Real-time laser scanning coupled with a fluorogenic probe is a new technique which enables us to quantify a large number of amplified products rapidly and accurately (7
). Using this system, it is possible to analyze more than 40 samples in 2 to 3 h even if they are tested in duplicate. We showed that this system was applicable to the quantitation of EBV load in patients with symptomatic EBV infections. This technique detected PCR inhibitors and estimated the efficiency of the extraction methods, both of which are particularly important to quantify the EBV load accurately and reproducibly. Furthermore, this system eliminates the precautions that must be taken with amplified products to avoid contamination because the technique is performed in completely sealed wells. This is a great improvement over the conventional PCR assays, which have considerable risks of carryover contamination. With its rapidness, accuracy, and ability to handle many samples, the real-time PCR assay should replace the quantitative PCR methods now in use.
It has been shown that heparin is an inhibitory factor for PCR (3
). Using the real-time PCR assay, we showed that heparin was not eliminated from plasma by using standard DNA extraction methods and that the yield of amplified products decreased 1 to 100 times. Heparin did not inhibit PCR when PBMNC were used as the templates, probably because any heparin was washed out from the PBMNC during the separation with Ficoll-Paque. In this study, we used the PBMNC fractions for most of the quantitation because of lack of inhibition. As EBV remains latent in lymphocytes, the PBMNC fraction is usually used to estimate the virus load in peripheral blood. In primary or chronic active infections, cell-free virus is produced and released from cells in lytic cycles (25
). We and others have previously reported that the presence of EBV-DNA in the plasma is diagnostic of primary EBV infection (4
), which was confirmed in the present study. Since serum and plasma are readily obtained, they may be better sources for the quantitation of the virus load. Serum and EDTA-treated plasma can be used because they do not inhibit the PCR. If only heparinized blood is available, heparinase is reported to be useful for eliminating heparin from the DNA extraction solution (16
Since even healthy individuals have latent EBV in their blood, the presence of EBV genomes does not always indicate an active EBV infection or EBV-related disease. For the diagnosis of EBV-related diseases, a significant virus load should be defined. When we used 102.5
copies/μg of PBMNC DNA as the criterion, the real-time PCR assay was specific enough to diagnose symptomatic EBV infections. All the patients with LPD and CAEBV had more than 102.5
copies/μg of DNA (Fig. ). In contrast, 2 of 14 (14%) posttransplant patients without these disease manifestations had levels higher than 102.5
copies/μg of DNA (Fig. ). To our knowledge, only two papers have defined the significant virus load in symptomatic EBV infections, and both of them stated that 500 copies/105
cells is sufficient to diagnose LPD (26
). In this study, we quantified the amount of DNA extracted from PBMNC and used a fixed amount of DNA in the real-time PCR assay (250 ng). Using a set volume of the DNA extraction solution from a fixed number of PBMNC is simpler but may produce a bias caused by differences in the extraction efficiency for each sample. If 105
lymphocytes produce 0.5 μg of DNA as suggested in the manufacturer’s handbook (QIAamp Blood Kit; QIAGEN), then 500 copies/105
PBMNC equals 103.0
copies/μg of DNA, which is slightly greater than our criterion, 102.5
copies/μg of DNA. Accordingly, we consider 102.5
copies/μg of DNA a suitable cutoff level for distinguishing EBV-related LPD or CAEBV from latent EBV infections or asymptomatic reactivation of the virus. Some patients with IM had fewer copies of EBV DNA than 102.5
copies/μg of DNA. Patients with IM may have less virus load in their peripheral blood compared with patients with CAEBV or LPD. Therefore, it might be inappropriate to use the above criterion for the diagnosis of IM. The other possible reason that the IM patients had fewer EBV DNA copies may be that some of these samples had been stored for more than 5 years.
In patients who have had a bone marrow or solid organ transplant, LPD is an acute, life-threatening disease. Its diagnosis is sometimes difficult, and the disease often progresses rapidly (5
). LPD is usually resistant to both chemotherapy and antiviral drugs (32
). However, recent papers report that infusions of donor leukocytes or EBV-specific CTL are useful for the treatment of LPD (8
). Rooney et al. stress the importance of early or preemptive administration of EBV-specific CTL in treating LPD (27
). We believe that the real-time PCR assay is a useful method for the rapid diagnosis of LPD and for monitoring the virus load to evaluate the efficacy of treatment.