Validation of a multiplex qPCR assay for detection and quantification of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythia in subgingival plaque samples. A comparison with anaerobic culture.

OBJECTIVE: To validate a multiplex real time qPCR (m-qPCR) assay for the simultaneous detection and quantification of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythia in subgingival samples, when compared with anaerobic culture. MATERIAL AND METHODS: Subgingival plaque samples were obtained from patients seeking periodontal treatment. Samples were processed in parallel by anaerobic culturing and by m-qPCR directed to the target bacterial species. Counts and frequency of detection were calculated and analyzed by Mann-Whitney U and chi-square tests, respectively. Contingency tables were constructed, and sensitivity, specificity, predictive values and Lin's correlation coefficients were calculated. RESULTS: Fifty-nine samples were included in the study. A good concordance was achieved between m-qPCR and culture for A. actinomycetemcomitans and P. gingivalis (net agreement, 94.92% and 91.53%, respectively). For T. forsythia, m-qPCR showed statistically significant higher counts than culture (p < 0.005), and low specificity (3.12%) and concordance (47.46%). High sensitivity (above 96.22%) was attained for the three target bacteria with m-qPCR. CONCLUSION: Compared to culture, the tested m-qPCR assay for subgingival plaque samples showed high degree of sensitivity in the simultaneous quantification of A. actinomycetemcomitans, P. gingivalis and T. forsythia.

Comparison of the detection of periodontal pathogens in bacteraemia after tooth brushing by culture and molecular techniques

BACKGROUND: The prevalence and amounts of periodontal pathogens detected in bacteraemia samples after tooth brushing-induced by means of four diagnostic technique, three based on culture and one in a molecular-based technique, have been compared in this study. MATERIAL AND METHODS: Blood samples were collected from thirty-six subjects with different periodontal status (17 were healthy, 10 with gingivitis and 9 with periodontitis) at baseline and 2 minutes after tooth brushing. Each sample was analyzed by three culture-based methods [direct anaerobic culturing (DAC), hemo-culture (BACTEC), and lysis-centrifugation (LC)] and one molecular-based technique [quantitative polymerase chain reaction (qPCR)]. With culture any bacterial isolate was detected and quantified, while with qPCR only Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans were detected and quantified. Descriptive analyses, ANOVA and Chi-squared tests, were performed. RESULTS: Neither BACTEC nor qPCR detected any type of bacteria in the blood samples. Only LC (2.7%) and DAC (8.3%) detected bacteraemia, although not in the same patients. Fusobacterium nucleatum was the most frequently detected bacterial species. CONCLUSIONS: The disparity in the results when the same samples were analyzed with four different microbiological detection methods highlights the need for a proper validation of the methodology to detect periodontal pathogens in bacteraemia samples, mainly when the presence of periodontal pathogens in blood samples after tooth brushing was very seldom

A randomized clinical trial on the clinical and microbiological efficacy of a xanthan gel with chlorhexidine for subgingival use.

BACKGROUND: The main indication of the adjunctive use of local antimicrobials lies around situations in which the outcome of non-surgical mechanical treatment results in a limited number of residual pockets. The purpose of this investigation was to evaluate the clinical and microbiological effects of the subgingival application of a xanthan-based 1.5% chlorhexidine (CHX) gel (Xan-CHX), adjunctive to scaling and root planing (SRP) in localized periodontitis. METHODS: Periodontitis patients with four to ten residual (after conventional SRP) or relapsing (during supportive periodontal treatment) pockets were recruited and randomized to receive SRP plus the subgingival application of (Xan-CHX) or SRP plus a placebo gel. Supragingival plaque, bleeding on probing (BOP), probing pocket depth (PPD), and clinical attachment level were evaluated with a computerized probe at baseline, and after 1, 3, and 6 months. Subgingival samples were also collected for the microbiological analysis. Statistical analysis used ANOVA and chi-square tests. RESULTS: Overall, the clinical results were better in the test group, with significant changes in BOP (between baseline and 3 months) and with a significant increase in the proportion of shallow pockets (1-3 mm) at 6 months. These results did not result in significant intergroup differences. The microbiological impact was limited in both treatment groups. CONCLUSION: The adjunctive use of Xan-CHX may improve, although to a limited extent, the clinical outcomes (BOP and PPD), in chronic periodontitis patients with "residual" or "relapsing" pockets, but no significant differences were detected between groups. No side effects, neither clinical nor microbiological, were detected after the use of the test product. CLINICAL RELEVANCE: Adjunctive use of slow-released chlorhexidine might be considered in the management of periodontal disease and gingival inflammation to reduce the need for periodontal surgery.

Microbiological effects of an antiseptic mouthrinsein irradiated cancer patients

Objective: To assess the microbiological effects of an antiseptic, non-alcohol based mouth-rinse containing chlorhexidineand cetylpyridinium chloride, in patients undergoing radiation therapy for head-and-neck cancer.Study Design: This was a parallel, double-blind, prospective, randomized clinical trial, including patients irradiatedas part of the therapy of head-and-neck cancer, aged 18-75, with at least 10 teeth, and willing to sign an informed consent. Cancer patients were randomly assigned to one of the two treatments (test mouth-rinse or a placebo).Three visits were scheduled (baseline, 14 and 28 days). Microbiological findings were evaluated in tongue,mucosa and subgingival samples, by means of culture. Microbiological variables were assessed by means of theMann-Whitney, Wilcoxon and chi-square tests.Results: 70 patients were screened and 36 were included. The detection of Candida species in mucosa and tonguesamples showed significant reductions in the test group. Total bacterial counts decreased in both groups frombaseline to the 2-week visit, while minor changes occurred between 2 and 4 weeks (effects on P. gingivalis, P.intermedia, C. rectus, E. corrodens).Conclusions: Within the limitations of the small sample size, this study suggests that the use of the tested mouthrinsemay lead to improvements in microbiological parameters in patients irradiated for head-and-neck cancer (AU)

Microbiological effects of an antiseptic mouthrinse in irradiated cancer patients.

OBJECTIVE: To assess the microbiological effects of an antiseptic, non-alcohol based mouth-rinse containing chlorhexidine and cetylpyridinium chloride, in patients undergoing radiation therapy for head-and-neck cancer. STUDY DESIGN: This was a parallel, double-blind, prospective, randomized clinical trial, including patients irradiated as part of the therapy of head-and-neck cancer, aged 18-75, with at least 10 teeth, and willing to sign an informed consent. Cancer patients were randomly assigned to one of the two treatments (test mouth-rinse or a placebo). Three visits were scheduled (baseline, 14 and 28 days). Microbiological findings were evaluated in tongue, mucosa and subgingival samples, by means of culture. Microbiological variables were assessed by means of the Mann-Whitney, Wilcoxon and chi-square tests. RESULTS: 70 patients were screened and 36 were included. The detection of Candida species in mucosa and tongue samples showed significant reductions in the test group. Total bacterial counts decreased in both groups from baseline to the 2-week visit, while minor changes occurred between 2 and 4 weeks (effects on P. gingivalis, P. intermedia, C. rectus, E. corrodens). CONCLUSIONS: Within the limitations of the small sample size, this study suggests that the use of the tested mouth-rinse may lead to improvements in microbiological parameters in patients irradiated for head-and-neck cancer.

Mucositis in irradiated cancer patients: Effects of an antiseptic mouthrinse

Objective: To assess the effects of an antiseptic, non-alcohol based mouth-rinse containing chlorhexidine and cetylpyridinium chloride, in preventing the oral complications associated to radiation therapy in head-and-neckcancer patients.Study design: This was a parallel, double blind, prospective, randomized clinical trial. Cancer patients were randomly assigned to one of the two treatments (test mouth-rinse or a placebo). Three visits were scheduled (baseline,14 and 28 days). Different outcome variables were evaluated: mucositis, plaque and gingival indices, stimulated saliva and salivary pH.Results: 70 patients were screened and 36 were included. The presence and the degree of mucositis significantly increased in both groups and no significant differences were detected between groups, although the median increase in the placebo group (1.81) at 2 weeks was higher than in the test group (1.20).Conclusions: Within the limitations of the small sample size, this study suggests that the use of the tested mouthrinsemay lead to some improvements in clinical parameters in patients irradiated for head-and-neck cancer (AU)

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Azithromycin as an adjunct to scaling and root planing in the treatment of Porphyromonas gingivalis-associated periodontitis: a pilot study.

OBJECTIVE: To evaluate the clinical and microbiological effects of systemic azithromycin as an adjunct to scaling and root planing (SRP) in the treatment of Porphyromonas gingivalis-associated chronic periodontitis. METHODS: Twenty-nine patients harbouring P. gingivalis were randomized into test and placebo groups. Test patients received SRP plus 500 mg of azithromycin per day (3 days), and control patients received SRP plus placebo. Clinical [plaque and bleeding indexes, probing pocket depth (PPD), clinical attachment level (CAL)] and microbiological data (four-sites pooled samples, processed by culture) were collected at baseline, and 1, 3 and 6 months, post-therapy. Clinical variables were compared by anova, and microbiological variables by chi-square, signed-rank and Wilcoxon tests. RESULTS: Fifteen test and 11 placebo patients completed the study. Mean PPD decreased 0.34 mm [95% confidence interval (CI) 0.19-0.49] in the placebo and 0.80 mm (CI 0.57-1.04) in the test group after 6 months. For mean CAL gain, the correspondent figures were 0.29 (CI 0.08-0.49) and 0.76 (CI 0.46-1.05), respectively. The frequency of detection of P. gingivalis decreased significantly (p0.01) in the test group after 1, 3 and 6 months. CONCLUSIONS: Within the limitations of this study, the adjunctive use of systemic azithromycin in the treatment of P. gingivalis periodontitis demonstrated significant clinical and microbiological benefits when compared with SRP plus placebo.

Mucositis in irradiated cancer patients: effects of an antiseptic mouthrinse.

OBJECTIVE: To assess the effects of an antiseptic, non-alcohol based mouth-rinse containing chlorhexidine and cetylpyridinium chloride, in preventing the oral complications associated to radiation therapy in head-and-neck cancer patients. STUDY DESIGN: This was a parallel, double blind, prospective, randomized clinical trial. Cancer patients were randomly assigned to one of the two treatments (test mouth-rinse or a placebo). Three visits were scheduled (baseline, 14 and 28 days). Different outcome variables were evaluated: mucositis, plaque and gingival indices, stimulated saliva and salivary pH. RESULTS: 70 patients were screened and 36 were included. The presence and the degree of mucositis significantly increased in both groups and no significant differences were detected between groups, although the median increase in the placebo group (1.81) at 2 weeks was higher than in the test group (1.20). CONCLUSIONS: Within the limitations of the small sample size, this study suggests that the use of the tested mouth-rinse may lead to some improvements in clinical parameters in patients irradiated for head-and-neck cancer.

Influence of sampling strategy on microbiologic results before and after periodontal treatment.

BACKGROUND: The aim of this study was to test whether different sampling strategies would influence the microbiologic outcomes by assessing the bacterial load and composition of the subgingival microbiota by means of anaerobic culturing before and after periodontal treatment. METHODS: The first study (1 versus 4 [1vs4]) included 33 patients with generalized chronic periodontitis. Two sampling strategies were compared, sampling one site from the deepest pocket in the mouth (M1) versus a pooled sample of four sites from the deepest pockets in each quadrant (M4). The second study (2 versus 4 [2vs4]) included 20 patients with generalized chronic periodontitis. The strategy M4 was compared to a pooled sample of two non-adjacent sites in the same quadrant (M2). All samples were processed identically by means of anaerobic culturing. In both studies, a pretreatment sampling was taken. However, in the second study (2vs4), subgingival samples were also taken at 1, 3, and 6 months after periodontal therapy. Quantitative data were compared between strategies by means of t test and signed-rank test; qualitative data were compared by means of 2 x 2 contingency tables. RESULTS: Pretreatment samples showed that total anaerobic counts were significantly higher for M4 compared to M1 (P <0.001) and M2 (P = 0.025). However, there were no significant differences in regard to percentage of microbiota and counts for each pathogen. Most of the qualitative differences between strategies were caused by false negatives in M1 and M2. Post-treatment samples showed a reduction in total counts and a limited impact in the frequency of detection of periodontal pathogens. M2 detected a significant decrease in the frequency of detection of Porphyromonas gingivalis, which was not confirmed by the M4 strategy. CONCLUSION: The criteria of selection and the number of sites selected when sampling the subgingival biofilm in patients with generalized chronic periodontitis may influence the detection and quantitation of periodontal pathogens when evaluated by culture especially after treatment.

A combined therapeutic approach to manage oral halitosis: a 3-month prospective case series.

BACKGROUND: Clinical research assessing different therapeutic protocols aimed at treating oral halitosis is scarce. The aim of this study was to evaluate the effects of a combined mechanical and pharmacological approach to treat oral halitosis on clinical and microbiological outcomes on patients followed for 3 months. METHODS: Nineteen subjects with oral malodor participated. At baseline, all subjects completed a questionnaire and carried out an examination including full-mouth organoleptic and volatile sulfur compound (VSC) levels and the Winkel tongue coating index. Standard periodontal outcome variables were assessed at six teeth. Standardized microbiological samples of subgingival plaque, unstimulated saliva, and tongue coating were obtained for culture analysis. The treatment protocol included supragingival prophylaxis; instructions in oral hygiene (toothbrushing, interproximal cleaning, and tongue scraping); and gargling with a mouthrinse containing chlorhexidine, cetylpiridinium chloride, and zinc lactate. The same outcome variables were registered 1 and 3 months after baseline. RESULTS: Statistically significant reductions in organoleptic scores (P <0.001), VSC levels (P <0.05), and tongue coating index (P <0.05) were observed after 1 and 3 months. Mean probing depth and plaque levels also demonstrated significant reductions after 3 months (P <0.05). Total anaerobic counts were significantly reduced at all three locations after 1 month (P <0.05), and in samples from tongue coating and subgingival plaque at 3 months (P <0.05). Aerobic counts were significantly reduced in saliva at 1 month (P <0.05), and the anaerobic/aerobic ratio significantly increased in the tongue samples. Among the selected pathogens evaluated, Porphyromonas gingivalis was the most affected of the three microflora evaluated. CONCLUSIONS: The evaluated therapeutic approach demonstrated its efficacy in the management of oral halitosis, demonstrating statistically significant improvements in both organoleptic and VSC values at 1 and 3 months. The proposed clinical protocol significantly affected the microbial composition in tongue coating, saliva, and subgingival microflora.

Comparative effects of different chlorhexidine mouth-rinse formulations on volatile sulphur compounds and salivary bacterial counts.

AIM: To compare five different commercial mouth rinses with chlorhexidine (CHX) with respect to their anti-halitosis effect and anti-microbial activity on salivary bacterial counts, following a standardised research protocol. And secondly, to validate the study model proposed in the evaluation of patients suffering from halitosis. PATIENTS AND METHODS: Ten volunteers, with a healthy oral status, were enrolled in a double-blind, cross-over design, using sterile saline as negative control and five CHX-containing mouth rinses: 0.12% CHX alone (CHX+NO), plus alcohol (CHX+ALC), plus 0.05% cetylpiridinium chloride (CHX+CPC), plus sodium fluoride (CHX+NaF), and 0.05% CHX plus 0.05% CPC, plus 0.14% zinc lactate (CHX+Zn). The levels of whole-mouth volatile sulphur compounds (VSCs) were measured by means of a sulphide monitor at baseline, 1 and 5 h after rinsing with the assigned product. Baseline measurements also included an organoleptic assessment and the recording of the tongue-coating index. Aerobic and anaerobic salivary bacterial counts were also obtained by collecting unstimulated saliva samples at the same evaluation times, and processed by culturing techniques. Analysis of variance was used to evaluate whether significant differences existed among groups, at each evaluation point, or in changes between evaluations. RESULTS: No significant differences were detected at baseline, with VSC levels ranging between 190 and 227 parts per billion (p.p.b.) After rinsing, VSC levels were reduced with all products (except saline), after 1 h. Significant differences at 1 h were detected (p=0.04), corresponding to a lower amount of p.p.b. (109) in (CHX+Zn) as compared with the other groups (except CHX+NO). At 5 h, VSC levels were lower for CHX+CPC and CHX+Zn (155 and 169, respectively), while the other groups showed levels higher than 220 p.p.b. With respect to aerobic salivary bacterial counts, CHX+CPC demonstrated the lowest percentage of survival (6% after 1 h and 18% after 5 h). For anaerobic bacterial counts, again CHX+CPC demonstrated the lowest percentage of survival (10% at 1 h and 23% at 5 h), together with CHX+ALC (18% of survival at 5 h). However, salivary counts and VSCs were only significantly correlated at baseline, but not after treatment. CONCLUSION: Important differences can be expected from different CHX formulations, in relation to both their anti-halitosis effect and anti-microbial activity in saliva. Formulations that combine CHX and CPC achieved the best results, and a formulation combining CHX with NaF resulted in the poorest.

A randomized clinical trial on the short-term clinical and microbiological effects of the adjunctive use of a 0.05% chlorhexidine mouth rinse for patients in supportive periodontal care.

OBJECTIVE: To evaluate the clinical and microbiological activity of a new mouth rinse formulation, used as an adjunct to oral hygiene, for patients in supportive periodontal care. PATIENTS AND METHODS: This was a randomized, placebo-controlled clinical trial with two groups: test group, rinsing twice per day with the test product (with 0.05% chlorhexidine and 0.05% cetylpyridinium chloride); and control group, rinsing with a placebo. Treated chronic periodontitis patients were included, and two visits were rendered, baseline, and after 15 days. Clinical outcome variables included plaque and gingival indices, and probing pocket depth. Subgingival samples were processed by culturing. Patient-based variables and adverse effects were also assessed. Outcome variables were compared by t-test, chi2, and Mann-Whitney test. RESULTS: The results belonged to 33 patients. Plaque and gingival indices, and the log of bacterial total counts were reduced in the test group (p < or = 0.01), but differences between groups were only statistically significant (p < 0.05) for plaque and bacterial counts. A significant reduction in the proportions of flora (p < 0.05) and frequency of detection (p = 0.01) of Porphyromonas gingivalis was observed in the test group. CONCLUSIONS: The newly formulated mouth rinse demonstrated short-term plaque-inhibitory activity. This was associated with a reduction in the total load of anaerobic subgingival microflora.