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Previous Article | Next Article 
Journal of Clinical Microbiology, February 2000, p. 613-619, Vol. 38, No. 2
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
High Levels of Epstein-Barr Virus DNA in Blood of
Solid-Organ Transplant Recipients and Their Value in Predicting
Posttransplant Lymphoproliferative Disorders
Fausto
Baldanti,1,2
Paolo
Grossi,3
Milena
Furione,1
Lavinia
Simoncini,1
Antonella
Sarasini,1
Patrizia
Comoli,4
Rita
Maccario,4
Roberto
Fiocchi,5 and
Giuseppe
Gerna1,*
Servizio di
Virologia,1 Laboratori Sperimentali di
Ricerca,2 Istituto di Malattie
Infettive,3 and Clinica
Pediatrica,4 Istituto di Ricovero e Cura a
Carattere Scientifico Policlinico San Matteo, Università di
Pavia, 27100 Pavia, and Divisione di Cardiochirurgia,
Ospedali Riuniti, 24100 Bergamo,5 Italy
Received 12 May 1999/Returned for modification 11 August
1999/Accepted 1 November 1999
 |
ABSTRACT |
Epstein-Barr virus (EBV) DNA was quantitated in peripheral blood
mononuclear cells (PBMC) from 25 healthy subjects, 105 asymptomatic solid-organ transplant (SOT) recipients, and 15 SOT recipients with
symptomatic EBV infections by using a newly developed quantitative-PCR technique. Patients with symptomatic EBV infections had significantly higher (P < 0.001) median EBV DNA levels than
asymptomatic SOT recipients and immunocompetent individuals. In SOT
recipients, the positive predictive value of EBV DNA levels of >1,000
genome equivalents (GE)/0.5 µg of total PBMC DNA was 64.7% for
symptomatic EBV infection, while the negative predictive value was
96.1%. In 19 of 32 (59.3%) asymptomatic SOT recipients, EBV DNA
levels were consistently below 1,000 GE for as long as 18 months, while 10 of 32 (31.2%) patients had 1,000 to 5,000 EBV GE at least once during follow-up. In a minority of patients (3 of 32; 9.3%), 
5,000 GE could be detected at least once during follow-up. Reduction of
immunosuppressive treatment decreased EBV DNA levels by 1 log10 unit in patients with symptomatic EBV infections.
Quantification of EBV DNA is valuable for the diagnosis and monitoring
of symptomatic EBV infections in SOT recipients.
| |
INTRODUCTION |
Posttransplant lymphoproliferative
disorders (PTLD) consist of a wide spectrum of manifestations ranging
from lymphoid hyperplasia to clonal malignancy that occur following
solid-organ and bone marrow transplantation (1, 2, 4, 9, 15, 16,
19, 27).
A strong correlation between Epstein-Barr virus (EBV) infection and the
development of PTLD has been recognized (14, 17, 20, 26,
31). In particular, uncontrolled EBV-induced B-cell proliferation
in these immunosuppressed patients may play a central role in the
pathogenesis of this disease (3, 11, 12, 28). In this
respect, it is generally assumed that there is a correlation between
the amount of immunosuppression and the risk of developing PTLD
(14, 18, 29).
PTLD may be reversible in solid-organ transplant (SOT) recipients
following reduction or discontinuation of immune suppression (27). Other therapeutic options include the administration
of antiviral drugs (acyclovir, famcyclovir, or foscarnet), alpha interferon, anti-B-cell monoclonal antibodies, and cytotoxic
chemotherapy (5, 7, 25). However, these approaches are
associated with high mortality rates in patients who do not respond to
treatment (15). Recently, the adoptive transfer of
EBV-specific cytotoxic T-cell clones has proven highly effective for
treatment of PTLD after bone marrow transplantation (BMT) with T-cell
depletion (22). Thus, timely detection of PTLD in transplant
recipients is necessary so that the appropriate therapy can be adopted.
The detection and quantification of EBV DNA in the blood have been utilized as prognostic markers for the development of
lymphoproliferative disorders (21, 24, 30), showing a
correlation between high levels of EBV DNA in the blood and the
development of PTLD (10, 21, 24). However, in two recent
papers very different threshold values were indicated as predictive for
PTLD, and large differences both in the prevalence and in the levels of
EBV DNA in the blood of control populations were reported (13,
23).
In the present study, we describe a novel and simple quantitative PCR
(Q-PCR) technique, developed according to an original design, which
allowed for reproducible DNA quantification in the range of
101 to 106 EBV genome equivalents (GE)/0.5 µg
of peripheral blood mononuclear cell (PBMC) DNA. EBV DNA was
comparatively quantitated in immunocompetent individuals as well as in
SOT recipients with asymptomatic and symptomatic EBV infections.
Threshold values predictive for PTLD were determined in a prospective
follow-up study.
| |
MATERIALS AND METHODS |
Study populations.
EBV DNA was quantitated in the blood of
three groups of individuals: (i) 25 immunocompetent healthy subjects,
(ii) 77 randomly selected asymptomatic adult SOT recipients (65 heart,
8 lung, and 4 liver transplant recipients), as well as 28 asymptomatic pediatric heart transplant recipients, and (iii) 12 adult SOT recipients (11 with heart transplants and 1 with a liver transplant) and 3 pediatric heart transplant recipients with symptomatic EBV infections.
The demographics of the study population were as follows. The median
age of the 25 immunocompetent subjects (25 females) studied was 27.0 years (range, 23 to 31 years). The median age of the 31 pediatric heart
recipients (17 males and 14 females) was 7.6 years (range, 0.2 to 17.3 years). Finally, for the 76 adult heart (64 males and 12 females), 8 lung (4 males and 4 females), and 5 liver (3 males and 2 females)
transplant recipients, the median ages were 46.7, 42.3, and 50.1 years,
respectively (ranges, 18.2 to 64.4, 22 to 60, and 28.3 to 60.5 years, respectively).
Asymptomatic SOT recipients were randomly enrolled 1 year or more after
transplantation concomitantly with a routine physical examination,
which was performed every 3 to 6 months, and blood samples were taken
and assayed by PCR for the presence of EBV DNA. EBV DNA-positive
patients were prospectively monitored every 1 to 3 months for as long
as 18 months. Given the very high prevalence of EBV seropositivity in
Italy, routine EBV serology was not performed for asymptomatic
patients. The virological follow-up of symptomatic patients was
initiated in the presence of clinical symptoms suggestive of PTLD.
Patients who experienced a febrile syndrome (body temperature of
>37.5°C for 2 weeks) without lymphadenopathy and for whom rejection, human cytomegalovirus infection, and other causes of fever
had been carefully ruled out, were defined as symptomatic. PTLD was
diagnosed as reported elsewhere (4, 11, 15, 20, 28), by
histologic examination of biopsy or autopsy tissue specimens from
patients with lymphadenopathy or disseminated disease. Sections were
evaluated for the degree of polymorphism, necrosis, plasmacytic differentiation, and number of large atypical cells. The cellular clonal status was determined by Southern blot analysis of lesional DNA.
The presence of EBV within the tumor cells was established by in situ
hybridization for EBV encoded RNA (EBER), performed on routinely
processed paraffin sections by using a fluorescein-conjugated EBER DNA
probe and a phytohemagglutinin in situ hybridization detection kit
(DAKO A/S, Glostrup, Denmark) according to the manufacturer's instructions.
DNA extraction from Ficoll-separated PBMC samples.
Aliquots
of 106 PBMC were lysed with proteinase K (25 mg/ml) for
1 h at 55°C. After enzyme inactivation at 94°C for 10 min, samples were centrifuged at 3,200 × g for 5 min.
Supernatants were collected and precipitated at 
80°C for 1 h
in the presence of 0.2 M sodium acetate and 3 volumes of 100% ethanol.
Then samples were centrifuged at 13,500 × g for 20 min at
4°C. The DNA pellets were washed three times with 70% ethanol and
dried at 55°C. Finally, DNA was resuspended in H2O, and
0.5-µg DNA aliquots, roughly corresponding to 105 PBMC,
were used in each PCR.
Construction of the internal control and external standards for
quantification of EBV DNA by Q-PCR.
Viral DNA in PBMC was
quantified by following a previously reported method (6) in
principle. Two recombinant DNA molecules, referred to as pP3 and pRM,
were constructed. pP3 was obtained by cloning into plasmid PCR 2000 (TA
cloning system; Invitrogen, Carlsbad, Calif.) a 269-bp region of the
EBV EBNA-1 gene (BamHI K region) which was amplified by the
primer pair 1-2 (primer 1, 5'-GTCATCATCATCCGGGTCTC-3';
primer 2, 5'-TTCGGGTTGGAACCTCCTTG-3'). (30). pRM was obtained by cloning into plasmid PCR
2000 a 330-bp recombinant DNA molecule consisting of a pGEM 3Z
plasmid (Promega, Madison, Wis.) sequence that was similar in its GC
and AT contents to the EBNA-1 amplified region sequence and had the
target sequences of primers specific for EBNA-1 artificially added to
its ends according to a previously reported PCR-directed mutagenesis
technique (32). Thus, pP3 and pRM were amplified by the same
set of primers but were different in size. Competition experiments were
performed in order to verify the PCR kinetics of the two molecules and
to select the most appropriate conditions for the Q-PCR. EBV DNA quantification in PBMC samples was achieved by amplifying serial amounts of pP3 (104, 103, 102,
101, and 100 copies, which were used as
external standards) in the presence of 0.5 µg of PBMC DNA from EBV
DNA-negative donors and in parallel with PBMC samples. A fixed amount
of pRM (100 copies) was added both to standards and to sample PCR
mixtures and was used as an internal amplification control.
PCR was carried out for 45 cycles (each consisting of 94°C for
30 s, 58°C for 45 s, and 72°C for 1 min plus 1 additional
second per cycle) in 1× PCR buffer (10 mM Tris-HCl [pH 8.3], 50 mM
KCl, 1.5 mM MgCl2, 0.001% gelatin) by using 25 pmol of
each primer and 2 U of Taq polymerase (Perkin-Elmer, Foster
City, Calif.) in a final volume of 50 µl. The pP3 and pRM
amplification products were differentiated by ethidium bromide staining
after agarose gel electrophoresis. Gel signals were then digitalized
and submitted to densitometric analysis (6).
Statistical analysis.
Since the sensitivity of our PCR
technique was 10 EBV DNA GE, median EBV DNA levels were calculated by
arbitrarily assigning the EBV DNA-negative (<10 GE) samples the value
of 5 EBV DNA GE. The prevalences of EBV DNA in immunocompetent
individuals, asymptomatic SOT recipients, and SOT recipients with
symptomatic EBV infections were compared by the Pearson chi-square
test, while differences in EBV DNA levels among the three groups of
individuals were analyzed by the Kolmogorov-Smirnov two-sample test for
nonparametric sample distribution (Systat for Macintosh; Systat Inc.,
Evanston, Ill.).
| |
RESULTS |
Quantification of EBV DNA by Q-PCR.
In repeated PCR
amplifications of serial dilutions of pP3 (external standard) and pRM
(internal control), it was shown that 10 copies of each molecule could
be reproducibly detected. Thus, this was defined as the cutoff of our
PCR method. In addition, by coamplifying serial concentrations of the
two molecules as well as fixed amounts of pRM and serial concentrations
of pP3, it was demonstrated that gel signals of identical intensity
were consistently obtained following PCR amplification of the same pP3
and pRM copy numbers (Fig. 1). Thus, the
two molecules showed comparable PCR kinetics and were amplified with
similar efficiency. EBV DNA was quantified according to a previously
reported method (6) by comparing the gel signals of the PCR
products from the clinical samples with those obtained from the
external standards. The use of a fixed amount of pRM (100 copies) as an
internal control of amplification allowed for detection of PCR
inhibitors in clinical samples and verification of PCR efficiency in
each tube, thus allowing normalization of the EBV DNA results during
densitometric analysis. The pP3/pRM (100 copies) gel signal ratios were
used to plot a standard curve for each test run. Quantification of EBV
DNA GE in clinical samples was obtained by interpolating EBV EBNA-1/pRM
(100 copies) gel signal ratios (Fig. 1). PBMC samples containing
104 GE were diluted 1:10 or 1:100 and then quantitated by
the same procedure. Thus, DNA quantification was also achieved in the
range of 104 to 106 GE.
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FIG. 1.
Construction of the standard curve. (A) pP3 copy number,
ranging from 10,000 to 1 (external standards); pRM, 100 copies for each
sample (internal control). MW, molecular weight markers (pBR322 cleaved
with HaeIII). (B) Standard curve obtained by densitometric
analysis of gel signals. (C) Example of EBV DNA quantification in
sequential PBMC samples from patient (pt) 5 (from left to right, 7,420, 11,520, and 70 GE) and pt 9 (from left to right, 1,200, 3,980, and
38,900 GE) (see Table 3).
|
|
EBV DNA prevalence and quantification.
Significant differences
in the prevalence of EBV DNA in the blood were found among the three
groups of subjects studied (Table 1). All
patients with symptomatic EBV infections (both adults and children)
were EBV DNA positive, whereas about half of the asymptomatic SOT
recipients had EBV DNA in their blood (P < 0.001). Among asymptomatic SOT recipients, pediatric patients showed a slightly
higher prevalence of EBV DNA than adults. However, this difference was
not significant. In addition, for asymptomatic patients, no difference
was observed in either the prevalence of DNA positivity or EBV DNA
levels with respect to time after transplantation. Specifically, at 
2
years, 2 to 5 years, and >5 years after transplantation, 11 of 21 (52.3%), 21 of 36 (58.3%), and 27 of 48 (56.2%) patients were
positive for EBV DNA, respectively. At the same times, median EBV DNA
values were 10 (range, <10 to 1,500), 10 (range, <10 to 800), and 10 (range, <10 to 3,000), respectively. In contrast, the rate of EBV DNA
positivity in the blood was very low in immunocompetent healthy
individuals compared to transplant patients (P < 0.001).
From a quantitative standpoint, marked differences in EBV DNA levels
were observed among the three groups of subjects (Fig. 2): median EBV DNA values in
immunocompetent individuals, asymptomatic SOT recipients, and SOT
recipients with symptomatic EBV infections (values at time of first
diagnosis) were <10 (range, <10 to 70), 10 (range, <10 to 3,000),
and 5,000 (range, 100 to 126,000), respectively (P < 0.001). No significant difference between EBV DNA onset and peak
levels (7,500; range, 100 to 126,000) was observed in symptomatic patients (P > 0.05). A detailed analysis of
quantitative EBV DNA levels in the three groups of subjects is shown in
Table 2. The large majority of
immunocompetent individuals were negative for EBV DNA, while the two
EBV DNA-positive subjects had very low EBV DNA levels (<100 EBV GE).
Fewer than half of the asymptomatic SOT recipients were EBV DNA
negative (46 of 105; 43.8%). However, the great majority of
asymptomatic EBV DNA-positive patients had low EBV DNA levels in their
blood (1,000 EBV GE). Only 6 of 105 (5.7%) asymptomatic SOT
recipients had >1,000 EBV GE. In contrast, the majority of symptomatic
patients (11 of 15; 73.3%) had >1,000 EBV GE in their blood at the
onset of clinical symptoms. On the other hand, 4 of 15 (26.6%)
symptomatic patients had low EBV DNA values (10 to 1,000 EBV GE). The
positive predictive value (PPV), which was calculated by comparing
patients with PTLD to asymptomatic transplant patients, increased with
increasing EBV DNA levels (Table 2). By combining data from all
patients with >1,000 EBV GE, the PPV was 64.7%, while the negative
predictive value (NPV) was 96.1%. However, the NPV was always
84.5%, thus confirming the direct correlation between the presence
of EBV DNA and the emergence of PTLD (Table 2).
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FIG. 2.
EBV DNA levels in the blood of the three population
groups studied. Median levels are indicated by horizontal lines.
Statistical differences between groups are given at the top.
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TABLE 2.
EBV DNA levels in immunocompetent individuals,
asymptomatic SOT recipients, and SOT recipients with symptomatic
EBV infections
|
|
Clinical outcome and virologic follow-up.
Virologic monitoring
of 32 asymptomatic SOT recipients who were EBV DNA positive at the time
of enrollment showed some fluctuations in EBV DNA levels over time
(Fig. 3). Nineteen of these 32 patients (59.3%) showed EBV DNA levels consistently below 1,000 GE for as long
as 16 months (Fig. 3A, lower curve), while 10 (31.2%) had at least one
sample with EBV DNA values in the range of 1,000 to 5,000 GE (Fig. 3A,
upper curve), and 3 (9.3%) showed 5,000 EBV GE in their blood at
least once during follow-up (Fig. 3B). In two of these patients, DNA
levels of 5,000 GE were often detected, and after the 1-year
follow-up one of these patients developed a febrile syndrome
concomitantly with the appearance of an EBV DNA level of 10,000 GE in
the blood (Fig. 3B).
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FIG. 3.
Follow-up of EBV DNA levels in 32 SOT recipients with
asymptomatic EBV infections. (A) EBV DNA levels (means ± standard
deviations) in 19 patients consistently showing <1,000 GE during
follow-up ( ) and in 10 patients with at least one blood sample in
the range of 1,000 to 5,000 GE during follow-up ( ). (B) Individual
fluctuations of EBV DNA levels in three patients showing 5,000 GE in
the blood at least once during follow-up.
|
|
A summary of the clinical and demographic characteristics of the 15 SOT
recipients with symptomatic EBV infections is given in Table
3. Six heart transplant recipients
developed febrile illness, and one liver transplant recipient
experienced a mild elevation of liver function test results. Patients
were diagnosed on the basis of EBV DNA positivity in the blood in the
absence of other possible causative agents, as assessed after extensive clinical, radiological, and microbiological analyses. Among the eight
patients with more-severe disease, five had non-Hodgkin's lymphoma
(NHL), two had disseminated lymphoproliferative disease with multiple
organ involvement, and one showed B-cell lymphoproliferation at
multiple node sites. The mean time to development of EBV-associated clinical symptoms was 5.3 (range, 1.1 to 9.9) years after
transplantation, and no significant difference was observed between
patients with mild and severe disease (5.9 ± 3.2 versus 4.7 ± 1.9 years). Although the seven patients with mild EBV infections had
lower mean levels of EBV DNA in their blood than the eight patients
with more-severe presentations (4,885.7 ± 4,880.7 versus
19,636.6 ± 32,640.0), the difference was not significant
(P > 0.05 by the Kolmogorov-Smirnov test).
The six patients with febrile illness and the single patient with liver
function test abnormalities were treated solely by reduction of their
immunosuppressive regimens, and all the patients improved clinically.
From the virological standpoint, modulation of immunosuppressive
treatment was effective in drastically reducing EBV DNA levels in the
blood of each patient. Four patients showed clearance of EBV DNA from
the blood, while one patient had a 2-log10 decrease and one
patient had a 1-log10 decrease in the level of EBV DNA in
the blood. The median time to clearance or 1-log10 decrease in the EBV DNA load was 3.2 (range, 1.1 to 5.8) months following reduction of the immunosuppressive regimen. Virological follow-up data for four of the most representative patients are shown
in Fig. 4 (patients 4, 5, 6, and 8).
Determination of FK506 plasma levels for patient 9 documented parallel
decreases in levels of the immunosuppressive drug and EBV DNA in the
blood (Fig. 5).
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FIG. 4.
Effect of modulation of immune-suppressive therapy on
EBV DNA levels in the blood of four SOT recipients with symptomatic EBV
infections (see Table 3). The vertical dotted line indicates the
initial time of reduction of the immunosuppressive regimen. pt,
patient.
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FIG. 5.
Parallel reductions in FK506 plasma levels and the EBV
DNA load in the blood of an SOT recipient (patient 9 [see Table 3])
with a symptomatic EBV infection.
|
|
All five patients with B-cell NHL (Table 3) were treated by reduction
of immunosuppressive medication in association with the administration
of acyclovir (patients 11, 13, and 14), alpha interferon (patient 3),
or chemotherapy (patient 15). Three of these patients died shortly
after diagnosis (median survival, 5.4 months; range, 1.1 to 12.6 months), while two patients who showed reductions in EBV DNA levels in
the blood were still alive 3.3 and 7.4 months after diagnosis,
respectively, with clinical improvement. One patient who developed a
PTLD presentation involving multiple lymph nodes (patient 12) was
treated by reduction of the immunosuppressive regimen and intravenous
(i.v.) administration of acyclovir (30 mg/kg of body weight/day) but
eventually died 12 days after diagnosis. Finally, two patients
(patients 7 and 10) developed disseminated disease which led to death
0.37 and 4.4 months after diagnosis, respectively, despite complete
withdrawal of immunosuppression and administration of i.v. acyclovir
for one (patient 7) and a marked decrease in immunosuppression and administration of alpha interferon for the other (patient 10). Although
clinically useless (both patients died), administration of alpha
interferon in association with modulation of the immunosuppressive regimen was followed by sharp decreases in EBV DNA levels (>1 log10 unit) in both patients.
| |
DISCUSSION |
PTLD is characterized by a wide spectrum of manifestations, and it
appears particularly important to diagnose PTLD when it is still in the
polyclonal phase. In fact, current therapeutic approaches include
reduction of immunosuppressive therapy in order to allow the
recipient's immune system to control EBV-induced proliferation
episodes, as occurs in immunocompetent individuals (27).
Treatment of EBV-induced clonal malignancies has proven to be more
difficult, since both the greater tumor load and possible variations in
the expression of EBV latency proteins may impair B-cell proliferation
control and result in poor immune surveillance. Recently, EBV-specific
cytotoxic T-cell lines expanded from pretransplant blood samples of SOT
recipients have been shown to be an effective therapeutic tool in the
posttransplant period (8). However, this treatment approach
appears more feasible in the BMT setting (13, 22), given the
high incidence of PTLD in T-cell-depleted BMT recipients in the early
posttransplant period (13, 14, 22). Generation, storage, and
utilization of EBV-specific cytotoxic T-cell lines for SOT recipients
appear more problematic, given the lower incidence and the later
appearance of PTLD in these patients (2, 4, 9, 15, 16, 19,
27). Thus, the availability of a simple and reliable assay for
timely detection of PTLD in transplant recipients is a pressing need.
In a previous study, Riddler et al. found an association between high
EBV DNA copy numbers and PTLD in SOT recipients (21). More
recently, a cutoff of >500 EBV GE/105 lymphocytes was
indicated as associated with PTLD in pediatric SOT recipients, while
asymptomatic SOT recipients were mostly EBV DNA negative or showed very
low (<200 GE) EBV DNA levels (23). In a third study, using
a semiquantitative PCR, it was shown that not all BMT patients with
elevated levels of EBV DNA developed PTLD, but all PTLD patients had
high EBV DNA levels (13). Although there might be
differences in the threshold values related to each method, the
emerging pattern indicates that PTLD is associated with a high EBV DNA
load in peripheral blood, as one would expect when considering the
proposed pathogenesis of this disease. This finding is clinically
relevant, and it appears most important to differentiate between
patients showing self-limiting reactivations and patients progressing
toward an aggressive disease in order to avoid unnecessary modification
in the immune-suppressive regimen. However, to date no definite cutoff
values predictive for the development of PTLD have been established for
SOT and BMT recipients.
We developed a simple and reproducible assay allowing EBV DNA
quantitation in the range of 101 to 106 EBV GE.
Striking differences in the prevalence of EBV DNA positivity in the
blood were observed among the three groups of subjects studied. In
fact, all symptomatic patients were EBV DNA positive, whereas about
half of the asymptomatic SOT recipients and nearly all the
immunocompetent individuals were EBV DNA negative. These results
confirm a close association between PTLD and the replication of
EBV-infected cells in peripheral blood and, together with data presented by Rowe et al. (23), indicate a marked influence
of long-term immune suppression on enhanced replication of EBV-infected cells. No difference was observed between adult and pediatric patients.
Quantitation of EBV DNA confirmed the finding by Lucas et al.
(13) that a few patients with elevated EBV GE counts did not
develop PTLD. Moreover, it was shown that a minority of patients with
symptomatic EBV infection had low EBV DNA copy numbers at the onset of
symptoms. However, the data clearly demonstrated that the PPV increased
with increasing levels of EBV DNA in the blood, reaching 64.7% with
EBV DNA levels of >1,000 GE. In addition, the virological follow-up of
a subset of asymptomatic EBV DNA-positive SOT recipients showed large
fluctuations in EBV DNA levels over time. However, levels were
consistently below 1,000 EBV GE in the majority of asymptomatic
patients, while about one-third of them showed transiently elevated
values. Finally, EBV DNA levels of >5,000 GE were detected repeatedly
in only a few asymptomatic patients (3 of 32; 9.3%), and one of these
patients developed a symptomatic EBV infection.
These results suggest that the extent of EBV replication in SOT
recipients is greater than that in immunocompetent individuals due to
long-term administration of immunosuppressive treatment. However, it
appears that the enhanced EBV-induced B-cell replication can still be
controlled by the patients' immune systems for a long time, at least
below a threshold level. Our results also indicate that this threshold
is apparently settled at 1,000 EBV GE and that a grey zone might be
identified between 1,000 and 5,000 EBV GE. Thus, patients with EBV DNA
values in this range may remain asymptomatic or, if the immune system
is no longer able to control EBV replication, may progress to PTLD.
Detection of these high-risk patients may be helpful for timely
therapeutic intervention. In fact, in our series, the best results with
immunosuppressive-regimen modulation were obtained for patients with
lower EBV DNA counts, in the absence of severe manifestations of PTLD.
In conclusion, our Q-PCR assay appears useful for the diagnosis and
monitoring of EBV-induced lymphoproliferative diseases in SOT
recipients. In addition, on the basis of our results, it appears
reasonable to suggest a routine prospective virologic follow-up of all
SOT recipients in order to better understand the kinetics of EBV
infection in these patients and to modulate the immunosuppressive
regimens accordingly in a timely manner. Adoptive transfer of
EBV-specific cytotoxic T lymphocytes may be attempted and may represent
the basis for the adoption of specific therapeutic strategies.
| |
ACKNOWLEDGMENTS |
We thank Lucia Chezzi, Cinzia Zanello, and Luca Dossena for
excellent technical assistance and Linda D'Arrigo for English revision.
This work was partially supported by IRCCS Policlinico S. Matteo,
Ricerca Corrente grant 820RCR95/01, Ricerca Finalizzata grant 390 RFM
97/01, and Progetto Finalizzato CNR, grant 97.01256.PF49.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Servizio di
Virologia, IRCCS Policlinico San Matteo, 27100 Pavia, Italy. Phone:
39-0382-502644/633. Fax: 39-0382-502599. E-mail:
g.gerna{at}smatteo.pv.it or
virology{at}ipv36.unipv.it.
| |
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Journal of Clinical Microbiology, February 2000, p. 613-619, Vol. 38, No. 2
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
