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 ).

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 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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