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`JOURNAL OF CLINICAL MICROBIOLOGY,
`0095-1137/99/$04.0010
`Copyright © 1999, American Society for Microbiology. All Rights Reserved.
`
`Jan. 1999, p. 132–136
`
`Vol. 37, No. 1
`
`Quantitative Analysis of Epstein-Barr Virus Load by Using
`a Real-Time PCR Assay
`HIROSHI KIMURA,1* MAKOTO MORITA,1 YUMI YABUTA,1 KIYOTAKA KUZUSHIMA,2
`KOJI KATO,3 SEIJI KOJIMA,3 TAKAHARU MATSUYAMA,3
`AND TSUNEO MORISHIMA1
`Department of Pediatrics, Nagoya University School of Medicine,1 Laboratory of Viral Oncology,
`Research Institute, Aichi Cancer Center,2 and Division of Hematology/Oncology, Children’s
`Medical Center, Japanese Red Cross Nagoya First Hospital,3 Nagoya, Japan
`
`Received 2 July 1998/Returned for modification 20 August 1998/Accepted 13 October 1998
`
`To measure the virus load in patients with symptomatic Epstein-Barr virus (EBV) infections, we used a real-
`time PCR assay to quantify the amount of EBV DNA in blood. The real-time PCR assay could detect from 2
`to over 107 copies of EBV DNA with a wide linear range. We estimated the virus load in peripheral blood mono-
`nuclear cells (PBMNC) from patients with symptomatic EBV infections. The mean EBV-DNA copy number in
`the PBMNC was 103.7 copies/mg of DNA in patients with EBV-related lymphoproliferative disorders, 104.1
`copies/mg of DNA in patients with chronic active EBV infections, and 102.2 copies/mg of DNA in patients with
`infectious mononucleosis. These numbers were significantly larger than those in either posttransplant patients
`or immunocompetent control patients without EBV-related diseases. In a patient with infectious mononucle-
`osis, the virus load decreased as the symptoms resolved. The copy number of EBV DNA in PBMNC from symp-
`tomatic EBV infections was correlated with the EBV-positive cell number determined by the in situ hybrid-
`ization assay (r 5 0.842; P < 0.0001). These results indicate that the real-time PCR assay is useful for
`diagnosing symptomatic EBV infection and for monitoring the virus load.
`
`Epstein-Barr virus (EBV) is the causative agent of infectious
`mononucleosis (IM) and EBV-related malignancies such as
`Burkitt’s lymphoma and nasopharyngeal carcinoma. During
`the primary infection, EBV infects and immortalizes B-lym-
`phocytes, which proliferate in the peripheral blood and lymph
`nodes. After the emergence of EBV-specific immunity, the
`number of EBV-infected B cells is regulated mainly by cyto-
`toxic T-lymphocytes (CTL) and remains at 1 to 105 B-lympho-
`cytes (25). In a limited number of individuals, the host immu-
`nity is unable to regulate the EBV-infected cells, and chronic
`active EBV infection (CAEBV) occurs (13, 24, 31). In immu-
`nocompromised hosts, such as AIDS patients or transplant
`recipients, EBV-infected cells proliferate again and cause op-
`portunistic B-cell lymphoma, a lymphoproliferative disorder
`(LPD) (6, 22).
`For the diagnosis of CAEBV and LPD, it is essential to
`measure the EBV load in either a biopsy specimen or the
`peripheral blood (19, 23, 24, 27, 33). A biopsy is invasive and
`labor-intensive, so several methods to detect the virus load
`in the peripheral blood for the early diagnosis of LPD and
`CAEBV have been developed. Spontaneous outgrowth of
`EBV-infected B cells in vitro (27, 30), in situ hybridization
`(ISH) using EBV-encoded small RNA (EBER) probe (18),
`and quantitative PCR assays are now used to determine the
`EBV load in peripheral blood mononuclear cells (PBMNC) (2,
`26, 27, 29). Previously, we reported that quantitative PCR was
`useful for diagnosing and monitoring EBV infections (37).
`However, the quantitative PCR assay requires at least 3 days to
`complete, since the assay includes gel electrophoresis and
`Southern hybridization steps. Moreover, the linear range of the
`quantitative PCR is too narrow to measure a variety of samples
`
`* Corresponding author. Mailing address: Department of Pediatrics,
`Nagoya University School of Medicine, 65 Tsuruma-cho, Showa-ku,
`Nagoya 466-8550, Japan. Phone: 81-52-744-2303. Fax: 81-52-744-2974.
`E-mail: hkimura@med.nagoya-u.ac.jp.
`
`because, in samples with a large amount of template, the
`amount of amplified product reaches a plateau after the log
`phase of the reaction (34).
`Recently, a novel real-time quantitative PCR was developed
`(7, 36). This method measures the accumulation of PCR prod-
`ucts with a fluorogenic probe and by real-time laser scanning in
`a 96-well plate. Since this assay does not require postsample
`handling, much faster assays are possible. The assay has a very
`large dynamic range of target molecule determination because
`the real-time measurement of the PCR product enables us to
`quantify the amplified products in the log phase of the reaction
`(7). In this study, the real-time PCR method was applied to the
`measurement of EBV DNA in peripheral blood. We measured
`the virus load in patients with symptomatic EBV infections and
`compared it with the load in patients without symptoms asso-
`ciated with EBV infection. The real-time PCR was also com-
`pared with the ISH and traditional qualitative PCR assays.
`
`MATERIALS AND METHODS
`
`Patients and samples. Eighteen patients who had symptomatic EBV infections
`were enrolled in this study (four with CAEBV, five with LPD, and nine with IM).
`These patients were 1 to 19 years old (mean age, 6.5 years). CAEBV was
`diagnosed according to previously published guidelines (19, 24). The clinical
`features of some of these cases were described elsewhere (10, 13, 15, 17). All the
`LPD patients except a 2-year-old boy with congenital immunodeficiency had had
`liver transplants. LPD was suspected in patients with lymphadenopathy, pulmo-
`nary infiltration, gastrointestinal tract bleeding, or unexplained allograft dysfunc-
`tion. The diagnosis of LPD was established by pathology or the detection of
`EBER by the ISH assay (18, 33). IM was diagnosed by clinical findings and
`serological examinations as follows: positive for anti-viral capsid antigen (VCA)
`immunoglobulin G (IgG) and/or IgM and negative for anti-EB nuclear antigen
`(EBNA) antibody. For controls, 10 patients who had had either a liver or a bone
`marrow transplantation were prospectively evaluated for EBV infections. These
`patients were 1 to 17 years old (mean age, 5.8 years) and had no symptoms
`characteristic of LPD. Either the recipient or the donor was seropositive for
`EBV. In addition, 13 immunocompetent patients (2 to 16 years old; mean age,
`6.7 years) who were initially suspected of having a primary EBV infection were
`enrolled as controls. These patients were positive for both anti-VCA IgG and
`EBNA antibodies, indicating that they had been previously infected with EBV.
`Either heparinized or EDTA-treated blood was taken from the patients, and
`
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`REAL-TIME PCR FOR EBV
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`FIG. 1. (A) Standard curve for real-time PCR. Serially diluted pGEM-
`BALF5 plasmid was amplified with or without DNA extraction solutions from
`blood and analyzed in real time with a model 7700 Sequence Detector. The CT
`values were plotted against copy number to construct the standard curve. Ex-
`planation of symbols: none, plasmid with EBV insert; PBMNC, PBMNC from an
`EBV-seronegative patient plus plasmid with EBV insert; plasma, plasma from an
`EBV negative patient plus plasmid with EBV insert. (B) Effect of heparin on
`quantitation of plasmid DNA. Serially diluted plasmid controls were amplified
`with or without various DNA extraction solutions and analyzed using a model
`7700 Sequence Detector. Explanation of symbols: none, plasmid with EBV in-
`sert; serum, serum from an EBV seronegative patient plus plasmid with EBV
`insert; serum 1 EDTA, serum and EDTA plus plasmid with EBV insert; se-
`rum 1 heparin, serum and heparin plus plasmid with EBV insert.
`
`marmoset cell line (B95-8), two Burkitt’s lymphoma cell lines
`(Raji and Daudi), and four lymphoblastoid cell lines. All the
`cell lines were positive for EBV DNA by the real-time PCR
`assay. From the standard curve, the estimated number of EBV
`DNA genomes ranged from 5.2 to 31 per cell in these cell lines,
`which approximately equals the previously reported values
`(25).
`Detection of an inhibitor in heparinized blood. Heparin
`inhibits the PCR (3, 16). Since some of our samples were
`heparinized blood, we performed reconstruction studies to
`confirm the removal of the inhibitor from these samples by the
`DNA extraction kit. Heparinized blood was taken from a pa-
`tient who was seronegative for EBV, and PBMNC and
`plasma were separated. DNA extraction solution from either
`the PBMNC or plasma fraction was added to serially diluted
`plasmid controls. The DNA-extraction solution from PBMNC
`did not inhibit the PCR. The solution from the plasma, how-
`
`the PBMNC and plasma were separated with Ficoll-Paque (Pharmacia Biotech,
`Piscataway, N.J.). For the PCR assay, the DNA was extracted from the PBMNC
`and plasma fraction by using a QIAamp Blood Kit (QIAGEN Inc., Chatsworth,
`Calif.) and stored at 220°C until use.
`Real-time quantitative PCR with a fluorogenic probe. The PCR primers for
`this assay were selected in the BALF5 gene encoding the viral DNA polymerase
`(1). The upstream and downstream primer sequences were 59-CGGAAGCCCT
`CTGGACTTC-39 and 59-CCCTGTTTATCCGATGGAATG-39, respectively.
`A fluorogenic probe (59-TGTACACGCACGAGAAATGCGCC-39) with a se-
`quence located between the PCR primers was synthesized by PE Applied Bio-
`systems (Foster City, Calif.). The PCR reaction was performed using the Taq-
`Man PCR kit (PE Applied Biosystems) as previously described (14). Briefly,
`either 250 ng of DNA from PBMNC or the extraction solution from 50 ml of
`plasma was added to a PCR mixture containing 10 mM Tris (pH 8.3), 50 mM
`KCl, 10 mM EDTA, 5 mM MgCl2, 100 mM dATP, dCTP, dGTP, and dTTP, 0.2
`mM each primer, 0.1 mM fluorogenic probe, and 1.25 U of AmpliTaq Gold (PE
`Applied Biosystems). Following an activation of the AmpliTaq Gold for 10 min
`at 95°C, 45 to 50 cycles of 15 sec at 95°C and 1 min at 62°C were carried out by
`a model 7700 Sequence Detector (PE Applied Biosystems). Real-time fluores-
`cence measurements were taken, and a threshold cycle (CT) value for each
`sample was calculated by determining the point at which the fluorescence ex-
`ceeded a threshold limit (10 times the standard deviation of the baseline) (7). For
`a positive control, a plasmid that contained the BALF5 gene was constructed
`from pGEM-T vector (Promega, Madison, Wis.) and termed pGEM-BALF5. A
`standard graph of the CT values obtained from serially diluted pGEM-BALF5
`was constructed. The CT values from clinical samples were plotted on the stan-
`dard curve, and the copy number was calculated automatically by Sequence
`Detector version 1.6 (PE Applied Biosystems), a software package for data
`analysis. Each sample was tested in duplicate, and the mean of the two values was
`shown as the copy number of the sample. Samples were defined as negative if the
`CT values exceeded 50 cycles.
`Qualitative PCR assay. The qualitative PCR assay using nested primers was
`performed with slight modifications of a previously described method (37).
`Briefly, the outer primers, which were designed in the BamHI W region of the
`EBV gene (positions 1544 to 1568 for the 59 primer and 1653 to 1677 for the 39
`primer), were used for the first round of PCR (12). The same volume of the DNA
`extraction solution used for the real-time PCR assay was added to a total of 50
`ml of reaction mixture containing 10 mM Tris (pH 8.3), 50 mM KCl, 1.5 mM
`MgCl2, 100 mM dATP, dCTP, dGTP, and dTTP, 0.2 mM each primer, and 1.25
`U ofTaq polymerase (TaKaRa, Ohtsu, Japan). Amplifications were carried out
`for 30 cycles with a PCR Thermal Cycler (TaKaRa). After the first amplification,
`2 ml of the amplified products was used for the second amplification, which
`consisted of 30 cycles using the inner primers (positions 1572 to 1591 for the 59
`primer and 1642 to 1661 for the 39 primer) (12). The amplified products were
`separated on a 1.2% agarose gel, stained with ethidium bromide, and visualized
`by UV light. The qualitative PCR assay could detect two copies of a plasmid
`control that contained the BamHI W fragment. Since the fragment is located in
`internal repeats and one EBV genome contains 10 or more BamHI fragments
`(12), the qualitative PCR was more sensitive than the real-time PCR assay. On
`the other hand, the region was not selected for the real-time quantitative assays
`since the number of the repeat was variable among EBV strains.
`ISH assay for the detection of EBER. The ISH assay was performed using
`the EBER probe as previously described (9). For the ISH assay, 105 separated
`PBMNC were spotted on silane-coated slides and dried. Each slide was hybrid-
`ized with the alkaline phosphatase-labeled EBER probe, washed, and reacted
`with 5-bromo-4-chloro-3-indoyl phosphate for visualization. Positive cells were
`counted and expressed as cells per 105 PBMNC.
`Statistical analysis. The software package Statview J 4.02 (Abacus Concepts
`Inc., Berkeley, Calif.) was used for data analysis. Student’s t test was used for the
`comparison of the mean copy numbers of EBV DNA in each group. The Pearson
`correlation coefficient was used to compare the real-time PCR and ISH assays.
`
`RESULTS
`
`Establishment of a real-time PCR assay for quantifying
`EBV-DNA. Serially diluted pGEM-BALF5 was tested by the
`real-time PCR assay, and a standard curve of the CT values
`obtained by using the positive control was constructed. A wide
`linear range (beginning at 10 copies and extending through 107
`copies of the control plasmid) was established (Fig. 1A). A
`minimum of two copies of the plasmid could be detected by the
`system (data not shown).
`To confirm the specificity of the primers and the probe, an
`EBV-negative lymphoma cell line, other human herpesviruses
`(herpes simplex virus type 1 and 2, cytomegalovirus, varicella-
`zoster virus, and human herpesvirus 8), and PBMNC from
`EBV-seronegative patients were tested by this system. All were
`negative for EBV DNA. Next, we examined an EBV-positive
`
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`FIG. 2. Quantitation of EBV DNA by real-time PCR. DNA was extracted from PBMNC obtained from patients with symptomatic EBV infections or control
`patients without EBV-related diseases. Two hundred fifty nanograms of DNA was used for the real-time PCR assay, and the EBV DNA copy numbers per microgram
`of DNA are shown. Multiple samples for some patients were tested because repeated evaluations were needed. Bars show the means and standard deviations for each
`group. The dotted line shows the detection limit of the assay.
`
`ever, inhibited the reaction, and the yield of amplified products
`decreased approximately 1- to 100-fold (Fig. 1A). For example,
`the CT value of 105 copies of the plasmid in the plasma fraction
`approximately equaled that of 107 copies of the plasmid alone.
`To confirm that the residual heparin in the plasma was the
`inhibitor, the DNA extraction solutions from serum, serum
`plus EDTA, and serum plus heparin were examined in the
`reconstruction study. EDTA or heparin was added to EBV-
`free serum at the standard concentrations used for anticoagu-
`lation (final concentrations, 3 mM and 25 U/ml, respectively).
`The DNA extraction kit was used in an attempt to remove
`these anticoagulants, and each extraction solution was mixed
`with the plasmid control. Only the serum-plus-heparin sample
`inhibited the PCR reaction (Fig. 1B). These results indicate
`that the heparin is the inhibitor and could not be removed
`from the plasma. The results also show that the real-time
`quantitative PCR assay is useful for determining the presence
`of inhibitors.
`Quantitation of EBV DNA in patients with symptomatic
`EBV infection. Next, we estimated the virus load in blood from
`patients with symptomatic EBV infections by the real-time
`PCR assay. Since the reconstruction study indicated that
`plasma from heparinized blood was unsuitable for the quanti-
`tation and blood from most patients with symptomatic EBV
`infections was drawn with heparin, PBMNC were used for the
`analysis. The mean number of EBV DNA genomes in the
`PBMNC was 103.7 copies/mg of DNA in patients with LPD,
`104.1 copies/mg of DNA in patients with CAEBV, and 102.2
`copies/mg of DNA in patients with IM (Fig. 2). These numbers
`were significantly larger than those for posttransplant patients
`without EBV-related diseases (101.3 copies/mg of DNA; P ,
`0.0001 for LPD and CAEBV, P 5 0.02 for IM) or in immu-
`nocompetent and EBV-seropositive controls (101.2 copies/mg
`of DNA; P , 0.0001 for LPD and CAEBV, P 5 0.004 for IM).
`Sequential samples from a 19-year-old female with IM were
`obtained and tested using the real-time PCR assay. Since the
`patient’s blood was drawn with EDTA, which did not interfere
`with the reaction, both PBMNC and plasma were used for the
`assay. The virus load decreased as the symptoms resolved (Fig.
`3).
`Specificity of real-time PCR assay. We hypothesized that
`102.5 copies per mg of PBMNC DNA were enough to diagnose
`
`symptomatic EBV infections, because all the patients with
`LPD and CAEBV had more copies of the EBV DNA genome
`than this number (Fig. 2). A total of 60 samples were used to
`evaluate the diagnostic performance of the real-time PCR as-
`say. When 102.5 copies per mg of PBMNC DNA was used as a
`criterion to diagnose symptomatic EBV infections, the real-
`time PCR assay was highly specific (both the specificity and the
`positive predictive value were 93%, as shown in Table 1). Next,
`this new quantitative method was compared with a qualitative
`method using conventional PCR. Blood samples tested by the
`real-time PCR assay were also evaluated by the qualitative
`PCR assay using PBMNC fraction, plasma fraction, or both.
`The qualitative PCR assay using PBMNC was very sensitive
`but its specificity was low because the assay was so sensitive
`that latent EBV in the PBMNC was detected in individuals
`without EBV-related diseases (Table 1). On the other hand,
`when plasma was used for the qualitative PCR assay, the assay
`was specific and as diagnostic as the real-time PCR assay (both
`the specificity and the positive predictive value were 92%, as
`
`FIG. 3. Change in the virus load in a patient with IM. Sequential samples
`from a patient with IM were obtained, and both the PBMNC and plasma were
`analyzed by the real-time PCR assay. The EBV DNA copy numbers are shown
`per microgram of DNA. The EBV DNA copy numbers are shown per milliliter
`of plasma. The dotted line shows the detection limit for each sample.
`
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`infection (4, 37), 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, 21).
`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 di-
`agnosis of EBV-related diseases, a significant virus load should
`be defined. When we used 102.5 copies/mg 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/mg of DNA (Fig.
`2). In contrast, 2 of 14 (14%) posttransplant patients without
`these disease manifestations had levels higher than 102.5 cop-
`ies/mg of DNA (Fig. 2). 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, 29). 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 num-
`ber 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 mg of DNA as suggested in the man-
`ufacturer’s handbook (QIAamp Blood Kit; QIAGEN), then
`500 copies/105 PBMNC equals 103.0 copies/mg of DNA, which
`is slightly greater than our criterion, 102.5 copies/mg of DNA.
`Accordingly, we consider 102.5 copies/mg 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/mg 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
`
`FIG. 4. Correlation of results of the real-time PCR and in situ hybridization
`assays. PBMNC were obtained from patients with symptomatic EBV diseases,
`and 20 samples were analyzed using both the real-time PCR and ISH assays. The
`copy numbers of EBV-DNA measured by the real-time PCR assay and EBV-
`positive cell numbers measured by the ISH assay were plotted, and the correla-
`tion coefficient (r) was calculated.
`
`TABLE 1. Performance of a real-time PCR assay for diagnosing
`symptomatic EBV infections and comparison
`with a qualitative PCR assay
`
`Assay type
`
`No. of
`samplesa
`
`Sensitivity
`(%)
`
`Specificity
`(%)
`
`Positive
`PVb (%)
`
`Negative
`PVb (%)
`
`Real-time PCRc
`Qualitative PCR
`PBMNC
`Plasma
`
`60
`
`51d
`54d
`
`85
`
`100
`82
`
`93
`
`57
`92
`
`93
`
`69
`92
`
`83
`
`100
`83
`
`a Samples were obtained from either patients with symptomatic EBV infec-
`tions (33 samples) or control patients without EBV-related diseases (27 sam-
`ples).
`b PV, predictive value.
`c 102.5 copies per mg of PBMNC DNA was regarded as the creiterion to diag-
`nose symptomatic EBV infections.
`d Some samples were not available for the qualitative PCR assays.
`
`shown in Table 1). This result agreed with our previous find-
`ings (37).
`To demonstrate the accuracy of the real-time PCR assay, we
`compared it with another quantitative method, the ISH assay.
`PBMNC from patients with symptomatic EBV infections were
`used for both the real-time PCR and ISH assays. The copy
`numbers of EBV DNA measured by the real-time PCR assay
`were highly correlated with the EBV-positive cell numbers by
`the ISH assay (r 5 0.842; P , 0.0001 [Fig. 4]). Taken together
`with the comparison with the qualitative PCR assay, these
`results showed that the real-time PCR assay was a sufficiently
`sensitive and specific method for diagnosing symptomatic EBV
`infections and for monitoring of the virus load.
`
`DISCUSSION
`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, 35). 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 de-
`tected PCR inhibitors and estimated the efficiency of the ex-
`traction methods, both of which are particularly important to
`quantify the EBV load accurately and reproducibly. Further-
`more, 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, 16). 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 prod-
`ucts 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
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`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 diag-
`nosis is sometimes difficult, and the disease often progresses
`rapidly (5, 11). 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, 20, 28). 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.
`
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