Periodontal Healthy Individuals Research Articles

The Influence of Circadian Rhythm on the Antioxidant Capacity of Saliva in Periodontal Diseases.

Background Saliva has a powerful antioxidant activity proposing that it might have a protective role in the oral cavity. It is yet unclear, how circadian rhythm might affect this activity. Objective The main goal of this study was to compare the antioxidant status of saliva in patients with periodontal diseases (PD) to that of healthy people on a diurnal basis. Material and methods A total of 18 periodontal healthy individuals and 18 patients with chronic periodontitis were chosen. Samples of saliva were collected in the morning between 6:00 and 8:00 and in the evening between 6:00 and 8:00 (both stimulated and non-stimulated). The amount of glutathione (GSH), malondialdehyde (MDA), and total antioxidant status (TAS) in the salivary samples were analyzed, and its flow was also assessed. In addition, the scavenging capacity of saliva was tested in three systems generating oxygen free radicals. Results Results showed that GSH and TAS concentrations in the evening saliva of healthy subjects were significantly higher than those in the morning saliva, while MDA levels decreased (p<0.05). Conversely, there was no significant increase in GSH and TAS levels in the evening saliva of subjects with PD, and lipid peroxidation remained constant. On the other hand, the evening saliva of healthy subjects but not of subjects with PD was significantly more potent in scavenging free radicals in vitro than the morning saliva, especially for the superoxide (O2.-)radical (p<0.05). Moreover, scavenging activity was higher in stimulated than non-stimulated saliva. This activity was higher in evening saliva compared to the morning one and greater in healthy subjects compared to patients with PD (p<0.05). Conclusion A balance exists between oxidative stress and antioxidant mechanisms to maintain homeostasis in the oral cavity. This balance is deregulated in patients with PD as their saliva is unable to properly scavenge free radicals that might potentially increase over the day. Antioxidant supplements may be used in accordance with the circadian rhythm to minimize oxidative damage.

Upregulated Vanins and their potential contribution to periodontitis

BackgroundAlthough Vanins are closely related to neutrophil regulation and response to oxidative stress, and play essential roles in inflammatory diseases with clinical significance, their contribution to periodontitis remains to be determined. This research was designed to assess the expression of Vanins in human gingiva, and to define the relationship between Vanins and periodontitis.MethodsForty-eight patients with periodontitis and forty-two periodontal healthy individuals were enrolled for gingival tissue sample collection. Expression levels of VNN1, VNN2 and VNN3 were evaluated by RT-qPCR and validated in datasets GSE10334 and GSE16134. Western blot and immunohistochemistry identified specific proteins within gingiva. The histopathological changes in gingival sections were investigated using HE staining. Correlations between Vanins and clinical parameters, PD and CAL; between Vanins and inflammation, IL1B; and between Vanins and MPO in periodontitis were investigated by Spearman's correlation analysis respectively. Associations between VNN2 and indicators of neutrophil adherence and migration were further validated in two datasets.ResultsVanins were at higher concentrations in diseased gingival tissues in both RT-qPCR and dataset analysis (p < 0.01). Assessment using western blot and immunohistochemistry presented significant upregulations of VNN1 and VNN2 in periodontitis (p < 0.05). The higher expression levels of Vanins, the larger the observed periodontal parameters PD and CAL (p < 0.05), and IL1B (p < 0.001). Moreover, positive correlations existed between VNN2 and MPO, and between VNN2 and neutrophil-related indicators.ConclusionOur study demonstrated upregulation of Vanins in periodontitis and the potential contribution of VNN2 to periodontitis through neutrophils-related pathological processes.

Effects of salivary microbiota on tryptophan-aryl hydrocarbon receptor signaling axis in mice with periodontitis

Objective: To study the effects of salivary microbiota in patients with periodontitis on the tryptophan-aryl hydrocarbon receptor (AhR) signaling axis in mice with periodontitis and to provide theoretical basis as well as new ideas for the influences of periodontitis on systemic metabolism. Methods: Salivary microbiota of 12 healthy individuals and 14 patients with periodontitis were collected in Nanjing Stomatological Hospital, Medical School of Nanjing University from June to December of 2020. According to the random number table method, twenty-four mice were randomly divided into three groups: Sham group (control group), P group (periodontitis patients' salivary microbiota group) and H group (periodontal healthy individuals' salivary microbiota group). The maxillary second molars of all mice were treated with silk thread ligation to induce periodontitis. Phosphate buffer as well as salivary microbiota of periodontal healthy individuals and periodontitis patients were gavaged into periodontitis mice for 2 weeks. The expression of inflammatory factors in mice serum were detected by enzyme linked immunosorbent assay, and the expression of tryptophan and indole metabolites in intestinal tract and serum were detected by liquid chromatography-mass spectrometry. The expression of AhR in intestinal tract of mice was detected by immunohistochemistry and quantitative real time-PCR while gut microbiota constitution was detected by 16S rRNA gene sequencing. The remaining saliva samples of periodontitis patients and periodontal healthy individuals were applied to detect the expression of tryptophan and indole metabolites themselves. Results: The salivary microbiota of periodontitis patients could induce the expression of interleukin-1β [P group: (162.38±39.46) pg/ml, H group: (82.83±20.01) pg/ml; t=4.40, P=0.001) and tumor necrosis factor-α [P group: (361.16±123.90) pg/ml, H group: (191.66±106.87) pg/ml; t=2.54, P=0.030) in serum of periodontitis mice, and reduce the expression of AhR in colon (P group: 1.18±0.05, H group:1.83±0.47; t=3.09, P=0.015) and ileum (P group: 0.80±0.13, H group: 1.18±0.11; t=4.93, P=0.001). After gavage of salivary microbiota of periodontitis patients to the mice, tryptophan (P group: (18.1±3.8)×107, H group: (26.6±6.6)×107; t=2.49, P=0.037] and indole lactic acid [P group: (1.9±0.7)×107, H group: (3.7±0.6)×107; t=4.49, P=0.002) in serum of periodontitis mice were significantly decreased, but was relatively disorder in intestinal tract. However, the expressions of tryptophan and indole metabolites in saliva of periodontitis patients were higher than those of periodontal healthy individuals. There were significant differences in indole propionic acid [P group: (1 239.39±818.72) nmol/L, H group: (56.96±38.33) nmol/L; t=2.83, P=0.022]. What we find noteworthy was that the expressions of indolelactic acid metabolism in saliva, serum and intestinal were consistent, and salivary microbiota of periodontitis patients could reduce the relative abundance of indolelactic acid-producing bacteria in the gut, suggesting that the salivary microbiota of periodontitis patients might affect the expression of AhR through gut microbiota disorder and indolelactic acid downregulation. Conclusions: Salivary microbiota in patients with periodontitis may affect the systemic inflammatory state through down-regulating the expression of tryptophan-AhR signal axis.

“Gingival Crevicular Fluid Levels of Interleukin-35 in Periodontal Health, Chronic Periodontitis &amp; the Effect of Scaling &amp; Root Planning on Interleukin-35 Levels in Chronic Periodontitis – A Randomized Controlled Clinical Study”

Background &amp; Objectives: Interleukin-35 (IL-35) is a newly identified anti-inflammatory cytokine generated by T regulatory cells. They are most effective at high inflammation sites and are a potent activator of T regulatory cells. Hence this study aimed to assess gingival crevicular fluid levels of IL-35 in healthy subjects and chronic periodontitis patients and to compare the levels of IL-35, before and after scaling and root planing in chronic periodontitis patients. Methods: Forty subjects were selected and categorised into 3 groups. Group 1: Periodontally healthy individuals, Group 2A: Chronic periodontitis patients and Group 2B: Group 2A patients who were evaluated 6 weeks after scaling and root planing. Gingival bleeding index, probing pocket depth and clinical attachment level were recorded. Gingival crevicular fluid samples were collected and stored at -800 C till they were subjected to analysis by Enzyme Linked Immunosorbent Assay (ELISA). Results: On intergroup comparison, the difference in mean IL-35 levels and clinical parameters were highly statistically significant between Group 1 and Group 2A while between Group 1 and Group 2B were not significant. On comparing Group 2A and Group 2B, the mean differences were highly statistically significant P (&lt;0.001). Significant reductions in all parameters were noted in Group 2B. Conclusion: Significant reduction of IL-35 levels in chronic periodontitis patients, 6-8 weeks after scaling and root planing and their presence in periodontally healthy subjects suggest the role of IL-35 in controlling the inflammation and their protective role in maintaining periodontal health. Key message: IL-35 has an effective role in controlling the inflammation and in maintaining periodontal health. IL-35 can be considered as a promising prognostic biomarker in periodontal diseases.

Probiotic Potential of Lactobacilli Isolated from Saliva of Periodontally Healthy Individuals.

To search for useful probiotics, we characterised antimicrobial lactic acid bacteria isolated from the oral cavities of 20 healthy volunteers from Nihon Univerisity School of Dentistry. Oral lactobacilli were isolated from the saliva of 20 periodontally healthy volunteers using the Dentocult LB dip-slide method. Primary antimicrobial screening of Lactobacillus isolates was performed using the paper disk method. Each suspected antimicrobial isolate was tested against Streptococcus mutans, S. sobrinus, and Porphyromonas gingivalis. Since P. gingivalis is considered as a keystone bacterium, we further analysed lactic acid bacteria that produced extracellular soluble antimicrobial agents that created an inhibitory zone of more than 12 mm against P. gingivalis. After two rounds of antimicrobial susceptibility testing, six isolates showing strong antibacterial effects against P. gingivalis were selected and identified as facultatively anaerobic, Gram-positive, non-spore-forming, non-capsule-forming, and catalase-negative bacilli. Selected isolates were identified to the species level through 16S rDNA sequence analyses. The antimicrobial substance produced by the isolated bacilli was inactivated slightly after catalase treatment, had significantly lower activity at pH levels above 9.0, and was also reduced by heat treatment above 100°C and autoclaving. The activity of the antimicrobial substance showed some resistance to lower levels of heat, as well as pepsin and proteinase K treatments. 16S rRNA sequencing identified these isolates as Lactobacillus casei, Lactobacillus fermentum, and Lactobacillus gasseri. These bacteria are antagonistic against potential periodontal pathogens and are good candidates for clinical application as probiotics.

Positive Correlation Between the Level of Interferon-Gamma and the Severity of Periodontitis

Objective: To examine the IFN- γ levels in patients with periodontitis and determine the difference in the levels of IFN- γ with the severity of the disease . Material and Methods: The study design was cross-sectional, and the sample consisted of 31 patients, aged between 18 and 64 years. Plaque index (PlI), calculus index (CI), and papillary bleeding index (PBI) were measured. Pocket depth (PD), recession, and clinical attachment loss (CAL) (mm) were measured at six sites per teeth. For mild/moderate periodontitis, pocket depth ≥4 mm in 1–3 sites was required, while the essential criteria for severe periodontitis were pocket depth ≥5 mm, clinical attachment loss >3 mm in more than 3 sites ≥2 quadrants. The IFN- γ levels were measured by performing enzyme-linked immunosorb ent assay with the gingival crevicular fluid (GCF) samples. The measurements were made in two different sites, and the severity of periodontitis was categorized based on the pocket depth, attachment loss, and the remaining natural teeth. Kruskal-Wallis test, Mann-Whitney test, and Spearman’s rank correlation coefficient were. Results: The levels of IFN- γ (pg/mL) were correlated with the severity of the periodontal status, with p <0.05. Clinical parameters of periodontitis also correlated with the level of IFN- γ (pg/mL). Conclusion: Subjects with periodontitis presented greater levels of IFN- γ ( pg/mL) in GCF than the periodontal healthy individuals. This result showed the role of IFN- γ in the inflammation .

IL-6 and TNF-α salivary levels according to the periodontal status in Portuguese pregnant women.

BackgroundPeriodontitis is associated with increased concentration of inflammatory markers and saliva has been proposed as a non-invasive diagnostic fluid in oral and systemic diseases. The levels of salivary biomarkers, such as cytokines, could potentially be used to distinguish periodontal healthy individuals from subjects with periodontal disease. The purpose of this study was to characterize the salivary levels of two inflammatory biomarkers associated with periodontitis, interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-α), in order to assess whether these cytokines salivary levels could potentially be used to complement periodontitis pregnant women diagnose.MethodsForty-four pregnant women were distributed into three groups, according to their periodontal status: healthy, mild/moderate periodontitis and severe periodontitis. Unstimulated saliva was collected and analysis of TNF-α and IL-6 salivary levels were performed with Immulite®.ResultsWomen with periodontitis exhibited significantly higher levels (p = 0.001) of salivary IL-6 and TNF-α compared with the healthy group: 25.1 (±11.2) pg/mL vs. 16.3 (±5.0) pg/mL and 29.7 (±17.2) pg/mL vs. 16.2 (±7.6) pg/mL, approximately 1.5 and 1.8 times more, respectively. Additionally, cytokines were significantly increased (p < 0.05) in severe periodontitis compared to periodontal healthy pregnant women.ConclusionsThese results revealed that IL-6 and TNF-α salivary biomarkers provide high discriminatory capacity for distinguishing periodontal disease from periodontal health in pregnant women.

A preliminary study on the effect of histatin 5 inhibiting Porphyromonas gingivalis and Fusobacterium nucleatum co-aggregation

Objective: To detect the inhibitory ability of histatin 5 on the auto-aggregation of Porphyromonas gingivalis (Pg), and the co-aggregation of Pg with Fusobacterium nucleatum (Fn); and to provide a theoretical basis for the role of oral innate immunity played in the inhibition of chronic periodontitis. Methods: Saliva and supragingival, subgingival plaque samples were collected from 49 chronic periodontitis patients in School of Stomatology, China Medical University and 27 periodontal healthy individuals. Enzyme linked immunosorbent assay was used to assess the amount of histatin 5 in saliva, absolute quantitative real-time PCR (qPCR) was applied to detect the DNA copies of Fn, Pg and total bacteria in supragingival and subgingival plaque samples. The effects of histatin 5 on auto- and co-aggregation were assessed by bacterial adhesion test and scanning electron microscopy. Hemagglutinin gene, arginine-gingipains gene in Pg and FomA gene in Fn were tested by relative qPCR. Independent samples t-test was used to calculate the significance between the experimental group and the control group. P-value<0.05 was considered statistically significant. Results: For chronic periodontitis patients, there was an inverse correlation between the concentration of histatin 5 and Fn and Pg in supragingival plaque samples (r=-0.379, r=-0.624). Similarly, an inverse correlation was also observed between the concentration of histatin 5 and subgingival Fn and Pg, respectively (r=-0.404, r=-0.314). As for periodontally healthy individuals, there was an inverse correlation between the concentration of histatin 5 and supragingival and subgingival Pg (r=-0.572, r=-0.533). Bacterial adhesion test and scanning electron microscopy certified that 25 mg/L histatin 5 inhibited the auto-aggregation of Pg-Pg and the co-aggregation of Pg-Fn. Results of qPCR showed that 25 mg/L histatin 5 up-regulated hemagglutinin gene by (14.52±3.25) fold and down-regulated FomA gene to (0.22±0.10) fold. Conclusions: Histatin 5 could inhibit the auto-aggregation of Pg-Pg and the co-aggregation of Pg-Fn by regulating hemagglutinin gene and FomA gene expression.

Matrix metalloproteinase-2, -9, and tissue inhibitor of MMP-2 gene polymorphisms in Taiwanese periodontitis patients

Background/purposeMatrix metalloproteinases (MMPs) and tissue inhibitor of MMPs (TIMPs) have been shown to play an important role in the pathogenesis of tissue destruction in periodontitis. The associations between single nucleotide polymorphisms (SNPs) in the promoter regions of MMP-2, MMP-9, and TIMP-2 genes and the risk of aggressive periodontitis (AgP) and chronic periodontitis (CP) were investigated in a Taiwanese population. Materials and methodsMMP-2 C-1306T, C-735T, T-790G, and MMP-9 C-1562T and TIMP-2 G-418C SNPs were genotyped by polymerase chain reaction-restriction fragment length polymorphism analysis in 69 patients and 129 patients with AgP and CP, respectively, and 117 periodontal healthy individuals who served as healthy controls (HC). Chi-square test and logistic regression analysis were used to investigate the possible association of genotypes with periodontitis. ResultsNo significant differences in the distributions of the C-1306T and C-735T variants between periodontitis and HC were detected. Patients with genotype of MMP-2 -790 TT or T allele of MMP-2-790T/G as compared to genotypes of GT + GG genotypes or G allele, were less susceptible to CP [odds ratio (OR) = 0.50, 95% confidence interval (CI) = 0.25–1.00 and OR = 0.52, 95% CI = 0.28–0.96, respectively]. The frequencies of TIMP-2 G-418C gene polymorphisms in nonsmokers were statistically significantly different among AgP, CP, and HC groups (P = 0.024). The nonalcohol drinking participants with C allele of MMP-9 C-1562T as compared to T allele, were less susceptible to AgP (adjusted OR = 0.4; 95% CI, 0.18–0.90). ConclusionIt is suggested that MMP-2 T-790G, MMP-9 C-1562T, and TIMP-2 G-418C gene polymorphisms might be associated with periodontitis in the Taiwanese Han population.

Ghrelin levels in chronic periodontitis patients

Ghrelin is a peptide hormone that has modulatory effects on the immune system. This study was designed to evaluate plasma ghrelin levels in patients with chronic periodontitis and to investigate if a relationship exists between ghrelin and periodontal parameters, serum cytokines, and bone turnover markers. Thirty-five chronic periodontitis patients (CP) and periodontal healthy individuals (C) were included in this study. Periodontal parameters were recorded. Blood samples were obtained to determine the levels of total and acylated ghrelin, interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), the soluble receptor activator nuclear factor kappaB ligand (sRANKL), alkaline phosphatase (ALP), and osteocalcin (OSC). Plasma levels of total and acylated ghrelin were significantly elevated in the CP group compared with the C group (p < 0.05). The difference was significant only between males in the two groups (groups were compared with respect to gender) (p < 0.05). There was no difference between the groups regarding the levels of serum sRANKL, TNF-α, and ALP. A relative increase in the serum levels of IL-1β and a decrease in the serum levels of OSC of the CP group were observed (p < 0.05). In addition, positive correlations between total ghrelin/ALP and total ghrelin/acylated ghrelin were discovered. We found no direct correlation between ghrelin levels and periodontal parameters. Our results indicate an increase of total and acylated ghrelin levels in patients with chronic periodontitis. Further, studies in larger populations (which could include ghrelin levels in gingival tissue, gingival crevicular fluid, and saliva) are needed in order to confirm the role of ghrelin in periodontal disease.

Ascorbate and α-tocopherol differentially modulate reactive oxygen species generation by neutrophils in response to FcγR and TLR agonists

Periodontitis, a ubiquitous chronic inflammatory disease, is associated with reduced antioxidant defences and neutrophil hyperactivity in terms of reactive oxygen species (ROS) generation. Its phenotype is thus characterized by oxidative stress. We have determined the effect of antioxidant micronutrients ascorbate and α-tocopherol on neutrophil ROS generation. Peripheral neutrophils from periodontally-healthy individuals (n = 20) were challenged with phorbol myristate acetate, IgG-opsonised Staphylococcus aureus, Fusobacterium nucleatum or PBS in the presence and absence of micronutrients (50 µM). Total and extracellular ROS were measured by luminol and isoluminol chemiluminescence respectively. Total and extracellular unstimulated, baseline ROS generation was unaffected by α-tocopherol, but inhibited by ascorbate and a combination of both micronutrients. Fcγ-receptor (Fcγ-R)-stimulated total or extracellular ROS generation was not affected by the presence of individual micronutrients. However, the combination significantly reduced extracellular FcγR-stimulated ROS release. Neither micronutrient inhibited TLR-stimulated total ROS, but the combination caused inhibition. Ascorbate and the micronutrient combination, but not α-tocopherol, inhibited extracellular ROS release by TLR-stimulated cells. Such micronutrient effects invivo could be beneficial in reducing collateral tissue damage in chronic inflammatory diseases, such as periodontitis, while retaining immune-mediated neutrophil function.

Microbiota around teeth and dental implants in periodontally healthy, partially edentulous patients: is pre-implant microbiological testing relevant?

This study aimed to assess the prevalence of seven periodontal marker pathogens, before implant placement and 1 yr after loading, in periodontally healthy individuals and to assess the long-term effectiveness of pre-implant reduction of pathogens to below threshold levels. In 93 individuals needing single tooth replacement, pooled subgingival microbiological samples from standard sites were cultured and analyzed before implant treatment and 1 yr after loading. Threshold levels commonly used in periodontology to predict periodontal breakdown were applied. Subjects with levels of pathogens above these thresholds received initial periodontal treatment including systemic antibiotics when indicated. At baseline, 49.5% of periodontally healthy subjects harboured one or more marker pathogens above threshold levels. Periodontal treatment reduced the pathogen levels below threshold values in 78.3% of these initially colonized subjects. In all cases Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis were reduced to below threshold. At 1 yr after loading, periodontal pathogens were present above threshold levels in 74.1% of all subjects. It is concluded that in almost half of periodontal healthy individuals the subgingival biofilm harbours periodontal pathogens above threshold values. Long-term effectiveness of pre-implant reduction of the selected marker pathogens appeared limited in our patient population, making pre-implant reduction unpredictive for post-implant levels of these pathogens. Thus, considering the applied microbiological criteria, generalized pre-implant microbiological testing is not contributory in periodontally healthy subjects.

Determinants of serum IgG responses to periodontal bacteria in a nationally representative sample of US adults

To assess the distribution of elevated antibody titres to multiple periodontal bacteria, including established/putative pathogens and health-related species, by selected demographic, behavioural, and oral- and general health-related characteristics. Data from 8153 >or=40-year-old participants from the third National Health and Nutrition Examination Survey were used, including 1588 edentulous individuals. We used checkerboard immunoblotting to assess serum IgG levels to 19 periodontal species. Thresholds for elevated antibody responses were defined for each species using the 90th percentile titre in periodontal healthy participants, using two alternative definitions of periodontitis. Edentulous individuals showed lower antibody responses than dentate participants, notably for titres to "red complex" species and Actinobacillus actinomycetemcomitans. Elevated titres to Porphyromonas gingivalis were twice as prevalent in participants with periodontitis than in periodontal healthy individuals. Non-Hispanic blacks and Mexican-Americans were more likely to display elevated titres for P. gingivalis compared with non-Hispanic whites (22.9%versus 19.4%versus 9.5%). Current smokers were significantly less likely to exhibit high titres to multiple bacteria than never smokers. Demographic, behavioural, and oral- and general health-related characteristics were strong determinants of systemic antibody responses to periodontal bacteria in a nationally representative sample of US adults.

Infection frequency of Epstein-Barr virus in subgingival samples from patients with different periodontal status and its correlation with severity of periodontal lesion

To detect the infection frequencies of different Epstein-Barr virus (EBV) genotypes in subgingival samples of chronic periodontitis, and the correlation among infection with different genotypes and the severity of periodontal lesion. Nested PCR (nPCR) with EBV-1 or EBV-2 specific primers was used to detect EBV-1 and EBV-2 in the subgingival samples from 65 chronic periodontitis patients, 65 gingivitis patients and 24 periodontal healthy individuals. The amplicons were further identified by RFLP with endonucleases Afa I and Stu I. By using periodontal attachment loss (AL) and gingival index (GI) as the observing in, the correlation of infection with different EBV genotypes and the severity of periodontal lesion were analyzed. 0.01 ng of EBV-1 DNA could be detectable by the established nPCR. All the samples showed the same detection results by two separated nPCR. All the EBV-1 amplification products (497 bp) by using endonuclease Afa I digestion could be divided into two fragments with 355 bp and 142 bp respectively. After endonuclease Stu I digestion, all the EBV-2 amplification products (165 bp) displayed two fragments with 118 bp and 47 bp respectively. In the samples of chronic periodontitis patients, gingivitis patients, and healthy periodontal tissues, the positive rates were 28.5% (74/260), 16.9% (44/260), and 14.6% (14/96) for EBV-1; and were 8.1% (21/260), 3.1% (8/260), and 0% for EBV-2 respectively, and the total EBV positive rates were 36.5% (95/260), 20.0% (52/260) and 14.6% (14/96) respectively. None of the positive samples was detectable for both the EBV-1 and EBV-2. The positive rates of EBV-1, EBV-2 and the total EBV positive rates in the chronic periodontitis samples were all higher than those in the gingivitis samples (all P < 0.05) and healthy periodontal tissue samples (all P < 0.01), without a significant difference between the gingivitis samples and healthy periodontal tissue samples (P > 0.05). Infection of EBV or EBV-1 or EBV-2 in CP patients could not be associated with AL or GI. Subgingival infection with either EBV-1 or EBV-2 is closely associated with chronic periodontitis. Infection of EBV may not correlate directly with severity of periodontitis.

Presence of Actinobacillus actinomycetemcomitans on the community Periodontal Index (CPI) Teeth in Periodontally Healthy Individuals

The aim of this study was to compare the prevalence of Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) located at Community Periodontal Index (CPI) teeth versus randomly selected teeth to determine if the CPI teeth are representative of this bacteria subgingival colonization in periodontally healthy young individuals. Forty-four individuals between 18 and 27 years of age were included in the study (mean age 23.11 +/- 2.91 years). Pooled subgingival plaque samples were collected with paper points from the mesio-buccal aspect of the CPI teeth (10 teeth for adults and 6 teeth for persons under 20 years of age) and transported in reduced Ringer's solution to the culture medium. Sixty days following the first microbial analysis, new pooled microbial samples were obtained from the mesio-buccal aspect of 10 or 6 randomly selected teeth. The presence of A. actinomycetemcomitans was determined using a selective culture medium. Microbiological data were assessed by the Wilcoxon test (p < 0.05). A border line significance (p=0.51) was observed between CPI teeth and randomly selected teeth in terms of detecting the subgingival occurrence of A. actinomycetemcomitans. CPI teeth showed to be representative of A. actinomycetemcomitans subgingival colonization. Therefore, these results suggest that in periodontally healthy young individuals, CPI teeth could be an appropriate source of samples for the subginginval detection of this pathogen.

Quantitative detection of periodontal pathogens using real-time polymerase chain reaction with TaqMan probes.

Quantitative analysis, with identification of periodontopathic bacteria, is important for the diagnosis, therapeutic evaluation and risk assessment of periodontal disease. We developed a highly sensitive and specific method using real-time polymerase chain reaction (PCR) to detect and quantify six periodontal bacteria: Porphyromonas gingivalis, Tannerella forsythia, Actinobacillus actinomycetemcomitans, Treponema denticola, Prevotella intermedia, and Prevotella nigrescens. Species-specific TaqMan probe/primer sets were designed according to 16S ribosomal RNA gene sequences. Plaque and tongue debris specimens were collected from 10 patients with advanced periodontitis and 10 periodontal healthy individuals and analyzed. All species, except for P. nigrescens, were detected in samples from diseased sites in significantly greater numbers than in those from healthy sites, whereas greater numbers of P. nigrescens were found in the controls. These results suggest that the present real-time PCR method with the designed probe/primer sets enabled sensitive detection of the six periodontal bacteria, and may also assist future microbial studies of periodontal diseases.

PCR reaction and dot-blot hybridization to monitor the distribution of oral pathogens within plaque samples of periodontally healthy individuals.

The purpose of this study was to determine the distribution of the putative periodontal pathogens Prevotella intermedia, Prevotella nigrescens, the three oral Capnocytophaga species (C. ochracea, C. sputigena, C. gingivalis), as well as Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans in plaque samples of periodontally healthy individuals. We chose a newly developed 16S rDNA directed polymerase chain reaction (PCR) and a previously described dot-blot hybridization assay to detect, differentiate, and quantify these bacteria directly in clinical samples. The subjects of these investigations were 66 sulcus fluid samples from 17 children (ages 3 to 5) attending a kindergarten, 48 sulcus fluid samples from 12 children (ages 9 and 10) from a primary school, and 25 subgingival plaque samples isolated from 6 different periodontally healthy dental students (ages 24 to 27). We were able to demonstrate the presence of P. nigrescens in 54 (kindergarten: 5; primary school: 33; students: 16) samples by PCR and quantified it by dot-blot hybridization. In addition, we found C. ochracea in 12 (kindergarten: 2; primary school: 10) samples by PCR reaction only. The other tested bacterial species were absent by the methods used. Furthermore we confirmed the specificity of our P. nigrescens-PCR in selected samples by enzyme electrophoresis.

Prevalence of Treponema denticola and Porphyromonas gingivalis in plaque from periodontally-healthy and periodontally-diseased sites

Intracrevicular plaque from periodontally-healthy individuals who had refrained from oral hygiene measures for 24 h prior to sampling, and subgingival plaque from diseased sites of patients with chronic periodontitis were screened by ELISA for the presence of Porphyromonas gingivalis and Treponema denticola. The samples were also subjected to the PerioScan test to detect the presence of enzymes capable of degrading N-benzoyl-DL-arginine-2-naphthylamide (BANA). Of the 141 samples from periodontally-healthy sites, 73% contained T. denticola antigens and 78% P. gingivalis antigens, compared to 43% and 59%, respectively, in plaque samples from the 159 diseased sites. A positive reaction in the PerioScan test was obtained in 89% of plaque samples from diseased sites and in 60% of those from healthy sites. The correlation between the results of the two assays was poor in the case of intracrevicular plaque from healthy sites. However, with plaque samples from diseased sites, the results of the PerioScan test showed very strong correlation with those obtained with the ELISA, suggesting that the former may be a useful, rapid means of indicating the presence of T. denticola and P. gingivalis in such plaque samples.