Oral human cytomegalovirus prevalence and its relationships with periodontitis and Porphyromonas gingivalis in Japanese adults: a cross-sectional study

Abstract Objective This study aimed to clarify the association between oral human cytomegalovirus (HCMV) and periodontitis in Japanese adults. Methodology In total, 190 patients (75 men and 115 women; mean age, 70.2 years) who visited Hiroshima University Hospital between March 2018 and May 2020 were included. Oral rinse samples were taken to examine the presence of HCMV DNA using real-time polymerase chain reaction (PCR). P. gingivalis was detected by semi-quantitative PCR analysis. Results HCMV DNA was present in nine of 190 patients (4.7%). There were significant associations between HCMV presence and the presence of ≥4-mm-deep periodontal pockets with bleeding on probing (BOP) (P<0.01) and ≥6-mm-deep periodontal pockets with BOP (P=0.01). However, no significant relationship was observed between HCMV presence and periodontal epithelial surface area scores. Logistic regression analysis revealed that the presence of ≥4-mm-deep periodontal pockets with BOP was significantly associated with HCMV (odds ratio, 14.4; P=0.01). Propensity score matching was performed between patients presenting ≥4-mm-deep periodontal pockets with BOP (i.e., active periodontitis) and patients without ≥4-mm-deep periodontal pockets with BOP; 62 matched pairs were generated. Patients who had ≥4-mm-deep periodontal pockets with BOP showed a higher rate of HCMV presence (9.7%) than those who lacked ≥4-mm-deep periodontal pockets with BOP (0.0%). There was a significant relationship between HCMV presence and ≥4-mm-deep periodontal pockets with BOP (P=0.03). A significant relationship was found between HCMV/P. gingivalis DNA presence and ≥4-mm-deep periodontal pockets with BOP (P=0.03). Conclusions Coinfection of oral HCMV and P. gingivalis was significantly associated with active periodontitis. Moreover, interactions between oral HCMV and P. gingivalis may be related to the severity of periodontal disease.

The oral cavity is a common site for primary human herpesvirus infection. HCMV prevalence has varied in patients with gingivitis or chronic periodontitis. 6,7 Periodontitis is a polymicrobial infectious disease affecting gingiva, tooth-supporting connective tissue, and alveolar bone. Periodontal disease destroys the crevicular epithelium, allowing HCMV to infect epithelial cells in the periodontal pocket.
Therefore, oral HCMV prevalence is associated with inflammation in periodontal tissue. HCMV has been found in unstimulated whole saliva from people with periodontitis, indicating that saliva may play a vital role in the transmission of HCMV to others. 8 To the best of our knowledge, little is known regarding the relationship of HCMV with inflamed periodontal pocket/ periodontal inflamed surface area (PISA) in Japanese adults. PISA is a useful indicator for evaluating the total amount of inflamed periodontal tissue. 9 In this crosssectional study, we investigated oral HCMV prevalence and its relationship with inflamed periodontal pockets.
Furthermore, we investigated the association of HCMV with periodontal disease-related bacteria (i.e., P. gingivalis). The fimA fimbria of P. gingivalis is a virulence factor for P. gingivalis. 10 Type II and IV fimA are closely associated with periodontal disease. 10 Therefore, we investigated the fimA genotype to clarify the association between the HCMV prevalence and fimA genotype. Furthermore, we performed propensity score matching to analyze the relationship of HCMV with active periodontitis. HCMV DNA detection by real-time PCR Real-time PCR analysis was performed to determine HCMV DNA copy number in samples using a CFX connect real-time PCR detection system (Bio-Rad, Hercules, CA, USA) in accordance with a previous study. 13 The CMV PPS Set (Nippon Genetics Co., Ltd., Tokyo, Japan) was used, including PCR primer, probe, and standard PCR products, for HCMV DNA detection.
HCMV strain AD169 was used to prepare the DNA control for PCR. Ten-fold dilutions of the standard PCR products (copy numbers ranging between 2.0 and 2.0×10 4 ) were used to generate a standard curve of HCMV DNA, in accordance with the manufacturer's protocol ( Figure 1). Real-time PCR analysis was performed using FastGene™ QPCR Probe Mastermix w/ROX (Nippon Genetics Co., Ltd.). Amplification was performed with the following thermocycler protocol: 95°C for 10 min; followed by 50 cycles of 95°C for 10 sec and 62°C for 1 min; and then 40°C for 30 sec. Copy numbers above the detection limit in a standard curve for HCMV DNA were considered as positive results. P. gingivalis detection and fimA genotyping by PCR In accordance with the method used in our previous study, P. gingivalis was detected by PCR with specific DNA primer sets. 14 Furthermore, the fimA genotype of P. gingivalis was examined using specific primers, as described previously. 15 PCR products were amplified with GoTaq ® Green Master Mix (Promega, Madison, WI, USA). Amplification was performed with the following thermocycler protocol: initial melting at 95°C for 2 min; followed by 30 cycles of 95°C for 1 min, 57°C for 1 min, and 72°C for 1 min. PCR products were electrophoresed on 2% agarose gels and stained with ethidium bromide. The presence of PCR product in the expected size was regarded as a positive result.

Statistical analysis
The Mann-Whitneys U test was used to compare differences in clinical parameters between groups.
When necessary χ 2 test or Fisher's exact test were used. We performed residual analysis to determine which cell had a significant difference in the χ 2 test for comparison between four groups. If the adjusted standardized residue was ≥1.96, the cells were considered to present significantly more subjects than   however, these differences were not statistically significant. Post-hoc statistical analysis revealed that the statistical power of the χ 2 test was ≥0.8 (i.e., high statistical power), but the statistical power of the Mann-Whitneys U-test was <0.8 (i.e., low statistical power). These results suggested a possible β error due to the low prevalence of HCMV. 11 Therefore, it was necessary to consider the probability of a β error when assessing the results of PCR, PISA, and PESA statistical analyses.
Associations of oral HCMV/P. gingivalis with periodontal health condition Associations of HCMV/P. gingivalis DNA presence with the plaque control score and periodontal health condition were assessed (    periodontitis in middle-aged and older Japanese people (mean age in this study: 70.2 years).

The association between PISA and oral HCMV
has not been fully elucidated so far. In this study, no significant association was found between PISA and oral HCMV prevalence. Oral HCMV may be involved in local periodontal inflammation rather than total periodontal inflammation. Additionally, the low prevalence of HCMV may have caused false negative results in the statistical analysis of PISA. A further study will be required to clarify the association between oral CMV and PISA using a large number of HCMV-positive cases. Furthermore, the presence of HCMV in inflamed periodontal pockets remains unclear.
Additional investigation using resected inflamed periodontal tissue or crevicular fluid is necessary to identify the existence of HCMV in periodontal pockets.

Regarding oral herpes viruses other than HCMV,
EBV is presumed to establish lifelong latent infections in the oral cavity. 16 The specific histological structure of the tonsillar region may facilitate EBV infection, for lymphoid tissue is abundant in the tonsillar region.
EBV can infect B lymphocytes through saliva. It can also infect epithelial cells. 16   However, the attachment level of the teeth was not measured in many participants. Therefore, the relationship between HCMV prevalence and the severity of periodontitis remains unclear. Furthermore, the small number of HCMV-positive cases may have affected the results of statistical analyses of plaque accumulation and the periodontal inflamed surface area.
In conclusion, oral HCMV was significantly associated with deep periodontal pockets with bleeding. We found that coinfection of oral HCMV and P. gingivalis is important for active periodontitis.
Moreover, interactions between oral HCMV and P.
Further prospective studies are necessary to confirm our findings and to clarify the association between persistent oral HCMV infection and periodontitis progression.

Funding
This study was financially supported by university grants from Hiroshima University.

Competing interests
The authors declare that they have no competing interests.