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Journal of Clinical Microbiology, May 2001, p. 1972-1974, Vol. 39, No. 5
Institute of Virology1
and Department of Medicine, Division of Nephrology and
Dialysis,2 University of Vienna, and
Pulmologisches Zentrum,3 Vienna, Austria
Received 13 October 2000/Returned for modification 4 January
2001/Accepted 5 March 2001
A line probe assay (INNO-LiPA HBV DR) detecting drug-resistant
hepatitis B virus (HBV) strains was evaluated. Results concordant with
sequence analysis were obtained with 48 of 56 serum samples from
HBV-infected patients undergoing lamivudine therapy. In eight cases,
additional minor subpopulations could be identified by the line probe assay.
Lamivudine, a nucleoside analogue,
has become a potent tool for the therapy of chronic hepatitis B virus
(HBV) infection (11). Studies performed with patients
suffering from chronic hepatitis have shown that treatment with
lamivudine leads to a decrease in the patient's HBV load in serum
(5). This effect was also observed in patients after liver
or renal transplantation (9, 10) and in patients with
simultaneous human immunodeficiency virus (HIV) infection
(14). It has been observed, however, that drug-resistant
HBV variants may emerge during lamivudine treatment. Such
lamivudine-resistant strains exhibit specific mutations, especially in
the YMDD region of the HBV polymerase gene, including either an M552V
mutation associated with an L528M mutation or an M552I mutation alone
(8).
The identification of such mutations is of increasing importance, also
in view of the use of alternative antiviral substances such as
penciclovir or famciclovir, for which promising data for the treatment
of chronic HBV infections have been presented (4, 7). The
identification of resistant HBV strains is usually performed by
sequence analysis. In addition to other alternative test systems, such
as restriction fragment length polymorphism assays (2), a
new test system using the line probe assay (LiPA) technology
(Innogenetics, Ghent, Belgium) (13) has recently been
presented for the detection of resistance mutations in the HBV
polymerase gene.
This INNO-LiPA HBV DR has now been evaluated by testing serum samples
from patients with chronic HBV infections and undergoing lamivudine
treatment. The results have been compared to those obtained by a
standard sequencing protocol that is routinely used for detection of
drug-resistant HBV strains.
In the present study 32 serum samples from 10 renal transplant patients
with chronic HBV infection were investigated at the onset and during
the follow-up of therapy with 100 mg of lamivudine per day in six
cases, 50 mg per day in two cases, and 25 mg per day in another two
cases. In addition, 22 serum samples from 13 HIV patients were
investigated during or after the completion of lamivudine therapy with
a 300-mg dose of lamivudine per day. Also, two serum samples from two
other patients with chronic HBV infection who were undergoing
lamivudine therapy were tested.
The sequence analysis was performed as previously described
(10), using the primers published by Bartholomew et al.
(3).
The INNO-LiPA HBV DR was performed as recommended by Innogenetics. A
part of HBV domains B and C of the pol gene is amplified. The biotinylated PCR fragments are reverse hybridized using
typing-membrane-based INNO-LiPA HBV DR strips. After hybridization,
streptavidin labeled with alkaline phosphatase was added and bound to
the previously formed biotinylated hybrids. Incubation with the
substrate BCIP (5-bromo-4-chloro-3-indolylphosphate)-nitroblue
tetrazolium chromogen results in a purple-brown color development.
The serum samples tested contained virus levels between 103
and 109 copies of HBV DNA as determined by a previously
described quantitative HBV DNA PCR assay (10). All samples
were easily amplified by the PCR steps of both genotyping assays and
yielded unambiguous lines on the LiPA strip and clear sequencing
results. In addition, we have further investigated the sensitivity of
the LiPA by applying it to samples from a proficiency panel for
quantitative HBV DNA testing, which had been distributed by the
European Union Quality Control Concerted Action (15). All
of the samples of this panel that contained HBV DNA down to a
concentration of 103 copies per ml were detected by the
LiPA (Fig. 1), indicating a high
sensitivity of the assay. This is of importance for the early detection
of resistant strains, especially in view of previously published data
that show that the appearance of resistance mutations may precede an
increase in viral load by several months (13).
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.5.1972-1974.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Comparison of Sequence Analysis and the INNO-LiPA HBV DR Line
Probe Assay for Detection of Lamivudine-Resistant Hepatitis B Virus
Strains in Patients under Various Clinical Conditions
ABSTRACT
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FIG. 1.
LiPA strips 1 to 4 show the development of resistant HBV
in a renal transplant patient during lamivudine therapy. Samples were
taken at the beginning of therapy (strip 1), after 3 and 6 months
(strips 2 and 3), and after 9 months, when a resistant strain was
detectable (strip 4). LiPA strips 5 and 6 show the results of patients
D and E, respectively. LiPA strip 7 shows the results found by testing
an HBV DNA standard with a low copy number (103 copies/ml).
Conj., conjugate; amp., amplification.
A comparison of the results obtained by the two methods is presented in
Table 1. Identical results were found
with 48 of the 56 samples (86%). With eight serum samples, however,
there were observed discrepancies between the results of LiPA and those of sequence analysis. In all of these cases, the LiPA indicated the
presence of additional virus species. This finding is statistically significant as shown by the McNemar chi-square test (P = 0.004) and is probably due to the presence of minor viral
subspecies that were missed by sequencing, which usually allows the
detection of minor virus variants only if they constitute about 20% of
the viral population (12). It is unlikely that the
detection of minor viral subspecies was due to nonspecific
hybridization because the selected probes in the LiPA have been shown
to be very specific (13) and no unusual mutations in the
polymerase gene were detected in any of the samples by sequence
analysis.
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The 8 discordant samples were taken from 3 of 32 renal transplant patient samples, from 4 of 22 sera taken from HIV patients, and from 1 of 2 samples taken from other patients with chronic hepatitis. This distribution between the patient groups was not statistically significant (chi-square test, P = 0.22).
The discrepant results are probably caused by the fact that, in patients during long-term therapy as well as after cessation of lamivudine treatment, various mixtures of wild-type and mutated virus strains may develop and are differentially detected by the two assays.
In two samples, the LiPA indicated the presence of additional mutations, which led in both cases to a combination of the M552I mutation with either the L528M mutation alone (patient D) or L528M together with the M552V mutation (patient E) (Fig. 1). In previous studies as well as in the present one, sequence analysis allowed in vivo the detection of either the M552I mutation only or the absolutely linked M552V and L528M mutations (1, 6, 8). In one published study it was possible only by cloning to show that both resistant variants were present in the same sample (8). The LiPA data shown here confirm that both mutational patterns can be present at the same time.
In conclusion, the INNO-LiPA HBV DR proved to be a sensitive tool for resistance testing and appears to be a technique which may be useful in the detection of HBV strains mutated at codons 528 and 552.
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ACKNOWLEDGMENTS |
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We thank Sylvia Malik, Thomas Urbanek, and Michaela Binder for excellent technical assistance and Steve Allison for critical reading of the manuscript.
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FOOTNOTES |
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* Corresponding author. Mailing address: Institute of Virology, University of Vienna, Kinderspitalgasse 15, A-1095 Vienna, Austria. Phone: 43 1 40490, ext. 79527. Fax: 43 1 406 21 61. E-mail: stephan.aberle{at}univie.ac.at.
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REFERENCES |
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Journal of Clinical Microbiology, May 2001, p. 1972-1974, Vol. 39, No. 5
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.5.1972-1974.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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