Microscopic observations of SARS-CoV-2 like particles in different oral samples.

The emerging coronavirus pneumonia epidemic caused by the SARS-CoV-2 infection has spread rapidly around the world. The main routes of transmission of SARS-CoV-2 are currently recognised as aerosol/droplet inhalation. However, the involvement of the oral cavity in coronavirus disease 2019 (COVID-19) is poorly known. The current data indicates the presence of viral RNA in oral samples, suggesting the implication of saliva in SARS-CoV-2 transmission, however, no direct observation of SARS-CoV-2 particles in different oral samples has been reported. In this study, we investigated whether particles of SARS-CoV-2 were present in oral samples collected from three symptomatic COVID-19 patients. Using scanning electron microscopy (SEM), the correlative strategy of light microscopy and electron microscopy and immunofluorescence staining, we showed the presence of SARS-like particles in RT-qPCR SARS-CoV-2-positive saliva, dental plaque and gingival crevicular fluid (GCF) samples. In the saliva samples, we demonstrated the presence of epithelial oral cells with morphogenetic features of SARS-CoV-2 infected cells. Inside those cells, vacuoles filled with nascent particles were observed, suggesting the potential infection and replication of SARS-CoV-2 in oral tissues. Our results corroborate previous studies and confirm that the oral cavity may be a potential niche for SARS-CoV-2 infection and a potential source of transmission.

Nexus between COVID-19 and periodontal disease.

Over the past several decades, studies have demonstrated the existence of bi-directional relationships between periodontal disease and systemic conditions. Periodontitis is a polymicrobial and multifactorial disease involving both host and environmental factors. Tissue destruction is primarily associated with hyperresponsiveness of the host resulting in release of inflammatory mediators. Pro-inflammatory cytokines play a major role in bacterial stimulation and tissue destruction. In addition, these cytokines are thought to underlie the associations between periodontitis and systemic conditions. Current research suggests that increased release of cytokines from host cells, referred to as the cytokine storm, is associated with disease progression in patients with coronavirus disease 2019 (COVID-19). An intersection between periodontitis and pulmonary disease is biologically plausible. Hence, we reviewed the evidence linking COVID-19, cytokines, and periodontal disease. Plaque control is essential to prevent exchange of bacteria between the mouth and the lungs, reducing the risk of lung disease. Understanding these associations may help identify individuals at high risk and deliver appropriate care at early stages.

Methods for Enrichment and Sequencing of Oral Viral Assemblages: Saliva, Oral Mucosa, and Dental Plaque Viromes.

The oral cavity is a major portal of entry for human pathogens including viruses. However, metagenomics has revealed that highly personalized and time-persistent bacteriophage assemblages dominate this habitat. Most oral bacteriophages follow lysogenic life cycles, deploying complex strategies to manage bacterial homeostasis. Although bacterial dysbiosis underlies common oral pathologies such as caries and periodontitis, the cause of these bacteria replacements remains obscure, and it is theorized that bacteriophages play an important role. The enormous sensitivity of metagenomics coupled with next-generation sequencing has made technically feasible to address the putative role of bacteriophages in oral dysbiosis and represents a valuable tool to discover new human viruses.This chapter proposes a workflow that consists of a simple viral enrichment protocol, two alternative random amplification methods, and next-generation sequencing to access virome composition in three oral environments: supragingival plaque, saliva, and mucosa. These protocols circumvent some well-known sources of bias, providing genomic information about DNA and RNA viral communities with minimal contamination from human and bacterial sources.

Characterization and serotype distribution of Aggregatibacter actinomycetemcomitans: Relationship of serotypes to herpesvirus and periodontal status in Indian subjects.

BACKGROUND: The virulence of Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) in any individual depends on the type of strain of this bacterium. To our knowledge, there have been no studies reported in Indian subjects about A. actinomycetemcomitans serotype occurrence, co-existence with herpes virus and the possible influence of such co-existence on periodontal pathology. METHODS: Subjects for this study were a subset of a larger study to identify the prevalence of A. actinomycetemcomitans in chronic periodontitis. A total of 63 subjects (12 periodontally healthy and 51 with chronic periodontitis) who were positive for A. actinomycetemcomitans were serotyped for strain-level identification. The presence of Human Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) was tested in subgingival plaque samples by polymerase chain reaction. RESULTS: All five serotypes a to e were detected. Of the samples analyzed 38.09% harbored a single serotype, 36.5% had two serotypes, 6.3% demonstrated three and 4.7% demonstrated four serotypes. None of the samples showed presence of JP2 strain. Serotypes b, c, and e were most frequently identified in these individuals (46.03%, 36.5% and 38.09% respectively). Presence of serotypes b and c and absence of serotype d was associated with increased PD and CAL. Among 63 samples analyzed, 11 samples had CMV, four samples had EBV and nine samples had both these viruses. The PD and CAL were significantly higher (p = 0.04) when a combination of CMV and one of the serotypes was present indicating a pathological role of the coexistence. CONCLUSION: Multiple serotypes are associated with chronic periodontitis in Indians, however, JP2 strains are not detectable in this cohort. Presence of multiple serotypes and a combination of any serotype with herpesvirus is associated with greater severity of the disease.

Role of Candida species from HIV infected children in enamel caries lesions: an in vitro study.

OBJECTIVES: This study analyzed the capacity of Candida spp. from dental biofilm of HIV infected (HIV+) children to demineralize primary molar enamel in vitro by Transversal Microhardness (TMH), Polarized Light Microscopy (PLM) and the quantity of calcium ions (Ca2+) released from the enamel. MATERIAL AND METHODS: Candida spp. samples were isolated from the supragingival biofilm of HIV+ children. A hundred and forty (140) enamel blocks were randomly assigned to six groups: biofilm formed by C. albicans (Group 1); mixed biofilm formed by C. albicans and C. tropicalis (Group 2); mixed biofilm formed by C. albicans and C. parapsilosis (Group 3); mixed biofilm formed by C. albicans, C. parapsilosis and C. glabrata (Group 4); biofilm formed by C. albicans ATCC (Group 5) and medium without Candida (Group 6). Enamel blocks from each group were removed on days 3, 5, 8 and 15 after biofilm formation to evaluate the TMH and images of enamel were analyzed by PLM. The quantity of Ca2+ released, from Groups 1 and 6, was determined using an Atomic Absorption Spectrophotometer. The SPSS program was used for statistical analysis and the significance level was 5%. RESULTS: TMH showed a gradual reduction in enamel hardness (p<0.05) from the 1st to 15th day, but mainly five days after biofilm formation in all groups. The PLM showed superficial lesions indicating an increase in porosity. C. albicans caused the release of Ca2+ into suspension during biofilm formation. CONCLUSION: Candida species from dental biofilm of HIV+ children can cause demineralization of primary enamel in vitro.

Role of Candida species from HIV infected children in enamel caries lesions: an in vitro study

Abstract Objectives This study analyzed the capacity of Candida spp. from dental biofilm of HIV infected (HIV+) children to demineralize primary molar enamel in vitro by Transversal Microhardness (TMH), Polarized Light Microscopy (PLM) and the quantity of calcium ions (Ca2+) released from the enamel. Material and Methods Candida spp. samples were isolated from the supragingival biofilm of HIV+ children. A hundred and forty (140) enamel blocks were randomly assigned to six groups: biofilm formed by C. albicans (Group 1); mixed biofilm formed by C. albicans and C. tropicalis (Group 2); mixed biofilm formed by C. albicans and C. parapsilosis (Group 3); mixed biofilm formed by C. albicans, C. parapsilosis and C. glabrata (Group 4); biofilm formed by C. albicans ATCC (Group 5) and medium without Candida (Group 6). Enamel blocks from each group were removed on days 3, 5, 8 and 15 after biofilm formation to evaluate the TMH and images of enamel were analyzed by PLM. The quantity of Ca2+ released, from Groups 1 and 6, was determined using an Atomic Absorption Spectrophotometer. The SPSS program was used for statistical analysis and the significance level was 5%. Results TMH showed a gradual reduction in enamel hardness (p<0.05) from the 1st to 15th day, but mainly five days after biofilm formation in all groups. The PLM showed superficial lesions indicating an increase in porosity. C. albicans caused the release of Ca2+ into suspension during biofilm formation. Conclusion Candida species from dental biofilm of HIV+ children can cause demineralization of primary enamel in vitro.

The role of bacteriophages in periodontal health and disease.

The human periodontium health is commonly compromised by chronic inflammatory conditions and has become a major public health concern. Dental plaque, the precursor of periodontal disease, is a complex biofilm consisting mainly of bacteria, but also archaea, protozoa, fungi and viruses. Viruses that specifically infect bacteria - bacteriophages - are most common in the oral cavity. Despite this, their role in the progression of periodontal disease remains poorly explored. This review aims to summarize how bacteriophages interact with the oral microbiota, their ability to increase bacterial virulence and mediate the transfer of resistance genes and suggests how bacteriophages can be used as an alternative to the current periodontal disease therapies.

Human cytomegalovirus-1 and Epstein-Barr virus-1 viral colonization of titanium and zirconia abutments: a split-mouth study.

AIM: Human cytomegalovirus-1 (HCMV-1) and Epstein-Barr virus-1 (EBV-1) detection in submarginal plaque is linked to diseased states of the periodontium. In the present study, we evaluated the viral colonization of titanium and zirconia abutments by HCMV-1 and EBV-1 in a split-mouth study. METHODS: Forty dental implant abutments placed in 20 non-smokers were evaluated retrospectively. Each participant had received at least one each of titanium and zirconia abutments (in function for at least 1 year). HCMV-1 and EBV-1 were evaluated in these clinically-healthy peri-implant sites' submarginal plaque biofilm at one titanium and one zirconia abutment, one healthy tooth site, and serum using polymerase chain reaction assays. Related-samples McNemar test and Wilcoxon signed-rank test were used to determine the differences in viral detection frequency and load, respectively. RESULTS: EBV-1 was detected at the titanium abutment in 60% of participants, but in none at their zirconia abutment (P = 0.04). HCMV-1 was detected at the titanium abutments in 90% of participants, and at the zirconia abutments in 70% of participants. This difference was not significant (P = 0.25). The differences in HCMV-1 viral load between the abutment types were insignificant (P = 0.075). CONCLUSION: EBV-1 did not colonize the biofilm at the zirconia abutments as opposed to the titanium abutments in the same participants. Abutment material could contribute to differences in biofilm characteristics.

Efficacy of the ND:YAG laser therapy on EBV and HSV1 contamination in periodontal pockets.

AIM: The aim of this retrospective multicenter study was to verify the efficacy of Nd:YAG laser in the treatment of periodontal pockets infected by Epstein-Barr Virus (EBV) and Herpes Simplex Virus 1 (HSV1). METHODS: Subgingival plaque samples of 291 Italian periodontal patients were analyzed by Real Time PCR to evaluate the frequency of both viruses before and after Nd:YAG laser-assisted periodontal treatment. RESULTS: Before treatment, EBV and HSV1 were observed in 29.9% and in 3.8% of periodontal patients respectively, while co-infection with both viruses was detected in 1.7% of cases. Periodontal Nd:YAG laser treatment ("Periodontal Biological Laser-Assisted Therapy", PERIOBLAST) produced statistical significant benefits, especially in EBV periodontal infection: 78.2% of EBV positive patients became EBV-negative following treatment. CONCLUSIONS: Results of this preliminary study highlight that EBV is found in periodontal pockets more frequently than HSV1, supporting the theory of the potential role of EBV in the onset and progression of periodontal disease. Moreover, our data showed that Nd:YAG laser-assisted periodontal treatment (Perioblast) is also effective in case of viral infection, validating evidences that it represents a successful alternative approach to traditional periodontal protocols.

Levels of HIV-1 in subgingival biofilm of HIV-infected patients.

AIM: The aims of the current study were to compare the levels of HIV-1 in the subgingival biofilm (SHVL) between detectable and undetectable plasmatic HIV-1 viral load (PHVL) in HIV-infected patients as well as to determine the association of SHVL with PHVL and clinical periodontal parameters. MATERIAL AND METHODS: Forty-one HIV-infected individuals were divided into two groups: detectable (21) and undetectable (20) PHVL. Subgingival biofilm samples were obtained for detection and quantification of HIV-1 by real-time RT-PCR. To estimate the effect of co-variables on the outcome undetectable SHVL, the Generalized Estimation Equation (GEE) was employed. RESULTS: Detectable SHVL was observed only in the detectable PHVL group and the detection of the HIV-1 was observed in 40% of these individuals. In the bivariate analysis between co-variables from the individual level and the outcome SHVL, significant difference was observed only for the CD4+ T lymphocytes levels (p = 0.017). The multiple logistic model demonstrated that only CD4+ T lymphocytes levels had a significant effect on the outcome undetectable SHVL [OR 8.85 (CI 3.6-9.2), p = 0.002]. CONCLUSION: HIV-1 can be detected and quantified in the subgingival biofilm of HIV-infected individuals, but these findings are not associated with PHVL and periodontal clinical parameters.

Characterization of bacteriophage communities and CRISPR profiles from dental plaque.

BACKGROUND: Dental plaque is home to a diverse and complex community of bacteria, but has generally been believed to be inhabited by relatively few viruses. We sampled the saliva and dental plaque from 4 healthy human subjects to determine whether plaque was populated by viral communities, and whether there were differences in viral communities specific to subject or sample type. RESULTS: We found that the plaque was inhabited by a community of bacteriophage whose membership was mostly subject-specific. There was a significant proportion of viral homologues shared between plaque and salivary viromes within each subject, suggesting that some oral viruses were present in both sites. We also characterized Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) in oral streptococci, as their profiles provide clues to the viruses that oral bacteria may be able to counteract. While there were some CRISPR spacers specific to each sample type, many more were shared across sites and were highly subject specific. Many CRISPR spacers matched viruses present in plaque, suggesting that the evolution of CRISPR loci may have been specific to plaque-derived viruses. CONCLUSIONS: Our findings of subject specificity to both plaque-derived viruses and CRISPR profiles suggest that human viral ecology may be highly personalized.

Failure to detect an association between aggressive periodontitis and the prevalence of herpesviruses.

BACKGROUND: Herpes simplex virus type 1 (HSV-1), human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) have been suspected to play a causal role in periodontitis pathogenesis. The aim of this study was to determine the prevalence of these viruses in subgingival plaque samples of Caucasian patients with generalized aggressive periodontitis compared to periodontally healthy controls. METHODS: A total of 65 patients with aggressive periodontitis and 65 unmatched controls from Germany were investigated in the study. Subgingival plaque samples were analysed for the presence of HSV-1, EBV and HCMV by quantitative real-time polymerase chain reaction assays. Viral antibody titres were determined quantitatively by immunosorbent assays. RESULTS: DNA of HSV-1 and HCMV were detected in 1.5% of the patients and controls, whereas EBV DNA was present in 10.8% and 13.9% respectively. Detection rates of serum IgG against HSV-1 (76.1% versus 73.9%), EBV (98.5% versus 96.9%), HCMV (47.7% versus 46.2%) and IgM levels against HSV-1 (6.2% versus 1.5%), EBV (0% versus 0%), HCMV (0% versus 1.5%) did not significantly differ between patients and controls. CONCLUSION: The data of our study do not suggest any contribution of HSV-1, EBV or HCMV to aggressive periodontitis in a German population. Ethnic and methodological aspects might have caused conflicting results of previous studies.

Comparative analysis of presence of Cytomegalovirus (CMV) and Epsteinbarr virus -1 (EBV-1) in cases of chronic periodontitis and aggressive periodontitis with controls.

AIM: The aim of the present study was to evaluate the presence of Cytomegalovirus (CMV) and Epsteinbarr virus -1 (EBV-1)viruses in sub gingival plaque of chronic periodontitis (groupA), aggressive periodontitis patients (group B), periodontally healthy controls (group C) and to compare the clinical parameters between virus negative and positive sites in each of these groups. MATERIALS AND METHODS: Sixty subjects were included in the study and equally divided into the 3 groups (group A - 20, group B - 20, group C - 20). Sub gingival plaque samples were obtained from the 3 deepest periodontal pocket sites in case of subjects suffering from periodontitis, and from one random bleeding site per quadrant in healthy groups. Clinical parameters like plaque index (PI), gingival index (GI), pocket depth (PD) and clinical loss of attachment (CAL) were recorded. Viral Deoxyribonucleic acid (DNA) was extracted using Proteinase-K DNA Extraction method, and the presence of CMV and EBV-1 was detected by polymerase chain reaction and 2% agarose gel. RESULTS: Results of our study showed a 45% prevalence of CMV and EBV-1 in Aggressive periodontitis cases. Prevalence of CMV in chronic periodontitis and healthy subjects was 20% and 10%, respectively; while for EBV-1 it was 25% and 0%, respectively. In terms of comparison of the clinical parameters with virus presence, both CMV and EBV-1 positive sites showed a significantly higher mean pocket depth compared to virus negative sites. CONCLUSION: Our studyshowed that the prevalence of EBV1 was higher in chronic and aggressive periodontitis subjects compared to controls and the prevalence of CMV was higher in aggressive periodontitis patients. The virus positive sites showed higher pocket depth compared to virus negative sites.

Genomic library screening for viruses from the human dental plaque revealed pathogen-specific lytic phage sequences.

Bacterial pathogenesis presents an astounding arsenal of virulence factors that allow them to conquer many different niches throughout the course of infection. Principally fascinating is the fact that some bacterial species are able to induce different diseases by expression of different combinations of virulence factors. Nevertheless, studies aiming at screening for the presence of bacteriophages in humans have been limited. Such screening procedures would eventually lead to identification of phage-encoded properties that impart increased bacterial fitness and/or virulence in a particular niche, and hence, would potentially be used to reverse the course of bacterial infections. As the human oral cavity represents a rich and dynamic ecosystem for several upper respiratory tract pathogens. However, little is known about virus diversity in human dental plaque which is an important reservoir. We applied the culture-independent approach to characterize virus diversity in human dental plaque making a library from a virus DNA fraction amplified using a multiple displacement method and sequenced 80 clones. The resulting sequence showed 44% significant identities to GenBank databases by TBLASTX analysis. TBLAST homology comparisons showed that 66% was viral; 18% eukarya; 10% bacterial; 6% mobile elements. These sequences were sorted into 6 contigs and 45 single sequences in which 4 contigs and a single sequence showed significant identity to a small region of a putative prophage in the Corynebacterium diphtheria genome. These findings interestingly highlight the uniqueness of over half of the sequences, whilst the dominance of a pathogen-specific prophage sequences imply their role in virulence.

Correlation between different genotypes of human cytomegalovirus and Epstein-Barr virus and peri-implant tissue status.

BACKGROUND: The purpose of this study was to estimate the prevalence of different genotypes of human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) in peri-implantitis and mucositis sites, and to evaluate the correlation between herpesvirus presence and clinical parameters. METHODS: A total of 80 dental implants (mean time of loading, 4.16 ± 1.8 years) were evaluated during the course of the study (30 peri-implantitis, 25 mucositis and 25 healthy peri-implant sites). The following clinical parameters were assessed: visible plaque index, bleeding on probing, suppuration and probing depth. A polymerase chain reaction (PCR) assay was used to identify the presence of different HCMV and EBV genotypes in peri-implant tissue plaque samples. RESULTS: HCMV-2 was detected in 53.3% and EBV-1 in 46.6% of the 30 peri-implantitis sites evaluated. By contrast, HCMV-2 was not detected in healthy periodontal sites and EBV-1 was detected in one healthy site. A statistically significant correlation was found between the presence of HCMV-2 and EBV-1 genotypes and clinical parameters of peri-implantitis. CONCLUSIONS: The results from the present study confirmed the high prevalence of HCMV-2 and EBV-1 in the peri-implant tissue plaque of peri-implantitis sites and suggests a possible active pathogenic role of the viruses in peri-implantitis.

Impact of Chikungunya virus infection on oral health status: an observational study.

BACKGROUND AND OBJECTIVE: Chikungunya fever outbreak started in December 2005 in India when the country experienced more than 13 lakhs of Chikungunya infected cases. We undertook this study to describe the impact of Chikungunya virus infection on oral health. MATERIALS AND METHODS: The confirmed seropositive patients were included for the study (N = 97). Oral hygiene index simplified, gingival index, plaque index were recorded. RESULTS: Of the 181 tested, 97 were confirmed seropositive for chikungunya infection. Pain and bleeding gums were seen in 55% of the subjects. Of them, 29.1% had poor oral hygiene, 42.27% had severe gingivitis, and 27.84% had severe plaque deposits. Severe gingivitis was observed in patients with chronic disease, this association was statistically significant (χ2 = 6.417, P = 0.040). CONCLUSION: Our findings showed that about more than half of the tested patients suffered severe pain and bleeding in the oral cavity thereby causing discomfort in chewing. About 1/3 patients had severe gingivitis and foul breath which caused discomfort in carrying out their day-to-day activities.

Relationship between herpesviruses and periodontopathogens in patients with HIV and periodontitis.

BACKGROUND: The purpose of the present study is to verify a possible association between herpesviruses and periodontal pathogens in individuals with human immunodeficiency virus (HIV) and periodontitis. METHODS: Twenty-seven patients with HIV and chronic periodontitis and 23 patients with HIV and gingivitis were included in the study. Probing depth, clinical attachment loss, gingival index, and plaque index were recorded. Blood, saliva, and subgingival plaque were processed for viral and bacterial identification. Bacteria were identified by 16S rRNA-based polymerase chain reaction and viruses by the nested polymerase chain reaction. RESULTS: For the chronic periodontitis group, Epstein-Barr (EBV)-1 (70.4%) and Tannerella forsythia (Tf) (51.8%) presented higher detection in subgingival plaque and saliva (81.5% and 40.7%, respectively) than in blood (22% and 0%, respectively) (P <0.005 and P <0.0001, respectively). Porphyromonas gingivalis (Pg) was more frequent in subgingival plaque (77.7%; P <0.0001). In the gingivitis group, Pg and human cytomegalovirus (HCMV) presented higher frequency in subgingival plaque (95.6% and 91.3%, respectively; P <0.0001 and P = 0.004). Tf and EBV-1 were detected more frequently in subgingival plaque (47.8% and 78.3%, respectively) and saliva (52.2% and 52.2%, respectively; P = 0.004 and P <0.005) than in blood. EBV-1, EBV-1-HCMV, and presence of different viruses presented an association with periodontitis in saliva. CONCLUSIONS: No association was detected for herpesviruses and periodontal pathogens in patients who are HIV-positive with periodontitis. EBV-1 and coinfection (EBV-1-HCMV) were associated with patients who are HIV-positive with periodontitis.

Subgingival Epstein-Barr and cytomegalovirus occurrence in pregnancy gingivitis.

BACKGROUND: Although recent studies focused on the role of human herpesviruses in various types of periodontal disease, there was a lack of information in these reports regarding the role of pregnancy gingivitis. The aim of this study is to determine the correlation between pregnancy and the subgingival virus presence and their relationship with clinical parameters. METHODS: Seventy pregnant and 40 non-pregnant women were examined for gingival and plaque indices, bleeding on probing (BOP), and clinical probing depths (PDs) from the whole dentition. Subgingival plaque samples were obtained from sites showing signs of gingivitis and healthy sites. The polymerase chain reaction methodology was used to detect cytomegalovirus (CMV) and Epstein-Barr virus (EBV) from plaque samples. RESULTS: Our results show that gingivitis lesions in 27 (38.6%) and 10 (14.3%) pregnant patients were positive for EBV and CMV, respectively. In the non-pregnant group, EBV and CMV were detected in six (15%) and eight (20%) lesions, respectively. A statistically significant difference (P <0.01) was found between the subgingival occurrence of EBV in the two groups. In gingivitis sites, clinical PDs were affected by gestation (P <0.001) and the occurrence of EBV (P <0.001). In healthy sites, clinical PDs were affected by gestation (P <0.05), and BOP was affected by the occurrence of CMV and EBV (P <0.001). CONCLUSION: Our data indicate that pregnancy increased the risk of the presence of subgingival EBV in pregnant women by 3.647 times more than in non-pregnant women.

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

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.