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JAC Advance Access originally published online on June 13, 2007
Journal of Antimicrobial Chemotherapy 2007 60(2):341-349; doi:10.1093/jac/dkm187
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Emergence of hepatitis B virus quasispecies with lower susceptibility to nucleos(t)ide analogues during lamivudine treatment

F. Moriconi1, P. Colombatto1, B. Coco1, P. Ciccorossi1, F. Oliveri1, D. Flichman1, A. M. Maina1, R. Sacco1, F. Bonino2 and M. R. Brunetto1,*

1 UO Gastroenterologia ed Epatologia Ospedaliera, University Hospital of Pisa, Pisa, Italy 2 Fondazione IRCCS Policlinico, Via Francesco Sforza 28, 20122 Milano, Italy

* Correspondence address. UO Gastroenterologia ed Epatologia Ospedaliera, Azienda Ospedaliero-Universitaria Pisana, Via Paradisa 2, 56124 Cisanello Pisa, Italia. Tel: +39-050-996857; Fax: +39-050-995456; E-mail: brunetto{at}med-club.com

Received 8 March 2007; returned 20 April 2007; revised 27 April 2007; accepted 1 May 2007


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Objectives: We studied the impact of hepatitis B virus (HBV) polymerase/reverse transcriptase (Pol/Rt) heterogeneity on adefovir rescue therapy in 34 consecutive chronic hepatitis B patients with viral breakthrough during lamivudine monotherapy.

Methods: The Pol/Rt A–F domains were directly sequenced in all patients at baseline, and 12 and 24 months. Response to therapy was evaluated at 3, 6, 12 and 24 months by quantitative HBV-DNA.

Results: Primary treatment failures did not occur. At 6 months 24/34 (70.6%) patients had viraemia < 104 copies/mL [initial viral response (IVR)]; at 12 and 24 months 23 (71.9%) and 26 (81.3%) of 32 had HBV-DNA < 200 copies/mL [complete viral response (CVR)]. IVR or CVR patients did not show viral breakthroughs, which occurred in one of the six remaining patients. All but three patients had baseline rtM204I/V substitutions associated with rtL180M in 23, rtL80I/V in 14, rtV173L in 4, rtT184S in 3, rtQ215S in 2 and rtA181S in 2 cases. rtA181S without rtM204I/V was found in one patient. Four of the six patients (67%) without 24 month CVR showed rtA181S or rtT184S substitutions either alone or with typical lamivudine resistance profiles. Baseline HBV-DNA levels were negatively associated with IVR (univariate analysis, P = 0.023). At least one of rtA181S and rtT184S substitutions correlated negatively with IVR and CVR (univariate analysis, P = 0.001) and was independently associated with absence of CVR (P = 0.016).

Conclusions: Lamivudine monotherapy favours the emergence of viral quasispecies that influence the response rate to adefovir rescue therapy independently from baseline viraemia and lower the susceptibility to other nucleos(t)ide analogues.

Keywords: antivirals , HBV , viral load , resistance


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Nucleos(t)ide analogues are a fundamental tool for treatment of hepatitis B virus (HBV) liver disease because of their potent antiviral activity in the absence of remarkable side effects and major contraindications.1–3 However, as long-term treatment is required to maintain viral suppression,3 the risk of emerging drug-resistant mutants, that increases with treatment duration, represents a major problem of nucleos(t)ide analogue therapy.4–6 The selection of resistant mutants is associated with recurrence of viral replication and has important clinical complications such as delisting from liver transplantation programmes,7 acute disease reactivations and accelerated disease progression to clinical decompensation in cirrhotic patients.8,9 The cumulative rate of lamivudine resistance is ~14% to 20% per year8,10 and up to 60% of patients with Child C cirrhosis who may experience life-threatening decompensation when hepatitis reactivates.9 The main mutation conferring lamivudine resistance is rtM204I/V: it can be associated with other amino acid substitutions (rtL80I, rtL180M, rtV173L, rtI169T, rtA181T, rtT184S and rtQ215S) that may act as compensatory mutations increasing either replication efficiency or antiviral resistance.8,11 Adefovir dipivoxil is a nucleotide analogue with activity against both wild-type and lamivudine-resistant HBVs: 57% of patients with lamivudine resistance cleared HBV-DNA by PCR after 1 year and their number increases with longer treatment.12,13 Lampertico et al.14 reported a further optimization of adefovir dipivoxil rescue therapy that can be obtained starting therapy when the viral load is lower than 106 copies/mL; 100% versus 62% of the patients cleared viraemia after 1 year according to baseline HBV-DNA, ≤ or > 106 copies/mL, respectively. Nevertheless, recent reports suggest that mutations such as rtA181T and rtQ215S can be selected by previous therapy and may reduce the susceptibility to adefovir dipivoxil or increase the risk of developing resistance against adefovir dipivoxil even in the absence of the classic rtN236T substitution.11,15,16

Our aim was to study the relationship between HBV polymerase/reverse transcriptase (Pol/Rt) heterogeneity at baseline and the response to adefovir dipivoxil rescue therapy in 34 consecutive chronic hepatitis B (CHB) patients with viral breakthroughs during lamivudine monotherapy.


   Patients and methods
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Patients

We studied 34 CHB patients (median age 56 years, range 40–73 years; 33 males, 1 female), who were consecutively enrolled in the Italian early expanded access programme to adefovir dipivoxil and followed-up for a median period of 39 months (range 7–77 months). Criteria for inclusion were: (i) viral breakthrough (HBV-DNA > 100 000 copies/mL) while on lamivudine treatment; (ii) elevated transaminases; or (iii) previous enrolment in the GS435 protocol (open-labelled study of safety and efficacy of adefovir dipivoxil in liver transplant patients with lamivudine resistance).7 The above studies were approved by the ethics committee of the Azienda Ospedaliera Universitaria Pisana and the patients signed a written informed consent prior to enrolment. In all patients, adefovir dipivoxil was added to lamivudine and the combination was continued. Four patients had a histological diagnosis of chronic hepatitis and 23 chronic hepatitis with cirrhosis (19 Child A, 2 Child B, 2 Child C; hepatocellular carcinoma was present in 2 patients). The remaining seven were transplant patients. All the patients were negative for antibodies against hepatitis C virus (HCV), hepatitis D virus (HDV) and human immunodeficiency virus (HIV). Patients were monitored for liver function tests and HBV-DNA levels monthly during the first 3 months of combination treatment and at least every 3 months thereafter. Clinical evaluations were performed every 3 months, ultrasound every 6 months in patients with cirrhosis, yearly in patients with chronic hepatitis.

Definitions

Viral response was evaluated at months: 3 [primary treatment failure (PTF): failure to achieve 1 log10 copies/mL HBV-DNA decline], 6 [initial viral response (IVR): HBV-DNA levels < 4 log10 copies/mL], and 12 and 24 [complete viral response (CVR): HBV-DNA < 200 copies/mL]. The HBV-DNA cut-off defining IVR was chosen assessing the best predictive value of different viraemia levels at week 24. Viral breakthrough was defined as a rebound of HBV-DNA levels of ≥ 1 log10 from nadir in patients who showed a response to antiviral treatment.5

Serology

Serum biochemistry included aspartate aminotransferases (AST) and alanine aminotransferase (ALT), gamma-glutamyl transpeptidase, alkaline phosphatase, albumin, globulins, total bilirubin, prothrombin time, alpha-1 fetoprotein, creatinine and BUN. HBsAg, anti-HBs, anti-HBc, HBeAg and anti-HBe, anti-HCV, anti-HDV and anti-HIV were detected by commercially available immunoassays (Abbott Laboratories, North Chicago, IL, USA). IgM anti-HBc levels were determined by CORE-M-IMx (Abbott Laboratories), using 0.200 and 0.100–0.200 IMx Index as cut-off and grey zone of chronic hepatitis, respectively.17 Serum levels of HBV-DNA were quantified by COBAS Amplicor Monitor 2.0 HBV assay (Roche Diagnostic Systems Inc., Mannheim, Germany) with a lower limit of detection of 200 copies/mL and linearity range from 200 to 200 000 copies/mL. For quantification, serum samples with HBV-DNA levels > 200 000 copies/mL were diluted and re-tested. Pol/Rt region (domains A, B, C, D, E and F) sequences were characterized by direct sequencing in all baseline serum samples, at 12 and 24 months, when HBV-DNA was detectable.

Direct sequencing of Pol/Rt region

After extraction by QIAGEN QIAamp DNA Mini Kit (Qiagen Inc., Chatsworth, CA, USA), 5 µL of eluted DNA was amplified by PCR in a 50 µL reaction volume. For the amplification of the entire Pol/Rt region, two overlapping PCRs were performed using primers SP8 (72-92 5'-GTT CAG GAA CAG TAA ACC CTG-3' sense)/LamSeq (819-797 5'-CAA AAG AAA ATT GGT AAC AGC GG-3' antisense) and primers Pol 2 (449-469 5'-GAC TAT CAA GGT ATG TTG CCC-3' sense)/LamEas (1017-997 5'-AAA GCC CAA AAG ACC CAC AAT-3' antisense), respectively.

PCR products were then purified using an ExoSAp Purification System and directly sequenced by the chain termination method using a CEQ 2000 Dye Terminator Cycle Sequencing Quick Start Kit (Beckman Coulter). Sequences obtained by the CEQ 2000 XL analysis System (Beckman) were analysed and corrected with the Chromas Lite version 2.0 software (www.technelysium.com.au). Translation into amino acid sequences was done by EMBOSS Transeq (www.ebi.ac.uk). Multiple alignments were performed using NPS@ Multalin (http://npsa-pbil.ibcp.fr). All sequences obtained with the sense primers were also confirmed using the antisense primers.

Statistical analysis

Quantitative data are expressed as median values and ranges. We analysed the relationship between treatment responses at 6 (IVR) and 24 months (CVR) and duration of previous lamivudine treatment, characteristics of the patients (gender and age), liver disease (chronic hepatitis versus cirrhosis versus liver transplant, and baseline ALT levels) and characteristics of viral infection: genotype, HBeAg status, baseline viral load (log10 copies/mL and HBV-DNA levels ≤ or > 6 log10 copies/mL), Pol/Rt amino acid substitutions [typical lamivudine resistance pattern, rtM204I/V ± rtL180M, alone or in combination with additional amino acid substitutions that have been previously described (rtL80V, rtV173L, rtQ215S) or we found in our patients without CVR (rtA181S, rtT184S)]. Univariate analysis was performed using corrected {chi}2 and Mann–Whitney exact test for categorical and continuous variables, respectively. P values < 0.05 were considered as statistically significant. Variables showing a statistical association or a trend (P ≤ 0.10) were included in the multivariate analysis by using multiple logistic regression. All others were default parameters of the statistical software package SPSS for windows, version 10.0 (SPSS Inc., Chicago IL, USA).

Nucleotide sequence accession numbers

Baseline nucleotide sequences are available in the GenBank database under accession numbers DQ995809 to DQ995842. Pre- and on-treatment nucleotide sequences have been deposited in the GenBank database under accession numbers DQ995843 to DQ995860. Comparisons of nucleotide sequences against the sequences in DNA Data Bank of Japan, EMBL Nucleotide Sequences Submission and GenBank, National Center for Biotechnology Information were done.


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Baseline characteristics

The baseline characteristics of the patients are reported in Table 1. Amino acid substitutions in the Pol/Rt domains (A–F), found in the patients at baseline when adefovir dipivoxil was added to lamivudine, are reported in Table 2. rtM204I/V substitutions were present in 31 of 34 patients; they were associated with rtL180M in 23 cases and with rtL80I in 14 cases. Additional amino acid substitutions were found: rtN123K or rtT128N or rtR138Q in one (3%) case each, rtV173L in four (12%) cases, rtA181S in two (6%) cases, rtT184S in three (9%) cases, rtQ215S in two (6%) cases and rtS256G in three (9%) cases. Two patients showed a complete wild-type sequence. The combination patterns of the amino acid substitutions are reported in Table 3. The rtA181S and rtT184S mutations resulted in amino acid changes [tryptophan to cysteine (sW172C) and leucine to phenylalanine (sL175F)] in the Surface (s) gene.


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  		Table 1. Demographic, clinical and virological baseline features of the patients

 


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  		Table 2. Amino acid substitutions of HBV-Pol/Rt (A–F domains) at baseline in the 34 patients

 


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  		Table 3. Sequence patterns of Pol/Rt

 
Follow-up

After 3 months of therapy at least 1 log10 viral load decline was observed in all patients; therefore none of them experienced primary treatment failure.

After 6 months, 24 of 34 (70.6%) patients had serum HBV-DNA levels < 4 log10 copies/mL (IVR), 14 with undetectable HBV-DNA (<200 copies/mL). In the remaining 10 patients without IVR, viral load ranged from 4.06 log10 to 6.42 log10 copies/mL with a median value of 4.84 log10 copies/mL. Two patients died after 7 and 9 months of follow-up because of liver failure and variceal bleeding, respectively: the former patient (patient no. 11) maintained active viral replication (HBV-DNA: 5.91 log10 copies/mL) and the latter (patient no. 13) had undetectable HBV-DNA.

After 12 months of therapy, 23 of 32 (71.9%) patients had HBV-DNA < 200 copies/mL and viraemia became undetectable by month 24 in 3 additional patients.

Overall, after 2 years of combined lamivudine + adefovir dipivoxil treatment 26 (81.3%) patients had viral loads < 200 copies/mL (CVR). The remaining six patients, none of them with IVR, maintained detectable viraemia, with HBV-DNA levels ranging from 3.70 log10 to 6.94 log10 copies/mL (median value of 4.71 log10 copies/mL). Two of them cleared viraemia at month 26 (Figure 1a and b), whereas the remaining four (Figures 2a and b, and 3a and b) remained viraemic throughout the follow-up (median 34 months, range 30–40 months).


Figure 1
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  		Figure 1. HBV-DNA and ALT levels in the two patients without 24 month CVR and typical lamivudine (3TC) resistance pattern. (a) Patient no. 17 had a viral breakthrough after 36 months of 3TC monotherapy; adefovir dipivoxil (ADV) was added 9 months later. A 2 log10 HBV-DNA reduction occurred in the first 2 months of combined treatment; viraemia remained steady for the following 16 months, thereafter showed a second decline and became undetectable 26 months after the beginning of therapy. (b) Patient no. 4 had a PTF to 3TC, after 30 months, when ADV was added, rtM204I + rtL180M + rtL80I substitutions were present. HBV-DNA levels showed a 2 log10 drop after 4 weeks of combination therapy. A slower decline of ~1 log10 occurred from the 2nd to the 15th month when an ALT flare-up occurred. Thereafter, ALT normalized and viraemia declined, becoming undetectable after 26 months of therapy.

 


Figure 2
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  		Figure 2. HBV-DNA and ALT levels in the two patients without 24 month CVR and rtT184S. (a) Patient no. 12 had a viral breakthrough after 51 months of lamivudine (3TC) monotherapy. When adefovir dipivoxil (ADV) was added (5 months later), the mutation pattern, that remained unchanged throughout treatment, was rtM204V + rtL180M + rtT184S. During the first 3 months of 3TC + ADV, HBV-DNA levels declined by 2 log10, then viraemia showed a slow reduction of a further 2 log10, stabilizing at 4 log10 copies/mL thereafter. (b) Patient no. 27 had a viral breakthrough after 48 months of 3TC and the mutational pattern was rtM204I + rtL80I. When (after 18 months) ADV was added, the amino acid substitution pattern was changed with a mixture of L and I at amino acid 80, rtL180M and a mixture of T and S at position 184. Selection of the rtT184S variant occurred after 10 months of ADV + 3TC treatment. Viraemia after an initial decline of 2 log10, remained steady at 4 log10 copies/mL throughout the follow-up (overall 37 months).

 
Viral breakthroughs were not seen in the patients with IVR or CVR, but one patient (no. 23, Figure 3b) who was still viraemic after 24 months of combined therapy.


Figure 3
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  		Figure 3. HBV-DNA and ALT levels in the two patients without 24 month CVR and rtA181S. (a) Patient no. 9 had a viral breakthrough after 14 months of lamivudine (3TC), when adefovir dipivoxil (ADV) was added (11 months later), the mutation pattern, that remained unchanged during treatment, was rtM204I + rtL80I + rtN123K + rtR138Q + rtA181S. Within the first 6 months of therapy, viraemia declined by 2.5 log10 with minor fluctuations in the following 30 months. (b) Patient no. 23 showed an initial viral response to 3TC without serum HBV-DNA clearance. A wild-type sequence was present at the nadir of viral load, after the 18th month HBV-DNA levels begun to increase and a single amino acid change, rtA181S substitution, was detected when ADV was added (after 46 months of 3TC monotherapy). The rtA181S was the only detectable amino acid substitution throughout treatment. At the last available follow-up sample, a mixture of rtA181S and rtA181T variants was detected. When ADV was given, viraemia declined by 1.5 log10 in 2 months; thereafter viraemia progressively increased by 2 log10, achieving a plateau of 6 log10 copies/mL, that was maintained throughout the follow-up.

 
Baseline Pol/Rt amino acid patterns and 24 month CVR

CVR was achieved by the patients without mutations in the Pol/Rt gene and all but two patients with typical lamivudine resistance patterns (A–D). The two patients (patterns A and B) without 24 month CVR cleared HBV-DNA at month 26.

Among the seven patients with additional mutations at positions 181, 184 and 215 of the Pol/Rt region (patterns E and G–I): both the patients with rtA181S (as single mutation in one case and associated with rtM204I + rtL80I + rtN123K + rtR138Q in the other case) and two of the three patients with rtT184S did not achieve CVR. Of three patients with rtT184S, two had mixtures of wild-type (T) and mutated (S) amino acids at baseline. The patient who achieved CVR maintained mixed viral populations, whereas the mutated strain was selected in the patient without CVR. Of the two patients with rtM204I + rtL80V ± rtL180M + rtQ215S, the former reached both IVR and CVR, whereas the latter (with rtS256G) died after 7 months when he was still viraemic.

Factors influencing response to therapy

IVR predicted the achievement of 24 month CVR with 88.5% sensitivity, 100% specificity, 100% positive predictive value, 66.7% negative predictive value and 90.6% diagnostic accuracy. At univariate analysis IVR was negatively associated with baseline viral load (log10 copies/mL HBV-DNA levels: P = 0.023; HBV-DNA levels > 6 log10 copies/mL: P = 0.017), rtT184S amino acid substitution (pattern E, P = 0.032) and at least one of the two amino acid substitutions detected in patients without CVR (rtA181S or rtT184S) (P = 0.001), but not at multivariate analysis (Table 4).


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  		Table 4. Univariate analysis of the factors influencing IVR and 24 month CVR

 
The lack of 24 month CVR was associated at univariate analysis with rtA181S alone (P = 0.035) as well as with at least one of the two amino acid substitutions detected in patients without CVR (rtA181S or rtT184S) (P = 0.001) (Table 4). The multivariate analysis confirmed the significant association between the presence of one of the two substitutions (rtA181S or rtT184S) and the lack of CVR (P = 0.016, odds ratio 0.038, 95% confidence interval 0.003–0.544).


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In our cohort of CHB patients with viral breakthroughs during lamivudine monotherapy, we confirmed the good response rate to adefovir dipivoxil rescue therapy, reported recently from our geographic area;14,18 71.9% and 81.3% of the patients had HBV-DNA < 200 copies/mL at 12 and 24 months, respectively. None of the 26 patients with 24 month CVR experienced viral breakthroughs, which occurred in 1 of the 6 patients who maintained detectable viraemia after 2 years of treatment. The low rate of adefovir dipivoxil resistance (1 of 32, 3.1% at 24 months) may be due to the combination of adefovir dipivoxil plus lamivudine, which was given to all of our patients.19 Baseline viral load influenced IVR (P = 0.023, univariate analysis) and patients with HBV-DNA levels ≤6 log10 copies/mL achieved CVR more rapidly than those with higher viraemia levels (median time intervals to reach HBV-DNA < 200 copies/mL 3 versus 7 months, P = 0.001). However, baseline viral load did not influence 24 month CVR, possibly because of low HBV-DNA levels present when adefovir dipivoxil was added to lamivudine. Nevertheless additional factors may influence the achievement of complete response: pharmacological properties of adefovir dipivoxil, compliance, genetic polymorphisms, phase and profile of HBV infection.8,20–22 Major changes in the host immune-control of HBV infection might have occurred in patients 4 and 17 (with typical lamivudine resistance patterns) who missed 24 month CVR but cleared viraemia within the 26th month (Figure 1). The remaining four patients without 24 month CVR remained viraemic throughout the follow-up. The common viral feature of these patients was the presence of one of the two substitutions (rtA181S in patients 9 and 23 and rtT184S in patients 12 and 27), which had never been described before in patients with a poor response to adefovir dipivoxil ± lamivudine treatment. Both the amino acid positions (181 and 184) were involved already in antiviral resistance, but with different amino acid substitutions or different analogues. rtA181T/V was described in patients with viral breakthroughs during lamivudine/adefovir dipivoxil treatment, and rtT184S alone or rtT184A/F/G/I/L/S in combination with M204V in patients with resistance either to lamivudine or entecavir.8,23–30

In vitro studies showed that rtA181T and rtA181V are associated with a 2- and 4-fold decrease in sensitivity to adefovir dipivoxil.31 The substitutions may be selected during either adefovir dipivoxil monotherapy (usually in combination with rtN236T) or adefovir dipivoxil + lamivudine combination (without rtM204I/V mutations). Overall data suggest that the 181 substitutions may significantly alter the interaction between HBV polymerase and both lamivudine and adefovir dipivoxil.19 Consistently, the emergence of rtA181S mutation in patient no. 23 was associated firstly with lamivudine resistance and then with a poor response to adefovir dipivoxil in the absence of any other classical lamivudine resistance pattern. Indeed, in our patient the decline of HBV-DNA upon adefovir dipivoxil administration could have been caused by the ongoing necrotic event rather than antiviral therapy (Figure 3b). Accordingly, the viral load increased rapidly to pre-treatment levels shortly after the transaminases flared up. Furthermore, during treatment this patient showed a further evolution of the viral quasispecies that brought about the emergence of the rtA181S/T mixture in the last available serum specimen. Our findings confirm previous reports suggesting that the inadequate inhibition of viral replication may favour the further selection of viral quasispecies with better fitness.11,19,31–33 Both the patients with rtA181S substitutions showed lower baseline HBV-DNA levels as compared with the other four patients without CVR (7 log10 versus 8 log10 copies/mL) suggesting that the rtA181S substitution might alter the overall replication efficiency of the HBV variant, in accordance with 3D modelling.34,35

The remaining two patients without CVR had the emergence of rtT184S substitutions during prolonged lamivudine treatments and in both patients, when adefovir dipivoxil was added, viraemia declined below 5 log10 with minor fluctuations thereafter. To our knowledge, this is the first finding of rtT184S mutations in patients with poor response to adefovir dipivoxil, whereas mutations at position 184 were involved in lamivudine and entecavir resistance.26

At univariate analysis, rtT184S and rtA181S were associated with the lack of IVR (P = 0.032) and 24 month CVR (P = 0.035), respectively. Overall, these substitutions (at least one of them) were negatively associated with both IVR and CVR at univariate analysis (P = 0.001) and at multivariate analysis for 24 month CVR as well (P = 0.016). These findings suggest that viral quasispecies selected during lamivudine treatment is a major factor influencing CVR at 24 months. At variance with previous reports, we found that baseline rtL80V/I or rtQ215S substitutions, associated with poor response to adefovir dipivoxil,36 did not influence either IVR/CVR (Table 4) or the kinetics of viraemia decline (data not shown).

In conclusion, lamivudine monotherapy may favour the emergence of HBV variants with cross-resistance to analogues of different chemical classes. We identified two amino acid substitutions, rtA181S and rtT184S, in hot spots of the B domain of Pol/Rt region that appear to influence the response to adefovir dipivoxil. Furthermore, the emergence of rtA181S without rtM204V/I resulted in viral breakthroughs during lamivudine treatment and poor response to adefovir dipivoxil. The evidence that mutations hampering the response to adefovir dipivoxil (and eventually to entecavir in the case of rtT184S) may be selected during lamivudine monotherapy supports the view that the treatment strategy of CHB in the single patient should always include a very careful analysis of the risk/benefit ratio before starting lamivudine monotherapy.


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None to declare.


   Acknowledgements
 
This work was funded in part by educational grants from the Italian Ministry of Health ('Progetto nazionale integrato per lo studio, la prevenzione e la cura dell'epatopatia cronica' contract no. 188–2002) and the Italian Ministry of Education, University and Research (FIRB—protocol RBNE013PMJ_004).


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14 Lampertico P, Viganò M, Manetti E, et al. Adefovir rapidly suppresses hepatitis B in HBeAg-negative patients developing genotypic resistance to lamivudine. Hepatology (2005) 42:1414–9.[CrossRef][ISI][Medline]

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16 Gerolami R, Bourliere M, Colson P, et al. Unusual selection of rtA181V HBV mutants cross-resistant to adefovir following prolonged lamivudine monotherapy: report of two cases. Antivir Ther (2006) 11:1103–6.[ISI][Medline]

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