Herpesviruses in chronic and aggressive periodontitis patients in an Indian population.

Abstract

Many recent studies have assessed the prevalence and role of herpesviruses in the etiopathogenesis of periodontal diseases, which has led to the realization of intricate interactions between viruses and bacteria within periodontal pockets. It has also been shown that the occurrence of herpesviruses may vary depending upon the age of the patient and the race of the population studied. Thus, the present study aimed at detecting herpes simplex virus type 1 and 2 (HSV 1 and 2), Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) in periodontal pockets of Indian patients with chronic and aggressive periodontitis. Subgingival plaque samples (n = 33) were collected from 19 randomly chosen chronic periodontitis and 14 aggressive periodontitis patients. Herpesviruses were detected using multiplex polymerase chain reaction technique. Chronic periodontitis patients revealed presence of HSV-1 in 19 (100%) samples, HSV-2 in 3 (15.7%), EBV in 15 (78.9%) and HCMV in 5 (26.31%) samples. Samples from aggressive periodontitis patients showed the presence of HSV-1 in 8 (57.14%), EBV in 4 (28.57%) and HCMV in 1 (7.14%), whereas HSV-2 was not detected in any specimen. In this population, herpesviruses were found more frequently in chronic periodontitis than in aggressive periodontitis patients and their prevalence may vary according to the age and race of the patient.

Full text

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Abstract: Many recent studies have assessed the
prevalence and role of herpesviruses in the
etiopathogenesis of periodontal diseases, which has
led to the realization of intricate interactions between
viruses and bacteria within periodontal pockets. It has
also been shown that the occurrence of herpesviruses
may vary depending upon the age of the patient and
the race of the population studied. Thus, the present
study aimed at detecting herpes simplex virus type 1
and 2 (HSV 1 and 2), Epstein-Barr virus (EBV) and
human cytomegalovirus (HCMV) in periodontal
pockets of Indian patients with chronic and aggressive
periodontitis. Subgingival plaque samples (n = 33)
were collected from 19 randomly chosen chronic
periodontitis and 14 aggressive periodontitis patients.
Herpesviruses were detected using multiplex
polymerase chain reaction technique. Chronic
periodontitis patients revealed presence of HSV-1 in 19
(100%) samples, HSV-2 in 3 (15.7%), EBV in 15
(78.9%) and HCMV in 5 (26.31%) samples. Samples
from aggressive periodontitis patients showed the
presence of HSV-1 in 8 (57.14%), EBV in 4 (28.57%)
and HCMV in 1 (7.14%), whereas HSV-2 was not
detected in any specimen. In this population,
herpesviruses were found more frequently in chronic
periodontitis than in aggressive periodontitis patients
and their prevalence may vary according to the age and
race of the patient. (J Oral Sci 51, 79-86, 2009)
Keywords: aggressive periodontitis; chronic
periodontitis; herpesviruses; polymerase
chain reaction.
Introduction
The etiopathogenesis of periodontal disease is a complex
process, involving the multifarious interaction between
microbial and host factors and a variety of disease-
modulating environmental factors. Bacterial etiology alone
has not been able to substantiate various aspects such as
i) rapid periodontal tissue breakdown with minimal plaque
(1), ii) phases of disease activity and quiescence (2), iii)
site specificity in periodontal disease (3) and, iv) progression
to advanced periodontal destruction which occurs in a
fraction of a given population (4).
Classically, microbiological research on human
Journal of Oral Science, Vol. 51, No. 1, 79-86, 2009
Original
Correspondence to Dr. Shivaprasad Bilichodmath, Department
of Periodontics, Rajarajeshwari Dental College and Hospital,
Bangalore, India
Tel: +91-80-26793637
Fax: +91-80-28437468
E-mail: drshivaprasad2008@yahoo.co.in
Herpesviruses in chronic and aggressive periodontitis
patients in an Indian population
Shivaprasad Bilichodmath1), Sachin B. Mangalekar2), Dileep C. G. Sharma3),
Ashok K. Prabhakar4), Sridhar B. Reddy5), Nagaraj B. Kalburgi6), Sudhir R. Patil6)
and Kishore Bhat7)
1)Department of Periodontics, Rajarajeshwari Dental College and Hospital, Bangalore, India
2)Department of Periodontics, Chattisgarh Dental College and Research Institute, Rajnandgaon,
Chattisgarh, India
3)Department of Periodontics, College of Dentistry, Al-Fateh University of Medical Sciences, Tripoli, Libya
4)Department of Periodontics, SJM Dental College and Hospital, Chitradurga, India
5)Department of Periodontics, HKDET Dental College and Hospital, Humnabad, India
6)Department of Periodontics, PMNM Dental College and Hospital, Bagalkot, India
7)Department of Microbiology, Maratha Mandal Dental College and Hospital, Belgaum, India
(Received 29 July and accepted 15 December 2008)

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periodontal disease has focused on bacteria, and to a minor
extent on yeasts and parasites. Many recent studies have
shown the prevalence of herpes viruses including herpes
simplex virus (HSV 1 and 2), Epstein-Barr virus (EBV)
and human cytomegalovirus (HCMV) in the periodontal
pockets and gingival tissue of periodontitis patients (5-9).
Association of herpesviruses with bacteria was also assessed
to improve our understanding of the etiopathogenesis of
periodontal diseases (10-12). Furthermore, recent studies
have quantified the herpesviruses in periodontal pockets
to show their positive association with severity of
periodontal disease (13). The prevalence and number of
herpesviruses in periodontal pockets may vary according
to the age (5, 14), ethnicity (14), type of periodontal disease
(8), immune status (15) and genetic predisposition (16) of
patients.
Thus, further studies in other races from different parts
of the world are deemed essential to establish herpesviruses
as a recognized etiologic (or co-etiologic) agent for
periodontitis. Accordingly, this is the first study conducted
in a South Indian population to assess the prevalence of
herpesviruses in periodontal pockets of different types of
periodontitis. The aim of the present study was to detect
HSV-1 and 2, EBV and HCMV in aggressive periodontitis
patients and compare their results with those of chronic
periodontitis in a South Indian population using a sensitive
multiplex polymerase chain reaction (PCR) technique.
Materials and Methods
The study population was selected from patients
attending the outpatient section of the Department of
Periodontics, PMNM Dental College and Hospital,
Bagalkot, Karnataka, India. Subgingival plaque samples
were collected from 19 randomly chosen patients with
chronic periodontitis (12 men and 7 women, age range 21-
57 years, mean age 43 ±7.3 years) and 14 with aggressive
periodontitis (11 men and 3 women, age range 21-29
years, mean age 25 ± 3.1 years).
Chronic and aggressive periodontitis were diagnosed
based on the criteria of American Academy of
Periodontology classification of periodontal diseases
(1999). Chronic periodontitis patients had periodontal
pockets greater than 5 mm and clinical attachment loss
greater than 3 mm in more than 20 teeth with moderate
to severe bone loss. Patients diagnosed as having aggressive
periodontitis exhibited probing attachment loss in the
excess of 5 mm in more than 14 teeth; at least 3 of which
were not first molars or incisors with moderate to severe
bone loss. All patients were systemically healthy and had
not received periodontal treatment or antibiotics for at
least 6 months prior to the clinical examination and
sampling.
Subjects who satisfied the inclusion criteria of the study
were selected and ethical approval was obtained from the
institutional review board and Rajiv Gandhi University of
Health Sciences, Karnataka, India. Furthermore, each
patient received a detailed explanation regarding the study
procedure, and written informed consent was obtained
from those who agreed to participate voluntarily in the
study.
The subjects for sampling were selected at random from
individuals scheduled for a routine oral examination.
Periodontal evaluation included the modified gingival
index (17), plaque index (18), probing pocket depth, and
probing attachment loss. A subgingival plaque sample
was taken from the deepest pockets of the dentition.
Subgingival sample collection
Supragingival plaque was gently removed with sterile
cotton pellets and sample sites were isolated with cotton
rolls and air-dried prior to sampling. Subgingival specimens
were collected using a sterile curette with a single stroke
after gentle insertion into the bottom of the sampling site.
From each periodontitis patient, a pooled specimen was
obtained from the two deepest pockets of the dentition (5-
10-mm probing depth). The specimens were suspended in
500 µl of TE buffer (10 mM Tris-hydrochloride, 1mM
EDTA, pH 8) and homogenized by vigorous mixing on a
vortex.
Nucleic acid extraction
Samples were stored at -20°C and centrifuged at 10,000
rpm for 5 min the following morning. The precipitate thus
obtained was washed three times with TE buffer. After the
third wash, the supernatant was discarded and the precipitate
was treated with 500 µl lysis buffer 1 (Tris HCl and Triton
x-100) for 5 min, centrifuged, and the supernatant was
discarded once again. The precipitate was treated with
100 µl lysis buffer 2 (Tris HCl, Nonidet p-40, Tween 200)
with 100 µg/ml freshly prepared proteinase K and kept at
60°C for 2 h followed by 95°C for 10 min and deep frozen
at -70°C until amplification (19).
DNA amplification by multiplex PCR technique
The primers used for polymerase chain reaction are
listed in Table 1. To optimize the multiplex PCR, a series
of titrations of primer concentrations and deoxynucleotide
triphosphate (dNTP) levels were performed. Primer
concentrations of 10, 25, 50 and 100 pmol from each
primer pair were titrated simultaneously with dNTP (0.1,
0.2 and 0.3 mM concentrations of each of the dNTPs).
Amplification was performed with the PCR system

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(Corbett research palm cycler version 2.2, imported by J.H.
BIO Innovations, Bangalore, India). Forty amplification
cycles of 30 s at 94°C, 40 s at 60°C and 50 s at 72°C were
carried out in a 50-µl final volume containing 5 µl of ×10
reaction buffer (Bangalore Genei, Bangalore, India), 0.2
mM concentrations of each dNTP, 10 pmol of each of the
12 primers, and 2.5 U of cloned Pfu DNA polymerase
(Bangalore Genei). Five microliters of appropriate DNA
sample was added to the reaction mixture. After the last
cycle, the samples were incubated for 15 min at 78°C to
complete the extension of primers (20). The molecular
weight of the individual PCR product was 147 bp (HSV
I), 227 bp (HSV2), 182 bp (EBV), and 256 bp (HCMV).
Ten µl of each amplified product was analyzed by
agarose gel electrophoresis on 3.5% agarose (Sigma-
Aldrich, Bangalore, India) containing 1µg of ethidium
bromide/ml in 1X TBE buffer and was visualized in a UV
transilluminator (Bioimaging systems, imported by JH
BIO Innovations, Bangalore, India).
Results
Tables 2, 3 and 4 show the distribution of viruses in the
study patients. Chronic periodontitis sites revealed HSV-
1 in 19 (100%) samples, HSV-2 in 3 (15.7%) samples, EBV
in 15 (78.9%) samples and HCMV in 5 (26.31%) samples
out of 12 specimens. Aggressive periodontitis sites revealed
HSV-1 in 8 (57.14%) samples, EBV in 4 (28.57%) samples
and HCMV in 1 (7.14%) sample. HSV-2 was not detected
in any specimen from the aggressive periodontitis patients.
Twenty-three males and 10 females were included in the
study. All males from the chronic periodontitis group,
and 5 males from the aggressive periodontitis were positive
for viruses. Herpesvirus (any one or more) was detected
in all the samples (n = 10) from females.
The distribution of viruses in 6 chronic periodontitis
patients belonging to the 31-40 years age group was as
follows: HSV-1 in 6, HSV-2 in 1, EBV in 5, and HCMV
in 1 sample. HSV-1 in 9, HSV-2 in 1, EBV in 7 and
HCMV in 4 samples were detected from 9 chronic
Table 2 Frequency of virus detection in periodontal pockets
of chronic and aggressive periodontitis patients
Table 1 Primers used in polymerase chain reaction to detect
human viruses
Table 3 Age and sex wise distribution of viruses in chronic periodontitis
patients

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periodontitis patients of the 41-50 years age group. Out
of the 3 chronic periodontitis patients belonging to the 51-
60 years age group, HSV-1 was present in 3, HSV-2 in 1,
and EBV in 3 samples (Table 5).
In the 11-20 years age group, HSV-1 was only present
in the sample from 1 aggressive periodontitis patient. One
sample from the lone chronic periodontitis patient in the
21-30 years age group revealed HSV-1. Thirteen patients
with aggressive periodontitis in the age group 21-30 years
expressed HSV-1 in 7, EBV in 4 and HCMV in 1 sample.
In the present study, there were no aggressive periodontitis
patients over 31 years of age (Table 5).
A combination of herpesviruses was seen in 16 samples
(out of 19) from chronic periodontitis patients. The
combination of HSV-1 and 2 was seen in 3 (15.7%)
specimens, HSV-1 and EBV in 15 (78.9%) samples, and
HSV-1 and HCMV in 6 (31.5%) specimens (Table 6).
The combined occurrence of EBV and HCMV was detected
Table 4 Age and sex wise distribution of viruses in aggressive
periodontitis patients
Table 5 Distribution of Herpes viruses in different age groups
Table 6 Incidence of Herpes virus combination in periodontal pockets

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in 4 (21%) specimens, HSV-1, EBV and HCMV in 4
(21%), and HSV-1, HSV-2 and EBV in 3 (15.7%) samples
from chronic periodontitis patients.
Combination of herpesviruses in periodontal pockets of
aggressive periodontitis patients was revealed in 5 of 14
samples. HSV-1 and EBV in grouping was found in 4
(28.5%) patients and HSV-1 and HCMV in 1 (7.1%)
patient only. The grouping of all the 4 viruses was not found
in any subgingival plaque specimen from chronic and
aggressive periodontitis patients.
Statistical analysis was done using Yates Chi-square test
(Intercooled STATA statistical software Version 9.2, STATA
corporation, Lakeway drive, College station, Texas, USA)
to compare the status of Herpesviruses in chronic and
aggressive periodontitis patients. The presence of HSV-1
(P= 0.0069), EBV (P= 0.0112) and HCMV (P= 0.0112)
was statistically significant in chronic periodontitis patient
but not in aggressive periodontitis patients (Tables 7 and
8).
Discussion
To the best of our knowledge, this is the first study
conducted in a South Indian population regarding detection
of herpesviruses in periodontal pockets of chronic
periodontitis patients compared with that in aggressive
periodontitis patients. Viral identification was done using
multiplex-PCR technique with which microorganisms of
different species may be detected simultaneously. Primer
pairs specific to each intended organisms are engaged in
a single-tube amplification process (21,22). PCR is a very
sensitive technique that can detect microorganisms in
smaller plaque samples than conventional and basic
microbiological techniques.
The present study detected a significantly higher
prevalence of herpesviruses in subgingival samples of
chronic periodontitis patients (HSV-1 100%, HSV-2 15.7%,
EBV 78.9%, and HCMV 26.31%) than in that of the
aggressive periodontitis patients (HSV-1 57.14%, HSV-2
0%, EBV 28.57%, and HCMV 7.14%). The results were
similar to the study conducted by Imbronito et al., in
which EBV-1 was detected in 45% and HCMV in 82.5%
of subgingival plaque samples (23).
The findings of the present study were in contrast to those
of studies conducted by Parra and Slots (5) and Contreras
and Slots (7). They demonstrated the presence of HCMV
or EBV in 90% of localized juvenile periodontitis lesions
(aggressive periodontitis) but only in 40-78% of adult
periodontitis lesions (chronic periodontitis). Kubar et al.
demonstrated HCMV DNA in 78% of subgingival samples
from aggressive periodontitis lesions, but only in 46% of
Table 7 Comparison of status of HSV-1 and HSV-2 in chronic and aggressive periodontitis
patients
Table 8 Comparison of status of EBV and HCMV in chronic and aggressive periodontitis
patients

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that of chronic periodontitis (8). EBV DNA was identified
in 89% of subgingival and 78% of gingival tissue samples
from aggressive periodontitis lesions, but only in 46% of
both subgingival and gingival tissue samples from chronic
periodontitis lesions (8). In another study, Kubar et al.
identified HCMV in 68.8% of aggressive periodontitis
lesions using real-time PCR (24). Also, Saygun et al. have
detected HCMV, EBV-1 and HSV-1 in 72-78% of the
subgingival samples from aggressive periodontitis patients
(10).
In the present study, herpesviruses were found more
frequently in chronic periodontitis than in aggressive
periodontitis patients. This variation in the results, compared
to previous studies, may be due to the age range of the
patients, ethnic differences within the study population,
the limited sample size, and difference in the severity of
periodontal disease in the selected group. Also, the
difference in the methodological approach used to detect
herpesviruses plays a vital role in the disparity between
the present study and the previous studies.
HSV-1 and 2, EBV and HCMV were found more
frequently in patients over 30 years of age (chronic
periodontitis) than those with age <30 years (aggressive
periodontitis) in the present study. This is in agreement with
one of the studies by Slots and Parra, which revealed
increased occurrence of CMV with increasing age (>45
years) (1). In contrast to the above findings, a study
conducted by Kubar et al (8) observed HCMV more
frequently (78%) in samples from aggressive periodontitis
lesions (ages 21-34 years). Only 46% of subgingival
samples from chronic periodontitis indicated the presence
of HCMV DNA.
The difference in the range of patients’ age between the
previous studies and the present study might be one of the
reasons for the variation in the findings. Patient age may
have a greater effect on the prevalence of herpesviruses
than type of periodontitis. As age increases, the prevalence
of herpesviruses in periodontal pockets may increase. If
age of the patient is not considered as one of the criteria,
the positive relationship between herpesviruses and
destructive periodontal disease might be overestimated.
Ling et al. demonstrated that HSV was related to the
severity of periodontal diseases in terms of clinical
attachment loss (25). In the present study, HSV-1 was
found most frequently than any other herpesviruses, in
100% of chronic periodontitis and 57.4% of aggressive
periodontitis patients. These results emphasize the presence
of HSV-1 in periodontal pockets but the tangible role of
HSV-1 in initiation and progression of periodontal diseases
has to be ascertained.
All female patients demonstrated the presence of
herpesviruses (any). All males except 6 aggressive
periodontitis patients revealed the presence of herpesviruses
in the subgingival specimens. However, a relationship
between sex and the prevalence of herpesviruses in
periodontal pockets was not established.
It has been estimated that, worldwide, 90% of the
population infected with EBV is asymptomatic. HCMV
was found to be prevalent in 50-80% of adolescents and
adults in developed countries, and 40-70% of the population
acquires antibodies against herpes simplex virus from the
first year of life to adolescence (5). These data suggest that
herpesviruses are usually present in the body in inactive
state. Reactivation of herpesviruses in periodontal sites
comprises an important pathogenic event in the devel-
opment of periodontitis (26).
Herpesviruses may cause direct cytopathic effects on
keratinocytes, fibroblasts, endothelial cells and inflam-
matory cells (27). Contreras et al have detected HCMV
in monocytes/macrophages and T lymphocytes, EBV-1 in
B lymphocytes, and HSV in T lymphocytes and
monocytes/macrophages (28). Infected immune cells may
not mount a proper immune response against peri-
odontopathogenic bacteria predisposing to microbial
superinfection. Herpesvirus induced defective poly-
morphonuclear neutrophils can increase the risk for
destructive periodontal disease. The study found
herpesviruses infecting various types of inflammatory
cells in periodontitis lesions (28). It was not known whether
the infected cells carry functional or latent herpesviruses.
Active herpesvirus infections would potentially be more
detrimental to the periodontium than latent herpesvirus
infections.
Herpesviruses existing in combination in periodontal
pockets may act in synergy against host tissues. Co-
existence of HSV-1 and EBV was found most frequently
in both chronic periodontitis (78.9%) and aggressive
periodontitis (28.5%) patients in the present study.
Genetic predisposition of the patient for disease
susceptibility and severity should also be contemplated
when considering viral etiopathogenesis. A patient may
be genetically predisposed to severe periodontal breakdown,
as shown in cases with genetic polymorphism of numerous
interleukins (29-31). Therefore, studies that detect or
quantify herpesviruses should also analyze the genetic
polymorphism to better understand the role of herpesviruses
in periodontitis.
In a study conducted by Ting et al, it was hypothesized
that active HCMV infection could be associated with the
initiation and progression of localized juvenile periodontitis
(9). In another study conducted by Contreras and Slots,
results suggested that active HCMV replication could

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occur in periodontal sites (32), but it remained unclear if
HCMV reactivation was related to the initiation or the
progression of destructive periodontal disease.
Despite circumstantial evidence of a role of herpesviruses
in destructive periodontal disease, a cause-and-effect
relationship remains to be established. Questions remain
as to whether active periodontal herpesvirus infection
gives rise to destructive periodontal disease or whether
destructive periodontal disease reactivates a latent viral
infection. Thus, more studies on viral pathogenesis from
different parts of the world are essential to establish
herpesviruses as an etiologic or co-etiologic agent of
periodontal diseases.
In conclusion, we found that herpesviruses (HSV-1 and
2, EBV, and HCMV) are associated more with chronic
periodontitis than aggressive periodontitis, and this finding
may be related to the age range of the patients, limited
sample size, and methodology used to detect the viruses
in this study. Also, prevalence of viruses in periodontal
pockets may vary according to ethnicity, type of periodontal
disease, immune status, and genetic predisposition of
patients. The present understanding of the potential role
of herpesviruses in etiopathogenesis of destructive
periodontal disease is credible but not decisive. Additional
studies in other populations are required to better understand
the association of viruses in periodontal diseases.
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