Eur Arch Otorhinolaryngol (2010) 267:143148

DOI 10.1007/s00405-009-0988-6

MI SCE LLANEOUS

The correlation between EBV viral load in the palatine tonsils

of patients with recurrent tonsillitis and concurrent serum titers
of VCAIgG
Bora Dogan Seyyal Rota Levent Gurbuzler
Gulendam Bozdayi Mustafa Nuri Ceyhan
Erdogan Inal

Received: 15 June 2008 / Accepted: 23 April 2009 / Published online: 13 May 2009
Springer-Verlag 2009

Abstract EpsteinBarr virus (EBV) infection usually

occurs in early childhood and can persist in palatine tonsil
lymphocytes to induce tonsillitis at a later date. We have
examined the presence of EBV in palatine tonsils and relationship between EBV-DNA quantity in tonsil tissues and
VCAIgG quantity in autologous sera. Tonsils were
obtained from 36 patients, male 20 (55.6%), female 16
(44.4%) (mean age 7.96 6.97 years), who underwent tonsils removal because of recurrent tonsillitis. Tissues were
processed for real-time PCR and patients sera were
assayed to determine VCAIgG by VCAIgG ELISA. In
27 out of 36 cases (75%), positive EBV-DNA reaction was
found. However, statistical analysis showed no correlation
between EBV-DNA quantity and VCAIgG quantity. We
conclude that tonsils of children can be colonized by EBV
and that virus may have a direct and indirect role in recurrent tonsillitis and nasopharyngeal carcinoma.

B. Dogan (&)
Department of Clinical Microbiology,
Polatl Duatepe State Hospital, Ankara, Turkey
e-mail: drbor@yahoo.com
S. Rota G. Bozdayi
Department of Clinical Microbiology,
Gazi University, Ankara, Turkey
L. Gurbuzler
Bitlis State Hospital, Bitlis, Turkey
M. N. Ceyhan
Elazf Provincial Director of Public Health Center, Elazig, Turkey
E. Inal
Department of Otorhinolaryngology,
Gazi University, Ankara, Turkey

Keywords EBV Tonsillitis Hypertrophy

Head and neck cancer

Introduction
EpsteinBarr virus (EBV) is a virus acquired more than
90% of the population and frequently seen during childhood. Primary infection is frequently asymptomatic and
rarely infectious mononucleosis (IM). EBV stays latent
after asymptomatic or symptomatic infection and may
cause transformation in various tissues. EBV was considered to be involved in the etiology of Burkitts lymphoma
(BL), nasopharyngeal carcinoma (NPC), Hodgkins disease, post transplantation lymphoproliferative disorders
(PTLD), gastric carcinoma and the presence of EBV was
displayed [3, 79, 18].
Infectious mononucleosis is primarily a disease of the
oropharynx and tonsils and characterized by cryptic tonsillitis. EBV gains access to the B lymphocytes, the main target, usually by the tonsil epithelium and stays latent here.
Furthermore, latently infected B lymphocytes may sometimes cause the occurrence of recurrent tonsillar infections
by the establishment of lytic replication and may contaminate other healthy individuals [20]. EBV structurally
encodes numerous transformation-related latent proteins.
These proteins play a role in the transformation of the cell.
Some proteins found in the structure of EBV genome play a
role in the escape from the immune system. For instance,
the protein encoded by BCRF-1 gene is similar to IL 10.
This situation may cause decrease in the regional resistance
and recurrence of bacterial infections [14, 24].
The aim of this study is to demonstrate the presence
and quantity of EBV in tonsil tissues of the patients
operated for recurrent tonsillitis using real-time PCR,

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which is a sensitive and speciWc method; furthermore, to

interrogate the relationship between the quantity of antibody response against EBV and the quantity of DNA in
the tissue.

Eur Arch Otorhinolaryngol (2010) 267:143148

Molecular assay
DNA extraction

Samples

Tissues were weighed and pre-puriWed and the values were

recorded to be used in the quantitative analysis. Tissue
extraction protocol of QIAamp DNA mini kit (QIAGEN,
Germany) was used for puriWcation of DNA according to
manufacturers instructions.

Tonsils

Real-time PCR assay

Samples from tonsils were collected in Gazi University

Faculty of Medicine from a total of 36 patients, male 20
(55.6%), female 16 (44.4%) (mean age 7.96
6.97 years), who were subjected to tonsillectomy with a
diagnosis of recurrent tonsillitis, and were stored in PBS
at 80C until the time of study. The demographic data,
otoscopic, rhinoscopy and throat examination Wndings
belonging of the patients were recorded. Viral capsid antigen IgM (VCAIgM) was detected in the sera obtained
from the patients simultaneously and acute infection was
eliminated. Quantitative real-time PCR method was used
to asses the viral load in tonsil tissue and the correlation
between the viral load and VCAIgG titers were also
investigated.

Roche Light-Cycler EBV-DNA QuantiWcation kit was used

to amplify DNA (Catalog Number 3 330 028) (Lot Number
11304920). The study was performed by following the
instructions of the producer Wrm and one negative control
and Wve standards were included to each study. AmpliWcation procedure was carried out with the Light-Cycler 2.0
(Roche, Germany) instrument.

Materials and methods

Evaluation of the data

Data were analyzed using LightCycler Software version
3.5.3. Melting peak degrees for EBV was accepted as
62 2C (Fig. 1), for internal control it was accepted as
68 2C (Fig. 2).

Sera
Results
Sera samples were obtained just before operation and
stored at 80C until the time of study.
Serological tests
VCAIgG and IgM ELISA
Patient sera were assayed to determine IgG and IgM levels
using EBV VCAIgG/IgM ELISA (IBL, Hamburg,
Germany) kits according to manufacturers instructions and
quantitative outcomes were obtained.

Fig. 1 The appearance of EBV

positivity in LC 2.0
(Tm 62 2C)

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After the examination, the patients received three diVerent

diagnosis, such as recurrent tonsillitis, adenoid vegetation
and obstructive sleep apnea syndrome (OSAS) and the
union of these diagnosis constituted subdiagnosis groups.
The distribution of the diagnosis patients is summarized in
Table 1).
The correlation between frequency of tonsillitis and the
degree of tonsillar hypertrophy was determined using
Kendalls tau-b method, but no signiWcant result was determined (P > 0.05).

Eur Arch Otorhinolaryngol (2010) 267:143148

145

Fig. 2 LC 2.0 appearance of

internal control (68 2C)

Table 1 Distribution of the patients according to their diagnosis

Table 2 Qualitative results of PCR and IgG belonging to the patients

Diagnosis

Rate (%)

PCR

Recurrent tonsillitis

25.0

Negative n (%) Positive n (%)

Recurrent tonsillitis + adenoid vegetation

36.1

Recurrent tonsillitis + adenoid vegetation + OSAS

27.8

Recurrent tonsillitis + OSAS

11.1

VCAIgG Negative 6 (50)

Positive
Total

Total n (%)

6 (50)

12 (100)

3 (12.5)

21 (87.5)

24 (100)

9 (25)

27 (75)

36 (100)

McNemar test P < 0.05

Results of VCAIgM and VCAIgG

VCAIgM was measured quantitatively in serum samples
of the patients prior to the others to eliminate acute infection and was found negative in all patients and active infection was not detected in the patients. VCAIgG positivity
was determined as mean 66.7% and was detected signiWcantly greater in female patients than in males (P < 0.05).
No diVerence was found in the results analyzed to determine the age-risk interval to Wnd the disease (P > 0.05).
Furthermore, correlation between the quantity of IgG, frequency of tonsillitis and tonsillar hypertrophy was investigated; however, no statistically signiWcant correlation was
determined (P > 0.05).

of the patients were found to be consistent with VCAIgG

results.
The correlation between quantitative EBV-VCAIgG
and EBV-DNA viral load tissue
Moreover, the correlation between the values of quantitative PCR and quantitative IgG was searched and when the
all group was examined a signiWcant correlation was determined between IU/ml and copy/mg (P < 0.05, R = 0.623)
but such a correlation was not seen in the group only with
positive IgG (P > 0.05). The correlation between the degree
of tonsillar hypertrophy and viral load was examined and
no statistical correlation was determined (P > 0.05).

Results of EBV-DNA PCR

EpsteinBarr virus DNA was studied in the tissue samples
obtained from the patients using quantitative real-time PCR
method and viral load per milligram of tissue was calculated. Internal control was determined positive in every
patient and included to the study. EBV-DNA was detected
negative in 9 (25%) and positive in 27 (75%) patients. No
correlation was found between the degree of tonsillar
hypertrophy, which was determined during examination,
and PCR positivity (P > 0.05). Sex diVerence seen in
VCAIgG could not be found in the results of the PCR
(P > 0.05).
Qualitative results of EBV-DNA PCR and VCAIgG
The results of the qualitative PCR and qualitative IgG are
summarized in Table 2. Polymerase chain reaction results

Discussion
EpsteinBarr virus is a virus occurring frequently worldwide and is found responsible for numerous diseases
changing from asymptomatic infection to cancer [2, 14,
24]. EBV infection is usually acquired during childhood in
underdeveloped countries; however in developed countries,
it occurs at advanced ages and results in more symptomatically. EBV infection begins with the infection in oropharyngeal epithelial cells or B lymphocytes found in the crypt
structures of the tonsils [2]. EBV carrying may persist for a
lifetime in consequence of the infection in oropharyngeal
epithelial cells. Although viral replication in the oropharynx is completely inhibited using acyclovir, the presence of
EBV-infected peripheral blood B lymphocytes is suYcient
for the carrying persistence [2]. Accordingly, the treatment

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of infection in the oropharynx for a certain period of time

inhibit virus carrying, consequently, the oropharynx brieXy
takes part again in the viral invasion. The virus is released
via the oropharynx and transmitted to other individuals as a
result of the indistinguishable periodic activations of the
lymphoid organs and reproduction of viruses in the tonsils
[13]. The spread of EBV among persons occurs via saliva,
which is contaminated due to viral replication in the oropharynx [2]. Various hypotheses have been suggested to
explain the recurrent tonsillitis. Decrease in the number of
B lymphocytes constituting J chain binding, which has a
role in producing secretory immunoglobulin, is among
these hypotheses [4]. Another theory is that viral IL10 protein, encoded by EBV, may cause secondary infections by
decreasing TH1 cytotoxin release and cytotoxic T lymphocyte (CTL) response. It has been claimed that EBV is found
latently in the tonsils and may provoke acute infection;
therefore, it may result in hypertrophy with respect to the
lymphoproliferative features [4]. EBV has been suggested
to play a role in various neoplastic diseases, such as Burkitt
lymphoma, NPC and gastric carcinoma. EBV was reported
to be seen in non-diVerentiated NPCs without being dependent on a geographical region [14].
In our study, VCAIgG positivity was determined to be
66.7% in patients with recurrent tonsillitis. VCAIgG positivity rate, despite changing with respect to ages, was found
to be 67.1% in a study performed by zsoy et al. [15] in
our country. Again in another study performed in our country by Tanyeli et al. [19] VCAIgG rate was calculated to
be above 50% in the group with lymphoma, and 22.8% in
the control group. EpsteinBarr nuclear antigen IgG
(EBNAIgG) positivity was determined to increase with
age and diVer between 35.4 and 72% in a study performed
by Morris et al. in England and in the Wales. Furthermore,
female patient group aged between 10 and 14 years signiWcantly had more positivity [12]. VCAIgG positivity below
5 years of age was determined to be 90% in a study by
Venkitaraman et al. [22] in India. In a study by Tsai et al.
[21] VCAIgG positivity was reported to increase with age
and the rate was demonstrated to increase above 90% after
4 years of age. In a study in Thailand VCAIgG positivity
was determined to be 72.7% in children between 6 months
and 15 years, in another study, the rate was found to be
68.4% in the same country [16, 17] In contrast to the general information, the highest rates were determined between
ages 0 and 2 years and above 25 years of age in a study performed in Bangladesh and again in the same study the rates
were found to be signiWcantly higher in females [10]. The
rate of positivity between 1 and 5 years of age was found to
be 70% in Iran and 56% in Germany in a simultaneous
study [1].
Although this study is beyond representing the society
due to the selected patient group and number of patients, it

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Eur Arch Otorhinolaryngol (2010) 267:143148

seems to be consistent with the study performed by zsoy

et al. Although this rate of positivity is similar to those
obtained in the developing countries, such as Iran, Brazil
and Thailand, it is above those obtained in the developed
countries such as Germany and below those obtained in the
countries such as India and Bangladesh where hygiene conditions are not good enough.
When the results of IgG were examined with respect to
the sex of the patients, the rate obtained from the female
patients was found to be signiWcantly higher than that of the
male patients, which was a remarkable result. Similar
results were obtained in the studies performed in England
and Bangladesh (P < 0.05). Despite variations due to sociological habits, this situation has been considered to originate from womens behavior diVerences and habit of
keeping close contact [12].
The tonsils (palatine, pharyngeal) are important structures of the Waldeyers ring and form an immunological
passage point. Its physiology cannot be entirely understood
[4]. Actually, hypertrophy response, developing as a
response to infection, should be expected to increase with
the rise in the number of infection. However, immune
response may diVer from person to person varying in levels.
We consider that the factor causing tonsillitis may also
aVect hypertrophy. No correlation between the number of
episodes of tonsillitis and degree of tonsillar hypertrophy
was determined.
Almost 5070% of the lymphocytes found in the structure of the tonsil are B lymphocytes. This rate is quite
higher than that of the periphery (723%). Some of the B
lymphocytes in the tonsils (20100/107) are latently
infected with EBV, exactly same as they are in the peripheral B lymphocytes (5500/107) [11].
In the study performed by Ikeda et al. [11] EBVs found
in the tonsillar tissue displayed latent protein expression
and BZLF-1 RNA, which is the transcription of lytic replication proteins, and the presence of replication in some
patients. Furthermore, tonsils were shown that they can be a
reservoir structure for EBV.
EpsteinBarr virus is known to cause adenotonsillar
hypertrophy, especially in organ transplant patients; it is
also known to cause even the occurrence of PTLD in such
patients [23].
In the studies performed recently, it has been shown
that the presence of immunosuppression is not required
for the occurrence of EBV-related adenotonsillar hypertrophy in non-immunosuppressive individuals. In a
study performed by Endo et al. adenoidectomy was performed using EBER immunohistochemical stain to children aged below 2 years of age and this rate was found
to be 33% [6] and in another study performed by Endo
et al. with patients between 2 and 13 years of age the rate
was found to be 72% [5].

Eur Arch Otorhinolaryngol (2010) 267:143148

Again in another study by Endo et al. reported that EBV

was found in the tonsils of individuals with recurrent tonsillitis and in hypertrophic tonsils. Authors suggested that
EBV may be colonized in the palatine tonsils of children
and may play the role of the pathogenesis of recurrent tonsillitis and tonsillar hypertrophy [6]. However, there was no
information on the EBV serology of the patients.
In a study performed by Nadal et al. [13] using real-time
PCR, positivity rate of EBV DNA was determined as 74%.
They also reported the presence of correlation of EBV with
VCAIgG values of the patients.
EpsteinBarr virus DNA positivity in tissues of the tonsil was found to be 75% in our study. Although it appears to
be too high when compared with the study performed by
Endo et al.; the diVerences in the techniques used and methods of tissue transfer might aVect the results. Endo et al.
performed their studies using paraYn blocks; whereas in
our study and in the study performed by Nadal et al. who
obtained values akin to ours, tissues were stored fresh after
they were collected and real-time PCR, which is a sensitive
test, was performed.
The presence of EBV in the tonsillar hypertrophy is an
unquestionable situation. Possible mechanisms, the presence of EBV in the B lymphocytes in the tonsils and the
lytic and latent infection here may cause hypertrophy in the
tonsillar structure like a recurrent infection or B lymphocytes, which are latently infected, may stimulate inner cell
transmission mechanisms and stimulate cell proliferation,
especially by LMP1 of the latent proteins.
In addition, EBV is known to synthesize a protein displaying amino acid sequence homology with human IL-10,
coded by BCRF-1. This protein is also called viral IL10.
IL10 is known to decrease TH1 cytotoxin release and CTL
response [11]. Probably, viral IL10 pressures the activity of
CTL in the EBV-infected regions and also sets the groundwork for the secondary infections while facilitating the stability and the replication of EBV.
In a recently performed study, it has been displayed that
the presence of EBV in the tonsils and lytic replication process may associate with the serum anti VCAIgG titers of
patients. There has been a correlation between IgG quantity
in the blood and the quantity of EBV genome in the tissue.
EBV DNA quantity in the tissue of the patients with IgG
titer of 1/160 and above is high and the quantity of VCA
IgG has been reported to provide foresight into EBV-associated lymphoproliferation [13]. In the study, we have
performed no correlation, which can be determined
between the quantitative values and quantitative tissue PCR
values of the patients with high IgG. Despite the lack of
correlation between the quantitative values of EBV-DNA
in the tonsillar tissue and the quantitative values of serum
VCAIgG, a 75% EBV-DNA rate in recurrent tonsillitis
may be evaluated as a positive Wnding in the role of EBV in

147

recurrent tonsillitis. Among patients with recurrent tonsillitis, EBV-DNA is positive in 87.5% of the patients with positive VCAIgG. This result may be an indicator for us to
evaluate this group of patients more carefully.
EpsteinBarr virus stays latent in B lymphocytes and
may cause reactivations from time to time. Tonsil structure
rich in B lymphocytes, which contains EBV episomes that
are important in immunity. Even though tonsillar epithelium transfers EBV infection to B lymphocytes in the initial
stage of infection, B lymphocytes can occasionally become
reactivated and transfer infection to epithelial cells and new
B lymphocytes. In a study on NPC, it was suggested that
EBV infection was not the Wrst step, but was a step before
the occurrence of invasive development. Perhaps, it
reminds us the question if the persistent presence and replication of EBV in the tonsillar tissue may be a triggering
factor or a cofactor for the epithelial cells that are appropriate for transformation. Under these circumstances, it can be
considered to ascertain the probable role of the tonsillar tissue transporting EBV in the carcinogenesis in more
detailed way.
In conclusion, the general eVect of EBV on the lymphatic tissues tends to proliferate. EBV rate determined in
our study has a tendency to support these data. However,
more molecular studies and studies targeting diVerences in
mechanisms for transmission of inner cell signal and latent
protein expressions are required to enlighten the real eVect
of EBV on the tonsillar tissue and proliferation causing
mechanisms.
ConXict of interest statement
conXict of interest.

The authors declare that they have no

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