Effects of initial periodontal therapy on the prevalence of Epstein-Barr virus DNA and Porphyromonas gingivalis in Japanese chronic periodontitis patients

doi:10.21203/rs.2.16315/v1

Background Initial periodontal therapy (IPT) is cornerstone of periodontal therapy and the first step to control of periodontal risk factors. Scaling and root planing are used to treat root surface irregularities and remove virulent factors caused by periodontal pathogens. This procedure also incorporated into periodontal surgery. To elucidate the effects of IPT on prevalence of Epstein-Barr virus (EBV) DNA and Porphyromonas gingivalis , we used subgingival plaque samples from chronic periodontitis (CP) patients.

Methods Seventeen CP patients were recruited and determined measured periodontal status clinical parameters such as by probing depth (PD), clinical attachment level (CAL), bleeding on probing (BOP) and an X-ray examination, and subgingival plaque samples were collected from two periodontal sites with PD of < 3 mm (healthy sites: HS) or > 5 mm (periodontitis sites: PS) at first visit and after IPT. Plaque samples were subjected to a real-time PCR to detect EBV DNA and P. gingivalis.

Results EBV DNA and P. gingivalis were detected 9 (52.9%) and 14 (82.3%) sites within the subgingival samples from HS, and 13 (76.5%) and 14 (82.3%) sites within the PS at first visit. After IPT, number of detections of EBV DNA and P. gingivalis were decreased to 5 (29.4%) and 13 (76.5%) sites within the subgingival samples from HS, and 9 (52.9%) and 10 (58.8%) sites within the PS. Significant improvements in PD and BOP were observed after IPT in PS. Coexistence of EBV DNA and P. gingivalis in the subgingival samples from PS at first visit (12 sites; 70.6%) were significantly decreased after IPT (6 sites; 35.3%).

Conclusion These results suggest that the IPT was effective in improvement of clinical parameters such as PD and BOP and reducing the coexistence of EBV and P. gingivalis in the subgingival plaque from PS. However, IPT could not eradicate the EBV and P. gingivalis . Further research would be necessary for improving the periodontal treatment strategy.

Head & Neck Surgery

Epstein-Barr virus

initial periodontal therapy

Porphyromonas gingivalis

periodontitis

prevalence

Patient education, training in oral hygiene, control of risk factors, removal of supra and subgingival bacterial plaque, scaling and root planing (SRP) are contents of implementation of initial periodontal therapy (IPT) [1, 2]. EBV is one of the most prevalent viruses in the world. It is estimated that over 90% of adults are EBV seropositive [3, 4]. Primary infections of infants with EBV are usually asymptomatic, but the infection of adolescence and young adult with EBV causes infectious mononucleosis, a self-limiting, lymphoproliferative disease. Spread within families is thought to be a common route of EBV transmission by salivary contact. The virus infects first within oropharyngeal epithelium, and later primarily within B lymphocytes are invaded via CD21 receptors, where it establishes a lifelong latent infection [5–9]. EBV has been linked to the development of several malignant tumors, including Burkitt’s lymphoma, Hodgkin’s disease certain forms of T-cell lymphoma, lymphoproliferative disease in immunosuppressed individuals, nasopharyngeal carcinoma and a proportion of gastric cancers [10–12]. ZEBRA is an early lytic protein of EBV encoded BZLF1 gene. In the latent state, hypoacetylation of histone in the BZLF1 promoter by histone deacetylases is involved in maintaining EBV latency. The reactivation of EBV from latent infection occurs frequently and multiplies with the epithelium cells of the pharyngeal and is exhausted in saliva [7, 13–15].

Association between EBV infection and periodontal disease has been reported [16, 17]. Therefore, we have examined the coexistence of EBV and Porphyromonas gingivalis which is a representative periodontal pathogen in the subgingival plaque from two periodontal sites with probing depth (PD) of <3 mm or >5 mm. EBV DNA and P. gingivalis were detected in higher copy numbers in deep periodontal pockets and showed a higher incidence of the coexistence as compared to shallow periodontal pockets [18, 19]. We showed that EBV may serve as a pathogenic factor leading to CP among Japanese patients. Periodontopathic anaerobic bacteria such as P. gingivalis could increase the virulence of EBV via reactivation of EBV through butyric acid [18, 19]. The findings of the meta-analysis suggest that EBV and human cytomegalovirus are significantly associated with CP [20]. EBV and P. gingivalis coinfection may promote the development of CP in pregnant women [21]. Therefore, it might be potential interactions between EBV and P. gingivalis in the etiopathogenesis of CP

The purpose of this study was to examine the effects of IPT on the prevalence of Epstein-Barr virus (EBV) DNA and P. gingivalis in the subgingival plaque.

Clinical examination and characteristics of participants

Periodontal examination comprising determination of PD, clinical attachment level (CAL), bleeding on probing (BOP) and an X-ray (intraoral radiograph) examination. At each point in time, periodontal examinations were performed by a trained periodontist (YO) using PCP11 probe (Hu-Friedy, Chicago, IL, USA) according to the method has been published previously [23]. CP patients were defined as the presence of at least two sites with PD >5 mm and CAL of more than 5 mm. Seventeen CP patients (mean age, 44.8 ± 14.9 years) were included in this study. All subjects were systemically healthy and had no history of periodontal treatment or any type of antibiotic therapy for at least 3 months prior to the present study. The Institutional Review Board at the Nihon University School of Dentistry at Matsudo approved the study (EC17–16–15–005–2). Written informed consent was obtained from each study subject after all experiments were fully explained. They received IPT, such as oral hygiene instructions, SRP and mechanical tooth cleaning (within 12 months) at Nihon University Hospital School of Dentistry at Matsudo, Japan.

Sampling

Seventeen subgingival plaque samples were collected from one periodontally healthy site (HS) of PD (<3 mm), and one periodontitis site (PS) of PD (>5 mm) among 17 CP patients at first visit and after initial periodontal therapy. Before sampling, supragingival plaque was removed with Gracey curette. Sterile paper points were inserted to the sample site (three times), retained for 30 sec, pooled in Eppendorf tubes, and then stored at –80 °C [18].

DNA extraction and real-time PCR

DNA samples from the subgingival plaque were prepared using High Pure Viral Nucleic Acid Kit (Roche Applied Science, Mannheim, Germany). Quantitative real-time polymerase chain reaction (PCR) was used to measure the copy numbers of EBV DNA and P. gingivalis in the samples, using the specific primer sets described previously. [19, 24]. The dynamic ranges of the real-time PCR assays were determined through serial dilution of DNA extracts either as AKATA cells or P. gingivalis TDC60 of the standards in the range of 10⁹∼10¹copies/ml [25, 26].

Statistical analysis

The chi squared test for independence, confirmed by Fisher’s exact probability test, was used to determine whether individual pathogens and BOP were changed by IPT. Significant differences between baseline values of PD and values after IPT were analyzed using one-way ANOVA.

The age, sex, PD, CAL and BOP of the patients are summarized in Table 1. Seven males and 10 females were included in this study. The mean PD of the HS and PS at first visit were 2.94 ± 0.24 mm and 7.35 ± 1.54 mm, and then they were changed to 2.82 ± 0.39 mm (HS) and 5.76 ± 1.68 mm (PS) after IPT. The mean CAL of the HS and PS at first visit were 3.65 ± 1.37 mm and 8.76 ± 1.92 mm, and then they were changed to 3.82 ± 1.51 mm (HS) and 7.47 ± 1.74 mm (PS) after IPT. BOP was detected in 2 (11.8%) HS and 17 (100%) PS at first visit, and then BOP could not detect in HS and detected in 11 (65%) PS after IPT. PD and BOP of the PS at first visit were significant improved after IPT. Table 2 and 3 show gender, age, clinical data and counts of EBV DNA and P. gingivalis (copies/ml) of each subject in the HS and PS at first visit and after IPT. EBV DNA and P. gingivalis were detected 9 sites (52.9%, range from 2.52×10² to 1.09×10⁴ copies/ml) and 14 sites (82.3%, range from 5.29×10¹ to 4.71×10⁸ copies/ml) in the subgingival samples from HS at first visit and changed to 5 sites (29.4%, range from 6.12×10² to 8.41×10³ copies/ml) and 13 sites (76.5%, range from 4.34 to 7.61×10⁷ copies/ml) from HS after IPT (Table 2). EBV DNA and P. gingivalis were detected 13 sites (76.5%, range from 3.78×10¹ to 2.55×10⁴ copies/ml) and 14 sites (82.3%, range from 2.28×10³ to 6.21×10⁹ copies/ml) in the subgingival samples from PS at first visit and changed to 9 sites (52.9%, range from 6.50×10² to 8.59×10³ copies/ml) and 10 sites (58.8%, range from 9.97×10¹ to 2.70×10⁹ copies/ml) from PS after IPT.

The prevalence of EBV DNA and P. gingivalis in the subgingival samples from HS or PS are listed in Table 4 and 5. Occurrence of EBV DNA and P. gingivalis in the HS or PS were decreased after IPT, but not statistically significant. Coexistence of EBV DNA and P. gingivalis in the PS at first visit (12; 70.6%) were significantly decreased after IPT (6; 35.3%) (Table 5). However, coexistence of EBV DNA and P. gingivalis in the HS did not decreased significantly after IPT (Table 4).

In this study, we demonstrated that higher levels of EBV DNA, P. gingivalis and coexistence of EBV DNA and P. gingivalis were detected in the HS and PS of Japanese CP patients and they were decreased by IPT. Especially, PD, BOP and coexistence of EBV DNA and P. gingivalis in the PS at first visit were significant decreased after IPT. The results suggest that IPT is effective in improvement of PD and BOP and reducing the coexistence of EBV and P. gingivalis in the subgingival plaque.

Periodontopathic bacteria is major risk factor for periodontal disease, however does not satisfied only by bacterial activity to explain several clinical characteristics of periodontitis, such as rapid bone resorption with minimal amount of plaque, site specific development, and presence of quiescence and active phase. Therefore, EBV could be involved in the etiology of several types of periodontitis [18, 27–29]. In this study, we examined whether higher prevalence of EBV DNA and P. gingivalis are associated with PS in Japanese CP patients and IPT could decrease the number of EBV and P. gingivalis in the subgingival plaque, because several studies suggest that EBV and P. gingivalis act synergistically to potentiate periodontal disease progression and tissue destruction [18, 19, 21, 29, 30]. In this study, EBV DNA and P. gingivalis coexist in the PS of CP patients at high frequency (70.6%). This value correlated with previous studies that showed higher levels (68%) of EBV DNA and P. gingivalis coexist in the deep PD (> 5 mm) in the CP patients [19]. EBV were associated with major periodontopathic bacteria and with the severity of periodontal disease [31, 32]. These reports suggested that high copy numbers of EBV DNA and P. gingivalis may correlate with periodontal disease state. In our previous report, detection rate and counts in PCR-positive deep PD >5 mm (PS) or sallow PD of <3 mm (HS) sites for EBV DNA (detected in 60 80% PS and 40% HC sites) were 3.74 x 10³ ~ 3.70 x 10⁷ and 4.37 x 10⁴ ~ 9.13 x 10⁶ copies/ml, and for P. gingivalis (detected in 80% PS and 36% HC sites) were 2.45 x 10⁷ ~ 1.15 x 10⁹ and 4.16 x 10⁶ ~ 2.13 x 10⁹ copies/ml [19]. The results showed that detection rate of EBV DNA and P. gingivalis in the PS were similar in this study, whereas detection rate in the HS were higher in this study (Table 2–5).

We have previously reported that immunostaining using B cell marker CD19 showed large number of B cells infiltrated into the inflamed gingival connective tissues [18]. And the results of in-situ hybridization using serial section by EBV-encoded small RNA (EBRE) showed a large number of B cells in the same location were EBER-positive [18].

Latent EBV could be induced into the lytic replication cycle by treatment with several inducers, such as anti-immunoglobulin, butyric acid, calcium ionophore, phorbol 12-myristate 13-acetate and transforming growth factor-β [7, 8, 33]. The EBV BZLF1 gene product ZEBRA is a regulator of the transition from latent form to the lytic replication cycle. Histone deacetylase (HDAC) induces hypoacetylation of histone in the BZLF1 promoter, and it is involved in the maintaining of EBV latency. Culture supernatant of P. gingivalis contains butyric acid which is an inhibitor of HDAC, increased histone acetylation and transcriptional activity of the BZLF1 gene [7, 8]. These findings suggest that periodontitis is risk factor for EBV reactivation in infected individuals.

Results of this study provides credence for potential interactions between EBV and P. gingivalis in the etiopathogenesis of periodontitis. EBV and periodontopathic bacteria co-existence apparently leads to additive effects and exacerbates the progress of periodontitis [33]. EBV-infected periodontium tends to harbor high levels of periodontopathic bacteria. Viral and bacterial co-existences were reported more frequently in deeper PD sites of CP patients [29, 30]. We have previously reported that coexistence of EBV DNA and P. gingivalis was significantly higher in CP patients with deeper PD sites (> 5 mm; 40%) than in those with shallow PD sites (< 3 mm; 14%) or healthy controls (13%) [18]. In addition, coexistence of EBV DNA and P. gingivalis was significantly higher in the deeper PD sites (> 5 mm) of CP patients (68%) than in the PD sites of the healthy controls (15%) and shallow PD sites (< 3 mm) of CP patients (12%) [19]. The data suggest that coexistence of EBV and P. gingivalis might increase the risk for developing periodontitis.

IPT was effective in reducing the coexistence of EBV and P. gingivalis in the subgingival plaque. Further studies are necessary to develop the new IPT procedure for keeping latency of EBV or reducing the coexistence of EBV and P. gingivalis.

IPT was effective in improvement of inflammatory conditions of periodontitis and reducing the coexistence of EBV and P. gingivalis in the subgingival plaque. However, IPT could not eradicate the EBV and P. gingivalis. Further research would be necessary for improving the periodontal treatment strategy.

BOP: Bleeding on probing; CP: Chronic periodontitis; EBV: Epstein-Barr virus; GAPDH: Glyceraldehyde–3-phosphate dehydrogenase; HDAC: Histone deacetylase; IPT: Initial periodontal therapy; PCR: Polymerase chain reaction; PD: Probing pocket depth.

Acknowledgements

Acknowledgement for cooperation in statistical analysis to Prof. Satoshi Horihata.

Funding

The study was supported in part by the Japan Society for the Promotion of Science KAKENHI Grants, Grant-in-Aid for Young Scientists (B); No.17K17361 and Scientific Research (C); No. 17K11994, No.19K10078 from the Ministry of Education, Science, Sports and Culture of Japan, and Nihon University Multidisciplinary Research Grant (17–019) for 2017–2018. The funding agencies were not involved in the design of the study; collection, analysis, and interpretation of data; or in writing the manuscript.

Availability of data and materials

The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

Authors’ contributions

AK, KI and YO were responsible for design and conception of the study. AK, YI and YO performed follow-up care of patients and sample collection. All authors were involved in the analysis and interpretation of data. AK and YO was responsible for drafting the manuscript. All authors revised it critically and approved the final manuscript.

Ethics approval and consent to participate

The study had been approved by the Institutional Review Board at the Nihon University School of Dentistry at Matsudo (EC17–16–15–005–2), in accordance with the Declaration of Helsinki. Verbal and written informed consent was received from each subject were then clinically examined.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Author details

¹Department of Periodontology and Research Institute of Oral Science, Nihon University School of Dentistry at Matsudo, Chiba, 271–8587, Japan. ²Department of Microbiology, Division of Immunology and Pathobiology, Dental Research Center, Nihon University School of Dentistry, Tokyo, 102–8310, Japan.

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Table 1 Characteristics of participants

CP patients (n =17)

First visit After IPT

Age (years) 44.8 ± 14.9

Males 7 (41.2%)

Females 10 (58.8%)

PD(mm) 2.94 ± 0.24(HS) 2.82 ± 0.39 (HS)

7.35 ± 1.54(PS) 5.76 ± 1.68 (PS)**

CAL (mm) 3.65 ± 1.37(HS) 3.82 ± 1.51 (HS)

8.76 ± 1.92(PS) 7.47 ± 1.74 (PS)

BOP 2 (11.8%) (HS) 0 (0%)

17 (100%) (PS) 11(65%)**

Chronic periodontitis (CP), initial periodontal therapy (IPT),

probing pocket depth (PD), bleeding on probing (BOP),

clinical attachment level (CAL), healthy sites (HS), periodontitis sites (PS)

Statistically significant; P<0.01**

Table 2 Clinical data and counts of EBV DNA and P. gingivalis in the HS at first visit and after IPT

First visit After IPT

Subject No.	Gender	Age	(≦3) PD (mm)	CAL (mm)	BOP	(Copies/ml) EBV	(Copies/ml) *P. gingivalis*	(≦3) PD (mm)	CAL (mm)	BOP	(Copies/ml) EBV	(Copies/ml) *P. gingivalis*
1	Female	27	3	3	－	ND	9.21 x 10³	3	3	－	ND	6.35 x 10³
2	Female	39	3	8	+	4.49 x 10³	4.71 x 10⁸	3	9	－	ND	7.61 x 10⁷
3	Female	28	2	2	－	ND	5.71 x 10⁷	3	3	－	ND	ND
4	Male	38	3	3	－	ND	ND	2	5	－	ND	ND
5	Female	41	3	3	－	4.48 x 10³	1.11 x 10⁴	3	3	－	ND	8.24 x 10³
6	Female	70	3	3	－	3.40x 10³	2.40x 10³	3	3	－	ND	ND
7	Female	31	3	4	－	ND	1.34 x 10³	3	4	－	1.03 x 10³	1.45x 10²
8	Female	42	3	3	－	ND	8.24 x 10³	3	3	－	ND	2.75 x 10³
9	Male	72	3	5	－	3.18x 10³	4.30 x 10⁴	2	3	－	ND	6.29 x 10⁴
10	Female	59	3	5	－	1.09 x 10⁴	3.61 x 10⁶	2	4	－	ND	9.50 x 10³
11	Male	47	3	3	－	8.34 x 10³	8.14 x 10⁵	3	4	－	7.94 x 10³	3.70 x 10⁴
12	Male	73	3	3	－	ND	5.29 x 10¹	3	3	－	ND	4.34
13	Female	46	3	4	－	2.52 x 10²	8.59 x 10²	3	3	－	6.12x 10²	1.65 x 10³
14	Male	24	3	3	－	ND	ND	3	3	－	ND	ND
15	Male	43	3	3	－	3.75 x 10³	ND	3	3	－	2.10 x 10³	1.23 x 10³
16	Male	37	3	3	－	4.56 x 10³	3.81 x 10³	3	4	－	8.41 x 10³	4.13 x 10¹
17	Female	44	3	4	+	ND	3.39 x 10⁵	3	5	－	ND	1.27 x 10³

not detectable (ND), initial periodontal therapy (IPT), Clinical attachment level (CAL)

Table 3 Clinical data and counts of EBV DNA and P. gingivalis in the PS at first visit and after IPT

First visit After IPT

Subject No.	Gender	Age	(≧5) PD (mm)	CAL (mm)	BOP	(Copies/ml) EBV	(Copies/ml) *P. gingivalis*	(≧5) PD (mm)	CAL (mm)	BOP	(Copies/ml) EBV	(Copies/ml) *P. gingivalis*
1	Female	27	6	7	+	2.98 x 10³	8.41 x 10⁸	6	7	－	ND	1.91 x 10⁸
2	Female	39	8	12	+	2.38 x 10³	3.56 x 10⁸	6	11	+	1.47 x 10³	2.70 x 10⁹
3	Female	28	8	9	+	8.36 x 10³	3.69 x 10⁸	6	7	+	8.59 x 10³	ND
4	Male	38	6	7	+	ND	ND	5	6	+	ND	ND
5	Female	41	8	10	+	ND	2.84 x 10⁹	8	9	+	1.78 x 10³	2.87 x 10⁸
6	Female	70	10	10	+	ND	2.28 x 10³	9	10	+	ND	ND
7	Female	31	6	8	+	9.04 x 10²	1.89 x 10⁴	5	7	+	6.03 x 10³	2.20 x 10⁵
8	Female	42	6	7	+	8.36 x 10³	1.89 x 10⁵	6	6	－	6.29 x 10³	1.53 x 10³
9	Male	72	8	11	+	5.65 x 10³	6.21 x 10⁹	6	8	－	6.50 x 10²	8.23 x 10⁵
10	Female	59	7	9	+	2.55 x 10⁴	4.43 x 10⁹	3	5	－	5.72 x 10³	7.66 x 10⁶
11	Male	47	6	7	+	3.78 x 10¹	1.42 x 10⁶	6	7	+	ND	1.05 x 10⁹
12	Male	73	8	10	+	5.88 x 10³	4.13 x 10⁶	3	7	+	1.15 x 10³	ND
13	Female	46	6	6	+	1.57 x 10²	2.14 x 10⁸	3	5	－	5.29 x 10³	ND
14	Male	24	10	11	+	2.37 x 10²	2.75 x 10⁷	6	8	+	ND	ND
15	Male	43	6	6	+	ND	ND	6	6	+	ND	ND
16	Male	37	6	8	+	8.89 x 10³	ND	6	8	－	ND	9.97 x 10¹
17	Female	44	10	11	+	2.03 x 10³	2.20 x 10⁸	8	10	+	ND	2.24 x 10⁴

not detectable (ND) , initial periodontal therapy (IPT) , Clinical attachment level (CAL)

Table 4 Occurrence of EBV DNA and P. gingivalis in the subgingival samples from HS at

first visit and after IPT

Detection frequency Significance (P-value)

Infectious agents First visit After IPT First visit vs After IPT

EBV 9 (52.9%) 5 (29.4%) 0.148

P. gingivalis 14 (82.3%) 13 (76.5%) 0.5

EBV + P. gingivalis 8 (47.1%) 5 (29.4%) 0.241

Healthy sites (HS), initial periodontal therapy (IPT)

Table 5 Occurrence of EBV DNA and P. gingivalis in the subgingival samples from PS at

first visit and after IPT

Detection frequency Significance (P-value)

Infectious agents First visit After IPT First visit vs After IPT

EBV 13 (76.5%) 9 (52.9%) 0.141

P. gingivalis 14 (82.3%) 10 (58.8%) 0.129

EBV + P. gingivalis 12 (70.6%) 6 (35.3%) 0.042*

Periodontitis sites (PS), initial periodontal therapy (IPT), Statistically significant; P<0.05*

Effects of initial periodontal therapy on the prevalence of Epstein-Barr virus DNA and Porphyromonas gingivalis in Japanese chronic periodontitis patients

Status:

Version 1

Abstract

background

methods

Clinical examination and characteristics of participants

Sampling

DNA extraction and real-time PCR

Statistical analysis

Results

discussion

conclusions

abbreviations

declarations

Acknowledgements

Funding

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