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Comparison of the new Abbott Real Time CMV assay and the Abbott CMV PCR Kit for the quantitation of plasma cytomegalovirus DNAemia
Diagnostic Microbiology and Infectious Disease 75 (2013) 207–209
Contents lists available at SciVerse ScienceDirect
Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Comparison of the new Abbott Real Time CMV assay and the Abbott CMV PCR Kit for the quantitation of plasma cytomegalovirus DNAemia María Ángeles Clari a, Dayana Bravo a, Elisa Costa a, Beatriz Muñoz-Cobo a, Carlos Solano b, María José Remigia b, Estela Giménez a, Omar J. BenMarzouk-Hidalgo c, Pilar Pérez-Romero c, David Navarro a, d,⁎ a
Microbiology Service, University Clinic Hospital, Valencia, Spain Hematology and Medical Oncology Service, University Clinic Hospital, Valencia, Spain c Unit of Infectious Disease, Microbiology, and Preventive Medicine, Institute of Biomedicine of Sevilla (IBiS), University Hospital Virgen del Rocio. Sevilla, Spain d Department of Microbiology, School of Medicine, University of Valencia, Spain b
a r t i c l e
i n f o
Article history: Received 21 August 2012 Received in revised form 12 October 2012 Accepted 13 October 2012 Available online 20 November 2012
a b s t r a c t CMV DNA loads measured by the new Abbott RealTime CMV PCR were significantly higher than those quantitated by the Abbott CMV PCR kit (approximately 1 log10), and provided a better estimate of the actual CMV load present in plasma specimens as inferred by the use of the WHO standard. © 2013 Elsevier Inc. All rights reserved.
Keywords: Cytomegalovirus Real-time PCR Plasma CMV DNAemia Abbott PCR assays
Quantitation of cytomegalovirus (CMV) DNA load in the blood compartment (CMV DNAemia) by quantitative real-time PCR (QRTPCR) has become a standard practice for the surveillance of active CMV infection and for monitoring the response to antiviral therapy in the transplantation setting (Solano and Navarro, 2010). Recently, a new QRT-PCR (Abbott RealTime CMV, Abbott Molecular Inc., Des Plaines, Il, USA), approved via the CE-labeling system in the European Union, has been launched to the market. This kit is intended to replace the Abbott CMV PCR Kit (produced by Qiagen GmbH, Hilde, Germany for Abbott Molecular Diagnostics) in laboratories using Abbott Molecular CMV reagents. In the current study we compared both assays for the quantitation of plasma CMV DNAemia. A total of 188 plasma specimens testing positive by the Abbott CMV PCR Kit in the m2000rt system (Abbott Molecular, Illinois, USA) were included in the study. These samples had been submitted to our laboratories for CMV DNA load monitoring and obtained from adult patients who underwent allogeneic hematopoietic stem cell transplantation at the Hospital Clínico Universtario of Valencia, or solid organ transplantation at the University Hospital Virgen del Rocío of Sevilla and were kept frozen at −20 °C after analysis. The 1st WHO International Standard for CMV for Nucleic Acid Amplification (NAT)-
⁎ Corresponding author. Tel.: +34-96-3864657; fax: +34-96-3864173. E-mail address: [email protected] (D. Navarro). 0732-8893/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.diagmicrobio.2012.10.010
Based Assays (National Institute for Biological Standards and Control, Hertfortshire, UK) was also assayed by both analytical methods. The specimens were retrieved and analyzed in parallel by both PCR methods. DNA extractions were performed from 500 μL of plasma using the Abbott mSample preparation system DNA Kit on the m2000sp instrument (Abbott Molecular, Illinois, USA) and eluted in a volume of 70 μL. According to the manufacturer, the sensitivity of the Abbott CMV PCR Kit for the measurement of CMV DNA load in plasma specimens is 64 copies/mL (95% confidence). No data on either the limit of CMV DNA quantitation or the linear range of quantitation of the assay are provided in the kit's insert. The analytical performance of this assay has been previously evaluated (Bravo et al., 2011; Caliendo et al., 2007; Gimeno et al., 2008; Gracia-Ahufinger et al., 2010). In this context, Caliendo et al., (2007) found that the limit of detection of the assay was 2.3 log10 copies/mL, and the linear range of quantitation 2.0 to 6.0 log10 copies/mL. In our experience, the limit of quantitation of the assay is 25 copies/mL (Gimeno et al., 2008; Bravo et al., 2011). The new Abbott RealTime CMV assay targets the UL34 and UL80.5 ORFs of the CMV genome. According to the manufacturer, the limit of detection of the assay is approximately 14 copies/mL (95% confidence), the linear dynamic range of quantitation between 1.40 to 8.5 log10 copies/mL and the intra-assay and inter-assay coefficient of variation for CMV DNA loads within the linear range of quantitation are below 0.5 log10 copies/mL. Amplification and and real-time detection of CMV DNA was performed on the m2000rt instrument.
M.Á. Clari et al. / Diagnostic Microbiology and Infectious Disease 75 (2013) 207–209
Abbott RealTime PCR assay (log10 copies/mL)
208
y=1.0677x+0.8665 r2=0.92 (95% C.I.: 0.90-0.94) P =< 0.0001
6,5
5,5
4,5
3,5
2,5
1,5 1
2
3
4
5
Abbott CMV PCR Kit (log10 copies/mL)
Differences in CMV DNA loads (log10 copies/mL)
2,5
2
1,5
1
0,5
0
-0,5 1
3
5
7
were performed using the SPSS 17.0 program (SPSS Inc. Chicago, USA); P values of ≤ 0.05 were considered statistically significant. CMV DNA loads in plasma specimens subjected to analysis ranged from 1.3 log10 to 5.0 log10 copies/mL, as quantitated by the Abbott CMV PCR kit. As shown in Fig. 1A, CMV DNA loads measured by both analytical systems correlated significantly (σ = 0.92; P = b0.0001); Nevertheless, the degree of such correlation varied depending on the magnitude of the CMV DNA load, being lowest at CMV DNA concentrations close to the limit of detection of the assays-1.3 to 2.0 log10 copies/mL- (σ=0.406; P = .017) and greatest for specimens with N3.0 log10 CMV DNA copies/mL (σ=0.733; P = b0.001). Overall, the new Abbott RealTime CMV assay yielded significantly higher CMV DNA loads than the Abbott CMV PCR kit (P = b0.001 in the Mann–Whitney U-test). Differences of around 1 log10 were observed for specimens with low to high content (1.3 to b4.0 log10 copies/mL). For specimens with very high CMV DNA loads (N4 log10 copies/mL) the differences were even higher (median, 1.54 log10 copies/mL) (Table 1; Fig. 1B). Three dilutions of the original WHO International standard preparation were assayed in triplicate in two different runs by both QRT-PCRs. In these experiments, the overall intra-assay and interassay coefficient of variation was b0.2 log10 for both methods. As shown in Table 1, the highest concentration of the WHO Standard (4.69 log10 IU/mL) yielded the smallest difference between the two methods, whereas the lowest concentration of the WHO standard (2.69 log10 IU/mL) gave the highest difference between the two QRTPCR methods. This is in contrast to what we found when assaying plasma specimens. Although speculative, the different conformation of CMV DNA present in plasma specimens, presumably highly fragmented (Boom et al., 2002) and in the WHO preparation (linear encapsidated DNA) might account in part for this finding. The overall conversion factor (copies/mL to IU/mL) provided by the manufacturer for the new Abbott RealTime PCR is 0.19 log10 (log10 copies/mL +0.19 log10 = log10 IU/mL). In our experience, the fitted regression lines between IU/mL and copies/mL was given by the following equation: log10 (IU/mL) =−0.4481 + 1.0096x (log10 copies/mL), suggesting that in our setting the conversion factor is 0.36 log10. The above difference may have been due to the limited number of concentrations of the WHO standard that were assayed in the current study. The overall conversion factor for the CMV PCR Kit has not been provided by the manufacturer. In our experience, it was
Average CMV DNA loads (log10 copies/mL) Fig. 1. Correlation and linear regression analysis of cytomegalovirus (CMV) DNA load values (log10 copies/mL) measured in plasma specimens by the new Abbott RealTime PCR Kit and the Abbott CMV PCR Kit following DNA extraction using the Abbott mSample preparation system DNA Kit on the m2000sp instrument.
Table 1 Cytomegalovirus (CMV) DNA values obtained with the new Abbott RealTime PCR assay and the Abbott CMV PCR Kit with plasma specimens and the 1st WHO International Standard for CMV for Nucleic Acid Amplification (NAT)-Based Assays. Specimen
The specimens were assayed in singlet in several consecutive runs. In order to normalize CMV DNA loads measured (in copies/mL) to International Units (IU)/mL, the 1st WHO International Standard for CMV for Nucleic Acid Amplification (NAT)-Based Assays was assayed by both analytical methods. This is a cell-free whole-virus (Merlin strain) preparation in an universal buffer comprising Tris–HCl and human serum albumin (Fryer et al., 2010; Pang et al., 2009). Three dilutions of the original preparation (2.69, 3.69 and 4.69 log10 IU/mL) were made in a matrix of human plasma with undetectable levels of CMV DNA in both QRT-PCR assays, were extracted in the m2000sp system, and analyzed by both QRT-PCR assays. The data (in copies/ mL) were log10 transformed prior to analysis. Differences between median CMV DNA loads measured with both assays were analyzed using the Mann–Whitney U-test. Quantitative correlations between the CMV DNA loads obtained using the two methods were evaluated using the Spearman correlation test. The method of Bland and Altman (1986) was used to assess the agreement between CMV DNA loads measured using the two analytical systems. Statistical calculations
Clinical samples (n = 188)a 1.3 to 2.0 log10 copies/mL (n = 36) N2.0 to b3.0 log10 copies/mL (n = 91) N3.0 to b4.0 log10 copies/mL (n = 50) N4.0 log10 copies/mL (n = 11) 1st WHO International Standardb 2.69 log10 IU/mL 3.69 log10 IU/mL 4.69 log10 IU/mL
CMV DNA load measured (log10 copies/mL or IU/mL) Abbott CMV PCR Kit
Abbott RealTime CMV
1.76 2.48 3.39 4.52
2.75 3.44 4.50 6.06
1.39 2.66 3.97
(1.3-2.0) (2.10-3.0) (3.01-3.98) (4.04-5.0)
(1.78-3.44) (2.19-4.77) (3.64-5.37) (4.90-6.63)
2.39 3.49 4.51
a Plasma specimens were arbitrarily grouped into four categories according to CMV DNA loads measured by the Abbott CMV PCR Kit assay (low, 1.3-2.0 log10 copies/mL; intermediate, N2.0 to b3.0 log10 copies/mL; high, N3.0 to b4.0 log10 copies/mL, and very high N4.0 log10 copies/mL). Median values and ranges are are given. b Data are reported as medians of two experiments. Each WHO dilution was assayed in triplicate in two runs performed in different days. The original material provided by National Institute for Biological Standards and Control in 10 nM Tris–HCl- human serum albumin (0.5%) pH, 7.4 to achieve a final concentration of 5x106 IU/mL. The dilutions of the reconstituted standard were prepared in a matrix of human plasma testing negative in the two QRT-PCRs employed in this study.
M.Á. Clari et al. / Diagnostic Microbiology and Infectious Disease 75 (2013) 207–209
1.61 log10, as given by the following fitted regression line: log10 IU/ mL=−2.086+1.20x (log10 copies/mL). Our data, nevertheless, suggested that the conversion factor in both PCR assays may vary upon the CMV DNA content, being higher for specimens with CMV DNA concentrations b3.69 log10 IU/mL than for samples with CMV DNA loads N 3.69 log10 IU/mL (Table 1); Further studies are nevertheless warranted to confirm this extent, and if it held true to determine whether it would have any clinical relevance. In summary, the data reported in this study indicated that CMV DNA loads measured by the new Abbott RealTime CMV PCR are significantly higher than those quantitated by the Abbott CMV PCR kit, and appeared to provide a better estimate of the actual CMV load present in plasma specimens, as inferred by the use of the WHO preparation as the standard for quantitation. The different nature of the gene targets in the two assays and the fact that the new Abbott assay has a dual gene target (UL34 and UL80.5) instead of a single one (UL122 in the old Abbott assay), most likely accounted for this difference, as both methods employ an identical real-time PCR chemistry and DNA was extracted employing the same platform. The information provided here may be of interest to the users of QRTPCR reagents commercialized by Abbott Molecular Diagnostics. Acknowledgments We thank Julia Garcia, Mónica Reig and Matilde Pastor for their technical assistance. We also thank Abbott Molecular Inc. for the donation of reagents employed in this study.
209
References Bland JM, Altman DG. Statistical method for assessing the agreement between two methods of clinical measurement. Lancet 1986;i:307–10. Boom R, Sol CJ, Schuurman T, Van Breda A, Weel JF, Beld M, et al. Human cytomegalovirus DNA in plasma and serum specimens of renal transplant recipients is highly fragmented. J Clin Microbiol 2002;40:4105–13. Bravo D, Clari MA, Costa E, Costa Muñoz-Cobo B, Solano C, Remigia MJ, et al. Comparative evaluation of three automated systems for DNA extraction in conjunction with three commercially available real-time PCR assays for quantitation of plasma Cytomegalovirus DNAemia in allogeneic stem cell transplant recipients. J Clin Microbiol 2011;49:2899–904. Caliendo AM, Ingersoll J, Fox-Canale AM, Pargman S, Bythwood T, Hayden MK, et al. Evaluation of real-time PCR laboratory- developed tests using analytespecific reagents for cytomegalovirus quantification. J Clin Microbiol 2007;45: 1723–37. Fryer JF, Heath AB, Anderson R, Minor PD, and the collaborative study group. Collaborative study to evaluate the proposed 1st WHO International Standard for human cytomegalovirus (HCMV) for nucleic acid amplification (NAT)-based assays. WHO ECBS Report 2010; WHO/BS/10.2138. Gimeno C, Solano C, Latorre JC, Hernández-Boluda JC, Clari MA, Remigia MJ, et al. Quantification of DNA in plasma by an automated real-time PCR assay (cytomegalovirus PCR kit) for surveillance of active cytomegalovirus infection and guidance of pre-emptive therapy for allogeneic hematopoietic stem cell transplant recipients. J Clin Microbiol 2008;46:3311–8. Gracia-Ahufinger I, Tormo N, Espigado I, Solano C, Urbano-Ispizua A, Clari MA, et al. Differences in cytomegalovirus plasma viral loads measured in allogeneic hematopoietic stem cell transplant recipients using two commercial real-time PCR assays. J Clin Virol 2010;48:142–6. Pang XL, Fox JD, Fenton JM, Miller GG, Caliendo AM, Preiksaitis JM, American Society of Transplantation Infectious Diseases Community of Practice, Canadian Society of Transplantation. Interlaboratory comparison of cytomegalovirus viral load assays. Am J Transplant 2009;9:258–68. Solano C, Navarro D. Clinical virology of cytomegalovirus infection following hematopoietic transplantation. Future Virol 2010;5:111–24.
Contents lists available at SciVerse ScienceDirect
Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Comparison of the new Abbott Real Time CMV assay and the Abbott CMV PCR Kit for the quantitation of plasma cytomegalovirus DNAemia María Ángeles Clari a, Dayana Bravo a, Elisa Costa a, Beatriz Muñoz-Cobo a, Carlos Solano b, María José Remigia b, Estela Giménez a, Omar J. BenMarzouk-Hidalgo c, Pilar Pérez-Romero c, David Navarro a, d,⁎ a
Microbiology Service, University Clinic Hospital, Valencia, Spain Hematology and Medical Oncology Service, University Clinic Hospital, Valencia, Spain c Unit of Infectious Disease, Microbiology, and Preventive Medicine, Institute of Biomedicine of Sevilla (IBiS), University Hospital Virgen del Rocio. Sevilla, Spain d Department of Microbiology, School of Medicine, University of Valencia, Spain b
a r t i c l e
i n f o
Article history: Received 21 August 2012 Received in revised form 12 October 2012 Accepted 13 October 2012 Available online 20 November 2012
a b s t r a c t CMV DNA loads measured by the new Abbott RealTime CMV PCR were significantly higher than those quantitated by the Abbott CMV PCR kit (approximately 1 log10), and provided a better estimate of the actual CMV load present in plasma specimens as inferred by the use of the WHO standard. © 2013 Elsevier Inc. All rights reserved.
Keywords: Cytomegalovirus Real-time PCR Plasma CMV DNAemia Abbott PCR assays
Quantitation of cytomegalovirus (CMV) DNA load in the blood compartment (CMV DNAemia) by quantitative real-time PCR (QRTPCR) has become a standard practice for the surveillance of active CMV infection and for monitoring the response to antiviral therapy in the transplantation setting (Solano and Navarro, 2010). Recently, a new QRT-PCR (Abbott RealTime CMV, Abbott Molecular Inc., Des Plaines, Il, USA), approved via the CE-labeling system in the European Union, has been launched to the market. This kit is intended to replace the Abbott CMV PCR Kit (produced by Qiagen GmbH, Hilde, Germany for Abbott Molecular Diagnostics) in laboratories using Abbott Molecular CMV reagents. In the current study we compared both assays for the quantitation of plasma CMV DNAemia. A total of 188 plasma specimens testing positive by the Abbott CMV PCR Kit in the m2000rt system (Abbott Molecular, Illinois, USA) were included in the study. These samples had been submitted to our laboratories for CMV DNA load monitoring and obtained from adult patients who underwent allogeneic hematopoietic stem cell transplantation at the Hospital Clínico Universtario of Valencia, or solid organ transplantation at the University Hospital Virgen del Rocío of Sevilla and were kept frozen at −20 °C after analysis. The 1st WHO International Standard for CMV for Nucleic Acid Amplification (NAT)-
⁎ Corresponding author. Tel.: +34-96-3864657; fax: +34-96-3864173. E-mail address: [email protected] (D. Navarro). 0732-8893/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.diagmicrobio.2012.10.010
Based Assays (National Institute for Biological Standards and Control, Hertfortshire, UK) was also assayed by both analytical methods. The specimens were retrieved and analyzed in parallel by both PCR methods. DNA extractions were performed from 500 μL of plasma using the Abbott mSample preparation system DNA Kit on the m2000sp instrument (Abbott Molecular, Illinois, USA) and eluted in a volume of 70 μL. According to the manufacturer, the sensitivity of the Abbott CMV PCR Kit for the measurement of CMV DNA load in plasma specimens is 64 copies/mL (95% confidence). No data on either the limit of CMV DNA quantitation or the linear range of quantitation of the assay are provided in the kit's insert. The analytical performance of this assay has been previously evaluated (Bravo et al., 2011; Caliendo et al., 2007; Gimeno et al., 2008; Gracia-Ahufinger et al., 2010). In this context, Caliendo et al., (2007) found that the limit of detection of the assay was 2.3 log10 copies/mL, and the linear range of quantitation 2.0 to 6.0 log10 copies/mL. In our experience, the limit of quantitation of the assay is 25 copies/mL (Gimeno et al., 2008; Bravo et al., 2011). The new Abbott RealTime CMV assay targets the UL34 and UL80.5 ORFs of the CMV genome. According to the manufacturer, the limit of detection of the assay is approximately 14 copies/mL (95% confidence), the linear dynamic range of quantitation between 1.40 to 8.5 log10 copies/mL and the intra-assay and inter-assay coefficient of variation for CMV DNA loads within the linear range of quantitation are below 0.5 log10 copies/mL. Amplification and and real-time detection of CMV DNA was performed on the m2000rt instrument.
M.Á. Clari et al. / Diagnostic Microbiology and Infectious Disease 75 (2013) 207–209
Abbott RealTime PCR assay (log10 copies/mL)
208
y=1.0677x+0.8665 r2=0.92 (95% C.I.: 0.90-0.94) P =< 0.0001
6,5
5,5
4,5
3,5
2,5
1,5 1
2
3
4
5
Abbott CMV PCR Kit (log10 copies/mL)
Differences in CMV DNA loads (log10 copies/mL)
2,5
2
1,5
1
0,5
0
-0,5 1
3
5
7
were performed using the SPSS 17.0 program (SPSS Inc. Chicago, USA); P values of ≤ 0.05 were considered statistically significant. CMV DNA loads in plasma specimens subjected to analysis ranged from 1.3 log10 to 5.0 log10 copies/mL, as quantitated by the Abbott CMV PCR kit. As shown in Fig. 1A, CMV DNA loads measured by both analytical systems correlated significantly (σ = 0.92; P = b0.0001); Nevertheless, the degree of such correlation varied depending on the magnitude of the CMV DNA load, being lowest at CMV DNA concentrations close to the limit of detection of the assays-1.3 to 2.0 log10 copies/mL- (σ=0.406; P = .017) and greatest for specimens with N3.0 log10 CMV DNA copies/mL (σ=0.733; P = b0.001). Overall, the new Abbott RealTime CMV assay yielded significantly higher CMV DNA loads than the Abbott CMV PCR kit (P = b0.001 in the Mann–Whitney U-test). Differences of around 1 log10 were observed for specimens with low to high content (1.3 to b4.0 log10 copies/mL). For specimens with very high CMV DNA loads (N4 log10 copies/mL) the differences were even higher (median, 1.54 log10 copies/mL) (Table 1; Fig. 1B). Three dilutions of the original WHO International standard preparation were assayed in triplicate in two different runs by both QRT-PCRs. In these experiments, the overall intra-assay and interassay coefficient of variation was b0.2 log10 for both methods. As shown in Table 1, the highest concentration of the WHO Standard (4.69 log10 IU/mL) yielded the smallest difference between the two methods, whereas the lowest concentration of the WHO standard (2.69 log10 IU/mL) gave the highest difference between the two QRTPCR methods. This is in contrast to what we found when assaying plasma specimens. Although speculative, the different conformation of CMV DNA present in plasma specimens, presumably highly fragmented (Boom et al., 2002) and in the WHO preparation (linear encapsidated DNA) might account in part for this finding. The overall conversion factor (copies/mL to IU/mL) provided by the manufacturer for the new Abbott RealTime PCR is 0.19 log10 (log10 copies/mL +0.19 log10 = log10 IU/mL). In our experience, the fitted regression lines between IU/mL and copies/mL was given by the following equation: log10 (IU/mL) =−0.4481 + 1.0096x (log10 copies/mL), suggesting that in our setting the conversion factor is 0.36 log10. The above difference may have been due to the limited number of concentrations of the WHO standard that were assayed in the current study. The overall conversion factor for the CMV PCR Kit has not been provided by the manufacturer. In our experience, it was
Average CMV DNA loads (log10 copies/mL) Fig. 1. Correlation and linear regression analysis of cytomegalovirus (CMV) DNA load values (log10 copies/mL) measured in plasma specimens by the new Abbott RealTime PCR Kit and the Abbott CMV PCR Kit following DNA extraction using the Abbott mSample preparation system DNA Kit on the m2000sp instrument.
Table 1 Cytomegalovirus (CMV) DNA values obtained with the new Abbott RealTime PCR assay and the Abbott CMV PCR Kit with plasma specimens and the 1st WHO International Standard for CMV for Nucleic Acid Amplification (NAT)-Based Assays. Specimen
The specimens were assayed in singlet in several consecutive runs. In order to normalize CMV DNA loads measured (in copies/mL) to International Units (IU)/mL, the 1st WHO International Standard for CMV for Nucleic Acid Amplification (NAT)-Based Assays was assayed by both analytical methods. This is a cell-free whole-virus (Merlin strain) preparation in an universal buffer comprising Tris–HCl and human serum albumin (Fryer et al., 2010; Pang et al., 2009). Three dilutions of the original preparation (2.69, 3.69 and 4.69 log10 IU/mL) were made in a matrix of human plasma with undetectable levels of CMV DNA in both QRT-PCR assays, were extracted in the m2000sp system, and analyzed by both QRT-PCR assays. The data (in copies/ mL) were log10 transformed prior to analysis. Differences between median CMV DNA loads measured with both assays were analyzed using the Mann–Whitney U-test. Quantitative correlations between the CMV DNA loads obtained using the two methods were evaluated using the Spearman correlation test. The method of Bland and Altman (1986) was used to assess the agreement between CMV DNA loads measured using the two analytical systems. Statistical calculations
Clinical samples (n = 188)a 1.3 to 2.0 log10 copies/mL (n = 36) N2.0 to b3.0 log10 copies/mL (n = 91) N3.0 to b4.0 log10 copies/mL (n = 50) N4.0 log10 copies/mL (n = 11) 1st WHO International Standardb 2.69 log10 IU/mL 3.69 log10 IU/mL 4.69 log10 IU/mL
CMV DNA load measured (log10 copies/mL or IU/mL) Abbott CMV PCR Kit
Abbott RealTime CMV
1.76 2.48 3.39 4.52
2.75 3.44 4.50 6.06
1.39 2.66 3.97
(1.3-2.0) (2.10-3.0) (3.01-3.98) (4.04-5.0)
(1.78-3.44) (2.19-4.77) (3.64-5.37) (4.90-6.63)
2.39 3.49 4.51
a Plasma specimens were arbitrarily grouped into four categories according to CMV DNA loads measured by the Abbott CMV PCR Kit assay (low, 1.3-2.0 log10 copies/mL; intermediate, N2.0 to b3.0 log10 copies/mL; high, N3.0 to b4.0 log10 copies/mL, and very high N4.0 log10 copies/mL). Median values and ranges are are given. b Data are reported as medians of two experiments. Each WHO dilution was assayed in triplicate in two runs performed in different days. The original material provided by National Institute for Biological Standards and Control in 10 nM Tris–HCl- human serum albumin (0.5%) pH, 7.4 to achieve a final concentration of 5x106 IU/mL. The dilutions of the reconstituted standard were prepared in a matrix of human plasma testing negative in the two QRT-PCRs employed in this study.
M.Á. Clari et al. / Diagnostic Microbiology and Infectious Disease 75 (2013) 207–209
1.61 log10, as given by the following fitted regression line: log10 IU/ mL=−2.086+1.20x (log10 copies/mL). Our data, nevertheless, suggested that the conversion factor in both PCR assays may vary upon the CMV DNA content, being higher for specimens with CMV DNA concentrations b3.69 log10 IU/mL than for samples with CMV DNA loads N 3.69 log10 IU/mL (Table 1); Further studies are nevertheless warranted to confirm this extent, and if it held true to determine whether it would have any clinical relevance. In summary, the data reported in this study indicated that CMV DNA loads measured by the new Abbott RealTime CMV PCR are significantly higher than those quantitated by the Abbott CMV PCR kit, and appeared to provide a better estimate of the actual CMV load present in plasma specimens, as inferred by the use of the WHO preparation as the standard for quantitation. The different nature of the gene targets in the two assays and the fact that the new Abbott assay has a dual gene target (UL34 and UL80.5) instead of a single one (UL122 in the old Abbott assay), most likely accounted for this difference, as both methods employ an identical real-time PCR chemistry and DNA was extracted employing the same platform. The information provided here may be of interest to the users of QRTPCR reagents commercialized by Abbott Molecular Diagnostics. Acknowledgments We thank Julia Garcia, Mónica Reig and Matilde Pastor for their technical assistance. We also thank Abbott Molecular Inc. for the donation of reagents employed in this study.
209
References Bland JM, Altman DG. Statistical method for assessing the agreement between two methods of clinical measurement. Lancet 1986;i:307–10. Boom R, Sol CJ, Schuurman T, Van Breda A, Weel JF, Beld M, et al. Human cytomegalovirus DNA in plasma and serum specimens of renal transplant recipients is highly fragmented. J Clin Microbiol 2002;40:4105–13. Bravo D, Clari MA, Costa E, Costa Muñoz-Cobo B, Solano C, Remigia MJ, et al. Comparative evaluation of three automated systems for DNA extraction in conjunction with three commercially available real-time PCR assays for quantitation of plasma Cytomegalovirus DNAemia in allogeneic stem cell transplant recipients. J Clin Microbiol 2011;49:2899–904. Caliendo AM, Ingersoll J, Fox-Canale AM, Pargman S, Bythwood T, Hayden MK, et al. Evaluation of real-time PCR laboratory- developed tests using analytespecific reagents for cytomegalovirus quantification. J Clin Microbiol 2007;45: 1723–37. Fryer JF, Heath AB, Anderson R, Minor PD, and the collaborative study group. Collaborative study to evaluate the proposed 1st WHO International Standard for human cytomegalovirus (HCMV) for nucleic acid amplification (NAT)-based assays. WHO ECBS Report 2010; WHO/BS/10.2138. Gimeno C, Solano C, Latorre JC, Hernández-Boluda JC, Clari MA, Remigia MJ, et al. Quantification of DNA in plasma by an automated real-time PCR assay (cytomegalovirus PCR kit) for surveillance of active cytomegalovirus infection and guidance of pre-emptive therapy for allogeneic hematopoietic stem cell transplant recipients. J Clin Microbiol 2008;46:3311–8. Gracia-Ahufinger I, Tormo N, Espigado I, Solano C, Urbano-Ispizua A, Clari MA, et al. Differences in cytomegalovirus plasma viral loads measured in allogeneic hematopoietic stem cell transplant recipients using two commercial real-time PCR assays. J Clin Virol 2010;48:142–6. Pang XL, Fox JD, Fenton JM, Miller GG, Caliendo AM, Preiksaitis JM, American Society of Transplantation Infectious Diseases Community of Practice, Canadian Society of Transplantation. Interlaboratory comparison of cytomegalovirus viral load assays. Am J Transplant 2009;9:258–68. Solano C, Navarro D. Clinical virology of cytomegalovirus infection following hematopoietic transplantation. Future Virol 2010;5:111–24.