Estabelecimento de protocolos de diagnóstico de agentes microbianos associados à periodontite

Abstract

Chronic periodontal diseases are one of the infectious conditions with bacterial etiology more prevalent in all mankind, mainly caused by pathogenic bacteria that grow in biofilms. The identification of specific microorganisms such as Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, can contribute to determine the risk of developing periodontal disease; to know the composition of the biofilm in periodontal health and also understand the behavior of the microflora after periodontal therapy. The present work aims to verify the feasibility of conducting an additional diagnostic exam in the UCP laboratories. This exam consists in the microbiological analysis of the presence of 2 microbial agents associated with periodontitis. The feasibility test of this exam consisted in the evaluation of procedures for sample collection, procedures for initial DNA extraction, PCR reaction procedures for results optimization, evaluation of the analysis requesters satisfaction about the collection procedures and use of results in periodontal diagnosis. To achieve the objectives proposed, a diagnostic protocol of microbial agents associated with periodontitis was developed, which included several stages from the elaboration of the request sheet, collection protocol, DNA protocol extraction and PCR protocol specific for each microorganism. To test the feasibility of the protocols, 59 samples, collected from 20 patients consulting the periodontology MIMD of CPU were analyzed. The operators, who participated in collecting these samples, completed a questionnaire that allowed analyzing globally the protocol provided and its usefulness in terms of the academic community of the CDU. We were unable to prove the feasibility of the protocol in the laboratories of the CPU since all samples proved negative for the two microorganisms. We considered some factors that could justify the fact that all samples were negative including: operation of the PCR reaction (by adding a positive control in all samples), presence of DNA (by agarose gel visualization of all samples after the extraction process), verification of the origin of DNA samples (by applying universal bacterial primers).The results of each of these assessments lead to some changes in the procedures of collecting samples and DNA isolation. Justifications are given for the results and suggested further changes to future investigations of the presence of these microorganisms in periodontal pockets.

Date of Award	Sept 2011
Original language	Portuguese
Awarding Institution	Universidade Católica Portuguesa
Supervisor	Maria José Correia (Supervisor) & Nuno Malta dos Santos (Co-Supervisor)