G Model
ARTICLE IN PRESS
JCV 3290 1–6
Journal of Clinical Virology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Evaluation of the COBAS® AmpliPrep/COBAS® TaqMan® HCV Test, v2.0 and comparison to assays used in routine clinical practice in an international multicenter clinical trial: The ExPECT study
1
2
3
4
Q2
5 6 7 8 9 10 11 12 13
Harald H. Kessler a,∗ , Bryan Cobb b , Heiner Wedemeyer c , Benjamin Maasoumy c , Veronique Michel-Treil d , Luca Ceccherini-Nelli e , Birgit Bremer c , Margit Hübner a , Anna Helander d , Hacene Khiri f , Gabrielle Heilek b , Christian Simon b , Kevin Luk b , Shagufta Aslam b , Philippe Halfon f a
Molecular Diagnostics Laboratory, IHMEM, Medical University of Graz, Graz, Austria Roche Molecular Systems, Inc., Pleasanton, CA, USA Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany d Covance Laboratories, Geneva, Switzerland e Università degli Studi di Pisa, Pisa, Italy f Laboratoire ALPHABIO, Hôpital Europeen, Marseille, France b c
14
15 26
a r t i c l e
i n f o
a b s t r a c t
16 17 18 19 20
Article history: Received 12 December 2014 Received in revised form 25 March 2015 Accepted 28 March 2015
21
25
Keywords: Hepatitis C virus RNA CAP/CTM HCV Test, v2.0 Performance
27
1. Background
22 23 24
28Q3 29 30 31 32 33
Background: The COBAS® AmpliPrep® /COBAS® TaqMan® HCV Test, v2.0 (CAP/CTM2) is used for HCV RNA viral load monitoring. Objectives: The performance of the CAP/CTM2 was compared to other widely used tests, including a manual version of the assay (the COBAS® TaqMan® HCV Test, v2.0 for use with the High Pure System, HPS/CTM2) predominantly used during phase III clinical trials for the new direct acting antiviral therapies. Study design: Low HCV RNA level comparisons were performed across tests (Abbott Realtime HCV Test, ART; COBAS® AmpliPrep® /COBAS® TaqMan® HCV Test, v1.0, CAP/CTM1; CAP/CTM2; and HPS/CTM2) using dilutions of the 2nd HCV WHO International Standard. Additionally, the clinical performance of the CAP/CTM2 was evaluated with 421 leftover HCV RNA-positive routine clinical samples. Results: All quantifiable WHO dilutions were within ±0.3 log10 IU/mL of the expected results across tests and the analytical sensitivity resulted in a limit of detection of 12 IU/mL (95% confidence interval, 10, 15). When clinical samples were tested the results for 87% (367 of 421) of all sample comparisons were within ±0.5 log10 IU/mL. When low viral load results (25–3500 IU/mL) were compared, values obtained by the ART assay were significantly lower (p < 0.0001) than those obtained with the CAP/CTM2. Conclusions: The new CAP/CTM2 showed good accuracy with comparable sensitivity to comparator assays. The new kit is well-suited for use in the routine diagnostic laboratory, especially for accurate monitoring of patients receiving triple therapy or interferone-free regimens. © 2015 Published by Elsevier B.V.
Accurate HCV RNA viral load (VL) monitoring is essential for the management of chronic hepatitis C patients to assess the virological response while on therapy. Furthermore, HCV RNA is also important to assess therapeutic outcome at the end of treatment and six months after the end of therapy [1–3]. A rapid virological response is a strong predictor of sustained virological response (SVR) for
∗ Corresponding author. Tel.: +43 316 380 4363. E-mail address: [email protected] (H.H. Kessler).
regimens including pegylated-interferon-alpha (Peg-IFN). The current EASL clinical practise guideline indicates HCV RNA detection and quantification should be made with a sensitive assay (lower limit of detection of 25 IU/mL at week 4, 12, or 24 of therapy [9]. All the registration trials of the currently approved triple therapy drug regimens used the manual extraction version of the Roche test, the COBAS® TaqMan® HCV Test, v2.0 for use with the High Pure System (HPS/CTM2). Automated tests that are widely used in routine clinical practise may show different performances, making it unclear whether the recommendations based on the pivotal trial data can be broadly applied. In order to provide a fast and easy test with a similar performance to the assay used in drug trials, a new assay was developed based on the HPS/CTM2 and the first generation automated test. This new test, the COBAS® AmpliPrep® /COBAS® TaqMan® HCV Test, v2.0 (CAP/CTM2) has a lower limit of HCV RNA quantification, (15 IU/mL) and optimized quantification across HCV genotypes, including challenging genotype 4 specimens [10–12]. This multi-center study was designed to investigate samples with low HCV viremia and compare the performance of the CAP/CTM2 to various HCV RNA assays used in routine clinical practise including the assay predominantly used in drug development clinical trials.
76
2. Objectives
46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74
77 78 79
The aim of this study was to assess the performance of the second generation CAP/CTM2 at different testing sites across Europe and to compare it with other widely used HCV RNA VL tests.
80
3. Study design
81
3.1. Study sites and molecular assays
(Molecular Diagnostics Laboratory, Medical University); Hannover, Germany (Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School); Marseille, France (Laboratoire Alphabio, Hospital Europeen); Pisa, Italy (Università degli Studi). All experiments were done in International Standard Organization (ISO) 9001:2008-certified or ISO 17,025:2005-accredited laboratories. All tests were performed according to the manufacturer´ıs package insert instructions. The characteristics of the molecular assays used in this study are summarized in Table 1. All assays have been in vitro diagnostics (IVD)/Conformité Europeéne (CE-IVD)-labeled and FDA approved. Results observed for each assay with an HCV RNA VL result were referred to as a numerical number (in IU/mL) if within the analytical measurement range, TND, or “HCV RNA detected, 0.5 log lower by the ART) were found throughout the range of the assay. Of the 3 samples that had VL differences that were lower by 1 log10 had an average result between the two assays of approximately 650, 45,000, and 4500,000 IU/mL. In all of these samples, the ART gave lower results. The mean difference between results obtained by the CAP/CTM2 and the HPS/CTM2 was −0.022 log10 unit. 91% (96/106) of the samples were found to be within ±0.5 log10 unit and 9% (10/106) of the samples were found to be within ±0.5 and ±1.0 log10 . Results that deviated more than 0.5 log10 were lower by the CAP/CTM2, all of which were observed with high-end values (exceeding 600,000 IU/mL). In paired patient sample results analyzed at different cutoffs, there were more results detected using the ART that were undetectable (TND) with the CAP/CTM2. At a 25 IU/mL cutoff (the threshold used for RGT in the clinical trials with faldaprevir, SPV, BOC and TVR and the recommended cut-off for treatment futility in the US SPV label), the HCV RNA result had an overall agreement of 87%. This agreement improved to 94% when using a lower 15 IU/mL cutoff (Table 6). When low VL results (25–3500 IU/mL) were compared in clinical samples, the mean difference was similar between the CAP/CTM2 and both the CAP/CTM1 and the HPS/CTM2 while
Table 4 Low level HCV RNA agreement across methods compared in this study using the WHO panel. HCV RNA Testa
Observed mean HCV RNA result IU/mL (log10 )b
Difference (nominal - observed, log10 IU/mL)
Standard deviation
Results obtained for WHO nominal titer of 100 IU/mL 102 (2.01) CAP/CTM1 128 (2.11) CAP/CTM2 ART 56 (1.75) 98 (1.99) HPS/CTM2
−0.01 −0.11 0.25 0.01
0.15 0.13 0.03 0.25
Results obtained for WHO nominal titer of 50 IU/mL CAP/CTM1 36 (1.56) 73 (1.86) CAP/CTM2 45 (1.65) ART HPS/CTM2 54 (1.73)
0.14 −0.16 0.05 −0.03
0.19 0.17 0.12 0.18
Results obtained for WHO nominal titer of 25 IU/mL – CAP/CTM1c 26 (1.41) CAP/CTM2 19 (1.29) ART 24 (1.38) HPS/CTM2
– -0.01 0.11 0.02
– 0.29 0.13 0.26
a b c
CAP/CTM2 testing was performed at Hannover, results across sites are compared in results section. Results are reported according to the manufacturer´ıs package insert. Panel member below the CAP/CTM1 tests lower limit of quantification (43 IU/mL), results cannot be calculated.
Please cite this article in press as: H.H. Kessler, et al., Evaluation of the COBAS® AmpliPrep/COBAS® TaqMan® HCV Test, v2.0 and comparison to assays used in routine clinical practice in an international multicenter clinical trial: The ExPECT study, J Clin Virol (2015), http://dx.doi.org/10.1016/j.jcv.2015.03.023
155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199
G Model JCV 3290 1–6
ARTICLE IN PRESS
4
H.H. Kessler et al. / Journal of Clinical Virology xxx (2015) xxx–xxx
Table 5 Limit of detection across methods compared in this study using the WHO panel. Testing site
Test
Assay LOD (IU/mL)a
Geneva Graz Hannover Marseille Combined Geneva Graz Marseille
CAP/CTM2 CAP/CTM2 CAP/CTM2 CAP/CTM2 CAP/CTM2 HPS/CTM2 CAP/CTM1 ART
15 15 15 15 15 20 15 12
No. tested 24 30 30 30 114 30 30 30
No. detected 21 30 30 29 110 30 29 29
Estimated limit of detection (95% CI) (IU/mL) 15 (10, 31) 18 (12, 28) 11 (9, 19) 12 (9, 20) 12 (10, 15) 10 (7, 16) 11 (8, 18) 8 (6, 15)
LOD, limit of detection. a According to the manufacturer´ıs package insert.
Fig. 1. Correlation between the results (IU/mL) for EDTA plasma or serum HCV RNA positive samples obtained from patients with chronic hepatitis C tested by the CAP/CTM HCV Test, v2.0 and comparator tests in Graz (A), Hannover (B), Marseille (C), and Pisa (D). Table 6 Comparative results above and below 25 IU/mL and 15 IU/mL for the CAP/CTM2 and ART in clinical specimens. CAP/CTM2
ART TND
TND
ARTICLE IN PRESS
JCV 3290 1–6
Journal of Clinical Virology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Evaluation of the COBAS® AmpliPrep/COBAS® TaqMan® HCV Test, v2.0 and comparison to assays used in routine clinical practice in an international multicenter clinical trial: The ExPECT study
1
2
3
4
Q2
5 6 7 8 9 10 11 12 13
Harald H. Kessler a,∗ , Bryan Cobb b , Heiner Wedemeyer c , Benjamin Maasoumy c , Veronique Michel-Treil d , Luca Ceccherini-Nelli e , Birgit Bremer c , Margit Hübner a , Anna Helander d , Hacene Khiri f , Gabrielle Heilek b , Christian Simon b , Kevin Luk b , Shagufta Aslam b , Philippe Halfon f a
Molecular Diagnostics Laboratory, IHMEM, Medical University of Graz, Graz, Austria Roche Molecular Systems, Inc., Pleasanton, CA, USA Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany d Covance Laboratories, Geneva, Switzerland e Università degli Studi di Pisa, Pisa, Italy f Laboratoire ALPHABIO, Hôpital Europeen, Marseille, France b c
14
15 26
a r t i c l e
i n f o
a b s t r a c t
16 17 18 19 20
Article history: Received 12 December 2014 Received in revised form 25 March 2015 Accepted 28 March 2015
21
25
Keywords: Hepatitis C virus RNA CAP/CTM HCV Test, v2.0 Performance
27
1. Background
22 23 24
28Q3 29 30 31 32 33
Background: The COBAS® AmpliPrep® /COBAS® TaqMan® HCV Test, v2.0 (CAP/CTM2) is used for HCV RNA viral load monitoring. Objectives: The performance of the CAP/CTM2 was compared to other widely used tests, including a manual version of the assay (the COBAS® TaqMan® HCV Test, v2.0 for use with the High Pure System, HPS/CTM2) predominantly used during phase III clinical trials for the new direct acting antiviral therapies. Study design: Low HCV RNA level comparisons were performed across tests (Abbott Realtime HCV Test, ART; COBAS® AmpliPrep® /COBAS® TaqMan® HCV Test, v1.0, CAP/CTM1; CAP/CTM2; and HPS/CTM2) using dilutions of the 2nd HCV WHO International Standard. Additionally, the clinical performance of the CAP/CTM2 was evaluated with 421 leftover HCV RNA-positive routine clinical samples. Results: All quantifiable WHO dilutions were within ±0.3 log10 IU/mL of the expected results across tests and the analytical sensitivity resulted in a limit of detection of 12 IU/mL (95% confidence interval, 10, 15). When clinical samples were tested the results for 87% (367 of 421) of all sample comparisons were within ±0.5 log10 IU/mL. When low viral load results (25–3500 IU/mL) were compared, values obtained by the ART assay were significantly lower (p < 0.0001) than those obtained with the CAP/CTM2. Conclusions: The new CAP/CTM2 showed good accuracy with comparable sensitivity to comparator assays. The new kit is well-suited for use in the routine diagnostic laboratory, especially for accurate monitoring of patients receiving triple therapy or interferone-free regimens. © 2015 Published by Elsevier B.V.
Accurate HCV RNA viral load (VL) monitoring is essential for the management of chronic hepatitis C patients to assess the virological response while on therapy. Furthermore, HCV RNA is also important to assess therapeutic outcome at the end of treatment and six months after the end of therapy [1–3]. A rapid virological response is a strong predictor of sustained virological response (SVR) for
∗ Corresponding author. Tel.: +43 316 380 4363. E-mail address: [email protected] (H.H. Kessler).
regimens including pegylated-interferon-alpha (Peg-IFN). The current EASL clinical practise guideline indicates HCV RNA detection and quantification should be made with a sensitive assay (lower limit of detection of 25 IU/mL at week 4, 12, or 24 of therapy [9]. All the registration trials of the currently approved triple therapy drug regimens used the manual extraction version of the Roche test, the COBAS® TaqMan® HCV Test, v2.0 for use with the High Pure System (HPS/CTM2). Automated tests that are widely used in routine clinical practise may show different performances, making it unclear whether the recommendations based on the pivotal trial data can be broadly applied. In order to provide a fast and easy test with a similar performance to the assay used in drug trials, a new assay was developed based on the HPS/CTM2 and the first generation automated test. This new test, the COBAS® AmpliPrep® /COBAS® TaqMan® HCV Test, v2.0 (CAP/CTM2) has a lower limit of HCV RNA quantification, (15 IU/mL) and optimized quantification across HCV genotypes, including challenging genotype 4 specimens [10–12]. This multi-center study was designed to investigate samples with low HCV viremia and compare the performance of the CAP/CTM2 to various HCV RNA assays used in routine clinical practise including the assay predominantly used in drug development clinical trials.
76
2. Objectives
46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74
77 78 79
The aim of this study was to assess the performance of the second generation CAP/CTM2 at different testing sites across Europe and to compare it with other widely used HCV RNA VL tests.
80
3. Study design
81
3.1. Study sites and molecular assays
(Molecular Diagnostics Laboratory, Medical University); Hannover, Germany (Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School); Marseille, France (Laboratoire Alphabio, Hospital Europeen); Pisa, Italy (Università degli Studi). All experiments were done in International Standard Organization (ISO) 9001:2008-certified or ISO 17,025:2005-accredited laboratories. All tests were performed according to the manufacturer´ıs package insert instructions. The characteristics of the molecular assays used in this study are summarized in Table 1. All assays have been in vitro diagnostics (IVD)/Conformité Europeéne (CE-IVD)-labeled and FDA approved. Results observed for each assay with an HCV RNA VL result were referred to as a numerical number (in IU/mL) if within the analytical measurement range, TND, or “HCV RNA detected, 0.5 log lower by the ART) were found throughout the range of the assay. Of the 3 samples that had VL differences that were lower by 1 log10 had an average result between the two assays of approximately 650, 45,000, and 4500,000 IU/mL. In all of these samples, the ART gave lower results. The mean difference between results obtained by the CAP/CTM2 and the HPS/CTM2 was −0.022 log10 unit. 91% (96/106) of the samples were found to be within ±0.5 log10 unit and 9% (10/106) of the samples were found to be within ±0.5 and ±1.0 log10 . Results that deviated more than 0.5 log10 were lower by the CAP/CTM2, all of which were observed with high-end values (exceeding 600,000 IU/mL). In paired patient sample results analyzed at different cutoffs, there were more results detected using the ART that were undetectable (TND) with the CAP/CTM2. At a 25 IU/mL cutoff (the threshold used for RGT in the clinical trials with faldaprevir, SPV, BOC and TVR and the recommended cut-off for treatment futility in the US SPV label), the HCV RNA result had an overall agreement of 87%. This agreement improved to 94% when using a lower 15 IU/mL cutoff (Table 6). When low VL results (25–3500 IU/mL) were compared in clinical samples, the mean difference was similar between the CAP/CTM2 and both the CAP/CTM1 and the HPS/CTM2 while
Table 4 Low level HCV RNA agreement across methods compared in this study using the WHO panel. HCV RNA Testa
Observed mean HCV RNA result IU/mL (log10 )b
Difference (nominal - observed, log10 IU/mL)
Standard deviation
Results obtained for WHO nominal titer of 100 IU/mL 102 (2.01) CAP/CTM1 128 (2.11) CAP/CTM2 ART 56 (1.75) 98 (1.99) HPS/CTM2
−0.01 −0.11 0.25 0.01
0.15 0.13 0.03 0.25
Results obtained for WHO nominal titer of 50 IU/mL CAP/CTM1 36 (1.56) 73 (1.86) CAP/CTM2 45 (1.65) ART HPS/CTM2 54 (1.73)
0.14 −0.16 0.05 −0.03
0.19 0.17 0.12 0.18
Results obtained for WHO nominal titer of 25 IU/mL – CAP/CTM1c 26 (1.41) CAP/CTM2 19 (1.29) ART 24 (1.38) HPS/CTM2
– -0.01 0.11 0.02
– 0.29 0.13 0.26
a b c
CAP/CTM2 testing was performed at Hannover, results across sites are compared in results section. Results are reported according to the manufacturer´ıs package insert. Panel member below the CAP/CTM1 tests lower limit of quantification (43 IU/mL), results cannot be calculated.
Please cite this article in press as: H.H. Kessler, et al., Evaluation of the COBAS® AmpliPrep/COBAS® TaqMan® HCV Test, v2.0 and comparison to assays used in routine clinical practice in an international multicenter clinical trial: The ExPECT study, J Clin Virol (2015), http://dx.doi.org/10.1016/j.jcv.2015.03.023
155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199
G Model JCV 3290 1–6
ARTICLE IN PRESS
4
H.H. Kessler et al. / Journal of Clinical Virology xxx (2015) xxx–xxx
Table 5 Limit of detection across methods compared in this study using the WHO panel. Testing site
Test
Assay LOD (IU/mL)a
Geneva Graz Hannover Marseille Combined Geneva Graz Marseille
CAP/CTM2 CAP/CTM2 CAP/CTM2 CAP/CTM2 CAP/CTM2 HPS/CTM2 CAP/CTM1 ART
15 15 15 15 15 20 15 12
No. tested 24 30 30 30 114 30 30 30
No. detected 21 30 30 29 110 30 29 29
Estimated limit of detection (95% CI) (IU/mL) 15 (10, 31) 18 (12, 28) 11 (9, 19) 12 (9, 20) 12 (10, 15) 10 (7, 16) 11 (8, 18) 8 (6, 15)
LOD, limit of detection. a According to the manufacturer´ıs package insert.
Fig. 1. Correlation between the results (IU/mL) for EDTA plasma or serum HCV RNA positive samples obtained from patients with chronic hepatitis C tested by the CAP/CTM HCV Test, v2.0 and comparator tests in Graz (A), Hannover (B), Marseille (C), and Pisa (D). Table 6 Comparative results above and below 25 IU/mL and 15 IU/mL for the CAP/CTM2 and ART in clinical specimens. CAP/CTM2
ART TND
TND
