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Journal of Clinical Microbiology, August 2001, p. 2850-2855, Vol. 39, No. 8
Medizinische Klinik II, Johann Wolfgang
Goethe-Universität, 60590 Frankfurt am Main,
Germany1; Bayer Diagnostics, Berkeley,
California 94702-04662; and
Hoffmann-LaRoche, Nutley, New Jersey 071103
Received 12 February 2001/Returned for modification 12 May
2001/Accepted 10 June 2001
Transcription-mediated amplification (TMA) is an isothermal,
autocatalytic target amplification method which has the potential to
detect less than 50 hepatitis C virus (HCV) RNA copies/ml (10 IU/ml).
The TMA assay was used to assess the presence of residual HCV RNA in
plasma from patients treated with polyethylene glycol-modified interferon Chronic hepatitis C virus (HCV)
infection is one of the major causes of the development of cirrhosis
and hepatocellular carcinoma (3, 18). The current standard
treatment of HCV infection with alpha interferon (IFN- Determination of virologic response is currently based on RT-PCR
methods for measurement of HCV RNA in serum or plasma with a lower
detection limit of about 100 HCV RNA copies/ml (~50 IU/ml). Transcription-mediated amplification (TMA) is an isothermal,
autocatalytic target amplification method, which has the
potential to detect less than 50 HCV RNA copies/ml (<10 IU/ml)
(21). The high sensitivity of the TMA-based assay may
allow for the detection of low HCV RNA levels in end-of-treatment
specimens from patients with subsequent virologic relapse. In a recent
pilot study, residual HCV RNA was detected by TMA at the end of
treatment in 36% of patients with virologic relapse after standard
therapy using IFN- (Part of this work was presented at the Annual Meeting of the European
Association for the Study of the Liver (EASL), Prague, Czech Republic,
18 to 22 April 2001.)
Patients.
Between December 1997 and November 1999, 531 IFN-naïve patients with chronic HCV infection were
prospectively enrolled in a global phase III open-label, parallel-dose,
randomized, multinational trial. Randomization for subcutaneous
treatment either with 180 µg of peginterferon
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.8.2850-2855.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Assessment, by Transcription-Mediated Amplification, of Virologic
Response in Patients with Chronic Hepatitis C Virus Treated with
Peginterferon
-2a
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
-2a (peginterferon -2a) who showed a virologic relapse after the end of therapy. Stored end-of-treatment and end-of-follow-up plasma samples from 177 of 267 patients treated with peginterferon -2a (S. Zeuzem et al., N. Engl. J. Med. 343:1666-1672,
2000) were available for retesting by TMA. Plasma samples from patients in the same study who exhibited virologic relapse after treatment with
standard interferon -2a served as controls. Virologic response during the trial was defined as HCV RNA that was undetectable using a
PCR-based test system with a sensitivity of 50 IU/mL (Cobas Amplicor
HCV version 2.0) and was compared with TMA-based retesting results
(VERSANT HCV RNA Qualitative Assay). Residual HCV RNA was detected in 4 of 60 cases (7%) by the TMA technology in end-of-treatment plasma
samples from patients who relapsed after receiving peginterferon -2a
and in 6 of 18 patients (33%) following therapy with standard interferon -2a. For peginterferon -2a-treated patients with sustained virologic response, HCV RNA was detectable by TMA in end-of-treatment samples in 3 of 78 cases but in none of the
end-of-follow-up samples. For all end-of-treatment and end-of-follow-up
plasma samples of virologic nonresponders, a complete concordance
between the PCR-based assay and TMA was observed. In conclusion, in
patients with virologic relapse after the end of therapy, according to PCR, who were treated with peginterferon -2a or standard interferon -2a, residual HCV RNA was detectable in end-of-treatment samples by
the TMA-based assay in 7 or 33% of cases, respectively. The lower rate
of residual HCV RNA detection by TMA for patients treated with
peginterferon -2a than that for patients treated with standard interferon -2a may be due to the maintained antiviral pressure of
the long-acting peginterferon -2a at the end-of-treatment visit.
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
) in
combination with ribavirin leads to virologic end-of-treatment response
in approximately 50% of cases. At the end of a 24-week follow-up
period after completion of therapy, approximately 40% of patients
achieve a sustained virologic response (13, 17). Thus, in
about one of five patients who achieve an end-of-treatment response, a
virologic relapse occurs. Rates of virologic relapse are even higher
for IFN- monotherapy than for combination therapy with IFN- plus
ribavirin. In patients treated with IFN- alone, HCV RNA cannot be
detected by reverse transcription-PCR (RT-PCR) at the end of treatment
and the end of follow-up in approximately 30 and 15% of cases,
respectively (4, 6, 7, 11, 12). Thus, ribavirin in the
current standard combination treatment enhances virologic
end-of-treatment response and reduces relapse rates (13,
16). Recently, treatment of patients with chronic hepatitis C
with long-acting polyethylene glycol-modified IFN (peginterferon)
resulted in an increase in virologic response rates (24,
25). In patients treated for 48 weeks with 180 µg of
peginterferon -2a, HCV RNA was undetectable by RT-PCR in 69 and 39%
of patients at the end of treatment and the end of a 24-week follow-up
period, respectively (25).
with or without ribavirin (20). In
the present study, end-of-treatment and end-of-follow-up plasma samples
were investigated by the TMA-based assay to test for residual HCV RNA
in a large cohort of patients who were chronically infected with HCV
and were treated with standard IFN -2a or peginterferon -2a.
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
-2a once per week
(PEGASYS; F. Hoffmann-La Roche, Ltd., Basel, Switzerland) for 48 weeks
or with IFN -2a (Roferon-A; F. Hoffmann-La Roche, Ltd.) at a dose of
6 million units thrice weekly for 12 weeks followed by 3 million units
thrice weekly for 36 weeks was performed. All patients were monitored until week 72 for determination of sustained virologic response. Written informed consent was obtained from each patient, and the ethics
committee at each site approved the study. Diagnosis of chronic HCV
infection was based on two elevated serum aminotransferase levels
within 6 months of treatment initiation, a positive anti-HCV antibody
test, an HCV RNA level above 2,000 copies/ml as determined by a
quantitative PCR-based assay, and a pretreatment liver biopsy result
consistent with a diagnosis of chronic hepatitis C. All patients were
negative for hepatitis B surface antigen and antibodies to human
immunodeficiency virus. Further details of study design and assessments
have been reported elsewhere (25).
-2a and 1 to 2 days
after the last dosing of standard IFN -2a. Blood samples drawn from
patients for HCV RNA tests were routinely centrifuged within 2 h at the
site and were sent on dry ice to the central laboratory. Virologic
response was defined as HCV RNA undetectable by RT-PCR at the central
laboratory. Additional plasma samples were stored at 
80°C.
-2a (n = 267) were selected for retesting by the TMA-based method. Plasma samples of patients who were treated with
standard IFN -2a in the same study and had a virologic relapse after
the end of treatment were retested by the TMA-based assay as controls.
PCR-based measurements of HCV RNA. Qualitative HCV RNA testing within the phase III trial was performed using the Cobas Amplicor HCV version 2.0 (Roche Diagnostics Inc., Mannheim, Germany) assay. In this test, cDNA is made from HCV RNA by RT and is then amplified by PCR under a single set of conditions using the DNA polymerase of Thermus thermophilus (a single-tube, single-enzyme, and single-primer-set process). Details of the assay have been described elsewhere (20). Cobas Amplicor HCV version 2.0 uses 200 µl of plasma for RNA extraction and achieves a sensitivity of 100 HCV RNA copies/ml (50 IU/ml). Compared with version 1.0, Amplicor HCV version 2.0 is independent of HCV genotypes (5, 10). Quantification of HCV RNA was performed by RT-PCR with an internal RNA standard derived from the 5' noncoding region of HCV (Amplicor HCV Monitor 2.0; Roche Diagnostics). All procedures were performed according to the manufacturer's instructions.
Analyses of plasma samples by TMA.
Stored plasma samples
from the end of treatment (week 48) and end of follow-up (week 72) were
available from 219 of 267 patients who received 180 µg of
peginterferon -2a once weekly. Plasma samples were not available
from patients who prematurely discontinued treatment or when the plasma
sample volume was insufficient after RT-PCR testing. In addition,
end-of-treatment and end-of-follow-up plasma samples from 18 of 23 virologic relapsers treated with standard IFN -2a in the same study
were available for TMA retesting. All samples were blinded and shipped
on dry ice from the study central laboratory for retesting by TMA.
Data analysis. Clinical and biochemical characteristics of patients are expressed as mean, median, and standard deviation as appropriate. Distribution of continuous, independent, nonparametric variables were analyzed by the Mann-Whitney U test (two-tailed). P values of <0.05 were considered significant.
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RESULTS |
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Materials and Methods
Results
Discussion
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Matched plasma samples from the end of treatment (week 48) and end
of follow-up (week 72) were available from 219 of 267 patients who
received peginterferon -2a treatment. Retesting by the TMA-based assay (VERSANT HCV RNA Qualitative Assay) was not possible due to
insufficient plasma volumes (<500 µl) in 21 week-48 and 20 week-72
samples. In addition, retesting by the TMA-based assay was determined
to be invalid due to unknown errors during the testing procedure in
three week-48 and three week-72 samples. In five week-48 samples with
insufficient volume for TMA retesting, an invalid test result was
obtained in the corresponding week-72 sample or vice versa. Thus,
altogether 42 patients had to be excluded from further analyses,
whereas for 177 of 219 patients, combined results from end-of-treatment
and end-of-follow-up plasma samples were available.
Virologic response was defined by PCR-based HCV RNA testing using the
Cobas Amplicor HCV version 2.0 assay. A sustained virologic response
with undetectable HCV RNA by RT-PCR at the end of treatment (week 48)
and at the end of follow-up 24 weeks after termination of therapy (week
72) was achieved in 78 of 177 patients (44%) treated with
peginterferon -2a. In 60 of 177 patients (34%), HCV RNA was not
detectable at the end of treatment, but a virologic relapse occurred
after discontinuation of therapy and 39 of 177 patients (22%) were
virologic nonresponders. Pretreatment clinical, biochemical, virologic,
and histological characteristics of the three groups are summarized in
Table 1.
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Among patients with sustained virologic response to treatment with
peginterferon -2a, residual HCV RNA was detected by the TMA-based
assay in 3 of 78 (4%) end-of-treatment samples and none of the 78 end-of-follow-up samples (Table 2). A
complete concordance between PCR-based results and TMA-based results
was observed in all virologic nonresponders, as HCV RNA was detectable
in all plasma samples from the 39 nonresponder patients at the end of treatment and the end of follow-up respectively (Table 2). In relapse
patients, according to PCR results, HCV RNA was detectable by the
TMA-based assay in 4 of 60 end-of-treatment plasma samples (7%) and in
all end-of-follow-up samples (Table 2). In patients with virologic
relapse after the end of treatment who were treated with standard IFN,
HCV RNA was detectable by TMA in 6 of 18 end-of-treatment samples
(33%) and in all end-of-follow-up plasma samples.
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All four TMA-positive peginterferon -2a-treated relapse patients
were infected with HCV genotype 1 (HCV-1). However, the majority of
peginterferon -2a-treated patients (44 of 60) (73%) were infected
with HCV-1 a/b subtypes, while only 16 of 60 peginterferon -2a-treated relapse patients (27%) were infected with non-subtype 1 genotypes. The six TMA-positive standard IFN-treated relapse patients
were infected with genotypes 1a (n = 3), 2c
(n = 1), and 3a (n = 2).
The differences in baseline alanine aminotransferase (ALT) levels
(mean ± standard deviation) among sustained virologic responders (156 ± 137 U/liter), relapsers (107 ± 73 U/liter) and
nonresponders (97 ± 59 U/liter) treated with peginterferon -2a
were not significant (Fig. 1). Mean
end-of-treatment ALT levels were similar for sustained responders
(44 ± 22 U/liter) and virologic relapsers (38 ± 19 U/liter), whereas mean ALT levels in virologic nonresponders (77 ± 48 U/liter) were significantly higher at the end of treatment than
in sustained responders (44 ± 22 U/liter; P < 0.0001) (Fig. 1). Mean (± standard deviation) ALT levels of the
four TMA-positive virologic relapse patients at the end of treatment
(55 ± 16 U/liter) were significantly higher than for TMA-negative
virologic relapse patients (37 ± 19 U/liter; P = 0.034) (Fig. 1). Interestingly, in 41 of 78 patients (53%)
treated with peginterferon -2a who achieved a sustained virologic
response, elevated ALT levels were observed at the end of treatment
(Fig. 1). At the end of follow-up, only 4 of 78 sustained virologic
responders (4%) showed ALT levels above the upper limit of normal,
whereas elevated ALT levels were detected in the majority of virologic
relapse (85%) and nonresponder (87%) patients (Fig. 1).
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DISCUSSION |
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Materials and Methods
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Discussion
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The underlying biological mechanisms for virologic relapse after antiviral therapy of patients with HCV infection are unknown. Generally two possibilities may be considered. Treatment leads to a complete replication arrest, but nonreplicating virions remain in hepatic or extrahepatic sites and resume replication after discontinuation of antiviral therapy. Alternatively, treatment does not completely suppress virus replication, and very low replication rates remain in hepatic or extrahepatic sites (1, 8, 9, 14, 15, 19, 22, 23). Testing the latter hypothesis requires the use of a highly sensitive assay to measure the residual viremia undetectable by currently used PCR-based techniques. TMA is an isothermal, autocatalytic method for detection of targets such as viral RNA. When different HCV RNA reference panels (World Health Organization, U.S. Food and Drug Administration, Paul Ehrlich Institute, and Pelypsy) are used, the TMA-based assay (VERSANT HCV RNA Qualitative Assay) has been shown to detect as few as 25 to 50 HCV RNA copies/ml (5 to 10 IU/ml) (21), compared with 100 copies/ml (50 IU/ml) by PCR-based methods (5).
In the present study, patients treated with a long-acting IFN
(peginterferon -2a) were investigated. The attachment of a 40-kDa
polyethylene glycol moiety to IFN -2a leads to generation of a drug
which has substantially more sustained absorption, lower clearance, and
a longer half-life than unmodified IFN-. As reported previously,
following therapy with peginterferon -2a, virologic end-of-treatment
and sustained responses were 69 and 39%, respectively (25). Thus, in a considerable proportion of patients
(43%), a virologic relapse occurred after discontinuation of therapy. Retesting of stored end-of-follow-up plasma samples by the TMA-based assay confirmed PCR-based results for virologic response at week 72 (end of follow-up). All end-of-follow-up plasma samples from patients
with sustained virologic response had no detectable HCV RNA, and all
end-of-follow-up plasma samples from patients with virologic relapse or
no response were HCV RNA positive by the TMA-based assay. Thus, the
sensitivity of currently available PCR-based assays is sufficient for
assessment of sustained virologic response 24 weeks after termination
of therapy.
HCV RNA was detected by TMA at the end of treatment in 3 of 78 (4%) plasma samples from patients with subsequent, sustained, virologic response. The results of retesting these samples, which scored HCV RNA positive by the TMA-based assay but HCV RNA negative by PCR, might be considered false positive. However, it is possible that the TMA results reflect low levels of viremia not detected by PCR-based techniques at the end of treatment. This residual viremia might reflect late responders or, possibly, replication-incompetent virions which cleared completely after discontinuation of therapy. Residual HCV RNA detected by PCR in end-of-treatment samples of patients with subsequent, sustained, virologic response has also been described previously (16). For virologic nonresponders a complete concordance between PCR-based results and TMA was observed at the end of treatment.
In 4 of 60 (7%) patients with virologic relapse, HCV RNA was detected
by TMA in end-of-treatment samples. In comparison, in patients who
exhibit virologic relapse after discontinuation of standard IFN -2a
therapy, 6 of 18 (33%) end-of-treatment plasma samples tested HCV RNA
positive by TMA. The latter data are in accordance with a previous
study showing that 36% of end-of-treatment plasma samples from
virologic relapse patients treated with standard IFN with or without
ribavirin tested HCV RNA positive by the TMA-based assay
(20).
The PCR testing in the previous (20) study and in the
present study was performed using Amplicor HCV version 2.0 and the automated Cobas Amplicor HCV version 2.0, respectively. Both test procedures achieve a similar sensitivity. Thus, the HCV RNA detection rate of the TMA-based assay in end-of-treatment plasma from virologic relapsers is apparently dependent on the applied IFN- preparation (7% versus 33 to 36% in patients treated with peginterferon -2a and standard IFN-, respectively). End-of-treatment plasma samples were obtained according to the study protocol 1 to 2 days after the
last standard IFN- injection and 7 days after the last peginterferon -2a dosing. The difference in half-life in serum between unmodified IFN- and peginterferon -2a is considerable (8 h versus 60 to 80 h) (2). Standard serum IFN- levels are
generally undetectable 24 to 36 h after the last injection,
whereas peginterferon -2a is still detectable in serum after a
48-week treatment period for up to 2 to 4 additional weeks of the
follow-up period. Thus, most likely the different pharmacokinetic
properties of the two drugs explain the differences in the detection
rate of the TMA-based assay in virologic relapsers in end-of-treatment
plasma samples (Fig. 2). In relapse
patients treated with standard IFN-, HCV RNA may be detectable by
TMA but not yet by PCR 1 to 2 days after treatment discontinuation. In
patients treated with peginterferon -2a, detection of a relapse will
require a longer posttreatment period before HCV RNA can be detected,
as peginterferon -2a will suppress viral replication as long as it
is circulating in the body (Fig. 2). Future studies should
prospectively investigate the pharmacokinetics of peginterferon -2a
after treatment discontinuation, and they should include serial HCV RNA
measurements by TMA and/or PCR techniques to define the time points of
possible HCV RNA detection in patients with relapse. According to the
terminal half-life of peginterferon -2a, detection of virologic
relapse could be delayed by several weeks compared with the result for
patients treated with standard IFN-.
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Interestingly, a discordance between the virologic and biochemical
response at the end of treatment was observed in many patients treated
with peginterferon -2a. In 53% (41 of 78) of patients with
undetectable HCV RNA at the end of treatment and subsequent, sustained,
virologic response, ALT levels above the upper normal limit were
observed at the end of treatment, compared with only 5% (4 of 78) of
patients at the end of follow-up (Fig. 1). However, patients who had a
virologic response but not a biochemical response at the end of
treatment had better overall virologic and biochemical responses at the
end of follow-up than did patients who had both virologic and
biochemical responses at the end of treatment (25).
In conclusion, residual HCV RNA can be detected by the TMA-based assay
(VERSANT HCV RNA Qualitative Assay) in 7 and 33% of end-of-treatment
samples from patients who exhibit a virologic relapse following therapy
with peginterferon -2a and standard IFN-, respectively. The lower
rate of detection of residual HCV RNA in patients treated with
peginterferon -2a may be due to the pharmacokinetics of this drug
leading to a maintained antiviral pressure on HCV replication beyond
the time point of treatment discontinuation. Future investigations have
to elucidate whether patients with PCR-negative and TMA-positive
results for HCV RNA at the end of treatment benefit from prolonged
treatment periods.
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FOOTNOTES |
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* Corresponding author. Mailing address: Medizinische Klinik II, Zentrum der Inneren Medizin, Klinikum der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany. Phone: 49-69-6301-5297. Fax: 49-69-6301-4807. E-mail: zeuzem{at}em.uni-frankfurt.de.
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Journal of Clinical Microbiology, August 2001, p. 2850-2855, Vol. 39, No. 8
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.8.2850-2855.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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47: 2872-2878
[Abstract] [Full Text] -
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Scott, J. D., Gretch, D. R.
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Germer, J. J., Harmsen, W. S., Mandrekar, J. N., Mitchell, P. S., Yao, J. D. C.
(2005). Evaluation of the COBAS TaqMan HCV Test with Automated Sample Processing Using the MagNA Pure LC Instrument. J. Clin. Microbiol.
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Barbeau, J. M., Goforth, J., Caliendo, A. M., Nolte, F. S.
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Germer, J. J., Lins, M. M., Jensen, M. E., Harmsen, W. S., Ilstrup, D. M., Mitchell, P. S., Cockerill, F. R. III, Patel, R.
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Gorrin, G., Friesenhahn, M., Lin, P., Sanders, M., Pollner, R., Eguchi, B., Pham, J., Roma, G., Spidle, J., Nicol, S., Wong, C., Bhade, S., Comanor, L.
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