qPCR Detection and Quantification of Aggregatibacter actinomycetemcomitans and Other Periodontal Pathogens in Saliva and Gingival Crevicular Fluid among Periodontitis Patients.

OBJECTIVE: The detection of special bacterial species in patients with periodontitis is considered useful for clinical diagnosis and treatment. The aim of this study was to investigate the presence of specific periopathogens and investigate whether there is a correlation between the results of different bacterial species in whole saliva and pooled subgingival plaque samples (healthy and diseased sites) from individuals with periodontitis and periodontally healthy subjects. MATERIALS AND METHODS: In total, 52 patients were recruited and divided into two groups: non-periodontitis and periodontitis patients. For each group, the following periodontal pathogens were detected using real-time polymerase chain reaction: A. actinomycetemcomitans JP2 clone, A. actinomycetemcomitans non JP2 clone, Porphyromonasgingivalis, and total eubacteria. RESULTS: Higher levels of the various studied bacteria were present in both saliva and plaque samples from the periodontitis group in comparison to non-periodontitis subjects. There were significant differences in P. gingivalis and A. actinomycetemcomitans JP2 clones in the saliva of periodontitis patient compared to the control group. Subgingival plaque of diseased sites presented a significant and strong positive correlation between A. actinomycetemcomitans and P. gingivalis. In saliva samples, there was a significant positive correlation between A. actinomycetemcomitans JP2 clone and P. gingivalis (p ≤ 0.002). CONCLUSION: Quantifying and differentiating these periodontal species from subgingival plaque and saliva samples showed a good potential as diagnostic markers for periodontal disease. Regarding the prevalence of the studied bacteria, specifically A. actinomycetemcomitans JP2 clone, found in this work, and the high rate of susceptibility to periodontal species in Africa, future larger studies are recommended.

The challenge of periimplantitis management: a case report.

INTRODUCTION: Implant therapy has changed tremendously the management of edentulism, and are a real alternative to conventional prosthesis. However, with the constant increase of the number of peri-implantitis, the question of therapeutic management arises. The first goal of the treatment is to control the infection, which requires periodontal debridement and decontamination of the implant surface. Secondary objectives are to promote bone regeneration and repair of the attachment around the implant. CASE REPORT: The patient was a 24-years-old female in good general health with no history of smoking. Intraoral and radiological examination revealed a peri-implantitis around the implant at the site of tooth 14. The peri-implantitis management involved a non-surgical treatment to reduce inflammation, followed by surgical treatment of the bone defect. This combined decontamination of the implant surface and bone regeneration. CONCLUSION: The heterogeneity of the surgical protocols does not suggest any true recommendations. Several treatments for periimplantitis are currently implemented. However, we still lack relevant data regarding the effectiveness of these therapies.

Occurrence of Candida albicans in Periodontitis.

BACKGROUND: Periodontal diseases are the result of an imbalance between the microbiota and immune defense. The role of yeast in the pathogenesis of these diseases has been studied. This study aims to assess the occurrence of Candida albicans in periodontitis. MATERIALS AND METHODS: Fifty subjects were recruited for the study (15 healthy individuals and 35 periodontitis subjects). The periodontal examination and plaque sampling were carried out for all patients. Candida albicans identification was based on culture, direct examination, and polymerase chain reaction. The statistical analysis was performed by SPSS 20 (SPSS Inc., Chicago, IL, USA). RESULTS: Twenty percent of the diseased group harbored Candida albicans which was slightly higher than in the healthy group (7%), suggesting that, under normal conditions, yeast does not grow easily in subgingival sites. However, no significant difference between the healthy and periodontitis groups (p=0.23) was found. Our results also indicated that the presence of Candida albicans was neither gender nor age related in the studied groups. CONCLUSION: The results of this study suggest that Candida albicans occurs in periodontitis. More studies are needed to clarify the potential role of this yeast in different stages and forms of the disease.

Association between periodontitis and the Mediterranean diet in young Moroccan individuals.

BACKGROUND & OBJECTIVES: Adherence to the Mediterranean diet (MedDiet) has been reported to be associated with a lower risk of various chronic diseases. This cross-sectional study aimed to investigate the potential association between adherence to the MedDiet and periodontitis, which is highly prevalent in young Moroccan individuals. METHODS: We evaluated 1075 Moroccan individuals (72% women, mean [standard deviation] age = 20.2 [1.5] years). Adherence to the MedDiet was assessed using the MedDiet score (MDS) based on the frequency of intake of eight food groups (vegetables, legumes, fruits, cereals or potatoes, fish, red meat, dairy products, and olive oil). A value of 0 (unhealthy) or 1 (healthy) was assigned to each food group, and the MDS (range, 0-8 points) was generated by adding the individual scores, with a higher score indicating better adherence to the MedDiet. The logistic regression model was used to evaluate the MDS (high [5-8 points]/low [0-4 points]) and each component score (1/0) with the presence of periodontitis, which was determined through full-mouth periodontal examinations. Age, sex, and oral health behavior were considered as potential confounders. RESULTS: In total, 693 (64.5%) study participants showed high MDSs. Periodontitis was observed in 71 (6.6%) participants. No significant association between MDS and periodontitis was observed. Nonetheless, olive oil consumption, a component of the MDS, showed a significant inverse association with periodontitis (adjusted odds ratio = 0.55; 95% confidence interval, 0.32-0.96). CONCLUSIONS: The MedDiet was not significantly associated with periodontitis among young Moroccans. However, frequent consumption of olive oil may have a protective effect against periodontitis, although the temporal association needs to be clarified in further studies.

High salivary levels of JP2 genotype of Aggregatibacter actinomycetemcomitans is associated with clinical attachment loss in Moroccan adolescents.

It has previously been shown that the presence of Aggregatibacter actinomycetemcomitans in subgingival plaque is significantly associated with increased risk for clinical attachment loss. The highly leukotoxic JP2 genotype of this bacterium is frequently detected in adolescents with aggressive forms of periodontitis. The aims of the study were to quantify the levels of JP2 and non-JP2 genotypes of A. actinomycetemcomitans in saliva of Moroccan adolescents with the JP2 genotype earlier detected in the subgingival plaque. The salivary concentrations of inflammatory proteins were quantified and linked to the clinical parameters and microbial findings. Finally, a mouth rinse with leukotoxin-neutralizing effect was administrated and its effect on the levels the biomarkers and A. actinomycetemcomitans examined. The study population consisted of 22 adolescents that previously were found to be positive for the JP2 genotype in subgingival plaque. Periodontal registration and sampling of stimulated saliva was performed at baseline. A mouth rinse (active/placebo) was administrated, and saliva sampling repeated after 2 and 4 weeks rinse. The salivary levels of JP2 and non-JP2 were analyzed by quantitative PCR and inflammatory proteins by ELISA. Both the JP2 and the non-JP2 genotype were detected in all individuals with significantly higher levels of the non-JP2. Enhanced levels of the JP2 genotype of A. actinomycetemcomitans was significantly correlated to the presence of attachment loss (≥3 mm). Salivary concentrations of inflammatory biomarkers did not correlate to periodontal condition or levels of A. actinomycetemcomitans. The use of active or placebo leukotoxin-neutralizing mouth rinse did not significantly interfered with the levels of these biomarkers. Saliva is an excellent source for detection of A. actinomycetemcomitans on individual basis, and high levels of the JP2 genotype were significantly associated with the presence of clinical attachment loss.

Use of traditional plants in management of halitosis in a Moroccan population.

INTRODUCTION: The use of medicinal plants was a very spread therapeutic way. At present, several studies are moving toward this ancestral option, seen the emergence of several bacterial resistance and for the large number of side effects of some synthetic drugs. OBJECTIVE: The objective of this study was to collect and evaluate information on medicinal plants commonly used in five Moroccan cities: Rabat, Salé, Témara, Khémisset, and Tiflet for the management of halitosis. METHODS: This is a cross-sectional survey; conducted among 171 herbalists. The tool of the study was a questionnaire filled by herbalists. SPSS in its version 13 was used for statistical calculations. Quantitative variables were expressed as a mean and standard deviation. Categorical variables were expressed as numbers and percentage. RESULTS: Analysis of the results of this study identified 23 plants that are used the most. The herbal knowledge herbalists prescribed on the toxicity of plants and their side effects were appreciated. CONCLUSIONS: Preliminary results presented in this work allow knowing the plants used by this population. This data could be the basis for experimental and clinical studies to promote the use of natural agents in the treatment of bad breath.

[Oral hygiene in Moroccan school children and their mothers]. / Hygiène bucco-dentaire d’enfants scolarisés marocains et de leurs mères.

OBJECTIVE: Parents play an important role in improving children's oral plaque control. The aim of this study was therefore to compare the oral hygiene of children and their mothers and to study the relationship with their oral health status. MATERIALS AND METHODS: Information was collected on oral hygiene habits (brushing, frequency and duration, equipment and methodology, frequency of changing toothbrushes) and various indices (CAF, plaque, bleeding). RESULTS: The sample was composed of 200 children and 200 mothers. The results of this study showed no statistically significant difference between mothers and their children in terms of the method of brushing and the frequency of changing toothbrushes. A statistically significant difference was found between mothers and their children concerning the use and change of toothbrushes and brushing frequency. A highly significant positive correlation was observed between the plaque index of mothers and their children. CONCLUSION: Low rates of toothbrush use were observed in this population and the brushing method was very inefficient. The correlation between the plaque index of mothers and their children suggests that mother's oral hygiene behaviours influence their children's oral health.

The JP2 genotype of Aggregatibacter actinomycetemcomitans and marginal periodontitis in the mixed dentition.

AIM: To perform a cross-sectional study on the carrier frequency of JP2 and non-JP2 genotypes of A. actinomycetemcomitans in Moroccan school children and relate the presence of these genotypes to the periodontal status in the mixed dentition. MATERIAL AND METHODS: A plaque sample from 513 children was analysed by PCR. JP2 genotype-positive subjects (n = 46), an equally sized group of non-JP2 genotype-positive subjects, and an A. actinomycetemcomitans-negative group were randomly chosen among the remaining subjects for clinical and radiographic examination. RESULTS: Among 513 children, 46 (9.0%) carried the JP2 genotype and 186 (36.3%) were positive for non-JP2 genotypes, whereas A. actinomycetemcomitans could not be detected in the remaining 281 subjects. Among 75 subjects with mixed dentition and selected for clinical examination, clinical attachment loss (CAL) ≥ 3 mm at two or more periodontal sites on primary teeth was found in the JP2 genotype-positive group only. In total, 6.7% of subjects with primary teeth present showed CAL ≥ 3 mm at two or more sites. CONCLUSIONS: The carrier frequency of the JP2 genotype of A. actinomycetemcomitans was at a comparable level to frequencies previously found in Moroccan adolescent populations. Clinical attachment loss, manifesting already in the primary dentition, was found only in the group of Moroccan children carrying the JP2 genotype of A. actinomycetemcomitans.

Bacterial profile of aggressive periodontitis in Morocco: a cross-sectional study.

BACKGROUND: Aggressive periodontitis (AgP) is one of the most severe forms of periodontal diseases. In Morocco, Aggregatibacter actinomycetemcomitans has been strongly associated with AgP, however limited knowledge is available about the implication of other periodontal pathogens in this entity. Therefore, the main aim of this study was to evaluate the composition of the subgingival microbiota in Moroccan patients with AgP. METHODS: Subgingival plaque samples were collected from 50 aggressive, 13 localized and 37 generalized periodontitis patients. Samples from 20 chronic periodontitis (ChP) patients were taken as controls. Samples collected from the four deepest periodontal pockets in each patient were pooled in pre-reduced transport fluid and examined by culture. RESULTS: A. actinomycetemcomitans was significantly more frequent (p = 0.004) in generalised AgP compared to ChP, and Porphyromonas gingivalis was less prevalent in localized AgP, when compared with generalized AgP (p = 0.040) or ChP (p = 0.016). Prevotella intermedia, Fusobacterium nucleatum and Tannerella forsythia were also frequently detected in all groups. Mean proportions of A. actinomycetemcomitans were significantly higher in AgP groups, when compared to ChP, and generalized AgP patients harbored significantly higher proportions of P. gingivalis and T. forsythia, when compared to localized AgP or ChP. CONCLUSIONS: A. actinomycetemcomitans, P. gingivalis, T. forsythia, P. intermedia and F. nucleatum were frequently detected in this Moroccan population with AgP. Differences in frequency of detection, counts and proportions of A. actinomycetemcomitans, P. gingivalis and T. forsythia suggests the presence of distinct microbiological profiles for localized AgP, generalized AgP and ChP patients.

Quantitative discrimination of Aggregatibacter actinomycetemcomitans highly leukotoxic JP2 clone from non-JP2 clones in diagnosis of aggressive periodontitis.

BACKGROUND: Aggregatibacter actinomycetemcomitans is the etiological agent of periodontitis, and there is a strong association between clone JP2 and aggressive periodontitis in adolescents of African descent. The JP2 clone has an approximately 530-bp deletion (∆530) in the promoter region of the lkt/ltx gene, which encodes leukotoxin, and this clone has high leukotoxic activity. Therefore, this clone is very important in aggressive periodontitis. To diagnose this disease, culture methods and conventional PCR techniques are used. However, quantitative detection based on qPCR for the JP2 clone has not been developed due to genetic difficulties. In this study, we developed a qPCR-based quantification method specific to the JP2 clone. METHODS: Based on our analysis of the DNA sequence of the lkt/ltx gene and its flanking region, we designed a reverse primer specific for the ∆530 deletion border sequence and developed a JP2-specific PCR-based quantification method using this primer. We also analyzed the DNA sequence of the ∆530 locus and found it to be highly conserved (97-100%) among 17 non-JP2 strains. Using the ∆530 locus, we designed a qPCR primer-probe set specific to non-JP2 clones. Next, we determined the numbers of JP2 and non-JP2 clone cells in the periodontal pockets of patients with aggressive periodontitis. RESULTS: The JP2-specific primers specifically amplified the genomic DNA of the A. actinomycetemcomitans JP2 clone and did not react with other bacterial DNA, whereas the non-JP2 specific primers reacted only with A. actinomycetemcomitans non-JP2 clones. Samples from the 88 periodontal sites in the 11 patients with aggressive periodontitis were analyzed. The bacterial cell numbers in 88 periodontal sites ranged from 0 to 4.8 × 10(8) (mean 1.28 × 10(7)) for JP2 clones and from 0 to 1.6 × 10(6) for non-JP2 clones (mean 1.84 × 10(5)). There were significant differences in the JP2 cell number between a clinical attachment level (CAL) ≤6 mm and a level ≥7 mm (p < 0.01). Our new qPCR-based JP2- and non-JP2-specific quantitative detection assay is applicable to the diagnosis of aggressive periodontitis with A. actinomycetemcomitans. CONCLUSIONS: We successfully developed a quantitative and discriminative PCR-based method for the detection of A. actinomycetemcomitans JP2 and non-JP2 clones. This technique will contribute to future analyses of the quantitative relationship between this organism and aggressive periodontitis.

The highly leukotoxic JP2 clone of Aggregatibacter actinomycetemcomitans in localized and generalized forms of aggressive periodontitis.

OBJECTIVE: To investigate the presence of A. actinomycetemcomitans, including the highly virulent JP2 clone, in young adult patients with aggressive periodontitis, and associate the findings with the two forms of the disease. MATERIALS AND METHODS: Seventy Moroccan subjects with aggressive periodontitis, aged less than 35 years, were recruited. Among these, 41 had LAgP and 29 had GAgP. Plaque samples were collected from periodontal pockets and examined using a PCR that detects the presence of A. actinomycetemcomitans and which differentiates between JP2 and non-JP2 genotypes of the bacterium. RESULTS: total of 58 (83%) from the 70 AgP patients were positive for A. actinomycetemcomitans, among whom 77% were positives for the JP2 clone. The JP2 clone was detected in 34 (83%) of the LAgP patients compared to 20 (69%) of the GAgP patients (p = 0.17). Fourteen (20%) of the patients harbored non-JP2 genotypes of A. actinomycetemcomitans, although most of these patients (10/14) also harbored the JP2 clone. CONCLUSIONS: The presence of the JP2 clone of A. actinomycetemcomitans is strongly associated with both LAgP and GAgP in young adults in Morocco. This implies that treatment of AgP in this population should include microbiological screening and aim at eradication of the bacterium when present.

Proteomic and immunoproteomic analysis of Aggregatibacter actinomycetemcomitans JP2 clone strain HK1651.

The proteome of Aggregatibacter actinomycetemcomitans HK1651 (JP2 clone) and immunoreactive antigens were studied by two-dimensional (2D) gel electrophoresis, matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS), and 2D immunoblotting. The highly leukotoxic JP2 clone of A. actinomycetemcomitans is strongly associated with aggressive periodontitis (AgP) in adolescents of North-West African descent and the pathogenicity of this bacterium is of major interest. Hence, we developed a comprehensive 2D proteome reference map of A. actinomycetemcomitans proteins with 167 identified spots representing 114 different proteins of which 15 were outer membrane proteins. To unravel immunoreactive antigens, we applied 2D-gel and subsequent immunoblotting analyses using sera from five individuals with A. actinomycetemcomitans infections and one healthy control. The analysis revealed 32 immunoreactive proteins. Antibodies to two outer membrane proteins, YaeT (85 kDa) and Omp39 (39 kDa), not previously described as immunoreactive, were found only in subjects with current or previous A. actinomycetemcomitans JP2 infection. Further proteome-based studies of A. actinomycetemcomitans combined with analyses of the humoral immune response and targeted against outer membrane proteins may provide important insight into the host relationship of this important pathogen.

Risk of aggressive periodontitis in adolescent carriers of the JP2 clone of Aggregatibacter (Actinobacillus) actinomycetemcomitans in Morocco: a prospective longitudinal cohort study.

BACKGROUND: Periodontitis is a loss of supporting connective tissue and alveolar bone around teeth, and if it occurs in an aggressive form it can lead to tooth loss before the age of 20 years. Although the cause of periodontitis in general remains elusive, a particular clone (JP2) of the gram-negative rod Aggregatibacter (Actinobacillus) actinomycetemcomitans is considered a possible aetiological agent of the aggressive form in adolescents living in or originating from north and west Africa, where the disease is highly prevalent. We did a population-based longitudinal study of adolescents to assess the role of the JP2 clone in the initiation of aggressive periodontitis. METHODS: A total of 700 adolescents from public schools in Rabat, Morocco, were enrolled in the study. We used PCR to detect A actinomycetemcomitans in plaque samples (taken from molar and incisor sites) and to differentiate between the JP2 clone and other non-JP2 genotypes of the bacterium. 18 individuals were found to already have periodontitis and were excluded. The 682 periodontally healthy adolescents (mean age 12.5 years; SD 1.0) were classified according to their A actinomycetemcomitans carrier status at baseline. After 2 years, 428 (62.8%) individuals returned for re-examination, which included recording of periodontal attachment loss measured from the cemento-enamel junction to the bottom of the periodontal pockets of all teeth present. FINDINGS: Individuals who carried the JP2 clone of A actinomycetemcomitans alone (relative risk 18.0; 95% CI 7.8-41.2, p<0.0001) or together with non-JP2 clones of A actinomycetemcomitans (12.4; 5.2-29.9, p<0.0001) had a significantly increased risk of periodontal attachment loss. A much less pronounced disease risk was found in those carrying non-JP2 clones only (3.0; 1.3-7.1, p=0.012). INTERPRETATION: The JP2 clone of A actinomycetemcomitans is likely to be an important aetiological agent in initiation of periodontal attachment loss in children and adolescents. Co-occurrence of non-JP2 clones of A actinomycetemcomitans reduces the risk of development of periodontitis, suggesting competition for the ecological niche between the JP2 and non-JP2 clones of this species.

Improved PCR for detection of the highly leukotoxic JP2 clone of Actinobacillus actinomycetemcomitans in subgingival plaque samples.

The JP2 clone of Actinobacillus actinomycetemcomitans is associated with early-onset periodontitis in certain ethnic populations of African origin. Here, we describe and evaluate a set of primers for PCR to assay for the presence of A. actinomycetemcomitans and to discriminate between JP2-like strains and other genotypes in subgingival plaque samples.