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Comparison between pp65 antigenemia assay and quantitative real-time polymerase chain reaction for detection of active cytomegalovirus infection in routine diagnostics
Available online at www.sciencedirect.com
Diagnostic Microbiology and Infectious Disease 65 (2009) 192 – 195 www.elsevier.com/locate/diagmicrobio
Notes
Comparison between pp65 antigenemia assay and quantitative real-time polymerase chain reaction for detection of active cytomegalovirus infection in routine diagnostics Raimond Lugerta,⁎, Utz Reicharda , Frank T. Hufertb , Uwe Großa a
Institute for Medical Microbiology, University Medical Center Göttingen, 37075 Göttingen, Germany b Institute of Virology, University Medical Center Göttingen, 37075 Göttingen, Germany Received 6 March 2009; accepted 28 May 2009
Abstract Real-time polymerase chain reaction (PCR) and pp65 antigenemia assay for the detection of active cytomegalovirus infection in immunocompromised patients experiencing neutropenia after bone marrow or kidney transplantation have been compared with a special focus on evaluability and embedment in daily routine diagnostics. Investigating 334 specimens from 97 patients, real-time PCR was shown to be the superior assay with regard to the parameters focused on. © 2009 Elsevier Inc. All rights reserved. Keywords: pp65 antigenemia assay; Quantitative real-time PCR; Cytomegalovirus
Testing of blood samples from immunocompromised patients for the presence of active cytomegalovirus infection is routinely performed in virologic diagnostic laboratories. Thereby, the pp65 antigenemia assay is still considered as the “gold standard” in many laboratories, although it is labor intensive, is subjective in reading, and may not be evaluable in case of neutropenia. As an alternative, quantitative realtime polymerase chain reaction (PCR) assays, either in-house or commercial, have emerged to the pp65 assay in recent years (Boeckh et al., 2004; Gouarin et al., 2007; Hanson et al., 2007; Herrman et al., 2004; Hong et al., 2004; Kalpoe et al., 2004; Pumannova et al., 2006; Yerly et al., 2007). We have routinely investigated 334 specimens comparing both assays with special regard to evaluability and the aim to embed these assays in daily laboratory diagnostics. The specimens were derived from 95 bone marrow and 2 kidney transplanted outpatients (63 male, 34 female) from the Department of Haematology and Oncology or inpatients from the Department of Nephrology and Rheumatology of the University Medical Center Göttingen, Göttingen, Germany.
⁎ Corresponding author. Tel.: +49-551-395869; fax: +49-551-395861. E-mail address: [email protected] (R. Lugert). 0732-8893/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2009.05.016
Thereby, 50 patients have been investigated only once, whereas 47 patients have been tested up to 19 times in periodic intervals for the detection of active cytomegalovirus infection. For pp65 antigenemia assays, 2-mL Lymphoprep (AxisShield Poc, Oslo, Norway) was overlaid with 5 mL of EDTA blood, and the samples were centrifuged at 300 × g for 10 min (Bøyum, 1968). Supernatants were removed and the pellets were resuspended with 10 mL of Gey's solution and incubated on ice for 10 min for lysis of red blood cells. After centrifugation, resuspension and incubation in Gey's solution were repeated twice. Purified granulocytes were resuspended and washed in 1-mL phosphate-buffered saline (PBS). Finally, the sediments were resuspended in 0.2-mL PBS and spotted onto a glass slide by using cytocentrifuge (500×g for 3 min). The slides were air-dried and fixed in PBS, containing 2% sucrose and 5% formaldehyde, for 5 min. After the slides were washed in PBS and air-dried, cells were incubated with 10 μL of VIR–cytomegalovirus (CMV) C10/ C11 antibody solution (IQ Products, Groningen, the Netherlands) for 30 min at 37 °C. Subsequently, the slides were washed twice with PBS and incubated with 10-μL VIR– fluorescin isothiocyanate secondary antibody solution (IQ Products) for further 30 min at 37 °C. Results were reported as the number of pp65-positive cells per 2 × 105 granulocytes.
R. Lugert et al. / Diagnostic Microbiology and Infectious Disease 65 (2009) 192–195 Table 1 Number of evaluable and CMV-positive blood samples of all 334 samples tested using quantitative real-time PCR and pp65 antigenemia assay Assay
Evaluable
Quantitative real-time PCR pp65 antigenemia assay
Positive
Number
%
Number
%
334/334 268/334
100 80.2
41/334 12/334
12 3.5
Recent data demonstrated that both whole blood and plasma are equally suitable for the detection of active CMV infection in recipients of allogenic hematopoietic stem cells (Caliendo et al., 2007; Kalpoe et al., 2004; Leruez-Ville et al., 2003). For real-time PCR, DNA was extracted from 200 μL of plasma using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Purified DNA was eluted in a volume of 200 μL, and 10 μL were applied for quantitative real-time PCR using the Artus CMV LC PCR Kit (Qiagen), which is licensed for diagnostic use. The CMV LC PCR Kit contains enzymes and reagents for the amplification and detection of a 105-bp fragment of the CMV major immediate early gene. In addition, external positive controls ranging from 10 to 104 CMV copies and an internal heterologous amplification system are supplied to determine the pathogen load and to identify possible PCR inhibition. The specificity of the PCR assay is assured by an oligonucleotide probe, which binds exclusively to the amplified product. Water controls have been carried along the process of purification and PCR amplification to rule out false-positive results.
193
Table 3 Monitoring of a kidney transplant recipient for the detection of active cytomegalovirus infection by pp65 antigenemia assay and quantitative realtime PCR Analysis
Days after transplantation
CMV genomes per milliliter plasma
pp65 positive per 200,000 cells
1 2 3 4 5 6 7 8 9 10
39 49 52 56 63 67 71 77 84 98
9.5 × 105 8.8 × 105 1.5 × 105 1.5 × 104 6.4 × 102 2.5 × 102 Negative 2.3 × 102 Negative Negative
Not evaluable 7 2 Not evaluable Negative Negative Negative Negative Negative Negative
We have investigated 334 specimens in routine diagnostics with both assays for the detection of active cytomegalovirus infection. Thereby, all samples could be evaluated by real-time PCR, because inhibition of the PCR assays was never observed. In contrast, 268 of 334 samples could be evaluated using pp65 antigenemia assay, which corresponds to only 80.2% of the whole number of samples tested. Because in the other 66 samples, the number of remaining cells was well below 2 × 105, an interpretation of the assay was not possible. In 12 samples, the detection of active cytomegalovirus succeeded with the pp65 antigenemia assay, whereas the number of CMV-positive samples in real-time PCR was 41, suggesting superior sensitivity of the DNA amplification technique. The results are summarized in Table 1. In addition, every sample tested positive in the pp65 assay was
Table 2 Comparison of positive pp65 diagnostic findings with quantitative real-time PCR Sample no.
pp65 positive per 200,000 cells
CMV genomes per milliliter plasma
Sample no.
pp65 positive per 200,000 cells
CMV genomes per milliliter plasma
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
0 Not evaluable 0 0 Not evaluable 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 9
1.5 × 102 1.5 × 102 1.8 × 102 1.9 × 102 2.2 × 102 2.3 × 102 2.3 × 102 2.5 × 102 4.5 × 102 4.8 × 102 5.0 × 102 5.1 × 102 5.6 × 102 5.6 × 102 5.8 × 102 6.4 × 102 6.6 × 102 6.7 × 102 8.1 × 102 9.4 × 102 1.4 × 103
22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41
not evaluable 0 7 0 0 0 3 9 0 6 0 Not evaluable 3 Not evaluable 7 2 2 28 7 Not evaluable
1.4 × 103 2.1 × 103 2.2 × 103 3.0 × 103 3.0 × 103 3.4 × 103 3.6 × 103 4.3 × 103 5.8 × 103 7.7 × 103 1.2 × 104 1.5 × 104 1.8 × 104 2.8 × 104 4.8 × 104 5.3 × 104 1.5 × 105 2.9 × 105 8.8 × 105 9.5 × 105
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also positive in the PCR assay, whereas none of the PCR negative samples yielded a positive result with the immunofluorescence test for detection of pp65. On the other hand, in 6 samples that could not be evaluated by the pp65 antigenemia assay, we detected CMV DNA using the real-time PCR with a virus load of up to 9.5 × 105 genomes/mL plasma. A comparison of the positive pp65 diagnostic findings with PCR titers is given in Table 2. An example for a typical but deficient CMV monitoring of a kidney transplant recipient by the pp65 assay is given in Table 3. Altogether, 10 specimens from this patient have been tested by pp65 assay and real-time PCR. Thereby, pp65 was not evaluable in the first and the fourth analysis, whereas real-time PCR detected high CMV loads of 9.5 × 105 and 1.5 × 104 copies/mL plasma. Furthermore, the fifth, sixth, and eigth analyses detected a CMV titers of 6.4 × 102, 2.5 × 102, and 2.3 × 102, but were pp65 negative, indicating the inferior sensitivity of this assay. Compared with real-time PCR, the pp65 antigenemia assay is more labor intensive and time consuming. Isolation of granulocytes, several washing steps, and incubation with 1st and secondary antibody solutions requires approximately 3 h. In addition, evaluation, if possible, needs at least further 10 min for every slide. Furthermore, simultaneous preparation of more than 10 samples is not easy to perform. Finally, the preparation of solutions required for the pp65 antigenemia assay is time consuming. In contrast, preparation of DNA for subsequent real-time PCR with the column-based Qiagen system can be accomplished in less than 1 h, even with sample numbers of more than 10. The following real-time PCR needs 1 further hour so that the results can be achieved in not more than 2 h. Another advantage of real-time PCR is the fact that plasma samples can be stored at −20 °C, whereas blood samples for pp65 antigenemia assay should be processed within 2 h. In addition, only 80.2% of the samples tested could be evaluated performing the pp65 antigenemia assay, whereas in the remaining 19.8%, the number of cells was not sufficient. This result has to be judged as critical because, particularly in patients experiencing neutropenia, active cytomegalovirus infection cannot be diagnosed in a relevant number of cases. Furthermore, the PCR assay is easier to integrate into daily routine of virologic diagnostic laboratories without significant additional expenses because many samples are investigated for more than 1 virus by PCR analysis, and thus, DNA has to be purified anyway. Moreover, many PCR assays can be carried out with the same PCR protocol, for example, Artus PCR assays for CMV, Epstein-Barr virus, herpes simplex virus-1/2, and varicella zoster virus. In recent years, several studies comparing the pp65 antigenemia assay with real-time PCR have been published. Thereby, real-time PCR were shown to be more sensitive than immunofluorescence and can detect CMV earlier than the pp65 antigenemia test (Bordils et al., 2005; Hernando et al., 2005; Koetz et al., 2001; Meyer-König et al., 2004,
2006; Sanghavi et al., 2008). It could also been shown that real-time PCR is very helpful for diagnosing CMV-induced diseases and monitoring the virus load in patients with CMV infection (Tanaka et al., 2000). In addition, real-time PCR is qualified to guide preemptive therapy for CMV and the clinical management of transplant recipients (Gimeno et al., 2008; Kalpoe et al., 2004; Leruez-Ville et al., 2003; Mori et al., 2002; Ruell et al., 2007). Real-time PCR was also a more reliable marker monitoring the clearance of the virus from the blood (Gimeno et al., 2008). In summary, it could be demonstrated that real-time PCR is less time consuming, is less labor intensive, and, in contrast to the pp65 antigenemia assay, was evaluable in every sample that was analyzed. Therefore, considerations should be made to replace the pp65 antigenemia assay by real-time PCR. References Boeckh M, Huang M, Ferrenberg J, Steven-Ayers T, Stensland L, Nichols WG, Corey L (2004) Optimization of quantitative detection of cytomegalovirus DNA in plasma by real-time PCR. J Clin Microbiol 42:1142–1148. Bordils A, Plumed JS, Ramos D, Beneyto I, Mascaros V, Molina JM, Cordoba J, Garcia J, Cruiz JM (2005) Comparison of quantitative PCR and antigenemia in cytomegalovirus infection in renal transplant patients. Transplant Proc 37:3756–3759. Bøyum A (1968) Separation of leucocytes from blood and bone marrow. Scand J Clin Lab Invest 21(Suppl 97):109. Caliendo AM, Ingersoll J, Fox-Canale AM, Pargman S, Bythwood T, Hayden MK, Bremer JW, Lurain NS (2007) Evaluation of real-time PCR laboratory developed test using analyte-specific reagents for cytomegalovirus quantification. J Clin Microbiol 45:1723–1727. Gimeno C, Solano C, Latorre JC, Hernandez-Boluda JC, Clari MA, Remigia MJ, Furio S, Calabuig M, Tormo N, Navarro D (2008) Quantification of DNA in plasma by an automated real-time PCR assay (cytomegalovirus PCR kit) for surveillance of active cytomegalovirus infection and guidance of preemptive therapy for allogeneic hematopoietic stem cell transplant recipients. J Clin Microbiol 46:3311–3318. Gouarin S, Vabret A, Scieux C, Aghalika F, Cherot J, Mengelle C, Debaack C, Petitjean J, Dina J, Freymuth F (2007) Multicentric evaluation of a new commercial cytomegalovirus real-time PCR quantification assay. J Virol Methods 146:147–154. Hanson KE, Reller LB, Kutzberg J, Horwitz M, Long G, Alexander BD (2007) Comparison of the Digene hybrid capture system cytomegalovirus (CMV) DNA (version 2.0), Roche CMV UL54 analyte-specific reagent, and Qiagen RealArt CMV LightCycler PCR reagent test using AcroMetrix OptiQuant CMV DNA quantification panels and specimens from allogenic-stem-cell transplant recipients. J Clin Microbiol 45:1973. Herrman B, Larsson VC, Rubin CJ, Sund F, Eriksson BM, Arvidson J, Yun Z, Bonderson K, Blomberg J (2004) Comparison of a duplex real-time PCR assay and the COBAS Amplicor CMV monitor test for the detection of cytomegalovirus. J Clin Microbiol 42:1909–1914. Hernando S, Folgueira L, Lumbreras C, San Juan R, Malsonado S, Prieto C, Babiano MJ, Delgado J, Andres A, Moreno E, Aquado JM, Otero JR (2005) Comparison of cytomegalovirus viral load measure by real-time PCR with pp65 antigenemia for the diagnosis of cytomegalovirus disease in solid organ transplant patients. Transplant Proc 37:4094–4096. Hong KM, Najjar H, Hawley M, Press RD (2004) Quantitative real-time PCR with automated sample preparation for diagnosis and monitoring of cytomegalovirus infection in bone marrow transplant patients. Clin Chem 50:846–856. Kalpoe JS, Kroes ACM, de Jong MD, Shinkel J, de Brouwer CS, Beersma MFC, Claas CJ (2004) Validation of clinical application of cytomegalovirus
R. Lugert et al. / Diagnostic Microbiology and Infectious Disease 65 (2009) 192–195 plasma DNA load measurement and definition of treatment criteria by analysis of correlation to antigen detection. J Clin Microbiol 42:1498–1504. Koetz AC, Delbrück R, Furtwängler A, Hufert FT, Neumann-Haeflin D, Kirste G, Meyer-König U (2001) Cytomegalovirus pp65 antigen-guided preemptive therapy with ganciclovir in solid organ transplant recipients: a prospective, double-blind, placebo-controlled study. Transplantation 72:1325–1327. Leruez-Ville M, Ouachée M, Delarue R, Sauget AS, Blanche S, Buzyn A, Rouzioux C (2003) Monitoring cytomegalovirus infection in adult and pediatric bone marrow transplant recipients by a real-time PCR assay performed with blood plasma. J Clin Microbiol 41:2040–2046. Meyer-König U, Weidmann M, Kirste G, Hufert FT (2004) Cytomegalovirus infection in organ-transplant recipients: diagnostic value of pp65 antigen test, qualitative polymerase chain reaction (PCR) and quantitative Taqman PCR. Transplantation 77:1692–1698. Meyer-König U, Romberg I, Schneider K, Weidmann M, Kern WV, Hufert FT (2006) Diagnostic value of reverse transcription-PCR for detection of cytomegalovirus pp67 in samples from solid-organ transplant recipients. J Clin Microbiol 44:3394–3396. Mori T, Okamoto S, Watanabe R, Yajima T, Iwao Y, Yamazaki R, Nakazato T, Sato N, Iguchi T, Nagayama H, Hibi T, Ikeda Y (2002) Dose-adjusted preemptive therapy for cytomegalovirus disease based on real-time
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polymerase chain reaction after allogeneic stem cell transplantation. Bone Marrow Transplant 29:777–782. Pumannova M, Rouibalova K, Vitek A, Saidova J (2006) Comparison of quantitative competitive polymerase chain reaction-enzyme linked immunosorbent assay with LightCycler-based polymerase chain reaction for measuring cytomegalovirus DNA in patients after hematopoietic stem cell transplantation. Diagn Microbiol Infect Dis 54:115–120. Ruell J, Barnes C, Mutton K, Foulkes B, Chang J, Cavet J, Guiver M, Menasce I, Dougal M, Chopra R (2007) Active CMV disease does not always correlate with viral load detection. Bone Marrow Transplant 40:55–61. Sanghavi SK, Abu-Elmagd K, Keightley MC, St. George K, Lewandowski K, Boes SS, Bullotta A, Dare R, Lassak M, Husain S, Kwak EJ, Paterson DL, Rinaldo CR (2008) Relationship of cytomegalovirus load assessed by real-time PCR to pp65 antigenemia in organ transplant recipients. J Clin Virol 42:335–342. Tanaka N, Kimura H, Iida K, Saito Y, Tsuge I, Yoshimi A, Matsuyama T, Morishima T (2000) Quantitative analysis of cytomegalovirus load using a real-time PCR assay. J Med Virol 60:455–462. 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 Microbiol 38:298–303.
Diagnostic Microbiology and Infectious Disease 65 (2009) 192 – 195 www.elsevier.com/locate/diagmicrobio
Notes
Comparison between pp65 antigenemia assay and quantitative real-time polymerase chain reaction for detection of active cytomegalovirus infection in routine diagnostics Raimond Lugerta,⁎, Utz Reicharda , Frank T. Hufertb , Uwe Großa a
Institute for Medical Microbiology, University Medical Center Göttingen, 37075 Göttingen, Germany b Institute of Virology, University Medical Center Göttingen, 37075 Göttingen, Germany Received 6 March 2009; accepted 28 May 2009
Abstract Real-time polymerase chain reaction (PCR) and pp65 antigenemia assay for the detection of active cytomegalovirus infection in immunocompromised patients experiencing neutropenia after bone marrow or kidney transplantation have been compared with a special focus on evaluability and embedment in daily routine diagnostics. Investigating 334 specimens from 97 patients, real-time PCR was shown to be the superior assay with regard to the parameters focused on. © 2009 Elsevier Inc. All rights reserved. Keywords: pp65 antigenemia assay; Quantitative real-time PCR; Cytomegalovirus
Testing of blood samples from immunocompromised patients for the presence of active cytomegalovirus infection is routinely performed in virologic diagnostic laboratories. Thereby, the pp65 antigenemia assay is still considered as the “gold standard” in many laboratories, although it is labor intensive, is subjective in reading, and may not be evaluable in case of neutropenia. As an alternative, quantitative realtime polymerase chain reaction (PCR) assays, either in-house or commercial, have emerged to the pp65 assay in recent years (Boeckh et al., 2004; Gouarin et al., 2007; Hanson et al., 2007; Herrman et al., 2004; Hong et al., 2004; Kalpoe et al., 2004; Pumannova et al., 2006; Yerly et al., 2007). We have routinely investigated 334 specimens comparing both assays with special regard to evaluability and the aim to embed these assays in daily laboratory diagnostics. The specimens were derived from 95 bone marrow and 2 kidney transplanted outpatients (63 male, 34 female) from the Department of Haematology and Oncology or inpatients from the Department of Nephrology and Rheumatology of the University Medical Center Göttingen, Göttingen, Germany.
⁎ Corresponding author. Tel.: +49-551-395869; fax: +49-551-395861. E-mail address: [email protected] (R. Lugert). 0732-8893/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2009.05.016
Thereby, 50 patients have been investigated only once, whereas 47 patients have been tested up to 19 times in periodic intervals for the detection of active cytomegalovirus infection. For pp65 antigenemia assays, 2-mL Lymphoprep (AxisShield Poc, Oslo, Norway) was overlaid with 5 mL of EDTA blood, and the samples were centrifuged at 300 × g for 10 min (Bøyum, 1968). Supernatants were removed and the pellets were resuspended with 10 mL of Gey's solution and incubated on ice for 10 min for lysis of red blood cells. After centrifugation, resuspension and incubation in Gey's solution were repeated twice. Purified granulocytes were resuspended and washed in 1-mL phosphate-buffered saline (PBS). Finally, the sediments were resuspended in 0.2-mL PBS and spotted onto a glass slide by using cytocentrifuge (500×g for 3 min). The slides were air-dried and fixed in PBS, containing 2% sucrose and 5% formaldehyde, for 5 min. After the slides were washed in PBS and air-dried, cells were incubated with 10 μL of VIR–cytomegalovirus (CMV) C10/ C11 antibody solution (IQ Products, Groningen, the Netherlands) for 30 min at 37 °C. Subsequently, the slides were washed twice with PBS and incubated with 10-μL VIR– fluorescin isothiocyanate secondary antibody solution (IQ Products) for further 30 min at 37 °C. Results were reported as the number of pp65-positive cells per 2 × 105 granulocytes.
R. Lugert et al. / Diagnostic Microbiology and Infectious Disease 65 (2009) 192–195 Table 1 Number of evaluable and CMV-positive blood samples of all 334 samples tested using quantitative real-time PCR and pp65 antigenemia assay Assay
Evaluable
Quantitative real-time PCR pp65 antigenemia assay
Positive
Number
%
Number
%
334/334 268/334
100 80.2
41/334 12/334
12 3.5
Recent data demonstrated that both whole blood and plasma are equally suitable for the detection of active CMV infection in recipients of allogenic hematopoietic stem cells (Caliendo et al., 2007; Kalpoe et al., 2004; Leruez-Ville et al., 2003). For real-time PCR, DNA was extracted from 200 μL of plasma using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Purified DNA was eluted in a volume of 200 μL, and 10 μL were applied for quantitative real-time PCR using the Artus CMV LC PCR Kit (Qiagen), which is licensed for diagnostic use. The CMV LC PCR Kit contains enzymes and reagents for the amplification and detection of a 105-bp fragment of the CMV major immediate early gene. In addition, external positive controls ranging from 10 to 104 CMV copies and an internal heterologous amplification system are supplied to determine the pathogen load and to identify possible PCR inhibition. The specificity of the PCR assay is assured by an oligonucleotide probe, which binds exclusively to the amplified product. Water controls have been carried along the process of purification and PCR amplification to rule out false-positive results.
193
Table 3 Monitoring of a kidney transplant recipient for the detection of active cytomegalovirus infection by pp65 antigenemia assay and quantitative realtime PCR Analysis
Days after transplantation
CMV genomes per milliliter plasma
pp65 positive per 200,000 cells
1 2 3 4 5 6 7 8 9 10
39 49 52 56 63 67 71 77 84 98
9.5 × 105 8.8 × 105 1.5 × 105 1.5 × 104 6.4 × 102 2.5 × 102 Negative 2.3 × 102 Negative Negative
Not evaluable 7 2 Not evaluable Negative Negative Negative Negative Negative Negative
We have investigated 334 specimens in routine diagnostics with both assays for the detection of active cytomegalovirus infection. Thereby, all samples could be evaluated by real-time PCR, because inhibition of the PCR assays was never observed. In contrast, 268 of 334 samples could be evaluated using pp65 antigenemia assay, which corresponds to only 80.2% of the whole number of samples tested. Because in the other 66 samples, the number of remaining cells was well below 2 × 105, an interpretation of the assay was not possible. In 12 samples, the detection of active cytomegalovirus succeeded with the pp65 antigenemia assay, whereas the number of CMV-positive samples in real-time PCR was 41, suggesting superior sensitivity of the DNA amplification technique. The results are summarized in Table 1. In addition, every sample tested positive in the pp65 assay was
Table 2 Comparison of positive pp65 diagnostic findings with quantitative real-time PCR Sample no.
pp65 positive per 200,000 cells
CMV genomes per milliliter plasma
Sample no.
pp65 positive per 200,000 cells
CMV genomes per milliliter plasma
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
0 Not evaluable 0 0 Not evaluable 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 9
1.5 × 102 1.5 × 102 1.8 × 102 1.9 × 102 2.2 × 102 2.3 × 102 2.3 × 102 2.5 × 102 4.5 × 102 4.8 × 102 5.0 × 102 5.1 × 102 5.6 × 102 5.6 × 102 5.8 × 102 6.4 × 102 6.6 × 102 6.7 × 102 8.1 × 102 9.4 × 102 1.4 × 103
22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41
not evaluable 0 7 0 0 0 3 9 0 6 0 Not evaluable 3 Not evaluable 7 2 2 28 7 Not evaluable
1.4 × 103 2.1 × 103 2.2 × 103 3.0 × 103 3.0 × 103 3.4 × 103 3.6 × 103 4.3 × 103 5.8 × 103 7.7 × 103 1.2 × 104 1.5 × 104 1.8 × 104 2.8 × 104 4.8 × 104 5.3 × 104 1.5 × 105 2.9 × 105 8.8 × 105 9.5 × 105
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also positive in the PCR assay, whereas none of the PCR negative samples yielded a positive result with the immunofluorescence test for detection of pp65. On the other hand, in 6 samples that could not be evaluated by the pp65 antigenemia assay, we detected CMV DNA using the real-time PCR with a virus load of up to 9.5 × 105 genomes/mL plasma. A comparison of the positive pp65 diagnostic findings with PCR titers is given in Table 2. An example for a typical but deficient CMV monitoring of a kidney transplant recipient by the pp65 assay is given in Table 3. Altogether, 10 specimens from this patient have been tested by pp65 assay and real-time PCR. Thereby, pp65 was not evaluable in the first and the fourth analysis, whereas real-time PCR detected high CMV loads of 9.5 × 105 and 1.5 × 104 copies/mL plasma. Furthermore, the fifth, sixth, and eigth analyses detected a CMV titers of 6.4 × 102, 2.5 × 102, and 2.3 × 102, but were pp65 negative, indicating the inferior sensitivity of this assay. Compared with real-time PCR, the pp65 antigenemia assay is more labor intensive and time consuming. Isolation of granulocytes, several washing steps, and incubation with 1st and secondary antibody solutions requires approximately 3 h. In addition, evaluation, if possible, needs at least further 10 min for every slide. Furthermore, simultaneous preparation of more than 10 samples is not easy to perform. Finally, the preparation of solutions required for the pp65 antigenemia assay is time consuming. In contrast, preparation of DNA for subsequent real-time PCR with the column-based Qiagen system can be accomplished in less than 1 h, even with sample numbers of more than 10. The following real-time PCR needs 1 further hour so that the results can be achieved in not more than 2 h. Another advantage of real-time PCR is the fact that plasma samples can be stored at −20 °C, whereas blood samples for pp65 antigenemia assay should be processed within 2 h. In addition, only 80.2% of the samples tested could be evaluated performing the pp65 antigenemia assay, whereas in the remaining 19.8%, the number of cells was not sufficient. This result has to be judged as critical because, particularly in patients experiencing neutropenia, active cytomegalovirus infection cannot be diagnosed in a relevant number of cases. Furthermore, the PCR assay is easier to integrate into daily routine of virologic diagnostic laboratories without significant additional expenses because many samples are investigated for more than 1 virus by PCR analysis, and thus, DNA has to be purified anyway. Moreover, many PCR assays can be carried out with the same PCR protocol, for example, Artus PCR assays for CMV, Epstein-Barr virus, herpes simplex virus-1/2, and varicella zoster virus. In recent years, several studies comparing the pp65 antigenemia assay with real-time PCR have been published. Thereby, real-time PCR were shown to be more sensitive than immunofluorescence and can detect CMV earlier than the pp65 antigenemia test (Bordils et al., 2005; Hernando et al., 2005; Koetz et al., 2001; Meyer-König et al., 2004,
2006; Sanghavi et al., 2008). It could also been shown that real-time PCR is very helpful for diagnosing CMV-induced diseases and monitoring the virus load in patients with CMV infection (Tanaka et al., 2000). In addition, real-time PCR is qualified to guide preemptive therapy for CMV and the clinical management of transplant recipients (Gimeno et al., 2008; Kalpoe et al., 2004; Leruez-Ville et al., 2003; Mori et al., 2002; Ruell et al., 2007). Real-time PCR was also a more reliable marker monitoring the clearance of the virus from the blood (Gimeno et al., 2008). In summary, it could be demonstrated that real-time PCR is less time consuming, is less labor intensive, and, in contrast to the pp65 antigenemia assay, was evaluable in every sample that was analyzed. Therefore, considerations should be made to replace the pp65 antigenemia assay by real-time PCR. References Boeckh M, Huang M, Ferrenberg J, Steven-Ayers T, Stensland L, Nichols WG, Corey L (2004) Optimization of quantitative detection of cytomegalovirus DNA in plasma by real-time PCR. J Clin Microbiol 42:1142–1148. Bordils A, Plumed JS, Ramos D, Beneyto I, Mascaros V, Molina JM, Cordoba J, Garcia J, Cruiz JM (2005) Comparison of quantitative PCR and antigenemia in cytomegalovirus infection in renal transplant patients. Transplant Proc 37:3756–3759. Bøyum A (1968) Separation of leucocytes from blood and bone marrow. Scand J Clin Lab Invest 21(Suppl 97):109. Caliendo AM, Ingersoll J, Fox-Canale AM, Pargman S, Bythwood T, Hayden MK, Bremer JW, Lurain NS (2007) Evaluation of real-time PCR laboratory developed test using analyte-specific reagents for cytomegalovirus quantification. J Clin Microbiol 45:1723–1727. Gimeno C, Solano C, Latorre JC, Hernandez-Boluda JC, Clari MA, Remigia MJ, Furio S, Calabuig M, Tormo N, Navarro D (2008) Quantification of DNA in plasma by an automated real-time PCR assay (cytomegalovirus PCR kit) for surveillance of active cytomegalovirus infection and guidance of preemptive therapy for allogeneic hematopoietic stem cell transplant recipients. J Clin Microbiol 46:3311–3318. Gouarin S, Vabret A, Scieux C, Aghalika F, Cherot J, Mengelle C, Debaack C, Petitjean J, Dina J, Freymuth F (2007) Multicentric evaluation of a new commercial cytomegalovirus real-time PCR quantification assay. J Virol Methods 146:147–154. Hanson KE, Reller LB, Kutzberg J, Horwitz M, Long G, Alexander BD (2007) Comparison of the Digene hybrid capture system cytomegalovirus (CMV) DNA (version 2.0), Roche CMV UL54 analyte-specific reagent, and Qiagen RealArt CMV LightCycler PCR reagent test using AcroMetrix OptiQuant CMV DNA quantification panels and specimens from allogenic-stem-cell transplant recipients. J Clin Microbiol 45:1973. Herrman B, Larsson VC, Rubin CJ, Sund F, Eriksson BM, Arvidson J, Yun Z, Bonderson K, Blomberg J (2004) Comparison of a duplex real-time PCR assay and the COBAS Amplicor CMV monitor test for the detection of cytomegalovirus. J Clin Microbiol 42:1909–1914. Hernando S, Folgueira L, Lumbreras C, San Juan R, Malsonado S, Prieto C, Babiano MJ, Delgado J, Andres A, Moreno E, Aquado JM, Otero JR (2005) Comparison of cytomegalovirus viral load measure by real-time PCR with pp65 antigenemia for the diagnosis of cytomegalovirus disease in solid organ transplant patients. Transplant Proc 37:4094–4096. Hong KM, Najjar H, Hawley M, Press RD (2004) Quantitative real-time PCR with automated sample preparation for diagnosis and monitoring of cytomegalovirus infection in bone marrow transplant patients. Clin Chem 50:846–856. Kalpoe JS, Kroes ACM, de Jong MD, Shinkel J, de Brouwer CS, Beersma MFC, Claas CJ (2004) Validation of clinical application of cytomegalovirus
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