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Real-Time PCR Assay Compared With Antigenemia Assay for Detecting Cytomegalovirus Infection in Kidney Transplant Recipients
Real-Time PCR Assay Compared With Antigenemia Assay for Detecting Cytomegalovirus Infection in Kidney Transplant Recipients D.J. Kim, S.J. Kim, J. Park, G.S. Choi, S. Lee, C.D. Kwon, C. Ki, and J. Joh ABSTRACT Human cytomegalovirus (CMV) infection is a major cause of morbidity and mortality among kidney transplant recipients. The CMVpp65 antigenemia assay has been used for preemptive therapy. Real-time polymerase chain reaction (PCR) technology for CMV DNA quantification in blood has demonstrated a good correlation with the currently employed CMV antigenemia assay. In this study, 90 renal transplant recipients were prospectively enrolled from July 2004 and May 2005. Monitoring of CMV infection was routinely performed with CMV antigenemia and real-time PCR assays. Real-time plasma PCR and CMV antigenemia assays were assessed on 797 samples. CMV antigenemia correlated with a positive CMV PCR (2 ⫽ 78.05; P ⬍ .0001). Not only the positive rate but also the number of positive cells correlated with the number of PCR DNA copies (F ⫽ 26.07, r2 ⫽ .25, P ⬍ .0001). To define an optimal cutoff value of CMV DNA load to initiate treatment in kidney transplant patients, we considered a CMV antigenemia titer of ⬎50 positive cells per 400,000 leukocytes as the gold standard in our previous study. The optimal cutoff value for the quantitative real-time PCR assay was predicted to be 86 copies/L. Thus, we observed that CMV real-time PCR assay would not completely replace antigenemia assay in kidney transplant recipients, but can be used complementarily to screen antigenemia and monitor preemptive therapy.
H
UMAN CYTOMEGALOVIRUS (CMV) infection is a major cause of morbidity and mortality among kidney transplant recipients. The success of therapy depends on the availability of sensitive, specific diagnostic tests for early detection of CMV infections. The CMV antigenemia assay has been used for pre-emptive therapy with considerable success.1 However, this disadvantages of the CMV antigenemia test are that it is labor intensive, time consuming, and requires processing of samples within a few hours. Real-time polymerase chain reaction (PCR) technology is simple, reliable, and cost effective.2 The results of these assays in CMV DNA quantification in blood have demonstrated a good correlation with those of the currently widely employed CMV antigenemia assay.3 In a previous study, we proposed that the CMV antigenemia titer can be used as a useful clinical guide line for preemptive treatment of HCMV infection after kidney transplantation.4 In this study, we compared the results of the two methods to define guidelines for preemptive treatment based on real-time PCR assays in kidney transplantation recipients. We also tried to determine the possibility of replacement of the CMV antigenemia assay.
PATIENTS AND METHODS A total of 90 renal transplant recipients were enrolled from July 2004 and May 2005. During hospitalization, all patients were monitored weekly for CMV infection by routine diagnostic testing: pp65antigenmia and quantitative real-time PCR assays. Out-patient follow-up was performed biweekly or monthly. HCMV Antigenemia Assay was performed using a commercially available monoclonal antibody against HCMV pp65 (Clonab CMV; Biotest Diagnostic Corporation, Denville, NJ). The results were expressed as the number of HCMV antigen-positive white blood cells per 400,000 cells. HCMV real-time PCR assay was performed on the LightCycler instrument (Roche Diagnostics, GmbH) with the RealArtTM CMV LC PCR Kit (Artus-Biotechs, Germany).
From the Department of Surgery, Transplant Division Sungkyunkwan University, Seoul, Republic of Korea. Address reprint requests to Dr. S.-J. Kim, Department of Surgery, Transplant Division, Sungkyunkwan University, #50 Ilwon Dong Kangnam Ku, Seoul 135-710, Republic of Korea.
0041-1345/07/$–see front matter doi:10.1016/j.transproceed.2007.01.088
© 2007 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
1458
Transplantation Proceedings, 39, 1458 –1460 (2007)
DETECTING CMV INFECTION
1459
Table 1. Patients Characteristics Characteristics
Age (yr) Median Range Gender (M/F) Cadaveric/living Immunosuppresant Cyclosporine A FK 506 Mycophenolate Prednisolone Antithymocyte golbulin HCMV IgG serostatus of donor/recipients D(⫹)/R(⫹) D(⫺)/R(⫹) D(⫺)/R(⫺) D(⫹)/R(⫺) Antignemia(⫹) at least once PCR(⫹) at least once Ganciclovir administration
No. of Patients (%)
42 15–65 51/39 (56.6/43.3) 22/68 (24.4/75.5) 57 (63.3) 33 (36.6) 90 (100) 90 (100) 10 (11.1) 82 (91.1) 5 (5.5) 1 (1.1) 2 (2.2) 32 (35.5) 60 (66.6) 14 (15.5)
Preemptive Therapy for Prevention of CMV Disease The decision to prescribe preemptive therapy was based on a positive antigenemia test (ⱖ50 antigen-positive cell/400,000 cells) or a diagnosis of CMV disease- or CMV infection-related symptoms. In this study, PCR results were not used to make decisions about preemptive therapy. Preemptive treatment involved intravenous infusion of ganciclovir (5 mg/kg every 12 hours for 1 to 3 weeks).
Statistical Analysis Mixed model and generalized estimating equations were used to assess the association between continuous variables, the number of antigen-positive cells, and level of CMV DNA in whole blood and in plasma. SAS version 8.2 was used to analyze the data. A P value of ⬍ .05 was accepted as statistically significant.
Comparisons of the CMV Antigenemia Assay With the CMV Real-Time PCR in Blood Plasma
Plasma real-time PCR and CMV antigenemia assay were assessed on 797 samples. While 605 samples were negative in both assays, 54 were positive in both tests. The sensitivity of the CMV antigenemia assay was much lower; 76 of 797 were antigenemia positive and 170 of 797 were PCR positive. One hundred sixteen samples were CMV PCR positive and CMV antigenemia negative; 22 samples were CMV PCR test negative, but CMV antigenemia assay positive. The results correlated significantly for the two diagnostic methods among the 797 samples. Among the 170 PCR-positive samples, the number of CMV DNA copies was between 0.5 and 1679 (mean value ⫽ 65.6 copies/L). Among the 76 CMV antigenemia-positive samples, the number of pp65-positive cells was between 2 and 248 (mean value ⫽ 22.7 positive cells/400,000 cells). A positive CMV antigenemia correlated with a positive CMV PCR (2 ⫽ 78.05; P ⬍ .0001 by generalized estimating equation). Not only the positive rate but also the numbers of positive cells correlated with the titer of PCR DNA copies (F value ⫽ 26.07; P ⬍ .0001 by mixed model; (Fig 1). Determination of CMV DNA Loads Threshold Values for Preemptive Therapy
As the CMV antigenemia assay has been used to guide CMV therapy in current clinical practice, it is important to establish the corresponding threshold for the CMV PCR assay. Our previous study4 suggested that CMV antigenemia assy of ⬎50 positive cells per 400,000 leukocytes warranteed preemptive treatment with ganciclovir. To define an optimal cutoff value of CMV DNA load to initiate treatment in kidney transplant patients, we considered and CMV antigenemia titer of ⬎50 positive cells per 400,000 leukocytes as the gold standard. The optimal cutoff value of quantitative real-time PCR assay was predicted to be 86 copies/L (P ⬍ .0001 by Bonferroni’s correction). If subse-
RESULTS Patients Characteristics
The characteristics of the 90 enrolled, consecutive kidney transplantation recipients are shown in Table 1. They were followed for a median of 26 weeks (range, 10 to 45), and a mean of 8.8 (range, 1 to 15) samples were analyzed per patient. Among 797 collected and tested samples HCMV infection was defined by a positive antigenemia assay in 32 patients (35.5%), and 60 patients were CMV PCR positive at least once. In 29 cases, both CMV PCR and CMV antigenemia assays were positive. Ganciclovir was administered to 14 patients (15.5%). We prescribed prophylactic treatment in 8, preemptive treatment in 5, according to our therapeutic guidelines, and CMV-related symptoms fever and hematologic abnormality in 1 patient. There was no organ-specific disease. All cases were treated successfully, without a mortality.
Fig 1. The correlation of CMV antigenemia assay and real-time PCR assay. Antigenemia assay (positive cells/400,000WBC), PCR assay (copies/uL). F Value ⫽ 26.07, P ⬍ 0.0001 by Mixed Model, R2 ⫽ 0.25, P ⬍ 0.001 by Pearson correlation Preemptive level of antigenemia (⬎ 50 predicted by real time PCR 86 copies/uL. P ⬍ 0.0001 by Bonferroni’s correction).
1460
quently a CMV DNA level of 86 copies/L was used as a threshold to initiate preemptive treatment with a CMV antigenemia assay of ⱖ50 antigen-positive cell/400,000 cells, the sensitivity and specificity were 62.5% and 97.4% respectively.
DISCUSSION
CMV diagnostic tests still lack standardization. There is no gold standard test to detect CMV disease. The antigenemia assay is still popular to monitor CMV infection. Therefore, every center has to determine its own clinically relevant cutoff value based on the antigenemia results.5 In this study, the evaluation was performed by comparing the quantitative real-time PCR assay of blood plasma with the CMV antigenemia assay as the reference method. Our study of kidney transplant patients also revealed a correlation between the number of antigen-positive cells and CMV DNA load in plasma (Fig 1). However, the relationship (r2 ⫽ 0.25; P ⬍ .001 by Pearson correlation) was relatively lower in our study than in other studies.6,7 Apart from these results, the number of positive PCR assays was greater than that of antigenemia tests, suggesting that the PCR assay is more sensitive. Dispite a wide discrepancy in the small numbers of samples, the results were generally well-correlated. Because the PCR assay was not positive in all specimens that were positive by antigenemia, there was a theoretical risk of missing some cases of progressive antigenemia. But, among our cases there were only three cases of negative serial PCR assays among 32 antigenemia-positive patients. If we apply our center’s guidelines from the antigenemia assay, the specificity of the PCR assay was high (97.3%) and all patients (n ⫽ 5) who needed preemptive treatment were PCR assay positive.
KIM, KIM, PARK ET AL
In conclusion, we compared the real-time PCR assay and antigenemia, deciding preemptive therapy on the basis of antigenemia. Moreover, only five patients received preemptive therapy. So the PCR assay seemed to lack clinical relevance. However, the PCR assay generally correlated with antigenemia and was more sensitive than antigenemia assay. We hypothesized that the CMV real-time PCR assay could not completely replace the antigenemia assay in kidney transplant recipients, but can be used complementarily to the antigenemia screen to monitor preemptive therapy, especially if examined serially. The PCR assay has some practical advantages, such as easy and rapid interpretation of the results, cost effectiveness, and reproducibility. REFERENCES 1. Kusne S, Grossi P, Irish W, et al: Cytomegalovirus pp65 antigenemia monitoring as a guide for preemptive therapy: a cost effective strategy for prevention of cytomegalo disease in adult liver transplant recipients. Transplantation 68:1125, 1999 2. Heid CA, Stevens J, Livak KJ, et al: Real-time quantitative PCR. Genome Res 6:986, 1996 3. Griscelli F, Barrois M, Chauvin S, et al: Quantification of human cytomegalovirus DNA in bone marrow transplant recipients by real-time PCR. J Clin Microbiol 39:4362, 2001 4. Kim CK, Song JH, Kim SM, et al: Clinical usefulness of human cytomegalovirus antigenemia assay after kidney transplantation. Transplantation 75:2151, 2003 5. Ursula MK, Manfred W, Gunter K, et al: Cytomegalovirus infection in organ-transplant recipients: diagnostic value of pp65 antigentest, qualitative polymerase chain reaction (PCR) and quantitative Taqman PCR. Transplantation 77:1692, 2004 6. Li H, Dummer JS, Estes WR, et al: Measurement of human cytomegalovirus load by quantitative real-time PCR for monitoring clinical intervention in transplant recipients. J Clin Microbiol 41:187, 2003 7. Boeckh M, Huang M, Ferrenberg J, et al: Optimization of quantitative detection of cytomegalovirus DNA in plasma by real-time PCR. J Clin Microbiol 42:1141, 2004
H
UMAN CYTOMEGALOVIRUS (CMV) infection is a major cause of morbidity and mortality among kidney transplant recipients. The success of therapy depends on the availability of sensitive, specific diagnostic tests for early detection of CMV infections. The CMV antigenemia assay has been used for pre-emptive therapy with considerable success.1 However, this disadvantages of the CMV antigenemia test are that it is labor intensive, time consuming, and requires processing of samples within a few hours. Real-time polymerase chain reaction (PCR) technology is simple, reliable, and cost effective.2 The results of these assays in CMV DNA quantification in blood have demonstrated a good correlation with those of the currently widely employed CMV antigenemia assay.3 In a previous study, we proposed that the CMV antigenemia titer can be used as a useful clinical guide line for preemptive treatment of HCMV infection after kidney transplantation.4 In this study, we compared the results of the two methods to define guidelines for preemptive treatment based on real-time PCR assays in kidney transplantation recipients. We also tried to determine the possibility of replacement of the CMV antigenemia assay.
PATIENTS AND METHODS A total of 90 renal transplant recipients were enrolled from July 2004 and May 2005. During hospitalization, all patients were monitored weekly for CMV infection by routine diagnostic testing: pp65antigenmia and quantitative real-time PCR assays. Out-patient follow-up was performed biweekly or monthly. HCMV Antigenemia Assay was performed using a commercially available monoclonal antibody against HCMV pp65 (Clonab CMV; Biotest Diagnostic Corporation, Denville, NJ). The results were expressed as the number of HCMV antigen-positive white blood cells per 400,000 cells. HCMV real-time PCR assay was performed on the LightCycler instrument (Roche Diagnostics, GmbH) with the RealArtTM CMV LC PCR Kit (Artus-Biotechs, Germany).
From the Department of Surgery, Transplant Division Sungkyunkwan University, Seoul, Republic of Korea. Address reprint requests to Dr. S.-J. Kim, Department of Surgery, Transplant Division, Sungkyunkwan University, #50 Ilwon Dong Kangnam Ku, Seoul 135-710, Republic of Korea.
0041-1345/07/$–see front matter doi:10.1016/j.transproceed.2007.01.088
© 2007 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
1458
Transplantation Proceedings, 39, 1458 –1460 (2007)
DETECTING CMV INFECTION
1459
Table 1. Patients Characteristics Characteristics
Age (yr) Median Range Gender (M/F) Cadaveric/living Immunosuppresant Cyclosporine A FK 506 Mycophenolate Prednisolone Antithymocyte golbulin HCMV IgG serostatus of donor/recipients D(⫹)/R(⫹) D(⫺)/R(⫹) D(⫺)/R(⫺) D(⫹)/R(⫺) Antignemia(⫹) at least once PCR(⫹) at least once Ganciclovir administration
No. of Patients (%)
42 15–65 51/39 (56.6/43.3) 22/68 (24.4/75.5) 57 (63.3) 33 (36.6) 90 (100) 90 (100) 10 (11.1) 82 (91.1) 5 (5.5) 1 (1.1) 2 (2.2) 32 (35.5) 60 (66.6) 14 (15.5)
Preemptive Therapy for Prevention of CMV Disease The decision to prescribe preemptive therapy was based on a positive antigenemia test (ⱖ50 antigen-positive cell/400,000 cells) or a diagnosis of CMV disease- or CMV infection-related symptoms. In this study, PCR results were not used to make decisions about preemptive therapy. Preemptive treatment involved intravenous infusion of ganciclovir (5 mg/kg every 12 hours for 1 to 3 weeks).
Statistical Analysis Mixed model and generalized estimating equations were used to assess the association between continuous variables, the number of antigen-positive cells, and level of CMV DNA in whole blood and in plasma. SAS version 8.2 was used to analyze the data. A P value of ⬍ .05 was accepted as statistically significant.
Comparisons of the CMV Antigenemia Assay With the CMV Real-Time PCR in Blood Plasma
Plasma real-time PCR and CMV antigenemia assay were assessed on 797 samples. While 605 samples were negative in both assays, 54 were positive in both tests. The sensitivity of the CMV antigenemia assay was much lower; 76 of 797 were antigenemia positive and 170 of 797 were PCR positive. One hundred sixteen samples were CMV PCR positive and CMV antigenemia negative; 22 samples were CMV PCR test negative, but CMV antigenemia assay positive. The results correlated significantly for the two diagnostic methods among the 797 samples. Among the 170 PCR-positive samples, the number of CMV DNA copies was between 0.5 and 1679 (mean value ⫽ 65.6 copies/L). Among the 76 CMV antigenemia-positive samples, the number of pp65-positive cells was between 2 and 248 (mean value ⫽ 22.7 positive cells/400,000 cells). A positive CMV antigenemia correlated with a positive CMV PCR (2 ⫽ 78.05; P ⬍ .0001 by generalized estimating equation). Not only the positive rate but also the numbers of positive cells correlated with the titer of PCR DNA copies (F value ⫽ 26.07; P ⬍ .0001 by mixed model; (Fig 1). Determination of CMV DNA Loads Threshold Values for Preemptive Therapy
As the CMV antigenemia assay has been used to guide CMV therapy in current clinical practice, it is important to establish the corresponding threshold for the CMV PCR assay. Our previous study4 suggested that CMV antigenemia assy of ⬎50 positive cells per 400,000 leukocytes warranteed preemptive treatment with ganciclovir. To define an optimal cutoff value of CMV DNA load to initiate treatment in kidney transplant patients, we considered and CMV antigenemia titer of ⬎50 positive cells per 400,000 leukocytes as the gold standard. The optimal cutoff value of quantitative real-time PCR assay was predicted to be 86 copies/L (P ⬍ .0001 by Bonferroni’s correction). If subse-
RESULTS Patients Characteristics
The characteristics of the 90 enrolled, consecutive kidney transplantation recipients are shown in Table 1. They were followed for a median of 26 weeks (range, 10 to 45), and a mean of 8.8 (range, 1 to 15) samples were analyzed per patient. Among 797 collected and tested samples HCMV infection was defined by a positive antigenemia assay in 32 patients (35.5%), and 60 patients were CMV PCR positive at least once. In 29 cases, both CMV PCR and CMV antigenemia assays were positive. Ganciclovir was administered to 14 patients (15.5%). We prescribed prophylactic treatment in 8, preemptive treatment in 5, according to our therapeutic guidelines, and CMV-related symptoms fever and hematologic abnormality in 1 patient. There was no organ-specific disease. All cases were treated successfully, without a mortality.
Fig 1. The correlation of CMV antigenemia assay and real-time PCR assay. Antigenemia assay (positive cells/400,000WBC), PCR assay (copies/uL). F Value ⫽ 26.07, P ⬍ 0.0001 by Mixed Model, R2 ⫽ 0.25, P ⬍ 0.001 by Pearson correlation Preemptive level of antigenemia (⬎ 50 predicted by real time PCR 86 copies/uL. P ⬍ 0.0001 by Bonferroni’s correction).
1460
quently a CMV DNA level of 86 copies/L was used as a threshold to initiate preemptive treatment with a CMV antigenemia assay of ⱖ50 antigen-positive cell/400,000 cells, the sensitivity and specificity were 62.5% and 97.4% respectively.
DISCUSSION
CMV diagnostic tests still lack standardization. There is no gold standard test to detect CMV disease. The antigenemia assay is still popular to monitor CMV infection. Therefore, every center has to determine its own clinically relevant cutoff value based on the antigenemia results.5 In this study, the evaluation was performed by comparing the quantitative real-time PCR assay of blood plasma with the CMV antigenemia assay as the reference method. Our study of kidney transplant patients also revealed a correlation between the number of antigen-positive cells and CMV DNA load in plasma (Fig 1). However, the relationship (r2 ⫽ 0.25; P ⬍ .001 by Pearson correlation) was relatively lower in our study than in other studies.6,7 Apart from these results, the number of positive PCR assays was greater than that of antigenemia tests, suggesting that the PCR assay is more sensitive. Dispite a wide discrepancy in the small numbers of samples, the results were generally well-correlated. Because the PCR assay was not positive in all specimens that were positive by antigenemia, there was a theoretical risk of missing some cases of progressive antigenemia. But, among our cases there were only three cases of negative serial PCR assays among 32 antigenemia-positive patients. If we apply our center’s guidelines from the antigenemia assay, the specificity of the PCR assay was high (97.3%) and all patients (n ⫽ 5) who needed preemptive treatment were PCR assay positive.
KIM, KIM, PARK ET AL
In conclusion, we compared the real-time PCR assay and antigenemia, deciding preemptive therapy on the basis of antigenemia. Moreover, only five patients received preemptive therapy. So the PCR assay seemed to lack clinical relevance. However, the PCR assay generally correlated with antigenemia and was more sensitive than antigenemia assay. We hypothesized that the CMV real-time PCR assay could not completely replace the antigenemia assay in kidney transplant recipients, but can be used complementarily to the antigenemia screen to monitor preemptive therapy, especially if examined serially. The PCR assay has some practical advantages, such as easy and rapid interpretation of the results, cost effectiveness, and reproducibility. REFERENCES 1. Kusne S, Grossi P, Irish W, et al: Cytomegalovirus pp65 antigenemia monitoring as a guide for preemptive therapy: a cost effective strategy for prevention of cytomegalo disease in adult liver transplant recipients. Transplantation 68:1125, 1999 2. Heid CA, Stevens J, Livak KJ, et al: Real-time quantitative PCR. Genome Res 6:986, 1996 3. Griscelli F, Barrois M, Chauvin S, et al: Quantification of human cytomegalovirus DNA in bone marrow transplant recipients by real-time PCR. J Clin Microbiol 39:4362, 2001 4. Kim CK, Song JH, Kim SM, et al: Clinical usefulness of human cytomegalovirus antigenemia assay after kidney transplantation. Transplantation 75:2151, 2003 5. Ursula MK, Manfred W, Gunter K, et al: Cytomegalovirus infection in organ-transplant recipients: diagnostic value of pp65 antigentest, qualitative polymerase chain reaction (PCR) and quantitative Taqman PCR. Transplantation 77:1692, 2004 6. Li H, Dummer JS, Estes WR, et al: Measurement of human cytomegalovirus load by quantitative real-time PCR for monitoring clinical intervention in transplant recipients. J Clin Microbiol 41:187, 2003 7. Boeckh M, Huang M, Ferrenberg J, et al: Optimization of quantitative detection of cytomegalovirus DNA in plasma by real-time PCR. J Clin Microbiol 42:1141, 2004