Purpose: The purpose is to evaluate whether the CariScreen meter is a reliable tool in quantifying cariogenic bacteria and a valuable addition to caries risk assessment.

Methods: Sixty children, aged 6-17 years, were recruited. Caries status was recorded utilizing ICDAS system. CariScreen scores were evaluated from two sites: the lingual surfaces of the mandibular anterior dentition per manufacturer’s instruction and the mandibular buccal surface of one posterior molar. Stimulated saliva samples were collected for bacterial enumeration (Mitis Salivarus plate for total oral streptococci, Mitis Salivarus Sucrose Bacitracin plates for Mutans Streptococci (MS), Rogosa tomato juice plates for Lactobacillus (LB), and Blood agar plates for total viable bacteria). Statistical analysis was conducted using SPSS 22.

Results: CariScreen scores from anterior and posterior sites revealed a moderate correlation (Correlation Coefficient = 0.42). CariScreen scores for both anterior and posterior sites revealed poor correlation with logarithmic counts of Mutans Streptococci, Total oral streptococci, Lactobacilli, and total viable bacteria (Pearson Correlation coefficient between -0.30 to 0.03, P>.02 for anterior sites and between -0.10 to 0.18, P>.18 for posterior sites). For anterior sites, caries-free subjects had higher log CariScreen scores than caries-active subjects (meanSE as 3.780.11 and 3.340.06, respectively, P=.12) while, for posterior sites, caries-free subjects’ log CariScreen scores were similar to caries-active subjects (meanSE as 2.950.19 and 3.060.08, respectively, P=.76). There were acceptable sensitivities (66-70%), but poor specificity (33-37%) and high false positive rate (45-75%) in diagnosing MS, LB and MS+LB levels compared with gold standard culture methods.

Conclusion: Compared to bacterial culture, CariScreen meter showed poor prediction of cariogenic bacteria levels. Furthermore, CarieScreen scores in anterior sites reveal a trend of negative correlation to cariogenic bacteria levels and caries status.

Purpose: To evaluate the effectiveness of a modified Caries Management by Risk Assessment (CAMBRA) model in a clinical trial amongst children between the ages of 5-9 years old, treated in a school-based community pediatric dental clinic setting.

Methods: In a one-year, randomized clinical trial, 66 children between the ages of 5-9 years old were randomized into control or intervention groups. The control group received conventional treatment, including exam, prophy, caries risk assessment and fluoride varnish at baseline, 6 months, and one year follow up. The intervention group, in addition to receiving conventional treatment, also received CAMBRA, if considered high caries risk, which consisted of additional fluoride treatments at 3 and 9 months as well as daily xylitol mint consumption. Saliva samples for mutans streptococci (MS) and lactobacilli (LB) enumerations (CFU/ml) and dmfs/DMFS scores were collected from each subject at baseline and one year examinations.

Results: Of the 66 participants, 60 subjects completed the study. At baseline there was no statistical differences in subject demographics, MS and LB levels, along with DMFS/dmfs scores between the two groups. At six months and one year, there were no statistically significant differences in logMS and logLB levels between the two groups (Student t- test p>0.05). Also, at one year, we did not find any significant difference in the change of decayed surfaces (DS/ds), smooth surface decay (SS-DS/ds) and overall decayed surfaces (DMFS/dmfs) between the intervention and the control groups (Student t-test, P>0.05). Although the difference is not statistically significant, the change of dental caries at one year in the intervention group displayed a consistent decrease in all scores compared to a consistent increase in the control group with a borderline statistically significant difference in SS-DS/ds (Student t- test, P=0.06). The caries risk assessment tools reviewed the oral habits of the children, such as oral hygiene and diet. An increase in tooth brushing frequency was reported from both control and intervention groups, with a decrease in snacking frequency per day. Although neither was statistically significant, it presents a positive trend towards a reduction of risk factors along with an increase in protective factors for both groups.

Conclusions: Although the modified CAMBRA intervention in children did not significantly reduce MS levels or change DMFS/dmfs scores, it did show positive trends towards reducing caries, especially on smooth surfaces. It has also shown decreased lactobacillus levels in children ages 5-9 years old. Due to limitations within the study, we were unable to recruit a sufficient number of subjects to meet the sample-size calculation. Future studies with sufficient number of subjects is needed to validate the success and positive benefits of the modified CAMBRA protocol in young children.

Authors: Alexandra Malebranche, Jing Cheng, Pamela Den Besten, Karen-Sokal-Gutierrez, Arup Roy-Burman, and Ling Zhan

Purpose: To evaluate feasibility and barriers for implementation of a novel oral-health and fluoride-varnish program for 0-5 year old children in vaccination clinics in Roatán, Honduras.

Methods: Administrators, physicians, nurses, and dentists involved in the oral-health and fluoride-varnish programs in vaccination clinics in Roatán were recruited for a mixed survey and interview study. Information regarding staff demographics, knowledge, confidence, attitudes and behavior, implementation barriers, as well as, suggestive solutions in providing oral health services in the vaccination clinic were collected.) The data were analyzed using SPSS Statistics 22.

Results: Twenty-five subjects were enrolled. The time that non-dental providers spent learning oral health education was between 2.5-5.3 hours. Of these individuals, more than 68% of the subjects correctly identified the frequency and method for application of fluoride-varnish, and cariogenicity of candy and soda. However only 32% subjects identified juice as cariogenic and the correct timing of fluoride-varnish application in children. Nine of the 25 subjects were confident that they could effectively provide fluoride-varnish and oral-health education. All subjects requested more training on oral-health with preference for hands-on-training and reading-material with pictures. Most subjects supported to providing basic oral health services during vaccination visits, such an oral exam (78-100%), nutrition counseling (71-88%), applying fluoride varnish (88-100%), or demonstrating brushing technique (100%). The most commonly reported barriers of the program were lack of supplies, family interest, staff time, and staff interest. Increased government support and improved training were common suggestions for program improvement.

Conclusions: The administration of fluoride varnish in vaccination clinics in Roatán, Honduras was largely supported by staff. Barriers to success were inadequate training in oral health, lack of interest by staff and families and inadequate supplies. Improving training and increased government support were identified as key elements to improve the program's effectiveness.

Purpose: to evaluate the value of CariScreen meter as a part of the caries risk assessment tool in new caries prediction compared to cariogenic bacteria enumeration by culture (gold standard).

Methods: Eighty 6-17 year-old children were recruited. At baseline and one year, oral bacterial levels were analyzed by CariScreen meter and culture (Mutans Streptoccoci (MS) and Lactobacilli) together with caries risk assessment (CRA) and caries scores were recorded using International Caries Detection an Assessment System (ICDAS). Correlations between the new caries at one-year and baseline Cariscreen or bacteria levels were computed. Incidence and severity of new caries were compared in risk-categories by Cariscreen or culture alone or combined with CRA at baseline using SPSS 22.

Results: Fifty-three subjects completed the one-year visit. 83.0% subjects were high risk at baseline with only 9 (17.0%) at low/moderate risk by CRA at baseline and 57% of them presented with new decay. CariScreen readings at baseline showed nearly no correlation with new decayed surfaces at one year (Spearman correlation coefficient of 0.04-0.05, P=0.73-0.79 for both sites). No trend of statistical significant differences was found for prevalence or severity of new decay at one year between high and low bacterial challenge at baseline measured by Cariscreen (P>0.67) while there was a trend of lower prevalence and severity of new decay at one year in low challenge by MS culture at baseline (P=0.15). The median for new decayed surfaces at one year were 0 and 1, respectively for low/moderate and high caries risk groups by CRA only (Kruskal-Wallis test, P = 0.11); 1 and 1 for low/moderate and high caries risk groups by CRA combined with Cariscreen (Kruskal-Wallis test, P=0.99); and 0 and 1 for low/moderate and high caries risk groups by CRA combined with MS levels (Kruskal-Wallis test, P = 0.10).

Conclusions: CariScreen meter measurement alone or combined with CRA at baseline showed no evidence in prediction of new decay after one year. CRA alone or combined with MS culture showed potential in predicting new caries in one year but this needs to be further confirmed with larger sample size study especially with more low/moderate risk subjects.

Periodontal disease is characterized by destruction of the hard and soft tissues that comprise the periodontium. This destruction translates to a degradation of the extracellular matrices (ECM), which is mediated by bacterial proteases, host-derived matrix metalloproteinases (MMPs), and other proteases released by host tissues and immune cells. Bacterial pathogens interact with host tissue and thereby trigger adverse cellular functions, including a heightened immune response, tissue destruction, and tissue migration. Oral spirochete, Treponema denticola (Td), is highly associated with periodontal disease. A Td outer membrane protein complex called dentilisin, contributes to the chronic activation of pro-MMP-2 in periodontal ligament (PDL) cells and triggers increased expression levels of activators and effectors of active MMP-2 in PDL cells. Despite these advances, there is no mechanism known for dentilisin-induced MMP-2 activation, PDL cytopathic behaviors leading to disease, or potential treatment. This thesis demonstrated that Td through dentilisin influences PDL cellular functions such as metabolism, contractility, migration, actin cytoskeletal reorganization, and mechanotransduction. Furthermore, we show dentilisin through an actin-RASA4-MMP-2 axis influences depolymerization and over expression and activity of MMP-2. Therapeutically, one possible avenue to combat these interactions is through the use of bacteriocin nisin. This thesis demonstrated that nisin delivered in a solid lipid nanoparticle (SLN-Nisin) is an effective anti-microbial against Td, with the potential to bacterial clearance inside and outside of the PDL cells. Proper clearance of Td may provide a more symbiotic microbiome and “reset” homeostatic conditions needed for repair, remodeling, or regeneration of the periodontium. Thus, the main hypotheses of this proposal are that T. denticola interactions with PDL cells mediate adverse effects on homeostasis and cellular functions leading to a compromised cellular phenotype. Additionally, nisin is a potential therapeutic for abrogating these effects. This hypothesis was tested using various functional assays, live imaging dyes and immunofluorescent staining via confocal microscopy, RNA sequencing and real-time quantitative polymerase chain reaction, western blotting, bacterial mutants, and functional gene knockdown approaches. The following dissertation provides molecular evidence for a mechanism of Td-mediated adverse cytopathic effects leading to cellular characteristics consistent with severe periodontitis; and evidence for nisin as a potential therapeutic for reducing or reversing Td-mediated periodontopathic processes in vitro.

Purpose: To determine whether Livionex Dental Gel affects oral hygiene, bacterial counts, and incidence of mucositis and bloodstream infections (BSI) in pediatric Human Stem Cell Transplant (HSCT) recipients.

Methods: This was a prospective, double-blinded, randomized-controlled trial pilot study. Subjects were randomized to Livionex or control (PreviDent 5000 Plus for children ≥6 years or Tom's and Maine Children's Toothpaste for children <6 years) groups in a 2:1 ratio. Gingival index (GI), plaque index (PI), relative amounts of mutans streptococci (MS), total streptococci (TS), Lactobacilli (LB), and total viable count (TVC), mucositis and BSI were recorded and compared using linear mixed effect models across timepoints. Brushing compliance was assessed with questionnaire responses and were compared using the Wilcoxon rank sum test.

Results: Twenty-six subjects (19 in Livionex and 7 in control) participated. MS counts decreased significantly in both groups. BSI incidence was not significantly different. There were no significant differences in GI, PI, incidence of mucositis, and questionnaire responses.

Conclusions: There is possible benefit from using Livionex in this population. Studies with larger sample-sizes are needed to further confirm the effect of Livionex on bacterial counts and BSI occurrences in HSCT patients.

Title: Effect of Xylitol-Wipe on Mutans Streptococci Virulence

Purpose: In a randomized clinical trial, we found a significant reduction of new caries in young children with daily use of xylitol-wipes over one year without reduction on mutans streptococci (MS) levels. The purpose of this study was to determine whether the use of xylitol-wipes for one year was due to selection of xylitol-resistant MS, altered the rate of acid production of MS and/or altered biofilm formation ability of MS.

Methods: Unique MS genotypes were isolated from the xylitol-wipe group and the placebo-wipe group at baseline and at 1 year. These isolates were examined for their xylitol sensitivity, rate of acid production and ability to form biofilm. Xylitol sensitivity was determined by comparing bacterial growth curves in TPY glucose broth with or without the addition of 1% xylitol. Acid production was monitored by the means of pH drop for 30 minutes after each isolate was grown in the presence of glucose. Sucrose dependent biofilm formation of each genotype was quantified using crystal violet assay.

Results: At one year, there was no significant difference in prevalence of xylitol-resistant MS genotypes between the xylitol-wipe and the placebo-wipe groups. There was significant decrease difference in plateau pH within each group from baseline to 1 year (p<0.05). The change in acid profile in the xylitol-wipe group from baseline to 1 year was similar to that observed in the caries vs. no caries group. There were no statistical significant differences in biofilm formation of MS isolated from the two wipe groups at baseline or one year (p>0.05).

Conclusions: This prospective randomized clinic trial has shown no correlation between caries reduction and selection of xylitol-resistant MS. Furthermore, xylitol-wipe use did not alter the ability of MS to form biofilm. However, there is evidence to suggest that after one year xylitol wipe use would select MS with decrease acid production ability. Future studies are needed to identify other possible mechanisms of caries prevention resulting from the use of xylitol-wipes in children.

This study aims to evaluate the impact of silver diamine fluoride (SDF) application(s) prior to dental treatment under general anesthesia (GA) on treatment types rendered to children under six years old with GA through a retrospective case control study.Baseline demographics/disease/planned treatment data and dental treatment for qualified patients were collected from electronic dental records. Patients were in divided into a SDF intervention group (N=335, 21.49%,) or non-SDF control group (N=1224, 78.51%) based on their receipt of SDF prior to GA. Statistical analysis was completed for baseline and outcome data using STATA. Student T tests or Mann-Whitney U Test were used appropriately to compare impact of pre-GA SDF application on treatment outcomes between the two groups without adjusting the baseline variable imbalance. Poisson, negative binomial, and linear regression were used in multivariate models to address the baseline variable imbalance on impact of pre-GA SDF application and analyze the effects of baseline and demographic variables on the GA treatment outcomes. There were significant differences in age, gender, pre-GA dental pain experience, pulp involvement, and planned treatment at baseline between the two groups. In a non-adjusted analysis, the SDF intervention group had significantly more crowns, but significantly fewer unplanned and total pulp therapy and dental extractions under GA (Mann-Whitney U test, P<0.05). A multi-variate model confirmed the negative association of pre-GA SDF application with total and unplanned pulp therapy and dental extractions under GA (p<0.05), but no association with crowns placed. The model also indicated a positive relationship between invasive dental treatment with patient age, planned treatment needs and GA wait-time. Our study supports SDF application prior to dental treatment under GA as a valuable tool to reduce invasive dental procedures under GA.

Purpose: to assess whether single or combinations of three novel antimicrobial peptide genes (NN2, PNS1, TKS1) of mutans Streptococci (MS) were associated with dental caries in 2-5 year old children.

Methods: Eighty-two caries-free (CF) and 37 severe early-childhood caries (S-ECC) 2-5 year-old children were recruited in a pilot and current studies. Caries status was recorded utilizing the International Caries Detection and Assessment System. Oral MS levels were enumerated from oral swab by selective culture. In the pilot study, MS were isolated from each subject with the genes identified by high-throughput illumina sequencing. In the current study, the genes were identified by SYBR green qPCR assays. The prevalence of the single gene or gene combinations were compared by Chi Square test/Fisher’s exact test between CF and S-ECC group and Odds Ratios were calculated. LogMS levels and decayed surfaces in subjects with or without the single gene or combination of the genes were compared by Student t and non-parametric tests.

Results: Thirty-nine CF and 36 S-ECC subjects had MS infection. The prevalence of any of the three genes was 21% in CF and 58% in S-ECC. In the individual gene analysis, PNS1 was the most commonly detected gene (38%) and was significantly more prevalent in S-ECC than in the CF group while NN2 and TKS1 were less prevalent (16% and 19% respectively) and showed no statistically significant difference in prevalence between S-ECC and CF groups. In analysis of combinations of any two or all three genes, the prevalence of all gene combinations with PNS1 were significantly higher in the S-ECC group combination (P<0.05) with the highest Odds ratio in PNS1 – TKS1 (9.17). MS levels between subjects with or without any single gene or combinations of genes were not significantly different (P>0.05). However, mean decayed surfaces were significantly higher in the presence of the genes or gene combinations except for NN2+TKS1.

Conclusion: PNS1 and combination of PNS1 with NN2 and/or TKS1 may contribute to caries development and risk. Future studies are needed to study the mechanism of these genes’ function to develop tools for caries prediction and prevention.

Caries Management by Risk Assessment in Children

Paul Aaron Johnson

Abstract

Purpose: To investigate the efficacy of a modified caries management by risk assessment (CAMBRA) protocol on reducing the cariogenic bacterial load, improving oral hygiene care and dietary habits on children aged 5-9 years old.

Methods: Sixty-six children aged 5-9 years old participated in a single blind randomized controlled clinical trial and were assigned to either the modified CAMBRA or regular treatment group. Parents of intervention group were asked to give child 6-8g of xylitol mints per day (8-12 mints/day) for a total of six months. These individuals were seen every 3 months for fluoride varnish application, oral hygiene and diet evaluation with counseling. Additional diet and oral hygiene information was sent home to parents. dmfs/DMFS scores, caries risk assessment, saliva samples were taken at baseline and at 6 months and MS, LB and TVC of bacterial levels tabulated.

Results: MS, LB and TVC at baseline and 6 month showed slight decrease but there was no statistical significance between the modified CAMBRA and regular treatment groups p<0.05. There was consistent trend of decreasing caries risk factors (tooth brush and snacking frequency, plaque scores) from baseline to 6 months however these changes between the two groups were not statistically significant as well.

Conclusion: 6-8g of daily xylitol mints show a minimal, but not statistically significant decrease in cariogenic bacterial load in the oral cavity. Increased frequency of professional oral hygiene and diet counseling shows a positive trend in decreasing caries risk behavior at six months in this population.