Adjunctive Steps for Disinfection of Teeth With Primary Endodontic Infections

Analysis of Metabolically Active Bacteria After Adjunctive Steps for Disinfection of Teeth With Primary Endodontic Infections: RNA- and DNA-based Molecular Study

This study aims to evaluate an endodontic treatment protocol using adjunctive steps for disinfection of teeth with primary endodontic infections.

Study Overview

• Status: Completed
• Conditions: Bacterial Infections; Periapical Periodontitis
• Intervention / Treatment: Procedure: Root canal preparation; Procedure: Intracanal medication

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 Type: Interventional
• Enrollment (Actual): 45
• Phase: Not Applicable

Contacts and Locations

Study Locations

• Brazil
  • São Paulo, Brazil, 05508000
    • University of Sao Paulo

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 18 years to 65 years (ADULT, OLDER_ADULT)
• Accepts Healthy Volunteers: No
• Genders Eligible for Study: All

Description

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.

Study Plan

How is the study designed?

Design Details

• Primary Purpose: TREATMENT
• Allocation: NON_RANDOMIZED
• Interventional Model: SINGLE_GROUP
• Masking: NONE

Number of Arms

2

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
EXPERIMENTAL: Total bacteria analysis after 1rst- and 2nd-visit procedures; DNA levels and activity (RNA/DNA ratio) of total bacteria after the first-visit procedures (root canal preparation with Reciproc System and NaOCl 2.5%, followed by final irrigation protocol using activation techniques: XP Endo-Finisher and ultrasonic activation) and the second-visit protocol (intracanal medication with calcium hydroxide paste, followed by an 2nd-visit root canal preparation). Additionally, the composition of the active microbiome will be assessed by Next Generation Sequencing (NGS) analysis of the root canal samples, and the success rate (apical repair) of the endodontic treatment after 1 follow-up period will be assessed by an intraoral radiograph and cone beam computed tomography (CBCT) analyses.	Procedure: Root canal preparation; First endodontic treatment session includes the root canal preparation with Reciproc System and NaOCl 2.5%, followed by final irrigation protocol using activation techniques: XP Endo-Finisher and ultrasonic activation.; Procedure: Intracanal medication; The second endodontic treatment includes the intracanal medication with calcium hydroxide paste, followed by an 2nd-visit root canal preparation
OTHER: Bacterial species analysis after root canal preparation; DNA levels and activity (RNA/DNA ratio) of Bacteroidaceae sp. 272 , Cutibacterium acnes, Selenomonas spp., and Enterococcus faecalis after root canal preparation.Additionally, the success rate (apical repair) of the endodontic treatment after 1 follow-up period will be assessed by an intraoral radiograph and cone beam computed tomography (CBCT) analyses.	Procedure: Root canal preparation; First endodontic treatment session includes the root canal preparation with Reciproc System and NaOCl 2.5%, followed by final irrigation protocol using activation techniques: XP Endo-Finisher and ultrasonic activation.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Metabolic Activity of Persistent Bacteria	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.	After 14 day treatment period

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Bacterial Levels	Quantitative data of total bacteria, Bacteroidaceae sp. HOT-272, Cutibacterium acnes, Selenomonas spp. and Enterococcus faecalis determined by DNA-based qPCR (quantitative polymerase chain reaction).	After 14 day treatment period
Bacterial Community Diversity	Composition of the active microbiome by Next Generation Sequencing (NGS) analysis of the root canal samples.	After 14 day treatment period

Other Outcome Measures

Outcome Measure	Measure Description	Time Frame
Endodontic Success	Absence of clinical signs/ symptoms and reduction/ absence of apical radiolucency in radiologic examination	1 year after endodontic treatment

Collaborators and Investigators

• Sponsor: University of Sao Paulo
• Investigators: Principal Investigator: Ericka T Pinheiro, PhD, University of Sao Paulo

Study record dates

Study Major Dates

• Study Start (ACTUAL): August 4, 2017
• Primary Completion (ACTUAL): May 29, 2020
• Study Completion (ACTUAL): May 29, 2020

Study Registration Dates

• First Submitted: May 15, 2018
• First Submitted That Met QC Criteria: May 15, 2018
• First Posted (ACTUAL): May 25, 2018

Study Record Updates

• Last Update Posted (ACTUAL): October 8, 2020
• Last Update Submitted That Met QC Criteria: September 14, 2020
• Last Verified: September 1, 2020

More Information

Terms related to this study

• Keywords: Bacterial Infections; Calcium Hydroxide; Real-Time Polymerase Chain Reaction; Periapical Periodontitis; Root Canal Preparation; RNA-Directed DNA Polymerase
• Additional Relevant MeSH Terms: Pathologic Processes; Disease Attributes; Stomatognathic Diseases; Periodontal Diseases; Mouth Diseases; Bacterial Infections and Mycoses; Jaw Diseases; Periapical Diseases; Periodontitis; Infections; Communicable Diseases; Periapical Periodontitis; Bacterial Infections
• Other Study ID Numbers: FAPESP 2016/15473-0

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No