- Email: [email protected]
COBAS® TaqMan® HCV test, v2.0 in comparison with FDA-approved nucleic acid tests
Accepted Manuscript Title: HCV RNA Detection in HCV Antibody-Positive Patients with the COBAS® AmpliPrep/COBAS® TaqMan® HCV Test, v2.0 in Comparison with FDA-approved Nucleic Acid Tests Author: Ann Butcher Shagufta Aslam Pari Hemyari Ula Cowen Gabrielle Heilek PII: DOI: Reference:
S1386-6532(14)00151-6 http://dx.doi.org/doi:10.1016/j.jcv.2014.04.018 JCV 3016
To appear in:
Journal of Clinical Virology
Received date: Revised date: Accepted date:
20-12-2013 16-4-2014 23-4-2014
Please cite this article as: Butcher A, Aslam S, Hemyari P, Cowen U, Heilek G, HCV RNA Detection in HCV Antibody-Positive Patients with the COBASregd AmpliPrep/COBASregd TaqManregd HCV Test, v2.0 in Comparison with FDA-approved Nucleic Acid Tests, Journal of Clinical Virology (2014), http://dx.doi.org/10.1016/j.jcv.2014.04.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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HCV RNA Detection in HCV Antibody-Positive Patients with the COBAS® AmpliPrep / COBAS® TaqMan® HCV Test, v2.0 in Comparison with FDA-approved Nucleic Acid Tests
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Ann Butcher, Shagufta Aslam, Pari Hemyari, Ula Cowen, and Gabrielle Heilek*
*Correspondent footnote:
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Gabrielle Heilek Ph.D.
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Roche Molecular Systems, Inc.
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4300 Hacienda Drive
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Pleasanton, CA 94588
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Tel: 925-730-8429
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Fax: 925-730-8988
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Email: [email protected]
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Abbreviations: CHC, chronic HCV infection; DAA, direct acting antiviral; HCV, hepatitis C virus; LLOD, lower limit of detection; LOD, limit of detection; NPA, negative percent agreement; PCR, polymerase chain reaction; PPA, positive percent agreement; RGT, response guided therapy; RMS, Roche Molecular Systems; SVR, sustained virologic response
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Keywords: Hepatitis C virus, nucleic acid testing, sustained virologic response, limit of detection
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Abstract: 199 words
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Text: 2500 words
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Highlights •
Detection of HCV RNA offers a highly sensitive means of detecting active viremia.
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The primary goal of HCV therapy is eradication of HCV infection.
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The TAQMAN v2.0is a sensitive test and accurately detects all genotype samples.
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The TAQMAN v2.0 demonstrated correlation with previously approved HCV RNA tests.
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Abstract
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Background: Analysis of hepatitis C virus (HCV) RNA levels is critical for assessing the
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efficacy of antiviral therapy and the achievement of a sustained virologic response.
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Objective and Study Design: This study evaluated the clinical performance of the COBAS®
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AmpliPrep / COBAS® TaqMan® HCV Quantitative Test, version 2.0 (TAQMAN v2.0) with the
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COBAS® AmpliPrep / COBAS® TaqMan® HCV Quantitative Test, version 1.0 (TAQMAN
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v1.0), the VERSANT® HCV Qualitative Assay (VERSANT), and the COBAS® AMPLICOR
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HCV Test, v2.0 (AMPLICOR) qualitative test for the detection of HCV RNA in serum or EDTA
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plasma from patients who are or have been infected with HCV and carry HCV antibodies.
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Results: A total of 277 participants were evaluable for the percent agreement analysis of the
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TAQMAN v2.0 with the VERSANT and with the AMPLICOR. The overall percent agreement
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between the TAQMAN v2.0 and the VERSANT or the AMPLICOR was 99.3% (95% CI:
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97.4%, 99.8%) or 98.9% (95% CI: 96.9%, 99.6%), respectively. The overall percent agreement
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between the TAQMAN v2.0 and the TAQMAN v1.0 when 267 of the original samples were
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assessed was 98.9 % (95% CI= 96.7%, 99.6%).
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Conclusion: The TAQMAN v2.0 demonstrated high correlation with the previously approved
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HCV RNA quantitative and qualitative tests.
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1
Background
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Hepatitis C virus (HCV) is a single-stranded, positive sense RNA virus and has a genome of
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about 10,000 nucleotides coding for 3,000 amino acids 1. The World Health Organization
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estimates that approximately 170 million persons worldwide are infected with HCV with the
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incidence in the United States (US) estimated at 1.6%, of which approximately 2.7 million are
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chronically infected with HCV2-4. The US guidelines recommend that the diagnosis of chronic
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HCV (CHC) infection be made through a stepwise process with initial screening for HCV
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antibodies performed using validated enzyme-linked immunoassays5-8. HCV antibody screening
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can yield positive results in patients who were infected with HCV but who subsequently have
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spontaneously cleared infection without antiviral therapy intervention. Detection of HCV RNA
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by polymerase chain reaction (PCR) nucleic acid amplification offers a highly sensitive and
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specific means of detecting active viremia and has been recommended as the confirmative test of
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choice following a positive result with HCV antibody detection 5.
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HCV infection is curable by antiviral therapy. Since 2011, the standard of care for the treatment
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of CHC genotype 1 infection is a triple therapy combination of direct acting antiviral (DAA)
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medicines, the protease inhibitors boceprevir and telaprevir, used in combination with pegylated
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interferon-alpha plus ribavirin9-12. The primary goal of HCV therapy is eradication of HCV
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infection, assessed at the sustained virologic response (SVR) timepoint, 12 to 24 weeks after
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cessation of anti-viral medication13,14. Analysis of HCV RNA levels is critical for assessing the
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efficacy of therapy for CHC and should be performed with a sensitive molecular method with a
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lower limit of detection (LOD) of <15 IU/ml13,15,16. Monitoring HCV RNA levels at defined
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timepoints is mandated and assures optimized outcomes; to qualify for the shortened treatment
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course (response guided therapy [RGT]), early achievement of undetectable HCV RNA (target
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not detected [TND] readout) while taking the triple combination regimen is required14,15,17.
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The COBAS® AmpliPrep / COBAS® TaqMan® HCV Quantitative Test, version 2.0
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(TAQMAN v2.0) offers an LOD = lower limit of quantification (LLOQ) of 15 IU/mL. Primers
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and probes have been modified from the first generation test to more accurately detect all
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genotype samples. Additional assay improvements include a novel dual probe approach, an
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improved sample preparation method, and a reduced sample input volume18,19.
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The LLOQ of 15 IU/mL test is similar to or lower than that of other FDA-approved qualitative
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assays used for the detection of HCV RNA: the VERSANT® HCV RNA Qualitative Assay
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(Siemens Healthcare Diagnostics Inc.; VERSANT) with an LOD of 5 IU/mL and the COBAS®
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AMPLICOR HCV Test, v2.0 (Roche Molecular Systems, Inc.; AMPLICOR) with an LOD of
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50 IU/mL. Additionally, the TAQMAN v2.0 is an improvement over its predecessor, the
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COBAS® AmpliPrep / COBAS® TaqMan® HCV Quantitative Test, version 1.0 (TAQMAN
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v1.0) which has an LLOQ of 43 IU/mL and can facilitate more accurate clinical interpretation in
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cases of HCV RNA levels below 43 IU/mL with its extended dynamic range 18,19.
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The aim of the present study was to evaluate the concordance between the TAQMAN v2.0 with
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that of the VERSANT, AMPLICOR, and TAQMAN v1.0 tests for the detection of HCV RNA in
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serum or EDTA plasma from patients who are or have been infected with HCV and carry HCV
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antibodies.
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Objectives
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Study Design
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3.1
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For this study, 282 plasma and 25 serum samples with sufficient volume for multiple assay
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testing were obtained from previous Institutional Review Board-approved Roche Molecular
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Systems (RMS) studies and included treatment-naïve subjects with chronic HCV infection or
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spontaneously-resolved infection and subjects who had been successfully treated with HCV
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antiviral therapy, as defined by undetectable HCV RNA following a minimum of 12 weeks after
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end of therapy, and from whom de-identified archived specimens from a clinical study were
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available. Samples were sent to a central laboratory for confirmatory HCV antibody testing.
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Samples that yielded HCV-antibody negative, indeterminate, or a missing result were excluded
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from the study analysis. The majority of participants in the analysis population were 40 years of
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age or older (78.7%) and White (77.3%); the analysis population had approximately equal
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Patient samples and test materials
numbers of male and female participants (Table 1). Out of 277 eligible participants included in
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the analysis, 165 (~60%) were treatment-naïve with established HCV RNA positive status. The
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rest were established as HCV RNA negative, either because of spontaneously resolved HCV
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infection or because they achieved SVR after HCV antiviral treatment. HCV genotyping was
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available from 125/165 HCV RNA positive patients. The majority of RNA positive patients
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(58.8%) carried genotype1; genotype 2 and 3 were represented at 6.7% and 9.7%, respectively.
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This distribution is in agreement with typical patient populations seen in the United States or
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Western Europe 20. Demographics and clinical characteristics of the analysis population are
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presented in Table 1.
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3.2
Viral load assays
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Samples were tested with the VERSANT, the AMPLICOR, the TAQMAN v1.0, and the
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TAQMAN v2.0 according to the manufacturer’s instructions and results for each test were
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interpreted according to the respective product package inserts. The TAQMAN v1.0 and
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TAQMAN v2.0 were carried out on the fully automated, docked COBAS® AmpliPrep /
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COBAS® TaqMan® configuration with either 48 or 96 processing capacity. Samples that
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yielded discrepant results between tests were retested from an additional aliquot if sample
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volume allowed. Performance characteristics of the assays used in this study are presented in
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Table 2.
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3.3
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For the calculation of agreement between the qualitative and quantitative tests, TAQMAN v2.0
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quantitative results were dichotomized into positive and negative. Negative was defined as
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having a result of ‘Target Not Detected’ (TND); any other valid result was defined as positive.
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The agreement of the TAQMAN v2.0 with any comparator assay (VERSANT, AMPLICOR, or
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TAQMAN v1.0) was calculated as shown below with confidence intervals (CIs) for positive
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percent agreement (PPA), negative percent agreement (NPA), and overall percent agreement
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calculated using the Wilson (score) CIs following the formulas described in the CLSI EP12-A2
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guideline 21,22.
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Statistical analysis
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The following statistics were calculated: Estimated positive percent agreement of the TAQMAN v2.0 with respect to comparator
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assay result = 100% × a/(a+c)
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Estimated negative percent agreement of the TAQMAN v2.0 with respect to comparator
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assay result = 100% × d/(b+d)
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Overall percent agreement of the TAQMAN v2.0 with respect to comparator assay result
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= 100% × (a+d)/(a+b+c+d)
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The 95% score CIs were calculated using PROC FREQ. All data analyses were performed using
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SAS/STAT® software (SAS Institute, Inc., Cary, NC).
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4
RESULTS
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4.1
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Of the 307 participants enrolled in this study, a final 277 participants (90.2%) were evaluable
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after review of inclusion and exclusion criteria for the percent agreement analysis of the
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TAQMAN v2.0 with the VERSANT and with the AMPLICOR. For comparison with the
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TAQMAN v1.0, 267 of the 277 original samples had sufficient volume and were assessed.
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4.2
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Concordance between the TAQMAN v2.0 and the composite comparator (VERSANT and the
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AMPLICOR) was evaluated by determining overall percent agreement (data not shown). Table 3
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shows the comparison of the results between the TAQMAN v2.0 and the VERSANT or the
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AMPLICOR. The overall percent agreements of the TAQMAN v2.0 with the VERSANT or the
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AMPLICOR were 99.3% (95% CI: 97.4%, 99.8%) or 98.9% (95% CI: 96.9%, 99.6%),
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respectively (Table 3).
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For two specimens, TAQMAN v2.0 results were positive and VERSANT results were negative;
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HCV RNA was detected with the TAQMAN v2.0 but was <15 IU/mL. Upon retesting of these
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two samples in duplicate on the TAQMAN v2.0 and the VERSANT, all four aliquots returned a
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negative result for the presence of HCV RNA, indicating that the initial TAQMAN v2.0 results
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were false positive (Table 4). One sample yielded an indeterminate composite result: HCV RNA
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positive by the AMPLICOR and HCV RNA negative by the VERSANT. This sample was also
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HCV RNA negative by the TAQMAN v2.0. Upon retesting this sample in duplicate with the
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Comparison of TAQMAN v2.0 to the VERSANT and AMPLICOR Qualitative Assays
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AMPLICOR, both retest results were negative, indicating that the initial AMPLICOR result was
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a false positive.
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4.3
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Concordance between the TAQMAN v2.0 and the first generation TAQMAN v1.0 was evaluated
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in 267 samples. Table 3 shows the comparison of results between the TAQMAN v2.0 and the
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TAQMAN v1.0; overall percent agreement between the two tests was 98.9% (95% CI= 96.7,
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99.6). The PPA and NPA of the TAQMAN v2.0 with the TAQMAN v1.0 was 99.4% and 98.1%,
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respectively. There were three discrepant results: one TAQMAN v2.0 negative and
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TAQMAN v1.0 positive and two TAQMAN v2.0 positive and TAQMAN v1.0 negative
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(Table 4). For these discrepant results the HCV RNA levels detected were below the LLOQ for
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all and represent sampling variability inherent in the extraction and amplification limits of all
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real time PCR assays.
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The one discrepant TAQMAN v1.0 positive sample, yielded negative results from the
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VERSANT and AMPLICOR (Table 4) and was not retested due to insufficient remaining sample
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volume. Overall, the data demonstrated high correlation between the TAQMAN v1.0 and the
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TAQMAN v2.0. Deming regression analysis was carried out in the HCV RNA positive samples
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across the linear range of the tests (n=161) and showed high comparability between the
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TAQMAN v1.0 and the TAQMAN v2.0 with an R2 value of 0.93 (Fig. 1).
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Comparison of TAQMAN v2.0 to TAQMAN v1.0
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Discussion
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The clinical utility of HCV RNA tests has been well-established. Initial HCV RNA tests were
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qualitative tests, detecting hepatitis C viremia to confirm active HCV infection in patients with
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antibodies to HCV. Once HCV treatments became available, quantitative tests were developed to
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monitor changes in viral load during treatment and to predict response to therapy. However, as
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the early quantitative tests were less sensitive (LOD ~600 IU/mL) than the qualitative tests (LOD
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~50 IU/mL), qualitative tests continued to be used to confirm “clearance” or persistence of HCV
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in patients with HCV RNA levels below 600 IU/mL. Qualitative HCV RNA tests therefore
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became widely used to determine clearance of viremia and to assess end of treatment response
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and confirmation of SVR5,6.
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As HCV treatment strategies progressed from pegylated-interferon alfa/ribavirin regimens to
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those including DAAs (such as telaprevir or boceprevir), information on the lower HCV RNA
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viral load levels has become increasingly important. A process involving continuous monitoring
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of therapy and the ability to distinguish and quantify low levels or absence of viremia both
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during and after therapy is now essential15. Futility rules have been established to predict success
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of treatment early-on during the regimen course14. In addition, the definition of viral clearance
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has become more stringent as therapies improve. The aim of triple therapy containing protease
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inhibitors is to achieve an undetectable HCV RNA level (TND) using a highly sensitive test
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(LLOQ < 15 IU/mL)13,15. Finally, guidelines have recognized that with the advent of more
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sensitive quantitative HCV RNA assays, qualitative assays are no longer considered necessary 6.
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In 2012, the Center for Disease Control and Prevention issued a recommendation that persons
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born during 1945 to 1965 should receive a one-time HCV test to better understand the overall
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epidemiology of HCV in the United States 23. To accomplish this large Hepatitis awareness
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effort, access to an accurate nucleic acid detection and quantitation tool is paramount.
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In the current study the performance of the TAQMAN v2.0 was compared to two FDA-approved
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qualitative HCV RNA tests and one quantitative test. The primary comparisons were to the
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VERSANT, a commercially available highly sensitive qualitative test (LOD = 5 IU/mL) and the
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AMPLICOR (LOD = 50 IU/mL). Additionally, the TAQMAN v2.0 was compared to the first
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generation TAQMAN v1.0. Clinical samples from a range of subjects with antibodies to HCV
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were tested in the current study; treatment naïve patients with spontaneously resolved or chronic
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HCV infection and previously treated patients who had achieved SVR. The TAQMAN v2.0
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results were similar to those of the comparator tests. HCV RNA was detected by the
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TAQMAN v2.0 in all samples (166/166) that were positive on the VERSANT and/or for which
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the composite result was positive. The TAQMAN v2.0 also detected HCV RNA in the single
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sample with an indeterminate composite result (VERSANT positive; AMPLICOR negative). In
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the HCV RNA negative samples, the TAQMAN v2.0 showed 98.2% (109/111) agreement with
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the VERSANT, 98.2% (108/110) agreement with the AMPLICOR, and 98.2% (108/110)
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agreement with the composite comparator. The specificity of the TAQMAN v2.0 was therefore
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similar to that of the VERSANT and the AMPLICOR.
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HCV RNA was detected in two samples at very low level (<15 IU/mL) on the TAQMAN v2.0
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but by neither comparator test. Upon retesting of a second aliquot, these samples returned a
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negative result for the presence of HCV RNA indicating that the initial TAQMAN v2.0 results
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were either false positive or were positive at a very low level where reproducible amplification is
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stochastically low. PCR amplification below the LOD = LLOQ of 15 IU/mL is a statistical event
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with decreasing probability of target amplification as the HCV RNA concentration nears the zero
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value. Residual HCV RNA has been reported to be detectable in blood mononuclear cells for
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extended periods of time, especially in early phases of viral clearance (eg, week 4 of strong DAA
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containing regimens) 24,25. Low level positive results in a quantitative assay should be considered
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in the context of other laboratory markers to achieve a final patient assessment.
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Ideally in an individual patient, the same HCV RNA test should be able to diagnose active
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infection, provide a baseline viral load, measure changes in viral load during treatment, and
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evaluate viral clearance at the end of treatment and during follow up. This may become of even
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more importance in the ‘test and treat’ era of HCV infection and therapy about to commence
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with the approval of several DAA regimens that obtain very high SVR rates 26-28. Such a test,
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also preferable to the clinician, would require sensitivity of LLOQ <15 IU/mL, a wide dynamic
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range of quantification, and similar performance across HCV genotypes. The TAQMAN v2.0
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was developed as a second generation assay to the TAQMAN v1.0 by using a novel dual-probe
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approach and an additional reverse primer to improve HCV genotype 4 detection18,19,29. With the
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TAQMAN v2.0 the “grey zone’ between the LOD and LLOQ present in the previous version of
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the TaqMan HCV test was eliminated, allowing clinicians and laboratories easier interpretation
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of results.
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In conclusion, in a range of patient groups (treated and treatment naïve, with or without active
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HCV infection), the TAQMAN v2.0 is highly concordant with other FDA-approved HCV RNA
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tests, the VERSANT, the AMPLICOR, and the TAQMAN v1.0, and has several improved
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performance features that provide accurate detection and quantitation of HCV RNA across HCV
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genotypes 1-6.
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242 243 Author’s contributions
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GH, AB, and UC conceived and designed the study. AB and UC coordinated study execution.
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PH and SA analyzed the data. AB and GH wrote the paper. All authors read and approved the
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final manuscript.
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Funding: This study was supported and funded by Roche Molecular Systems, Inc., Pleasanton,
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CA.
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Competing interests: Ann Butcher, Shagufta Aslam, Pari Hemyari, Ula Cowen, and Gabrielle
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Heilek are Roche employees.
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Ethical approval: Institutional Review Board
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Acknowledgments
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Support in preparation of the manuscript was provided by A. C. Jacobson of InClin, Inc.
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Peiffer KH, Sarrazin C. The importance of HCV RNA measurement for tailoring treatment
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Sofosbuvir for hepatitis C genotype 2 or 3 in patients without treatment options. N Engl J Med
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2013;368:1867-77. 27.
321 322
Lawitz E, Mangia A, Wyles D, Rodriguez-Torres M, Hassanein T, Gordon SC, et al. Sofosbuvir
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for previously untreated chronic hepatitis C infection. N Engl J Med 2013;368:1878-87. 28.
Zeuzem S, Berg T, Gane E, Ferenci P, Foster GR, Fried MW, et al. Simeprevir Increases Rate of
323
Sustained Virologic Response Among Treatment-Experienced Patients with HCV Genotype-1
324
Infection: a Phase IIb Trial. Gastroenterology 2013.
325
29.
Chevaliez S, Bouvier-Alias M, Rodriguez C, Soulier A, Poveda JD, Pawlotsky JM. The Cobas
326
AmpliPrep/Cobas TaqMan HCV test, version 2.0, real-time PCR assay accurately quantifies
327
hepatitis C virus genotype 4 RNA. J Clin Microbiol 2013;51:1078-82.
328 329 17 Page 17 of 23
329 Figure Legends
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Fig. 1. Deming regression analysis of HCV RNA levels determined by the TAQMAN v2.0 and
332
the TAQMAN HCV Test in RNA positive clinical samples (n=161). The solid line represents the
333
calculated trajectory from the samples and the dotted line represents the line of unity.
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Table 1 Demographics and Clinical Characteristics of Analysis Populations HCV Antibody Positive Participants N=277 n (%)
Age Category
112 (40.4%)
Detectable HCV RNA
165 (59.6%)
Male
151 (54.5%)
Female
126 (45.5%)
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Sex
Undetectable HCV RNA
<40
59 (21.3%)
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Subjects Enrolled
>=40
218 (78.7%)
Black / African-American
Race
214 (77.3%)
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White Other Hispanic or Latino Not Hispanic or Latino Not Reported/Unknown
3 (1.1%)
95 (34.3%)
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Ethnicity
a
60 (21.7%)
165 (59.6%) 17 (6.1%)
HCV Genotype/Subtype
Total Genotype 1 1 1B
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1A
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Participants with Detectable HCV RNA (N=165)
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Genotype 2
9 (5.5%) 62 (37.6%) 26 (15.8%) 11 (6.7%)
Genotype 3
16 (9.7%)
Genotype 4
1 (0.6%)
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a
97 (58.8%)
40 (24.2%)
Category unknown includes participants for whom the corresponding information is not available.
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Table 2 Performance Characteristics of Assays Assay VERSANT AMPLICOR TAQMAN v1.0 TAQMAN v2.0
LOD 5 IU/mla
LLOQ N/A
50 IU/mLa 14 IU/mLa 15 IU/mLa
N/A 43 IU/mL 15 IU/mL
Manufacturer Siemens Medical Solutions Diagnostics, Tarrytown, NY Roche Molecular Systems, Pleasanton, CA Roche Molecular Systems, Pleasanton, CA Roche Molecular Systems, Pleasanton, CA
a
LOD for plasma determined in comparison to World Health Organization Standard LOD = limit of detection; LLOQ = lower limit of quantitation; N/A = not applicable
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Table 3 Comparison of the TAQMAN v2.0 with Individual HCV Qualitative Assays VERSANT Positive
Negativea
Total
166
2
168
Negative Total Positive Percent Agreement (95% exact CI)
0
109
109
166
111
277
100.0% (97.7%, 100.0%) 98.2% (93.7%, 99.5%)
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Negative Percent Agreement (95% exact CI)
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Positive
Overall Percent Agreement (95% exact CI) AMPLICOR
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TAQMAN v2.0
99.3% (97.4%, 99.8%)
Negativeb
Total
Positive
166
2
168
Negative
1
108
109
110
277
Total
167 99.4% (96.7%, 99.9%)
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Positive Percent Agreement (95% exact CI)
Negative Total
Positive Percent Agreement (95% exact CI)
Negative Percent Agreement (95% exact CI) Overall Percent Agreement (95% exact CI) a
te
Positive
98.9% (96.9%, 99.6%)
TAQMAN v1.0
Positivec
Negativec
Total
161
2
163
1
103
104
162
105
267
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TAQMAN v2.0
98.2% (93.6%, 99.5%)
d
Negative Percent Agreement (95% exact CI) Overall Percent Agreement (95% exact CI)
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Positive
TAQMAN v2.0
99.4% (96.6%, 99.9%) 98.1% (93.3%, 99.5%) 98.9% (96.7%, 99.6%)
For two specimens with TAQMAN v2.0 results positive and VERSANT results negative, the HCV RNA was detected on the TAQMAN v2.0, but was <15 IU/mL. b For two specimens with TAQMAN v2.0 results positive and AMPLICOR results negative, HCV RNA was detected on the TAQMAN v2.0 but was <15 IU/mL c For two specimens with TAQMAN v2.0 results positive and TAQMAN v1.0 results negative, HCV RNA was detected on the TAQMAN v2.0 but was <15 IU/mL CI = confidence interval.
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Table 4 Discordant Test Results Results TAQMAN v1.0b Negative
VERSANT Negative
AMPLICOR Negative
Re-test Negativec
200-0371
Positive
Negative
Negative
Negative
Negativec
200-0465
Negative
Negative
Negative
Positive
Negatived
200-0767
Negative
Positive
Negative
Negative
NA
Positive indicates a valid test result below the lower limit of quantification (<15 IU/mL) Positive indicates a valid test result below the lower limit of quantification (<43 IU/mL) c Re-test with TAQMAN v2.0 and VERSANT in duplicate d Re-test with AMPLICOR in duplicate ID = identification; IU = international units; NA= not available; b
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TAQMAN v2.0a Positive
Sample ID 200-0130
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S1386-6532(14)00151-6 http://dx.doi.org/doi:10.1016/j.jcv.2014.04.018 JCV 3016
To appear in:
Journal of Clinical Virology
Received date: Revised date: Accepted date:
20-12-2013 16-4-2014 23-4-2014
Please cite this article as: Butcher A, Aslam S, Hemyari P, Cowen U, Heilek G, HCV RNA Detection in HCV Antibody-Positive Patients with the COBASregd AmpliPrep/COBASregd TaqManregd HCV Test, v2.0 in Comparison with FDA-approved Nucleic Acid Tests, Journal of Clinical Virology (2014), http://dx.doi.org/10.1016/j.jcv.2014.04.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 2
HCV RNA Detection in HCV Antibody-Positive Patients with the COBAS® AmpliPrep / COBAS® TaqMan® HCV Test, v2.0 in Comparison with FDA-approved Nucleic Acid Tests
3 4
Ann Butcher, Shagufta Aslam, Pari Hemyari, Ula Cowen, and Gabrielle Heilek*
*Correspondent footnote:
7
Gabrielle Heilek Ph.D.
8
Roche Molecular Systems, Inc.
9
4300 Hacienda Drive
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Pleasanton, CA 94588
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Tel: 925-730-8429
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Fax: 925-730-8988
13
Email: [email protected]
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Abbreviations: CHC, chronic HCV infection; DAA, direct acting antiviral; HCV, hepatitis C virus; LLOD, lower limit of detection; LOD, limit of detection; NPA, negative percent agreement; PCR, polymerase chain reaction; PPA, positive percent agreement; RGT, response guided therapy; RMS, Roche Molecular Systems; SVR, sustained virologic response
19
Keywords: Hepatitis C virus, nucleic acid testing, sustained virologic response, limit of detection
20
Abstract: 199 words
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Text: 2500 words
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Highlights •
Detection of HCV RNA offers a highly sensitive means of detecting active viremia.
25
•
The primary goal of HCV therapy is eradication of HCV infection.
26
•
The TAQMAN v2.0is a sensitive test and accurately detects all genotype samples.
27
•
The TAQMAN v2.0 demonstrated correlation with previously approved HCV RNA tests.
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Abstract
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Background: Analysis of hepatitis C virus (HCV) RNA levels is critical for assessing the
30
efficacy of antiviral therapy and the achievement of a sustained virologic response.
31
Objective and Study Design: This study evaluated the clinical performance of the COBAS®
32
AmpliPrep / COBAS® TaqMan® HCV Quantitative Test, version 2.0 (TAQMAN v2.0) with the
33
COBAS® AmpliPrep / COBAS® TaqMan® HCV Quantitative Test, version 1.0 (TAQMAN
34
v1.0), the VERSANT® HCV Qualitative Assay (VERSANT), and the COBAS® AMPLICOR
35
HCV Test, v2.0 (AMPLICOR) qualitative test for the detection of HCV RNA in serum or EDTA
36
plasma from patients who are or have been infected with HCV and carry HCV antibodies.
37
Results: A total of 277 participants were evaluable for the percent agreement analysis of the
38
TAQMAN v2.0 with the VERSANT and with the AMPLICOR. The overall percent agreement
39
between the TAQMAN v2.0 and the VERSANT or the AMPLICOR was 99.3% (95% CI:
40
97.4%, 99.8%) or 98.9% (95% CI: 96.9%, 99.6%), respectively. The overall percent agreement
41
between the TAQMAN v2.0 and the TAQMAN v1.0 when 267 of the original samples were
42
assessed was 98.9 % (95% CI= 96.7%, 99.6%).
43
Conclusion: The TAQMAN v2.0 demonstrated high correlation with the previously approved
44
HCV RNA quantitative and qualitative tests.
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1
Background
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Hepatitis C virus (HCV) is a single-stranded, positive sense RNA virus and has a genome of
48
about 10,000 nucleotides coding for 3,000 amino acids 1. The World Health Organization
49
estimates that approximately 170 million persons worldwide are infected with HCV with the
50
incidence in the United States (US) estimated at 1.6%, of which approximately 2.7 million are
51
chronically infected with HCV2-4. The US guidelines recommend that the diagnosis of chronic
52
HCV (CHC) infection be made through a stepwise process with initial screening for HCV
53
antibodies performed using validated enzyme-linked immunoassays5-8. HCV antibody screening
54
can yield positive results in patients who were infected with HCV but who subsequently have
55
spontaneously cleared infection without antiviral therapy intervention. Detection of HCV RNA
56
by polymerase chain reaction (PCR) nucleic acid amplification offers a highly sensitive and
57
specific means of detecting active viremia and has been recommended as the confirmative test of
58
choice following a positive result with HCV antibody detection 5.
59
HCV infection is curable by antiviral therapy. Since 2011, the standard of care for the treatment
60
of CHC genotype 1 infection is a triple therapy combination of direct acting antiviral (DAA)
61
medicines, the protease inhibitors boceprevir and telaprevir, used in combination with pegylated
62
interferon-alpha plus ribavirin9-12. The primary goal of HCV therapy is eradication of HCV
63
infection, assessed at the sustained virologic response (SVR) timepoint, 12 to 24 weeks after
64
cessation of anti-viral medication13,14. Analysis of HCV RNA levels is critical for assessing the
65
efficacy of therapy for CHC and should be performed with a sensitive molecular method with a
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lower limit of detection (LOD) of <15 IU/ml13,15,16. Monitoring HCV RNA levels at defined
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timepoints is mandated and assures optimized outcomes; to qualify for the shortened treatment
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course (response guided therapy [RGT]), early achievement of undetectable HCV RNA (target
69
not detected [TND] readout) while taking the triple combination regimen is required14,15,17.
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The COBAS® AmpliPrep / COBAS® TaqMan® HCV Quantitative Test, version 2.0
71
(TAQMAN v2.0) offers an LOD = lower limit of quantification (LLOQ) of 15 IU/mL. Primers
72
and probes have been modified from the first generation test to more accurately detect all
73
genotype samples. Additional assay improvements include a novel dual probe approach, an
74
improved sample preparation method, and a reduced sample input volume18,19.
75
The LLOQ of 15 IU/mL test is similar to or lower than that of other FDA-approved qualitative
76
assays used for the detection of HCV RNA: the VERSANT® HCV RNA Qualitative Assay
77
(Siemens Healthcare Diagnostics Inc.; VERSANT) with an LOD of 5 IU/mL and the COBAS®
78
AMPLICOR HCV Test, v2.0 (Roche Molecular Systems, Inc.; AMPLICOR) with an LOD of
79
50 IU/mL. Additionally, the TAQMAN v2.0 is an improvement over its predecessor, the
80
COBAS® AmpliPrep / COBAS® TaqMan® HCV Quantitative Test, version 1.0 (TAQMAN
81
v1.0) which has an LLOQ of 43 IU/mL and can facilitate more accurate clinical interpretation in
82
cases of HCV RNA levels below 43 IU/mL with its extended dynamic range 18,19.
83
2
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The aim of the present study was to evaluate the concordance between the TAQMAN v2.0 with
85
that of the VERSANT, AMPLICOR, and TAQMAN v1.0 tests for the detection of HCV RNA in
86
serum or EDTA plasma from patients who are or have been infected with HCV and carry HCV
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antibodies.
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Objectives
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3
Study Design
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3.1
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For this study, 282 plasma and 25 serum samples with sufficient volume for multiple assay
91
testing were obtained from previous Institutional Review Board-approved Roche Molecular
92
Systems (RMS) studies and included treatment-naïve subjects with chronic HCV infection or
93
spontaneously-resolved infection and subjects who had been successfully treated with HCV
94
antiviral therapy, as defined by undetectable HCV RNA following a minimum of 12 weeks after
95
end of therapy, and from whom de-identified archived specimens from a clinical study were
96
available. Samples were sent to a central laboratory for confirmatory HCV antibody testing.
97
Samples that yielded HCV-antibody negative, indeterminate, or a missing result were excluded
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from the study analysis. The majority of participants in the analysis population were 40 years of
99
age or older (78.7%) and White (77.3%); the analysis population had approximately equal
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Patient samples and test materials
numbers of male and female participants (Table 1). Out of 277 eligible participants included in
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the analysis, 165 (~60%) were treatment-naïve with established HCV RNA positive status. The
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rest were established as HCV RNA negative, either because of spontaneously resolved HCV
103
infection or because they achieved SVR after HCV antiviral treatment. HCV genotyping was
104
available from 125/165 HCV RNA positive patients. The majority of RNA positive patients
105
(58.8%) carried genotype1; genotype 2 and 3 were represented at 6.7% and 9.7%, respectively.
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This distribution is in agreement with typical patient populations seen in the United States or
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Western Europe 20. Demographics and clinical characteristics of the analysis population are
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presented in Table 1.
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3.2
Viral load assays
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Samples were tested with the VERSANT, the AMPLICOR, the TAQMAN v1.0, and the
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TAQMAN v2.0 according to the manufacturer’s instructions and results for each test were
112
interpreted according to the respective product package inserts. The TAQMAN v1.0 and
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TAQMAN v2.0 were carried out on the fully automated, docked COBAS® AmpliPrep /
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COBAS® TaqMan® configuration with either 48 or 96 processing capacity. Samples that
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yielded discrepant results between tests were retested from an additional aliquot if sample
116
volume allowed. Performance characteristics of the assays used in this study are presented in
117
Table 2.
118
3.3
119
For the calculation of agreement between the qualitative and quantitative tests, TAQMAN v2.0
120
quantitative results were dichotomized into positive and negative. Negative was defined as
121
having a result of ‘Target Not Detected’ (TND); any other valid result was defined as positive.
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The agreement of the TAQMAN v2.0 with any comparator assay (VERSANT, AMPLICOR, or
123
TAQMAN v1.0) was calculated as shown below with confidence intervals (CIs) for positive
124
percent agreement (PPA), negative percent agreement (NPA), and overall percent agreement
125
calculated using the Wilson (score) CIs following the formulas described in the CLSI EP12-A2
126
guideline 21,22.
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The following statistics were calculated: Estimated positive percent agreement of the TAQMAN v2.0 with respect to comparator
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assay result = 100% × a/(a+c)
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Estimated negative percent agreement of the TAQMAN v2.0 with respect to comparator
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assay result = 100% × d/(b+d)
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Overall percent agreement of the TAQMAN v2.0 with respect to comparator assay result
133
= 100% × (a+d)/(a+b+c+d)
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The 95% score CIs were calculated using PROC FREQ. All data analyses were performed using
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SAS/STAT® software (SAS Institute, Inc., Cary, NC).
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4
RESULTS
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4.1
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Of the 307 participants enrolled in this study, a final 277 participants (90.2%) were evaluable
140
after review of inclusion and exclusion criteria for the percent agreement analysis of the
141
TAQMAN v2.0 with the VERSANT and with the AMPLICOR. For comparison with the
142
TAQMAN v1.0, 267 of the 277 original samples had sufficient volume and were assessed.
143
4.2
144
Concordance between the TAQMAN v2.0 and the composite comparator (VERSANT and the
145
AMPLICOR) was evaluated by determining overall percent agreement (data not shown). Table 3
146
shows the comparison of the results between the TAQMAN v2.0 and the VERSANT or the
147
AMPLICOR. The overall percent agreements of the TAQMAN v2.0 with the VERSANT or the
148
AMPLICOR were 99.3% (95% CI: 97.4%, 99.8%) or 98.9% (95% CI: 96.9%, 99.6%),
149
respectively (Table 3).
150
For two specimens, TAQMAN v2.0 results were positive and VERSANT results were negative;
151
HCV RNA was detected with the TAQMAN v2.0 but was <15 IU/mL. Upon retesting of these
152
two samples in duplicate on the TAQMAN v2.0 and the VERSANT, all four aliquots returned a
153
negative result for the presence of HCV RNA, indicating that the initial TAQMAN v2.0 results
154
were false positive (Table 4). One sample yielded an indeterminate composite result: HCV RNA
155
positive by the AMPLICOR and HCV RNA negative by the VERSANT. This sample was also
156
HCV RNA negative by the TAQMAN v2.0. Upon retesting this sample in duplicate with the
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Comparison of TAQMAN v2.0 to the VERSANT and AMPLICOR Qualitative Assays
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AMPLICOR, both retest results were negative, indicating that the initial AMPLICOR result was
158
a false positive.
159
4.3
160
Concordance between the TAQMAN v2.0 and the first generation TAQMAN v1.0 was evaluated
161
in 267 samples. Table 3 shows the comparison of results between the TAQMAN v2.0 and the
162
TAQMAN v1.0; overall percent agreement between the two tests was 98.9% (95% CI= 96.7,
163
99.6). The PPA and NPA of the TAQMAN v2.0 with the TAQMAN v1.0 was 99.4% and 98.1%,
164
respectively. There were three discrepant results: one TAQMAN v2.0 negative and
165
TAQMAN v1.0 positive and two TAQMAN v2.0 positive and TAQMAN v1.0 negative
166
(Table 4). For these discrepant results the HCV RNA levels detected were below the LLOQ for
167
all and represent sampling variability inherent in the extraction and amplification limits of all
168
real time PCR assays.
169
The one discrepant TAQMAN v1.0 positive sample, yielded negative results from the
170
VERSANT and AMPLICOR (Table 4) and was not retested due to insufficient remaining sample
171
volume. Overall, the data demonstrated high correlation between the TAQMAN v1.0 and the
172
TAQMAN v2.0. Deming regression analysis was carried out in the HCV RNA positive samples
173
across the linear range of the tests (n=161) and showed high comparability between the
174
TAQMAN v1.0 and the TAQMAN v2.0 with an R2 value of 0.93 (Fig. 1).
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Comparison of TAQMAN v2.0 to TAQMAN v1.0
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5
Discussion
177
The clinical utility of HCV RNA tests has been well-established. Initial HCV RNA tests were
178
qualitative tests, detecting hepatitis C viremia to confirm active HCV infection in patients with
179
antibodies to HCV. Once HCV treatments became available, quantitative tests were developed to
180
monitor changes in viral load during treatment and to predict response to therapy. However, as
181
the early quantitative tests were less sensitive (LOD ~600 IU/mL) than the qualitative tests (LOD
182
~50 IU/mL), qualitative tests continued to be used to confirm “clearance” or persistence of HCV
183
in patients with HCV RNA levels below 600 IU/mL. Qualitative HCV RNA tests therefore
184
became widely used to determine clearance of viremia and to assess end of treatment response
185
and confirmation of SVR5,6.
186
As HCV treatment strategies progressed from pegylated-interferon alfa/ribavirin regimens to
187
those including DAAs (such as telaprevir or boceprevir), information on the lower HCV RNA
188
viral load levels has become increasingly important. A process involving continuous monitoring
189
of therapy and the ability to distinguish and quantify low levels or absence of viremia both
190
during and after therapy is now essential15. Futility rules have been established to predict success
191
of treatment early-on during the regimen course14. In addition, the definition of viral clearance
192
has become more stringent as therapies improve. The aim of triple therapy containing protease
193
inhibitors is to achieve an undetectable HCV RNA level (TND) using a highly sensitive test
194
(LLOQ < 15 IU/mL)13,15. Finally, guidelines have recognized that with the advent of more
195
sensitive quantitative HCV RNA assays, qualitative assays are no longer considered necessary 6.
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In 2012, the Center for Disease Control and Prevention issued a recommendation that persons
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born during 1945 to 1965 should receive a one-time HCV test to better understand the overall
198
epidemiology of HCV in the United States 23. To accomplish this large Hepatitis awareness
199
effort, access to an accurate nucleic acid detection and quantitation tool is paramount.
200
In the current study the performance of the TAQMAN v2.0 was compared to two FDA-approved
201
qualitative HCV RNA tests and one quantitative test. The primary comparisons were to the
202
VERSANT, a commercially available highly sensitive qualitative test (LOD = 5 IU/mL) and the
203
AMPLICOR (LOD = 50 IU/mL). Additionally, the TAQMAN v2.0 was compared to the first
204
generation TAQMAN v1.0. Clinical samples from a range of subjects with antibodies to HCV
205
were tested in the current study; treatment naïve patients with spontaneously resolved or chronic
206
HCV infection and previously treated patients who had achieved SVR. The TAQMAN v2.0
207
results were similar to those of the comparator tests. HCV RNA was detected by the
208
TAQMAN v2.0 in all samples (166/166) that were positive on the VERSANT and/or for which
209
the composite result was positive. The TAQMAN v2.0 also detected HCV RNA in the single
210
sample with an indeterminate composite result (VERSANT positive; AMPLICOR negative). In
211
the HCV RNA negative samples, the TAQMAN v2.0 showed 98.2% (109/111) agreement with
212
the VERSANT, 98.2% (108/110) agreement with the AMPLICOR, and 98.2% (108/110)
213
agreement with the composite comparator. The specificity of the TAQMAN v2.0 was therefore
214
similar to that of the VERSANT and the AMPLICOR.
215
HCV RNA was detected in two samples at very low level (<15 IU/mL) on the TAQMAN v2.0
216
but by neither comparator test. Upon retesting of a second aliquot, these samples returned a
217
negative result for the presence of HCV RNA indicating that the initial TAQMAN v2.0 results
218
were either false positive or were positive at a very low level where reproducible amplification is
219
stochastically low. PCR amplification below the LOD = LLOQ of 15 IU/mL is a statistical event
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with decreasing probability of target amplification as the HCV RNA concentration nears the zero
221
value. Residual HCV RNA has been reported to be detectable in blood mononuclear cells for
222
extended periods of time, especially in early phases of viral clearance (eg, week 4 of strong DAA
223
containing regimens) 24,25. Low level positive results in a quantitative assay should be considered
224
in the context of other laboratory markers to achieve a final patient assessment.
225
Ideally in an individual patient, the same HCV RNA test should be able to diagnose active
226
infection, provide a baseline viral load, measure changes in viral load during treatment, and
227
evaluate viral clearance at the end of treatment and during follow up. This may become of even
228
more importance in the ‘test and treat’ era of HCV infection and therapy about to commence
229
with the approval of several DAA regimens that obtain very high SVR rates 26-28. Such a test,
230
also preferable to the clinician, would require sensitivity of LLOQ <15 IU/mL, a wide dynamic
231
range of quantification, and similar performance across HCV genotypes. The TAQMAN v2.0
232
was developed as a second generation assay to the TAQMAN v1.0 by using a novel dual-probe
233
approach and an additional reverse primer to improve HCV genotype 4 detection18,19,29. With the
234
TAQMAN v2.0 the “grey zone’ between the LOD and LLOQ present in the previous version of
235
the TaqMan HCV test was eliminated, allowing clinicians and laboratories easier interpretation
236
of results.
237
In conclusion, in a range of patient groups (treated and treatment naïve, with or without active
238
HCV infection), the TAQMAN v2.0 is highly concordant with other FDA-approved HCV RNA
239
tests, the VERSANT, the AMPLICOR, and the TAQMAN v1.0, and has several improved
240
performance features that provide accurate detection and quantitation of HCV RNA across HCV
241
genotypes 1-6.
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242 243 Author’s contributions
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GH, AB, and UC conceived and designed the study. AB and UC coordinated study execution.
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PH and SA analyzed the data. AB and GH wrote the paper. All authors read and approved the
247
final manuscript.
248
Funding: This study was supported and funded by Roche Molecular Systems, Inc., Pleasanton,
249
CA.
250
Competing interests: Ann Butcher, Shagufta Aslam, Pari Hemyari, Ula Cowen, and Gabrielle
251
Heilek are Roche employees.
252
Ethical approval: Institutional Review Board
253
Acknowledgments
254
Support in preparation of the manuscript was provided by A. C. Jacobson of InClin, Inc.
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329 Figure Legends
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Fig. 1. Deming regression analysis of HCV RNA levels determined by the TAQMAN v2.0 and
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the TAQMAN HCV Test in RNA positive clinical samples (n=161). The solid line represents the
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calculated trajectory from the samples and the dotted line represents the line of unity.
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Table 1 Demographics and Clinical Characteristics of Analysis Populations HCV Antibody Positive Participants N=277 n (%)
Age Category
112 (40.4%)
Detectable HCV RNA
165 (59.6%)
Male
151 (54.5%)
Female
126 (45.5%)
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Undetectable HCV RNA
<40
59 (21.3%)
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Subjects Enrolled
>=40
218 (78.7%)
Black / African-American
Race
214 (77.3%)
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White Other Hispanic or Latino Not Hispanic or Latino Not Reported/Unknown
3 (1.1%)
95 (34.3%)
an
Ethnicity
a
60 (21.7%)
165 (59.6%) 17 (6.1%)
HCV Genotype/Subtype
Total Genotype 1 1 1B
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1A
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Participants with Detectable HCV RNA (N=165)
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Genotype 2
9 (5.5%) 62 (37.6%) 26 (15.8%) 11 (6.7%)
Genotype 3
16 (9.7%)
Genotype 4
1 (0.6%)
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Not Reported
a
97 (58.8%)
40 (24.2%)
Category unknown includes participants for whom the corresponding information is not available.
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Table 2 Performance Characteristics of Assays Assay VERSANT AMPLICOR TAQMAN v1.0 TAQMAN v2.0
LOD 5 IU/mla
LLOQ N/A
50 IU/mLa 14 IU/mLa 15 IU/mLa
N/A 43 IU/mL 15 IU/mL
Manufacturer Siemens Medical Solutions Diagnostics, Tarrytown, NY Roche Molecular Systems, Pleasanton, CA Roche Molecular Systems, Pleasanton, CA Roche Molecular Systems, Pleasanton, CA
a
LOD for plasma determined in comparison to World Health Organization Standard LOD = limit of detection; LLOQ = lower limit of quantitation; N/A = not applicable
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Table 3 Comparison of the TAQMAN v2.0 with Individual HCV Qualitative Assays VERSANT Positive
Negativea
Total
166
2
168
Negative Total Positive Percent Agreement (95% exact CI)
0
109
109
166
111
277
100.0% (97.7%, 100.0%) 98.2% (93.7%, 99.5%)
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Negative Percent Agreement (95% exact CI)
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Positive
Overall Percent Agreement (95% exact CI) AMPLICOR
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TAQMAN v2.0
99.3% (97.4%, 99.8%)
Negativeb
Total
Positive
166
2
168
Negative
1
108
109
110
277
Total
167 99.4% (96.7%, 99.9%)
M
Positive Percent Agreement (95% exact CI)
Negative Total
Positive Percent Agreement (95% exact CI)
Negative Percent Agreement (95% exact CI) Overall Percent Agreement (95% exact CI) a
te
Positive
98.9% (96.9%, 99.6%)
TAQMAN v1.0
Positivec
Negativec
Total
161
2
163
1
103
104
162
105
267
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TAQMAN v2.0
98.2% (93.6%, 99.5%)
d
Negative Percent Agreement (95% exact CI) Overall Percent Agreement (95% exact CI)
an
Positive
TAQMAN v2.0
99.4% (96.6%, 99.9%) 98.1% (93.3%, 99.5%) 98.9% (96.7%, 99.6%)
For two specimens with TAQMAN v2.0 results positive and VERSANT results negative, the HCV RNA was detected on the TAQMAN v2.0, but was <15 IU/mL. b For two specimens with TAQMAN v2.0 results positive and AMPLICOR results negative, HCV RNA was detected on the TAQMAN v2.0 but was <15 IU/mL c For two specimens with TAQMAN v2.0 results positive and TAQMAN v1.0 results negative, HCV RNA was detected on the TAQMAN v2.0 but was <15 IU/mL CI = confidence interval.
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Table 4 Discordant Test Results Results TAQMAN v1.0b Negative
VERSANT Negative
AMPLICOR Negative
Re-test Negativec
200-0371
Positive
Negative
Negative
Negative
Negativec
200-0465
Negative
Negative
Negative
Positive
Negatived
200-0767
Negative
Positive
Negative
Negative
NA
Positive indicates a valid test result below the lower limit of quantification (<15 IU/mL) Positive indicates a valid test result below the lower limit of quantification (<43 IU/mL) c Re-test with TAQMAN v2.0 and VERSANT in duplicate d Re-test with AMPLICOR in duplicate ID = identification; IU = international units; NA= not available; b
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TAQMAN v2.0a Positive
Sample ID 200-0130
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