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Journal of Clinical Microbiology, August 2000, p. 2933-2939, Vol. 38, No. 8
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Clinical Evaluation of the Automated COBAS AMPLICOR
HCV MONITOR Test Version 2.0 for Quantifying Serum Hepatitis C Virus
RNA and Comparison to the Quantiplex HCV Version 2.0 Test
Ming-Lung
Yu,1,2
Wan-Long
Chuang,1
Chia-Yen
Dai,1,2
Shinn-Cherng
Chen,1
Zu-Yau
Lin,1
Ming-Yuh
Hsieh,1
Liang-Yen
Wang,1 and
Wen-Yu
Chang1,*
Hepatobiliary Division, Department of
Internal Medicine, Kaohsiung Medical
University,1 and Department of
Internal Medicine, Municipal Hsiao Kang
Hospital,2 Kaohsiung, Taiwan
Received 7 December 1999/Returned for modification 21 February
2000/Accepted 19 May 2000
 |
ABSTRACT |
A second-generation hepatitis C virus (HCV) quantitative assay
(COBAS AMPLICOR HCV MONITOR Test, version 2.0; COBAS HCM-2) has been
developed, with the intention of achieving equivalent quantification of
all HCV genotypes and improving assay performance. To evaluate the
clinical performance of COBAS HCM-2 and its utility in predicting the
response to alpha interferon treatment, sera from 215 chronic hepatitis
C patients were analyzed and the results were compared with those
obtained by the Quantiplex bDNA HCV RNA, version 2.0, assay (bDNA-2).
The COBAS HCM-2 had significantly greater sensitivity than bDNA-2 (94.9 versus 88.4%; P < 0.001) when performed with sera
from chronic hepatitis C patients who were viremic by a qualitative PCR
test. The standard deviations for the within-run and between-run
reproducibilities of COBAS HCM-2 were <0.1 and <0.2, respectively,
and it showed an improved linear range between genotypes with the
threefold serial dilutions tested (r2 = 0.986 to 0.995). The COBAS HCM-2 results were positively correlated with the bDNA-2 results, but the values for COBAS HCM-2 were on average
0.96 log lower than the values for bDNA-2. The mean difference in
quantification values between these two assays did not differ among
samples with different genotypes (0.70 to 1.00 log). No genotype-dependent difference in viral load was observed. The pretreatment viral load was significantly lower in complete responders. By using multivariate analysis, the viral load 2 weeks after the initiation of alpha interferon treatment was the strongest predictor of
a complete response. In conclusion, COBAS HCM-2 demonstrated good
sensitivity, linearity, and reproducibility and efficiency equal to
that of bDNA-2 for the quantification of HCV genotypes 1 and 2. Hence,
this assay provides a rapid and reliable method for the quantification
of HCV RNA in serum and is useful for the planning of interferon treatment.
| |
INTRODUCTION |
Hepatitis C virus (HCV) is the major
etiologic agent in parenterally transmitted non-A, non-B hepatitis and
frequently causes persistent infection, which leads to chronic liver
disease and primary hepatocellular carcinoma (2, 3). Since
serum HCV RNA levels (viral load) may correlate with clinical
manifestations and virologic characteristics (10, 11) and
have been reported to be an important factor predictive of the response
to alpha interferon (IFN-
) therapy in patients with chronic HCV
infection (8, 28), easy, reliable, and standardized tests
with good reproducibilities are needed for routine clinical use.
Several systems have been developed for quantitation of HCV RNA,
including competitive reverse transcription (RT)-PCR (10,
11), the branched DNA (bDNA) assay (Quantiplex HCV RNA assay;
Bayer, Emeryville, Calif.) (5, 9, 13, 17, 28), a
noncompetitive RT-PCR assay (AMPLICOR HCV MONITOR assay;
Roche, Branchburg, N.J.) (4, 17, 28), and a real-time RT-PCR
technique (15). Differences in the performances of these
systems due to fundamentally different technologies and variations in
the efficiency of hybridization of HCV RNA to complementary nucleotide
sequences in these assays may lead to method-related discrepancies in
absolute quantification values and in the correlation between HCV
genotypes and viral load (9, 14, 28).
We have previously undertaken a comparative study to examine the
performance characteristics and clinical utility of the current version of the bDNA assay (the Quantiplex HCV RNA, version 2.0, assay,
referred to as bDNA-2) and the first version of the AMPLICOR HCV
MONITOR (AMPLICOR, version 1.0) assay (28). In agreement with other reports, significant differences in the efficiencies of
detection of genotype 1 and non-genotype 1 isolates were observed with
the first version of the AMPLICOR assay (9, 14, 17). Recently, a second version of the AMPLICOR HCV MONITOR Test
(COBAS AMPLICOR HCV MONITOR Test, version 2.0, referred to as
COBAS HCM-2; Roche, Branchburg, N.J.) has been produced. The assay has
been placed onto the automated COBAS AMPLICOR system (19)
with the intention of achieving more equivalent quantification of all
HCV genotypes, improving assay performance, and saving hands-on time. In addition, previous studies with the qualitative AMPLICOR HCV test
have demonstrated that the automated COBAS AMPLICOR system, with the
incorporation of the uracil-N-glycosylase procedure for the
prevention of carryover contamination, provides a reliable and specific
RT-PCR method for the detection of HCV RNA (1, 6).
The objectives of the current study were (i) to investigate the
clinical sensitivity, reproducibility, and linearity of COBAS HCM-2 and
to examine the correlation of the results of COBAS HCM-2 and bDNA-2
with serum samples from patients with different HCV genotypes and
various clinical settings and (ii) to evaluate the clinical utility of
COBAS HCM-2 in predicting the response to IFN- treatment before and
during therapy. Because specificity studies were performed by the
manufacturers during the test registration procedure, specificity was
not a focus of the present study.
| |
MATERIALS AND METHODS |
Patients.
Two hundred fifteen Taiwanese patients with
chronic hepatitis C (126 men and 89 women; age range, 15 to 73 years;
mean age, 46.3 ± 12.0 years) were enrolled in the study. Among
the patients included 57 had chronic persistent hepatitis, 115 had
chronic active hepatitis, and 43 had liver cirrhosis. Histologic
diagnosis of chronic hepatitis was made on the basis of standard
criteria (12). Sixty-two patients (28.8%) had a history of
blood transfusion.
Seventy-nine patients with chronic hepatitis C were treated with 6 million units (MIU) of recombinant IFN- (IFN- 2b) thrice weekly
for 24 weeks, followed by 3 MIU thrice weekly for the next 12 weeks.
The presence of HCV RNA in serum was assessed by qualitative RT-PCR
every 3 months for 21 months. Complete responders were defined as
patients showing normal alanine aminotransferase levels and clearance
of serum HCV RNA at the end of the therapy and for 12 months after the
cessation of therapy, as measured by qualitative COBAS AMPLICOR HCV
Test, version 2.0 (Roche). All other patients were classified as nonresponders.
Clinical samples.
Serum samples from 215 chronic hepatitis C
patients were analyzed for their HCV RNA levels. Serum was separated
from whole blood collected in a serum separation tube at the time that
a liver biopsy was performed and was immediately stored at 
70°C in
several aliquots. All the samples were reactive for antibodies to HCV
by using the second-generation HCV antibody enzyme immunoassay (Abbott
Laboratories, North Chicago, Ill.) and were positive for HCV RNA by
qualitative COBAS AMPLICOR HCV Test, version 2.0.
Samples were available at 2 and 4 weeks for a subset of 30 unselected
patients treated with IFN-
. These 60 samples were analyzed
for serum
HCV RNA levels by COBAS HCM-2. Samples with viral loads
under the
detection limit of COBAS HCM-2 (<1,000 copies/ml) were
further tested
for the presence of HCV RNA by using the qualitative
COBAS AMPLICOR HCV
Test, version 2.0.
Detection, quantification, and genotyping of HCV RNA in
serum.
Detection of HCV RNA in serum was performed by a
standardized, automated, qualitative RT-PCR assay (COBAS AMPLICOR HCV
Test, version 2.0; Roche) (6). The detection limit was 100 copies/ml.
HCV genotypes were determined by amplification of the core region with
the genotype-specific primers that distinguish between
genotypes 1a,
1b, 2a, 2b, and 3a described by Okamoto et al. (
18).
Two commercial assays for the quantification of serum HCV RNA were
used: COBAS HCM-2 and bDNA-2. Both assays were performed
strictly in
accordance with the manufacturers' instructions. HCV
RNA was detected
directly in bDNA-2 by a series of probe hybridizations
that boost the
signal coming from each HCV RNA strand. The relative
intensity of the
signal was compared with that on a curve prepared
with an external
standard, giving a quantification range from
0.2 million to 120 million
equivalents (MEq) of HCV RNA per
ml.
COBAS HCM-2 is based on RT and amplification of HCV RNA with primers
that target a 244-base region located within the highly
conserved 5'
noncoding region of the HCV genome. An internal quantitation
standard
(HCM QS) was added to every sample, via the lysis buffer,
and was
coamplified with the HCV RNA target. The HCM QS consists
of a
noninfectious RNA transcript that has primer binding regions
identical
to those of the HCV target sequence and that generates
a product of the
same length and base composition as that generated
by the HCV target
RNA but a probe binding region different from
that of the target
amplicon. Both the amplified products of the
internal standard and
sample RNA were serially diluted and detected
by probe
hybridization.
Briefly, a known concentration of HCM QS and 100 µl of patient serum
were coincubated with a lysis buffer; and the RNA was
precipitated with
isopropanol, pelleted by centrifugation, washed
once with 70% ethanol,
and resuspended in 1 ml of specimen diluent.
The isolated RNA was then
added to a master mix solution that
contained r
Tth DNA
polymerase and dimethyl sulfoxide, which decreases
the inter- and
intrastrand reanealing (
25), resulting in equal
access and
amplification of the target sequences (
24). This
was then
loaded onto the COBAS AMPLICOR system, which performed
the remainder of
the procedure. After RT and amplification of
the specimen or the
control in the thermal cycler section, the
COBAS AMPLICOR system
denatured and serially diluted the double-stranded,
biotinylated
amplified products (four HCV amplicon dilutions and
two HCM QS amplicon
dilutions) and used a suspension of magnetic
particles coated with
multiple copies of an oligonucleotide probe
specific for HCV (or HCM
QS) to capture the amplified amplicons.
Unbound material was removed by
washing, and the biotinylated
amplicon was detected with an
avidin-horseradish peroxidase conjugate-tetramethylbenzidine-hydrogen
peroxide colorimetric reaction. The absorbance (660 nm) at each
dilution was recorded, and the COBAS AMPLICOR system automatically
selected the optical density and dilution, according to the
manufacturer's
criteria, to be used for the calculation of the copy
number for
each specimen and control. The manufacturer's stated
quantification
range was 10
3 to 10
6 HCV RNA
copies/ml.
Samples with titers outside the linear range by either of the two
quantitative assays were retested following dilution 1/10
and 1/100.
To investigate the reproducibility of COBAS HCM-2, three samples (low-,
medium-, and high-titer sera) were tested 10 times
in one test run and
then one time in each of five test runs. A
separate aliquot of each
sample was processed for each test; thus,
the variation between results
includes variation due to sample
processing, amplification, and
detection. To assess the linearity
of HCV RNA quantification for
different genotypes, four dilution
panels were made by seven threefold
serial dilutions of high-titer
serum samples from patients infected
with genotype 1b, 2a, 2b,
or both 1b and 2a, which are the common
genotypes in
Taiwan.
Statistical analysis.
Data were expressed as means ± standard deviations after logarithmic transformation of the original
values. The chi-square test with Yates' correction, the chi-square
test with linear correlation, Fisher's exact test, Student's
t test, analysis of variance, Spearman's rank correlation
coefficient, simple linear regression, stepwise multiple linear
regression, and multiple logistic regression were used. For the purpose
of analyzing the data with suitable statistical methods, we assigned a
nominal value of 0.1 MEq/ml to samples that were negative for HCV RNA
by bDNA-2 but positive for HCV RNA by qualitative RT-PCR and a nominal
value of 500 copies/ml to samples which were COBAS HCM-2 negative but
positive by qualitative RT-PCR. For the 60 serum samples collected
during IFN- treatment, a nominal value of 50 copies/ml was assigned
to those negative for HCV RNA by both COBAS HCM-2 and qualitative
RT-PCR.
| |
RESULTS |
Performance characteristics of COBAS HCM-2.
The performance
characteristics of COBAS HCM-2 and bDNA-2 were analyzed with samples
collected at the time that liver biopsy was performed but before the
initiation of therapy. Of the 215 patients, HCV RNA was quantifiable in
204 patients (94.9%) by COBAS HCM-2 and in 190 patients (88.4%) by
bDNA-2. The difference was significant (P < 0.001;
chi-square test). One hundred eighty-six (86.5%) samples were
quantifiable by both of the assays. Seven (3.3%) had HCV RNA levels
below the detection limits of both assays but were positive by the
qualitative COBAS AMPLICOR HCV Test, version 2.0. The quantitative
range observed with clinical samples was 1 × 103 to
3.88 × 106 copies/ml for COBAS HCM-2 and 0.2 to 62.93 MEq/ml for bDNA-2. Six (5.9%) of the 101 genotype 1b-infected samples,
2 (3.1%) of the 65 genotype 2a-infected samples, 1 (3.7%) of the 27 genotype 2b-infected samples, 1 (9.1%) of the 11 samples infected with mixed genotypes, and 1 (10%) of the 10 samples infected with
unclassified genotypes had viral loads below the detection limit of
COBAS HCM-2. Fifteen (14.9%) genotype 1b-infected samples, six (9.2%)
genotype 2a-infected samples, one (9.1%) sample infected with mixed
genotypes, and three (30%) samples infected with unclassified
genotypes had viral loads below the detection limit of bDNA-2. The
clinical sensitivities of COBAS HCM-2 and bDNA-2 for the detection of
HCV RNA did not differ for the samples infected with different genotypes.
Serum HCV RNA levels, tested 10 times in the same round for within-run
reproducibility of COBAS HCM-2, showed that the standard
deviations
were 0.04, 0.06, and 0.09 for low-, medium- and high-titer
sera,
respectively (Table
1). Those tested five
times in five
different rounds to determine the between-run
reproducibility
of COBAS HCM-2 showed that the standard deviations were
0.03,
0.09, and 0.12 for low-, medium-, and high-titer sera,
respectively.
Because a separate sample aliquot was processed for each
test
performed, these variations represent the total variability of
the
assay.
The linearity of HCV RNA quantification was assessed with dilution
panels of high-titer sera infected with different HCV genotypes.
The
undiluted high-titer samples contained HCV genotypes 1b, 2a,
2b, and
both 1b and 2a and had viral loads of 6.44, 6.11, 6.49,
and 6.46 logs,
respectively, as determined by COBAS HCM-2 and
viral loads of 7.80, 6.83, 7.74, and 6.95 logs, respectively,
as determined by bDNA-2. As
shown in Fig.
1, COBAS HCM-2 showed
a
good linear response (
r2 = 0.986 to 0.995)
in the threefold serial dilutions, although
the undiluted high-titer
serum samples did demonstrate some underquantification
(especially in
the sample with genotype 2a infection). However,
this is to be
expected, as the viral loads were above the upper
detection limit of
the assay.
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FIG. 1.
Analysis of HCV RNA in four dilution panels of
high-titer sera infected with different HCV genotypes by COBAS HCM-2.
|
|
For 186 specimens positive by both COBAS HCM-2 and bDNA-2, HCV RNA
quantification values by COBAS HCM-2 were positively correlated
with
those measured by bDNA-2 [log(bDNA-2) = 0.47 × log(COBAS
HCM-2) + 3.86;
n = 186; Spearman's rank
correlation coefficient
r = 0.693;
P < 0.0001].
The values obtained by COBAS HCM-2 were
on average 0.96 log lower than
the values obtained by bDNA-2 (Table
2).
Nonparametric tests of correlation gave a high correlation
coefficient
upon pairwise comparison of the results for samples
with different
genotypes (for genotypes 1b, 2a, and 2b,
P < 0.0001;
for unclassified genotypes and mixed genotypes,
P < 0.001). The
mean difference in quantification values between these
two assays
ranged from 0.70 to 1.00 log. The mean difference in the
values
between the two assays did not differ for samples infected with
different genotypes.
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TABLE 2.
Correlation between HCV genotypes and quantification
values by COBAS HCM-2 and bDNA-2 with 186 specimens positive by
both COBAS HCM-2 and bDNA-2
|
|
Viral load and clinical manifestations.
To evaluate the
relationship between the HCV load and the clinical manifestations of
HCV infection, patient age, sex, history of transfusion, liver
biochemistry, liver histology, and viral genotype were analyzed (Table
3). By univariate and multivariate analyses, none of the factors analyzed was found to be correlated with
viral load, as measured by both COBAS HCM-2 and bDNA-2.
Pretreatment HCV RNA levels and response to IFN- treatment.
Of the 79 patients who received IFN- therapy at 6 MIU thrice weekly
for 24 weeks, followed by 3 MIU thrice weekly for the next 12 weeks, 27 (34.2%) were complete biochemical and virological responders. The mean
pretreatment concentration of HCV RNA was significantly higher in the
52 nonresponders than in the 27 complete responders (5.21 ± 0.87 versus 4.71 ± 1.08 logs for the COBAS HCM-2 results; P < 0.05) (Fig. 2). After the
patients were placed into three groups with low (
3 logs), medium (3 to 5 logs) and high (>5 logs) viral loads, the percentage of complete
responders was observed to decrease with higher pretreatment serum HCV
RNA concentrations (3 of 3 [100%], 14 of 30 [46.7%], and 10 of 46 [21.7%] for those with low, medium, and high viral loads,
respectively; P < 0.01 by the chi-square test with
linear correlation).
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FIG. 2.
Pretreatment levels of HCV RNA in sera of responders
(CR) and nonresponders (NR) to IFN- therapy at 6 MIU thrice weekly
for 24 weeks plus 3 MIU thrice weekly by COBAS HCM-2. Of 79 patients,
27 were responders. The solid lines represent the mean pretreatment
serum HCV RNA level in each group.
|
|
The dynamic changes in serum HCV RNA levels before and at 2 and 4 weeks
after IFN-
treatment of 30 unselected patients (8
complete
responders and 22 nonresponders) are shown in Fig.
3.
Twelve (40%) of 30 serum samples
collected 2 weeks after the initiation
of IFN-
treatment had loads
below the detection limit of COBAS
HCM-2. Of these, three were positive
for HCV RNA by qualitative
RT-PCR. The complete response rate was
significantly higher among
patients with negative COBAS HCM-2 results
at 2 weeks after the
initiation of treatment than among those with
positive results
(7 of 12 [58.3%] versus 1/18 [5.6%];
P < 0.01). Nineteen (63.3%)
of 30 serum samples
collected 4 weeks after the initiation of
IFN-
treatment were COBAS
HCM-2 negative, and 4 of these were
positive by qualitative RT-PCR.
None of the 11 patients with quantifiable
viral loads at 4 weeks after
the initiation of treatment became
complete responders. In contrast, 8 of the 19 (42.1%) patients
with negative COBAS HCM-2 results 4 weeks
after the initiation
of treatment became complete responders
(
P < 0.05). By univariate
analysis, sex, history of
transfusion, levels of HCV RNA in serum
before and at 2 and 4 weeks
after the initiation of treatment,
and the ratio of the reduction of
the viral load 2 weeks after
the initiation of treatment were
significant factors associated
with the HCV response to IFN-
treatment (Table
4). By further
analysis
by using multiple logistic regression, the serum HCV
RNA level 2 weeks
after the initiation of treatment was the only
significant factor
associated with the HCV response to IFN-
treatment,
with an odds
ratio and a 95% confidence interval of 0.14 and 0.03
to 0.67, respectively.
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FIG. 3.
Dynamic change in serum HCV RNA levels determined by
COBAS HCM-2 before and at 2 and 4 weeks after initiation of IFN-
treatment for 8 complete responders and 22 nonresponders. Black circles
linked by bold lines represent complete responders. Solid dots linked
by regular lines represent nonresponders.
|
|
| |
DISCUSSION |
In the present study, the automated COBAS HCM-2 was shown to be an
easy, reliable, standardized test with good performance for routine
clinical use. Compared to the manual AMPLICOR microwell plate system,
version 1.0, the integrated AMPLICOR HCV assay based on the COBAS
AMPLICOR system allowed a significant reduction of hands-on time
(1, 19, 28). The turnaround time for the complete COBAS
HCM-2 including specimen preparation and the amplification and
detection procedures was 8 h for a batch of 21 specimens plus 3 controls. The hands-on time required to perform the manual extraction was about 2 h, so that the overall workload time was 6 min/specimen, whereas it was 21 min/specimen for the manual test.
In comparison to bDNA-2 and the first version of the AMPLICOR HCV
MONITOR assay (23, 28), COBAS HCM-2 was found to have greater clinical sensitivity (95%) and was shown to be able to detect
genotypes 1b, 2a, and 2b and mixtures of genotypes, which are the
common genotypes in Taiwan, with equal sensitivities. In this study,
repeat testing of low-, medium-, and high-titer sera in the same run
and in different runs revealed standard deviations of less than 0.1 and
0.2 for within-run and between-run tests of COBAS HCM-2, respectively,
in contrast to the larger variability found in the manual first version
of the AMPLICOR assay observed in previous studies (9, 14).
The standard deviations of 0.1 to 0.2 imply that the test can reliably
detect 0.5 log10 differences in viral loads. The good
reproducibility of this integrated quantitative PCR system for the
detection of HCV, regardless of viral load, will enable the routine
diagnostic laboratory and the clinician to better define the clinical
utilities of these tests. By using four dilution panels of high-titer
sera infected with different HCV genotypes, the linearity of COBAS
HCM-2 was quite reliable with respect to its dynamic range of
103 to 106 copies/ml, whatever the genotype.
For high concentrations, the less linear results may be indicative of a
saturation effect in the RT-PCR. However, the relatively poor linearity
for high-titer sera reported in previous studies that evaluated the
first and second versions of the manual AMPLICOR assay (14,
17) was not observed in the present study.
The mean difference in quantification values between bDNA-2 and COBAS
HCM-2 was, on average, 1.00 log for samples infected with genotype 1 and 0.93 log for samples infected with a genotype other than genotype
1, in contrast to previously reported differences of about 2 logs
between bDNA-2 and the first version of the AMPLICOR HCV MONITOR assay
for non-genotype 1-infected specimens (23, 28). Although a
significant linear relationship between the results of COBAS HCM-2 and
bDNA-2 was observed in the present study, the Spearman rank correlation
coefficient r was only 0.693. Hence, these two assays, based
on fundamentally different technologies, should not to be used
interchangeably, as observed in previous studies (9, 14, 17, 23,
28).
In the current study, sex, patient age, mode of transmission, liver
enzyme levels, and severity of liver disease did not correlate with
serum HCV RNA levels, as measured by either COBAS HCM-2 or bDNA-2. This
was consistent with the results of previous studies, which used
different quantitative HCV technologies (9, 10, 13, 21, 28).
Also, consistent with the results of other studies of bDNA-2 and
version 2.0 of the manual AMPLICOR assay (9, 17, 23, 28), no
genotype-dependent differences in viral load were observed in the
present study. Our study demonstrated that, by COBAS HCM-2, the
discrepancies in the correlation between HCV genotypes and viral load
that have been reported with the first version of the AMPLICOR assay
(9, 14, 17, 23, 28) and competitive RT-PCR assays
(10) no longer exist. Since underestimation of the viral
load for non-genotype 1-infected samples with the first version of the
AMPLICOR assay might be due to the genotype-specific differences in the
strength of base pairings and in the secondary structure of the 5'
noncoding region in which the PCR primers anneal (22),
introduction of 16% dimethyl sulfoxide into the amplification
kit to reduce the secondary structure appears to improve the
amplification efficiency for non-genotype 1 HCV RNA, resulting in more
accurate quantitation. Since genotype and pretreatment viral load have
been shown to be two major prognostic markers for chronic hepatitis C
patients receiving IFN- treatment (8, 16, 27, 28, 29),
genotype-specific differences in the efficiency of quantitation of HCV
RNA may interfere with the roles of these two factors in the outcome of
IFN- treatment. As it is genotype independent, COBAS HCM-2 could
provide, prior to and during therapy, a proper assessment of the HCV
load and be useful for predicting the outcome of IFN- treatment in
chronic hepatitis C patients. However, further clinical evaluation of
COBAS HCM-2 is necessary to document that it is equally efficient for
the quantification of HCV genotypes other than genotypes 1 and 2 to extend its usefulness worldwide.
Similar to other reports (8, 16, 28), a correlation between
the pretreatment viral load by COBAS HCM-2 and the response to IFN-
was observed. In the 30 patients whose viral loads were monitored
during treatment, serum HCV RNA levels 2 weeks after the initiation of
IFN- treatment were shown to be the strongest predictor of a
complete response by using multivariate analysis. None of 11 patients
with quantifiable viral loads at 4 weeks of therapy achieved a complete
response, suggesting that IFN- treatment may be terminated in this
patient group. These results were similar to those in other reports
that failure to clear HCV RNA, as determined by qualitative RT-PCR, at
2 or 4 weeks of therapy (7, 26) or an initial decline in
serum HCV RNA levels of less than 3 logs in the first 4 weeks of
treatment (30) are strongly and independently associated
with a very low probability of a complete response to IFN-. In this
limited study, the predictive value of the HCV load at 2 weeks of
therapy clearly exceeds the significance of other predictors, such as
pretreatment viremia, viral load at 4 weeks of therapy, ratio of viral
load reductions at 2 and 4 weeks after therapy, and genotype (8,
16, 28, 29, 30). Further larger studies are needed to elucidate
this issue. Initial results indicate that viral load measurements will
also provide useful prognostic information for patients receiving the
more potent combination therapy (20).
In conclusion, COBAS HCM-2 was found to have greater sensitivity,
linearity, and reproducibility than the first version of the AMPLICOR
test and quantified HCV genotypes 1 and 2 with equal efficiencies. The
ability to automate the test not only improves the assay performance
but also allows cost savings in terms of the hands-on time required.
Use of COBAS HCM-2 to monitor the HCV load before and during the early
phase of treatment appears to be very useful in the planning of IFN-
treatment. On the basis of the present results, the automated COBAS
HCM-2 system, which saves hands-on time in the amplification and
detection procedures, is a rapid and reliable PCR method for routine
quantification of HCV RNA in serum. Further clinical evaluation of
COBAS HCM-2 with HCV genotypes other than genotypes 1 and 2 is
necessary to extend its usefulness worldwide.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Hepatobiliary
Division, Department of Internal Medicine, Kaohsiung Medical
University, No. 100, Shih-Chuan 1st Rd, Kaohsiung 807, Taiwan. Phone:
886-7-3121101, ext. 6014. Fax: 886-7-3123955. E-mail:
fishya{at}ms14.hinet.net.
| |
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Journal of Clinical Microbiology, August 2000, p. 2933-2939, Vol. 38, No. 8
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
