Analysis of Metabolically Active Bacteria After Adjunctive Steps for Disinfection of Teeth With Primary Endodontic Infections: RNA- and DNA-based Molecular Study
Trial Snapshot
- Phase
- Not Applicable
- Status
- Completed
- Sponsor
- University of Sao Paulo
- Enrollment
- 45
- Locations
- 2
- Primary Endpoint
- Metabolic Activity of Persistent Bacteria
Study Overview
Brief Summary
This study aims to evaluate an endodontic treatment protocol using adjunctive steps for disinfection of teeth with primary endodontic infections.
Detailed Description
The success of endodontic treatment in teeth with necrotic pulp and apical periodontitis depends on the reduction of viable microorganisms of root canals by endodontic disinfection procedures. This study aims to evaluate by molecular methods the effect of different disinfection procedures on reduction, diversity and microbial metabolic activity. Patients with necrotic pulps and apical periodontitis in single-rooted teeth will be selected. Microbiological samples will be taken from root canals after access cavity (S1), after chemomechanical preparation with Reciproc System and 2.5% NaOCl (S2), after irrigant activation using the XP-Endo Finisher instrument (S3a) and ultrasonic activation (S3b); and after intracanal medication with calcium hydroxide for 14 days (S4), followed by 2nd-visit root canal preparation (S5). DNA and RNA will be extracted from root canal samples, and complementary DNA (cDNA) synthetized using reverse transcription reaction. The effect of treatment protocols on total microbiota levels will be determined by DNA-based quantitative polymerase chain reaction (qPCR) using universal primers for Bacteria domain. RNA of root canal samples will be used to determine the diversity of metabolically active microbiota by Reverse Transcriptase Reaction (RT), followed by amplification (PCR) and high throughput sequencing of the hypervariable region V4-V5 of 16S rRNA gene. The metabolic activity of the most prevalent species / taxa will be calculated by rRNA- and DNA-based molecular methods. Ratios between rRNA and DNA levels will be calculated to search for active bacteria (rRNA/DNA ≥ 1) in root canal samples. Data will be analyzed by statistical tests, with 5% significance level.
Study Design
- Study Type
- Interventional
- Allocation
- Non Randomized
- Intervention Model
- Single Group
- Primary Purpose
- Treatment
- Masking
- None
Eligibility Criteria
- Ages
- 18 Years to 65 Years (Adult, Older Adult)
- Sex
- All
- Accepts Healthy Volunteers
- No
Inclusion Criteria
- •Teeth with necrotic pulps and asymptomatic apical periodontitis
Exclusion Criteria
- •patients who had received antibiotics during the previous 3 months or had any general disease,
- •teeth that could not be properly isolated with rubber dam,
- •non-restored teeth,
- •periodontal pockets depths greater than 4 mm,
- •previous endodontic treatment,
- •open apex,
- •crown/root fracture
- •root resorption or calcifications.
Outcomes
Primary Outcomes
Metabolic Activity of Persistent Bacteria
Time Frame: After 14 day treatment period
The metabolic activity (rRNA/DNA ratio) of total bacteria, Bacteroidaceae sp. 272, Cutibacterium acnes, Selenomonas spp. and Enterococcus faecalis in root canal samples taken before and after endodontic procedures.
Secondary Outcomes
- Bacterial Community Diversity(After 14 day treatment period)
- Bacterial Levels(After 14 day treatment period)
Investigators
Ericka Tavares Pinheiro
Professor
University of Sao Paulo