Prevalence of human cytomegalovirus and Epstein-Barr virus in subgingival plaque at peri-implantitis, mucositis and healthy sites. A pilot study

Abstract

This study evaluated the prevalence of human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) in peri-implantitis and mucositis sites and the correlation between herpesvirus and clinical parameters. Fifty-six dental implants (mean time of loading, 4.27 ± 1.6 years) were evaluated (20 peri-implantitis, 18 mucositis, 18 healthy peri-implant sites.) The clinical parameters assessed were: visible plaque index (PI), bleeding on probing (BOP), suppuration (SUP), probing depth (PD). A polymerase chain reaction assay identified HCMV and EBV in subgingival plaque samples. The percent of sites with plaque and BOP was significantly higher around mucositis and peri-implantitis compared with healthy implants ( p < 0.05). The mean PD around the implants was significantly higher in peri-implantitis, followed by mucositis and healthy implants ( p < 0.05). HCMV was detected in 13 (65%) and EBV in 9 (45%) of the 20 peri-implantitis sites. HCMV was found in 1 of the 18 (6%) healthy periodontal sites and EBV in 2 (11%). A statistically significant correlation was found between presence of HCMV and EBV subgingivally and clinical parameters of peri-implantitis and healthy sites. These results confirm the high prevalence of HCMV and EBV in subgingival plaque of peri-implantitis sites and suggest the viruses have a possible active pathogenic role in peri-implantitis.

Peri-implant disease following successful integration of an endosseous implant is the result of a discrepancy between the bacterial challenge and the host response. Numerous studies show that bacterial infection plays the most important role in the failure of dental implants . Mucositis is a reversible inflammatory reaction limited to the soft tissues around osseointegrated implants; peri-implantitis is characterized by soft tissue inflammation and bone loss around an osseointegrated implant in occlusal function . The microbial profile of peri-implantitis is similar to that found in sites with chronic periodontitis, with high numbers of periodontal pathogens such as Porphyromons gingivalis , Tanerella forsythia , Treponema denticola and Prevotella intermedia in supra and subgingival biofilms .

It has been suggested that herpes viruses contain an important source for triggering periodontal tissue destruction. Studies on a viral cause for periodontitis mark a turning point in periodontal research, which until recently was centred almost exclusively on a bacterial etiology. Epstein-Barr virus (EBV) and cytomegalovirus (HCMV) are the most commonly studied viruses in periodontology, and more than one million herpesvirus genome-copies can be present in a single periodontitis site . Significantly more copies of HCMV and EBV DNA have been detected in deep periodontal pockets than in shallow periodontal pockets . Periodontitis is primarily initiated by microbial accumulation, so the interaction of herpesviruses (HCMV, EBV) with periodontopathic microorganisms presents an important area of investigation. It has been proposed that local immune suppression mediated predominantly by EBV and HCMV permits the overgrowth of periodontopathic microorganisms . HCMV infects periodontal monocytes/macrophages and T-lymphocytes, and EBV infects periodontal B-lymphocytes . HCMV and EBV infected inflammatory cells produce tissue-destroying cytokines and may harm the body’s ability to defend itself against a periodontopathogenic bacterial challenge . Periodontal sites demonstrating HCMV and EBV also have a tendency to exhibit elevated levels of P. gingivalis and Actinobacillus actinomycetemcomitans , important periodontopathogens .

If these viruses possess the biological mechanisms to promote periodontal tissue destruction then the role and influence of herpesviruses in peri-implantitis has to be evaluated carefully. To the authors’ knowledge no study has been published evaluating the association between peri-implantitis and mucositis with HCMV and EBV viruses.

The understanding of the etiopathogenesis of peri-implantitis is incomplete, so the present pilot study was designed to investigate the hypothesis that peri-implantitis is associated with HCMV and EBV viruses. The aim of the study was to evaluate the presence of HCMV and EBV viruses in peri-implantitis and mucositis sites and to investigate any correlation of virus presence with clinical parameters. A polymerase chain reaction (PCR) assay was used to identify HCMV and EBV in subgingival plaque samples from 20 peri-implantitis, 18 mucositis and 18 healthy peri-implant sites.

Materials and methods

Fifty-six patients were consecutively enrolled for this clinical study. The University Ethical Committee approved the protocol for human subjects. Inclusion criteria were subjects treated with at least one Biohorizons ® (Birmingham, AL, USA) tapered implant that had been functioning for at least 1 year. The subjects were systemically healthy non-smokers. Patients were periodontally healthy or, if they presented a history of periodontal disease, they should have been periodontally treated and be engaged in supportive periodontal therapy. Exclusion criteria were the use of systemic antibiotics, anti-inflammatory drugs or oral antimicrobial agents within the preceding 6 months and the presence of herpetic infection (self-reported) during the last 6 months. Implant exclusion criteria were mobility, previous peri-implant treatment and unscrewed prostheses. Subjects were informed of the characteristics of the study and gave their written consent to the procedures described.

All clinical examinations were performed by the same trained and calibrated operator (P.M.D.). The following parameters were assessed at six peri-implant sites (mesio, medio, disto/buccal and lingual) using a periodontal probe (North Carolina–Hu-Friedy, Chicago, IL, USA): visible plaque index (PI), presence or absence of plaque along the mucosal/gingival margin; bleeding on probing (BOP), presence or absence of bleeding of up to 15 s after gentle probing; suppuration (SUP), presence or absence of spontaneous or after probing SUP; probing depth (PD), distance (mm) between the mucosal/gingival margin and the bottom of the sulcus/pocket.

According to clinical and radiographic data, the implants were classified in one of the three following groups according to M áximo et al. Healthy, PD <4 mm without BOP, SUP and radiographic bone loss; mucositis, BOP, absence of radiographic bone loss or SUP; and peri-implantitis, PD ≥5 mm with BOP and/or SUP and concomitant radiographic bone loss (bone loss ≥3 threads until half of implant length). If the same subject had healthy implants and implants with mucositis or peri-implantitis, he/she was included in only one group based on the worst diagnosis as follows: peri-implantitis, mucositis and healthy. All the implants within the same clinical diagnosis per subject were included in the study.

Clinical parameters (PI, BOP, SUP, PD) and frequency of HCMV and EBV detection, were compared between the groups using the Mann–Whitney U -test. Statistical correlation between presence of HCMV or EBV subgingivally and clinical parameters of peri-implantitis was computed using Fisher’s exact test. For all the above, the significance level was set at 0.05.

After removing the supragingival plaque with sterile cotton pellets, the sites were isolated with cotton rolls and gently dried with an air syringe. Subgingival material was collected using a sterile curette. After gently inserting the curette into the bottom of the periodontal site, a single stroke was taken to remove subgingival debris.

DNA extraction and PCR for the detection of HCMV and EBV was carried out according to S aygun et al. The primer set for HCMV consisted of 5′-GAGCGCGTCCACAAAGTCTA-3′ and 5′-GTGATCCGACTGGGCGAAAA-3′, which generated a 264-bp PCR amplification product . Primers for EBV-1 were 5′-AGGGATGCCTGGACACAAGA-3′ and 5′-GCCTCGGTTGTGACAGAG-3′, which generated a 256-bp PCR product . The PCR mixture (50 μl) consisted of 10 pmol of each primer, 1× PCR buffer (Bioron GmbH, Hannover, Germany), 2 units Taq polymerase (Bioron GmbH), 2.5 mM MgCl 2 (Bioron GmbH), 0.1 mM dNTPs (Sigma–Aldrich Chemie Gmbh, Munich, Germany) and 10 μl sample DNA.

Samples were initially denatured at 94 °C for 5 min, followed by 30 cycles, which included denaturation for 30 s at 94 °C, annealing for 30 s at 59 °C, and extension for 30 s at 72 °C, with a final extension at 72 °C for 5 min. Controls included HCMV and EBV positive and negative cell lines. Specificity was confirmed by determining the size of the amplicons and retesting the positive samples. PCR products were detected by electrophoresis in a 1.5% agarose gel containing 0.5 μg/ml ethidium bromide. Gels were analysed using Quantity One software (BioRad Laboratories, Hercules, CA, USA).

Results

The study population consisted of 56 individuals (23 females, 33 males, mean age 49.16 ± 14.5 years). Fifty-six dental implants (mean time of loading 4.27 ± 1.6) were evaluated during the study. Twenty peri-implantitis, 18 mucositis and 18 healthy peri-implant sites were evaluated. No significant differences were observed for demographic and clinical parameters among the three clinical groups at baseline ( Table 1 ).

Table 1
Demographic and clinical description of the study population.
Characteristics Healthy Mucositis Peri-implantitis
Number of patients 18 18 20
Age (years; mean ± SD) 45.1 ± 9.6 50.6± 12 51.8 ± 11.5
Gender; F (female)/M (male) F 8, M 10 F 6, M 12 F 9, M 11
Number of implants 18 18 20
Time of loading (years/mean ± SD) 3.8 ± 1.8 4.4 ± 2.1 4.6 ± 1.5

The mean clinical parameters of all the implants evaluated (mucositis and peri-implantitis and healthy group) are presented in Table 2 . The percentage of sites with plaque and BOP were significantly higher around mucositis and peri-implantitis when compared with healthy implants ( p < 0.05). Mean PD detected in the peri-implantitis group was 5.95 ± 0.89 mm. Recorded mean PD in the mucositis and healthy group was 2.67 ± 0.76 mm and 1.50 ± 0.51 mm. The mean PD around the implants was significantly higher in peri-implantitis, followed by mucositis and healthy implants ( p < 0.05). Sites showing suppuration upon probing in the peri-implantits group were recorded in 65%.

Table 2
Clinical characteristics.
Clinical variables Peri-implantitis Mucositis Healthy
Mean probing pocket depth in mm 5.95 ± 0.89 * 2.67 ± 0.76 * 1.50 ± 0.51
% sites showing bleeding upon probing 100% 100% * 0%
% sites showing suppuration upon probing 65% * 0% 0%
Mean plaque index 1.5 ± 0.6 * 0.6 ± 0.3 0.3 ± 0.2

* Statistically significant p < 0.05.

Table 3 shows the distribution of herpesviruses in the study patients. HCMV was detected in 13 (65%) and EBV-1 in 9 (45%) of the 20 peri-implantitis sites evaluated. HCMV was found in only 1 of the 18 (6%) healthy periodontal sites and EBV-1 in 2 (11%). Co-infection by HCMV and EBV was recorded in 8 (40%) of the peri-implantitis sites. In the mucositis group, HCMV was detected in 4 (22%) and EBV in 6 (33%) of the 18 evaluated sites. No co-infection was detected in the mucositis and healthy groups. The difference between the distribution of HCMV and EBV in peri-implantitis and healthy sites was significant ( p < 0.05). The same relations were recorded between peri-implantitis and mucositis groups ( p < 0.05). There was a significantly higher percentage of sites showing absence of HCMV and EBV in the healthy group (83%) compared with the peri-implantitis group (25%). The same relationship was detected between sites showing absence of HCMV and EBV in the mucositis group (44%) and the healthy group.

Table 3
Occurrence of HCMV and EBV in 20 peri-implantitis, 18 mucositis and 18 peri-implantitis healthy sites.
Items No. (%) of sites showing HCMV No. (%) of sites showing EBV No. (%) of sites showing HCMV–EBV co-infection No. (%) of sites showing neither HCMV nor EBV
Peri-implantitis( n = 20) 13 (65) 9 (45) 8 (40) 5 (25)
Mucositis ( n = 18) 4 (22.2) 6 (33.3) 0 (0) 8 (44.4)
Healthy sites ( n = 18) 1 (5.55) 2 (11.1) 0 15 (83.3)
Peri-implantitis/mucositis p = 0.012 * p = 0.027 * p = 0.097
Mucositis/healthy sites p = 0.200 p = 0.131 p = 0.045 *
Peri-implantitis/healthy sites p = 0.001 * p = 0.038 * p = 0.001 *
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Feb 7, 2018 | Posted by in Oral and Maxillofacial Surgery | Comments Off on Prevalence of human cytomegalovirus and Epstein-Barr virus in subgingival plaque at peri-implantitis, mucositis and healthy sites. A pilot study
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