mutation may result in FCH. With combination of adefovir and high-dose HBIG, however, long-term survival can be achieved after retransplantation. (Liver Transpl 2004;10:557–563.)

Liver transplantation in patients with chronic hepatitis B virus (HBV) infection is associated with a high risk of graft reinfection. A unique variant of recurrent hepatitis with a distinct histological and severe clinical manifestation has been termed fibrosing cholestatic hepatitis (FCH).1–2 The disease is characterized histologically by hepatocyte ballooning degeneration with severe cholestasis and periportal fibrosis, but only mild inflammatory infiltrates. There is a markedly increased viral replication with intracellular overexpression and massive accumulation of HBV antigens, which may result in direct cytopathic liver damage. Patients with FCH develop rapidly progressive liver failure, and without treatment, the condition is universally fatal within weeks of onset.

The introduction of nucleoside analogues, notably lamivudine, has revolutionized the management of HBV infection in liver transplant recipients. Lamivudine, either alone,3–4 or in combination with hepatitis B immunoglobulin (HBIG),5 has been shown to be effective in reducing HBV reinfection and improving graft survival. Lamivudine treatment has also been demonstrated to be effective in patients with recurrent hepatitis B after liver transplantation,6 and even in the occasional patients with FCH.7–8 The emergence of lamivudine resistance due to viral mutants is a serious concern, but recurrent disease is usually mild with a benign course,9 and there has been only 1 case report of FCH due to lamivudine-resistant mutant.10 In this article, we report 2 cases of FCH due to lamivudine-resistant escape mutation in precore or core promoter HBV variant after liver transplantation. There was rapid onset of graft failure, and both patients underwent successful retransplantation using combination of adefovir dipivoxil and HBIG.

Case 1

  1. Top of page
  2. Abstract
  3. Case 1
  4. Case 2
  5. Discussion
  6. References

A 37-year-old Chinese man suffered from HBV-related cirrhosis complicated by recurrent episodes of spontaneous bacterial peritonitis and intractable pleural effusion. He has been receiving lamivudine therapy (100 mg daily) since August 1997. The pretreatment serum HBeAg was negative, anti-HBe positive, and HBV-DNA level was 35.8 × 106 copies/mL. Three months later, his serum HBV-DNA decreased to 287 copies/mL and he underwent liver transplantation on November 7, 1997, using a cadaver liver graft from a donor with positive serology for anti-HBc. His postoperative course was uneventful. Lamivudine was continued at a dose of 100 mg daily and HBIG was not used. Postoperative immunosuppression consisted of tacrolimus (target trough level 10–15 ng/ml) and steroid (intravenous methylprednisolone 200 mg/day tapered to oral prednisolone 20 mg/day from day 7). The tacrolimus trough level was lowered to 5 to 10 ng/ml 1 month after transplantation, and the dosage of prednisolone was reduced to 10 mg/day at 2 months. In February 1998, he had an episode of acute cellular rejection confirmed on histology, and he responded well to 2 doses of intravenous steroid pulse consisting of 1 g of methylprednisolone each. His graft function then remained normal.

His serum HBsAg became undetectable from day 10 after transplantation and serum anti-HBs titer rose spontaneously from 6.3 mIU/mL on day 9 to a maximum of 750 mIU/mL on day 35 after transplantation. Serum HBV DNA level was less than 200 copies/mL from day 7 after transplantation. In November 1998, (12 months after transplantation), his serum HBsAg became positive. Serum HBV-DNA level rose to 1.02 × 106 copies/mL, but HBeAg was negative and his liver functions remained normal. Six weeks later, in December 1998, his liver function deteriorated with serum alanine aminotransferase rising to 305 IU/L (normal range: 6–53 IU/L), total bilirubin 79 μmol/L (normal range: 7–19 μmol/L), prothrombin time 19.4 seconds (normal range: 11.3–13.2 seconds) and international normalized ratio 1.6. His serum HBV DNA level had escalated rapidly with a 3 log10 increase to 3,290 × 106 copies/mL (Fig. 1) but serum HBeAg remained negative. Doppler ultrasound examination showed that all vessels were patent and a trucut liver biopsy showed features consistent with FCH (Fig. 2). Oral famciclovir 500 mg 3 times daily was added to lamivudine therapy in December 1998, but there was no response, and the HBV DNA level remained high at 1,926 × 106 copies/mL.

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Figure 1. Serial changes in serum alanine aminotransferase, total bilirubin and HBV DNA with time in Case 1. Treatment with lamivudine and adefovir dipivoxil is indicated by the horizontal bar.

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Figure 2. Liver biopsy in Case 1 13 months after transplantation. (A) There was periportal fibrosis and the hepatocytes showed extensive ballooning degeneration. Cytoplasmic and canalicular cholestasis was prominent. (Hematoxylin and eosin) (B) Immunohistochemical staining showed frequent membranous or cytoplasmic positivity for HBsAg and (inset) cytoplasmic positivity for HBcAg.

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On February 25, 1999, famciclovir was taken off and adefovir dipivoxil at a dose of 30 mg daily with L-carnitine 250 mg daily was added to lamivudine therapy. There was a 2 log10 reduction in serum HBV-DNA from 819.8 × 106 copies/mL to 8.9 × 106 copies/mL after 8 days. The liver function, however, continued to deteriorate with bilirubin rising to 936 μmol/L, prothrombin time 46.6 seconds, and international normalized ratio 4.1 Retransplantation with a cadaver graft was performed on March 5, 1999. Intravenous HBIG was given at a dose of 10,000 u during anhepatic phase and daily for 6 days. Repeated doses were given to maintain an anti-HBs titer above 100 mIU/mL. Adefovir with L-carnitine and lamivudine were continued. Histologic examination of the explanted liver confirmed the diagnosis of FCH.

His postoperative course was complicated by hepatic venous outflow obstruction that was relieved after a reoperation for revision of the suprahepatic vena cava anastomosis on day 25. Immunosuppression was continued with steroid and tacrolimus. As a result of renal function impairment after operation with creatinine rising to 263 μmol/L (normal range: 80–140 μmol/L), the dosage of adefovir dipivoxil was reduced to 10 mg daily and L-carnitine was discontinued in April 1999. He remained well with a combination of adefovir, lamivudine and periodic doses of HBIG injection to maintain anti-HBs greater than 100 mIU/mL. The dosage of adefovir was reduced further to 10 mg every alternate day when he developed transient renal dysfunction in August 2002. As on the date of his latest follow-up on April 2, 2003 (48 months after retransplantation), his serum alanine aminotransferase was 26 IU/L, total bilirubin 20 μmol/L, albumin 45 g/L, prothrombin time 12.7 seconds, and creatinine 130 μmol/L. His serum was negative for HBsAg and positive for anti-HBsAb. There was no detectable HBV DNA (< 200 copies/mL) in serum.

HBV gene sequence alignment showed genotype C with core promoter variant A1762T/G1764A and precore stop codon variant G1896A in both serum samples before lamivudine treatment and after breakthrough reinfection (Table 1). Comparison of the HBV polymerase gene from the 2 specimens showed a mutation resulting in a methionine-to-isoleucine substitution at codon 552 (M552I) at recurrence. There was no mutation at codon 528.

Table 1. HBV Genotype, Polymerase, Core Promoter and Precore Gene Sequences Before Treatment and at Recurrence
 HBV GenotypeBefore TreatmentAt Recurrence
Core Promoter nt 1762-1764Precore nt 1896PolymeraseCore Promoter nt 1762-1764Precore nt 1896Polymerase
aa 528aa 552aa 528aa 552
  1. Abbreviations: nt, nucleotide; aa, amino acid; HBV, hepatitis B virus; T, thymine; G, guanine; A, adenine; L, leucine; M, methionine; I, isoleucine.

Case 1CTGAALMTGAALI
Case 2CTGAGLMTGAGLI

Case 2

  1. Top of page
  2. Abstract
  3. Case 1
  4. Case 2
  5. Discussion
  6. References

A 32-year-old Chinese man with HBV-related cirrhosis presented with intractable ascites and pleural effusion in March 2000. He developed spontaneous bacterial peritonitis and hepatic encephalopathy in August 2000. His serum was negative for HBeAg and HBV DNA level was 43,400 copies/mL. Lamivudine 100 mg daily was started in October 2000. Serum HBV DNA level decreased to 240 copies/mL (Fig. 3), and living donor liver transplantation, using a right lobe graft from his father who was seropositive for anti-HBC, was performed on November 20, 2000. Lamivudine was continued after operation and HBIG was not used. Postoperative immunosuppression consisted of tacrolimus and steroid similar to case 1 and steroid was weaned off in June 2001 (7 months after transplantation).

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Figure 3. Serial changes in serum alanine aminotransferase, total bilirubin, and HBV DNA with time in Case 2. Treatment with lamivudine and adefovir dipivoxil is indicated by the horizontal bar.

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He cleared HBsAg from serum 1 day after transplantation and had transient anti-HBs seroconversion with a maximum titer of 36 mIU/mL 3 months after transplantation. Graft function was normal and serum HBV DNA was less than 200 copies/mL until September 2001 (10 months after transplantation) when his HBV DNA level rose and both serum HBsAg and HBeAg became positive. His liver function deteriorated since November 2001 and doppler ultrasound examination showed that all vessels were patent. Trucut liver biopsy showed features typical of FCH (Fig. 4). Adefovir dipivoxil 10 mg daily was added on December 1, 2001, and lamivudine was continued. Despite a 3 log10 reduction in serum HBV DNA level from 2,235 × 106 copies/mL to 3.6 × 106 copies/mL in 2 weeks, his liver function continued to deteriorate with bilirubin rising to 760 μmol/L, prothrombin time 47.5 seconds and international normalized ratio 4.3, and he developed hepatic encephalopathy. Retransplantation with a right lobe liver graft from a close friend was performed on December 24, 2001. Intravenous HBIG was given at a dose of 10,000 u during anhepatic phase and daily for 6 days. Repeated doses were given to maintain an anti-HBs titer above 100 mIU/mL. Adefovir and lamivudine were continued. Histologic examination of the explanted liver confirmed the presence of FCH. Both the donor and recipient recovered uneventfully after operation. The donor was discharged on day 8 and the recipient on day 21. As on the date of latest follow-up on March 26, 2003 (15 months after retransplantation), the recipient's serum alanine aminotransferase was 45 IU/mL, total bilirubin 15 μmol/mL, albumin 46 g/L, prothrombin time 12.3 seconds, and creatinine 87 μmol/L. His serum was negative for HBsAg, positive for anti-HBS, and negative for HBV DNA (< 200 copies/mL).

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Figure 4. Liver biopsy in Case 2 12 months after transplantation. (A) There was marked periportal fibrosis with prominent proliferation of bile ductules (lower left). The hepatocytes were swollen with ground-glass transformation. Cytoplasmic cholestasis was present. (Hematoxylin and eosin) (B) Diffuse and strong cytoplasmic expression of HBsAg by immunohistochemistry was present in almost every hepatocyte. (inset) There was moderate cytoplasmic expression and occasional simultaneous strong unclear expression of HBcAg.

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HBV genotype C with core promoter variant A1762T/G1764A was detected in the prelamivudine and postrecurrence serum samples, but there was no precore stop codon variant (Table 1). As for the tyrosine, methionine, aspartic acid, and aspartic acid (YMDD) motif, the wild type virus before lamivudine treatment was replaced by the M552I mutant after breakthrough reinfection occurred.

Laboratory Methods

Tests for serum HBsAg, HBeAg, and anti-HBe were performed by commercially available enzyme immunoassays (Abbott Laboratories, Chicago, IL). Serum HBV DNA level was initially measured by branched-chain DNA assay (Quantiplex, Chiron Diagnostics, Emeryville, CA) with a lowest detection limit of 700,000 copies/mL. Negative samples were subsequently tested by quantitative polymerase chain reaction (PCR) assay (Cobas Amplicor, Roche Diagnostics, Basel, Switzerland) with a lowest detection limit of 200 copies/mL.

HBV genotyping and analysis of sequence variations in precore/core promoter as well as polymerase gene were performed by PCR and direct sequencing. For analysis of polymerase gene and precore/core promoter region, DNA was extracted from 200 μl of serum, using QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. DNA in the purification column was eluted with 50 μl of water, and 1μl of serum DNA was subjected to 35 cycles of PCR amplification. Reaction volume of 25 μl contained 2.5 ρmole of each primer, 62.5 μM of each dNTP, 2 μM MgCl2 and 1.25 unit of AmpliTaqGold DNA polymerase (Applied Biosystems, Foster City, CA) in buffer as provided. Reaction profile included denaturation at 95°C for 5 minutes, 35 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 60 seconds, and final extension at 72°C for 10 minutes. The sequence of the YMDD region of the polymerase gene was amplified (sense, 5′-CCAGGAACATCAACCACCAG-3′; antisense, 5′-ACATATCCCATGAAGTTAAGGGA-3′) followed by direct sequencing with internal primer (sense 5′-TCGGACGGAAACTGCAC-3′) using the BigDye Terminators Cycle Sequencing Kit (Applied Biosystems) with the ABI Prism 3100 genetic analyzer system (Applied Biosystems). The sequence of the precore/core promoter region was analyzed similarly with annealing temperature of 58°C by PCR amplification (sense 5′-AGGAGTTGGGGGAGGAG-3′; antisense 5′-CCTCCCGATACAGAGCAG-3′), and direct sequencing. Genotyping PCR was performed by amplifying HBV genome between nucleotide 256 and 796 using primers P7 and P811 and followed by direct sequencing.

Discussion

  1. Top of page
  2. Abstract
  3. Case 1
  4. Case 2
  5. Discussion
  6. References

The present report illustrates 2 important points. First, reinfection of a liver graft by precore/core promoter variant with lamivudine-resistant escape mutation may result in rapid graft loss from FCH. Second, with combination of adefovir dipivoxil and high-dose HBIG, long-term survival can be achieved after retransplantation for FCH secondary to lamivudine-resistant mutant.

Lamivudine has been increasingly used for prophylaxis or treatment of recurrent hepatitis B after liver transplantation. With prolonged usage, resistance to lamivudine is frequently encountered3, 6 and is associated with the development of specific mutations in the polymerase gene encoding the YMDD motif of the reverse transcriptase. Two well-defined mutations at codon 552 with substitution of methionine by isoleucine (M552I) or valine (M552V) have been described, occasionally with an additional leucine-to-methionine (L528M) mutation at codon 528.12 Recurrent graft infection by the escape mutant is generally mild9 probably because the mutant virus has a reduced competence to replicate.13 FCH, which develops in immunocompromised patients when rapid viral replication leads to massive intracellular accumulation of HBV antigens, is, therefore, rare with the escape mutant. There has been only 1 report on the development of FCH due to a lamivudine-resistant mutant with M552V and L528M double mutations after liver transplantation.10 Our report is the first to show that reinfection with the M552I mutant can also lead to FCH. We continued lamivudine therapy after resistance development to avoid recurrence of the wild-type virus that might be more virulent. The presence of the M552I mutant only and the absence of the wild type virus on direct sequencing of the polymerase gene of HBV in both patients' serum after resistance development suggested that the M552I mutant was the predominant HBV population responsible for the FCH.

The very high viral replication and development of FCH secondary to lamivudine-resistant escape mutant in the present 2 cases may be related to genetic variance in other parts of the HBV genome. It has been shown that HBV precore variant may be associated with more severe disease in nontransplant patients with chronic hepatitis B.14 In liver transplant recipients, reinfection with precore variant is associated with more severe recurrence with early graft loss and the development of FCH.15–16 Core promoter variant has also been reported to show a higher viral replication17 and more severe liver damage.18 In addition, HBV genotype C is associated with more active liver disease than genotype B.19 In the present report, both patients had HBV genotype C with core promoter and/or precore variant detected by direct sequencing in the serum samples before lamivudine treatment and when recurrence occurred. Hence, the precore/core promoter HBV variant was the predominant strain at the very beginning, and the M552I mutation with lamivudine resistance in the preexisting precore/core promoter variant led to the development of FCH.

FCH is a disease with high mortality from progressive graft failure and effective treatments are lacking. Isolated cases of successful treatment by suppressing viral replication and reducing intracellular viral antigen expression with nucleoside analogues such as lamivudine or adefovir dipivoxil have been reported.7, 8, 10 The graft damage, however, may progress so rapidly that despite successful control of the viral replication, graft failure has already established. Retransplantation for FCH is rarely indicated because of an accelerated recurrence of the same syndrome.2 This risk is expected to be even worse if the FCH is related to a known mutant virus with resistance to lamivudine as lamivudine is the nucleoside analogue most frequently used to treat FCH.7 Adefovir dipivoxil is a nucleotide analogue that has been shown to be effective for the treatment of chronic hepatitis B.20–21 Adefovir has been shown to have in vitro as well as in vivo activities against lamivudine-resistant mutants,22–24 and resistance to adefovir has not been observed after treatment for up to 60 weeks.25 Our 2 patients had extremely high levels of viral replication in excess of 109 copies/mL when FCH developed shortly after the emergence of escape mutants. “Add-in” treatment with adefovir dipivoxil in each case successfully suppressed viral replication with a 2 to 3 log10 reduction in viral load within 2 weeks. Graft failure, however, was advanced and both patients would definitely not have survived without retransplantation. As a result of the failure of monotherapy prophylaxis in the first transplant and in the presence of an extremely active viral replication, we decided to use a combination of adefovir and HBIG in the retransplant. With such a regimen and the removal of the explant, which contained a huge load of the virus, a more than 9 log10 total reduction in HBV DNA was achieved. HBV DNA rapidly became undetectable even with quantitative PCR and the suppression of HBV DNA was sustained after a follow-up of 15 and 48 months after retransplantation. Our experience indicates that this combination of prophylaxis offers effective long-term protection against graft reinfection despite the presence of a very high pretransplant viral load and lamivudine-resistant HBV mutants. FCH should not be considered a contraindication for retransplantation even when lamivudine resistance has developed.

References

  1. Top of page
  2. Abstract
  3. Case 1
  4. Case 2
  5. Discussion
  6. References
  • 1
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