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Journal of Clinical Microbiology, September 1999, p. 2793-2797, Vol. 37, No. 9
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Automated Quantitative Analysis of Hepatitis B
Virus DNA by Using the Cobas Amplicor HBV Monitor Test
Ulrika
Noborg,1
Annkatrin
Gusdal,1
Eva K.
Pisa,2
Anders
Hedrum,2 and
Magnus
Lindh1,*
Department of Clinical Virology,
Göteborg University, Göteborg,1 and
Sangtec Medical, Stockholm,2 Sweden
Received 21 January 1999/Returned for modification 16 April
1999/Accepted 15 June 1999
 |
ABSTRACT |
A highly sensitive method of quantitative analysis of hepatitis B
virus (HBV) DNA in serum, the Cobas Amplicor HBV Monitor (Cobas-AM)
test, was evaluated. Following a manual extraction of viral DNA,
amplification, colorimetric detection, and quantitative determination
are all automatically performed in the Cobas analyzer. Serially diluted
samples with known HBV DNA concentrations were analyzed blindly. All
samples with a virus concentration of 400 copies/ml and 83% of samples
with a virus concentration of 100 copies/ml could be detected. A linear
correlation between input HBV DNA and measured HBV DNA was seen in the
range from 100 to 105 copies/ml. The mean coefficient of
variation was 29.6% for all input levels and 18.9% for HBV DNA
concentrations above 400 copies/ml. Samples with an HBV DNA level above
109 copies/ml could be reproducibly measured after
predilution to 10
4 or 106 in negative
serum; however, the level was underestimated if target DNA after
dilution was still above the linear range of the assay. Quantitative
results of the Cobas-AM test were interchangeable with measurements by
the manual microwell plate version of Amplicor HBV Monitor (MWP-AM);
the mean ratio for log Cobas-AM results/log MWP-AM results was 0.97 (standard error of the mean, 0.007) when serum samples from 153 chronic
carriers were analyzed. The test should be of value for clinical
assessment of chronic carriers and for monitoring the response to
antiviral treatment. A limitation is the relatively narrow linear range
of the assay, requiring predilution of high-titer (mainly hepatitis B
e-antigen-positive) samples.
| |
INTRODUCTION |
There are about 300 million chronic
carriers of hepatitis B virus (HBV) worldwide, some of whom develop
severe liver damage, including cirrhosis and hepatocellular carcinoma.
New antiviral strategies for the treatment of chronic hepatitis B have
emphasized the need for high-sensitivity detection of viral load, both
for monitoring the effect on HBV replication and for identifying
breakthroughs indicating drug resistance (8, 12). Measuring
of HBV DNA levels in serum may also prove useful for the clinical
staging of chronic infection (6, 13), including assessment
of infectivity and prognosis.
Recently, methods for analyzing HBV DNA by quantitative PCR have been
developed (5, 6, 9, 13, 14), and one such method has been
introduced on the market (Amplicor HBV Monitor test, manufactured by
Roche Molecular Systems, Branchburg, N.J.). This test is based on
coamplification of HBV template and an internal quantitation standard
(QS), and subsequent enzyme-linked immunosorbent assay detection of
captured amplicons (9). The HBV DNA is then calculated by
comparing HBV/QS ratios of optical density values to a standard curve
set up for each run. The assay has a wide linear range but is rather
laborious, as it requires much hands-on time, and carries risk for
technical errors.
In the present study, we have evaluated a new version of the Amplicor
HBV Monitor test, adapted for automated processing by a Cobas analyzer
(4).
| |
MATERIALS AND METHODS |
Quantitative PCR.
Analysis of HBV DNA in serum was performed
by using the Amplicor HBV Monitor test (Roche Molecular Systems)
according to the manufacturer's instructions and by using the Cobas
Amplicor HBV Monitor test (Cobas-AM) as follows.
(i) Specimen preparation.
HBV DNA was manually isolated from
100 µl of serum by polyethylene glycol precipitation followed by
virion lysis and neutralization. A known number of QS molecules was
introduced into each specimen and was carried through the specimen
preparation, amplification, and detection steps subsequently used for
quantification of HBV DNA in the specimen.
(ii) PCR amplification.
Processed specimens (50 µl,
corresponding to 22 µl of serum) were added to 50 µl of
amplification mixture in amplification tubes (A tubes) and loaded onto
the Cobas analyzer. A 104-bp segment of the highly conserved
precore-core region was amplified by using one biotinylated primer
(HBV-104UB) and one nonbiotinylated primer (HBV-104D). The QS was
amplified with the same primers as target HBV, generating a 104-bp amplicon.
(iii) Detection.
After amplification for 30 cycles,
denaturation solution was automatically added to the A tubes to
chemically denature HBV and QS amplicons to form single-stranded DNA.
The denatured amplicon was then serially diluted in detection cups (D
cups) to achieve quantitative results over a broad dynamic range.
Magnetic particles coated with oligonucleotide probes specific for
target HBV and QS sequences, respectively, were added to the D cups and
hybridized to the biotin-labeled amplicon. Following hybridization the
Cobas analyzer washed the magnetic beads and added avidin-horseradish peroxidase conjugate, and upon addition of TMB (3,3',5,5'
tetramethylbenzidine), a color complex, the absorbance (A)
of which was measured at 660 nm, was formed.
(iv) HBV DNA quantification.
Within the linear range of the
assay, A of each D cup is proportional to the amount of HBV
or QS amplicon in the cup. The Cobas analyzer calculates a total
absorbance by multiplying the absorbance of the D cup by the amplicon
dilution factor of that cup. The amount of HBV DNA in each specimen was
calculated from the ratio of the total HBV absorbance to the total QS
absorbance and the input number of QS molecules by using the following
algorithm: HBV DNA/ml = total HBV
A660/total QS A660 × input QS copies × 45
where total HBV A660 is
calculated total HBV absorbance, total QS A660
is calculated total QS absorbance, input QS copies is the number of QS
copies in each PCR mixture (lot specific), and 45 is a factor to
convert copies per PCR to copies per milliliter.
Evaluation of the test.
The study evaluated the linearity,
reproducibility, and precision of Cobas-AM and the correlation of the
Cobas-AM results with the results of the microwell plate version of the
Amplicor Monitor (MWP-AM) test. Panels of serial dilutions of a
reference sample were analyzed blindly. The HBV DNA concentrations of
the panel members had previously been determined at a different
laboratory (Sangtec Medical) by multiple analysis using the MWP-AM.
(i) Linearity.
Duplicates of 12 samples with known HBV DNA
concentrations were analyzed on 3 consecutive days, i.e., each sample
was analyzed six times.
(ii) Precision.
Quadruplicates of samples representing six
different HBV DNA concentrations were analyzed by each of two
technicians on 5 consecutive days. Thus, each sample was analyzed 20 times by each technician. Within-run variation and overall variation
were analyzed.
In addition, on 5 consecutive days triplicates of serum samples
collected from four highly viremic carriers were analyzed after
predilution in HBV-negative serum to 1:104 or
1:106. Thus, each sample was analyzed 15 times at each dilution.
(iii) Correlation.
Duplicates of the 12 samples in the
linearity analysis were also examined by the MWP-AM on 3 consecutive
days, each sample thus being analyzed six times. Mean values and
variation were compared with results from Cobas-AM.
In addition, serum samples from 153 HBV carriers (35 hepatitis B e
antigen [HBeAg]-positive and 118 HBeAg-negative carriers)
at various
clinical stages of chronic infection were analyzed
once using the
Cobas-AM. The genotypes of HBV in these samples
as determined by
methods described previously (
10,
11) were
A in 32 samples,
B in 22 samples, C in 13 samples, D in 77 samples,
and E in 3 samples
(6 were not genotypable). The results were
compared with mean values
from repeated analyses (two or three
analyses) using the MWP-AM.
Because the Cobas-AM test has a narrower
detection range, high-titer
samples were prediluted in negative
serum, based on the previous MWP-AM
results, according to the
following criteria: samples were not
prediluted if the HBV DNA
concentration was below 80,000 copies/ml;
they were diluted 1:50
if it was 80,000 to 3.2 million copies/ml, 1:900
if it was 3.2
to 40 million copies/ml, and 1:40,000 if it was above 40 million
copies/ml in previous MWP-AM
analysis.
(iv) Clinical samples and negative controls.
To evaluate the
clinical value of Cobas-AM for monitoring viremia during therapy, serum
samples from an HBeAg-positive 31-year-old Korean woman with chronic
active hepatitis and developing cirrhosis, treated consecutively with
interferon and lamivudine, were analyzed. She was first given alpha
interferon at 15 MU/week for 6 months, resulting in alanine
aminotransferase (ALT) normalization and HBeAg seroconversion. Because
of a relapse 3 months after discontinuation of interferon she was given
150 mg of lamivudine twice daily for one month and then 150 mg of
lamivudine daily. Serum samples drawn regularly during treatment were
analyzed. Samples with high HBV DNA levels were reanalyzed after
predilution to 102 or 104. Serum samples
from 10 blood donors negative for HBV markers (HBsAg, anti-HBc
antibodies, and anti-HBs antibodies) were analyzed to confirm specificity.
| |
RESULTS |
Samples analyzed by the Cobas-AM assay had 100, 91, and 83%
detection rates when the HBV DNA levels in serum were 400, 200, and 100 copies/ml, respectively. In the MWP-AM assay, a level of 825 copies/ml
was needed for 100% detection and when the levels were 400 and 200 copies/ml the detection rates were 50 and 17%, respectively. The sera
from 10 HBV-negative controls were all nonreactive.
Precision and reproducibility.
Figure
1 shows the observed HBV DNA values
(means ± standard errors of the means [SEMs] from 20 analyses) obtained by each of the two technicians plotted against the
HBV input. The correlation coefficient, r, for mean HBV DNA
values was 1.00 for both technicians. Table
1 shows the mean, range, and coefficient
of variation (CV) for observed HBV DNA concentrations at each HBV input
level.
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|
FIG. 1.
Reproducibility and precision of Cobas-AM for detection
of HBV DNA at levels in the range from 200 to 100,000 copies/ml. Each
sample was analyzed 20 times by each of two technicians; mean values
and SEMs are shown.
|
|
Figure
2 shows the levels of HBV DNA
obtained by analysis of high-titer samples after dilution in negative
serum (to 10
4 or 10
6). Each sample was
analyzed 15 times; CV ranged from 8.8 to 36.3%
(mean CV, 18.2%).
However, a predilution to 10
4 resulted in an
underestimation of the HBV DNA level in samples
with very high HBV
input (10
10 copies/ml), because the target HBV DNA level
after predilution
was still above the linear range of detection (i.e.,
above 10
5 copies/ml). On the other hand, a dilution to
10
6 caused samples with moderately high HBV input
(10
7 copies/ml) to become nonreactive.
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FIG. 2.
Measurements by Cobas-AM of four high-titer serum
samples diluted in negative serum to 1:104 (Dil  4) and
1:106 (Dil 6) prior to analysis. Each sample was analyzed
15 times; mean values and SEMs are shown.
|
|
Linearity and correlation.
Table
2 shows the mean, CV, and range of
observed HBV DNA level at each HBV input level as measured by Cobas-AM.
As shown in Fig. 3, there was a linear
correlation between observed HBV DNA and HBV input in the range from
100 to 100,000 copies/ml. All 153 clinical samples were reactive by
both MWP-AM and Cobas-AM, showing a good correlation (b = 1.16, r = 0.97) when samples were diluted prior to analysis
as described in the Materials and Methods section (Fig.
4). In agreement, the mean ratio of log
HBV DNA level obtained by Cobas-AM to that obtained by MWP-AM for each sample was 0.97 (SEM = 0.007), and there were minor differences among genotypes (the ratios were 0.95, 0.98, 1.01, and 0.97 for genotypes A, B, C, and D, respectively). The median HBV DNA level for
the HBeAg-positive samples was 108.6 copies/ml, and that
for the HBeAg-negative samples was 104.2 copies/ml. Of the
35 HBeAg-positive samples, 85% showed HBV DNA level above
107.0 copies/ml, and 8.3% showed level below
106.0 copies/ml.
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FIG. 3.
Linearity in the detection of HBV DNA in the range from
100 to 200,000 copies/ml by Cobas-AM. Each sample was analyzed six
times; mean values and SEMs are shown.
|
|
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FIG. 4.
Correlation between Cobas-AM and the MTW-AM in the
analysis of HBV DNA in serum samples from 153 chronic HBV carriers (36 HBeAg-positive carriers and 118 HBeAg-negative carriers). The mean of
repeated (two or three analyses) MWP-AM analyses is plotted against a
single value from analysis by Cobas-AM after predilution in negative
serum as described in the Materials and Methods section (r = 0.97, b = 1.17).
|
|
Clinical monitoring.
Figure 5
shows the HBV DNA levels of the patient with chronic active hepatitis
in relation to the course of ALT level and therapy. At the end of
interferon treatment ALT had normalized, HBeAg had been replaced by
anti-HBe antibody, and the HBV DNA level was reduced to
104.8 copies/ml. Six weeks after discontinuation of
interferon treatment, HBV DNA level had increased to 107.0
copies/ml, but the ALT level was only slightly above the upper reference value (URV). Six weeks later a severe relapse of hepatitis occurred; the ALT level was 20× URV, and the HBV DNA level was 107.5 copies/ml. Lamivudine treatment resulted in a
decrease of HBV DNA levels to 103.7 and 102.8
copies/ml after 8 and 16 weeks, respectively, paralleled by ALT normalization.
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FIG. 5.
ALT and HBV DNA levels measured by Cobas-AM in a patient
with chronic active hepatitis treated with alpha interferon and
lamivudine.
|
|
| |
DISCUSSION |
The present study demonstrates that the Cobas-AM test has high
sensitivity and reproducibility. The virus in all samples with an input
above 400 copies/ml and in 83% of samples with input of 100 copies/ml
was detected. The CV ranged from 8.8 to 36.3%, and the most variation
was observed for samples with very low HBV DNA concentrations.
In comparison with the MWP-AM (7, 9), the automation of
amplification, detection, and calculation reduced the hands-on time and
risk for technical or computational errors. The sensitivity was higher
than that for MWP-AM, but the detection range was narrower as the upper
limit of detection was reduced from 107 to 105
copies/ml. Thus, the range of detection is particularly suitable for
measuring low-level viremia, as seen in most HBeAg-negative patients
(13) and during therapy. Recently, new antiviral therapies, such as lamivudine, have been introduced (8). The advantage of such antiviral treatment is that it is given orally and has few side
effects. On the other hand, viremia tends to reappear at
discontinuation of therapy, and breakthrough due to drug resistance has
recently been described (1, 3, 12). Therefore, in the future
combination therapy may prove the most effective strategy, and as in
the management of human immunodeficiency virus, monitoring viral load
with highly sensitive methods may prove essential for control of HBV
infection (2). As regards treatment with interferon, which
probably acts through both immunomodulation and antiviral effects, the
value of monitoring HBV DNA remains to be demonstrated. In the present
study, monitoring of a patient consecutively treated with interferon
and lamivudine showed that the (transient) response to interferon
(including ALT normalization and HBeAg seroconversion) was accompanied
by a reduction of viral load to 6 × 104 copies/ml,
and the clinical response to lamivudine included reduction of viral
levels to around 700 copies/ml during treatment. Of interest, the ALT
peaks, both the flare during interferon treatment and the flare at the
relapse after interferon treatment, were preceded by increasing HBV DNA
levels, indicating that HBV DNA monitoring may be useful in identifying
and (when further antiviral drugs emerge) reducing the hepatocellular
damage caused by viral activations and treatment breakthroughs. Further
investigations are needed to find out if suppression of viremia below a
certain limit is enough or if eradication of viral replication is
required for preventing further liver damage and minimizing the risk
for viral resistance and/or reactivation of hepatitis when therapy is discontinued.
There was a good correlation between results from Cobas-AM and those
from MWP-AM. Analysis of 153 clinical samples by both methods showed a
regression coefficient of 1.17 and a mean ratio of 0.97 for individual
log HBV DNA results measured by Cobas and results measured by MWP.
Thus, the ratio for Cobas:MWP results was approximately 1:1, indicating
that results of the tests are interchangeable. The MWP-AM has the
advantage of a broader detection range, allowing quantification of
virus at relatively high levels as well. However, our data indicate
that for samples with HBV DNA levels close to or above 107
copies/ml the levels are underestimated by MWP-AM, unless they are
diluted in negative serum prior to analysis (not shown).
In HBeAg-positive samples, the median HBV DNA concentration was
108.6 copies/ml, i.e., far above the upper limit of
detection by Cobas-AM. Therefore, predilution is required for analysis
of serum samples from HBeAg-positive carriers (and HBeAg-negative
carriers if they are suspected to be highly viremic). Our data indicate
that the most suitable predilution for HBeAg-positive samples is
1:105. This moves the detection range of Cobas-AM from
102 to 105 copies/ml to 107 to
1010 copies/ml and should allow detection and
quantification of more than 90% of HBeAg-positive samples, less than
5% of which would be categorized as containing more than
1010 copies/ml.
In summary, HBV DNA quantification by Cobas-AM is highly sensitive and
reproducible but has a range of detection that requires predilution of
high-titer samples. It should be particularly suitable for monitoring
response to antiviral therapy.
| |
ACKNOWLEDGMENTS |
We thank Peter Horal for reviewing the manuscript, and Roche
Molecular Systems for supplying reagents.
The project was supported by grants from the Swedish Medical Research
Council and the Swedish Medical Society.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Clinical Virology, Göteborg University, Guldhedsgatan 10B, 413 46 Göteborg, Sweden. Phone: 46 31 3424976. Fax: 46 31 827032. E-mail: magnus.lindh{at}microbio.gu.se.
| |
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Journal of Clinical Microbiology, September 1999, p. 2793-2797, Vol. 37, No. 9
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
