Comparison of Quantitative Cytomegalovirus Real-time PCR in Whole Blood and pp65 Antigenemia Assay: Clinical Utility of CMV Real-time PCR in Hematopoietic Stem Cell Transplant Recipients

Cytomegalovirus (CMV) disease continues to be an important cause of morbidity and mortality in hematopoietic stem cell transplantation (HSCT) recipients. We have been conducting a preemptive strategy based on the CMV pp65 antigenemia assay for the prevention of CMV infection and disease in a risk-adapted manner (1). Although the antigenemia assay has been used extensively as a tool to monitor CMV infections, there are several limitations, including 1) the need for immediate sample processing, 2) the nonstandardization of sample processing between laboratories, 3) the time-consuming and laborious procedure, and 4) the lack of objectivity of quantification (2-4). Furthermore, as the detection of antigenemia is applicable only to blood specimens (only virus associated with cells), it requires an adequate number of leukocytes.

Recently, several studies have demonstrated that real-time quantitative polymerase chain reaction (RQ-PCR) for CMV is useful for the rapid diagnosis of CMV infection and for the monitoring of clinical responses to antiviral therapy (5-11). However, there is, as yet, no consensus regarding the cutoff level and the appropriate blood compartment to be tested by RQ-PCR assay in HSCT recipients.

In this study, we compared RQ-PCR with the pp65 antigenemia assay for the monitoring of CMV infection in allogeneic HSCT recipients. Additionally, we have determined the proper cutoff level of CMV DNA load for preemptive therapy, in order to switch the monitoring method from the antigenemia assay to an RQ-PCR method.

Quantitative detection of CMV DNA using real-time PCR has become an increasingly popular method for the monitoring of CMV reactivation and for the initiation of antiviral therapy in allogeneic stem cell transplant recipients (4-11, 13-19). It has been suggested that RQ-PCR has several advantages over the antigenemia assay, including an increased sensitivity for the detection of CMV reactivation, the reliable detection of CMV reactivation during severe neutropenia in the early post-transplant period, the shorter time required for the procedure, and the convenient processing of large numbers of specimens (2, 3). For all of these reasons, we attempted to replace the antigenemia assay with RQ-PCR for the monitoring of CMV reactivation.

However, the replacement of the antigenemia assay with RQ-PCR is not a simple matter. The first task was the determination of the optimal specimen for RQ-PCR. There have been several reports addressing the proper specimen for RQ-PCR (3, 9, 15, 16, 20). Most of these studies have utilized plasma, whole blood, and leukocytes, and reported that these specimens correlated well with one another. Although CMV is closely associated with leukocytes, CMV DNA can also be detected in the plasma while the virus is undergoing active viral replication. However, inconsistencies in the storage procedures for blood samples and the separation of plasma from whole blood can result in artificially increased CMV viral loads secondary to cell lysis. Inhomogeneity of leukocyte lysates can lead to variability and inaccuracies in results. Additionally, blood is simpler to process, as it requires one less centrifugation step than does plasma preparation. Due to its higher sensitivity and higher CMV DNA yield, whole blood has been accepted more recently as an optimal sample for CMV DNA quantification (3, 15, 16, 18, 20). Thus, we elected to test whole blood rather than plasma or leukocyte lysates for CMV viral load detection.

The next step was the establishment of precise relationship between the two methods. Most of all, we observed a qualitative and quantitative correlation between the pp65 antigenemia value and the viral load estimated by RQ-PCR. Additionally, in the longitudinal analysis of individual patients, the follow-up curves of RQ-PCR and pp65 antigenemia were relatively uniform and well correlated in response to antiviral therapy. The majority of the discordant samples were either in patients receiving anti-CMV therapy, in whom the DNA versus protein levels appeared to decline at different rates, or in the patients with very low viral burdens, in whom we suspect that sampling error or variable lower limits of detection may have contributed to the discrepancy. There have been some reports asserting that increasing or persistently high levels of antigenemia during treatment with GCV were associated with decreasing or persistently low levels of DNA load and viremia, thus revealing an unexplained dissociation (21, 22). Additionally, the pp65 antigen tends to disappear at a later time than CMV DNA after anti-CMV treatment. Thus, the determination of cutoff values for preemptive treatment was conducted only with the results of samples naive to the anti-CMV agents. Unusually, sensitivity of the antigenemia assay was found to be higher than that of RQ-PCR for the detection of CMV reactivation in this study. We thought that anti-CMV therapy might have affected these results. About half of 77 specimens determined to be positive on antigenemia assay but negative on RQ-PCR, were under anti-CMV therapy. Additionally, false positivity of antigenemia caused by cytoplasmatic background staining might also affect the results (23, 24). On the other hand, there was a report that purer nuclear acid extracts and a better depletion of PCR inhibitors by the automated nucleic acid isolation could additionally improve PCR performance and increase the sensitivity of RQ-PCR (13, 15). Thus, we began to use automated nucleic acid isolation after this study. As our study was conducted during a limited designated period, specimens were not fully collected from the initial reactivation of CMV after engraftment in every patient. In some patients, only samples from the middle of antiviral therapy or after the discontinuation of anti-CMV drugs were included. We surmised that the characteristics of this uneven sample also influenced the results. And because of this reason, we could not compare the point that which method could detect reactivation of CMV earlier than the other. We could analyze the data of 18 selected patients, whose serial samples were collected from the initial increase of CMV viral load to the termination of anti-CMV therapy. Subgroup analysis revealed that the first detection time of reactivation did not differ between the two methods and also the cutoff value did not differ from that of the overall group (data not shown). As the kinetics of the CMV DNA viral load differ from that of antigenemia, we think that the consistency in serial values between the two methods is more important than the concordance at a point.

Although RQ-PCR assays have become quite popular, guidelines for antiviral therapy based on these methods have yet to be established. Like the antigenemia assay, RQ-PCR assays also lack standardization. Different institutions have developed their own assays and local guidelines, and these vary depending on the type of specimen and the assay design. In our HSCT center, pp65 antigenemia guided preemptive therapy has been conducted in a risk adapted manner (1). Patients were classified into low and high risk groups in accordance with HSCT type and the grade of acute GVHD. High risk patients were defined as those who had unrelated donors and any grade of acute GVHD, or who had matched related donors and grade II to IV acute GVHD. The patients with matched related donors and grade 0 to I acute GVHD were classified as low risk patients. GCV treatment was initiated when 5 or more CMV antigen-positive cells were detected in the low risk patients and when any antigen-positive cells were detected in the high risk patients. Thus, considering an antigenemia value of more than 5 antigen-postivie cells or more than 1 antigen-positive cell as a standard, respectively, the optimal cutoff value was provided in Fig. 4. We suggested that the tentative cutoff value was 20,000 copies/mL for the high risk patients and 30,000 copies/mL for the low risk group. Usually, RQ-PCR was introduced in order to allow for the more sensitive, rapid, and accurate diagnosis of CMV reactivation in HSCT recipients, which allowed for preemptive therapy to be administered as early as possible. However, in this study, RQ-PCR showed lower sensitivity than antigenemia. So, we defined present cutoff value as the "tentative" one, because of the reasons described above. At the same time, we were concerned that these cutoff values were somewhat higher than those suggested previously (9, 17). Lilleri et al. suggested a cutoff value of 10,000 copies/mL for the initiation of preemptive therapy in the HSCT recipients, and that group conducted randomized clincal trials comparing RQ-PCR and antigenemia guided preemptive therapy (17, 25, 26). However, they did not conduct preemptive therapy in a risk adapted fashion. Additionally, Kalpoe et al. also suggested that preemptive therapy could be initiated at CMV DNA load of 10,000 copies/mL or 1,000 copies/mL, and then increased by 10-fold per week at the first episode of CMV reactivation (9). We believe that, in addition to the viral DNA load, the kinetics of the DNA load and the pattern of changes in the viral load are crucial and should be taken into consideration when making treatment decisions. Further study to correct present cutoff value and to validate the optimal cutoff value for the initiation of preemptive therapy is currently underway at our HSCT center. On the other hand, we thought that high negative predictive value (87.9% in the high risk group and 96.5% in the low risk group) could allow us to avoid unnecessary preemptive therapy with a fair aliquot of patients. Additionally, we suggested that the duration of preemptive therapy might be shortened by RQ-PCR guided manner, because more rapid negative conversion of viral reactivation was detected using RQ-PCR as previously reported (27).

In summary, we observed a correlation between the pp65 antignemia assay and the viral load estimated by RQ-PCR in this study. Additionally, we realized that anti-CMV therapy might influence the results of the two assays. Based on a comparison with the established antigenemia assay, the tentative threshold at which preemptive anti-CMV therapy initiated was determined. The quantification of CMV DNA load from whole blood using RQ-PCR appears to be applicable to the clinical practice. An optimal cutoff value for guiding timely preemptive therapy should be clinically validated in future studies.