Microbiological testing of clinical samples before and after periodontal treatment. A comparative methodological study between real-time PCR and real-time-PCR associated to propidium monoazide.

OBJECTIVES: The aim of the present methodological study was to evaluate the discrepancies in the detection of a number of periodontally involved pathogenic bacteria obtained from clinical samples by two methods: the quantitative Polymerase Chain Reaction (qPCR) and the qPCR combined with pre-treatment by Propidium Monoazide (PMA). MATERIAL AND METHODS: Plaque and saliva samples were obtained from 30 subjects: 20 subjects with chronic or aggressive periodontitis in need of periodontal therapy with or without antibiotics and 10 subjects in Supportive Periodontal Treatment (SPT). The clinical samples taken before treatment (BL) and 1 month later (M1), were divided in two aliquots: one was immediately treated with PMA while the other was left untreated. All samples were further analyzed with qPCR after DNA extraction, for the detection of Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf), Treponema denticola (Td), Parvimonas micra (Pm), and Prevotella intermedia (Pi). RESULTS: Large inter-individual variations were observed in the concentration of the studied bacteria. At both instances (BL and M1) and for the three groups, significantly lower counts of bacteria were depicted when plaque and saliva samples were pre-treated with PMA as compared to those without treatment. Treatment resulted in significant decreases in the number of bacteria, mainly in the plaque samples. However, these changes were almost similar in the three groups independently of the method of detection used (PMA-qPCR vs. q-PCR). CONCLUSION: Removal of DNA from non-viable cells with PMA treatment is an easily applied step added to the classical qPCR that could give accurate information on the presence of viable bacterial load and evaluate the response to periodontal treatment.

Gingival crevicular fluid biomarkers in type 1 diabetes mellitus: A case-control study.

OBJECTIVE: The aim of the study is to compare the levels of Gingival Crevicular Fluid (GCF) interleukin 8 (IL-8), matrix metalloproteinase 8 (MMP-8) and advanced glycated-end products (AGEs) in a cohort of type 1 diabetic (T1D) subjects and healthy controls. MATERIAL AND METHODS: GCF samples and periodontal examination were assessed in 50 subjects with T1D (30 males and 20 females; mean age: 35.2 years) recruited from the Diabetology Unit of the Geneva University Hospitals and in 50 control subjects matched for gender, age and smoking status. Samples were assessed for IL-8 and MMP-8 using a bead array multianalyte detection system and for AGEs the ELISA. The two groups were compared using the Wilcoxon signed rank test. RESULTS: The mean HbA1c differed significantly between the groups (8.3% for the T1D group vs. 5.2% for the control group, p < 0.001). T1D subjects had significantly more plaque and gingival inflammation and presented more sites with bleeding on probing compared to the controls. The GCF levels of IL-8, MMP-8 and AGEs did not differ significantly between the groups. Further analysis of the GCF markers in younger (<40 years) and older (≥40 years) cohorts, revealed no significant differences between younger diabetics and controls or between older diabetics and controls. When the groups were divided according to their glycemic status (HbA1c 6.1-8, and > 8%), again no significant differences could be identified for any of the biochemical markers. CONCLUSIONS: T1D subjects, particularly the younger ones, exhibited more inflammation compared to the matched healthy controls. Results on the GCF expression of IL-8, MMP-8 and AGEs did not differ between the groups. The diabetic population of our cohort was for the most part fairly-controlled, with little if any complications and with presence of only mild type of periodontal disease, as 68% had gingivitis.