Clinical evaluation of the COBAS Ampliprep™/COBAS TaqMan™ for HCV RNA quantitation in comparison with the branched-DNA assay

Article (PDF Available)inJournal of Medical Virology 80(2):254-60 · February 2008with27 Reads
DOI: 10.1002/jmv.21073 · Source: PubMed
Abstract
Diagnosis and monitoring of HCV infection relies on sensitive and accurate HCV RNA detection and quantitation. The performance of the COBAS AmpliPrep/COBAS TaqMan 48 (CAP/CTM) (Roche, Branchburg, NJ), a fully automated, real-time PCR HCV RNA quantitative test was assessed and compared with the branched-DNA (bDNA) assay. Clinical evaluation on 576 specimens obtained from patients with chronic hepatitis C showed a good correlation (r = 0.893) between the two test, but the CAP/CTM scored higher HCV RNA titers than the bDNA across all viral genotypes. The mean bDNA versus CAP/CTM log10 IU/ml differences were -0.49, -0.4, -0.54, -0.26 for genotype 1a, 1b, 2a/2c, 3a, and 4, respectively. These differences reached statistical significance for genotypes 1b, 2a/c, and 3a. The ability of the CAP/CTM to monitor patients undergoing antiviral therapy and correctly identify the weeks 4 and 12 rapid and early virological responses was confirmed. The broader dynamic range of the CAP/CTM compared with the bDNA allowed for a better definition of viral kinetics. In conclusion, the CAP/CTM appears as a reliable and user-friendly assay to monitor HCV viremia during treatment of patients with chronic hepatitis. Its high sensitivity and wide dynamic range may help a better definition of viral load changes during antiviral therapy.

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Journal of Medical Virology 80:254260 (2008)
Clinical Evaluation of the COBAS
AmpliPrep
TM
/COBAS TaqMan
TM
for
HCV RNA Quantitation in Comparison
With the Branched-DNA Assay
Fabrizia Pittaluga,
1
Tiziano Allice,
1
Maria Lorena Abate,
2,3
Alessia Ciancio,
2,3
Francesco Cerutti,
1
Silvia Varetto,
1
Giuseppe Colucci,
4
Antonina Smedile,
2,3
and Valeria Ghisetti
1
*
1
Laboratory of Microbiology, Molinette Hospital, University of Turin, Turin, Italy
2
Gastro-Hepatology Department, Molinette Hospital, University of Turin, Turin, Italy
3
Department of Internal Medicine, University of Turin, Turin, Italy
4
Scientific Affairs, Roche Molecular Systems, Rotkreuz, Switzerland
Diagnosis and monitoring of HCV infection relies
on sensitive and accurate HCV RNA detection
and quantitation. The performance of the COBAS
AmpliPrep
TM
/COBAS TaqMan
TM
48 (CAP/CTM)
(Roche, Branchburg, NJ), a fully automated, real-
time PCR HCV RNA quantitative test was
assessed and compared with the branched-
DNA (bDNA) assay. Clinical evaluation on 576
specimens obtained from patients with chronic
hepatitis C showed a good correlation (r ¼ 0.893)
between the two test, but the CAP/CTM scored
higher HCV RNA titers than the bDNA across
all viral genotypes. The mean bDNA versus CAP/
CTM log
10
IU/ml differences were 0.49, 0.4,
0.54, 0.26 for genotype 1a, 1b, 2a/2c, 3a, and
4, respectively. These differences reached sta-
tistical significance for genotypes 1b, 2a/c, and
3a. The ability of the CAP/CTM to monitor
patients undergoing antiviral therapy and cor-
rectly identify the weeks 4 and 12 rapid and
early virological responses was confirmed.
The broader dynamic range of the CAP/CTM
compared with the bDNA allowed for a better
definition of viral kinetics. In conclusion, the CAP/
CTM appears as a reliable and user-friendly assay
to monitor HCV viremia during treatment of
patients with chronic hepatitis. Its high sensitivity
and wide dynamic range may help a better
definition of viral load changes during antiviral
therapy. J. Med. Virol. 80:254 260, 2008.
ß 2007 Wiley-Liss, Inc.
KEY WORDS: HCV RNA quantitation; real-
time PCR; automated system;
early and rapid virologic res-
ponse; antiviral therapy
INTRODUCTION
HCV viral load plays a major role in the management
of chronic hepatitis C for stratifying and monitor-
ing patient response to antiviral therapy. HCV RNA
changes at weeks 4 and 12 during Interferon þ Ribavirin
combination therapy predict the sustained virological
response through the definition of end points for anti-
viral treatment [Mangia et al., 2005; Lukasiewicz et al.,
2007]. The study of viral kinetic with mathematical
models during the first 3 months of therapy has led to
the development of algorithms for an early prediction of
the virological response in order to avoid unnecessary
therapy in patients with little chance of sustained
virological response [Zeuzem et al., 2001; Perelson
et al., 2005]. The rate of sustained virological response
depends on HCV genotype, being much higher (>80%) in
genotype 2 and 3 infected patients than in those infected
by HCV genotype 1, 4, and 6 (<40%) [Manns et al., 2001;
Zeuzem et al., 2001; Mangia et al., 2005; Perelson
et al., 2005; Lukasiewicz et al., 2007]. A <2 log viral load
decrease from baseline at week 12 (early virological
response) has a high predictive value for a virological
nonresponse (>98%) in genotype 1, 4, and 6 infected
patients [Manns et al., 2001; Zeuzem et al., 2001, 2006;
Berg et al., 2003; Davis et al., 2003; Perelson et al., 2005].
On the other hand, a HCV RNA negative result at week 4
None to declare Authors are grateful to Roche Diagnostics, Italy
for the kind support of kits and technical assistance with the
COBAS
1
AMPLIPREP
TM
/COBAS
1
TaqMan
1
instrumentation.
*Correspondence to: Valeria Ghisetti, MD, Laboratory of
Microbiology, Molinette Hospital, Corso Bramante 88/90, 10126
Torino, Italy. E-mail: vghisetti@molinette.piemonte.it
Accepted 9 October 2007
DOI 10.1002/jmv.21073
Published online in Wiley InterScience
(www.interscience.wiley.com)
ß 2007 WILEY-LISS, INC.
(rapid virological response), identifies patients with a high
probability of sustained virological response who may
benefit from shorter treatment schedules [Zeuzem et al.,
2004; Mangia et al., 2005; Perelson et al., 2005; Herrmann
et al., 2006; Lukasiewicz et al., 2007].
Both signal amplification and gene amplification
techniques, based on the branched DNA (bDNA)
and polymerase chain reaction (PCR) technology,
respectively, can be used to quantify and monitor
HCV viral load with different detection limit and
dynamic ranges. The more sensitive qualitative PCR
and transcription-mediated-amplification (TMA) assays
are used for detecting residual and low levels of HCV
RNA at the end of treatment and follow-up [Pawlotsky
et al., 2000; Strader et al., 2004; Gerotto et al., 2006;
Herrmann et al., 2006; Zeuzem et al., 2006]. Real-time
PCR provides a high sensitivity and a broad dynamic
range being suitable for both a qualitative and quanti-
tative HCV RNA determination, thus simplifying
current laboratories workflow. However, the virus
genetic variability and the efficiency of nucleic acid
extraction may influence the performances of PCR-
based assays [Gardner et al., 2003].
The real-time PCR system COBAS TaqMan
TM
48 (Roche Molecular Systems, Inc., Branchburg, NJ)
was evaluated for HCV RNA quantitation with different
low and high throughput extraction platforms [Barbeau
et al., 2004; Germer et al., 2005; Konnick et al., 2005;
Caliendo et al., 2006; Gelderblom et al., 2006]. More
recently, the combination of the COBAS TaqMan
TM
48 Analyzer with a fully automated extraction platform,
the COBAS Ampliprep
TM
(CAP/CTM), has been intro-
duced [Sarrazin et al., 2006; Halfon et al., 2006] for large
routine series. This configuration has been adapted to
the CAP/CTM HCV test, an automated real-time PCR
assay based on a dual labeled hybridization probe
targeting the 5
0
NC region of HCV.
In the present study, the performance of the CAP/
CTM was evaluated using specimens from patients with
chronic hepatitis C in comparison with the bDNA
signal amplification assay. Correlation and differences
in RNA quantitation were studied according to HCV
genotypes. An additional collection of samples from well
characterized patients undergoing antiviral therapy
was used to assess the ability of the test to detect
patient responses at weeks 4 and 12 and the treatment
outcomes.
METHODS
Clinical Specimens
Plasma specimens from 400 patients with HCV-
related chronic disease, referred for routine HCV RNA
quantitation were stored at 808C for up to 3 days
and then analyzed in parallel with the CAP/CTM
and the bDNA techniques. HCV genotyping was 1a in
42 patients, 1b in 197, 2a/2c in 81, 3a in 53, and 4 in 27.
Both the assays were also assessed using an
additional collection of plasma samples from 31 patients
with chronic hepatitis C treated with PEG-Interferon
(IFN) þ Ribavirin combination therapy. PEG-IFN alfa-
2b (PegIntron, Schering-Plough Corp., Kenilworth, NJ)
at a dosage of 1.5 mg/Kg/week and Ribavarin (Rebetol,
Schering-Plough Corp.) at a dosage of either 1,000 mg/
day (body weight <75 Kg) or 1,200 mg/day (body
weight >75 Kg) were administered for 6 (genotype 2/3)
or 12 months (genotype 1/4). A total of 176 serial samples
were collected at day 15, month 1, 3, 6, 12, and 18 during
and after treatment and stored at 808C.
Rapid and early virological responses were defined as
undetectable HCV RNA (sensitivity: 50 IU/ml) or 2 log
viral load decline at weeks 4 and 12, respectively.
Nonresponders and relapsers were patients HCV RNA
positive at week 24 or at the end of treatment (EOT) after
an initial clearance of viremia. Sustained responders
were those negative for HCV RNA (sensitivity: 50 IU/ml)
at the end of treatment and of follow-up.
HCV RNA Quantitative Assays
COBAS AmpliPrep
TM
/COBAS TaqMan
TM
HCV
(CAP/CTM HCV). One thousand fifty microliters of
each plasma sample was processed in automation
for RNA extraction and real-time PCR amplification
by the CAP/CTM HCV, following the instruction
of the manufacturer. In brief, after virion lysis and
glass-particle-mediated RNA capture and purification,
each specimen was transferred from the Cobas
1
AmpliPrep
TM
to the Cobas TaqMan
TM
for real-time
amplification. An internal quantitation standard (QS)
is added to each sample to monitor the efficiency of
the process. The QS is a noninfections armored RNA
containing fragments of HCV sequences with primer
binding regions identical to those of the HCV target
sequence, but with a different detection probe. Preven-
tion of carry-over contamination is ensured by the use of
Amperase
1
.
The sensitivity of the system is 15 IU/ml with a
dynamic range from 43 to 6.9 10
7
IU/ml and a 2 hr
turnaround time for 24 plasma specimens [Germer
et al., 2005; Caliendo et al., 2006; Sarrazin et al., 2006].
At the time of the introduction of the system in the
Laboratory, a commercially available panel of HCV
standards at concentration of 5,000,000; 500,000;
50,000; 5,000; 500; 50 and 0 IU/ml, (OptiQuant HCV
RNA; Acrometrix Corp., Benicia, CA) calibrated on
the 1st International WHO Standard, was used to
evaluate CAP/CTM performances. A single sample of
each standard was analyzed in four sequential days by
CAP/CTM and mean interassay variation was deter-
mined. CAP/CTM observed results were very close to the
expected ones for each standard with an excellent
correlation (r ¼ 0.995) and linearity (R
2
¼ 0.992). Inter-
assay CV for standards 50 IU, 500 IU, 5 10
3
IU, 5 10
4
IU, 5 10
5
IU and 5 10
6
IU, were, respectively, 43%,
32%, 27%, 19%, 1.7%, and 9.2%.
Branched-DNA (bDNA)
The signal amplification bDNA test (Versant HCV 3.0,
Bayer Diagnostic Corporation, Tarrytown, NY) was
used following the instruction of the manufacturer. The
J. Med. Virol. DOI 10.1002/jmv
HCV RNA Quantitation for Clinical Evaluation 255
assay has a detection limit of 615 IU/ml and a dynamic
range up to 8 10
6
IU/ml [Ross et al., 2002].
HCV RNA Qualitative Assay
Qualitative detection of HCV RNA was performed
using the COBAS Amplicor HCV system (Roche Molec-
ular Systems, Inc., Branchburg, NJ) (sensitivity equal to
50 IU/ml) to assess rapid, early, end of treatment and
sustained virological responses.
HCV Genotying
HCV genotypes and subtypes were determined with a
reverse hybridization line probe assay (INNO-LIPA,
Innogenetics, Ghent, Belgium) after nested-PCR ampli-
fication of the 5
0
NC viral region.
HCV Serologic Test
Antibody to HCV was detected in sera with a
chemiluminescent assay (Architect, Abbott Laboratories,
Abbott Park, IL).
Statistical Analysis
Mean, median, standard deviation were calculat-
ed using conventional statistical tests. Correlation
between CAP/CTM and bDNA was determined by linear
regression analysis and mean differences in quantita-
tion for averaged logs by the BlandAltman plot. HCV
RNA results were expressed as log
10
IU/ml. Quantita-
tion differences between the two assays across HCV
genotype 14 were considered statistically significant
for P-value <0.05 (Student t-test) and when above 2SD
by the BlandAltman analysis. Predictors of virological
response were assessed with Fisher exact test for
categorical variables.
RESULTS
The performance of CAP/CTM was assessed and
compared with the bDNA on 576 plasma specimens,
400 from anti-HCV positive patients undergoing routine
HCV RNA testing and 176 samples from 31 patients
treated with PEG-Interferon þ Ribavarin combination
therapy and monitored at week 4, 12, at the end of
therapy and after 6 months of follow-up.
CAP/CTM and bDNA Comparison for HCV RNA
Quantitation on Routine Specimens
Branched-DNA and CAP/CTM tests detected HCV
RNA in 388 and 399 samples, respectively, with a
97.2% concordance. Eleven bDNA-negative CAP/CTM-
positive samples included 3 genotype 1a, 5 genotype 1b,
and 3 genotype 2a/2c, with HCV RNA levels ranging
from 43 to 5,540 IU/ml, being in eight samples lower
than the bDNA detection limit.
A good correlation (r ¼ 0.893) was observed between
HCV RNA values obtained with the bDNA and the CAP/
CTM; the correlation varied if stratified by viremia
range, being 0.786 for HCV RNA levels between 4.1 and
6 logs, but significantly lower for levels below 4 log and
above 6 logs (r ¼ 0.431 and 0.427, respectively) (data not
shown). When data were analyzed according to HCV
genotypes, correlation coefficients were excellent for
genotype 1a (r ¼ 0.923), 1b (r ¼ 0.900) and 3a (r ¼ 0.954)
and slightly lower for genotype 2a/2c (r ¼ 0.845) and
4(r¼ 0.852) (data not shown).
The CAP/CTM showed significantly higher HCV RNA
titers across all viral genotypes, with a wider standard
deviation (range 0.71.4 log
10
) than the bDNA
(range 0.61 log
10
). As shown in Table I, mean differ-
ences in quantitation between the bDNA and the CAP/
CTM ranged from 0.26 log
10
for genotype 4 to
0.54 log
10
for genotype 3a. These differences reached
statistical significance for genotypes 1b (P < 0.0001),
2a/2c (P ¼ 0.01) and 3a (P ¼ 0.02), while quantitation
difference in HCV genotype 1a and 4 were not signi-
ficant. In genotype 1a infected patients, the bDNA
detected significantly lower HCV RNA levels than in
genotype 1b (P ¼ 0.03), but such difference was not
observed with the CAP/CTM test.
BlandAltman analysis showed that differences
between the CAP/CTM and the bDNA were within 1
log
10
IU/ml of the averaged log
10
results of the two tests
for 95% of the tested specimens (for genotype 1a: 0.49,
0.74/1.72; genotype 1b: 0.45, 1.26/0.37; genotype
J. Med. Virol. DOI 10.1002/jmv
TABLE I. HCV RNA Concentrations Measured by the bDNA and the CAP/CTM
According to Genotype 14
Genotype N
Mean SD
Mean difference
bDNA-CAP/CTM P-value*
bDNA
(log
10
IU/ml)
CAP/CTM
(log
10
IU/ml)
1a 42 5.12 1.0
a
5.61 1.4 0.49 NS
1b 197 5.45 0.8
a
5.90 0.9 0.45 <0.0001
2a/2c 81 5.60 0.9 6.00 1.0 0.40 0.01
3a 53 5.46 0.9 6.00 0.9 0.54 0.002
4 27 5.44 0.6 5.70 0.7 0.26 NS
Results are expressed as HCV RNA log
10
IU/ml.
NS, not significant.
a
P ¼ 0.03.
*Student t-test.
256 Pittaluga et al.
2a/2c: 0.4, 1.56/0.75; genotype 3a: 0.54, 1.1/0.01;
genotype 4: 0.26, 0.98/0.46) (Fig. 1ae).
HCV RNA quantitation by the CAP/CTM,
Rapid and Early Virological Response
A total of 176 samples from 31 patients were stratified
in three groups according to therapy outcome as follows:
responders (n ¼ 17, 8 genotype 1b, 3 genotype 2a/2c,
5 genotype 3a and 1 genotype 4), nonresponder (n ¼ 6,
3 genotype 1b, 1 genotype 1a, 1 genotype 3a and
1 genotype 4) and relapsers (n ¼ 8, 5 genotype 1b,
2 genotype 2a/2c and 1 genotype 3a).
HCV RNA was quantified previously by the bDNA,
then retrospectively tested with the CAP/CTM and
results analyzed according to the rapid and early
virologic responses at weeks 4 and 12. For each patient,
serial samples were quantified at baseline, day 15, week
4, week 12, at the end of treatment and after 6 months of
follow-up. Mean levels of HCV RNA as detected with
both the CAP/CTM and the bDNA are shown in Figure 2
for each time-point for all the studied patients. As shown
by Figure 2, the extent of virus decay was better shown
by the CAP/CTM that could monitor viral load when it
fell below the bDNA detection limit.
Data obtained at week 4 by the CAP/CTM were used to
estimate its ability to determine the rapid virological
response. As shown in Figure 3, at week 4 HCV viral
load showed a decline of 2 log from baseline or a
negative result in 21 patients, of whom 15 were sustained
virological responders (positive predictive value, PPV ¼
71%), whereas 8 out of 10 patients who did not achieve a
rapid response, were nonresponders (negative predictive
value, NPV ¼ 80%). At week 12, 17 out of 26 patients who
were early virological responders, achieved a sustained
virological response (PPV ¼ 65%) while all those who did
not show the early response, were nonresponders
(NPV ¼ 100%). The positive predictive value for the
sustained virological response differed significantly
between respondersversus relapserplus non- responders
(P ¼ 0.00181, Fisher’s Exact test).
DISCUSSION
The ideal molecular test for HCV RNA detection and
quantitation has to be sensitive, accurate, genotype
J. Med. Virol. DOI 10.1002/jmv
Fig. 1. ae: BlandAltman analysis of genotype specific mean differences in HCV RNA quantitation
between the bDNA and the CAP/CTM.
HCV RNA Quantitation for Clinical Evaluation 257
independent and have a broad dynamic range to monitor
viral load changes as those seen during anti-viral
therapy and support informed clinical decision [Zeuzem
et al., 2001; Berg et al., 2003; Gerotto et al., 2006;
Sarrazin et al., 2006]. Real-time PCR technology
associated with automated extraction systems has the
potential to satisfy these requirements combining high
sensitivity and reproducibility with a linear amplifica-
tion over a wide dynamic range.
In the present study, the performance of a fully
automated system was evaluated for nucleic acid
extraction from plasma, the COBAS AmpliPrep (CAP),
in combination with the CTM HCV test, an automated
HCV quantitative assay based on the real-time PCR
technology (CAP/CTM). The CAP/CTM has been
reported to be extremely sensitive (15 IU/ml) [Germer
et al., 2005; Caliendo et al., 2006; Sarrazin et al., 2006;
Chevaliez et al., 2007] with a linear dynamic range up to
7 logs [Konnick et al., 2005; Sizmann et al., 2007]. The
combined system allows for a more standardized HCV
RNA quantitation than the same real-time PCR assay
associated with different extraction procedures based on
either manual or semi-automated platforms [Barbeau
et al., 2004; Germer et al., 2005; Caliendo et al., 2006;
Gelderblom et al., 2006]. Recent clinical observations
showed that quantitation of HCV RNA by the CAP/
CTM is adequate for the management of anti-HCV
therapeutic responses, identifying reliably responders
from nonresponder patients at both weeks 4 and 12 of
treatment [Halfon et al., 2006a,b; Sarrazin et al., 2006].
Routine anti-HCV positive samples and serial speci-
mens taken from patients undergoing antiviral therapy
were tested with the CAP/CTM assay and results were
compared to those obtained with the signal amplifica-
tion branched-DNA assay. The overall concordance
between the two assays was good; however, as expected
due to the higher sensitivity of the CAP/CTM, low levels
of viremia (<5,000 IU/ml) were detected in most of the
bDNA-negative samples. This is an important finding
due to the fact that residual HCV viremia in patients
undergoing anti-HCV therapy are associated with a
high chance of relapse [Gerotto et al., 2006].
A good correlation between the two tests was observed
only for medium-high level of viremia (between 4 and
6 log
10
IU/ml) while, in lower and higher viral loads, the
CAP/CTM scored constantly better results than the
bDNA. Differences across HCV genotypes 14 varied
from 0.26 to 0.54 log
10
IU/ml (bDNA—CAP/CTM),
the largest and most significant observed within
genotype 1b, 2a/2c, and 3a. This may reflect different
methods in the calibration of the assays since both
manufacturers use the first international WHO Stand-
ard (96/790) as their reference. The branched-DNA
values were also significantly lower for subtype 1a than
for 1b, a finding not shown by the CAP/CTM.
Previously published studies comparing standardized
conventional PCR and not-PCR based assays with real-
time PCR have shown wide differences among results
[Germer et al., 2005; Konnick et al., 2005; Caliendo et al.,
2006; Sarrazin et al., 2006] with an almost constant
lower quantitation by the bDNA assay across all reports.
Small differences were found when the CAP/CTM was
compared with the end-point PCR assay Cobas Amplicor
Monitor 2.0 (CAM), while larger differences were
observed between bDNA and CAM, suggesting the
possibility of a constant underestimation of HCV RNA
levels by the non-PCR based techniques, at least for
HCV genotype 1, 2 and 3 [Caliendo et al., 2006; Sarrazin
et al., 2006; Sizmann et al., 2007]. Recently, differences
ranging from 1.61 to 0.03 logs were reported from a
study comparing the CAP/CTM and the CAM across
genotype 1, 2, 3, and 4 [Colson et al., 2006]. Under-
estimation of HCV RNA by the Cobas TaqMan HCV in
comparison with the bDNA has only been reported
for low volume (less than 500 ml) plasma samples
[Gelderblom et al., 2006]. These findings suggest that
the nucleic extraction method is crucial for HCV RNA
quantitation, affecting both the assay sensitivity and
the efficiency of quantitation. The poor RNA stability
in clinical samples may also play a role, but it can be
controlled by using the same sample aliquot for system
comparison reducing repeated freezing/thawing.
The results are in agreement with those published
by Sarrazin et al. [2006], who reported a constant
J. Med. Virol. DOI 10.1002/jmv
0
1
2
3
4
5
482412420
Weeks
HCV RNA IU/ml log
CAP/CTM
bDNA
bDNA LOD
CA P/CTM LOD
Fig. 2. Mean viral load levels measured by the CAP/CTM and the bDNA at frequent time points during
antiviral therapy in all the studied patients. Results are expressed as HCV RNA log
10
IU/ml. LOD ¼ limit of
detection of the two methods for assessing HCV RNA.
258 Pittaluga et al.
underestimation of HCV RNA by the bDNA test across
HCV genotype 14. Differences in HCV RNA quantita-
tion may be due to the sample extraction methods
and the viral load quantitation technology, gene versus
signal amplification and homogeneous versus, multiple
phases. On the other hand, Chevaliez et al. [2007]
reported recently an underestimation of genotype 2 and
4 in 15% and 30% of their samples and speculated about
possible sequence variation not detected by the CAP/
CTM. While only a slight underestimation of genotype 4
has been reported by some authors [Sarrazin et al., 2006;
Sizmann et al., 2007], it is possible that some specific
genotype 2 samples harbor virus strains that may not be
efficiently quantified. However, no information was
ever published on the CAP/CTM primers and probe
sequences and any hypothesis about possible mis-
matches remains unconfirmed.
In this study, the ability of the CAP/CTM was
investigated to determine viral load changes during
antiviral treatment. In this respect, a viral load decline
2 log IU/ml or a negative HCV RNA result as assessed
by CAP/CTM at week 4 was significantly predictive of
a sustained virological response in patients stratified
according to therapy outcome (Responders vs. Non-
Responders plus Relapsers). In addition, five out of eight
patients who experienced a relapse after therapy
discontinuation, showed high levels of viremia at week
4. The high sensitivity and wide dynamic range of the
assay allows for the detection of early virological
responses useful to individualize treatment for patients
with chronic hepatitis C. In this respect, the lower
sensitivity of the bDNA test limits its utility in moni-
toring individual treatment response, particularly in
the assessment of the rapid virological response
at week 4.
Taken together, these data suggest that the CAP/
CTM assay can be used to quantify HCV RNA in routine
clinical samples and monitor viral load in patients
undergoing antiviral therapy.
CONTRIBUTORS
Valeria Ghisetti was in charge of the evaluation and
comparison between the CAP/CTM and the bDNA on
routine samples for HCV RNA monitoring; she designed
the project, drafted the paper and supervised the
technical laboratory work and data analysis, while
Fabrizia Pittaluga, Tiziano Allice and Silvia Varetto
carried out the laboratory work. Francesco Cerutti
performed statistical analysis of the laboratory data.
Antonina Smedile was in charge of the clinical manage-
ment of the patients included in the study, with Maria
lorena Abate and Alessia Ciancio as collaborators.
Giuseppe Colucci contributed to the study design and
the editing of the manuscript.
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260 Pittaluga et al.
    • "[3,4] Qualitative and quantitative assessment of HCV RNA were performed using the " COBAS Amplicor HCV system " (sensitivity 50 IU/mL, Roche Molecular Systems, INC., Branchburg, NJ) and the bDNA signal amplification test (sensitivity 615 IU/mL, Branched- DNA version 3.0, Bayer Diagnostics Corporation, Tarritown, NY), respectively, in the period 2002- 2007, and the qualiquantitative method COBAS AmpliPrepTM-COBAS TaqManTM (CAP/CTM HCV; sensitivity 15 IU/mL) since 2008. [5][6][7][8]Both HCV RNA genotype and subtype were assessed by reverse hybridization line probe assay (INNO-LIPA, Innogenetics, Ghent, Belgium). [9] Hepatic steatosis, assessed by ultrasound, was defined as an increased liver parenchyma echogenicity compared to the spleen or to the right kidney, the attenuation of the ultrasound beam in depth tissues and the loss of echoes in the portal veins walls according to the following grade scoring system: grade 0, normal echogenicity, absence of differences between echogenicity of liver and kidney; grade 1, mild steatosis with increased echogenicity of liver compared to kidney, absence of attenuation of the ultrasound beam, possibility to explore the depth of hepatic parenchyma; grade 2, moderate increase of steatosis with higher echogenicity of the liver, attenuation of ultrasound beam in depth, loss of echoes from the peripheral portal branches; and grade 3, advanced steatosis with marked increase in echogenicity, attenuation of ultrasound beam in depth and loss of echoes from the major portal branches. "
    Article · May 2016
    • "Participants were diagnosed with HCV infection on the basis of anti-HCV detected in a serum or plasma sample by 3 rd generation enzyme-linked immunosorbent assay and confirmed by recombinant immunoplot assay or HCV RNA [21, 22]. All samples were tested for RNA levels in the real-time polymerase chain reaction (PCR) system COBAS AmpliPrep/COBAS TaqMan HCV (CAP/ CTM HCV) [23]; the detection limit was 15 IU/ml. HCV genotype was determined with genotype specific primers from the 5' noncoding region of the virus by RT-PCR [24] or by sequence analysis of RT-PCR generated fragments using C/E1 and NS5B-specific primers [25, 26]. "
    [Show abstract] [Hide abstract] ABSTRACT: Liver fibrosis has been associated with hepatitis C virus (HCV) genotype and genetic variation near the interleukin 28B (IL28B) gene, but the relative contribution is unknown. We aimed to investigate the relation between HCV genotypes, IL28B and development of liver stiffness. This cross-sectional study consists of 369 patients with chronic hepatitis C (CHC). Liver stiffness was evaluated using transient elastograhy (TE). Factors associated with development of liver fibrosis were identified by logistic regression analysis. We identified 369 patients with CHC. 235 were male, 297 Caucasians, and 223 had been exposed to HCV through intravenous drug use. The overall median TE value was 7.4 kPa (interquartile range (IQR) 5.7-12.1). HCV replication was enhanced in patients carrying the IL28B CC genotype compared to TT and TC (5.8 vs. 5.4 log10 IU/mL, p = 0.03). Patients infected with HCV genotype 3 had significantly higher TE values (8.2 kPa; IQR, 5.9-14.5) compared to genotype 1 (6.9 kPa; IQR, 5.4-10.9) and 2 (6.7 kPa; IQR, 4.9-8.8) (p = 0.02). Within patients with genotype 3, IL28B CC genotype had the highest TE values (p = 0.04). However, in multivariate logistic regression, using various cut-off values for fibrosis and cirrhosis, only increasing age (odds ratio (OR) 1.09 (95% confidence interval (CI), 1.05-1.14 per year increment)), ALT (OR 1.01 (95% CI, 1.002-1.011), per unit increment) and HCV genotype 3 compared to genotype 1 (OR 2.40 (95% CI, 1.19-4.81), were consistently associated with cirrhosis (TE>17.1 kPa). Age, ALT and infection with HCV genotype 3 were associated with cirrhosis assessed by TE. However, IL28B genotype was not an independent predictor of fibrosis in our study.
    Full-text · Article · Dec 2014
    • "Serum samples were stored at −80°C until they were analyzed. HCV RNA levels were measured using a quantitative real-time PCR-based method (COBAS AmpliPrep/ COBAS TaqMan HCV Test) [29,30]. The reduction in HCV RNA 4 and 12 weeks after initiation of therapy was calculated. "
    [Show abstract] [Hide abstract] ABSTRACT: Background The importance of the reduction in hepatitis C virus (HCV) RNA levels 4 and 12 weeks after starting peginterferon (PEG-IFN) and ribavirin combination therapy has been reported to predict a sustained virologic response (SVR) in patients infected with HCV genotype 1. We conducted a multicenter study to validate this importance along with baseline predictive factors in this patient subpopulation. Methods A total of 516 patients with HCV genotype 1 and pretreatment HCV RNA levels ≥5.0 log10 IU/mL who completed response-guided therapy according to the AASLD guidelines were enrolled. The reduction in serum HCV RNA levels 4 and 12 weeks after starting therapy was measured using real-time PCR, and its value in predicting the likelihood of SVR was evaluated. Results The area under the receiver operating characteristics (ROC) curve was 0.852 for 4-week reduction and 0.826 for 12-week reduction of HCV RNA levels, respectively. When the cut-off is fixed at a 2.8-log10 reduction at 4 weeks and a 4.9-log10 reduction at 12 weeks on the basis of ROC analysis, the sensitivity and specificity for SVR were 80.9% and 77.9% at 4 weeks and were 89.0% and 67.2% at 12 weeks, respectively. These variables were independent factors associated with SVR in multivariate analysis. Among 99 patients who showed a delayed virologic response and completed 72-week extended regimen, the area under ROC curve was low: 0.516 for 4-week reduction and 0.482 for 12-week reduction of HCV RNA levels, respectively. Conclusions The reduction in HCV RNA levels 4 and 12 weeks after starting combination therapy is a strong independent predictor for SVR overall. These variables were not useful for predicting SVR in patients who showed a slow virologic response and experienced 72-week extended regimen.
    Full-text · Article · Nov 2012
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