- Email: [email protected]
Cytomegalovirus quantification in plasma with Abbott RealTime CMV and Roche Cobas Amplicor CMV assays
Journal of Virological Methods 225 (2015) 1–3
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
Cytomegalovirus quantification in plasma with Abbott RealTime CMV and Roche Cobas Amplicor CMV assays Maxime-Antoine Tremblay a,∗ , Marc-André Rodrigue b , Louise Deschênes c , Guy Boivin d , Jean Longtin b a
CHU de Québec, 2705 Boulevard Laurier, Room F-00413, Québec, QC, Canada G1V 4G2 CHU de Québec, 2705 Boulevard Laurier, Québec, QC, Canada G1V 4G2 c CHU de Québec, 11 Côte du Palais, Québec, QC, Canada G1R 2J6 d Research Center in Infectious Diseases, CHU de Québec and Université Laval, 2705 Boulevard Laurier, Québec, QC, Canada G1V 4G2 b
a b s t r a c t Article history: Received 6 February 2015 Received in revised form 12 August 2015 Accepted 15 August 2015 Available online 1 September 2015 Keywords: Cytomegalovirus Quantitative PCR Plasma Standardization Abbott
We assessed the performance of Abbott RealTime CMV assay (ARC) compared to Roche Cobas Amplicor CMV Monitor Test (RCM) for quantification of CMV in plasma of transplant patients. Commercial panels were used to test linearity, precision and interference and 83 clinical samples were used for the accuracy and precision analyses. All 43 RCM-positive clinical samples tested positive by ARC. The overall concordance between the two tests was good (98%). Based on 17 samples, the inter-assay median coefficient of variation was 13%. A linearity panel ranging from approximately 1 to 7 log10 copies/mL was used to confirm linearity (R2 = 0.99). CMV viral load measurement was not affected by different concentrations of HSV-1 or EBV DNA. We conclude that The Abbott RealTime CMV assay offers good sensitivity, precision and linearity and is suitable for monitoring CMV viral loads in transplant recipients. Standardization with the WHO CMV standard allows for comparison with other assays. © 2015 Elsevier B.V. All rights reserved.
Cytomegalovirus (CMV) infection is an important cause of morbidity and mortality in hematopoietic stem cell and solid organ transplant recipients. An early diagnosis and subsequent preemptive treatment have been shown to decrease the incidence of CMV disease (Einsele et al., 1995; Kalil et al., 2005). In recent years, polymerase chain reaction (PCR) assays have taken an increasingly greater place in the diagnosis of CMV infection, in part because of their good sensitivity, which in theory allows for an earlier detection of viremia. Real-time PCR assays have the advantages of being less time-consuming, of carrying a lower risk of amplicon contamination and of having a wider linear range as compared with conventional end-point PCR (Cockerill, 2003; Kotton et al., 2013). Unfortunately many of these tests are not standardized, impairing comparison between hospitals and the possibility to conduct multicenter trials. In 2010 the World Health Organization (WHO) proposed the first international standard for CMV nucleic acid amplification (NAAT)-based assays (Fryer et al., 2010). This
∗ Corresponding author. E-mail addresses: [email protected] (M.-A. Tremblay), [email protected] (M.-A. Rodrigue), [email protected] (L. Deschênes), [email protected] (G. Boivin), [email protected] (J. Longtin). http://dx.doi.org/10.1016/j.jviromet.2015.08.010 0166-0934/© 2015 Elsevier B.V. All rights reserved.
standard allows conversion of copies to international units per ml, setting ground for normalization. The Abbott RealTime CMV assay (ARC) and the Roche Cobas Amplicor CMV Monitor Test (RCM) are quantitative CMV viral load measurement methods used in clinical laboratories (Caliendo et al., 2000, 2001; Piiparinen et al., 2001). The RCM is FDA-approved for clinical use. ARC is standardized against WHO standard with a conversion factor of 1.56 copy/mL for 1 IU/mL of plasma. It is Health Canada approved and CE marked, but pending FDA approval. The objective of this study was to compare the performance of ARC and RCM methods for the quantification of CMV. Eighty-three clinical samples previously tested with the RCM (43 positives and 40 negatives) were used for the accuracy and precision verification steps. Positive samples were chosen by a single investigator (LD) to represent a range of low and high values. The clinical samples originated from hematopoietic stem cell or solid organ transplant recipients and were tested by ARC in a blinded fashion, between August and October 2012. Inter-assay coefficient of variation (CV) was calculated by duplicate or triplicate testing of 17 samples, performed by two different technicians on different days. Linearity was verified with the PTC801 CMV DNA Linearity Panel (BBI Diagnostics, SeraCare Life Sciences, Milford, MA, USA) composed of one negative and 9 positive samples ranging from 30 to 107 copies/mL. We verified a clinical limit of detection (LOD) of 100 IU/mL by
M.-A. Tremblay et al. / Journal of Virological Methods 225 (2015) 1–3
Difference of CMV DNA loads (RCM minus ARC) (log10 copies/mL)
applying a 1:250 dilution to six commercial 25,200 IU/mL CMV standards (NATCMV-ERCL AD-169, Zeptometrix). ARC assay was performed according to the manufacturer’s instructions. DNA extraction, amplification and detection were performed on the automated m2000 RealTime System (Abbott Molecular Inc, Des Plaines, IL, USA). The assay targets DNA sequences from UL34 and UL80.5 CMV genes. The reported limits of detection (LOD) and of quantification (LOQ) are 31.2 IU/mL (20 copies/mL). An internal control is included in each run to assess for the presence of PCR inhibition. This internal control is derived from pumpkin DNA, added to the lysis buffer during extraction. RCM is an end-point quantitative PCR assay. DNA extraction, amplification and detection were performed according to the product monograph on a Cobas Amplicor Analyser (Roche Diagnostics Canada, Laval, QC, Canada). The amplified DNA sequence is a 365bp N-terminal segment of the CMV polymerase gene. The reported lower limit of detection (LOD) is 400 copies/mL and the linear range of quantification is 600–100,000 copies/mL. The assay is also provided with an internal control, amplified using the same primer binding sites as the CMV target sequence, but is detected by a different probe. Data were analyzed using R (R Core Team, 2012) and Microsoft® Excel, including calculations of means, medians, standard deviations, variation coefficients and correlation coefficient. Trueness of agreement was evaluated through a Bland–Altman analysis. The difference between the measurements by the two methods was plotted against their mean with a 95% limit of agreement defined as bias ±1.96 SD. For linearity analysis, experimental data were plotted as log10 copies/mL against expected values and a correlation coefficient (R2 ) was calculated. Of the 83 clinical plasma samples, 40 were negative by both ARC and RCM. All RCM-positive samples were also positive by ARC. Closeness of agreement was evaluated through a Bland–Altman analysis as shown in Fig. 1. Concordance between the two assays was 98% (42/43). Deming regression yielded a slope and intercepts of 1.1669 and 0.4655 respectively. Overall viral load results obtained with ARC were on average 0.1 log10 copies/mL (SD 0.32) higher than those obtained with RCM. The 100 IU/mL clinical LOD was corroborated on the ARC, which yielded average results of 83 ± 17 IU/mL. The median inter-assay coefficient of variation was 13% (range 8–23%, SD 6%).
1.00 0.80 0.60
0.55
0.40 0.20 0.00
-0.10
-0.20 -0.40 -0.60 -0.80 -1.00
-0,74 2
3
4
5
Mean of CMV DNA loads (ARC and RCM) (log10 copies/mL) Fig. 1. Bland–Altman analysis of the CMV RealTime assay and the Cobas Amplicor CMV Monitor Test for the 43 CMV positive clinical samples. Each dot corresponds to one sample. A good concordance is met if ≥95% of the dots are found between ±1.96 SD (dashed lines) from the mean difference on the y axis. In this case, 42/43 (98%) meet this criterion.
7.5
ARC CMV DNA load (log10 copies/mL)
2
7.0 6.5 6.0
y = 1.0223x - 0.3795 R² = 0.9926
5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
Commercial panel CMV DNA load (log10copies/mL) Fig. 2. Linearity shown by a simple linear regression plot between ARC measurements and expected values from the commercial CMV Linearity Panel PTC-801 (as measured by RCM by the manufacturer). Values shown in copies/mL because there is no IU/mL conversion factor available for the commercial panel used.
The linear range for plasma samples specified by the manufacturer runs from 31.2 to 1.56 × 108 IU/mL (20–108 copies/mL). As shown in Fig. 2, the assay showed a good linearity (R2 = 0.9926) between expected values and ARC measurements. We tested for interference with related herpesviridae HSV-1 and EBV. Dilutions of HSV-1 cultures were added to four commercial CMV positive samples (Zeptometrix), ranging from 1000 to 10,000 CMV copies/mL. For EBV interference, we used 6 clinical EBV positive samples (200–13,500) copies/mL, supplemented with known CMV concentration. In both cases CMV viral load was measured in duplicates and compared to a pure CMV standard. CMV detection and quantification were not influenced and remained unchanged by the presence of HSV-1 or EBV. In this study we assessed the performance of the nucleic acidbased Abbott RealTime CMV assay compared to the Roche Cobas Amplicor CMV Monitor Test. The ARC showed good concordance with RCM, as well as good inter-assay precision and linear range. This high concordance is consistent with another study (Yerly et al., 2007) which compared ARC with a modified (ultrasensitive) Cobas Amplicor CMV Monitor. Precision of the assay is also an important clinical asset as the absolute viral load and its kinetics are predictors of active disease (Kraft et al., 2012). We believe standardization of the assay against the WHO international standard is a major advantage. This will allow researchers to conduct multicenter trials and cohort studies which could potentially lead to evidence-based international viral load thresholds for preemptive treatment initiation and cessation in transplant recipients. Direct patient care will also be facilitated as we can now compare results obtained through different laboratories. The assay claims a linear range of 20–108 copies/mL. We verified a smaller range reflecting clinical practice which spanned from 30 to 107 copies/mL. Very high CMV viremia in the range of 107 copies/mL are plausible but rarely seen (Kraft et al., 2012). However the test’s high limit of quantification would be deemed useful for these high viral load samples since it would preclude the need to prepare dilutions. The greater sensitivity of the assay is of unknown significance. From a clinical standpoint earlier detection of CMV viremia may result in earlier treatment and better outcome. On the other hand, a very sensitive DNA detection test may also lengthen treatment duration and raise the risk of adverse effects, since antiviral drugs are most often withdrawn only when CMV viremia becomes undetectable (Boeckh and Ljungman, 2009; Kotton et al., 2013; Kraft
M.-A. Tremblay et al. / Journal of Virological Methods 225 (2015) 1–3
et al., 2012; Ljungman et al., 2004, 2011; Razonable and Humar, 2013; Tomblyn et al., 2009). One limit of our study is that the comparator assay is an older end-point technology being gradually phased and replaced with the Roche COBAS AmpliPrep/COBAS TaqMan CMV Test. However our evaluation reflects the reality of most clinical laboratories upgrading their current system. Another limit is the small number of patients, although it did not prevent obtaining a good correlation between the two tests. Furthermore, the study population included hematopoietic cell and solid organ transplant recipients, but the latter were restricted to kidney transplants. In conclusion, the Abbott RealTime CMV assay offers a valuable alternative to the older end-point PCR assays, such as the Cobas Amplicor CMV Monitor Test. Its good analytical performance and its standardization could, along with similarly standardized assays, open new opportunities for researchers and clinicians in the future. Acknowledgements We thank Nathalie Boucher and Line Vézina for technical work. Ethical approval and patient consent were not required since this was a diagnostic test verification study, which included anonymized retrospective samples, implying there could be no impact for patient care. This practice is in agreement with the Clinical Laboratory Improvement Amendments (CLIA) federal regulatory standards (Burd, 2010). References Abbott RealTime CMV Package Insert, (2011 September), Abbott Molecular Inc, Des Plaines, IL, USA. http://www.abbottmolecular.com (accessed on 06.02.15). 2005, March. BBI Diagnostics CMV DNA Linearity Panel PTC801. BBI Diagnostics, SeraCare Life Sciences, Milford, MA, USA. Boeckh, M., Ljungman, P., 2009. How we treat cytomegalovirus in hematopoietic cell transplant recipients. Blood 113 (June (23)), 5711–5719, http://dx.doi.org/ 10.1182/blood-2008-10-143560. Burd, E.M., 2010. Validation of laboratory-developed molecular assays for infectious diseases. Clin. Microbiol. Rev. 23 (3), 550–576, http://dx.doi.org/10. 1128/CMR.00074-09. Caliendo, A.M., St. George, K., Kao, S.Y., Allega, J., Tan, B.H., LaFontaine, R., Bui, L., Rinaldo, C.R., 2000. Comparison of quantitative cytomegalovirus (CMV) PCR in plasma and CMV antigenemia assay: clinical utility of the prototype AMPLICOR CMV MONITOR test in transplant recipients. J. Clin. Microbiol. 38 (June (6)), 2122–2127. Caliendo, A.M., Schuurman, R., Yen-Lieberman, B., Spector, S.A., Andersen, J., Manjiry, R., Crumpacker, C., Lurain, N.S., Erice, A., 2001. Comparison of quantitative and qualitative PCR assays for cytomegalovirus DNA in plasma. J. Clin. Microbiol. 39 (April (4)), 1334–1338, http://dx.doi.org/10.1128/JCM.39.4. 1334-1338.2001.
3
Cockerill Jr., F.R., 2003. Application of rapid-cycle real-time polymerase chain reaction for diagnostic testing in the clinical microbiology laboratory. Arch. Pathol. Lab. Med. 127 (September (9)), 1112–1120. Einsele, H., Ehninger, G., Hebart, H., Wittkowski, K.M., Schuler, U., Jahn, G., Mackes, P., Herter, M., Klingebiel, T., Löffler, J., Wagner, S., Müller, C.A., 1995. Polymerase chain reaction monitoring reduces the incidence of cytomegalovirus disease and the duration and side effects of antiviral therapy after bone marrow transplantation. Blood 86 (October (7)), 2815–2820. Fryer, J.F., Heath, A.B., Anderson, R., Minor, P.D., The Collaborative Study Group, 2010. Collaborative Study to Evaluate the Proposed 1st WHO International Standard for Human Cytomegalovirus (HCMV) for Nucleic Acid Amplification (NAT)-Based Assays. WHO/BS/10.2138. World Health Organization, Geneva, Switzerland, http://apps.who.int/iris/handle/10665/70521?locale=en (accessed on 06.02.15). Kalil, A.C., Levitsky, J., Lyden, E., Stoner, J., Freifeld, A.G., 2005. Meta-analysis: the efficacy of strategies to prevent organ disease by cytomegalovirus in solid organ transplant recipients. Ann. Intern. Med. 143 (December (12)), 870–880. Kotton, C.N., Kumar, D., Caliendo, A.M., Asberg, A., Chou, S., Danziger-Isakov, L., Humar, A., Transplantation Society International CMV Consensus Group, 2013. Updated international consensus guidelines on the management of cytomegalovirus in solid-organ transplantation. Transplantation 96 (August (4)), 333–360, http://dx.doi.org/10.1097/TP.0b013e31829df29d. Kraft, C.S., Armstrong, W.S., Caliendo, A.M., 2012. Interpreting quantitative cytomegalovirus DNA testing: understanding the laboratory perspective. Clin. Infect. Dis. 54 (June (12)), 1793–1797, http://dx.doi.org/10.1093/cid/cis212. Ljungman, P., Reusser, P., de la Camara, R., Einsele, H., Engelhard, D., Ribaud, P., Ward, K., European Group for Blood and Marrow Transplantation, 2004. Management of CMV infections: recommendations from the infectious diseases working party of the EBMT. Bone Marrow Transplant. 33 (June (11)), 1075–1081, http://dx.doi.org/10.1038/sj.bmt.1704505. Ljungman, P., Hakki, M., Boeckh, M., 2011. Cytomegalovirus in hematopoietic stem cell transplant recipients. Hematol. Oncol. Clin. N. Am. 25 (February (1)), 151–169, http://dx.doi.org/10.1016/j.hoc.2010.11.011. Piiparinen, H., Höckerstedt, K., Grönhagen-Riska, C., Lappalainen, M., Suni, J., Lautenschlager, I., 2001. Comparison of plasma polymerase chain reaction and pp65-antigenemia assay in the quantification of cytomegalovirus in liver and kidney transplant patients. J. Clin. Virol. 22 (August (1)), 111–116. R Core Team, 2012. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria, http://www.R-project. org/ (accessed on 06.02.15). ISBN 3-900051-07-0. Razonable, R.R., Humar, A., 2013. AST infectious diseases community of practice, cytomegalovirus in solid organ transplantation. Am. J. Transplant. 13 (March (Suppl 4)), 93–106, http://dx.doi.org/10.1111/ajt.12103. Roche Cobas Amplicor CMV Monitor Test Product Information. Roche Diagnostics Canada, Laval, QC, Canada. http://molecular.roche.com/assays/Pages/ COBASAMPLICORCMVMONITORTest.aspx (accessed 06.02.15). Tomblyn, M., Chiller, T., Einsele, H., Gress, R., Sepkowitz, K., Storek, J., Wingard, J.R., Young, J.A., Boeckh, M.J., 2009. Guidelines for preventing infectious complications among hematopoietic cell transplantation recipients: a global perspective. Biol. Blood Marrow Transplant. 15 (October (10)), 1143–1238, http://dx.doi.org/10.1016/j.bbmt.2009.06.019. Yerly, S., Perrin, L., Van Delden, C., Schaffer, S., Thamm, S., Wunderli, W., Kaiser, L., 2007. Cytomegalovirus quantification in plasma by an automated real-time PCR assay. J. Clin. Virol. 38, 298–303, http://dx.doi.org/10.1016/j.jcv.2007.01. 003. ZeptoMetrix NATtrolTM Cytomegalovirus External Run Controls Strain: AD-169 (NATCMV-ERCL) Product Insert. ZeptoMetrix Corporation, Buffalo, NY, USA. http://www.zeptometrix.com/docs/PINATCMV-ERCL.pdf?1365017153 (accessed on 06.02.15).
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
Cytomegalovirus quantification in plasma with Abbott RealTime CMV and Roche Cobas Amplicor CMV assays Maxime-Antoine Tremblay a,∗ , Marc-André Rodrigue b , Louise Deschênes c , Guy Boivin d , Jean Longtin b a
CHU de Québec, 2705 Boulevard Laurier, Room F-00413, Québec, QC, Canada G1V 4G2 CHU de Québec, 2705 Boulevard Laurier, Québec, QC, Canada G1V 4G2 c CHU de Québec, 11 Côte du Palais, Québec, QC, Canada G1R 2J6 d Research Center in Infectious Diseases, CHU de Québec and Université Laval, 2705 Boulevard Laurier, Québec, QC, Canada G1V 4G2 b
a b s t r a c t Article history: Received 6 February 2015 Received in revised form 12 August 2015 Accepted 15 August 2015 Available online 1 September 2015 Keywords: Cytomegalovirus Quantitative PCR Plasma Standardization Abbott
We assessed the performance of Abbott RealTime CMV assay (ARC) compared to Roche Cobas Amplicor CMV Monitor Test (RCM) for quantification of CMV in plasma of transplant patients. Commercial panels were used to test linearity, precision and interference and 83 clinical samples were used for the accuracy and precision analyses. All 43 RCM-positive clinical samples tested positive by ARC. The overall concordance between the two tests was good (98%). Based on 17 samples, the inter-assay median coefficient of variation was 13%. A linearity panel ranging from approximately 1 to 7 log10 copies/mL was used to confirm linearity (R2 = 0.99). CMV viral load measurement was not affected by different concentrations of HSV-1 or EBV DNA. We conclude that The Abbott RealTime CMV assay offers good sensitivity, precision and linearity and is suitable for monitoring CMV viral loads in transplant recipients. Standardization with the WHO CMV standard allows for comparison with other assays. © 2015 Elsevier B.V. All rights reserved.
Cytomegalovirus (CMV) infection is an important cause of morbidity and mortality in hematopoietic stem cell and solid organ transplant recipients. An early diagnosis and subsequent preemptive treatment have been shown to decrease the incidence of CMV disease (Einsele et al., 1995; Kalil et al., 2005). In recent years, polymerase chain reaction (PCR) assays have taken an increasingly greater place in the diagnosis of CMV infection, in part because of their good sensitivity, which in theory allows for an earlier detection of viremia. Real-time PCR assays have the advantages of being less time-consuming, of carrying a lower risk of amplicon contamination and of having a wider linear range as compared with conventional end-point PCR (Cockerill, 2003; Kotton et al., 2013). Unfortunately many of these tests are not standardized, impairing comparison between hospitals and the possibility to conduct multicenter trials. In 2010 the World Health Organization (WHO) proposed the first international standard for CMV nucleic acid amplification (NAAT)-based assays (Fryer et al., 2010). This
∗ Corresponding author. E-mail addresses: [email protected] (M.-A. Tremblay), [email protected] (M.-A. Rodrigue), [email protected] (L. Deschênes), [email protected] (G. Boivin), [email protected] (J. Longtin). http://dx.doi.org/10.1016/j.jviromet.2015.08.010 0166-0934/© 2015 Elsevier B.V. All rights reserved.
standard allows conversion of copies to international units per ml, setting ground for normalization. The Abbott RealTime CMV assay (ARC) and the Roche Cobas Amplicor CMV Monitor Test (RCM) are quantitative CMV viral load measurement methods used in clinical laboratories (Caliendo et al., 2000, 2001; Piiparinen et al., 2001). The RCM is FDA-approved for clinical use. ARC is standardized against WHO standard with a conversion factor of 1.56 copy/mL for 1 IU/mL of plasma. It is Health Canada approved and CE marked, but pending FDA approval. The objective of this study was to compare the performance of ARC and RCM methods for the quantification of CMV. Eighty-three clinical samples previously tested with the RCM (43 positives and 40 negatives) were used for the accuracy and precision verification steps. Positive samples were chosen by a single investigator (LD) to represent a range of low and high values. The clinical samples originated from hematopoietic stem cell or solid organ transplant recipients and were tested by ARC in a blinded fashion, between August and October 2012. Inter-assay coefficient of variation (CV) was calculated by duplicate or triplicate testing of 17 samples, performed by two different technicians on different days. Linearity was verified with the PTC801 CMV DNA Linearity Panel (BBI Diagnostics, SeraCare Life Sciences, Milford, MA, USA) composed of one negative and 9 positive samples ranging from 30 to 107 copies/mL. We verified a clinical limit of detection (LOD) of 100 IU/mL by
M.-A. Tremblay et al. / Journal of Virological Methods 225 (2015) 1–3
Difference of CMV DNA loads (RCM minus ARC) (log10 copies/mL)
applying a 1:250 dilution to six commercial 25,200 IU/mL CMV standards (NATCMV-ERCL AD-169, Zeptometrix). ARC assay was performed according to the manufacturer’s instructions. DNA extraction, amplification and detection were performed on the automated m2000 RealTime System (Abbott Molecular Inc, Des Plaines, IL, USA). The assay targets DNA sequences from UL34 and UL80.5 CMV genes. The reported limits of detection (LOD) and of quantification (LOQ) are 31.2 IU/mL (20 copies/mL). An internal control is included in each run to assess for the presence of PCR inhibition. This internal control is derived from pumpkin DNA, added to the lysis buffer during extraction. RCM is an end-point quantitative PCR assay. DNA extraction, amplification and detection were performed according to the product monograph on a Cobas Amplicor Analyser (Roche Diagnostics Canada, Laval, QC, Canada). The amplified DNA sequence is a 365bp N-terminal segment of the CMV polymerase gene. The reported lower limit of detection (LOD) is 400 copies/mL and the linear range of quantification is 600–100,000 copies/mL. The assay is also provided with an internal control, amplified using the same primer binding sites as the CMV target sequence, but is detected by a different probe. Data were analyzed using R (R Core Team, 2012) and Microsoft® Excel, including calculations of means, medians, standard deviations, variation coefficients and correlation coefficient. Trueness of agreement was evaluated through a Bland–Altman analysis. The difference between the measurements by the two methods was plotted against their mean with a 95% limit of agreement defined as bias ±1.96 SD. For linearity analysis, experimental data were plotted as log10 copies/mL against expected values and a correlation coefficient (R2 ) was calculated. Of the 83 clinical plasma samples, 40 were negative by both ARC and RCM. All RCM-positive samples were also positive by ARC. Closeness of agreement was evaluated through a Bland–Altman analysis as shown in Fig. 1. Concordance between the two assays was 98% (42/43). Deming regression yielded a slope and intercepts of 1.1669 and 0.4655 respectively. Overall viral load results obtained with ARC were on average 0.1 log10 copies/mL (SD 0.32) higher than those obtained with RCM. The 100 IU/mL clinical LOD was corroborated on the ARC, which yielded average results of 83 ± 17 IU/mL. The median inter-assay coefficient of variation was 13% (range 8–23%, SD 6%).
1.00 0.80 0.60
0.55
0.40 0.20 0.00
-0.10
-0.20 -0.40 -0.60 -0.80 -1.00
-0,74 2
3
4
5
Mean of CMV DNA loads (ARC and RCM) (log10 copies/mL) Fig. 1. Bland–Altman analysis of the CMV RealTime assay and the Cobas Amplicor CMV Monitor Test for the 43 CMV positive clinical samples. Each dot corresponds to one sample. A good concordance is met if ≥95% of the dots are found between ±1.96 SD (dashed lines) from the mean difference on the y axis. In this case, 42/43 (98%) meet this criterion.
7.5
ARC CMV DNA load (log10 copies/mL)
2
7.0 6.5 6.0
y = 1.0223x - 0.3795 R² = 0.9926
5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
Commercial panel CMV DNA load (log10copies/mL) Fig. 2. Linearity shown by a simple linear regression plot between ARC measurements and expected values from the commercial CMV Linearity Panel PTC-801 (as measured by RCM by the manufacturer). Values shown in copies/mL because there is no IU/mL conversion factor available for the commercial panel used.
The linear range for plasma samples specified by the manufacturer runs from 31.2 to 1.56 × 108 IU/mL (20–108 copies/mL). As shown in Fig. 2, the assay showed a good linearity (R2 = 0.9926) between expected values and ARC measurements. We tested for interference with related herpesviridae HSV-1 and EBV. Dilutions of HSV-1 cultures were added to four commercial CMV positive samples (Zeptometrix), ranging from 1000 to 10,000 CMV copies/mL. For EBV interference, we used 6 clinical EBV positive samples (200–13,500) copies/mL, supplemented with known CMV concentration. In both cases CMV viral load was measured in duplicates and compared to a pure CMV standard. CMV detection and quantification were not influenced and remained unchanged by the presence of HSV-1 or EBV. In this study we assessed the performance of the nucleic acidbased Abbott RealTime CMV assay compared to the Roche Cobas Amplicor CMV Monitor Test. The ARC showed good concordance with RCM, as well as good inter-assay precision and linear range. This high concordance is consistent with another study (Yerly et al., 2007) which compared ARC with a modified (ultrasensitive) Cobas Amplicor CMV Monitor. Precision of the assay is also an important clinical asset as the absolute viral load and its kinetics are predictors of active disease (Kraft et al., 2012). We believe standardization of the assay against the WHO international standard is a major advantage. This will allow researchers to conduct multicenter trials and cohort studies which could potentially lead to evidence-based international viral load thresholds for preemptive treatment initiation and cessation in transplant recipients. Direct patient care will also be facilitated as we can now compare results obtained through different laboratories. The assay claims a linear range of 20–108 copies/mL. We verified a smaller range reflecting clinical practice which spanned from 30 to 107 copies/mL. Very high CMV viremia in the range of 107 copies/mL are plausible but rarely seen (Kraft et al., 2012). However the test’s high limit of quantification would be deemed useful for these high viral load samples since it would preclude the need to prepare dilutions. The greater sensitivity of the assay is of unknown significance. From a clinical standpoint earlier detection of CMV viremia may result in earlier treatment and better outcome. On the other hand, a very sensitive DNA detection test may also lengthen treatment duration and raise the risk of adverse effects, since antiviral drugs are most often withdrawn only when CMV viremia becomes undetectable (Boeckh and Ljungman, 2009; Kotton et al., 2013; Kraft
M.-A. Tremblay et al. / Journal of Virological Methods 225 (2015) 1–3
et al., 2012; Ljungman et al., 2004, 2011; Razonable and Humar, 2013; Tomblyn et al., 2009). One limit of our study is that the comparator assay is an older end-point technology being gradually phased and replaced with the Roche COBAS AmpliPrep/COBAS TaqMan CMV Test. However our evaluation reflects the reality of most clinical laboratories upgrading their current system. Another limit is the small number of patients, although it did not prevent obtaining a good correlation between the two tests. Furthermore, the study population included hematopoietic cell and solid organ transplant recipients, but the latter were restricted to kidney transplants. In conclusion, the Abbott RealTime CMV assay offers a valuable alternative to the older end-point PCR assays, such as the Cobas Amplicor CMV Monitor Test. Its good analytical performance and its standardization could, along with similarly standardized assays, open new opportunities for researchers and clinicians in the future. Acknowledgements We thank Nathalie Boucher and Line Vézina for technical work. Ethical approval and patient consent were not required since this was a diagnostic test verification study, which included anonymized retrospective samples, implying there could be no impact for patient care. This practice is in agreement with the Clinical Laboratory Improvement Amendments (CLIA) federal regulatory standards (Burd, 2010). References Abbott RealTime CMV Package Insert, (2011 September), Abbott Molecular Inc, Des Plaines, IL, USA. http://www.abbottmolecular.com (accessed on 06.02.15). 2005, March. BBI Diagnostics CMV DNA Linearity Panel PTC801. BBI Diagnostics, SeraCare Life Sciences, Milford, MA, USA. Boeckh, M., Ljungman, P., 2009. How we treat cytomegalovirus in hematopoietic cell transplant recipients. Blood 113 (June (23)), 5711–5719, http://dx.doi.org/ 10.1182/blood-2008-10-143560. Burd, E.M., 2010. Validation of laboratory-developed molecular assays for infectious diseases. Clin. Microbiol. Rev. 23 (3), 550–576, http://dx.doi.org/10. 1128/CMR.00074-09. Caliendo, A.M., St. George, K., Kao, S.Y., Allega, J., Tan, B.H., LaFontaine, R., Bui, L., Rinaldo, C.R., 2000. Comparison of quantitative cytomegalovirus (CMV) PCR in plasma and CMV antigenemia assay: clinical utility of the prototype AMPLICOR CMV MONITOR test in transplant recipients. J. Clin. Microbiol. 38 (June (6)), 2122–2127. Caliendo, A.M., Schuurman, R., Yen-Lieberman, B., Spector, S.A., Andersen, J., Manjiry, R., Crumpacker, C., Lurain, N.S., Erice, A., 2001. Comparison of quantitative and qualitative PCR assays for cytomegalovirus DNA in plasma. J. Clin. Microbiol. 39 (April (4)), 1334–1338, http://dx.doi.org/10.1128/JCM.39.4. 1334-1338.2001.
3
Cockerill Jr., F.R., 2003. Application of rapid-cycle real-time polymerase chain reaction for diagnostic testing in the clinical microbiology laboratory. Arch. Pathol. Lab. Med. 127 (September (9)), 1112–1120. Einsele, H., Ehninger, G., Hebart, H., Wittkowski, K.M., Schuler, U., Jahn, G., Mackes, P., Herter, M., Klingebiel, T., Löffler, J., Wagner, S., Müller, C.A., 1995. Polymerase chain reaction monitoring reduces the incidence of cytomegalovirus disease and the duration and side effects of antiviral therapy after bone marrow transplantation. Blood 86 (October (7)), 2815–2820. Fryer, J.F., Heath, A.B., Anderson, R., Minor, P.D., The Collaborative Study Group, 2010. Collaborative Study to Evaluate the Proposed 1st WHO International Standard for Human Cytomegalovirus (HCMV) for Nucleic Acid Amplification (NAT)-Based Assays. WHO/BS/10.2138. World Health Organization, Geneva, Switzerland, http://apps.who.int/iris/handle/10665/70521?locale=en (accessed on 06.02.15). Kalil, A.C., Levitsky, J., Lyden, E., Stoner, J., Freifeld, A.G., 2005. Meta-analysis: the efficacy of strategies to prevent organ disease by cytomegalovirus in solid organ transplant recipients. Ann. Intern. Med. 143 (December (12)), 870–880. Kotton, C.N., Kumar, D., Caliendo, A.M., Asberg, A., Chou, S., Danziger-Isakov, L., Humar, A., Transplantation Society International CMV Consensus Group, 2013. Updated international consensus guidelines on the management of cytomegalovirus in solid-organ transplantation. Transplantation 96 (August (4)), 333–360, http://dx.doi.org/10.1097/TP.0b013e31829df29d. Kraft, C.S., Armstrong, W.S., Caliendo, A.M., 2012. Interpreting quantitative cytomegalovirus DNA testing: understanding the laboratory perspective. Clin. Infect. Dis. 54 (June (12)), 1793–1797, http://dx.doi.org/10.1093/cid/cis212. Ljungman, P., Reusser, P., de la Camara, R., Einsele, H., Engelhard, D., Ribaud, P., Ward, K., European Group for Blood and Marrow Transplantation, 2004. Management of CMV infections: recommendations from the infectious diseases working party of the EBMT. Bone Marrow Transplant. 33 (June (11)), 1075–1081, http://dx.doi.org/10.1038/sj.bmt.1704505. Ljungman, P., Hakki, M., Boeckh, M., 2011. Cytomegalovirus in hematopoietic stem cell transplant recipients. Hematol. Oncol. Clin. N. Am. 25 (February (1)), 151–169, http://dx.doi.org/10.1016/j.hoc.2010.11.011. Piiparinen, H., Höckerstedt, K., Grönhagen-Riska, C., Lappalainen, M., Suni, J., Lautenschlager, I., 2001. Comparison of plasma polymerase chain reaction and pp65-antigenemia assay in the quantification of cytomegalovirus in liver and kidney transplant patients. J. Clin. Virol. 22 (August (1)), 111–116. R Core Team, 2012. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria, http://www.R-project. org/ (accessed on 06.02.15). ISBN 3-900051-07-0. Razonable, R.R., Humar, A., 2013. AST infectious diseases community of practice, cytomegalovirus in solid organ transplantation. Am. J. Transplant. 13 (March (Suppl 4)), 93–106, http://dx.doi.org/10.1111/ajt.12103. Roche Cobas Amplicor CMV Monitor Test Product Information. Roche Diagnostics Canada, Laval, QC, Canada. http://molecular.roche.com/assays/Pages/ COBASAMPLICORCMVMONITORTest.aspx (accessed 06.02.15). Tomblyn, M., Chiller, T., Einsele, H., Gress, R., Sepkowitz, K., Storek, J., Wingard, J.R., Young, J.A., Boeckh, M.J., 2009. Guidelines for preventing infectious complications among hematopoietic cell transplantation recipients: a global perspective. Biol. Blood Marrow Transplant. 15 (October (10)), 1143–1238, http://dx.doi.org/10.1016/j.bbmt.2009.06.019. Yerly, S., Perrin, L., Van Delden, C., Schaffer, S., Thamm, S., Wunderli, W., Kaiser, L., 2007. Cytomegalovirus quantification in plasma by an automated real-time PCR assay. J. Clin. Virol. 38, 298–303, http://dx.doi.org/10.1016/j.jcv.2007.01. 003. ZeptoMetrix NATtrolTM Cytomegalovirus External Run Controls Strain: AD-169 (NATCMV-ERCL) Product Insert. ZeptoMetrix Corporation, Buffalo, NY, USA. http://www.zeptometrix.com/docs/PINATCMV-ERCL.pdf?1365017153 (accessed on 06.02.15).