Quantification of Human Cytomegalovirus DNA in Bone Marrow Transplant Recipients by Real-Time PCR

Abstract

A real-time PCR assay was developed to quantify human cytomegalovirus (CMV) DNA in peripheral blood leukocytes (PBLs) of bone marrow transplantation patients. Unlike other teams, we quantified CMV and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene using a plasmid containing both sequences as an external standard. Tenfold serial dilutions of this plasmid yielded overlapping standard curves that allowed the quantification of CMV and GAPDH gene copies in an efficient and accurate manner. Sequential blood samples (164 specimens) were collected from 16 patients. PBLs were tested by the pp65 antigenemia assay and quantitative CMV and GAPDH gene PCRs. CMV DNA was detected by PCR in 13 patients a mean of 15 days prior to the appearance of antigenemia. The administration of anti-CMV drugs led to a rapid decrease in the numbers of viral copies and positive nuclei. Real-time PCR assay results correlated with those of the CMV pp65 antigenemia assay (P < 0.00001). The TaqMan assay may be a useful tool for rapid quantification of CMV infection and for monitoring of CMV reactivation in bone marrow transplantation recipients.Human cytomegalovirus (CMV) is a well-known cause of mortality in blood and bone marrow transplantation (BMT) patients. Monitoring of CMV reactivation from latency is critical for these patients. Prophylaxis against CMV disease with ganciclovir (8, 9) or foscarnet (16) in asymptomatic BMT recipients has been shown to dramatically reduce the incidence of CMV disease (8, 17). As ganciclovir and foscarnet are myelotoxic and nephrotoxic, respectively, full treatment is often started at the time of documented CMV reactivation. The key to efficient and effective management of CMV infection in these patients is a test capable of rapidly monitoring and quantifying the presence of CMV in the blood. This is particularly essential for the identification of subjects at high risk of developing CMV disease, e.g., patients receiving steroid or immunosuppressive compounds for accelerated graft-versus-host disease and also for the application and monitoring of preemptive antiviral therapeutic strategies. The CMV assays presently available and frequently used in this setting include shell vial culture (7), the CMV antigenemia assay (2), PCR for CMV DNA (3), hybrid capture assay for quantitation of CMV DNA (10), and detection of CMV RNA by nucleic acid sequencebased amplification (1).Among white blood cells, peripheral blood leukocytes (PBLs) are the main CMV carriers during active CMV infection. The detection of CMV antigenemia in PBLs has been shown to be an early marker of CMV infection. A monoclonal antibody is used to detect pp65, the CMV lower matrix phosphoprotein in PBLs, and this test is widely used to monitor BMT recipients. Although a correlation was found between the number of pp65-positive PBLs and the development of clinical symptoms, this method (6, 20) poses a number of problems. It is difficult to perform before engraftment because the number of leukocytes is limited and false-negative results are ...

Influence of antithymocyte globulin dose on outcome in cytomegalovirus‐seropositive recipients of partially T cell‐depleted stem cell grafts from matched‐unrelated donors

“…Until April 2001, CMV monitoring was performed using the CMV pp65 antigenaemia assay, thereafter monitoring was based on a real-time TaqMan TM CMV DNA PCR. This method is known to be more sensitive (Machida et al, 2000;Griscelli et al, 2001;Yakushiji et al, 2002) compared with the antigenaemia assay, however, the incidence of CMV reactivations was still lower in the recent group compared with the historical group. In conclusion, CMVseropositive MUD recipients of partial TCD grafts treated since 1999 show survival rates comparable to CMVseronegative recipients.…”

Monitoring cytomegalovirus infection by antigenemia assay and two distinct plasma real-time PCR methods after hematopoietic stem cell transplantation

“…[12][13][14][18][19][20][21] However, they utilized different methods including regions for primers and TaqMan probe, and the source of DNA extraction (plasma, whole blood or leukocyte). Therefore, we must carefully interpret the results of real-time PCR.…”

“…10 Although it might be too sensitive for discrimination of high-risk patients for CMV disease, 11 the development of real-time PCR has made it possible to quantitatively evaluate the virus load. [12][13][14] However, the threshold of the virus load to discriminate high-risk patients has not been determined, although the threshold of the antigenemia value has been reported. 15,16 Serially monitoring the virus load without administering ganciclovir is the only method to address this issue, but it is unethical.…”

Once daily ganciclovir as initial pre-emptive therapy delayed until threshold CMV load ⩾10000 copies/ml: a safe and effective strategy for allogeneic stem cell transplant patients

“…The recent development of quantitative PCR (QPCR) for CMV offers the possibility of developing a threshold level for initiating treatment that would minimize unnecessary exposure to antiviral agents. [9][10][11]13,14 However, there is currently no agreement of what type of blood specimen should be tested (whole blood, plasma, leukocyte preparation) nor is there agreement on the optimal threshold level for initiating pre-emptive therapy.…”