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Abstract

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. REFERENCES

Viral differences among lamivudine resistant hepatitis B (HBV) genotypes have not been yet investigated. Therefore, we analyzed the characteristics of these viral strains in vivo. Forty-one patients carrying lamivudine resistant HBV were enrolled. Twenty-six patients (63%) carried resistant HBV genotype A (group A) and 15 patients (37%) carried resistant HBV genotype D (group D). The rate of reverse transcriptase 204I mutants was significantly higher in group D (67%) compared with group A (19%), whereas rt204V mutants (81% in group A vs 33% in group D; P = .006) and rt180M mutants (81% in group A vs 40% in group D, P = .015) prevailed in group A. The median time of shift from rt204I to rt204V mutants was significantly shorter in group A (4 months in group A, >12 months in group D, P < .001). Additional resistance associated mutations were detected exclusively in group D (P = .004). In a multivariate analysis, HBV genotype (P = .039) and pretreatment serum HBV DNA (P = .001) were independently associated with emerging rt204I or rt204V mutants, respectively. Serum HBV copy numbers after emergence of resistance were higher in group A (mean log10 6.99 copies/ml; range 3–9) compared with group D (mean log10 6.1 copies/ml; range 3.3–8; P = .04). There was no difference between both groups regarding core promoter/precore mutations, viral turnover, and number of flares or disease progression during follow-up. In conclusion, the mutational pattern during selection of lamivudine resistant HBV strains differs between genotypes A and D. This may have consequences for a salvage regimen initiated for treatment of lamivudine resistant HBV. (HEPATOLOGY 2004;39:42–50.)

The emergence of drug resistant hepatitis B virus (HBV) during lamivudine treatment for chronic hepatitis B is a major problem with an incidence of 14–36% after 1 year of treatment.1–4 This frequency increases to 38%, 49%, and 66% after 2, 3, and 4 years of treatment, respectively.5–7 Lamivudine resistant HBV is characterized by amino acid variations in the reverse transcriptase domain of the HBV polymerase. In particular, an exchange of the methionine within the YMDD motif by an isoleucine or a valin (rtM204I/V mutants) is associated with lamivudine resistance. Breakthrough of these drug-resistant HBV mutants leads to a viral rebound to baseline levels,8, 9 to a decrease in the rate of loss of hepatitis B e antigen (HBeAg),10 a high rate of relapses of serum alanine transaminase (ALT) levels,11, 12 and worsening liver histology.13 Therefore, the emergence of viral resistance is one of the critical issues in the long-term outcome of patients treated for chronic hepatitis B. On the other hand, lamivudine resistant HBV is considered to have reduced viral fitness due to less replication efficiency in vitro14 and lower ALT levels in vivo as compared with baseline levels.4, 15, 16 This led to the recommendation to continue lamivudine treatment despite the emergence of resistant variants as long as benefit to the patient is maintained.17 Taken together, it would be useful to identify factors which are associated with a better outcome of lamivudine treatment after the emergence of resistance.

In previous studies we found that, compared with HBV genotype A, genotype D showed a significantly better response with respect to HBV DNA decrease during the first 12 months of lamivudine treatment as measured by quantitative real-time polymerase chain reaction (PCR).18 Also, the incidence of lamivudine resistance after 18 months was lower in patients carrying HBV genotype D.18, 19 Since differences between HBV genotypes B and C have been demonstrated to influence the response during treatment with interferon alfa,20 there is increasing evidence that HBV genotypes are potential viral factors that influence the outcome of antiviral therapy for chronic hepatitis B. This prompted us to investigate whether lamivudine resistant HBV variants with genotypes A and D, which are the most prevalent genotypes in our cohort, exhibit different viral features in vivo. We retrospectively analyzed the pattern of resistance-associated mutations, core promoter (CP)/precore mutations, virus levels, and viral turnover of lamivudine resistant hepatitis B genotypes A and D.

Patients and Methods

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. REFERENCES

Patients, Study Design, and Definitions

In a retrospective study, all consecutive patients were analyzed who underwent lamivudine monotherapy for chronic hepatitis B for at least 12 months between December 1995 and February 2003 at the University Hospitals of Hamburg and Vienna. Seventy-nine patients were included. The median time of treatment in these patients was 18 months (range 12–60). Thirteen patients (16%) had undergone renal transplantation, 15 patients (19%) had received a liver transplant, and 51 (65%) were immunocompetent patients. Forty-one patients (53%) developed lamivudine resistant HBV during a median treatment duration of 12 months (range 6–29). Lamivudine resistance was defined as detection of a rt204I or a rt204V HBV variant with or without additional resistance associated mutations within the HBV polymerase. These 41 patients (35 men and 6 women) were further analyzed. All patients had been positive for hepatitis B surface antigen (HBsAg) for at least 6 months before treatment. They had received 100 mg lamivudine daily and continued lamivudine therapy after emergence of resistance. The study design is shown in Fig. 1. Serum samples were obtained every 3 to 4 months during treatment before resistance and after resistance had emerged. In case of a positive result for serum HBV DNA in the polymerase chain reaction (PCR), the HBV polymerase was sequenced for resistance associated mutations by population-based sequencing. In our analysis, the first sample of each patient after breakthrough of lamivudine resistant HBV was investigated cross-sectionally, respectively. After the first detection of resistant HBV, patients were followed for 12 months during continuous lamivudine treatment. The endpoint of this follow-up was set as disease progression (defined as ALT > 2× the upper limit of normal (ULN) and serum HBV DNA levels >log 7/ml in at least 2 samples making it necessary to change antiviral treatment). Flares were defined as ALT level >10× ULN.17 For calculation of the turnover of resistant HBV, HBV DNA was quantified in the final serum sample with lamivudine sensitive HBV and compared with the HBV DNA concentration detected in the first sample with resistant HBV.

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Figure 1. Design of this retrospective study.

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Serological Testing

From every serum sample HBV surface antigen (HBsAg), HBeAg, anti-HBs, anti-HBe and anti-HBc were determined by EIA (Axsym, Abbott Laboratories, Wiesbaden, Germany).

Quantitation of HBV DNA, Lamivudine Resistance Testing, and HBV Genotyping

HBV copy numbers were quantified by real time PCR (LightCycler-DNA Master SYBR GreenI, Roche Diagnostics, Basel, Switzerland) targeting a conserved region of the HBV genome, which overlaps the genes encoding the X-protein and DNA-polymerase.21 Primer pairs HBV1F (5′-CCGTCTGTGCCTTCTCATCTG-3′) and HBV1R (5′-AGTCCAAGAGTYCTC TTATGYAAGACCTT-3′) were used, and amplicons were quantified by fluorescent activities compared with serial dilutions of an external standard preparation (cloned HBV, plasmid pHBV991), which was calibrated with two Eurohep reference samples containing HBV DNA of genotype A and D, respectively.22 The detection limit of the PCR was shown to be 102 HBV genomes/ml serum (20 WHO U/ml serum) for both genotypes with a dynamic range up to 109 genomes/ml. The reproducibility was high as shown by repeated measurements of 40 sera (correlation coefficient r = .96, P < .0001), obtained from chronic HBV carriers. These sera were tested independently in triplicate, respectively (data not shown).

To investigate resistance-associated mutations, a recently published nomenclature was used.23 Samples with a positive result in the real-time PCR assay were submitted to sequencing of the HBV polymerase region. This region was amplified by a nested PCR with primers 252 (5′-AGACTCGTGGTGGACTTCTCT-3′)/1309 (5′-AGAATGTTTGCTCCAGACC-3′) as external primers and 377 (5′-GGATGTGTCTGCGGCGTTT-3′)/840 (5′-ACCCCATCTT TTTGTTTTGTTAGG-3′) as internal primers spanning the polymerase region from codon rt103 to codon rt244.24 Both strands of the amplification products were sequenced for resistance-associated mutations (rtF166L, rtV173L, rtL180M, rtA200V, rtM204I, rtM204V, and rtV207I) by BigDye termination chemistry (Applied Biosystems) using an automated sequencer (AbiPrism). The identical region and procedure was used for the determination of HBV genotypes.25 A phylogenetic analysis of all sequences was performed using MEGA 2.126 with reference sequences of different HBV genotypes (GenBank accession numbers: genotype A: V00866; genotype B: D23677; genotype C: D00630; genotype D: V01460; genotype E: X75657; genotype F: X75658; genotype G: AF160501).

Population Analysis of Resistant HBV by Cloning and Sequencing

Thirteen patients carried a rt204I variant as the predominant strain in population-based sequencing (5 patients with HBV genotype A and 8 patients with genotype D). In these patients consecutive follow-up samples were investigated for 12 months. The endpoint was set as shift to a rt180M/rt204V mutant as the predominant variant (>50% of the viral population) or disease progression. A total of 38 samples were analyzed. The polymerase region was amplified from each sample. Amplicons were cloned into Escherichia coli using the TOPO-TA cloning system (Invitrogen, Karlsruhe, Germany) and 12 to 20 clones were sequenced and analyzed on an automatic sequencer (Beckmann CEQ2000) as described above.

Analysis of Precore/Core Promoter Mutations

Core promoter (CP) and precore sequences of 27 patients were amplified using a nested PCR as described previously.27 The external primers were 5′-CATAAGAGGACTCTTGGACT-3′ (sense, nt 1,653 to 1,672) and 5′-GGCGAGGGAGTTCTTCTTCTAGGGG-3′ (antisense, nt 2,394 to 2,369). Internal primers were 5′-AATGTCAACGACCGACCTTG-3′ (sense, nt 1,679 to 1,698) and 5′-AGCTGAGGCGGTGTCGAGGAGATC-3′(antisense, nt 1,985 to 2,009), which were also used as sequencing primers. The sequences were analyzed for CP mutations with nucleotide exchanges at positions A1762T and G1764A and for precore mutations at codon 28 (M2 stop codon mutant with the nucleotide exchange G1896A) and codon 29 (M4 mutant with the nucleotide exchange G1899A/G1900C).

Calculation of Turnover Rates

During breakthrough of resistant HBV the serum HBV DNA concentration regularly increases. Therefore, the turnover of resistant HBV can be calculated for each patient using the kinetics of HBV DNA levels between the time of the last serum sample with lamivudine sensitive HBV and the first serum sample showing breakthrough of resistant HBV (Fig. 1). An HBV load below the limit of detection of the PCR was considered to represent lamivudine sensitive HBV. A linear model was used for the calculation of turnover rates.28 Briefly, the turnover z(t) was calculated by dividing the concentration (c) change of HBV DNA by the time in days between the measurements of the last serum sample with sensitive HBV and the first sample in which resistant HBV could be detected (tsens and tres) according to the formula:

  • equation image

Assuming that 1 copy of HBV DNA is located within 1 infectious particle of HBV, the number reflects the turnover of virions per milliliter per day. The assumption was made in this formula that there was a change of a 100% wild type HBV to a 100% resistant HBV. However, since there is a gradual shift from predominantly wild type to predominatly resistant HBV in reality, we performed a second calculation. In this calculation, we took into account that the detection limit for minor populations is 20–30% in our population-based sequencing assay. Therefore, we adjusted c(tsens) by the factor .3 assuming that already 30% of HBV was resistant in the population tested as sensitive. Also, c(tres) was adjusted by the factor .7 assuming that 30% of HBV still was wild type virus within the resistant population. This led to the adjusted formula:

  • equation image

The assumption of a constant proportion of 30% minor species is a “worst case scenario” and may be not precise in every patient. However, this is a systematic mistake and should apply for both genotype groups. Therefore, the turnover values are not absolute but allow comparison of resistant HBV genotypes with each other.

Statistical Analysis

For statistical analysis the WinStat, Statistica, and SPSS software package were used. Categoric data were analyzed using the 2-sided Fisher exact test and the Yates' corrected chi-square test where appropriate. Continuous variables were compared with the Mann-Whitney-Wilcoxon test. A Kaplan-Meier estimate and the Cox-Mantel log-rank test were used to calculate the median time and significance for 1) the selection of resistant variants from baseline, and 2) for the shift of rt204I to rt204V mutants. Stepwise logistic regression (outcome variable: mutational pattern at codon rt204) and the Cox's proportional hazard model (outcome variable: time to lamivudine resistance) were used for multivariate analysis. The split plot repeated measures model was performed to compare the longitudinal HBV DNA levels during follow-up in both genotype groups. A P-value of less than .05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. REFERENCES

Patient Characteristics

Forty-one patients selected resistant HBV after a median treatment duration of 12 months (range 6–29). The subgroup analysis revealed that 6 of 13 (46%) renal transplant patients, 10 of 15 (67%) liver transplant patients, and 25 of 51 (49%) immunocompetent patients developed lamivudine resistance. These differences were not significant. Twenty-six (63%) patients carried a resistant HBV mutant with genotype A (group A) and 15 (37%) patients carried resistant HBV with genotype D (group D). The median time to lamivudine resistance was 12 months in group A (range 6–26) and 16 months in group D (range 8–29). This difference was statistically significant in a univariate analysis using either the Mann-Whitney-Wilcoxon test (P = .01) involving only patients with lamivudine resistance or the Cox-Mantel log-rank test (P = .04) including the 79 patients of the total cohort. A multivariate analysis (including the covariates pretreatment HBV DNA and ALT levels, HBeAg status, and HBV genotype) revealed that only low pretreatment ALT levels were slightly, but significantly associated with a shorter time to lamivudine resistance (OR .989, 95%CI .981–.996, P = .003). There was a trend for a more rapid selection of lamivudine resistance in HBV genotype A (OR 1.6, 95%CI .8–3.3, P = .18).

Demographic characteristics of all patients were comparable with respect to age and sex. Also, the HBeAg status at baseline and after emergence of resistance, and the immune status was comparable between group A and group D (Table 1). The time between the last serum sample with lamivudine sensitive HBV and first serum sample with resistant HBV did not differ significantly.

Table 1. Characteristics of Patients Carrying Resistant Hepatitis B Virus
CharacteristicsGenotype A (n = 26)Genotype D (n = 15)P
  • Abbreviations: HBeAg, hepatitis B e antigen; wt, wildtype HBV; n.s., not significant; CP, core promoter.

  • *

    Core promoter (CP) and precore sequences were available for 27 patients; P > 0.05, respectively.

Baseline (pretreatment)   
 Male/female21 (81%)/5 (19%)14 (93%)/1 (7%)n.s.
 Age (years, mean ± SD)46.3 ± 9.646.2 ± 15.2n.s.
 HBeAg positive/negative22 (85%)/4 (15%)9 (60%)/6 (40%)n.s.
 Serum HBV DNA (log10copies/mL, mean ± SD)7.08 ± 1.16.57 ± 0.9n.s.
 ALT (U/L, mean ± SD)57.5 ± 39.769.4 ± 42.7n.s.
First sample with resistant HBV   
 Median time to resistant HBV from baseline in months (range)12 (6–26)16 (8–29)0.01
 Time between samples with last sensitive and first resistant HBV (days, mean ± SD)111.8 ± 18.0123.9 ± 30.9n.s.
 HBeAg positive/negative22 (85%)/4 (15%)8 (53%)/7 (47%)n.s.
 Serum HBV DNA (log10copies/mL, mean ± SD)6.99 ± 1.466.1 ± 1.270.03
 Virus turnover (virions/mL/day, mean ± SD)5.5 × 105 ± 2 × 1032 × 105 ± 1.5 × 103n.s.
 Virus turnoveradjusted (virions/mL/day, mean ± SD)3.3 × 105 ± 1.5 × 1031 × 105 ± 3 × 103n.s.
 Polymerase mutational pattern   
  rt173wt/rt173L26 (100%)/0 (–)12 (80%)/3 (20%)0.04
  rt180wt/rt180M5 (19%)/21 (81%)9 (60%)/6 (40%)0.015
  rt200wt/rt200V26 (100%)/0 (–)13 (87%)/2 (13%)n.s.
  rt204I/rt204V5 (19%)/21 (81%)10 (67%)/5 (33%)0.006
 CP mutations (nt1762T/nt1764A)*4/12 (33%)8/15 (53%)n.s.
 Precore mutation (nt1896 Stop)*3/12 (25%)8/15 (53%)n.s.
Follow-up for 12 monthsn = 22n = 12 
 HBeAg positive/negative18/46/6n.s.
  Loss of HBeAg0/180/6n.s.
  Relapse of HBeAg0/41/6n.s.
 Serum HBV DNA (log10copies/mL, mean ± SD) (no. of samples available)7.13 ± 1.3 (80)5.79 ± 2.0 (46)0.04
 Number of flares (>10 × ULN)1/220/12n.s.
 Disease progression8/225/12n.s.

Patterns of Resistance Associated Mutations

There were significant differences between group A and D with respect to the mutational pattern within the YMDD motif. Codon rt204I mutants (n = 15) were more frequent in group D (n = 10), while rt204V mutants (n = 26) were more prevalent in group A (n = 21; P = .006). Also, a higher incidence of the rt180M substitution was found in group A (81% vs. 40% in group D; P = .015). Additional mutations within the polymerase gene were found in 5 patients of group D (3 patients with rtV173L, 2 patients with rtA200V), but in none of group A (P = .004). A multivariate analysis revealed that HBV genotype (P = .039) and pretreatment serum HBV DNA (P = .01) were independently associated with the emerging mutational pattern at codon rt204. HBV genotype A had a 6-fold higher probability of selecting a valin substitution at this codon compared to genotype D (95% CI 1.1-33.6, P = .039) (Table 2).

Table 2. Influence of Baseline Parameters on the Emerging Pattern of Resistance Associated Mutations
Patients ParameterUnivariate P-ValueMultivariate
OR (95% CI)P-Value
  • –, not included in the multivariate analysis.

  • *

    HBeAg was excluded from the analysis during stepwise logistic regression.

Age0.22 
Sex0.18 
Immune status0.46 
Pretreatment ALT level0.4 
Pretreatment HBV DNA0.0033.3 (1.3–8.3)0.01
HBV genotype0.0086.0 (1.1–33.6)0.039
Pretreatment HBeAg status0.02* 

At baseline, the HBV DNA levels did not differ significantly between genotypes A and D. However, after resistance had emerged the HBV DNA level was significantly higher in group A. The mean serum HBV copy number was log10 6.99 copies/ml (range 3–9) in group A compared with log10 6.1 copies/ml (range 3.3–8) in group D (P = .04). After stratification for the mutational patterns at codons rt180 and rt204, a significant difference could not be observed for HBV copies between groups A and D. The viral turnover of resistant variants was not different in group A and D (5.5 × 105 virions/ml/day vs. 2 × 105 virions/ml/day; P = .2). Also, the adjusted turnover values did not differ significantly (3.3 × 105 virions/ml/day in group A and 1 × 105 virions/ml/day in group D, Table 1).

Follow-up

Patients were followed for 12 months while under lamivudine. Samples (n = 126) were available in median intervals of 4 months (range 1–9). Seven patients were lost for follow-up. Hence, a total of 34 patients were evaluated (genotype A: n = 22; genotype D: n = 12). Twenty-one patients carried a rt204V mutant (genotype A: n = 17, genotype D: n = 4) and 13 patients were infected with a rt204I mutant (genotype A: n = 5, genotype D: n = 8). The mean level of serum HBV DNA was significantly higher in group A during the 12 month period (P = .04)(Table 1). There was no difference in the number of flares between both groups (group A: n = 1, group D: n = 0). Also, the rates of disease progression were similar in both genotype groups (genotype A: n = 8, genotype D: n = 5). Disease progression was independent of the mutational pattern at codon rt204: 5 of 13 patients (38%) carrying a rt204I mutant and 8 of 21 patients (38%) infected with a rt204V mutant had disease progression during the follow-up period. Loss of HBeAg was not detected in any patient after 12 months. Relapse of HBeAg occurred in 1 patient of group D who was infected with a rt204I mutant.

Stability of the rt204V Mutation

Population-based sequencing was performed from each sample drawn from the patients infected with a rt204V mutant. None of these patients lost the rt204V mutant or selected a rt204I mutant, neither as a minor population within a mixture nor as the predominant strain.

Stability of the rt204I Mutation

Patients carrying an HBV variant with a rt204I mutation were monitored for additional 12 months to further analyze the changes of the mutational patterns during continuous lamivudine treatment. Follow-up samples were not available for 2 patients so that a total of 38 serum samples of 13 patients were investigated. Five of these patients had disease progression during follow-up so that lamivudine was either discontinued (n = 2) or famciclovir (n = 1) or adefovir (n = 2) was added to lamivudine. Further analysis of the mutational pattern was not carried out after this change of treatment. In population based-sequencing, 4 of 5 patients with genotype A shifted from the rt204I variant to the rt204V within a median time of 3 months (range 1–12) (Table 2). On the other hand, a shift was noted in only 1 of 8 patients carrying an HBV genotype D with a rt204I mutation after 12 months (Table 3). This difference was significant using either the Fisher's exact test (P = .03) or the Cox-Mantel log-rank test (P < .001).

Table 3. Follow-up of Patients Carrying a Codon rt204I Variant
Patient No.HBV Genotypert180M at Baseline*Shift From rt180wt to rt180MShift From rt204I to rt204VTime (months)No. Samples Sequenced
  • Abbreviations: wt, wildtype HBV; n.s., not significant. Patient no. identical to Fig. 2.

  • *

    Baseline was set as the first serum sample with breakthrough of resistant HBV.

  • Patients with disease progression.

  • Kaplan Meier estimate for the median time to the shift from rtM204I to rtM204V.

  • §

    Kaplan Meier estimate not possible.

1ANoNoNo32
2AYesYes12
3AYesYes124
4ANoYesYes42
5ANoYesYes124
6DYesNo122
7DYesNo124
8DNoNoNo32
9DNoNoNo123
10DNoNoNo124
11DNoNoNo92
12DNoNoNo124
13DNoYesYes123
TotalA2/52/34/5314
 D2/81/61/8§24
P-value n.s.n.s.0.030.0004 
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Figure 2. Proportion of wild type HBV and lamivudine-resistant variants as determined by cloning and sequencing of the HBV polymerase derived from consecutive samples. Five patients had disease progression (DP). Further analysis of the mutational pattern was not possible (n.p.) in these patients.

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To investigate the shift of resistant HBV populations in more detail we performed cloning experiments from these longitudinal samples. Sequencing of 12–20 cloned HBV genomes isolated from each serum sample showed that in 15/38 (39%) of these samples mixtures of wild type HBV and resistant variants or mixtures of resistant variants with different mutational patterns were present (Fig. 2). The only HBV variants which were likely to be selected as a major variant during continous lamivudine treatment were rt180M/rt204I and rt180M/rt204V. Again, the shift of a major rt204I population to a major rt204V population occurred more rapidly in HBV genotype A compared with genotype D.

CP and Precore Mutations

CP/precore sequences could be amplified from 27 patients. Precore mutations were detected in 11 (41%) and CP mutations in 12 (44%) of these 27 patients. Although there was a trend towards a higher rate of precore mutations in patients carrying HBV genotype D, the differences of CP/precore mutations between both genotype groups did not reach statistical significance (Table 1). Precore mutations were detected exclusively in patients negative for HBeAg (n = 11). All of these patients showed either a nt1896 stop codon mutant (n = 5), or a nt1896 stop codon mutant combined with a G1899A mutant (n = 6). CP mutations (n = 12) were found in 9 patients negative for HBeAg and in 3 patients positive for HBeAg. Serum HBV DNA concentrations were not significantly different between patients carrying HBV variants with CP mutations or precore mutations compared with patients without these mutations.

Discussion

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. REFERENCES

The results of our study show for the first time that the emergence of resistance-associated mutations during lamivudine treatment follows different patterns in HBV genotype A and D. In previous studies28–30 it was shown that the first mutation which appears during selection of lamivudine resistant HBV is an isoleucine exchange at codon rt204 (rtM204I) within the YMDD motif of the HBV polymerase. If lamivudine treatment is continued, the isoleucine is replaced in most patients by a valine (rtM204V), often in parallel with a methionine substitution at codon rt180 (rtL180M). Since the rtM204I variant is replaced completely by the rtL180M/rtM204V variant, this succession has been interpreted as a gain of viral fitness for resistant HBV in vivo. After emergence of resistance, the rtL180M/rtM204V mutant has been described to represent a stable viral population during continuous lamivudine treatment.30 In our investigation, the frequency of the rtM204I mutation was significantly higher in group D, while the rtM204V mutation was more prevalent in group A. It can be concluded from this cross-sectional analysis that HBV genotype A has a preference for a valin at codon rt204 (YVDD mutant) during the emergence of lamivudine resistance. This is further confirmed by the longitudinal observation that the mean time for a shift of the rtM204I mutant to a rtM204V mutant was significantly shorter in genotype A carriers. Our data strongly indicate that the rtL180M/rtM204V variants represent the most stable virus population in HBV genotype A infection after emergence of resistance. However, hepatitis B genotype D populations persist for long periods with a stable rtM204I mutation as the predominant viral strain. In this context, it is interesting to note that in a recent study,31 involving exclusively HBV genotype D carriers, 22/30 (73%) patients were infected with a rtM204I variant, a rate similar high to that found in our study (67%). Taken together, the succession of resistance-associated mutations from rtM204I to rtM204V appears to occur mainly in genotype A carriers, but to a significantly lesser extent in genotype D carriers.

The underlying mechanism for these different prevalences of the mutational patterns in HBV genotypes A and D is unclear. HBV genotypes show the greatest genetic diversity in the surface gene. The surface gene, however, overlaps with the reading frame of the HBV polymerase. Hence, mutations within the polymerase gene may change the antigenicity of the HBsAg in different ways in genotypes A and D. This could have impact on the succession of resistance-associated mutations. This was postulated by our group using Chou-Fasman predictions for the hydrophobicity of the HBsAg in lamivudine sensitive and resistant HBV with genotypes A and D, respectively.19 In this analysis, the HBsAg of genotype D became more hydrophilic after the emergence of lamivudine resistance at codon 198 which affects a conformational region of the HBsAg protein.32 This was not found for the HBsAg of resistant hepatitis B genotype A. In a recent study,33 it has been shown that lamivudine selects for mutations that change the antigenicity of the HBsAg, leading to reduced binding of HBsAg to anti-HBs antibodies. We conclude that, besides other reasons, immunological effects may have great impact on the succession of resistance-associated mutations.

The incidence of lamivudine resistance has been demonstrated to be higher in patients who are positive for HBeAg compared with HBeAg negative patients.4 A loss of HBeAg is often due to the emergence of HBV variants with CP/precore mutations.34 It has been shown in vitro, that precore mutants can compensate for the replication deficiency in lamivudine resistant HBV.35 These mutations have also been shown to be associated with genotype D.36 However, our in vivo data suggest that CP/precore mutations in lamivudine resistant HBV are neither associated with certain mutations within the HBV polymerase nor with higher HBV-DNA yield. Hence, CP/precore mutations appear to have no influence on the mutational pattern arising in the HBV polymerase during lamivudine treatment in vivo.

The most prevalent HBV genotype in North America and Europe is genotype A.37 An important aspect of our study is that this genotype is associated with complex mutational patterns during lamivudine treatment inducing cross-resistance to other antiviral drugs, such as the rtL180M mutation. This mutation has been shown to be a key mutation associated with famciclovir resistance.38 Hence, it can be expected from the epidemiological data that the prevalence of patients carrying HBV with mutations inducing cross-resistance will increase dramatically in regions in which lamivudine is used widely as a monotherapeutic agent. In particular, the ability to transmit rtL180M/rtM204V mutants even to persons receiving high-dose lamivudine39 raises concerns about the use of lamivudine as a first-line drug. This may apply particularly to patients infected with HBV genotype A. However, studies involving a greater number of patients are required to assess the optimal strategy for the initial treatment of chronic hepatitis B.

In conclusion, the results of this study show for the first time that viral differences exist in vivo between lamivudine resistant hepatitis B genotypes A and D. In particular, the HBV genotype seems to determine the emerging pattern of resistance-associated mutations, the stability of the mutations at codon rt204, and the level of viremia after selection of lamivudine resistant HBV. Our data suggest that the selection of resistant HBV during lamivudine treatment is a multifactorial process in which the replicative capacity of the virus (as measured by pretreatment HBV DNA), the host immune response (reflected by pretreatment ALT values), and the HBV genotype play important roles. The different mutational patterns of resistant HBV genotypes A and D may have impact on treatment strategies for lamivudine resistant HBV.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. REFERENCES

The authors thank Anja Koppe, Petra Schillemeit, and Bianca Lepping for excellent technical assistance, and Volker Schoder (Institute for Data Sciences in Medicine, Hamburg) for performing the multivariate analysis.

REFERENCES

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. REFERENCES
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    Dienstag JL, Schiff ER, Wright TL, Perillo PR, Hann HWL, Goodman Z, Crowther L, et al. for the U.S. Lamivudine Investigator Group. Lamivudine as initial treatment for chronic hepatitis B in the United States. N Engl J Med 1999; 341: 12561263.
  • 3
    Schalm SW, Heathcote J, Cianciara J, Farrell G, Sherman M, Willems B, Dhillon A, et al. Lamivudine and alpha interferon combination treatment of patients with chronic hepatitis B infection: a randomised trial. Gut 2000; 46: 562568.
  • 4
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