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Objective: The aim of this study was to optimize the sensitivity, specificity and cost-effectiveness of the RNA-Oligonucleotide Quantification Technique (ROQT) in order to identify periodontal pathogens that remain unrecognized or uncultured in the oral microbiome., Design: Total nucleic acids (TNA) were extracted from subgingival biofilm samples using an automated process. RNA, DNA and Locked Nucleic Acid (LNA) digoxigenin-labeled oligonucleotide probes targeting 5 cultivated/named species and 16 uncultivated or unnamed bacterial taxa were synthesized. Probe specificity was determined by targeting 96 oral bacterial species; sensitivity was assessed using serial dilutions of reference bacterial strains. Different stringency temperatures were compared and new standards were tested. The tested conditions were evaluated analyzing samples from periodontally healthy individuals, and patients with moderate or severe periodontitis., Results: The automated extraction method at 63⁰C along with LNA-oligunucleotides probes, and use of reverse RNA sequences for standards yielded stronger signals without cross-reactions. In the pilot clinical study, the most commonly detected uncultivated/unrecognized species were Selenomonas sp. HMT 134, Prevotella sp. HMT 306, Desulfobulbus sp. HMT 041, Synergistetes sp. HMT 360 and Bacteroidetes HMT 274. In the cultivated segment of the microbiota, the most abundant taxa were T. forsythia HMT 613 and Fretibacterium fastidiosum (formerly Synergistetes) HMT 363., Conclusions: In general, samples from severe patients had the greatest levels of organisms. Classic (T. forsythia, P. gingivalis) and newly proposed (F. alocis and Desulfobulbus sp. HMT 041) pathogens were present in greater amounts in samples from severe periodontitis sites, followed by moderate periodontitis sites., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Objective: The aim of this study was to evaluate the levels of Interleukin-1α (IL-1α), Interleukin-1β (IL-1β), Interleukin-1 receptor antagonist (IL-1Ra), Interleukin-10 (IL-10), Interleukin-13 (IL-13), Vascular endothelial growth factor (VEGF), Granulocyte-colony stimulating factor (G-CSF), and Growth related oncogene (GRO) in the peri-miniscrew implant crevicular fluid (MICF) under orthodontic loading., Design: The study sample comprised 14 miniscrews immediately loaded and 17 unloaded ones. A load of 200gF was immediately applied to the miniscrews in the loaded group after the placement surgery. Peri-miniscrew implant crevicular fluid was collected at baseline, at day 7, and at day 21. The levels of the biomarkers were measured using a multiplexed bead immunoassay. Intergroup comparisons were made using Mann-Whitney test. Friedman and Dunn's multiple comparison tests were used to evaluate intragroup differences over time., Results: Although no statistical differences were observed between the groups at any time point for any of the 8 biomarkers evaluated, there was a statistically significant increase (p < 0.02) in the levels of all the biomarkers over time on both groups., Conclusions: An immediate loading of 200gF does not alter the balance in the inflammatory response in peri-miniscrew tissues., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Antibodies to leukotoxin A are markers that link Aggregatibacter actinomycetemcomitans -associated periodontitis and rheumatoid arthritis., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Aim: To compare the microbiome of healthy (H) and diseased (P) peri-implant sites and determine the core peri-implant microbiome., Materials and Methods: Submucosal biofilms from 32 H and 35 P sites were analysed using 16S rRNA sequencing (MiSeq, Illumina), QIIME and HOMINGS. Differences between groups were determined using principal coordinate analysis (PCoA), t tests and Wilcoxon rank sum test and FDR-adjusted. The peri-implant core microbiome was determined., Results: PCoA showed partitioning between H and P at all taxonomic levels. Bacteroidetes, Spirochetes and Synergistetes were higher in P, while Actinobacteria prevailed in H (p < .05). Porphyromonas and Treponema were more abundant in P while Rothia and Neisseria were higher in H (p < .05). The core peri-implant microbiome contained Fusobacterium, Parvimonas and Campylobacter sp. T. denticola, and P. gingivalis levels were higher in P, as well as F. alocis, F. fastidiosum and T. maltophilum (p < .05)., Conclusion: The peri-implantitis microbiome is commensal-depleted and pathogen-enriched, harbouring traditional and new pathogens. The core peri-implant microbiome harbours taxa from genera often associated with periodontal inflammation., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Chronic periodontitis is controlled without antibiotics by scaling and root planing (SRP) to remove dental biofilm. It has been previously reported that the epithelial barrier to bacterial proinflammatory products is impaired when biofilm lysine falls below the minimal content of normal blood plasma. Aims were to examine whether being refractory and requiring antibiotics to supplement SRP were associated with low biofilm lysine contents., Methods: Sixteen patients with periodontitis and six periodontally healthy volunteers (HVs) (respective mean ages: 57 ± 6 and 36 ± 8 years) were examined. Patients with periodontitis received SRP and surgery, and HVs received prophylaxis. At quarterly maintenance or prophylaxis visits during the subsequent year, therapeutic response was good (GR, n = 9) or poor (PR, n = 7; including five cigarette smokers). Biofilm cadaverine, lysine, and other amino acid (AA) contents were determined by liquid chromatography. Cadaverine mole fraction of lysine plus cadaverine (CF) indicated biofilm lysine decarboxylase activity., Results: Biofilm lysine was 0.19 ± 0.10 and 0.20 ± 0.09 μmol/mg in GRs and HVs, but 0.07 ± 0.03 μmol/mg in PRs (Kruskal-Wallis: P <0.01). All AAs were depleted in biofilm from smokers, but only lysine was depleted in biofilm from non-smokers. CF was inversely associated with clinical attachment level (CAL) at baseline before therapy in all patients (R 2 = 0.28, P <0.01) and with CAL change after therapy in GR (R 2 = 0.49, P <0.05). Lysine and cadaverine contents discriminated PRs from GRs and HVs (Wilks' λ = 0.499, P <0.012)., Conclusions: Refractory responses requiring antibiotic therapy result from smoking and/or microbial infections that starve the biofilm and epithelial attachment of lysine. Biofilm CF is associated with periodontitis severity pretherapy and extent of therapeutic response post-therapy.

A bacterial etiology of rheumatoid arthritis (RA) has been suspected since the beginnings of modern germ theory. Recent studies implicate mucosal surfaces as sites of disease initiation. The common occurrence of periodontal dysbiosis in RA suggests that oral pathogens may trigger the production of disease-specific autoantibodies and arthritis in susceptible individuals. We used mass spectrometry to define the microbial composition and antigenic repertoire of gingival crevicular fluid in patients with periodontal disease and healthy controls. Periodontitis was characterized by the presence of citrullinated autoantigens that are primary immune targets in RA. The citrullinome in periodontitis mirrored patterns of hypercitrullination observed in the rheumatoid joint, implicating this mucosal site in RA pathogenesis. Proteomic signatures of several microbial species were detected in hypercitrullinated periodontitis samples. Among these, Aggregatibacter actinomycetemcomitans (Aa), but not other candidate pathogens, induced hypercitrullination in host neutrophils. We identified the pore-forming toxin leukotoxin A (LtxA) as the molecular mechanism by which Aa triggers dysregulated activation of citrullinating enzymes in neutrophils, mimicking membranolytic pathways that sustain autoantigen citrullination in the RA joint. Moreover, LtxA induced changes in neutrophil morphology mimicking extracellular trap formation, thereby releasing the hypercitrullinated cargo. Exposure to leukotoxic Aa strains was confirmed in patients with RA and was associated with both anticitrullinated protein antibodies and rheumatoid factor. The effect of human lymphocyte antigen-DRB1 shared epitope alleles on autoantibody positivity was limited to RA patients who were exposed to Aa These studies identify the periodontal pathogen Aa as a candidate bacterial trigger of autoimmunity in RA., (Copyright © 2016, American Association for the Advancement of Science.)

Aim: The objective of this cross-sectional study was to examine the potential of peri-implant crevicular fluid (PICF) analytes to discriminate between peri-implant health and disease using a multi-biomarker approach., Methods: We collected PICF samples from the mesio-buccal site of every implant (n = 145) from 52 subjects with peri-implantitis and measured the levels of 20 biomarkers using Luminex. We grouped implants and subjects based on the clinical characteristic of the sampled sites and implants into: healthy sites from healthy implants (HH), diseased sites from diseased implants (DD) and healthy sites from diseased implants (HD). The significance of the differences between the HH and DD groups was determined using general linear models controlling for false discovery rate. We used logistic regression to determine the best multi-biomarker models that could distinguish HH from DD subjects and HH from HD subjects., Results: There were statistically significant differences between HH and DD groups for 12/20 biomarkers. Logistic regression resulted in a 6-biomarker model (Flt-3L, GM-CSF, IL-10, sCD40L, IL-17 and TNFα) that discriminated HH from DD subjects (AUC = 0.93) and a 3-biomarker model (IL-17, IL-1ra and vascular endothelial growth factor) that distinguished HH from DD subjects (AUC = 0.90)., Conclusion: PICF biomarkers might help discriminate peri-implant health from disease., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Introduction: The purpose of this study was to combine multiple displacement amplification and checkerboard DNA-DNA hybridization to qualitatively and quantitatively evaluate the microbiota present in infections refractory to endodontic treatment., Methods: The subjects of this study were 40 patients presenting with periapical lesions refractory to endodontic treatment. Samples were taken by scraping or filing root canal walls with a #10 K-type hand file. Sample DNA was amplified by multiple displacement amplification, and the levels of 107 bacterial taxa were analyzed by checkerboard DNA-DNA hybridization. The taxa were divided into 3 distinct microbial populations depending on their mean proportion in samples (% DNA probe counts ± standard error of the mean) as follows: dominant (≥3.0%), subdominant (>1.6%-3.0%), and residual (≤1.6%) populations. The significance of differences was determined using the Mann-Whitney test., Results: The taxa present with the highest mean proportions (constituting the dominant population) were Corynebacterium diphtheriae (8.03 ± 0.98), Porphyromonas gingivalis (5.42 ± 2.09), Streptococcus sobrinus (5.33 ± 0.69), and Stenotrophomonas maltophilia (4.72 ± 1.73). Among the subdominant population were Eubacterium saphenum (3.85 ± 1.06), Helicobacter pylori (3.16 ± 0.62), Dialister pneumosintes (3.12 ± 1.1), Clostridium difficile (2.74 ± 0.41), Enterobacter agglomerans (2.64 ± 0.54), Salmonella enterica (2.51 ± 0.52), Mobiluncus mulieris (2.44 ± 0.6), and Klebsiella oxytoca (2.32 ± 0.66). In the population of bacteria present at the lowest mean proportions (the residual population), Bacteroides ureolyticus (0.04 ± 0.01), Haemophilus influenzae (0.04 ± 0.02), and Prevotella oris (0.01 ± 0.01) were found at the lowest mean proportions. Enterococcus faecalis was detected in the residual population (0.52 ± 0.26)., Conclusions: The microbial climax community in teeth refractory to endodontic treatment not only harbors medically important species but also contains distinct microbial consortia present with different population levels., (Copyright © 2016. Published by Elsevier Inc.)

Aim: This exploratory randomized, controlled clinical trial sought to evaluate anti-inflammatory and -microbial effects of triclosan during experimental gingivitis as assessed by host response biomarkers and biofilm microbial pathogens., Materials and Methods: Thirty participants were randomized to triclosan or control dentifrice groups who ceased homecare for 21 days in an experimental gingivitis (EG) protocol. Plaque and gingival indices and saliva, plaque, and gingival crevicular fluid (GCF) were assessed/collected at days 0, 14, 21 and 35. Levels and proportions of 40 bacterial species from plaque samples were determined using checkerboard DNA-DNA hybridization. Ten biomarkers associated with inflammation, matrix degradation, and host protection were measured from GCF and saliva and analysed using a multiplex array. Participants were stratified as "high" or "low" responders based on gingival index and GCF biomarkers and bacterial biofilm were combined to generate receiver operating characteristic curves and predict gingivitis susceptibility., Results: No differences in mean PI and GI values were observed between groups and non-significant trends of reduction of host response biomarkers with triclosan treatment. Triclosan significantly reduced levels of A. actinomycetemcomitans and P. gingivalis during induction of gingivitis., Conclusions: Triclosan reduced microbial levels during gingivitis development (ClinicalTrials.gov NCT01799226)., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective: To examine levels of matrix metalloproteinases (MMPs)-1, -2, -3, -7, -8, -12, and -13 in the gingival crevicular fluid (GCF) of periodontally compromised teeth at different time points during orthodontic movement., Materials and Methods: Ten controlled periodontitis subjects were submitted to orthodontic treatment. One dental arch was subjected to orthodontic movement, and teeth in the opposite arch were used as controls. GCF samples were collected from the lingual sites of two movement and two control incisors 1 week before orthodontic activation (-7 d), immediately after orthodontic activation, and after 1 hour, 24 hours, and 7, 14, and 21 days. Multiplexed bead immunoassay was used to measure MMPs in GCF. Data were analyzed using Friedman and Wilcoxon statistical tests., Results: The only significant change found over time was in the levels of MMP-1 in the movement group (P < .05). When the two groups were compared after activation, the only statistically significant difference found was in levels of MMP-12 24 hours after activation (P < .05)., Conclusions: Our findings suggested that the orthodontic movement of periodontally compromised teeth without active pockets did not result in significant changes in the GCF levels of MMPs.

Aim: To evaluate CD4(+) CD28(+) and CD8(+) T-cell genes and the gene expression of IFN-γ, TNF-α, IL-1-β, IL-17A, IL-10, CCL-2/MCP-1, CCL-4, CCL-5 (RANTES), CXCR4, CCR5 and RANKL from cells in the periapical interstitial fluid from root canal infections in healthy patients (HIV-) and HIV-positive individuals (HIV+)., Methodology: Subjects included 20 HIV- and 23 HIV+ patients referred to the School of Dentistry at the Universidade Federal de Minas Gerais (Belo Horizonte, MG, Brazil). Almost all HIV+ patients were undergoing highly active antiretroviral therapy (HAART). Clinical samples were taken from teeth with pulp necrosis, and no patients had acute periapical symptoms at the time of the appointments. After cleaning and drying, 3 paper points were introduced into the root canal, passing passively through the root apex (2 mm) into the periapical tissues for 1 min. The samples were collected immediately after root canal cleaning and 7 days later (restrained root canal bacterial load) to characterize those gene expressions using real-time PCR., Results: Significantly higher levels of CD4(+) CD28(+) and CD8(+) T cells in teeth with restrained bacterial loads (second collection) compared with the first collection were observed in both HIV- and HIV+ samples. In HIV- patients, an increase in IL-10 and CXCR4 expression was demonstrated as well as a decrease in pro-inflammatory cytokines such as RANKL, IFN-γ, IL1-β and CCL5. However, in HIV+ patients an increase in cytokines IFN-γ, IL-1-β, TNF-α and IL-17A, and chemokines CCL-2, CXCR4 and CCR5 were observed. The chemokine CCL-5 was not detected in HIV+ individuals., Conclusions: These findings suggest that after reducing the root canal bacterial load in HIV- individuals an anti-inflammatory response is generated whilst in HIV+ patients a pro-inflammatory response is sustained in the periapical area., (© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Purpose: Periodontitis, a chronic inflammatory response to pathogenic bacteria in the oral microbiome, is common among adults. It is associated with several medical conditions, including cardiovascular diseases, and potentially with esophageal, lung, oral, and pancreatic cancer. One of the proposed mechanisms behind these associations is systemic inflammation, which has also been implicated in ovarian cancer etiology. The aim of this study was to evaluate association between ovarian cancer and periodontal bone loss., Methods: The association between periodontal bone loss, a marker of periodontitis, and risk of epithelial ovarian cancer was estimated among 60,560 participants of the prospective Nurses' Health Study using Cox proportional hazards analysis. Competing risks analysis was used to estimate association by histologic subtype., Results: We did not observe an increased risk of ovarian cancer among participants with periodontal bone loss (HR 0.86, 95% CI 0.64-1.15). Among women younger than 69 years, periodontal bone loss was associated with a 40 % (HR 0.60, 95% CI 0.36-0.98) decreased ovarian cancer risk, while there was no association in women older than 69 (HR 1.09, 95% CI 0.75-1.58), although this difference did not reach statistical significance (p-heterogeneity = 0.06). We observed a suggestive decreased risk for serous tumors (HR 0.76, 95% CI 0.53-1.09). The number of natural teeth and root canals, other metrics of oral health, were not associated with ovarian cancer risk., Conclusion: Our results do not support an increased ovarian cancer risk in women with periodontal bone loss; however, there was a significant decrease in risk in women younger than 69. Given the unexpected association between periodontal bone loss and ovarian cancer risk in younger women, further research is warranted.

Biofilm accumulation on implant surfaces is one of the most important factors for early and late implant failure. Because of the related clinical implications, the aim of this in vitro study was to compare the bacterial cell attachment of a four-species oral biofilm on titanium discs of purity grade 2 and 4, with machined surfaces and etched-thermochemically modified with Avantblast®. The in vitro biofilm model was composed of early (Actinomyces naeslundii, Streptococcus gordonii), secondary (Veillonella parvula), and intermediate (Fusobacterium nucleatum ssp. polymorphum) colonizers of tooth surfaces. A total of 36 discs were divided into four groups: Tigr2-c (titanium grade 2, machined surface), Tigr2-t (titanium grade 2, modified surface with Avantblast®), Tigr4-c (titanium grade 4, machined surface), Tigr4-t (titanium grade 4, modified surface with Avantblast®). The experiment was repeated three times. Biofilm viability was tested with 1% 2, 3, 5-triphenyltetrazolium chloride solution and bacterial cell quantification by checkerboard DNA-DNA hybridization. Descriptive analysis was performed to evaluate biofilm composition and differences between groups were checked with the Mann-Whitney test (p < 0.05). After one week, multispecies biofilms showed a similar pattern of bacterial composition on all analyzed implant surfaces. The most prevalent bacterium was V. parvula (∼50% of the total biomass), followed by S. gordonii (∼30%), F. nucleatum ssp. polymorphum (∼10%) and A. naeslundii (<5%). Total bacterial biomass was significantly higher in both grade-4-titanium surfaces (p < 0.05). The results demonstrated that not only implant surface treatment, but also titanium purity, influence early bacterial colonization.

This randomized split-mouth study aimed to examine the levels of matrix metalloproteinases (MMPs) -1, -2, -3, -7, -8, -12, and -13 in the gingival crevicular fluid (GCF) at different time points during orthodontic tooth movement. A total of 16 healthy orthodontic subjects (7 females, 9 males; mean age, 17.7 years) who needed their first upper premolars extracted were enrolled. One randomly chosen maxillary canine was subjected to a distalizing force and was considered to be the test side. The contralateral canine, which was not subjected to any force but was included in the orthodontic appliance, was used as a control side. GCF sampling was performed at both the mesial (tension) and distal (pressure) test and control sites at baseline, immediately before applying the orthodontic appliance, and after 1 and 24 hours and 7, 14, and 21 days. A multiplexed bead immunoassay was used to analyse the GCF samples. The mean levels of the MMP-1, -2, -3, -7, -8, -12, and -13 were not significantly different between the test and control groups in each time showed. The comparisons between the tension and pressure sites were also not significantly different at each individual time. A few variations focused on MMP-1 and -3, but the expression of MMP-8 was higher than that of the other MMPs. MMPs are released in sufficient quantities such that tooth movement occurs but with no significant increase in GCF levels.

Aim: To evaluate the clinical effects of the adjunctive use of metronidazole (MTZ) and amoxicillin (AMX) in the treatment of generalized aggressive periodontitis (GAgP)., Methods: Thirty subjects were randomly assigned to receive scaling and root planing (SRP) alone or combined with MTZ (400 mg/TID) and AMX (500 mg/TID) for 14 days. Subjects were clinically monitored at baseline, 6 months and 1 year post-therapies., Results: Both therapies led to a statistically significant improvement in all clinical parameters at 1 year post-therapy (p < 0.05). Subjects receiving MTZ plus AMX exhibited the deepest reductions in mean probing depth (PD) and gain in clinical attachment between baseline and 1 year post-therapy in the full-mouth analysis and in initially intermediate (PD 4-6 mm) and deep (PD ≥ 7 mm) sites (p < 0.01). In addition, the antibiotic group presented lower mean number of residual sites with PD ≥ 5 or 6 mm as well as fewer subjects still presenting nine or more sites with PD ≥ 5 mm or three or more sites with PD ≥ 6 mm at the end of the study period., Conclusion: The non-surgical treatment of GAgP is markedly improved by the adjunctive use of MTZ+AMX, up to 1 year post-treatment., (© 2012 John Wiley & Sons A/S.)

Introduction: The use of calcium hydroxide is an effective step in killing bacteria that remain after cleaning and shaping procedures. It also induces hard-tissue formation and is effective for stopping inflammatory exudates., Methods: The aim of this study was to assay and to compare the influence of calcium hydroxide on periapical interstitial fluid from human root canals. The mRNA expression levels of the cytokines interferon (IFN)-γ, tumor necrosis factor-α, interleukin (IL)-1β, IL-17A, and IL-10 as well as the chemokine MCP-1 were assayed by real-time polymerase chain reaction immediately after root canal cleaning and 15 days later., Results: Levels of IL-1β, IFN-γ, IL-10, and the chemokine CCL2/MCP-1 were increased in teeth without endodontic dressings. With calcium hydroxide interappointment dressings, no statistically significant changes were observed in cytokine mRNA expression. However, when comparing teeth that received the medication with those that did not, expression levels of IL-1β, IFN-γ, and IL-10 were statistically lower in those teeth that received calcium hydroxide., Conclusions: Analyses of cytokines and the chemokine CCL-2/MCP-1 demonstrated the benefits of calcium hydroxide as a root canal dressing because it impedes the increase of all mediators during the experimental time., (Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Background: The aim of this study is to explore relationships among serum adipokines, vitamin D, and clinical and microbial parameters of chronic periodontitis before and after treatment., Methods: Weight, height, and smoking status were recorded for 56 patients with chronic periodontitis. Plaque, gingivitis, bleeding on probing, suppuration, probing depth, and clinical attachment level were measured at all teeth present. Subgingival biofilm samples from each tooth were analyzed for levels of 40 bacterial species using checkerboard DNA-DNA hybridization. Serum levels of interleukin-6 (IL-6), tumor necrosis factor-α, adiponectin, leptin, resistin, and vitamin D were measured at baseline. Sample collection was then performed in a subset of the population 6 months after therapy (n = 17). Serum samples were analyzed using enzyme-linked immunosorbent assay and immunoassays. Differences in clinical, microbial, and serum factors among groups were sought using the Mann-Whitney U test. Correlations among factors were evaluated using regression analysis. Effects of therapy were sought using the Wilcoxon signed rank test., Results: There were positive correlations between adiponectin/vitamin D and between IL-6/leptin, negative correlations between IL-6/vitamin D and leptin/vitamin D, but no associations between serum analytes and clinical or microbial parameters. Sex and body mass index were associated with levels of adipokines. Periodontal therapy improved clinical and microbiologic parameters but did not influence the levels of serum analytes., Conclusion: Adipokines and IL-6 levels were affected by sex and body mass index. Serum analytes were not influenced by periodontal therapy.

Objective: To compare the microbiota of endodontic infections in necrotic pulp from HIV-negative and HIV-positive subjects., Materials and Methods: Root canal samples from necrotic pulp were collected from 40 HIV- and 20 HIV+ subjects. Pulps were amplified using multiple displacement amplification (MDA). Then, checkerboard DNA-DNA hybridization was employed to assess the levels of 107 microbial taxa. The percentage of DNA probe count and the percentage of teeth colonized by each test species were investigated. Significant differences between groups regarding proportions of taxa and prevalence of the test species were sought using the Mann-Whitney test and the Chi-square analysis, respectively., Results: The most prevalent taxa detected were Dialister pneumosintes, Stenotrophomonas maltophilia, Streptococcus sobrinus, Corynebacterium diphteriae, and Helicobacter pylori among HIV- subjects and D. pneumosintes, Prevotella tannerae, Porphyromonas gingivalis, Parvimonas micra, Prevotella nigrescens, and Corynebacterium diphtheriae among HIV+ individuals. D. pneumosintes, C. diphtheria, and C. albicans were the most abundant species in the HIV- group, whereas the predominant taxa in HIV+ samples were P. tannerae, D. pneumosintes and Olsenella uli. P. tannerae, O. uli, Veilonella dispar, Bacteroides fragilis, and Actinomyces meyeri were significantly more abundant in HIV+ samples., Conclusions: There were significant differences in the prevalence and proportions of specific microbial taxa between HIV- and HIV+ individuals. The root canal microbiota may represent a reservoir of important oral and medical pathogens, mainly in HIV+ individuals., (© 2012 John Wiley & Sons A/S.)

Background: Surfaces and fluids can affect oral bacterial colonization. The aim of this study is to compare redeveloping biofilms on natural teeth and dentures., Methods: Supragingival plaque samples were taken from 55 dentate individuals and the denture teeth of 62 edentulous individuals before and after professional cleaning. Also, samples from seven "teeth" (samples included dentures) in randomly selected quadrants were collected after 1, 2, 4, and 7 days of no oral hygiene. Samples were analyzed using checkerboard DNA-DNA hybridization. Counts and proportions of 41 bacterial taxa were determined at each time point, and significant differences were determined using the Mann-Whitney U test. Ecological succession was determined using a modified moving window analysis., Results: Mean total DNA probe counts were similar precleaning but were higher in dentate individuals at all post-cleaning visits (P <0.01). Precleaning edentate biofilms had higher counts and proportions of Streptococcus mitis, Streptococcus oralis, and Streptococcus mutans, whereas dentate individuals had higher proportions of Tannerella forsythia, Selenomonas noxia, and Neisseria mucosa. By day 2, mean counts of all taxa were higher in natural teeth, and most remained higher at day 7 (P <0.01). Succession was more rapid and complex in dentate individuals. Both groups demonstrated increased proportions of S. mitis and S. oralis by day 1. N. mucosa, Veillonella parvula, and Eikenella corrodens increased in both groups, but later in samples from edentate individuals., Conclusions: "Mature" natural and denture teeth biofilms have similar total numbers of bacteria but different species proportions. Post-cleaning biofilm redevelopment is more rapid and more complex on natural teeth than on denture teeth.

Some individuals make contributions so vital to their field of knowledge that their names become almost synonymous with that field. This is the case of Sig Socransky and the field of periodontal microbiology. Sig Socransky, or simply Sig, was born in Toronto, Canada and received his DDS degree from the University of Toronto in 1957. He studied microbiology and periodontology at Harvard, receiving a certificate in 1961. That same year he was recruited to work as a Research Associate at the Forsyth Dental Center. In 1968, he was nominated Senior Member of the Staff and Head of the Department of Periodontology. During his 50-year career at Forsyth, Sig published over 300 manuscripts, keeping an average of 7 publications per year. His work had an indelible impact in the fields of periodontology and oral microbiology. All these accomplishments pale in comparison with the impact that Sig had on a personal level. We have collected testimonials from some of his former students, closest collaborators, and friends in an attempt to give readers an insight into Sig's personality. We hope we can offer those who knew him through his work a glimpse of how it felt to interact with this remarkable individual.

Introduction: Lymphocytes, among many cells, express different sets of cytokines, chemokines, and receptors, which are considered important mediators of periapical immune response to infection., Methods: The aim of this study was to evaluate the mRNA expression of CD4(+)CD28(+) and CD8(+) T genes and the gene expression of interferon-γ, tumor necrosis factor-α, interleukin (IL)-1β, IL-17A, IL-10, CCL2/MCP-1, CCL4, CCL5, CXCR4, CCR5, and receptor activator for nuclear factor kappa B ligand (RANKL) in periapical interstitial fluid from human root canal infections. The samples were collected immediately after root canal cleaning and 7 days later (restrained root canal bacterial load) to characterize those gene expressions., Results: Real-time polymerase chain reaction demonstrated significantly higher levels of CD4(+)CD28(+) and CD8(+) T-cell markers in the former root canal condition and an increase of IL-10 and CXCR4, followed by a decrease of proinflammatory cytokines such as RANKL, interferon-γ, IL-1β, and CCL5., Conclusions: Analyses of T-lymphocyte and cytokine expression in periapical area were able to show that distinct root canal conditions might play regulatory roles in controlling local immune/inflammatory processes., (Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Aim: To examine changes in levels of gingival crevicular fluid (GCF) cytokines, after periodontal therapy of generalized aggressive periodontitis (GAgP)., Materials and Methods: Twenty-five periodontally healthy and 24 GAgP subjects had periodontal clinical parameters measured and gingival crevicular fluid (GCF) samples collected from up to 14 sites/subject. GCF samples were analysed using multiplex bead immunoassay for: GM-CSF, IFN-γ, IL-10, IL-1β, IL-2, IL-6 and TNF-α. Aggressive periodontitis subjects were randomly assigned to either scaling and root planing (SRP) alone or SRP plus systemic amoxicillin (500 mg) and metronidazole (400 mg) 3 times a day for 14 days. Clinical parameters and GCF cytokines were re-measured 6 months after treatment. Differences over time were analysed using the Wilcoxon test and between groups using the Mann-Whitney test., Results: Significant reductions in GCF GM-CSF, IL-1β and the ratio IL-1β/IL-10 and increases in GCF IL-6 were detected after therapy. The mean change in GCF cytokines did not differ significantly between groups., Conclusions: Periodontal therapy improved GCF cytokine profiles by lowering IL-1β and increasing IL-10 levels. The reduction in GCF GM-CSF after therapy implicates this cytokine in the pathogenesis of GAgP. There was no difference between therapies in changes of GCF cytokines., (© 2011 John Wiley & Sons A/S.)

Background and Objective: The development of dental biofilms after professional plaque removal is very rapid. However, it is not clear whether most bacterial species return at similar rates in periodontally healthy and periodontitis subjects or if there are differences in bacterial recolonization between supragingival and subgingival biofilms in periodontal health and disease., Material and Methods: Supragingival and subgingival plaque samples were taken separately from 28 teeth in 38 healthy and 17 periodontitis subjects immediately after professional cleaning. Samples were taken again from seven teeth in randomly selected quadrants after 1, 2, 4 and 7 d of no oral hygiene and analyzed using checkerboard DNA-DNA hybridization. The percentage of DNA probe counts were averaged within subjects at each time-point. Ecological succession was determined using a modified moving-window analysis., Results: Succession in supragingival biofilms from subjects with periodontitis and from healthy individuals was similar. At 1 d, Streptococcus mitis and Neisseria mucosa showed increased proportions, followed by Capnocytophaga gingivalis, Eikenella corrodens, Veillonella parvula and Streptococcus oralis at 1-4 d. At 4-7 d, Campylobacter rectus, Campylobacter showae, Prevotella melaninogenica and Prevotella nigrescens became elevated. Subgingival plaque redevelopment was slower and very different from supragingival plaque redevelopment. Increased proportions were first observed for S. mitis, followed by V. parvula and C. gingivalis and, at 7 d, by Capnocytophaga sputigena and P. nigrescens. No significant increase in the proportions of periodontal pathogens was observed in any of the clinical groups or locations., Conclusion: There is a defined order in bacterial species succession in early supragingival and subgingival biofilm redevelopment after professional cleaning., (© 2011 John Wiley & Sons A/S.)

Background: The aim of this human investigation is to explore the relationship of gingivitis with salivary biomarkers, periodontal pathogens, and interleukin (IL)-1 polymorphism after a transient inflammatory burden., Methods: Thirty healthy human participants were randomized by IL-1 genotype status to control for potential influences of this particular single nucleotide polymorphism on the inflammatory profile. Oral hygiene practices ceased for 21 days to induce gingivitis (induction), after which home care was reinstated until 35 days (resolution). Clinical parameters included plaque (PI) and gingival (GI) indices and papillary bleeding score (PBS). Levels and proportions of 40 subgingival bacteria were determined using checkerboard DNA-DNA hybridization. Saliva was analyzed using a multiplex protein array for 30 biomarkers associated with host defense, inflammation, tissue destruction, and angiogenesis., Results: Mean PI, GI, and PBS values were significantly increased during induction and decreased during resolution as measured at 35 days (P <0.01), although no differences were observed between IL-1 groups. Participants were stratified as either "high" or "low" responders based on inflammatory response (high: GI >1.5; low: GI ≤1.5). Baseline levels of salivary IL-6 and IL-8 demonstrated the highest ability to discriminate between high and low responders (area under the curve [AUC] of 0.81 and 0.72, respectively). Salivary biomarkers, matrix metalloproteinases (MMPs), and bacterial biofilm were combined to generate receiver operating characteristic curves. High levels of IL-6 and MMP-1 at baseline demonstrated the strongest ability to predict high responders (AUC of 0.89; odds ratio of 17.0; 95% confidence interval, 1.7 to 171.7)., Conclusion: In this proof-of-concept investigation, we identified specific biomarker and microbial signatures that are associated with gingival inflammation (ClinicalTrials.gov number NCT00980525).

Matrix metalloproteinases (MMPs) and monocyte chemoattractants are key modulators of the biological mechanisms triggered in the periodontium by mechanical forces. The gingival crevicular fluid (GCF) provides a non-invasive method to assess longitudinally the release of inflammatory mediators during orthodontic tooth movement. The goal of this study was to examine the GCF levels of MMP-3, MMP-9, and MMP-13 and of the chemokines macrophage inflammatory protein (MIP)-1β, monocyte chemoattractant protein (MCP)-1, and regulated on activation normal T cells expressed and secreted (RANTES) at different time points during orthodontic tooth movement. Fourteen subjects (three males and 11 females, 18.8 ± 4.8 years of age; range from 12 to 28 years) had their maxillary canines retracted. Thirty-second GCF samples were collected from the tension and pressure sides 7 days prior to the activation of the orthodontic appliance, on the day of activation, and after 1 and 24 hours, and 14, 21, and 80 days of constant force application. The volume of GCF was measured and samples analysed using a multiplexed bead immunoassay for the content of the six target molecules. Differences in the mean GFC volumes and mean level for each analyte over time were assessed using the Friedman test, and differences between the tension and pressure sides at each time point with the Mann-Whitney test. The mean levels of the three MMPs changed significantly over time but only at the compression side (P < 0.05, Friedman test). The GCF levels of the three chemokines were not affected by the application of mechanical stress. The levels of MMPs in GCF at the pressure side are modulated by the application of orthodontic force.

Background: Our goal was to examine differences in clinical, microbiologic, and immunologic responses to non-surgical mechanical therapy in patients with generalized chronic periodontitis (GCP) and generalized aggressive periodontitis (GAgP)., Methods: Twenty patients with GCP and 14 patients with GAgP were evaluated. Clinical data, gingival crevicular fluid (GCF), and subgingival plaque samples were collected at baseline and 3 months after non-surgical periodontal treatment. Levels of 40 subgingival species were measured using checkerboard DNA-DNA hybridization. GCF interleukin (IL)-1β, -4, and -8 and interferon-γ (IFN-γ) were analyzed using a multiplexed bead immunoassay, and elastase activity was measured using an enzymatic assay. The significance of changes with time was examined using the Wilcoxon rank sum test. Changes in clinical, microbiologic, and immunologic parameters after therapy were compared between groups using the Mann-Whitney U test., Results: After periodontal therapy, we found significant improvements for all clinical parameters in both groups. We also observed significant reductions in elastase activity in shallow and deep sites from the GAgP group and in deep sites from the GCP group. Microbiologic data showed significant reductions in proportions of orange and red complexes and an increase in proportions of Actinomyces species in both clinical groups. When the clinical, microbiologic, and immunologic responses after therapy were compared between groups, only minor differences were found., Conclusion: This study fails to show any significant differences between severe forms of GCP and GAgP in response to non-surgical periodontal treatment.

Aim: To monitor microbial shifts during dental biofilm re-development., Materials and Methods: Supra- and subgingival plaque samples were taken separately from 28 teeth in 38 healthy and 17 periodontitis subjects at baseline and immediately after tooth cleaning. Samples were taken again from seven teeth in randomly selected quadrants during 1, 2, 4 and 7 days of no oral hygiene. Samples were analysed using checkerboard DNA-DNA hybridization. Species counts were averaged within subjects at each time point. Significant differences in the counts between healthy and periodontitis subjects were determined using the Mann-Whitney test., Results: The total supra- and subgingival counts were significantly higher in periodontitis on entry and reached or exceeded the baseline values after day 2. Supragingival counts of Veillonella parvula, Fusobacterium nucleatum ss vincentii and Neisseria mucosa increased from 2 to 7 days. Subgingival counts were greater for Actinomyces, green and orange complex species. Significant differences between groups in supragingival counts occurred for 17 of 41 species at entry, 0 at day 7; for subgingival plaque, these values were 39/41 taxa at entry, 17/41 at day 7., Conclusions: Supragingival plaque re-development was similar in periodontitis and health, but subgingival species recolonization was more marked in periodontitis., (© 2011 John Wiley & Sons A/S.)

Approximately 35% of the species present in subgingival biofilms are as yet uncultivated, so their role in periodontal pathogenesis is unknown. The aim of the present study was to develop a high throughput method to quantify a wide range of cultivated and uncultivated taxa in subgingival biofilm samples associated with periodontal disease or health. Oligonucleotides targeting the 16S ribosomal DNA gene were designed, synthesized and labeled with digoxigenin. These probes were hybridized with the total nucleic acids of pure cultures or subgingival biofilm samples. Target species included cultivated taxa associated with periodontal health and disease, as well as uncultivated species, such as TM7 sp. OT 346, Mitsuokella sp. OT 131 and Desulfobulbus sp. OT 041. Sensitivity and specificity of the probes were determined. A Universal probe was used to assess total bacterial load. Sequences complementary to the probes were used as standards for quantification. Chemiluminescent signals were visualized after film exposure or using a CCD camera. In a pilot clinical study, 266 subgingival plaque samples from eight periodontally healthy people and 11 patients with periodontitis were examined. Probes were specific and sensitivity reached 10(4) cells. Fusobacterium nucleatum ss. polymorphum and Actinomyces gerencseriae were the most abundant cultivated taxa in clinical samples. Among uncultivated/unrecognized species, Mitsuokella sp. OT 131 and Prevotella sp. OT 306 were the most numerous. Porphyromonas gingivalis and Desulfobulbus sp. OT 041 were only detected in patients with periodontitis. Direct hybridization of total nucleic acids using oligonucleotide probes permitted the quantification of multiple cultivated and uncultivated taxa in mixed species biofilm samples., (© 2011 John Wiley & Sons A/S.)

Aims: To evaluate the microbiota of endodontic infections in deciduous teeth by Checkerboard DNA-DNA hybridization after uniform amplification of DNA in samples by multiple displacement amplification (MDA)., Methodology: Forty samples from the root canal system of deciduous teeth exhibiting pulp necrosis with or without radiographically detectable periradicular/interradicular bone resorption were collected and 32 were analysed, with three individuals contributing two samples; these were MDA-amplified and analysed by Checkerboard DNA-DNA hybridization for levels of 83 bacterial taxa. Two outcome measures were used: the percentage of teeth colonized by each species and the mean proportion of each bacterial taxon present across all samples., Results: The mean amount of DNA in the samples prior to amplification was 5.2 (±4.7) ng and 6.1 (±2.3) μg after MDA. The mean number of species detected per sample was 19 (±4) (range: 3-66) to the nearest whole number. The most prevalent taxa were Prevotella intermedia (96.9%), Neisseria mucosa (65.6%), Prevotella nigrescens (56.2%) and Tannerella forsythia (56.2%). Aggregatibacter (Haemophilus) aphrophilus and Helicobacter pylori were not detected. P. intermedia (10%), Prevotella tannerae (7%) and Prevotella nigrescens (4.3%) presented the highest mean proportions of the target species averaged across the positive samples., Conclusion: Root canals of infected deciduous teeth had a diverse bacterial population. Prevotella sp. were commonly found with P. intermedia, Prevotella tannerae and Prevotella nigrescens amongst the most prominent species detected., (© 2010 International Endodontic Journal.)

Background: This study determines the gingival crevicular fluid (GCF) levels of interleukin (IL)-1 beta, IL-2, IL-4, IL-8, interferon (IFN)-gamma and elastase activity in inflamed shallow and deep periodontal sites from patients with generalized chronic (GCP) and generalized aggressive periodontitis (GAgP), and to compare them to shallow sites from subjects with gingivitis. A secondary aim analyzes the microbiologic profile of these subjects., Methods: Cross-sectional clinical data were obtained from 20 GCP, 17 GAgP, and 10 gingivitis subjects. GCF samples were collected with paper strips and the levels of IL-1 beta, IL-2, IL-4, IL-8, and IFN-gamma were measured using a multiplexed bead immunoassay. Elastase activity was assessed by an enzymatic assay. Subgingival plaque samples were analyzed using checkerboard DNA-DNA hybridization. Significance of differences among groups for immunologic and microbiologic data was examined using Kruskal-Wallis adjusting for multiple comparisons., Results: Mean clinical parameters and GCF volumes were higher in patients with GCP and GAgP compared to the gingivitis group. Higher levels of IL-1 beta and higher elastase activity were found in deep sites compared to shallow sites in both periodontitis groups (P <0.05). The microbiologic data showed significantly higher levels of the red complex species in patients with GCP and GAgP compared to gingivitis (P <0.05). There were no statistically significant differences in levels of GCF biomarkers and in levels of subgingival bacterial species between subjects with GCP and GAgP., Conclusion: There were no statistically significant differences in the measured immunologic and microbiologic parameters between subjects with GCP and GAgP.

Aim: To examine relationships between subgingival biofilm composition and levels of gingival crevicular fluid (GCF) cytokines in periodontal health and generalized aggressive periodontitis (GAP)., Materials and Methods: Periodontal parameters were measured in 25 periodontally healthy and 31 GAP subjects. Subgingival plaque and GCF samples were obtained from 14 sites from each subject. Forty subgingival taxa were quantified using checkerboard DNA-DNA hybridization and the concentrations of eight GCF cytokines were measured using Luminex. Cluster analysis was used to define sites with similar subgingival microbiotas in each clinical group. Significance of differences in clinical, microbiological and immunological parameters among clusters was determined using the Kruskal-Wallis test., Results: GAP subjects had statistically significantly higher GCF levels of interleukin-1beta (IL-1beta) (p<0.001), granulocyte-macrophage colony-stimulating factor (GM-CSF) (p<0.01) and IL-1beta/IL-10 ratio (p<0.001) and higher proportions of Red and Orange complex species than periodontally healthy subjects. There were no statistically significant differences in the mean proportion of cytokines among clusters in the periodontally healthy subjects, while the ratio IL-1beta/IL-10 (p<0.05) differed significantly among clusters in the aggressive periodontitis group., Conclusions: Different subgingival biofilm profiles are associated with distinct patterns of GCF cytokine expression. Aggressive periodontitis subjects were characterized by a higher IL-1beta/IL-10 ratio than periodontally healthy subjects, suggesting an imbalance between pro- and anti-inflammatory cytokines in aggressive periodontitis.

Background: It was demonstrated that implants restored according to a platform-switching concept presented less crestal bone loss than implants restored with standard protocols. The aim of this study is to examine differences between the composition of the peri-implant microbiotas associated with implants restored with the platform-switching approach and implants restored with a standard internal connection protocol., Methods: A total of 48 implants were examined in 18 subjects: 33 implants were restored with platform switching, and 15 implants were restored using the traditional approach. Thirty-six months after prosthetic loading, subgingival plaque samples were taken from the mesio- and disto-buccal aspects of each implant and from one tooth adjacent to one of the implants in each subject. The levels of 40 subgingival species were measured using checkerboard DNA-DNA hybridization. Microbiologic parameters were averaged within each subject and across subjects in each clinical group (platform switching versus control) and site category (implants versus teeth) separately. The significance of differences between clinical groups and site categories was determined using the Mann-Whitney test and the Wilcoxon test, respectively., Results: There were no statistically significant differences between groups for any of the species. The platform-switching group showed a small trend for lower levels of early colonizer members of the Actinomyces, purple and yellow complexes, Campylobacter species, Tannerella forsythia (previously T. forsythensis), and Porphyromonas gingivalis. Teeth and implants presented similar microbial profiles., Conclusion: The results of the study suggest that the difference in bone crest resorption between implants restored with platform switching compared to traditionally restored implants is not associated with differences in the peri-implant microbiota.

Objective: To perform a systematic review of published data with the aim of evaluating the levels of IgA antibodies to Candida albicans in HIV-infected adult patients., Methods: The search strategy was based on PubMed, Web of Science, Google Scholar, Cochrane and EMBASE databases. Also, the reference lists of included studies were searched. All abstracts found by electronic searches were independently scrutinized by two reviewers. To be eligible for review, the controlled studies had to present the following characteristics: samples of both HIV-infected adults and noninfected adults; appropriate HIV-diagnostic tests for both patient groups (case and control); IgA-diagnostic test applied to a similar population sample., Results: Of 144 studies found, only six met the initial eligibility criteria, but three were excluded after a thorough analysis. To assess the methodological quality of the three remaining studies, they were categorized according the risk of bias. The three selected studies revealed that the levels of C. albicans-specific IgA antibody were higher in HIV-infected individuals compared with the control group., Conclusion: Adequate IgA antibody responses to C. albicans appear to be maintained, since the levels of these antibodies were higher in all studies selected. Although the findings of this systematic review are encouraging, the scientific evidence should be interpreted carefully because there are only a few reports in the literature, mostly because of the lack of important methodological details or the varying methodologies employed.

Background and Objective: Little is known regarding the factors that affect the microbial composition of supragingival biofilms. This study was designed to test the hypothesis that tooth location affects the microbial composition of supragingival plaque beyond the effect due to plaque mass as reflected by total DNA probe count., Material and Methods: Supragingival plaque samples were taken from the mesiobuccal aspect of each tooth in 187 subjects (n = 4745 samples). All samples were individually analyzed for their content of 40 bacterial species using checkerboard DNA-DNA hybridization. Significance of differences in mean species counts and proportions were determined among tooth surfaces and six tooth type categories: molars, bicuspids, incisors/canines in the mandible and maxilla separately using the Kruskal-Wallis test. Stepwise multiple linear regression was employed to examine the relationship between species proportions and total DNA probe count, tooth location, periodontal and smoking status, age and sex., Results: All species differed significantly among tooth types and among the six tooth categories. Higher plaque levels were seen on molars and lower incisors. Some differences observed between tooth types could be partly explained by the level of plaque. Teeth with high plaque mass exhibited high levels of Capnocytophaga gingivalis, Actinomyces naeslundii genospecies 2, Campylobacter rectus and Campylobacter showae. However, certain species, such as Veillonella parvula and Streptococcus sanguinis, differed significantly at different tooth locations despite similarities in plaque mass. Twenty of the test species exhibited a significant association with tooth location after adjusting for total DNA probe count and subject level factors., Conclusion: While plaque mass was associated with differences in proportions of many species in supragingival biofilms, tooth location also was strongly associated with species proportions in both univariate and multivariate analyses.

Background and Objective: Little is known about the factors that affect the microbial composition of supragingival biofilms. This study was designed to examine the relationship between total DNA probe counts of supragingival biofilm samples, clinical parameters and supragingival biofilm composition., Material and Methods: Supragingival plaque samples were taken from 187 systemically healthy adult subjects (n = 4745 samples). All samples were individually analyzed for their content of 40 bacterial species using checkerboard DNA-DNA hybridization. The relationship between total DNA probe counts and microbial composition was examined by subsetting the data into 10 groups based on 10 percentile increments of the total DNA probe counts. Differences among groups in terms of species counts and proportions were sought, as well as relationships of total plaque DNA probe count and clinical parameters., Results: There was a wide distribution in mean total DNA probe counts among the 187 subjects. With increasing total plaque levels there was a change in the proportions of individual species and microbial complexes. 'Small plaques' were characterized by high proportions of species in the yellow, orange, purple and 'other' complexes; plaques of moderate mass were characterized by high proportions of Actinomyces and purple complex species, while 'large plaques' exhibited increased proportions of green and orange complex species. Measures of gingival inflammation, pocket depth and recession were significantly positively associated with total DNA probe counts. Increased plaque numbers were related to increased pocket depth irrespective of presence or absence of gingival inflammation., Conclusion: The proportions of individual species and microbial complexes in supragingival biofilms are influenced by the total numbers of organisms in the biofilm.

Objectives: The aim was to examine the impact of antiretroviral therapy on the prevalence of oral candidiasis, recovery of oral Candida spp. , and salivary levels of total secretory immunoglobulin A (SIgA) and Candida-specific SIgA in human immunodeficiency virus (HIV)-infected children., Study Design: Sixty-six HIV+ and 40 HIV- children were cross-sectionally examined for the presence of oral lesions. Whole stimulated saliva samples were collected for the identification of Candida spp. using culture and measurement of total and specific SIgA using enzyme-linked immunosorbent assay (ELISA)., Results: The HIV+ children had a higher prevalence of oral candidiasis (P < .05), higher frequency of detection of Candida spp. (P < .05), and higher levels of total (P < .05) and Candida-specific SIgA (P < .001) than the HIV- children. Among the HIV+ subjects, antiretroviral users had lower viral loads (P < .001) and lower levels of Candida spp. (P < .05) and total SIgA (P < .05) compared with antiretroviral nonusers., Conclusions: The use of antiretroviral therapy was associated with decreases in the prevalence of oral candidiasis. This diminished exposure to Candida spp. was accompanied by decreases in levels of total and Candida-specific SIgA.

Background and Objective: Saliva has been proposed as a noninvasive diagnostic fluid that could be used in the diagnosis of oral and systemic diseases. The levels of salivary biomarkers, such as cytokines, could potentially be used as a surrogate to distinguish periodontally healthy individuals from subjects with periodontitis. Therefore, the goal of the present investigation was to determine if the levels of 10 different cytokines in saliva differed between a group of periodontally healthy individuals and a group of subjects with periodontitis. Correlations between the concentrations of these 10 cytokines and clinical parameters of periodontal disease were also examined., Material and Methods: In this cross-sectional study, 74 subjects with chronic periodontitis and 44 periodontally healthy individuals were periodontally examined and had the levels of granulocyte-macrophage colony-stimulating factor, interleukin-1beta, interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin-8, interleukin-10, interferon-gamma and tumor necrosis factor-alpha measured in whole saliva using a multiplexed bead immunoassay (Luminex). Significance of statistical differences in the levels of salivary cytokines between groups was determined using nonparametric analysis of covariance, adjusting for age and smoking status. The Spearman rank correlation coefficient was used to explore associations between the mean levels of salivary cytokines and mean clinical parameters., Results: There were no statistically significant differences between groups for any of the cytokines. There were weak, statistically significant positive associations between salivary interleukin-8 and pocket depth (r(s) = 0.2, p < 0.05) and bleeding on probing (r(s) = 0.2, p < 0.05), and weak negative correlations between salivary interleukin-10 and attachment level (r(s) = -0.2, p < 0.05) and bleeding on probing (r(s) = -0.3, p < 0.001)., Conclusion: Mean salivary levels of granulocyte-macrophage colony-stimulating factor, interleukin-1beta, interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin-8, interleukin-10, interferon-gamma and tumor necrosis factor-alpha could not discriminate between periodontal health and disease.

Background: The aim of this study was to describe the development and validation of the checkerboard immunoblotting (CBIB) technique for the high-throughput quantification of multiple inflammatory mediators in gingival crevicular fluid (GCF) samples., Methods: Monoclonal antibodies were used to bind GCF interleukin (IL)-1beta and -8 and matrix metalloproteinase (MMP)-8 to the surface of membranes. Biotinylated antibodies were used to detect bound antigens in a checkerboard format. Signals were developed using chemiluminescence, captured on film, and quantified using software for array analysis. The assay was tested for potential cross-reactions among the three pairs of antibodies. Eleven CBIBs were processed to determine the analytical sensitivity of the assay. Forty GCF samples were analyzed using CBIB and enzyme-linked immunosorbent assay (ELISA) in parallel, and the significance of the correlations among the results was tested using the Pearson correlation coefficient. Nine hundred thirty-one GCF samples were collected from 20 periodontally healthy subjects and 20 periodontitis subjects and analyzed using CBIB to test the assay's sensitivity and dynamic ranges using clinical samples., Results: The CBIB was capable of distinguishing among the three analytes. The sensitivity and dynamic ranges of the assay were suitable for the detection of the three targets in the majority of GCF samples. There were highly statistically significant (P <0.0001) positive correlations between CBIB and ELISA data for all three biomarkers. The periodontitis subjects had statistically significantly higher mean levels of IL-1beta and -8 compared to healthy subjects., Conclusion: The CBIB technique is a sensitive and specific assay for the high-throughput quantification of MMP-8 and IL-8 and -1beta in GCF.

Objective: The purpose of the present investigation was to determine if antimicrobial mouthrinses with different formulations could affect the composition of the subgingival microbiota and clinical parameters of adjacent tissues in periodontal maintenance subjects., Methods: One-hundred and sixteen subjects, who had been treated for chronic periodontitis and were in a maintenance program, were randomly assigned one of four mouthrinses, to be used twice daily for three months. The mouthrinses were herbal 1, herbal 2, essential oil, and chlorhexidine. Clinical measurements and subgingival plaque samples were taken at baseline and at three months. Plaque samples were individually evaluated for 18 test species/taxa using checkerboard DNA-DNA hybridization. Significance of differences between baseline and three months for both microbiological and clinical parameters were determined using the Wilcoxon Signed Ranks test. Significance of difference among groups for change in clinical and microbiological parameters was determined using analysis of covariance (ANCOVA), adjusting for baseline values., Results: Shifts in species proportions differed significantly for 9/18 test species/taxa among the four mouthrinse groups. Streptococcus and Capnocytophaga species were reduced most in the herbal rinse groups, while Veillonella parvula was reduced most in the essential oil and chlorhexidine groups. Actinomyces were also markedly reduced in the chlorhexidine group. Mean Plaque (PI) and Gingival Indices (GI) were reduced between baseline and three months in each group. Results emphasize that chlorhexidine (p < 0.001) and herbal (p < 0.05) rinses significantly reduced PI. Some subjects in each group responded better than others., Conclusion: All four mouthrinses tested produced shifts in the composition of subgingival microbiota, although the results differed among the groups. The observed microbial changes were accompanied by improvements in clinical parameters in the periodontal maintenance subjects.

The control of biofilm accumulation on teeth has been the cornerstone of periodontal disease prevention for decades. However, the widespread prevalence of gingivitis suggests the inefficiency of self-performed mechanical plaque control in preventing gingival inflammation. This is particularly relevant in light of recent evidence suggesting that long standing gingivitis increases the risk of loss of attachment and that prevention of gingival inflammation might reduce the prevalence of mild to moderate periodontitis. Several antimicrobials have been tested as adjuncts to mechanical plaque control in order to improve the results obtained with oral home care. Recent studies, including meta-analyses, have indicated that home care products containing chemical antimicrobials can provide gingivitis reduction beyond what can be accomplished with brushing and flossing. Particularly, formulations containing chlorhexidine, mouthrinses containing essential oils and triclosan/copolymer dentifrices have well documented clinical antiplaque and antigingivitis effects. In vivo microbiological tests have demonstrated the ability of these antimicrobial agents to penetrate the biofilm mass and to kill bacteria growing within biofilms. In addition, chemical antimicrobials can reach difficult-to-clean areas such as interproximal surfaces and can also impact the growth of biofilms on soft tissue. These agents have a positive track record of safety and their use does not seem to increase the levels of resistant species. Further, no study has been able to establish a correlation between mouthrinses containing alcohol and oral cancer. In summary, the adjunct use of chemical plaque control should be recommended to subjects with well documented difficulties in achieving proper biofilm control using only mechanical means.

Background: The aim of this study was to determine whether the rate of attachment loss in periodontally healthy subjects in a prevention regimen would differ from the rate of disease progression in periodontitis subjects enrolled in a maintenance program., Methods: Fifty-five periodontally healthy subjects and 57 periodontitis subjects were clinically and microbiologically monitored at baseline and at 1, 2, and 3 years. Clinical parameters measured at six sites per tooth included bleeding on probing, visible plaque, probing depth, and attachment level. Subgingival plaque samples were taken from the mesio-buccal aspect of every tooth and were analyzed for the levels of 40 bacterial species using checkerboard DNA-DNA hybridization. The significance of differences over time in the clinical parameters was determined using repeated-measures analysis of variance, whereas the significance of differences between groups was determined using the unpaired t test. The Mann-Whitney test was used for microbial analyses, and P values were adjusted for multiple comparisons., Results: Mean clinical parameters improved for both groups over time. By the end of the study, 4% of the sites in maintenance subjects lost > or =2 mm of attachment, whereas in the prophylaxis subjects only 1% of the sites lost > or =2 mm of attachment. Maintenance subjects lost attachment primarily at shallow buccal and lingual sites. The maintenance subjects harbored significantly higher levels of most test species throughout the study. The maintenance program did not reduce the levels of red complex species to those typical of healthy subjects., Conclusions: Treated periodontitis subjects under maintenance displayed more rapid attachment loss than periodontally healthy subjects in a preventive regimen. The greater propensity to disease progression may be related to an elevated exposure to periodontal pathogens.

Aim: This study aimed to investigate the prevalence and levels of salivary microorganisms in HIV-positive children, and their correlation to HIV status, oral lesions, and salivary IgA levels., Design: Forty-two HIV-positive and 36 control children were clinically examined, had their saliva collected and processed for the microbiological analysis of 38 bacterial taxa by the checkerboard method, and salivary IgA quantification by ELISA., Results: The majority of the species tested were more prevalent in control children than in the HIV group. Mean concentration of total salivary IgA was similar in both groups. High levels of Veillonella parvula were found in children with cheilitis and herpes. Tannerella forsythia, Eikenella Corrodens, and Propionibacterium acnes were prevalent in children with gingivitis, while Fusobacterium periodonticum, Streptococcus gordonii, and Streptococcus oralis were significantly more frequent in children with no oral lesions. Significant negative correlations between salivary IgA levels and Eubacterium nodatum and oral streptococci were observed (P < 0.05)., Conclusion: HIV-seropositive children presented significantly lower prevalence and levels of several bacterial species in saliva; HIV-positive children are able to mount a mucosal immune response; HIV-seropositive children under highly active antiretroviral therapy presented low prevalence of oral lesions.

Background: Adjunctive locally delivered antibiotics during maintenance may favor the control of periodontal infections. This study evaluated the long-term clinical and microbiologic effects of yearly locally delivered controlled-release doxycycline as an adjunct to mechanical debridement., Methods: A total of 128 periodontal maintenance patients having at least four teeth with probing depth (PD) > or =5 mm were randomly assigned to local application of doxycycline gel at baseline and 1 and 2 years as an adjunct to mechanical debridement (test) or mechanical debridement only (control). Supportive periodontal therapy (mechanical debridement, polishing, and oral hygiene reinforcement) was provided every 6 months. Plaque, bleeding on probing (BOP), PD, and relative attachment level (RAL) were scored at baseline; 3 months; and 1, 2, and 3 years. Subgingival plaque samples were taken at each examination and analyzed for their content of 40 bacterial species. Data analyses were performed on an intention-to-treat basis with the subject as the statistical unit., Results: Significant reductions in BOP, PD, RAL, and the mean counts of a number of target species between baseline and 3 years were documented for both treatment groups, whereas plaque scores remained unchanged. A statistically significant difference in favor of the adjunctive doxycycline therapy was found between the two groups only at the 3-month examination for BOP, PD, and RAL and for a minority of bacterial species at 2 years., Conclusion: Although short-term effects on clinical parameters were found with the adjunctive use of locally delivered doxycycline, repeated applications annually had no clinical or microbiologic effects beyond those observed with mechanical debridement alone in maintenance patients.

Introduction: This study aimed to measure the levels of interleukin-18 (IL-18) in inflamed shallow sites and inflamed deep sites in patients with periodontitis and to compare the data with results from inflamed shallow sites in patients with gingivitis. A secondary aim was to examine the composition of the subgingival microbiota in the sampled sites., Methods: Gingival crevicular fluid was collected from five gingivitis sites and five periodontitis sites from 18 patients with chronic periodontitis, and from five gingivitis sites from 15 patients with gingivitis. Samples from each site category were pooled and IL-18 levels were measured using an enzyme-linked immunosorbent assay. The subgingival microbiota was analyzed by checkerboard DNA-DNA hybridization., Results: All clinical parameters and gingival crevicular fluid volumes were higher in periodontitis sites compared with gingivitis sites from patients with periodontitis and gingivitis. The total amount of IL-18 was higher in periodontitis sites than gingivitis sites in both periodontitis (P = 0.018) and gingivitis (P = 0.002) patients and was higher in gingivitis sites from periodontitis patients than in those from gingivitis patients (P = 0.015). There were higher levels of Tannerella forsythia, Porphyromonas gingivalis, and Treponema denticola (red complex species) in periodontitis sites compared with gingivitis sites in both the periodontitis and gingivitis patients (P < 0.001)., Conclusion: Levels of IL-18 were higher in patients with chronic periodontitis compared with patients with gingivitis, even at sites with similar pocket depths. The presence of similar levels of red complex species in gingivitis sites from periodontitis patients and from gingivitis patients suggested that the higher levels of IL-18 were not associated with a different microbial challenge.

Aim: To test the hypothesis of a superior clinical and microbiological effect of the combined use of powered toothbrush+triclosan-containing dentifrice compared with manual toothbrush+regular fluoride-containing dentifrice in periodontal maintenance patients., Material and Methods: A total of 128 periodontitis subjects involved in recall programmes were randomized to use either powered toothbrush with triclosan-dentifrice (test) or manual toothbrush and standard dentifrice (control). Supportive periodontal treatment was provided at baseline and every 6 months. Plaque, bleeding on probing (BoP), probing pocket depth (PPD) and relative attachment level (RAL) were scored at baseline, 1, 2 and 3 years. Subgingival plaque samples were taken and analysed for their content of 40 bacterial species at each examination interval. All analyses were performed by "intention-to-treat" protocol., Results: Both groups showed significant reduction in BoP, PPD and in mean total counts of the 40 bacterial species between baseline and 3 years, while plaque score and RAL remained almost unchanged. No significant differences between the two prevention programmes were found for any of the clinical outcome variables or in mean counts of the various bacterial species., Conclusions: The study failed to demonstrate superior clinical and microbiological effects of powered toothbrush+triclosan dentifrice compared with manual toothbrush+standard fluoride-dentifrice in periodontitis-susceptible patients on regular maintenance therapy.

Background: Different means are available for self-performed oral hygiene. The aim of this study was to evaluate the clinical and microbiologic effects of a preventive homecare program including the combined use of a powered toothbrush and a triclosan/copolymer-containing dentifrice., Methods: A total of 160 adult subjects without signs of destructive periodontal disease were recruited for this 3-year randomized controlled trial. The subjects were assigned to a homecare program using an oscillating/rotating powered toothbrush and a triclosan/copolymer/fluoride-containing dentifrice (test) or a manual toothbrush and a standard fluoride-containing dentifrice (control). Supragingival polishing and reinforcement of homecare procedures were provided every 6 months. Plaque, bleeding on probing (BOP), and probing depth (PD) were scored at baseline and after 1, 2, and 3 years. Subgingival plaque samples were taken from the mesial aspect of each tooth at baseline and after 1, 2, and 3 years and were analyzed for their content of 40 bacterial species using checkerboard DNA-DNA hybridization. All data analyses were based on "intention-to-treat" with the subject as the statistical unit., Results: Compared to baseline, no significant changes in clinical parameters were observed during the 3 years, except for a reduction in the mean PD at the 2- and 3-year follow-up examinations (P <0.05). No significant differences were found between the two groups with regard to plaque, BOP, or PD or in the mean counts of the 40 species at any time point., Conclusion: The study failed to prove additional benefits of the combined use of a powered toothbrush and a triclosan/copolymer-containing dentifrice in adult subjects without signs of destructive periodontal disease.

Multiple-displacement amplification (MDA) has been used to uniformly amplify bacterial genomes present in small samples, providing abundant targets for molecular analysis. The purpose of this investigation was to combine MDA and checkerboard DNA-DNA hybridization to examine the microbiota of endodontic infections. Sixty-six samples were collected from teeth with endodontic infections. Nonamplified and amplified samples were analyzed by checkerboard DNA-DNA hybridization for levels and proportions of 77 bacterial taxa. Counts, percentages of DNA probe counts, and percentages of teeth colonized for each species in amplified and nonamplified samples were computed. Significance of differences for each species between amplified and nonamplified samples was sought with Wilcoxon signed-rank test and adjusted for multiple comparisons. The amount of DNA in the samples ranged from 6.80 (+/- 5.2) ng before to 6.26 (+/- 1.73) mug after MDA. Seventy of the 77 DNA probes hybridized with one or more of the nonamplified samples. All probes hybridized with at least one sample after amplification. Most commonly detected species at levels of >10(4) in both amplified and nonamplified samples were Prevotella tannerae and Acinetobacter baumannii at frequencies between 89 and 100% of samples. The mean number of species at counts of >10(4) in amplified samples was 51.2 +/- 2.2 and in nonamplified samples was 14.5 +/- 1.7. The endodontic microbiota was far more complex than previously shown, although microbial profiles at teeth with or without periradicular lesions did not differ significantly. Species commonly detected in endodontic samples included P. tannerae, Prevotella oris, and A. baumannii.

Aim: To investigate whether the clinical benefits obtained with a periodontal prevention programme in subjects with periodontal health or minimal disease were accompanied by beneficial changes in the subgingival microbiota., Material and Methods: One hundred and twenty-four subjects completed the study. Subjects were clinically and microbiologically monitored at baseline, 1, 2 and 3 years. Subgingival plaque samples were taken from the mesiobuccal aspect of every tooth and were analysed for the levels of 40 bacterial species using checkerboard DNA-DNA hybridization (total samples=13,477). The mean counts of each of the 40 test species were calculated for each subject at each time point. Significance of differences over time was sought using the Friedman test. p values were adjusted for multiple comparisons., Results: All clinical parameters, at the microbiologically sampled sites, improved over time. The clinical changes were accompanied by statistically significant decreases in the mean counts of 35 of the 40 test species. Major reductions occurred by year 2 for Actinomyces, Capnocytophaga, Campylobacter, Fusobacterium and Prevotella species. At year 3, there was a modest re-growth of the majority of the species., Conclusions: The clinical improvements obtained through preventive measures were accompanied by a shift to a more host-compatible subgingival microbiota.

Background: The purpose of the present investigation was to compare the levels, proportions and percentage of sites colonized by 40 bacterial species in subgingival plaque samples from periodontally healthy subjects and patients with chronic periodontitis to seek possible pathogens other than the consensus pathogens Porphyromonas gingivalis and Tannerella forsythia., Method: Subgingival plaque samples were taken from the mesial aspect of each tooth in 635 subjects with chronic periodontitis and 189 periodontally healthy subjects. The samples were individually analyzed for their content of 40 bacterial species using checkerboard DNA-DNA hybridization (total samples = 21,832). Mean counts, % DNA probe counts and percentage of sites colonized at >10(5) were determined for each species in each subject and then averaged in each clinical group. Significance of difference between groups was determined using the Mann-Whitney test. Association between combinations of species and periodontal status was examined by stepwise logistic regression analysis. Analyses were repeated using a subset of subjects from both clinical groups who had proportions of P. gingivalis plus T. forsythia less than the median (4.42%) found in periodontally healthy subjects. All analyses were adjusted for multiple comparisons., Results: For the 824 subjects the consensus pathogens P. gingivalis and T. forsythia as well as Eubacterium nodatum and Treponema denticola had significantly higher mean counts, proportions and percentage of sites colonized in samples from subjects with periodontitis than from periodontally healthy subjects. There were significantly more Capnocytophaga gingivalis, Streptococcus gordonii and Veillonella parvula in periodontally healthy subjects. E. nodatum, T. denticola, Streptococcus oralis, Streptococcus intermedius, Fusobacterium nucleatum ssp. vincentii all had higher counts and proportions in diseased than healthy subjects who had low proportions of P. gingivalis and T. forsythia. Logistic regression analysis indicated that the same species groups were associated with disease status after adjusting for the proportions of the other species., Conclusions: This investigation confirmed the strong association of P. gingivalis and T. forsythia with chronic periodontitis and emphasized a strong association of E. nodatum and T. denticola with periodontitis whether in the presence or absence of high levels of the consensus pathogens. Other species, including S. oralis, Eikenella corrodens, S. intermedius and F. nucleatum ssp. vincentii, were associated with disease when P. gingivalis and T. forsythia were present in low proportions.