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.

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.

Periapical Periodontitis, Bacterial Infections, Root Canal Preparation, Calcium Hydroxide, Real-Time Polymerase Chain Reaction, RNA-Directed DNA Polymerase

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.

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.

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.

The second endodontic treatment includes the intracanal medication with calcium hydroxide paste, followed by an 2nd-visit root canal preparation

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.

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).

Composition of the active microbiome by Next Generation Sequencing (NGS) analysis of the root canal samples.

Absence of clinical signs/ symptoms and reduction/ absence of apical radiolucency in radiologic examination

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.