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Keywords:

  • Adefovir;
  • famciclovir;
  • hepatitis B immune globulin (HBIG);
  • hepatitis B virus (HBV);
  • hepatitis B virus surface antigen (HBsAg);
  • lamivudine;
  • liver transplant;
  • polymerase;
  • surface (S) gene;
  • YMDD

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We evaluated serum samples from 18 chronic hepatitis B virus (HBV) patients who underwent liver transplantation for the presence of HBV polymerase and S gene mutations and HBV genotype using a new commercially available sequencing assay. All three patients with hepatitis B immune globulin (HBIG) treatment failure followed by nucleoside analogue treatment failure were infected with HBV genotype C; a pre-existing HBV S antigen (HBsAg) mutation (sD144A) was identified in one patient pretransplant, while sG145R mutations emerged in the other two patients post-transplant. These HBsAg mutations persisted for the duration of the study (5–6 years), despite the absence of HBIG administration for a 4–5-year period. Significant viral polymerase mutations (rtL180M and rtM204I/V) also emerged in all of these patients following treatment with lamivudine and/or famciclovir. Four of six patients with HBIG breakthrough without nucleoside analogue treatment failure yielded potentially significant HBsAg mutations post transplant. These data do not support previous reports highlighting the disappearance of HBsAg mutants in liver transplant recipients after discontinuation of HBIG. Determination of HBV genotype, as well as identification of HBV polymerase and S gene mutations in liver transplant candidates may be warranted to optimize HBV management strategies post transplant.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Hepatitis B virus (HBV) is one of the most common infectious disease agents in the world. The complete viral particle (Dane particle) is a sphere approximately 42 nm in diameter, consisting of an outer envelope or surface antigen (HBsAg) surrounding the nucleocapsid, which in turn contains HBV DNA (1). The viral genome is partially double-stranded, approximately 3200 base pairs in length, and encodes four overlapping open reading frames: the surface (S), core, polymerase, and X genes (1,2). Although HBV is a DNA virus, replication occurs through an RNA intermediate; thus the presence of an active viral reverse transcriptase as well as a viral DNA polymerase is required (3,4). The viral polymerase/reverse transcriptase of HBV has been shown to lack the proof-reading ability characteristic of many other polymerases. As a result, HBV exhibits a mutational rate during chronic infection estimated at approximately 1.4–3.2 × 10−5 substitutions/site/year, at least 10-fold higher than that of many other DNA viruses (5). Following liver transplantation, the mutational frequency can be as much as 100-fold higher than in other settings (6). Under the selective pressure of lamivudine or famciclovir, this increase in the mutation rate can lead to the development of polymerase mutations, resulting in lamivudine- or famciclovir-resistant strains of HBV. Under the selective pressure of hepatitis B immune globulin (HBIG), HBsAg mutations can also occur, leading to the selection of HBIG escape mutants.

HBV had previously been classified into four major subtypes by serological methods (7). However, more recent molecular studies of viral strains collected around the world have led to the development of a classification system consisting of seven genotypes (A–G) (8–11). A number of studies have demonstrated different clinical manifestations of HBV in patients infected with different genotypes (12–19). While the current classification scheme is based upon intergenotypic divergence over the entire genome, sequence analysis of subgenomic elements has also been useful in determination of viral genotype. Recent studies have shown the potential clinical utility of HBV genotype determination based upon nucleotide differences found within the S gene (20).

Encoded by the S gene, HBsAg is highly antigenic and is directly involved in humoral immune response to the virus. Primary neutralizing epitopes of HBsAg have been located within the region defined as the ‘a’ determinant (21). The ‘a’ determinant, extending from amino acid (aa) s121 to s149 of HBsAg, induces strong neutralizing antibodies and is the primary target of recombinant vaccines as well as passive immune prophylaxis with HBIG (22,23). While a variety of point mutations within the ‘a’ determinant have been associated with vaccine and HBIG breakthrough, mutations at aa s144 and s145 comprise the majority of HBsAg escape mutants reported to date (21,24–27).

Despite the fact that HBV vaccine efficacy has been shown to be as high as 95%, high-risk infants treated with HBV vaccine and HBIG have an HBV carriage rate of as high as 5–10% (28–30). In addition, with HBIG monotherapy, patients undergoing liver transplant for chronic HBV infection may become re-infected with HBV (31). This may result from low anti-HBsAg levels, and/or HBV S gene mutations (32). Amino acid s144 or s145 escape mutants may have the capacity to become dominant strains and to cause chronic HBV infection in vaccinated populations (33–35). In addition, mutations affecting the HBsAg ‘a’ determinant region can potentially render HBsAg undetectable by some laboratory tests (36–38).

The overlapping nature of the HBV genome results in a frame-shifted overlap of the open reading frames of the S and polymerase genes. As a result, naturally occurring and immunologically induced mutations in the S gene can result in either silent or expressed mutations in the polymerase gene. The impact of these S gene mutations on the polymerase gene is largely unknown. In addition to these mutations, the use of nucleoside analogue therapy has been shown to induce drug resistance mutations in the polymerase gene. In turn, these mutations can potentially result in changes in HBsAg. Recent reports have demonstrated the coexistence of HBsAg and polymerase mutants in patients receiving sequential treatment with these regimens following liver transplantation (39,40). The most commonly reported drug resistance mutations have been shown to occur in the reverse transcriptase portion of the polymerase gene, and confer high-level resistance to the nucleoside analogues lamivudine and famciclovir (41–44). Due to the lack of a universally accepted nomenclature for antiviral drug-related resistance mutations in the reverse transcriptase (rt) domain of HBV, a new numbering system has recently been proposed by Stuyver et al. (45).

The existence of HBsAg and polymerase mutants not only has been shown to significantly impact individual patient management, but also raises long-term public health concerns. These strains could potentially be transmitted to immunized individuals and be associated with nucleoside analogue (lamivudine and famciclovir) treatment failure, respectively. For these reasons, there is an increasing need to provide clinicians with timely information regarding the potential pre-existence or selection of HBsAg and polymerase mutants in HBV-infected patients. In this study, we used the TRUGENE HBV Genotyping Kit in conjunction with the OpenGene DNA Sequencing System (Visible Genetics Inc., Toronto, Ontario, Canada) and the INNO-LiPA HBV DR Kit (Innogenetics N.V., Ghent, Belgium) to detect and characterize the evolution of HBV polymerase and HBsAg (for the TRUGENE HBV Genotyping Kit only) mutants in patients undergoing liver transplantation.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Patients

Patients with chronic HBV infection who underwent liver transplantation at our institution (Mayo Clinic, Rochester, MN, USA) between January 1987 and October 1999 were studied. Retrospectively collected serum specimens, stored at −70 °C, were analyzed. The patients were divided into three groups as follows. Nine to 11 serum specimens were obtained from each of three patients treated initially with HBIG alone, who subsequently broke through and were then treated with a nucleoside analogue with resultant HBV breakthrough (group I). HBV breakthrough resulted in detectable serum HBV DNA at multiple time-points during the post-transplant period (Figures 1–3). For each of these three patients, a pretransplant specimen and additional specimens selected on the basis of modifications to HBV therapy and specimen availability were evaluated. For six additional patients with HBIG treatment failure post transplant (group II), a pretransplant specimen and a single post-transplant specimen (collected at or following the time of HBIG treatment failure) were studied. For these six patients, additional specimens following HBIG treatment failure were not studied, because either the patients died (n = 3), subsequently responded to lamivudine therapy (n = 1), or additional specimens were unavailable for testing (n = 2). For an additional nine patients (group III), a single pretransplant specimen was studied; these nine patients responded to HBIG with or without lamivudine and had no evidence of HBV recurrence following transplantation.

imageimageimage

Figure 1–3. Sequential results of serial genotyping studies for group I patients with HBIG and subsequent lamivudine/famciclovir treatment failure. HBsAg and polymerase mutations of known significance are indicated in bold. Polymerase mutations resulting from the selection of HBsAg mutants during HBIG therapy and HBsAg mutants resulting from the selection of polymerase mutants during lamivudine therapy are indicated in italics, while the selected mutations are underlined. Failed amplification reactions are indicated with an asterisk. INR, international normalized ratio; ALT, alanine aminotransferase.

Immunosuppression consisted of cyclosporine, prednisone and azathioprine until 1994, at which time a tacrolimus-based regimen was introduced, initially as part of the FK506 Primary Immunosuppression Trial (46). In 1997, mycophenolate mofetil (MMF) was introduced as part of a randomized clinical trial comparing azathioprine and MMF. Since 1999, the typical immunosuppression regimen has been tacrolimus, prednisone and MMF. Patients who developed biopsy-proven acute cellular rejection were treated with three boluses of methylprednisolone (1000 mg) on alternate days.

A 10 000 IU dose of intravenous HBIG was administered during the anhepatic phase of transplantation and on days 1 through 7, 14, and 28, and months 2 through 6, to maintain an anti-HBsAg level of greater than 500 IU/mL. After 6 months post transplant, HBIG was administered monthly (or as needed) to maintain an anti-HBsAg level of greater than 250 IU/mL.

HBV DNA extraction

Serum specimens were thawed and extracted in batches consisting of 10 specimens along with an HBV positive and negative control. Specimens were extracted utilizing the QIAamp® DNA Blood Mini Kit: Blood and Body Fluid Spin Protocol (Qiagen Inc., Valencia, CA, USA). Briefly, 20 μL of Protease, 200 μL of serum, and 200 μL of Buffer AL (lysis buffer) were combined in 1.5-mL microcentrifuge tubes, mixed, and heated to 56 °C for 10 min. Tubes were removed from the heat block, 200 μL of ethanol was added, and the contents were mixed prior to transfer of the mixture to QIAamp spin columns. Spin columns were centrifuged at 6000 × g for 1 min binding the DNA to the columns. Each column containing the bound DNA was then washed twice prior to the removal of residual wash solution through an additional high-speed spin. DNA was eluted in 200 μL of Buffer AE and used as input material for each of the genotyping assays described below.

TRUGENE HBV Genotyping Kit and OpenGene DNA sequencing system

Approximately 1200 base pairs of the HBV genome encoding both the HBsAg and the active site of the HBV polymerase were amplified using the TRUGENE HBV Genotyping kit and directly sequenced using the OpenGene DNA Sequencing System following the manufacturer's recommended test procedures. Following amplification, reaction products were utilized as substrate in TRUGENE HBV CLIP sequencing reactions. CLIP sequencing utilizes two different dye-labeled primers (Cy5 and Cy5.5) to generate both forward and reverse sequences simultaneously. The products generated in the CLIP reaction encompass the region extending from (approximately aa rt112 to rt277) of the HBV polymerase gene. This region also encodes a portion of the frame-shifted HBV S gene extending from aa s102 through the end of the gene. Ultimately, 509 base pairs of sequence information were utilized in the genotypic analysis. The analysis was performed by combining and editing the forward and reverse sequences prior to comparison to a consensus sequence of each of the HBV genotypic groups using the GeneObjects version 3.2 alpha software and HBV GeneLibrarian module of the OpenGene DNA Sequencing System (Visible Genetics Inc.). Both the S (genotype) and polymerase genes of each HBV sequence were analyzed simultaneously with respect to the seven consensus genotype sequences (A–G) utilizing the HBV GeneLibrarian module. Viral genotype was determined by a best-fit algorithm represented by the score of the sample sequence compared to the consensus sequences utilizing the HBV GeneLibrarian module. Similarly, the software was used to analyze the S and polymerase genes for specific aa changes that have previously been associated with vaccine and HBIG escape (sG145R) or resistance to either lamivudine and/or famciclovir [rtV173L (V521L), rtL180M (L528M), rtM204V (M552V), rtM204I (M552I), and rtV207I (V555I)]. Final genotyping reports contained all nucleotide mutations found within the analyzed portions of the S and polymerase genes in addition to viral genotype.

INNO-LiPA HBV DR kit

The manufacturer's recommended test procedures were modified as follows. HBV DNA was amplified using an in-house developed PCR assay amplifying a 615 base pair segment of the HBV polymerase gene extending from aa rt26 to rt230. The use of both forward and reverse primer pools (Table 1) was employed in addition to positioning of the primers within highly conserved regions of the HBV genome. This strategy was used in order to ensure efficient amplification of all known HBV genotypes and variants. Briefly, PCR products were generated in 50 μL reactions containing 50 mm KCl, 10 mm Tris-HCl (pH 8.3), 2.0 mm MgCl2, 10% glycerol (v/v), 200 μm deoxynucleotide triphosphates (including a 100-μm dUTP/dTTP substitution), 2.5 units of Amplitaq Gold™ DNA polymerase (Applied Biosystems, Foster City, CA, USA), 0.5 units of HK UNG Thermolabile Uracil N-Glycosylase (Epicentre Technologies, Madison, WI, USA), 1 μm each of forward and reverse primer pools, and 10 μL of DNA extract. Thermocycling was performed using a GeneAmp® PCR System 9600 thermal cycler (Applied Biosystems) and the following profile: 50 °C for 5 min, 95 °C for 10 min followed by 45 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 45 s, with a final extension step of 72 °C for 5 min.

Table 1. : HBV amplification primers
For 15′-biotin-GGGTTTTTCTTGTTGACA-3′
For 25′-biotin-GTGTGTTTCTTGTTGACA-3′
Rev 15′-biotin-AAGACAAAAGAAAATTGGT-3′
Rev 25′-biotin-CAGATAACAGAAAATTGGT-3′
Rev 35′-biotin-AAGACAAAAGAAAATTGGA-3′

Amplification reactions were screened by agarose gel electrophoresis and ethidium bromide staining. All amplification products yielding a visible band of the appropriate size by agarose gel electrophoresis were subjected to further analysis using the INNO-LiPA HBV DR Kit. Testing was performed with the aid of the AutoBlot 2000 instrument (MedTec, Inc., Chapel Hill, NC, USA). In brief, 10 mL of biotinylated amplification product was denatured by mixing with an equal volume of Denaturation Solution, and placed in the upper portion of an individual reaction trough along with a test strip containing immobilized HBV-specific oligonucleotide probes. After a 5-min denaturation step, 2 mL of prewarmed Hybridization Solution was added to each trough. The trays containing the test strips were gently mixed and placed in a 50 °C shaking waterbath for 60 min (approximately 80 r.p.m.). Upon completion of the 50 °C incubation, the trays containing the test strips were placed on the AutoBlot 2000 instrument. The Hybridization Solution was aspirated from each trough by the instrument, and each strip was rinsed twice with 2 mL of Stringent Wash Solution prior to the final addition of 2 mL of Stringent Wash Solution. Trays were again placed in a 50 °C shaking waterbath for 30 min, then moved back to the AutoBlot 2000 for the remainder of the test procedure. Each strip was rinsed twice with 2 mL of Rinse Solution prior to the addition of 2 mL of diluted conjugate. The trays were incubated for another 30 min at room temperature (with gentle agitation), rinsed twice with 2 mL of Rinse Solution, rinsed with 2 mL of Substrate Buffer, and incubated for another 30 min (with gentle agitation) after the addition of 2 mL of diluted Substrate Solution. Reactions were stopped by rinsing them twice with 2 mL of Rinse Solution. Strips were allowed to air dry prior to the interpretation of results according to the manufacturer's directions. This assay was designed for the detection of specific mutations previously associated with drug resistance occurring at codons rt180 (528), rt204 (552), and rt207 (555) of the HBV polymerase gene.

HBV quantification

HBV viral load was retrospectively determined for all specimens studied. The second-generation Digene HBV Test utilizing Hybrid Capture® II technology (Digene Corp., Gaithersburg, MD, USA) was performed according to the manufacturer's directions. The dynamic range of this assay extends from 142 000 to 1.7 × 109 copies/mL.

Statistical analysis

A comparison of the sensitivities for INNO-LiPA HBV DR Kit (modified procedure) and the TRUGENE HBV Genotyping Kit was made using a sign test. Exact 95% binomial confidence intervals were calculated. Statistical analysis was performed by means of a McNemar test. A p-value of = 0.05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Patient group I

Results derived from the three patients with HBIG and subsequent nucleoside analogue treatment failure (group I) are shown in Figures 1–3. The three patients exhibited detectable serum HBV DNA at multiple time points during the post-transplant period, and were determined, using the TRUGENE HBV Genotyping Kit, to be infected with HBV genotype C. HBsAg mutations previously associated with immunoprophylactic breakthrough (sG145R or sD144A) were identified in all three patients. These mutations persisted and remained for the duration of the study period (5–6 years) despite discontinuation of HBIG administration for a 4–5-year period. The sD144A mutation present in patient 2 was detected in the pretransplant specimen as well as all subsequent specimens from the patient. A sT189I mutation was also detected in the pretransplant specimen obtained from patient 2; this mutation has previously been reported in a liver transplant recipient with HBIG breakthrough (47). All group I patients had at least one documented anti-HBsAg level of <200 IU/mL while receiving HBIG. Polymerase mutations associated with high-level lamivudine/famciclovir resistance were also readily detected in multiple specimens obtained from these patients using both methodologies. Despite the discontinuation of lamivudine therapy for almost 1 year, rtL180M and rtM204I polymerase mutations were detectable in multiple specimens obtained from patient 1 while receiving adefovir monotherapy (Figure 1). While being treated with adefovir, HBV DNA and hepatitis B e antigen both became undetectable and this patient subsequently developed hepatitis B e antibody positivity. The elapsed time from transplant to HBIG breakthrough ranged from 0 to 530 days among these patients, while the time from the start of lamivudine therapy until the detection of lamivudine resistance-associated mutations ranged from 203 to 915 days.

Although polymerase mutations were associated with increases in HBV DNA levels in all three group I patients, as shown in Figures 1–3, these mutations were not associated with substantial changes in biochemical or synthetic profiles. Allograft biopsies were carried out following the emergence of detectable levels of HBV DNA in all three group I patients. All biopsies demonstrated HBV hepatitis as determined by staining positively for HBV core Ag by immunohistochemistry and exhibiting necroinflammatory activity with progressive fibrosis (stages 2–4). Follow-up biopsies are pending in patients 1 and 3 who had >2 log reductions in HBV DNA levels following initiation of adefovir. Patient 2 had resolution of necroinflammatory activity and no progression of fibrosis stage following 1 year of combined lamivudine and adefovir therapy.

Patient group II

Genotyping results derived from the six patients with HBIG treatment failure post transplant (group II) are shown in Table 2. Group II included: three genotype D, two genotype A, and one genotype C infected patients as determined by the TRUGENE HBV Genotyping Kit. With the exception of patient 2, all group II patients yielded both HBsAg and polymerase mutations post transplant. Among the group II patients, two of the six patients (patients 5 and 6) exhibited the emergence of the HBsAg sG145R mutation previously associated with HBIG and vaccine breakthrough. Another group II patient (patient 3) exhibited the emergence of less-well-characterized HBsAg mutations at aa s144 and s145 (sE/D144G and sG145E) (24). A fourth group II patient (patient 4) demonstrated the emergence of a sT189I mutation within the HBsAg. In addition, a sT118A HBsAg mutation was identified in patient 6 both pre- and post-transplant. The sT118A mutation has been predicted to have a destabilizing effect on structural integrity of the ‘a’ determinant and also to alter the antigenicity profile of the mutant HBsAg in an HBsAg-seronegative chronic liver disease patient (48). All group II patients had at least one documented anti-HBsAg level of <200 IU/mL while receiving HBIG and prior to the date of the post-transplant specimen analyzed. No polymerase mutations were identified in any group II patient using the INNO-LiPA HBV DR Kit.

Table 2. : Results of HBV mutation analysis using the TRUGENE HBV Genotyping Kit, and the INNO-LiPA HBV DR Kit for group II and III patients. HBsAg mutations of known significance are indicated in bold. HBsAg mutations resulting in polymerase mutations are underlined, while associated polymerase mutations are indicated in italics
Kit Digene HBV Test (Hybrid Capture II)TRUGENE HBV Genotyping Kit  INNO-LiPA HBV DR
    Amino acidAmino acidAmino acid changes
PatientSpecimenHBV DNA (copies/mL×103)Genotypechanges in HBsAgchanges in polymerase implicated in drug resistance
  • *

    Specimen collected 1 year following HBIG breakthrough and discontinuation of HBIG.

  • **

    Did not amplify.

Group II
1Pre-transplant50 177DsS207R, sS210S/RrtQ149Q/K, rtQ215S, rtS219S/ANone
 Post-transplant (366 days)254 434DsI110L, sT116N, sS207R, sS210RrtN118T, rtN/H/Y124Q, rtQ215SrtS219ANone
       
2Pre-transplant≥1 700 000AsA194VNoneNone
 Post-transplant (24 days)146 856AsA194VNoneNone
     NoneNone
3Pre-transplant268DsI110I/L, sY134Y/F, sS207RrtN118N/T, rtN/H/Y124D, rtL164M, rtQ215P/S, rtN238N/H, rtY245Y/H, rtS/C256GNone
 Post-transplant≥1 700 000DsY134F, sE/D144D/G, sG145G/E,rtL157L/M, rtL164M, rtQ215P/S,None
 (386 days)  sS207RrtN238H, rtS/C256G 
4Pre-transplant675 392AsP203P/RNoneNone
 Post-transplant* (399 days)857 527AsT189T/INoneNone
5Pre-transplant390CsY200Y/F, sF212F/SrtY124Y/HNone
 Post-transplant2758CsG145R/KrtR/W153QNone
  (498 days)     
6Pre-transplant2691DsT118A, sT/P127T/SrtN118D, rtH126R, rtY/S135Y/F, rtQ149Q/KNone
       
 Post-transplant (730 days)46 017DsT118A, sT126T/I, sT/P127S, sG145RrtN118N/D, rtH126R, rtY/S135F, rtQ149Q/K, rtR/W153Q, rtR/I/G/V266TGNone
       
       
Group III
1Pre-transplant1 015 057AsY161F, sP203P/QNoneNone
2Pre-transplant101 578AsP135P/A, sS204RrtS143S/C, rtS213T, rtH271DNone
3Pre-transplant5645AsT118T/A, sP203P/RrtY126Y/CNone
4Pre-transplant143 138AsG119G/R, sM133M/IrtG127G/E, rtV142V/INone
5Pre-transplant1 519 466ANonertH271QNone
6Pre-transplant<142AsT140T/IrtH271Q**
7Pre-transplant595CNoneNoneNone
8Pre-transplant<142DsT126T/SrtD134D/V, rtQ149K, rtP237TNone
9Pre-transplant161CsY200Y/S/FNoneNone

Patient group III

Group III consisted of six genotype A, two genotype C, and one genotype D infected patients (Table 2). Seven of these patients yielded HBV with S gene mutations and six yielded HBV with polymerase gene mutations with respect to the appropriate genotype consensus sequences used by the TRUGENE HBV Genotyping Kit. However, none of the well-characterized vaccine or HBIG breakthrough or nucleoside analogue resistance-associated mutations were detected in this patient group using the TRUGENE HBV Genotyping Kit. As in group II, a single patient (patient 3) exhibited a T118A S gene mutation. No polymerase mutations were identified using the INNO-LiPA HBV DR Kit.

INNO-LiPA HBV DR and direct DNA sequence analysis

Generally, similar polymerase results were obtained from both assays evaluated in this study. However, the INNO-LiPA HBV DR Kit (modified procedure) lacked the sensitivity of the TRUGENE HBV Genotyping Kit. This was evident by increased failure to amplify HBV products, particularly at relatively low viral loads (10/11, 90.9%, 95% CI [58.7–99.8%] vs. 4/11, 36.4%, 95% CI [10.9–69.2%] at viral loads <300 000 copies/mL; p = 0.031). For group I patients, the INNO-LiPA HBV DR Kit yielded increased detection of mixed populations with respect to polymerase gene codons rt180 or rt204 as compared to the TRUGENE HBV Genotyping Kit (9/20, 45.0%, 95% CI [23.1–68.5%] vs. 3/20, 15.0%, 95% CI [3.2–37.9%]; p = 0.031).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

HBsAg mutants have been described in association with passive and/or active vaccination, in the ‘missed’ diagnosis of acute HBV infection, and in association with failure of HBIG prophylaxis after liver transplantation. Our study (Figures 1–3) shows that HBsAg mutants may persist for years as the predominant strain in liver transplant recipients despite discontinuation of HBIG. This may be considered contrary to the findings of prior studies. For example, Ghany et al. reported that sG145R HBsAg mutants selected or induced by HBIG following liver transplantation tend to revert back to their pretransplant sequences after withdrawal of HBIG (49). Since the overall similarity of HBsAg mutations arising under high-dose HBIG immune pressure and following vaccination suggests identical immune escape phenomena, and since in congenitally infected children, HBsAg mutants, including sG145R, have been shown to persist for years (27,50), it is our opinion that our results are expected. The sG145R HBsAg mutation has recently been described in a survey of nonimmunized Chinese patients with chronic hepatitis B virus infection (35), further demonstrating the long-term viability of such mutant strains. Detailed review of the findings of Ghany et al. reveals that amongst the three patients with sG145R HBsAg mutants studied, mutant virus did in fact remain detectable 5, 9 and 24 months after discontinuing HBIG (in 9/9, 5/13 and 4/10 subclones sequenced from each of the three patients) (49). Therefore, both our data and existing data support persistence, despite withdrawal of HBIG, of sG145R HBsAg mutants selected or induced by HBIG following liver transplantation. These mutant viruses may theoretically infect individuals who have ‘protective’ anti-HBsAg levels as a result of currently available HBV vaccines, including healthcare workers caring for and sexual contacts of transplant recipients (34,51). These findings emphasize the need for development of more effective HBV vaccination strategies (51), an increasing need to screen for HBV vaccine/HBIG escape mutants in chronically infected individuals prior to antiviral therapy and liver transplantation, and the need for effective combination (HBIG and nucleoside analogue) therapies for use in the post-transplant setting.

The existence of a potentially significant HBsAg mutation (sD144A) prior to liver transplantation in group I patient 2 likely explains HBV breakthrough in this particular patient following liver transplantation despite HBIG immunoprophylaxis (Figure 2). Patients in whom a well-characterized HBsAg escape mutant is present prior to transplant are very likely at high risk for HBV recurrence using HBIG monotherapy. Whether the use of combination (HBIG and nucleoside analogue) therapy in such patients would improve the outcome following liver transplantation is unknown. While HBsAg mutations at aa s144 and s145 comprise the majority of the escape mutants reported to date, a variety of other mutations have been described in association with HBIG breakthrough following liver transplantation. In group II patients, HBsAg mutations of known significance were not detected in all cases. It is likely that in addition to the emergence of HBsAg escape mutants, other factors may contribute to HBV re-infection. For example, patients with active HBV replication pretransplant (i.e. hepatitis B e antigen and HBV DNA positive) have been shown to have higher rates of re-infection despite HBIG immunotherapy (52). Additionally, a fixed dose of HBIG may be insufficient to maintain therapeutic levels of anti-HBsAg in all patients. Early re-infection in some patients may have been related to overwhelming quantities of circulating virus, release of virus from extrahepatic reservoirs, or increased viral replication. While both HBsAg and polymerase mutants were detected in the majority of group III patients pretransplant, no mutations previously associated with either HBIG escape or lamivudine resistance were detected in this patient group. Despite our incomplete understanding of all of the factors involved in HBIG breakthrough, the detection of well-characterized vaccine/HBIG escape mutants in liver transplant candidates prior to transplant or in transplant recipients following HBIG breakthrough may put these patients at increased risk for HBV recurrence and preclude the use of passive and/or active immunization strategies in these patient groups.

It has recently been shown that HBsAg mutations located at aa positions s195 and s196 (Figures 1–3), resulting from the selection of HBV polymerase mutants during lamivudine therapy, demonstrate reduced binding to anti-HBs antibody, raising concerns about the selection of HBsAg mutants during long-term nucleoside analogue therapy (53). These findings also suggest that HBV mutation analysis may have clinical utility in predicting the effectiveness of HBIG or HBV vaccination in liver transplant recipients with previous nucleoside analogue exposure.

Previous reports have documented decreased replicative capacity of polymerase mutants (54–57). In contrast to these findings, rtL180M and rtM204I polymerase mutations were readily detectable in group I patient 1 following the discontinuation of lamivudine therapy and while being treated with adefovir alone (Figure 1). These mutations were detectable by both methodologies studied in multiple specimens obtained over a 1-year period. To date, the use of adefovir has not been associated with any resistance-associated polymerase mutations (58,59). Interestingly however, the polymerase mutation rtR/W153Q, which has been recently shown to restore replication competence to rtL180M/M204V mutants (60), was detected in this patient. Selection of the rtR/W153Q mutation as a compensatory mutation in response to HBIG exposure followed by selection of rtL180M/M204I mutations may have resulted in selection of a replication-competent L180M/M204I strain in this patient.

In addition to simply restoring replication fitness, another recent report demonstrated that severe and fatal HBV infection can occur during lamivudine therapy with certain HBV variants exhibiting combinations of HBsAg (sP120T and sG145R) and polymerase (rtL180M plus rtM204I) mutations selected during sequential HBIG and lamivudine therapy in the post-transplant setting (61). Not only did these compensatory mutations restore replication competence, but they appeared to result in increased levels of replication in the presence of lamivudine, thus suggesting that the continuation of lamivudine therapy in some patients may result in an exacerbation of liver disease.

The persistence of lamivudine-resistance mutants in a nontransplant patient has recently been described (62) and further emphasizes the clinical utility of screening for HBV polymerase mutants in liver transplant candidates previously exposed to nucleoside analogues (62).

To the best of our knowledge, there are no previously published evaluations of the TRUGENE HBV Genotyping Kit. We identified several advantages of the TRUGENE HBV Genotyping Kit as compared to the INNO-LiPA HBV DR Kit. The former identifies all polymerase mutations (within the region analyzed), including those that are identified by the latter as well as other, less-well-characterized mutations. As our understanding of the significance of these less-well-characterized mutations expands and new nucleoside analogue therapies become available, it may be advantageous to have an assay available that is capable of readily identifying these mutations. A further strength of the TRUGENE HBV Genotyping Kit is that HBsAg mutations are identified and viral genotype is determined concurrently. Recent studies also suggest that determination of HBV genotype may predict both therapeutic response (63), and clinical outcome (12,14,18,19).

The INNO-LiPA HBV DR Kit provides information regarding the presence or absence of specific polymerase gene mutations at aa rt180 (528), rt204 (552) and rt207 (555); other polymerase gene mutations, albeit of undetermined significance, are not identified (64). A recent report described discrepancies between the results obtained using the INNO-LiPA HBV DR Kit and direct sequencing that could be attributed to HBV polymorphisms not covered by the INNO-LiPA HBV DR Kit probes (65). On the other hand, the strength of the INNO-LiPA HBV DR Kit is the reverse hybridization probe design, which enables enhanced detection of mixed populations of mutant and wild-type virus, and potentially therefore, earlier detection of mutant virus. Consistent with our findings, several recent evaluations of the INNO-LiPA HBV DR Kit, as compared to direct sequence analysis, have shown that the former identified minor subpopulations not identified by the latter (65,66). The sensitive detection of viral subpopulations may be important in unraveling the dynamics of emerging HBV resistance and may improve the monitoring of antiviral therapy.

In this study, the overall sensitivity of the INNO-LiPA HBV DR Kit (modified procedure) was less than that of the TRUGENE HBV Genotyping Kit. It is important to note, however, that nested PCR amplification (as recommended by the manufacturer) was not utilized in this study due to concerns regarding PCR amplification reaction cross-contamination, which are especially relevant in clinical microbiology laboratories. The INNO-LiPA HBV DR Kit does not provide information regarding HBV genotype or HBsAg mutants, although the former could potentially be determined using the same amplification products and techniques (20).

In summary, while the number of patients included in this study is small, the data presented herein suggest that HBsAg mutations can persist for extended periods (at least 5 years) in the absence of HBIG administration, and therefore do not support prior results highlighting the disappearance of HBsAg mutants in liver transplant recipients after discontinuation of HBIG. HBV DNA detection and quantification can be important in determining the presence or absence of active disease, assessing the likelihood of HBV recurrence post liver transplant, and monitoring for viral breakthrough or the emergence of nucleoside analogue resistance. Our results suggest that the ability to detect pre-existing HBsAg mutations and/or nucleoside analogue resistance mutations as well as determine HBV genotype in potential liver transplant candidates may be important in more accurately assessing the likelihood of HBV recurrence and determining the most appropriate therapy for these patients. Regarding the clinical relevance of polymerase mutations which emerge post transplant, we observed histological recurrence of chronic HBV infection in the absence of substantial biochemical or synthetic profile changes, perhaps reinforcing the need for histological evaluation of patients who develop HBV recurrence/breakthrough. As therapeutic strategies for liver transplant recipients continue to evolve and new therapeutic agents become available for the treatment of HBV, the need for rapid and reliable detection of emerging HBV mutants, including polymerase and HBsAg mutants, while patients are on therapy will likely become increasingly important in the selection and modification of therapeutic drug combinations. The TRUGENE HBV Genotyping Kit and the INNO-LiPA HBV DR Kit can provide clinicians with timely information regarding the existence of HBV polymerase mutants. The TRUGENE HBV Genotyping Kit also simultaneously determines HBV genotype and identifies HBsAg mutants. Based on lessons learned from other microbes (e.g. human immunodeficiency virus, Mycobacterium tuberculosis), identifying antimicrobial resistance is key to successful therapy.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We acknowledge Mimi Healy and Joe T. Huong (Visible Genetics Inc., Suwanee, Georgia) for technical assistance, Visible Genetics Inc. and INNOGENETICS N.V. (Ghent, Belgium) for provision of reagents, Ms. Gretchen A. Thomason for excellent secretarial assistance, Mr P. Shawn Mitchell for his thoughtful review of the manuscript, and Mr William Scott Harmsen for assistance with statistical analysis. This study was reviewed and approved by the Mayo Institutional Review Board (#956–00).

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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