Abstract
ObjectivesThe 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 methodsPlaque 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).ResultsLarge 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).ConclusionRemoval 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.
Highlights
For the detection and quantification of microbial pathogens in clinical specimens, culture techniques have long been considered the gold standard
This was a methodological study for optimized microbiological analysis of clinical samples before and after periodontal therapy
DNA is slowly degrading and amplifiable DNA may be present long after treatment (Brundin et al, 2010). This could explain the detection of periodontal pathogens after effective periodontal treatment in several studies, despite the clinical resolution of periodontitis (Cionca et al, 2010; Mombelli et al, 2017) and in patients in maintenance care (Moëne et al, 2010; Müller Campanile et al, 2015)
Summary
For the detection and quantification of microbial pathogens in clinical specimens, culture techniques have long been considered the gold standard. Clinical trials used these methods extensively to evaluate antimicrobial effects of periodontal therapy (Loomer, 2004). Culture techniques were found to be fairly reproducible and consistent in demonstrating reductions of bacterial counts after various types of periodontal treatment (Mombelli et al, 1989). Thorough analyses required advanced technical skills and specific equipment to assure the survival and growth of the microorganisms in vitro. Anaerobic bacteria that were thought to play important roles in periodontal diseases (Haffajee & Socransky, 1994) were especially difficult to cultivate. Bacteria that could not be grown under laboratory conditions were ignored (Loesche et al, 1992)
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