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Analytical performance of the VERIS MDx system HCV assay for detecting and quantifying HCV RNA
Journal of Clinical Virology 84 (2016) 7–11
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Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Analytical performance of the VERIS MDx system HCV assay for detecting and quantifying HCV RNA Dr Karine Sauné a,b,∗ , Florence Abravanel a,b , Catherine Haslé b , Jérôme Boineau b , Catherine Mengelle b , Jacques Izopet a,b a b
INSERM, U1043, Centre de Physiopathologie de Toulouse Purpan, Toulouse F-31300, France CHU Toulouse, Hôpital Purpan, Laboratoire de virologie, Institut fédératif de biologie de Purpan, F-31300, France
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Article history: Received 25 February 2016 Received in revised form 2 August 2016 Accepted 7 September 2016 Keywords: Hepatitis C virus HCV RNA quantification VERIS MDx system HCV assay COBAS Ampliprep COBAS Taqman
a b s t r a c t Background: The diagnosis of HCV relies on the detection of viral RNA. Objective: To evaluate the performance of the VERIS/MDx System HCV Assay, a new automated system for quantifying HCV RNA, and to compare with the COBAS® Ampliprep/COBAS® TaqmanTM (CAPCTM) HCV Test version 2.0. Study design: The limit of detection was determined by Probit analysis with the 3rd International WHO HCV standard and precision by assaying in duplicate control samples with HCV RNA concentrations of 7.9; 5.0; 3.4; 1.6 and 0 log IU/ml over 20 days. Analytical specificity was assessed by assaying 180 samples from negative anti-HCV and HCV RNA blood donors and linearity with replicates of serial dilutions of a clinical plasma (6.4–0.6 log IU/ml). We compared the VERIS MDx HCV and CAPCTM HCV assays by testing 209 samples. Results: The limit of detection was 6.1 IU/ml [CI 95%: 5.0–8.3] and the precision, given by the standard deviation, was ≤0.11 log IU/ml. Specificity was 100%. The linearity ranged from 1.5 to 6.4 log IU/ml. Passing-Bablok regression analysis gave: VERIS log IU/ml = −0.33 + [1.04× CAPCTM] log IU/ml, with biases for the 25th, 50th, 75th percentiles of 0.18, −0.10 and −0.06 log IU/ml. The two assays were well correlated ( = 0.92, p < 0.001) and Bland-Altman analysis gave biases of 0.12, log IU/ml for genotype 1, −0.19 for genotype 2, −0.26 for genotype 3, and −0.77 for genotype 4. Conclusion: The VERIS MDx HCV assay performed well. But, we observed an under-quantification of the genotype 4 samples. © 2016 Published by Elsevier B.V.
1. Background Chronic hepatitis C virus (HCV) infections are associated with an increased risk of cirrhosis and hepatic decompensation, and are a leading cause of liver-related deaths. The diagnosis and management of HCV infections have recently been revised in Europe and the USA: a major factor was the approval for clinical use of directacting antivirals (DAAs) [1,2]. Certain populations, selected because of their demography, prior exposures, high-risk behavior, and medical conditions, should be tested for HCV. Patients with anti-HCV antibodies should have their HCV RNA determined by a sensitive molecular method to identify those with an on-going infection [1,2]. The recommendations for monitoring patients on antiviral
∗ Corresponding author at: INSERM, U1043, Centre de Physiopathologie de Toulouse Purpan, Toulouse, F-31300, France. E-mail address: [email protected] (K. Sauné). http://dx.doi.org/10.1016/j.jcv.2016.09.003 1386-6532/© 2016 Published by Elsevier B.V.
therapy have also evolved [1,2]. Four classes of DAAs are now available in industrialized countries for treating HCV infections: NS3 protease inhibitors, NS5B nucleoside polymerase inhibitors, NS5B non-nucleoside polymerase inhibitors, and NS5A inhibitors. DAAs and ribavirin give significantly better sustained virological response (SVR) rates than does therapy with pegylated-interferon alpha (Peg-IFN) [3–16]. Monitoring the HCV RNA of patients on these new drugs is mainly used to ensure medication adherence and to identify any adverse events and drug–drug interactions [1,2,17]. The methods used to detect HCV RNA include targeted end-point polymerase chain reactions (PCR), transcription-mediated amplification (TMA), and real-time PCR that quantifies HCV RNA during the exponential phase of amplification, allowing RNA concentrations of less than 10 IU/ml to be detected and concentrations of up to approximately 108 IU/ml to be accurately quantified [18–20]. The American Association for the Study of the Liver Diseases (AASLD) and the European Association for the study of the Liver (EASL)
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guidelines recommend the use of real-time PCR-based assays for monitoring HCV RNA during and after therapy because of their dynamic range (lower limit of detection ≤15 IU/ml) [1,2]. 2. Objectives We have assessed the analytical performance of a new, real-time PCR-based, fully automated sample processing system, the VERIS MDx System HCV Assay (Beckman-Coulter). We used a panel of specimens, including different HCV genotypes, to compare the HCV RNA concentrations obtained with this assay and those obtained using the Cobas Ampliprep-Cobas TaqMan (CAPCTM) HCV RNA vs 2.0 assay (Roche). 3. Study design 3.1. Quality control materials and clinical samples Sensitivity, specificity, linearity and reproducibility were assessed by the same operator using both test panels and clinical samples. The samples were (i) 180 anti-HCV antibody-negative plasma samples from blood donors; (ii) 36 replicates of each of five HCV RNA concentrations (0, 2, 6, 10 and 20 IU/ml) prepared by diluting in HCV-negative plasma a commercially available 3rd International WHO HCV standard (NIBSC code 06/100); (iii) 4 replicates of a panel of seven serial 10 fold-dilutions (6.35, 5.35, 4.35, 3.35, 2.35, 1.35, 0.13 log IU/ml HCV RNA) derived from a single clinical HCV genotype 1b diluted in negative plasma; (iv) 40 replicates of 5 samples HCV genotype 1a with a target HCV RNA concentrations of 7.9; 5.0; 3.4; 1.6 and 0 log IU/ml, tested twice for during 20 days. We also compared the VERIS MDx HCV and CAPCTM HCV RNA vs 2.0 assays by testing (vi) a total of 209 plasma samples from HCV-infected patients eligible for antiviral therapy. The HCV genotype of 172 of the samples quantified with both assays was known (G1, n = 85; G2, n = 23; G3, n = 29; G4, n = 31; G5, n = 2; G6, n = 2). Genotypes were determined by phylogenetic analysis after sequencing the NS5b region [21]. The genotype 1 subtypes were: 1a (n = 49) and 1b (n = 36). All HCV RNA concentrations are reported in IU/ml. 3.2. Plasma HCV RNA assays 3.2.1. VERIS MDx system HCV assay The whole process (automated nucleic acid extraction and realtime PCR) was performed on the VERIS MDx system according to the manufacturer’s instructions, using a plasma input volume of 1.0 ml. The VERIS assay is not yet validated for serum samples. 3.2.2. CAPCTM HCV RNA vs 2.0 assay Nucleic acids were extracted automatically using the Cobas Ampliprep/Cobas TaqMan HCV vs 2.0 assay on the Cobas Ampliprep instrument with a plasma input volume of 0.65 ml. Real-Time PCR was performed on the “docked” configuration of the Cobas TaqMan 96 instrument, according to the manufacturer’s instructions. The stated quantification range of the current version is 15–100,000,000 IU/ml. The stated LOD is 15 IU/ml. 3.3. Data analysis HCV RNA quantities were log10 transformed prior to analysis. Differences between replicates and runs are given for each HCV RNA as standard deviations (SD). Linearity was estimated by linear regression of data for 4 replicates of serial dilutions (6.3–0.6 log IU/ml) of a clinical plasma sample. The limit of detection (LOD) was determined by Probit analysis of data from serial
dilutions of the 3rd WHO standard HCV sample: 36 replicates for each RNA concentration per day for 3 days. The data obtained with the VERIS MDx HCV and CAPCTM HCV assays were analyzed by Passing-Bablok linear regression. A Bland-Altman analysis (scatter plot of the differences between the paired measurements plotted against their means) of positive samples obtained by the two methods was used to assess graphically the magnitude of disagreement between the methods and estimate the overall bias. A Kruskal-Wallis test was used to determine whether the bias varied according to the HCV genotype and Spearman’s correlation coefficient was used to assess the strength of linear association. 4. Results 4.1. Analytical performances of the Veris DNx HCV assay 4.1.1. Sensitivity Probit analysis of the 108 replicates of the diluted 3rd HCV WHO standard gave a LOD of 6.1 [CI 95%: 5.0–8.3] log IU/ml. The WHO standard at 20 UI/ml, a concentration, close to virologic failure cutoff used in clinical practice, was tested in 36 replicates, it gave a mean value of 20.4 ± 5.6 UI/ml and only one sample was negative. 4.1.2. Specificity All the samples from 180 seronegative blood donors tested negative for HCV RNA. 4.1.3. Reproducibility The data obtained by testing 4 replicates of a single HCV genotype 1b clinical sample diluted 10-fold (6.35, 5.35, 4.35, 3.35, 2.35, 1.35, 0.13 log IU/ml HCV RNA) gave a mean within-run log standard deviation of 0.05 log IU/ml (0.03–0.09 log IU/ml). The sample at 0.13 log UI/ml was never detected. Between-run reproducibility (log standard deviation), determined by testing 40 replicates of 5 samples of HCV genotype 1a (7.9; 5.0; 3.4; 1.6 and 0 log UI/ml HCV RNA) tested over 20 days was 0.09 log IU/ml (0.06–0.11 log IU/ml). The reproducibility at 7.9 log IU/ml was 0.06 log UI/ml and the highest standard deviation was observed for the 1.6 log IU/ml target concentration (i.e. reproducibility = 0.11 log UI/ml). The sample at 0 log UI/ml was never detected. 4.1.4. Linearity The linear range of the VERIS MDx assay was [1.4–6.3] log IU/ml. Linear regression analysis of the quantitative results plotted against the expected concentrations yielded the coefficient of determination R2 = 0.99 (Fig. 1). Spearman analysis showed a significant correlation between the observed and expected values ( = 0.99, p < 0.001). 4.2. Comparison of VERIS MDx and CAPCTM We also evaluated the capacity of the VERIS MDx assay to accurately quantify HCV RNA in clinical samples by comparing the results it gave for 209 clinical samples with those obtained with the CAPCTM vs 2.0 assay. Both tests gave positive values for all the samples. The VERIS MDx assay gave a mean HCV RNA concentration of 4.87 log IU/ml and the CAP/CTM assay gave a value of 5.00 log IU/ml. Passing Bablok regression analysis (Fig. 2) and statistical analysis of pairs of log10 concentrations indicated that the two tests were well correlated (Spearman, r = 0.92, p < 0.001). The average deviation between the VERIS MDx and CAP/CTM results was −0.13 log IU/ml. The Bland Altman analysis (Fig. 3) illustrates the differences between the two assays. Both gave very similar values for 168/209 (80%) of the samples (less than 0.5 log-unit difference). Only 41 paired results ([VERIS MDx-CAPCTM]) (20%) differed by more than 0.5 log IU/ml. The VERIS MDx test underquantified 31/41
K. Sauné et al. / Journal of Clinical Virology 84 (2016) 7–11
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7.0
Observed values (log IU/ml)
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y = 1.01x – 0.18 R² = 0.99
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5.21
5.0 4.10
4.0 3.10
3.0 2.03
2.0 1.40 1.0 0 0
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3.0
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Expected values (log IU/ml) Fig. 1. Linearity of the VERIS MDx assay.
Fig. 3. Bland Altman plot of data for all the clinical samples showing the bias between the VERIS MDx and CAPCTM assays. Continuous blue line represents the mean bias between the two tests and dashed blue lines represent the 95% confidence interval. The mean bias is given above the chart with the 95% confidence interval in brackets. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
influence of the subtype on HCV RNA quantification was analyzed for genotypes 1 and 4. The mean [VERIS MDx −CAP/CTM] difference for subtype 1a was 0.05 log IU/ml and that for subtype 1b was 0.23 log IU/ml (not significant). The mean difference observed for the subtype 4a was −0.83 log UI/ml and it was −1 log UI/ml for the subtype 4d (not significant). 5. Discussion
Fig. 2. Passing Bablok regression analysis of data obtained with the VERIS MDx and CAPCTM assays. Red line reprensents the regression line. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
(76%) of these samples. The mean HCV RNA concentration from these 41 samples were 5.47 log UI/ml (range: 1.98–7.2 log UI/ml) and theses samples belong to genotype 1 (n = 12), genotype 2 (n = 2), genotype 3 (n = 7); genotype 4 (n = 19) and genotype 5 (n = 1). Thus is not associated with a single genotype. A few (11/209; 5.2%) results differed by more than 1.0 log IU/ml. The VERIS MDx test under-quantified 10 samples compared with the CAPCTM test: 3 samples were genotype 1a, 1 sample was genotype 3a and 1 sample was genotype 4a and 5 samples were genotype 4d. Only one sample collected in a patient infected with HCV genotype 1b had an HCV RNA quantification >1 log IU/ml with the VERID MDx test. The Bland-Altman analysis indicated that the mean [VERIS MDx −CAP/CTM] differences were 0.12 log IU/ml for genotype 1, −0.19 for genotype 2, −0.26 for genotype 3, and −0.77 for genotype 4 (Fig. 4). Genotype 4 infections resulted in discrepant results with a bias more elevated than for the other genotypes (p = 0.001). The
We find that the VERIS MDx HCV RNA quantification assay is extremely specific, precise and reproducible. Quantification is linear over the full dynamic range, which covers virus concentrations observed in all presently known clinical situations. The analytical specificity of the VERIS MDx assay was excellent (100%), as good as that of other HCV assays presently available [18,22]. The intra-assay and inter-assay variations were good and close to those reported for other molecular assays, both automated and manual [23–25]. The manufacturer’s stated dynamic range for the real-time VERIS MDx is 12 to 10,000,000 IU/ml. We confirmed this using serial dilutions of clinical sample. The detection limit, determined by Probit analysis of the results for serial dilutions of the third WHO HCV RNA standard, was 6.1 [CI 95%: 5.0–8.3] log IU/ml, less than that stated by the manufacturer (12 IU/ml) and less than the values for other commercially available assays. Hence, the VERIS MDx assay is suitable for use wherever great sensitivity is essential. We find that the precision, linearity, and sensitivity of the Veris assay are all similar to the values obtained in other laboratories and testing environments [26–28]. Like several real-time RT-PCR assays, the VERIS MDx test target the 5 non-coding region of the HCV genome as this region is highly conserved. The quantification is based on HCV WHO international standards. Despite this strategy, discrepancies between assays still persist and it is therefore recommended to monitor individual patients with the same test throughout treatment [25,29]. The HCV RNA concentrations produced by the VERIS MDx test were all a little lower than those given by the CAPCTM vs 2.0 assay. While there was a linear correlation between the HCV RNA concentrations determined with the two assays, regardless of genotype, a genotype-specific analysis of the HCV RNA concentrations indicated that the VERIS MDx test under-quantifies HCV genotype 4 strains. Previous studies using the CAPCTM version 1.0 assay have reported similar observations [30]. A dual-probe approach can be
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Fig. 4. Bland Altman plot of data for the 85 genotype 1 clinical samples (a), the 23 genotype 2 clinical samples (b), the 29 genotype 3 clinical samples (c), and the 31 genotype 4 clinical samples (d) showing the bias between the VERIS MDx and CAPCTM assays. Continuous blue line represents the mean bias between the two tests and dashed blue lines represent the 95% confidence interval. The mean bias is given above the chart with the 95% confidence interval in brackets. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
used to solve this problem of under-estimation of certain HCV genotypes [22,23,26,31]. This under-quantification was not associated with any genotype 4 subtype. A recent evaluation of the VERIS assay compared with the Abbott RealTime HCV assay was performed by Haim-Boukobza et al. [32]. They have also observed an underquantification of the genotype 4 samples. But, their analysis of the 5 UTR target sequence from samples with and without underquantification did not identified significant differences between samples [32]. Further studies are thus needed to assess the origin of this under-quantification. It was suggested that genotype 1-infected, treatment-naive patients without cirrhosis and with HCV RNA concentration <6 million UI/ml could be treated with ledipasvir/sofosbuvir for 8 weeks. However, no recommendation exists for genotype 4-infected patients and this threshold is highly questioned due to the variability of the different HCV RNA assays [29] or to statistical bias [33]. There has been substantial progress in the antiviral treatment of HCV. HCV RNA quantification is still recommended at week 4 and at the end of treatment. But, no futility rules have been defined for the most recent treatment regimens, while some guidelines now recommend more limited monitoring. A recent report indicates that HCV RNA is no longer a predictive on-therapy marker of treatment outcome [34]. AASLD and EASL recommend testing for HCV RNA during therapy to check for drug-drug interactions and to improve adherence to treatment [1,2]. This indicates that the under-quantification of genotype 4 HCV RNA by the VERIS MDx assay will have less impact on clinical management with current
therapies than it would have on monitoring treatment with older antiviral regimens. Current treatment regimens are simpler and more potent than previous ones, which makes diagnostic testing more limited. It is still required for identifying people with active HCV infection, checking adherence to therapy and evaluating treatment success. The VERIS MDx assay appears to be sufficiently specific, reproducible, sensitive, and broad-ranged for use in this setting. It is therefore suitable for the routine diagnostic determination of HCV RNA. The quantification of HCV genotype 4 should be improved in future versions of the assay. Conflict of interest None to declare. Funding Reagents were provided by Beckman Coulter. References [1] Panel AIHG, Hepatitis C guidance: AASLD-IDSA recommendations for testing, managing, and treating adults infected with hepatitis C virus, Hepatology 62 (3) (2015) 932–954. [2] European Association for Study of L. EASL Recommendations on treatment of hepatitis C 2015, J. Hepatol. 63 (1) (2015) 199–236.
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Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Analytical performance of the VERIS MDx system HCV assay for detecting and quantifying HCV RNA Dr Karine Sauné a,b,∗ , Florence Abravanel a,b , Catherine Haslé b , Jérôme Boineau b , Catherine Mengelle b , Jacques Izopet a,b a b
INSERM, U1043, Centre de Physiopathologie de Toulouse Purpan, Toulouse F-31300, France CHU Toulouse, Hôpital Purpan, Laboratoire de virologie, Institut fédératif de biologie de Purpan, F-31300, France
a r t i c l e
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Article history: Received 25 February 2016 Received in revised form 2 August 2016 Accepted 7 September 2016 Keywords: Hepatitis C virus HCV RNA quantification VERIS MDx system HCV assay COBAS Ampliprep COBAS Taqman
a b s t r a c t Background: The diagnosis of HCV relies on the detection of viral RNA. Objective: To evaluate the performance of the VERIS/MDx System HCV Assay, a new automated system for quantifying HCV RNA, and to compare with the COBAS® Ampliprep/COBAS® TaqmanTM (CAPCTM) HCV Test version 2.0. Study design: The limit of detection was determined by Probit analysis with the 3rd International WHO HCV standard and precision by assaying in duplicate control samples with HCV RNA concentrations of 7.9; 5.0; 3.4; 1.6 and 0 log IU/ml over 20 days. Analytical specificity was assessed by assaying 180 samples from negative anti-HCV and HCV RNA blood donors and linearity with replicates of serial dilutions of a clinical plasma (6.4–0.6 log IU/ml). We compared the VERIS MDx HCV and CAPCTM HCV assays by testing 209 samples. Results: The limit of detection was 6.1 IU/ml [CI 95%: 5.0–8.3] and the precision, given by the standard deviation, was ≤0.11 log IU/ml. Specificity was 100%. The linearity ranged from 1.5 to 6.4 log IU/ml. Passing-Bablok regression analysis gave: VERIS log IU/ml = −0.33 + [1.04× CAPCTM] log IU/ml, with biases for the 25th, 50th, 75th percentiles of 0.18, −0.10 and −0.06 log IU/ml. The two assays were well correlated ( = 0.92, p < 0.001) and Bland-Altman analysis gave biases of 0.12, log IU/ml for genotype 1, −0.19 for genotype 2, −0.26 for genotype 3, and −0.77 for genotype 4. Conclusion: The VERIS MDx HCV assay performed well. But, we observed an under-quantification of the genotype 4 samples. © 2016 Published by Elsevier B.V.
1. Background Chronic hepatitis C virus (HCV) infections are associated with an increased risk of cirrhosis and hepatic decompensation, and are a leading cause of liver-related deaths. The diagnosis and management of HCV infections have recently been revised in Europe and the USA: a major factor was the approval for clinical use of directacting antivirals (DAAs) [1,2]. Certain populations, selected because of their demography, prior exposures, high-risk behavior, and medical conditions, should be tested for HCV. Patients with anti-HCV antibodies should have their HCV RNA determined by a sensitive molecular method to identify those with an on-going infection [1,2]. The recommendations for monitoring patients on antiviral
∗ Corresponding author at: INSERM, U1043, Centre de Physiopathologie de Toulouse Purpan, Toulouse, F-31300, France. E-mail address: [email protected] (K. Sauné). http://dx.doi.org/10.1016/j.jcv.2016.09.003 1386-6532/© 2016 Published by Elsevier B.V.
therapy have also evolved [1,2]. Four classes of DAAs are now available in industrialized countries for treating HCV infections: NS3 protease inhibitors, NS5B nucleoside polymerase inhibitors, NS5B non-nucleoside polymerase inhibitors, and NS5A inhibitors. DAAs and ribavirin give significantly better sustained virological response (SVR) rates than does therapy with pegylated-interferon alpha (Peg-IFN) [3–16]. Monitoring the HCV RNA of patients on these new drugs is mainly used to ensure medication adherence and to identify any adverse events and drug–drug interactions [1,2,17]. The methods used to detect HCV RNA include targeted end-point polymerase chain reactions (PCR), transcription-mediated amplification (TMA), and real-time PCR that quantifies HCV RNA during the exponential phase of amplification, allowing RNA concentrations of less than 10 IU/ml to be detected and concentrations of up to approximately 108 IU/ml to be accurately quantified [18–20]. The American Association for the Study of the Liver Diseases (AASLD) and the European Association for the study of the Liver (EASL)
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guidelines recommend the use of real-time PCR-based assays for monitoring HCV RNA during and after therapy because of their dynamic range (lower limit of detection ≤15 IU/ml) [1,2]. 2. Objectives We have assessed the analytical performance of a new, real-time PCR-based, fully automated sample processing system, the VERIS MDx System HCV Assay (Beckman-Coulter). We used a panel of specimens, including different HCV genotypes, to compare the HCV RNA concentrations obtained with this assay and those obtained using the Cobas Ampliprep-Cobas TaqMan (CAPCTM) HCV RNA vs 2.0 assay (Roche). 3. Study design 3.1. Quality control materials and clinical samples Sensitivity, specificity, linearity and reproducibility were assessed by the same operator using both test panels and clinical samples. The samples were (i) 180 anti-HCV antibody-negative plasma samples from blood donors; (ii) 36 replicates of each of five HCV RNA concentrations (0, 2, 6, 10 and 20 IU/ml) prepared by diluting in HCV-negative plasma a commercially available 3rd International WHO HCV standard (NIBSC code 06/100); (iii) 4 replicates of a panel of seven serial 10 fold-dilutions (6.35, 5.35, 4.35, 3.35, 2.35, 1.35, 0.13 log IU/ml HCV RNA) derived from a single clinical HCV genotype 1b diluted in negative plasma; (iv) 40 replicates of 5 samples HCV genotype 1a with a target HCV RNA concentrations of 7.9; 5.0; 3.4; 1.6 and 0 log IU/ml, tested twice for during 20 days. We also compared the VERIS MDx HCV and CAPCTM HCV RNA vs 2.0 assays by testing (vi) a total of 209 plasma samples from HCV-infected patients eligible for antiviral therapy. The HCV genotype of 172 of the samples quantified with both assays was known (G1, n = 85; G2, n = 23; G3, n = 29; G4, n = 31; G5, n = 2; G6, n = 2). Genotypes were determined by phylogenetic analysis after sequencing the NS5b region [21]. The genotype 1 subtypes were: 1a (n = 49) and 1b (n = 36). All HCV RNA concentrations are reported in IU/ml. 3.2. Plasma HCV RNA assays 3.2.1. VERIS MDx system HCV assay The whole process (automated nucleic acid extraction and realtime PCR) was performed on the VERIS MDx system according to the manufacturer’s instructions, using a plasma input volume of 1.0 ml. The VERIS assay is not yet validated for serum samples. 3.2.2. CAPCTM HCV RNA vs 2.0 assay Nucleic acids were extracted automatically using the Cobas Ampliprep/Cobas TaqMan HCV vs 2.0 assay on the Cobas Ampliprep instrument with a plasma input volume of 0.65 ml. Real-Time PCR was performed on the “docked” configuration of the Cobas TaqMan 96 instrument, according to the manufacturer’s instructions. The stated quantification range of the current version is 15–100,000,000 IU/ml. The stated LOD is 15 IU/ml. 3.3. Data analysis HCV RNA quantities were log10 transformed prior to analysis. Differences between replicates and runs are given for each HCV RNA as standard deviations (SD). Linearity was estimated by linear regression of data for 4 replicates of serial dilutions (6.3–0.6 log IU/ml) of a clinical plasma sample. The limit of detection (LOD) was determined by Probit analysis of data from serial
dilutions of the 3rd WHO standard HCV sample: 36 replicates for each RNA concentration per day for 3 days. The data obtained with the VERIS MDx HCV and CAPCTM HCV assays were analyzed by Passing-Bablok linear regression. A Bland-Altman analysis (scatter plot of the differences between the paired measurements plotted against their means) of positive samples obtained by the two methods was used to assess graphically the magnitude of disagreement between the methods and estimate the overall bias. A Kruskal-Wallis test was used to determine whether the bias varied according to the HCV genotype and Spearman’s correlation coefficient was used to assess the strength of linear association. 4. Results 4.1. Analytical performances of the Veris DNx HCV assay 4.1.1. Sensitivity Probit analysis of the 108 replicates of the diluted 3rd HCV WHO standard gave a LOD of 6.1 [CI 95%: 5.0–8.3] log IU/ml. The WHO standard at 20 UI/ml, a concentration, close to virologic failure cutoff used in clinical practice, was tested in 36 replicates, it gave a mean value of 20.4 ± 5.6 UI/ml and only one sample was negative. 4.1.2. Specificity All the samples from 180 seronegative blood donors tested negative for HCV RNA. 4.1.3. Reproducibility The data obtained by testing 4 replicates of a single HCV genotype 1b clinical sample diluted 10-fold (6.35, 5.35, 4.35, 3.35, 2.35, 1.35, 0.13 log IU/ml HCV RNA) gave a mean within-run log standard deviation of 0.05 log IU/ml (0.03–0.09 log IU/ml). The sample at 0.13 log UI/ml was never detected. Between-run reproducibility (log standard deviation), determined by testing 40 replicates of 5 samples of HCV genotype 1a (7.9; 5.0; 3.4; 1.6 and 0 log UI/ml HCV RNA) tested over 20 days was 0.09 log IU/ml (0.06–0.11 log IU/ml). The reproducibility at 7.9 log IU/ml was 0.06 log UI/ml and the highest standard deviation was observed for the 1.6 log IU/ml target concentration (i.e. reproducibility = 0.11 log UI/ml). The sample at 0 log UI/ml was never detected. 4.1.4. Linearity The linear range of the VERIS MDx assay was [1.4–6.3] log IU/ml. Linear regression analysis of the quantitative results plotted against the expected concentrations yielded the coefficient of determination R2 = 0.99 (Fig. 1). Spearman analysis showed a significant correlation between the observed and expected values ( = 0.99, p < 0.001). 4.2. Comparison of VERIS MDx and CAPCTM We also evaluated the capacity of the VERIS MDx assay to accurately quantify HCV RNA in clinical samples by comparing the results it gave for 209 clinical samples with those obtained with the CAPCTM vs 2.0 assay. Both tests gave positive values for all the samples. The VERIS MDx assay gave a mean HCV RNA concentration of 4.87 log IU/ml and the CAP/CTM assay gave a value of 5.00 log IU/ml. Passing Bablok regression analysis (Fig. 2) and statistical analysis of pairs of log10 concentrations indicated that the two tests were well correlated (Spearman, r = 0.92, p < 0.001). The average deviation between the VERIS MDx and CAP/CTM results was −0.13 log IU/ml. The Bland Altman analysis (Fig. 3) illustrates the differences between the two assays. Both gave very similar values for 168/209 (80%) of the samples (less than 0.5 log-unit difference). Only 41 paired results ([VERIS MDx-CAPCTM]) (20%) differed by more than 0.5 log IU/ml. The VERIS MDx test underquantified 31/41
K. Sauné et al. / Journal of Clinical Virology 84 (2016) 7–11
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7.0
Observed values (log IU/ml)
6.34
y = 1.01x – 0.18 R² = 0.99
6.0
5.21
5.0 4.10
4.0 3.10
3.0 2.03
2.0 1.40 1.0 0 0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
Expected values (log IU/ml) Fig. 1. Linearity of the VERIS MDx assay.
Fig. 3. Bland Altman plot of data for all the clinical samples showing the bias between the VERIS MDx and CAPCTM assays. Continuous blue line represents the mean bias between the two tests and dashed blue lines represent the 95% confidence interval. The mean bias is given above the chart with the 95% confidence interval in brackets. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
influence of the subtype on HCV RNA quantification was analyzed for genotypes 1 and 4. The mean [VERIS MDx −CAP/CTM] difference for subtype 1a was 0.05 log IU/ml and that for subtype 1b was 0.23 log IU/ml (not significant). The mean difference observed for the subtype 4a was −0.83 log UI/ml and it was −1 log UI/ml for the subtype 4d (not significant). 5. Discussion
Fig. 2. Passing Bablok regression analysis of data obtained with the VERIS MDx and CAPCTM assays. Red line reprensents the regression line. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
(76%) of these samples. The mean HCV RNA concentration from these 41 samples were 5.47 log UI/ml (range: 1.98–7.2 log UI/ml) and theses samples belong to genotype 1 (n = 12), genotype 2 (n = 2), genotype 3 (n = 7); genotype 4 (n = 19) and genotype 5 (n = 1). Thus is not associated with a single genotype. A few (11/209; 5.2%) results differed by more than 1.0 log IU/ml. The VERIS MDx test under-quantified 10 samples compared with the CAPCTM test: 3 samples were genotype 1a, 1 sample was genotype 3a and 1 sample was genotype 4a and 5 samples were genotype 4d. Only one sample collected in a patient infected with HCV genotype 1b had an HCV RNA quantification >1 log IU/ml with the VERID MDx test. The Bland-Altman analysis indicated that the mean [VERIS MDx −CAP/CTM] differences were 0.12 log IU/ml for genotype 1, −0.19 for genotype 2, −0.26 for genotype 3, and −0.77 for genotype 4 (Fig. 4). Genotype 4 infections resulted in discrepant results with a bias more elevated than for the other genotypes (p = 0.001). The
We find that the VERIS MDx HCV RNA quantification assay is extremely specific, precise and reproducible. Quantification is linear over the full dynamic range, which covers virus concentrations observed in all presently known clinical situations. The analytical specificity of the VERIS MDx assay was excellent (100%), as good as that of other HCV assays presently available [18,22]. The intra-assay and inter-assay variations were good and close to those reported for other molecular assays, both automated and manual [23–25]. The manufacturer’s stated dynamic range for the real-time VERIS MDx is 12 to 10,000,000 IU/ml. We confirmed this using serial dilutions of clinical sample. The detection limit, determined by Probit analysis of the results for serial dilutions of the third WHO HCV RNA standard, was 6.1 [CI 95%: 5.0–8.3] log IU/ml, less than that stated by the manufacturer (12 IU/ml) and less than the values for other commercially available assays. Hence, the VERIS MDx assay is suitable for use wherever great sensitivity is essential. We find that the precision, linearity, and sensitivity of the Veris assay are all similar to the values obtained in other laboratories and testing environments [26–28]. Like several real-time RT-PCR assays, the VERIS MDx test target the 5 non-coding region of the HCV genome as this region is highly conserved. The quantification is based on HCV WHO international standards. Despite this strategy, discrepancies between assays still persist and it is therefore recommended to monitor individual patients with the same test throughout treatment [25,29]. The HCV RNA concentrations produced by the VERIS MDx test were all a little lower than those given by the CAPCTM vs 2.0 assay. While there was a linear correlation between the HCV RNA concentrations determined with the two assays, regardless of genotype, a genotype-specific analysis of the HCV RNA concentrations indicated that the VERIS MDx test under-quantifies HCV genotype 4 strains. Previous studies using the CAPCTM version 1.0 assay have reported similar observations [30]. A dual-probe approach can be
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Fig. 4. Bland Altman plot of data for the 85 genotype 1 clinical samples (a), the 23 genotype 2 clinical samples (b), the 29 genotype 3 clinical samples (c), and the 31 genotype 4 clinical samples (d) showing the bias between the VERIS MDx and CAPCTM assays. Continuous blue line represents the mean bias between the two tests and dashed blue lines represent the 95% confidence interval. The mean bias is given above the chart with the 95% confidence interval in brackets. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
used to solve this problem of under-estimation of certain HCV genotypes [22,23,26,31]. This under-quantification was not associated with any genotype 4 subtype. A recent evaluation of the VERIS assay compared with the Abbott RealTime HCV assay was performed by Haim-Boukobza et al. [32]. They have also observed an underquantification of the genotype 4 samples. But, their analysis of the 5 UTR target sequence from samples with and without underquantification did not identified significant differences between samples [32]. Further studies are thus needed to assess the origin of this under-quantification. It was suggested that genotype 1-infected, treatment-naive patients without cirrhosis and with HCV RNA concentration <6 million UI/ml could be treated with ledipasvir/sofosbuvir for 8 weeks. However, no recommendation exists for genotype 4-infected patients and this threshold is highly questioned due to the variability of the different HCV RNA assays [29] or to statistical bias [33]. There has been substantial progress in the antiviral treatment of HCV. HCV RNA quantification is still recommended at week 4 and at the end of treatment. But, no futility rules have been defined for the most recent treatment regimens, while some guidelines now recommend more limited monitoring. A recent report indicates that HCV RNA is no longer a predictive on-therapy marker of treatment outcome [34]. AASLD and EASL recommend testing for HCV RNA during therapy to check for drug-drug interactions and to improve adherence to treatment [1,2]. This indicates that the under-quantification of genotype 4 HCV RNA by the VERIS MDx assay will have less impact on clinical management with current
therapies than it would have on monitoring treatment with older antiviral regimens. Current treatment regimens are simpler and more potent than previous ones, which makes diagnostic testing more limited. It is still required for identifying people with active HCV infection, checking adherence to therapy and evaluating treatment success. The VERIS MDx assay appears to be sufficiently specific, reproducible, sensitive, and broad-ranged for use in this setting. It is therefore suitable for the routine diagnostic determination of HCV RNA. The quantification of HCV genotype 4 should be improved in future versions of the assay. Conflict of interest None to declare. Funding Reagents were provided by Beckman Coulter. References [1] Panel AIHG, Hepatitis C guidance: AASLD-IDSA recommendations for testing, managing, and treating adults infected with hepatitis C virus, Hepatology 62 (3) (2015) 932–954. [2] European Association for Study of L. EASL Recommendations on treatment of hepatitis C 2015, J. Hepatol. 63 (1) (2015) 199–236.
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