Indian Journal of Dental Sciences

: 2023  |  Volume : 15  |  Issue : 1  |  Page : 1--6

Herpes simplex virus-1, epstein–barr virus, and human cytomegalovirus in the saliva of periodontally diseased pregnant and nonpregnant women: A cross-sectional analytical study

Paramjit Kaur Khinda1, Dinesh Rao2, Surender Pal Singh Sodhi3, Anita Mehta4, Sanjeev Kumar Salaria5,  
1 Department of Preventive Dentistry, College of Dentistry, King Saud Bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia; Research Scholar, Pacific Dental College, Pacific Academy of Higher Education and Research University, Udaipur, Rajasthan, India
2 Department of Pediatric Dentistry, Pacific Dental College, Pacific Academy of Higher Education and Research University, Udaipur, Rajasthan, India
3 Department of Oral and Maxillofacial Surgery, Dasmesh Institute of Dental Sciences and Research, Faridkot, Punjab, India
4 Department of Periodontics, Dasmesh Institute of Dental Sciences and Research, Faridkot, Punjab, India
5 Department of Periodontics, Kalka Dental College, Meerut, Uttar Pradesh, India

Correspondence Address:
Paramjit Kaur Khinda
Faculty of Periodontology, College of Dentistry, King Saud Bin Abdulaziz University for Health Sciences, Riyadh


Context: Periodontal disease is commonly found among pregnant women. Viruses may play a key role in the etiology of periodontal disease through a variety of mechanisms that work alone or in tandem. Many researchers have explored the possible role of herpes viruses in periodontal disease, but extremely limited studies have evaluated the effect of pregnancy on herpes viruses in women with periodontal disease. Aims and Objectives: To study the effect of pregnancy on herpes simplex virus-1, Epstein–Barr virus (EBV), and human cytomegalovirus in women with periodontal disease and to evaluate the presence of herpes viruses within the saliva of pregnant and nonpregnant women with periodontal disease. Settings and Design: The study design involves a cross-sectional analytical study. Materials and Methods: The study consisted of 50 females aged between 18 and 35 years. Group I comprised pregnant women at any gestational age with periodontal disease and Group II comprised nonpregnant women with periodontal disease. The clinical parameters assessed were plaque index, gingival index, bleeding on probing, probing pocket depth, and clinical attachment loss. Nonstimulated whole saliva was utilized for the detection of three herpes viruses by multiplex polymerase chain reaction. Statistical Analysis Used: The categorical variables and research parameters were compared using the Chi-squared test and the Student's t-test. Results: Both groups tested positive for the three herpes viruses, but only a significant increase was observed in EBV in Group I participants on the intergroup comparison. Conclusions: The presence of EBV in the saliva of women with periodontal disease was found to be more likely during pregnancy.

How to cite this article:
Khinda PK, Rao D, Singh Sodhi SP, Mehta A, Salaria SK. Herpes simplex virus-1, epstein–barr virus, and human cytomegalovirus in the saliva of periodontally diseased pregnant and nonpregnant women: A cross-sectional analytical study.Indian J Dent Sci 2023;15:1-6

How to cite this URL:
Khinda PK, Rao D, Singh Sodhi SP, Mehta A, Salaria SK. Herpes simplex virus-1, epstein–barr virus, and human cytomegalovirus in the saliva of periodontally diseased pregnant and nonpregnant women: A cross-sectional analytical study. Indian J Dent Sci [serial online] 2023 [cited 2023 Nov 28 ];15:1-6
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Periodontal disease is one of the commonly occurring diseases found in humans. Gingivitis is caused by the accumulation of biofilm on the tooth surface, which causes irritation of the marginal tissue. If left untreated, this can evolve into periodontitis, which is characterized by attachment loss and bone resorption, eventually leading to tooth movement and loss. Microbiological, host, and environmental factors all have a role in periodontal disease.[1] Periodontal disease is caused by specific bacteria and their products according to conventional etiological studies.[2] However, the clinicopathological features observed in the disease cannot be fully explained by the bacterial etiology. Moreover, multiple studies have found a paucity of putative periodontal bacteria in periodontal disease, and the bacterial presence between healthy and diseased periodontium was found to be similar.[3],[4],[5],[6] Viruses may have a significant role in the etiopathogenesis of periodontal disease acting alone or in combinations.[7] There is a frequent presence of human cytomegalovirus (HCMV) within the crevicular samples with patients having chronic periodontitis, which indicates a strong association between HCMV and Epstein Barr virus (EBV) detection in subgingival areas and attachment loss measurements.[8]

Periodontal disease is commonly found among pregnant women. It has been suggested that the underlying periodontal inflammation may be aggravated by alterations in sex hormone levels during pregnancy.[9] Research has shown that periodontal disease affects fetal health as well as increases the risk of low birth weight and preterm labor.[10],[11],[12],[13] Research pertaining to the etiology of maternal periodontitis is vital to successfully prevent and treat this condition. The studies pertaining to the presence of herpes viruses in pregnant patients with periodontitis are still inconclusive. Therefore, the present cross-sectional analytical study was planned with the aim to evaluate whether pregnancy has an influence on herpes viruses (herpes simplex virus-1 [HSV-1]), EBV, and HCMV in patients with periodontitis in comparison to nonpregnant patients with periodontitis.

 Materials and Methods

The present cross-sectional analytical study was carried out in an institutional outpatient department of periodontology and maternity hospital, India, as per ethical standards in the 1964 Declaration of Helsinki as revised in 2008. The study synopsis was duly approved by the Research Board of the University vide letter no. PAHER/DPGS/R/2018/022 dated 25/3/2018. The study was executed after approval from the institutional head vide letter no. DIRDS/2018-2019 dated July 21, 2018 and institutional ethical committee clearance vide letter no. DIRDS/2018 dated December 14, 2018. Sixty-seven female subjects, of which 33 nonpregnant with periodontitis and 34 pregnant subjects with periodontitis who were systemically healthy and free from any previous history of herpes infection were selected on the basis of the inclusion and exclusion criterion from a maternity hospital and institutional outpatient clinic. Fifty-eight participants (30 pregnant subjects at any gestational age with periodontitis and 28 nonpregnant subjects with periodontitis) submitted written signed consent. The sample size was calculated by using the following formula (Charan and Biswas, 2013).[14]

n = Z1-α/22 × SD2/d2Z1-α/2 = Power of the studyStandard deviation (SD): Assumed SDd: Absolute error (mean difference)n = (1.96)2 × (1.5 × 1.5)/(0.7 × 0.7) =18.

(Assuming a 80% power, a 5% significance level with a 95% confidence interval, and a standard deviation of 1.5)

Therefore, out of 58 participants, a total of 50 participants with ages ranging between 18 and 35 (equal in number for pregnant participants at any gestational age with periodontitis and nonpregnant participants with periodontitis) were selected by coin toss and allocated to Group I and II, respectively.

The criteria of periodontitis were taken as defined by the American Academy of Periodontology Classification of Periodontal Diseases (2017).[15] The inclusion criteria were that the participants in both groups should have retained at least 20 natural teeth (excluding the third molars). The study excluded participants with systemic illness, periodontal therapy, or long-term use of systemic antibiotics in the previous 3 months.

Participants were informed about the study protocol and unstimulated salivary sample was collected by the patient under the observation of clinician, followed by the recording of clinical parameters such as plaque index (PI),[16] gingival index (GI),[17] bleeding on probing (BOP), probing pocket depth (PPD), and clinical attachment loss (CAL) by the clinician.

DNA extraction

The extraction of DNA was performed using the Modified proteinase-K method. After transferring the obtained samples to a tube containing T. E. buffer, they were centrifuged at 5000 rpm for 5 min. Following this, the supernatant was removed and 500 μlfresh T. E. buffer was added. This process was repeated 3–4 times using fresh T. E. buffer each time. After discarding the supernatant, 50 μl lysis buffer was added and vortexed and kept again for 5 min. After this, 50 μl lysis buffer II and 10 μlproteinase-K (10 mg/ml) were added and vortexed vigorously. Later, it was placed in a water bath at 600°C for 2 h before being placed in a boiling water bath for 10 min.

Finally, the supernatant containing the DNA was poured into a clean tube and stored at –20°C.[18]

Multiplex polymerase chain reaction procedure

The following polymerase chain reaction (PCR) primers, which are unique to the organisms in question, were employed.

(1) Primer sequences for HSV-1 5'-CGT ACC TGC GGC TCG TGA AGT-3' as forward 5'-AGC AGG GTG CTC GTG TAT GGG C-3' as reverse (2) Primer sequences for HCMV 5'-ACG TGT TAC TGG CGG AGT CG-3' as forward 5'-TTG AGT GTG GCC AGA CTG AG-3' as reverse (3) Primer sequences for EBV 5'-AGC ACT GGC CAG CTC ATA TC-3' as forward 5'-TTG ACG TCA TGC CAA GGC AA-3' as reverse AMPLIQON RED 2X Mastermix containing the reagents Tris-HCL pH 8.5, (NH4)2SO4, 3 mM MgCl2, 0.2% Tween 20.0.4 mM of each dNTP, 0.2 units/μl Ampliqon Taq DNA Polymerase was used. Amplified products were subjected to electrophoresis through 2% Agarose gel containing 1× TAE (Tris Acetate EDTA buffer) which was performed at 25V for 2 h. The gel was stained with ethidium bromide (0.5 g/ml) and viewed using a UV light illuminator. Gel Documentation System was used to capture and analyze the gel picture (Major Science, USA). Each organism has a specific band size based on the primer sequences selected. HSV-1 has amplified product of 271 base pair, CMV has amplified product of 368 base pair and EBV gives amplified product of 326 base pair. Using a DNA ladder, bands of known sizes were obtained simultaneously with each gel for locating band positions in the test samples as shown in [Figure 1]. Total lab software (UK) was used to obtain the quantification of positive bands based on the intensity of the amplified products. The gel was uploaded into the software and quantification was obtained.[19] The results are presented in frequencies, percentages, and mean ± SD. Chi-squared test and Student's t-test were used to compare the categorical variables and study parameters between the groups. A value of P < 0.05 was considered statistically significant. The data were analyzed using the SPSS 22.0 version (Chicago, Inc., USA).{Figure 1}


The mean age of the study participants in Group I and II was 26.44 ± 5.15 and 28.20 ± 4.33, respectively. Majority of study participants in both groups belonged to the middle-income category and brushed their teeth once daily [Table 1]. Out of PI, GI, BOP, PPD, and CAL; only GI and BOP were found to be statistically significant P < 0.0001 [Table 2] on intergroup comparison. Although all three herpes viruses were detected in both groups, statistically significant (P <.009) changes were observed among EBV in pregnant participants with periodontitis in comparison to nonpregnant participants with periodontitis on intergroup comparison [Table 3].{Table 1}{Table 2}{Table 3}


Periodontal disease results from the complex interplay between microbial, environment, and host factors and is commonly found in pregnant women. Although pregnancy itself does not cause periodontal disease, the alteration in sex hormones levels may intensify the existing periodontal inflammation.[9] The herpes viruses have been proposed to be a pathogenic factor for various periodontal diseases since the 1990s.[20] Slots suggested that modern periodontopathogenic models should be based on coinfection of herpesvirus and presumed periodontal bacteria, rather than a simple sum of the roles of two microorganisms.[21] As a result, the goal of this study was to see how pregnancy affected HSV-1, EBV, and HCMV levels in saliva.

Subgingival plaque, gingival crevicular sites, biopsy, saliva are the different specimens from which herpes viruses can be isolated, although, the richest concentration of herpes viruses has been detected in biopsy specimens in comparison to other samples.[22] The detection rate of herpes viruses might be lower than the actual infection rate but salivary molecules may aid in the diagnosis of varieties of hormonal disorders, cancers, pathogenic viruses, etc.[23],[24] Giving due consideration to ethical issues during pregnancy, unstimulated saliva was considered as specimen for herpes viruses detection in the present study as it is painless, noninvasive, time-saving and allows easy sample collection without imparting special training to the patient, clinicians, attendant or parents.[25]

Periodontitis and diabetes are chronic noncommunicable diseases. Periodontitis is considered to be the sixth complication of diabetes mellitus (DM).[26] and DM2 is an established risk factor for periodontal disease.[27] Increased oxidative stress, interleukin (IL) 1 β, tumor necrosis factor α, IL6, expression of toll-like receptors, etc., may be a link between these two diseases.[28] Herpesviruses can play a biological role in the pathogenesis of the periodontal disease, including cytopathic effects on various types of periodontal and inflammatory cells. These cells may carry a potential herpesvirus that can be reactivated when host defense is compromised. Therefore, it is suggested that active herpes virus infection can cause periodontal tissue destruction, and the host's immune response to this infection may play a role in the pathogenesis of the periodontal disease.[29],[30] The onset of periodontal disease is a series of infectious processes that progress from bacteria to herpesviruses to bacteria.[31] The role of periodontal disease and various systemic disorders and the effect of periodontal therapy in reducing the microbial load is well established. A significant association between DM and HSV-I has been observed.[32] Periodontal therapy leads to the reduction of herpes viruses at infected periodontal sites.[33] Therefore, patients with systemic diseases and who had undergone periodontal therapy in the past 3 months were excluded from the study.

Both the groups were similar in respect of age, oral hygiene methods used, and socioeconomic status. On comparing the periodontal parameters, there was no difference between the scores of PI, PPD, and CAL between the two groups. However, there was a difference between the scores of GI and BOP, with Group I (pregnant patients with periodontal disease) presenting with significantly higher scores. Few epidemiological studies have been done on pregnancy and tooth loss which suggest that the extent and severity of gingivitis increases during pregnancy.[34],[35],[36],[37],[38],[39],[40] A study done by Raber et al. evaluated gingival inflammation in pregnant women and at 6 months postpartum. The study reported that women during pregnancy had more severe gingival inflammation although the differences in plaque scores during pregnancy and at 6 months postpartum were not significant.[41] A recent review by Slots and Slots has shown a higher prevalence of herpes viruses in periodontal disease.[42] However, the percentage of different types of herpes viruses differs greatly in different studies. Kazi and Bhardwaj found that HSV-1 (46.6%) was the most common herpes virus, followed by HSV-2 (34.6%), EBV (30.6%) and HCMV (30.0%) in participants with chronic periodontitis.[43] Imbranito et al. reported the detection of HSV-1, HCMV, and EBV-1 as 40.0%, 50.0%, and 46.7%, respectively.[44] Rotola et al. found EBV in 50.0% of gingival samples of patients with periodontitis.[45] The detection rate of HSV1, EBV, and CMV in the control group (nonpregnant patients with periodontal disease) was 12%, 20%, and 12%. Differences in detection rates of herpes viruses among various studies may be attributed to study participants' characteristics (ethnicity, age, gender, and socioeconomic status), type of sample (saliva, subgingival plaque, gingival crevicular fluid, and gingival biopsy), and method of detection (PCR, real-time PCR, and nested PCR). In the present study, the detection rates of HSV1, EBV, and CMV in the case group (pregnant patients with periodontal disease) were 20%, 56%, and 20%, respectively. On comparing the detection rates of three herpes viruses in both groups, it was found that the pregnant participants with periodontal disease had a significant increase in EBV as compared with nonpregnant participants with periodontal disease. This is in agreement with a study by Eres et al. who found the presence of subgingival EBV in pregnant women by 3.647 times higher than in nonpregnant women with gingivitis.[46] A study by Lu et al. compared the detection of three herpes viruses in pregnant women with and without periodontitis and did not find any significant difference between the two groups.[25] Pregnancy is associated with a reduced cellular immune response associated with T-helper lymphocyte type I (Th I). This is accompanied by antibody-mediated immune responses via T-helper type (Th-2) lymphocytes, which may be responsible for antibody-generating B-cell multiplication and activation. Changes in estrogen, progesterone, and chorionic gonadotropin levels during pregnancy contribute to a shift in the Th 1/Th2 balance, which is not totally understood. The changes in immune response do not limit to changes in Th1/Th2 balance. Myeloperoxidase, phagocytosis, and respiratory burst activities are all significantly lower in neutrophils observed in the peripheral circulation of pregnant women. These alterations and shifts in the Th1/Th2 balance lead to enhanced susceptibility to viral infections in pregnant women.[47]

Coinfection with herpesvirus and bacteria is associated with particularly serious illnesses in humans and animals,[21] but the exact mechanism of pathogenesis is still unclear. Synergistic cooperation between active herpesviruses and bacterial pathogens can be considered to be the cause of severe periodontal disease.[48] Porphyromonas gingivalis in the microbial plaque triggers gingival inflammation which may lead to latent herpes viruses embedded with in the DNA of various defense cells such as macrophages, T-lymphocytes, and B-lymphocytes to gain access to the periodontium.[22],[31] Herpes viruses are capable enough to evade the immune system and cause immune pathogenicity, cytopathogenic effects, latency, tissue tropism, and reactivation.[49] They also have the potential to alter the structural and host defense cells in periodontal tissues and ultimately reduce their ability to resist microbial insult.[22]

Therapies employing antiviral agents along with periodontal debridement might be more efficient in the treatment of periodontal disease. Interferon delivered in a low dose has been found to be successful in reducing gingival inflammation in a canine model.[50] In addition, vaccination against herpes viruses may also prove to be effective in controlling periodontopathic bacteria. Limited sample size, variation in ethnicity of the study population, and lack of biopsy specimen's analysis for the detection of herpes viruses were the limitations of the study.

Future perspectives

The involvement of herpes viruses in periodontal etiology, particularly in pregnant women, may provide a novel direction for periodontal management. Therefore, giving due consideration to the limitations mentioned above, randomized controlled clinical trials in relevant population have been proposed to assess the role of herpes viruses in periodontitis and also the effectiveness of various antimicrobials and/or nonsurgical periodontal therapy on the same.


On the basis of observations drawn from the study, it was suggested that pregnant participants with periodontitis might have enhanced EBV susceptibility which may facilitate the progression of periodontal disease in pregnant women. Nevertheless, more research is warranted to unravel the precise mechanism of interplay between herpes viruses and periodontopathic microbiota before establishing conclusion.

Ethical clearance

The study was approved by the Institutional Ethical Committee of Dasmesh Institute of Dental Sciences and Research, Faridkot, Punjab with approval number DIRDS/2018.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


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