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Blood, Vol. 93 No. 11 (June 1), 1999:
pp. 3949-3955
Direct Epstein-Barr Virus (EBV) Typing on Peripheral Blood Mononuclear
Cells: No Association Between EBV Type 2 Infection or
Superinfection and the Development of Acquired Immunodeficiency
Syndrome-Related Non-Hodgkin's Lymphoma
By
Debbie van Baarle,
Egbert Hovenkamp,
Marie José Kersten,
Michèl R. Klein,
Frank Miedema, and
Marinus H.J. van Oers
From the Department of Hematology, Academical Medical Center,
Amsterdam; the Department of Clinical Viro-Immunology, CLB, Sanquin
Blood Supply Foundation, Laboratory for Experimental and Clinical
Immunology, Academical Medical Center, University of Amsterdam,
Amsterdam; the Department of Immunology and Medical Oncology,
Netherlands Cancer Institute, Antoni van Leeuwenhoek Ziekenhuis,
Amsterdam; and the Department of Human Retrovirology, Academical
Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
 |
ABSTRACT |
In the literature, a correlation has been suggested between the
occurrence of acquired immunodeficiency syndrome (AIDS)-related non-Hodgkin's lymphomas (NHL) and Epstein-Barr virus (EBV) type 2 infection. To further investigate a possible role for EBV type 2 infection in the development of AIDS-NHL, we developed a sensitive and
type-specific nested polymerase chain reaction (PCR) assay and analyzed
EBV types directly on peripheral blood mononuclear cells (PBMC) in
three subgroups of human immunodeficiency virus (HIV)-1 infected
individuals: 30 AIDS-NHL patients, 42 individuals progressing to
AIDS without lymphoma (PROG), either developing opportunistic infections (AIDS-OI) or Kaposi's sarcoma (AIDS-KS), and
18 long-term asymptomatic individuals (LTA). Furthermore, EBV type
analysis was performed on PBMC samples obtained from AIDS-NHL patients
in the course of HIV-1 infection. The results showed that: (1) direct
analysis of PBMC is superior to analysis of B-lymphoblastoid cell lines
(B-LCL) grown from the same PBMC samples; (2) in HIV-1 infected
individuals, there is a high prevalence of EBV type 2 infection (50%
in LTA, 62% in progressors, and 53% in AIDS-NHL) and superinfection
with both type 1 and 2 (24% in LTA, 40% in progressors, and 47% in
AIDS-NHL); (3) EBV type 2 (super)infection is not associated with an
increased risk for development of AIDS-NHL; (4) type 2 infection can be found early in HIV-1 infection, and neither type 2 infection nor superinfection correlates with a failing immune system.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
EPSTEIN-BARR VIRUS (EBV) is a widespread
human gamma herpes virus, which selectively infects two types of target
cells, ie, squamous epithelial cells in the oropharynx and B
lymphocytes.1 After primary infection, which usually occurs
asymptomatically, the virus persists for life in a latent form in the B
lymphocytes.2 Reactivation of these latently infected B
lymphocytes is controlled by specific cytotoxic T-lymphocyte
responses.3 During immunodeficiency, reactivation of
EBV-infection can lead to uncontrolled
lymphoproliferation.4 In human immunodeficiency virus
(HIV)-1-infected individuals, the majority of acquired
immunodeficiency syndrome (AIDS)-related diffuse large cell
non-Hodgkin's lymphomas (NHL) is EBV-positive and is thought to arise
because of loss of EBV-specific T-cell immunity.5-7
Two different EBV types are distinguished based on polymorphisms in the
genes encoding the nuclear antigens EBNA-2,8,9 which is
required for transformation of B lymphocytes, and EBNA-3A, -3B, and
-3C.10 These different virus types have been classified as
type 1 and 2 EBV and show distinct biological
differences.8,11 These differences are reflected in the
reduced transforming capacity of type 2 viruses.12
In healthy individuals, both virus strains can be found in epithelial
cells in the oropharynx.13 Yet, it has been reported that
only one strain is present in the peripheral blood.13,14 Type 1 strains are more prevalent in Caucasian and Asian
populations, whereas both types are common in Africa and New
Guinea.9,11,15,16 However, in HIV-1-infected individuals,
it has been shown that the peripheral blood B lymphocytes frequently
harbor EBV type 2 and that a high percentage of the AIDS patients has a
dual infection with type 1 and 2.17,18 A cross-sectional
study in patients with AIDS-related NHL has shown an equal prevalence
of type 1 and 2 in the tumors.19 It has been suggested that
loss of EBV-specific T-cell immunity may predispose immunocompromised
individuals to superinfection with other EBV strains.17
To study persistent EBV infection in normal and immunocompromised
individuals, biological assays have been used that are based on the
spontaneous outgrowth of EBV-transformed B
lymphocytes.20,21 These EBV-transformed B-lymphoblastoid
cell lines (B-LCL) can be EBV-typed by polymerase chain reaction (PCR)
amplification across the polymorphic region of EBNA-2 or
EBNA-3A.10 Because type 2 strains have a reduced
transforming capacity, these biological assays most likely
underestimate type 2 prevalence.22 In addition, these
assays are time-consuming and vary widely in sensitivity. Therefore, we
developed a sensitive and EBV type-specific PCR assay that can be used
directly on peripheral blood mononuclear cell (PBMC) samples without
the need of prior culture. Using this assay, we have determined EBV
types in HIV-1 seropositive individuals directly on PBMC and compared
the results with conventional EBV-typing on spontaneously established
B-LCL.
We have used the sensitive and type-specific PCR assay to investigate
the role of type 2 EBV and superinfection in the development of
AIDS-NHL. First, in a cross-sectional analysis, we investigated EBV-type prevalence in three groups of HIV-1-infected individuals: AIDS-NHL patients, progressors to AIDS without lymphoma, and long-term asymptomatic individuals. Second, in the longitudinal part of the
study, EBV type analysis was performed on PBMC samples obtained from
AIDS-NHL patients early and late in the course of HIV-1 infection.
| |
MATERIALS AND METHODS |
Study population.
This study was performed on participants of the Amsterdam Cohort
studies on AIDS and HIV-1 infection and HIV-1-infected individuals visiting the Academical Medical Center (AMC). Blood samples from these
(homosexual) individuals at risk for HIV-1 infection were collected
every 3 months for HIV-1 serology and immunologic studies. In addition,
at all time points PBMC were cryopreserved. Individuals who were
HIV-seronegative at entry of the cohort study and seroconverted during
follow-up, were classified as seroconverters. Patients from the AMC and
individuals who were already HIV-seropositive at entry in the cohort
were classified as seroprevalent individuals.
We analyzed 30 patients with AIDS-related diffuse large cell NHL
(median follow-up, 49 months). In comparison, 42 cohort participants who progressed to AIDS (classification of the Centers for Disease Control 1993) without a lymphoma (progressors, [PROG]) within 7 years
after seroconversion (median seropositive follow-up, 54 months) were
studied. Of these progressors, 29 developed an opportunistic infection
(AIDS-OI), and 13 developed Kaposi's sarcoma (AIDS-KS). Furthermore,
18 HIV-1 seropositive long-term asymptomatic individuals (LTA) with
CD4+ T-cell counts above 500/µL during more
than 8 years of seropositive follow-up (median follow-up, 148 months)
were studied. Characteristics of the HIV-1-infected individuals are in
part described elsewhere23 and are summarized in
Table 1.
B-cell lines.
Spontaneous EBV-transformed B-LCL were established as previously
described.7,24,25 Briefly, PBMC were thawed, resuspended in
RPMI 1640 (GIBCO-BRL, Gaithersburg, MD) supplemented with L-glutamine, antibiotics, 10% fetal calf serum (FCS, Hyclone, UT) and cyclosporin A
(CsA, final concentration 0.1 µg/mL; Sandoz, Basel,
Switzerland) and cultured in limiting dilution at 6 serial
dilutions with concentrations ranging from 0.5 × 106
to 1.5 × 104 cells per well (6 replicate cultures per
dilution) in a 96-well microtiter plate. Cells were fed weekly with
RPMI/10% FCS supplemented in the first weeks with CsA. Wells that
showed outgrowth of EBV-transformed B lymphocytes, as monitored
microscopically, were expanded.
The B95.8 cell line and the Ag876 cell line were used as EBV type 1 and
2 positive controls, respectively. The EBV-negative B-cell line BJAB
was used as a negative control.
Anti-CD3 proliferation assay.
T-lymphocyte reactivity to CD3 monoclonal antibody (CLB-T3/4E, CLB,
Amsterdam, The Netherlands) was determined in a whole-blood lymphocyte
culture assay and expressed as cpm per 103 CD3+
T lymphocytes.26
DNA extraction.
Cells were collected, washed with phosphate-buffered saline (PBS), and
lysed by addition of L6-lysis buffer.27 After incubation for 30 minutes at room temperature, DNA was precipitated by addition of
isopropanol, washed twice with 70% ethanol, and dissolved in dH2O. DNA concentration was measured by optical densimetry
at 260 nm.
PCR for EBV typing on B-LCL and PBMC.
Genomic DNA extracted from B-LCL and PBMC (100 ng in case of DNA from
B-LCL and control cell lines, 1 or 2 µg in case of DNA from PBMC) was
amplified in 50-µL reactions containing 5.0 µL 10X PCR buffer (100 mmol/L Tris-HCl pH 8.3, 500 mmol/L KCl, and 0.01% wt/vol gelatin), 1.5 mmol/L MgCl2, 10 mmol/L deoxy nucleoside triphosphate
(dNTPs; Promega, Madison, WI; 2.5 mmol/L each), 1 U DNA
Taq polymerase (Promega), and 100 ng of the EBNA-2 primers. For the
amplification of EBNA-2-specific DNA extracted directly from PBMC, a
nested PCR was performed using primers EBNA-2I and EBNA-2F in the first
reaction. The region within the EBNA-2 gene discriminating between EBV
type 1 and 2 was amplified with the nested primers EBNA-2C and EBNA-2G
for type 1 and EBNA-2C and EBNA-2B (kindly provided by A.B. Rickinson,
CRC Institute for Cancer Studies, University of Birmingham,
Birmingham) for type 2. For the amplification of
EBNA-2-specific DNA from B-LCL, a single PCR was performed using the
nested primers only. For primer sequences, see
Table 2.
For the first reaction of this nested PCR, denaturation was performed
at 94°C for 1 minute, the primers annealed at 52°C for 90 seconds, and extension was performed at 72°C for 4 minutes. For the
second reaction of the nested PCR, denaturation was performed at
94°C for 30 seconds, primer annealing at 52°C for 1 minute, and
extension at 72°C for 2 minutes. The cycles were repeated 35 times
followed by a final extension time of 10 minutes. The whole procedure
was automated using a thermal cycler (Amersham [Roosendaal, The
Netherlands] and Perkin Elmer [Foster City, CA]). After
PCR, the identity of the amplified EBV fragment, 250 bp for EBV type 1 and 300 bp for EBV type 2, was confirmed by Southern blot analysis.
Southern blot analysis.
PCR products were separated on 2% agarose gels and transferred to
Genescreen plus (NEN Life Science Products, Boston, MA) membranes.
Membranes were hybridized with either an EBNA-2 type 1-specific probe,
EBNA-2.1, or an EBNA-2 type 2-specific probe, EBNA-2.2, labeled with
 32P-deoxyadenosine triphosphate (dATP). Sequences of
both oligonucleotide probes (kindly provided by A.B. Rickinson) are
depicted in Table 2.
Statistical analysis.
For statistical analysis,  2 tests and the Kruskal-Wallis
test were performed using the software program SPSS version 7.5 for Windows (SPSS Inc, Chicago, IL).
| |
RESULTS |
Sensitivity and type-specificity of the nested PCR.
To analyze the EBV type directly on PBMC, a nested PCR assay was
developed. Using the EBV type 1 and 2 positive cell lines, B95.8 and
Ag876, the optimal conditions for the PCR assay were determined.
Dilution of both cell lines in an EBV-negative background gave an
estimation of the sensitivity. Type 2 DNA from the Ag876 cell line
could still be detected at a dilution of 10 cells in a background of 2 × 105 EBV-negative cells
(Fig 1A). Type 1 DNA from the B95.8 cell
line, which contains more EBV copies per cell than the Ag876 cell line, could still be detected at a dilution of 1 cell in a background of 2 × 105 EBV-negative cells (Fig 1B). Similar data were
obtained when diluting the B95.8 cell line in a type 2 background or
diluting the Ag876 cell line in a type 1 background (data not shown).
On all occasions, EBV type 1 DNA was not detected in the type 2 PCR and
type 2 DNA was not detected in the type 1 PCR. Negative controls (EBV-negative B-cell lines, PBMC from an EBV-negative individual) were
indeed always negative.
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| Fig 1.
Sensitivity of the nested PCR for EBV-typing on PBMC. PCR
analysis was performed on sequential dilutions of the EBV-positive cell
lines, B95.8 and Ag876. (A) Dilution of Ag876 (number of cells) in a
total of 2 × 105 EBV-negative background cells and (B)
dilution of B95.8 (number of cells) in a total of 2 × 105
EBV-negative background cells.
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Comparative analysis of EBV types in B-LCL and PBMC.
A total of 76 B-LCL were grown from PBMC of nine HIV-1-infected
individuals and typed by PCR amplification across the polymorphic locus
of EBNA-2 using a single PCR assay. In parallel, PBMC samples obtained
at the same time point from the same individuals were analyzed for the
presence of EBV type 1 and 2 using the nested PCR assay.
EBV type analysis of B-LCL showed that 7 of 9 HIV-1-infected
individuals harbored only type 1 EBV; 1 patient harbored only EBV type
2 and 1 showed dual infection with both types
(Table 3). In contrast, EBV type analysis
performed on PBMC showed that only 4 of these HIV-1-infected subjects
harbored only EBV type 1. The other 5 individuals harbored both type 1 and type 2.
Analysis of B-LCL would have suggested that only 2 of 9 individuals had
a type 2 infection, whereas direct EBV-typing of PBMC showed that 5 of
9 individuals were infected with EBV type 2. Interestingly, in the
dually infected AIDS-NHL patient N319, the type 1 strain detected in
PBMC could not be shown by analyzing EBV types in B-LCL. Thus, using
B-LCL not only the EBV type 2 prevalence would have been
underestimated, but also the prevalence of superinfection.
EBV type 2 infection and superinfection in different groups of
HIV-infected individuals.
Using the sensitive and type-specific nested PCR, EBV-typing was
performed on PBMC from different groups of HIV-1-infected individuals:
patients with AIDS-NHL (n = 30), progressors to AIDS (OI/KS, n = 42),
and LTA (n = 18). From the LTA, the PBMC samples analyzed had been
obtained in the ninth year of follow-up when CD4+ T-cell
counts of 10 individuals were still above 500/µL. In case of
progressors and AIDS-NHL patients, PBMC samples were analyzed either at
AIDS-OI/KS diagnosis or AIDS-NHL diagnosis, respectively, or in the
year preceding diagnosis.
A total of 90 HIV-1-infected individuals was studied. Patient
characteristics of all groups are summarized in Table 1. In 6% of the
individuals, no EBV could be detected. As shown in
Fig 2A, only EBV type 1 was found in 44%
of the LTA, 33% of the progressors, and 40% of the AIDS-NHL patients.
Only EBV type 2 infection was found in 28% of the LTA, 21% of the
progressors, and in 7% of AIDS-NHL patients. The total percentage of
type 2 infected individuals, which includes superinfected individuals,
was comparable between the three groups (50% in LTA, 62% in
progressors, and 53% in AIDS-NHL). However, the percentage of
superinfected individuals was significantly higher both in AIDS-NHL
(47%) and progressors (40%) when compared with LTA (24%, P < .005, 2). There was no difference in superinfection
between the total group of progressors and AIDS-NHL patients.
Interestingly, when the progressors to AIDS were subdivided into
separate groups: AIDS-OI (n = 29) and AIDS-KS (n = 13), the percentage
of superinfected individuals was significantly higher in AIDS-KS, as
compared with AIDS-OI (P = .017, 2, see Fig 2B).
The percentage of superinfected individuals was similar in AIDS-OI and
LTA, whereas in both AIDS-KS and AIDS-NHL, the percentage of
superinfected individuals was significantly higher than in LTA
(P < .001).
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| Fig 2.
EBV types in subgroups of HIV-1-infected individuals.
(A) Contribution of EBV type 1 ( ), EBV type 2 ( ), and dual
infection ( ) in the total percentage of EBV-positive individuals in
different groups of HIV-1-infected subjects (LTA, PROG, and NHL).
EBV-type assessment was performed by type-specific PCR analysis
directly on PBMC as described in Materials and Methods. (B)
Contribution of EBV type 1 (), EBV type 2 (), and dual infection
() in the total percentage of EBV-positive individuals in two groups
of progressors to AIDS (AIDS-OI and AIDS-KS). EBV-type assessment was
performed by type-specific PCR analysis directly on PBMC as described
in Materials and Methods.
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To analyze whether the occurrence of type 2 infection or superinfection
is related to the degree of immunodeficiency of the HIV-1-infected
individuals, we studied both CD4+ T-cell counts and T-cell
function.28,29 CD4+ T-cell counts for LTA were
higher than for progressors to AIDS (OI/KS and NHL, P = .015, Kruskal-Wallis test). When CD4+ T-cell counts
at the time point of EBV typing were plotted against the EBV strain(s)
that was (were) detected, there was no statistically significant
difference between CD4+ T-cell counts of type 1 (mean, 0.44 ± 0.21), type 2 (mean, 0.45 ± 0.24), and superinfected
individuals (mean, 0.41 ± 0.16), as shown in
Fig 3A.
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| Fig 3.
EBV type infection in relation to immune status. (A) CD4
counts are depicted for every HIV-1-infected individual harboring EBV
type 1 (type 1), EBV type 2 (type 2), or harboring both type 1 and 2 (type 1+2). Mean CD4 count per group is depicted by the long thin
lines, standard deviation by the short thin lines. No statistically
significant difference was found between the groups (Kruskal-Wallis).
(B) Anti-CD3 responses expressed as cpm/1,000 T cells are depicted for
48 HIV-1-infected individuals harboring EBV type 1 (type 1), EBV type
2 (type 2), or harboring both type 1 and 2 (type 1+2). Mean anti-CD3
response per group is depicted by the long thin lines, standard
deviation by the short thin lines. No statistically significant
difference was found between the groups (Kruskal-Wallis). ( ), LTA;
( ), progressors; ( ), NHL.
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Because the T-cell response to mitogens may be a more sensitive marker
for HIV-1-induced immune dysfunction than CD4+ T-cell
counts,28,29 the anti-CD3 response was studied in vitro from 47 of the 90 individuals at the time point of EBV typing. In Fig
3B, the anti-CD3 responses (expressed as cpm per 1,000 T lymphocytes)
at the time point of EBV-typing were plotted against the EBV strain(s)
that was (were) detected. No difference was found between the anti-CD3
response of type 1 (mean, 49 ± 33), type 2 (mean, 66 ± 49), and
superinfected individuals (mean, 33 ± 24).
EBV superinfection in the course of HIV-1 infection.
From 19 individuals who developed AIDS-NHL, PBMC samples obtained from
early and late time points in the course of HIV-1 infection were
studied to investigate a possible role of type 2 (super)infection in
the development of AIDS-NHL. In case of the seroconverter, PBMC samples
were studied at HIV seroconversion (early) and at AIDS-NHL diagnosis
(late). In case of seroprevalent individuals, PBMC samples obtained
either at entry in the Amsterdam Cohort Studies or at the first
hospital visit (early) and at AIDS-NHL diagnosis (late) were studied.
EBV typing using the nested PCR assay showed that a considerable
proportion of the individuals studied (50%) were infected with type 2 EBV already early in HIV-1 infection (Fig
4). Moreover, half of the type 2 infected subjects had a superinfection
already early. The percentage of AIDS-NHL patients that were
superinfected early was comparable to the percentage of superinfected
LTA individuals. In the course of time, there was a statistically
significant increase in superinfection (P = .009), with either
type 1 or type 2, depending on the strain present initially: 3 patients
harbored a type 2 strain early in HIV-1 infection and were
superinfected with a type 1 strain late in HIV-1 infection; 2 patients
harbored a type 1 strain at an early time point and were superinfected
with a type 2 strain at a late time point in HIV-1 infection. No loss of EBV strains was observed. Thus, superinfection in the course of
HIV-1 infection does occur, but can involve both type 1 and type 2.
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| Fig 4.
Longitudinal analysis of EBV types in AIDS-NHL patients.
Contribution of EBV type 1 (), EBV type 2 (), and dual infection
() in the total percentage of EBV-positive AIDS-NHL patients in
early and late PBMC samples in the course of HIV-1 infection. EBV type
discrimination was performed by type-specific PCR analysis on PBMC as
described in Materials and Methods.
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| |
DISCUSSION |
In the present study, we have used a sensitive and type-specific nested
PCR to analyze EBV types in subgroups of HIV-1-infected individuals.
We have reached the following conclusions: (1) direct analysis of PBMC
may be superior to analysis of B-LCL grown from the same PBMC samples;
(2) in HIV-1-infected individuals, there is a high prevalence of EBV
type 2 infection and superinfection with both type 1 and 2; (3) there
is no evidence for an association between EBV type 2 infection or
superinfection and an increased risk for development of AIDS-NHL; and
(4) EBV type 2 infection or acquisition of superinfection is not merely
a reflection of a failing immune system in HIV-1-infected individuals.
The EBV type 2 and superinfection prevalence found in the present study
is higher than reported in studies analyzing EBV-types on
B-LCL.17,18,30 The underestimation of EBV type 2 found by
analysis of B-LCL is probably due to culture bias because the transforming capacity of type 2 strains is reduced. This finding stresses the importance of using direct PCR assays for EBV detection in
clinical samples. This issue has also recently been addressed by Haque
et al.31 Furthermore, if we would have studied B-LCL only,
we would have found a possible correlation between EBV type 2 infection
and AIDS-NHL because the two type 2-infected individuals identified in
that assay were both AIDS-NHL patients (see Table 3). This would have
been in agreement with the literature.6 In contrast,
EBV-type analysis on PBMC of these same patients showed that 3 of the 5 type 2-infected individuals were AIDS-NHL patients and 2 were AIDS-OI
patients, a result not suggestive of a correlation between EBV type 2 infection and the development of AIDS-NHL. Therefore, studies performed
on B-LCL, which suggest an association between EBV type 2 and the
development of lymphoma, may be of limited value.
Furthermore, to our knowledge, this is the first study evaluating the
possible pathogenic role of EBV type 2 infection and superinfection by
comparing three large subgroups of HIV-1-infected individuals by
direct EBV type analysis on PBMC: AIDS-NHL patients, progressors to
AIDS-OI/KS, and LTA individuals. By studying these groups, no relation
could be found between either type 2 infection or superinfection and
the development of AIDS-NHL.
In the longitudinal study, five AIDS-NHL patients showed acquisition of
superinfection, either with type 1 or 2. If this finding would be
viewed separately, it might be tempting to conclude that superinfection
is the result of immunodeficiency. However, considering the other data
obtained in this study, it is clear that immunodeficiency at most
contributes to, but does not cause the superinfection. The conclusion
that immunodeficiency does not cause superinfection is based on the
fact that (1) no difference could be found in mean CD4+
T-cell counts and mean T-cell response in vitro between EBV type 1 carriers, EBV type 2 carriers, and superinfected individuals; (2) LTA
individuals, who were asymptomatic for more than 8 years with
CD4+ T-cell counts higher than 500 per µL, already showed
high prevalence of type 2 infection and superinfection; (3)
analysis of EBV-types at early and late time points in the course of
HIV-1 infection showed that the majority of the patients already harbor
EBV type 2 early in HIV-1 infection, with some of them already showing superinfection; and (4) we found high EBV type 2 and superinfection prevalence in AIDS-KS patients, who develop KS when CD4+
T-cell counts are still high (median CD4+ T-cell counts = 520/µL).32
Most likely the high type 2 prevalence found (early) in HIV-1 infection
indicates a higher type 2 prevalence in homosexual populations,
offering a new view on the epidemiology of EBV infection. This would be
in agreement with Yao et al,33 who recently
suggested a higher type 2 prevalence in homosexual populations based on the comparison of EBV-type infection in B-LCL from HIV-infected homosexuals (31% total type 2 infection) and HIV-infected
(heterosexual) hemophiliacs (10% total type 2 infection). Moreover,
the finding that EBV-type 2 prevalence is very high in AIDS-KS patients
also suggests that these infections are related to sexual behavior because KS is believed to be caused by human herpesvirus 8 (HHV8), which has been shown to be sexually transmitted.34
Until now, B-LCL-analysis has shown that 1% to 3% of healthy
Caucasian EBV carriers in Europe harbor a type 2 strain. Our data
suggest that type 2 infections are much more prevalent, especially among homosexual men. To elucidate EBV epidemiology in western Europe,
it is necessary to perform a detailed study of EBV strains in
HIV-negative homosexual and heterosexual populations using direct PCR
analysis of PBMC.
| |
ACKNOWLEDGMENT |
This study was part of the Amsterdam Cohort Studies on AIDS and HIV-1
infection, a collaboration of the Municipal Health Service, the
Academical Medical Center, and the CLB. We thank Dr M. Roos and
collaborators for immunological data.
| |
FOOTNOTES |
Submitted October 9, 1998; accepted January 23, 1999.
Supported by Grant No. 96-1168 from the Dutch Cancer Society and Grant
No. 1007 from the Dutch AIDS Fund.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Debbie van Baarle, MSc,
Department of Clinical Viro-Immunology, CLB, Sanquin Blood Supply
Foundation, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands;
e-mail: D_van_Baarle{at}clb.nl.
| |
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