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Journal of Clinical Microbiology, September 1999, p. 3013-3016, Vol. 37, No. 9
Laboratoire de Virologie, Service
d'Hépato-Gastro-Entérologie, Service des Maladies
Infectieuses, Groupe Hospitalier Pitié-Salpêtrière,
75013 Paris, France
Received 11 January 1999/Returned for modification 8 April
1999/Accepted 7 June 1999
Mutations associated with hepatitis B virus (HBV) resistance to
lamivudine have not been extensively addressed in human
immunodeficiency virus (HIV)-HBV coinfection. We have studied the HBV
polymerase sequences from nine coinfected patients who experienced HBV
recurrence while under lamivudine treatment. In seven of these nine
patients, Met550, belonging to the highly conserved YMDD
motif, was mutated to Val and was associated with a substitution of Met
for Leu526 in each case. In the two remaining patients, we
found a Met550-to-Ile change that was associated in only
one case with a Leu526-to-Met mutation. No mutation was
observed in three control patients not receiving lamivudine. This study
demonstrates the emergence of particular genetic profiles in
HBV-HIV-coinfected patients experiencing a loss of control of HBV
infection despite high doses of lamivudine.
Hepatitis B virus (HBV) and human
immunodeficiency virus (HIV) share the same routes of transmission.
Chronic infection with HBV affects about 5% of the population
worldwide and as many as 20% of patients infected with HIV
(14). The nucleoside analog lamivudine
(2',3'-dideoxy-3'-thiacytidine) has been shown to be very efficient in
reducing both HIV and HBV replication by inhibiting the reverse
transcriptase enzyme (5, 16). HIV resistance to lamivudine
occurs rapidly within the first months of treatment and mostly involves
mutations in the conserved YMDD motif of the reverse transcriptase of
the virus (18, 19). Recent studies of patients treated with
lamivudine for chronic hepatitis or post liver transplantation have
suggested that mutations of the HBV polymerase could also confer
resistance to this drug (1, 4, 6, 11, 13, 17). However,
these HBV polymerase mutations have not been described in
HIV-HBV-coinfected patients. These patients usually receive several
antiviral drugs, and some of them may have impaired immune functions.
To investigate the HBV mutations possibly associated with lamivudine
resistance in HIV-HBV-coinfected patients, we studied the nucleotide
sequence of the HBV polymerase gene from patients receiving lamivudine
as part of their HIV infection treatment.
Patients.
Patients belonged to a cohort of 226 HIV-seropositive and hepatitis B surface antigen (HBsAg)-positive
subjects monitored by the Liver Disease Department of our institution.
All of these patients have had at least four visits per year for
clinical examinations, biological tests, CD4 measurements, HBV
serological marker analysis, serum HBV DNA evaluation, and HIV viral
load measurements. Sixty-six of them received lamivudine, 150 mg twice
daily, as part of their antiretroviral therapy. The recommended
lamivudine dosage for HBV therapy is currently 100 mg/day; however,
these patients received 300 mg/day, as recommended for HIV infection.
Nine of these 66 HIV-HBV-coinfected patients were retrospectively
selected on the basis of HBV replication recurrence during therapy and
the availability of serum samples at the time of interest. Replication
recurrence or breakthrough was defined as an initial drop in viral
replication after initiation of therapy, leading to an undetectable HBV
DNA level (threshold, 2.5 pg/ml), followed by a subsequent increase while therapy was maintained. Three patients who were not treated with
lamivudine and whose serum had persistent high levels of HBV DNA (more
than 2,000 pg/ml) served as controls. These 12 patients were homosexual
men with a mean age of 36 (range, 31 to 47) years.
Biological markers.
CD4 lymphocytes were counted by dual-color
flow cytometry. HIV antibodies were detected with both Sanofi Pasteur
Diagnostics Genescreen HIV1/2 and Abbott HIV 1/2 Recombigen 3+. HIV
loads were assessed by using a Roche Monitor reverse transcription-PCR kit (Roche, Neuilly, France). The detection threshold of the test is
200 RNA copies/ml. HBV serological markers were determined by using
Axsym (HBsAg, core antibody, hepatitis B e antigen [HBeAg], and
hepatitis B e antibody [HBeAb]) Abbott tests (Abbott, Les Ulis,
France) in accordance with the manufacturer's specifications. HBV DNA
levels in serum were assessed by Digene Hybrid Capture test (Murex,
Châtillon, France). The range of detection was 2.5 to 2,000 pg/ml.
Sequencing procedures.
For each patient, two serum samples
were studied, one at the beginning of lamivudine treatment and one at
breakthrough. For the three control patients, two sera were also
studied at 9-month intervals. After viral DNA purification (QIAamp
blood kit; Qiagen) starting from 200 µl of each serum, we amplified a
667-nucleotide fragment by using primers VT366
(5'-TGGTTATCGCTGGATGTGTC) and VT1015
(5'-CCCAAAAGACCCACAATTC). Sequencing of the purified PCR products was conducted on a Vistra DNA sequencer by using two inner
primers labeled with Texas red at the 5' end (VT462R
[5'-TATGTTGCCCGTTTGTCCTC] and VT842R
[5'-AGGGTTC/TAAATGTATACCC]). These two sets of conserved primers located in the polymerase gene have been designed from a
consensus sequence generated from published HBV gene sequences and
accessible from GenBank. Analysis of the sequences was accomplished with Geneworks software (Intelligenetics, Oxford, United Kingdom). There are a variety of amino acid numbering systems for HBV; we decided
to use that of Pillay et al. (15).
Results.
The nine selected patients undergoing lamivudine
therapy had their treatment started in association with another
nucleoside analog, either zidovudine (n = 5) or
stavudine (n = 4). None of them was receiving an HIV
protease inhibitor at the time of initiation of therapy. At
breakthrough, six patients were receiving a protease inhibitor, either
indinavir (n = 3), ritonavir (n = 2),
or nelfinavir (n = 1), in association with lamivudine
and another nucleoside analog, either zidovudine (n = 2) or stavudine (n = 4). Median HIV loads, alanine
aminotransferase (ALT) levels in serum, CD4 lymphocyte counts,
serological HBV markers, and HBV DNA levels in serum are summarized in
Table 1. All patients but one were HBeAg
positive and HBeAb negative throughout the study. Interestingly, patient 8, who had HBeAbs at the beginning of lamivudine treatment, was
found to be HBeAg positive at breakthrough. In the sera of all patients
but patient 2, HBV DNA was undetectable after 2 months of lamivudine
therapy and HBV replication recurrence, suggesting the emergence of
resistance, occurred after a mean delay of 17 (range, 4 to 33) months
(Fig. 1). At the time of initiation of therapy, the sequences of the HBV polymerase gene were identical to
those previously published for the wild-type virus (Fig. 1). When viral
resistance emerged, HBV DNA sequence analysis revealed mutations mostly
at position 550 of the polymerase, where a methionine was replaced with
a valine (n = 7 of 9) or an isoleucine (n = 2 of 9) (Fig. 1). HBV 550M
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Copyright © 1999, American Society for Microbiology. All rights reserved.
Hepatitis B Virus (HBV) Mutations Associated with
Resistance to Lamivudine in Patients Coinfected with HBV and Human
Immunodeficiency Virus
ABSTRACT
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V or 550MI mutations were
associated with an LM substitution at position 526 in eight of nine
cases. Only one patient (no. 9) had a unique mutation at position 550 (MI) without any substitution at position 526. As expected, for the
three patients not receiving lamivudine, sequences were similar to
those of the wild-type virus throughout the study (data not shown).
TABLE 1.
Main characteristics of nine HIV-HBV-coinfected patients
receiving lamivudine therapy and experiencing HBV relapse
View larger version (35K):
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FIG. 1.
Course of HBV DNA during lamivudine treatment and
resistance occurrence. HBV DNA from each of nine patients was measured
at different time points and is represented by a specific dot. The time
point of sequence analysis is represented by an arrowhead above each
graph with the position of each residue.
V or 550MI mutations are associated with an
LM substitution at position 526 in eight of nine cases. The 550MV
substitution associated with 526LM has been commonly described. In
our study, the mutation 550MI was also detected; in one case it was
a single mutation, and in the other it was associated with 526LM.
The mutation 550MI could correspond, as it has been reported for
HIV, to transient variants which will eventually evolve toward more fit
species with the 550MV-526LM double mutation (3).
These findings may also indicate the selection of progressive
conformational rearrangements of the polymerase toward more
replication-efficient resistant strains under drug pressure. In vitro
studies assessing replication efficiency in the presence of 3TC are
necessary to confirm this hypothesis. It is noteworthy that the
replication level of YMDD-mutated strains usually increases slowly
after breakthrough and only sometimes reaches the pretreatment level.
However, many parameters may influence HBV replication, especially in
coinfected patients. Although a larger study is needed to strengthen
our findings, CD4 counts did not seem to correlate with the occurrence of breakthrough, nor did the HIV level of replication.
Other mutations, particularly at position 519, have been described in
non-HIV-infected patients; however, none of these mutations was found
in the nine patients studied here (1, 4, 8, 11, 15).
Interestingly, although most of these HIV-infected patients were
treated for several months with other nucleoside analogs, none showed
particular mutations in the HBV polymerase catalytic site before
lamivudine introduction. These findings agree with the absence of
activity against HBV of the other nucleoside analogs and therefore
confirm the absence of HBV resistance selection by these molecules.
Although we studied only three patients not treated with lamivudine, it
is highly unlikely that the mutations found were solely due to a high
level of replication over time. Indeed, most of the patients have been
chronic HBV carriers for several years and polymerase sequences before
lamivudine therapy were found analogous to that of the wild type.
Furthermore, several in vitro studies have clearly shown that mutations
occurring within the polymerase B and C domains can confer resistance
to lamivudine (2, 4, 7, 10, 12, 20).
It is of note that although underlying liver disease could not be
precisely evaluated due to the lack of liver biopsies, the low level of
ALT in serum at breakthrough indicates a low activity of HBV hepatitis
in the context of lamivudine resistance and might suggest impaired
replication of the virus.
In summary, in HIV-HBV-coinfected patients, treatment with high daily
doses of lamivudine does not prevent the emergence of HBV-resistant
strains carrying mutations within the polymerase B and C domains. These
mutations also confer resistance to other nucleoside analogs
(10). Hence, the selection of lamivudine-resistant HBV
mutants raises concern about the future of hepatitis B management in
both HIV-infected and non-HIV-infected persons.
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ACKNOWLEDGMENTS |
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We are sincerely grateful to Jack Gauldie for English revision of this article.
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FOOTNOTES |
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* Corresponding author. Mailing address: Groupe Hospitalier Pitié-Salpêtrière, Laboratoire de Virologie-CERVI, 83 Bd. de l'hôpital, 75013 Paris, France. Phone: 33 1 42 17 74 26. Fax: 33 1 42 17 74 11. E-mail: vincent.thibault{at}psl.ap-hop-paris.fr.
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Journal of Clinical Microbiology, September 1999, p. 3013-3016, Vol. 37, No. 9
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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