Periapical Periodontitis, Bacterial Infections
Conditions
Keywords
Periapical Periodontitis, Bacterial Infections, Root Canal Preparation, Calcium Hydroxide, Real-Time Polymerase Chain Reaction, RNA-Directed DNA Polymerase
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.
Interventions
PROCEDURERoot 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.
PROCEDUREIntracanal medication
The second endodontic treatment includes the intracanal medication with calcium hydroxide paste, followed by an 2nd-visit root canal preparation
Sponsors
University of Sao Paulo
Study design
Allocation
NON_RANDOMIZED
Intervention model
SINGLE_GROUP
Primary purpose
TREATMENT
Masking
NONE
Eligibility
Sex/Gender
ALL
Age
18 Years to 65 Years
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.
Design outcomes
Primary
| Measure | Time frame | Description |
|---|---|---|
| Metabolic Activity of Persistent Bacteria | 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
| Measure | Time frame | Description |
|---|---|---|
| Bacterial Community Diversity | After 14 day treatment period | Composition of the active microbiome by Next Generation Sequencing (NGS) analysis of the root canal samples. |
| Bacterial Levels | After 14 day treatment period | 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). |
Other
| Measure | Time frame | Description |
|---|---|---|
| Endodontic Success | 1 year after endodontic treatment | Absence of clinical signs/ symptoms and reduction/ absence of apical radiolucency in radiologic examination |
Countries
Brazil
Participant flow
Pre-assignment details
Samples from 20 patients were used for the analysis of total bacteria in 1st and 2nd visits. Additional 25 patients were necessary for the analysis of specific bacterial species in post-instrumentation samples (n=45 patients).
Participants by arm
| Arm | Count |
|---|---|
| Root Canal Preparation and Medication 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.
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.
Intracanal medication: The second endodontic treatment includes the intracanal medication with calcium hydroxide paste. | 45 |
| Total | 45 |
Baseline characteristics
| Characteristic | Root Canal Preparation and Medication |
|---|---|
| Age, Categorical <=18 years | 0 Participants |
| Age, Categorical >=65 years | 0 Participants |
| Age, Categorical Between 18 and 65 years | 45 Participants |
| Age, Continuous | 41 years |
| Number of participants with infected root canals | 45 Participants |
| Race (NIH/OMB) American Indian or Alaska Native | 0 Participants |
| Race (NIH/OMB) Asian | 0 Participants |
| Race (NIH/OMB) Black or African American | 0 Participants |
| Race (NIH/OMB) More than one race | 0 Participants |
| Race (NIH/OMB) Native Hawaiian or Other Pacific Islander | 0 Participants |
| Race (NIH/OMB) Unknown or Not Reported | 45 Participants |
| Race (NIH/OMB) White | 0 Participants |
| Region of Enrollment Brazil | 45 participants |
| Sex: Female, Male Female | 22 Participants |
| Sex: Female, Male Male | 23 Participants |
Adverse events
| Event type | EG000 affected / at risk |
|---|---|
| deaths Total, all-cause mortality | 0 / 0 |
| other Total, other adverse events | 0 / 0 |
| serious Total, serious adverse events | 0 / 0 |
Outcome results
Primary
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.
Time frame: After 14 day treatment period
Population: There was a lost of RNA samples from 5 cases.
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Total Bacteria Analysis After 1rst- and 2nd-visit Procedures | Metabolic Activity of Persistent Bacteria | Total bacteria at baseline (S1) | 4.748 RNA/DNA ratio | Standard Deviation 6.2904 |
| Total Bacteria Analysis After 1rst- and 2nd-visit Procedures | Metabolic Activity of Persistent Bacteria | Total bacteria after root canal Preparation (S2) | 2.57 RNA/DNA ratio | Standard Deviation 3.21 |
| Total Bacteria Analysis After 1rst- and 2nd-visit Procedures | Metabolic Activity of Persistent Bacteria | Total bacteria after adjunctive procedures (S3) | 2.614 RNA/DNA ratio | Standard Deviation 2.978 |
| Total Bacteria Analysis After 1rst- and 2nd-visit Procedures | Metabolic Activity of Persistent Bacteria | Total bacteria after intracanal medication (S4) | 4.0008 RNA/DNA ratio | Standard Deviation 4.374 |
| Total Bacteria Analysis After 1rst- and 2nd-visit Procedures | Metabolic Activity of Persistent Bacteria | Total bacteria after 2nd-visit preparation (S5) | 3.7875 RNA/DNA ratio | Standard Deviation 2.9727 |
| Bacterial Species Analysis After Root Canal Preparation | Metabolic Activity of Persistent Bacteria | Bacteroidaceae sp. HOT-272 at S1 | 1.136 RNA/DNA ratio | Standard Deviation 0.1318 |
| Bacterial Species Analysis After Root Canal Preparation | Metabolic Activity of Persistent Bacteria | Bacteroidaceae sp. HOT-272 at S2 | 1.123 RNA/DNA ratio | Standard Deviation 0.1033 |
| Bacterial Species Analysis After Root Canal Preparation | Metabolic Activity of Persistent Bacteria | Cutibacterium acnes at S1 | 1.411 RNA/DNA ratio | Standard Deviation 0.3693 |
| Bacterial Species Analysis After Root Canal Preparation | Metabolic Activity of Persistent Bacteria | Cutibacterium acnes at S2 | 1.489 RNA/DNA ratio | Standard Deviation 0.3547 |
| Bacterial Species Analysis After Root Canal Preparation | Metabolic Activity of Persistent Bacteria | Selenomonas spp. at S1 | 1.092 RNA/DNA ratio | Standard Deviation 0.09859 |
| Bacterial Species Analysis After Root Canal Preparation | Metabolic Activity of Persistent Bacteria | Selenomonas spp. at S2 | 1 RNA/DNA ratio | Standard Deviation 0 |
| Bacterial Species Analysis After Root Canal Preparation | Metabolic Activity of Persistent Bacteria | Enterococcus faecalis at S1 | 0.7433 RNA/DNA ratio | Standard Deviation 0.1882 |
| Bacterial Species Analysis After Root Canal Preparation | Metabolic Activity of Persistent Bacteria | Enterococcus faecalis at S2 | 0 RNA/DNA ratio | Standard Deviation 0 |
Secondary
Bacterial Community Diversity
Composition of the active microbiome by Next Generation Sequencing (NGS) analysis of the root canal samples.
Time frame: After 14 day treatment period
Population: Root canal samples from 10 patients were included in the sequencing analysis.
| Arm | Measure | Group | Value (NUMBER) |
|---|---|---|---|
| Total Bacteria Analysis After 1rst- and 2nd-visit Procedures | Bacterial Community Diversity | Firmicutes | 39.7 relative abundance of phyla (percentage) |
| Total Bacteria Analysis After 1rst- and 2nd-visit Procedures | Bacterial Community Diversity | Actinobacteria | 1.4 relative abundance of phyla (percentage) |
| Total Bacteria Analysis After 1rst- and 2nd-visit Procedures | Bacterial Community Diversity | Proteobacteria | 15.8 relative abundance of phyla (percentage) |
| Total Bacteria Analysis After 1rst- and 2nd-visit Procedures | Bacterial Community Diversity | Spirochaetes | 0.7 relative abundance of phyla (percentage) |
| Total Bacteria Analysis After 1rst- and 2nd-visit Procedures | Bacterial Community Diversity | Bacteroidetes | 39 relative abundance of phyla (percentage) |
| Bacterial Species Analysis After Root Canal Preparation | Bacterial Community Diversity | Spirochaetes | 0.7 relative abundance of phyla (percentage) |
| Bacterial Species Analysis After Root Canal Preparation | Bacterial Community Diversity | Firmicutes | 39.8 relative abundance of phyla (percentage) |
| Bacterial Species Analysis After Root Canal Preparation | Bacterial Community Diversity | Bacteroidetes | 39.1 relative abundance of phyla (percentage) |
| Bacterial Species Analysis After Root Canal Preparation | Bacterial Community Diversity | Proteobacteria | 15.7 relative abundance of phyla (percentage) |
| Bacterial Species Analysis After Root Canal Preparation | Bacterial Community Diversity | Actinobacteria | 1.4 relative abundance of phyla (percentage) |
| Bacterial Composition After Intracanal Medication | Bacterial Community Diversity | Bacteroidetes | 38.6 relative abundance of phyla (percentage) |
| Bacterial Composition After Intracanal Medication | Bacterial Community Diversity | Spirochaetes | 0.6 relative abundance of phyla (percentage) |
| Bacterial Composition After Intracanal Medication | Bacterial Community Diversity | Actinobacteria | 1.6 relative abundance of phyla (percentage) |
| Bacterial Composition After Intracanal Medication | Bacterial Community Diversity | Firmicutes | 39.8 relative abundance of phyla (percentage) |
| Bacterial Composition After Intracanal Medication | Bacterial Community Diversity | Proteobacteria | 16 relative abundance of phyla (percentage) |
Secondary
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).
Time frame: After 14 day treatment period
Population: There was a lost of RNA samples from 5 cases
| Arm | Measure | Group | Value (MEDIAN) |
|---|---|---|---|
| Total Bacteria Analysis After 1rst- and 2nd-visit Procedures | Bacterial Levels | Total bacteria at baseline (S1) | 178500 DNA copies (Bacterial levels) |
| Total Bacteria Analysis After 1rst- and 2nd-visit Procedures | Bacterial Levels | Total bacteria after root canal Preparation (S2) | 7580 DNA copies (Bacterial levels) |
| Total Bacteria Analysis After 1rst- and 2nd-visit Procedures | Bacterial Levels | Total bacteria after adjunctive procedures (S3) | 3815 DNA copies (Bacterial levels) |
| Total Bacteria Analysis After 1rst- and 2nd-visit Procedures | Bacterial Levels | Total bacteria after intracanal medication (S4) | 15550 DNA copies (Bacterial levels) |
| Total Bacteria Analysis After 1rst- and 2nd-visit Procedures | Bacterial Levels | Total bacteria after 2nd-visit preparation (S5) | 0 DNA copies (Bacterial levels) |
| Bacterial Species Analysis After Root Canal Preparation | Bacterial Levels | Bacteroidaceae sp. 272 at S1 | 0 DNA copies (Bacterial levels) |
| Bacterial Species Analysis After Root Canal Preparation | Bacterial Levels | Bacteroidaceae sp. 272 at S2 | 0 DNA copies (Bacterial levels) |
| Bacterial Species Analysis After Root Canal Preparation | Bacterial Levels | Cutibacterium acnes at S1 | 0 DNA copies (Bacterial levels) |
| Bacterial Species Analysis After Root Canal Preparation | Bacterial Levels | Cutibacterium acnes at S2 | 0 DNA copies (Bacterial levels) |
| Bacterial Species Analysis After Root Canal Preparation | Bacterial Levels | Selenomonas spp. at S1 | 4090 DNA copies (Bacterial levels) |
| Bacterial Species Analysis After Root Canal Preparation | Bacterial Levels | Selenomonas spp. at S2 | 0 DNA copies (Bacterial levels) |
| Bacterial Species Analysis After Root Canal Preparation | Bacterial Levels | Enterococcus faecalis at S1 | 0 DNA copies (Bacterial levels) |
| Bacterial Species Analysis After Root Canal Preparation | Bacterial Levels | Enterococcus faecalis at S2 | 0 DNA copies (Bacterial levels) |
Other Pre-specified
Endodontic Success
Absence of clinical signs/ symptoms and reduction/ absence of apical radiolucency in radiologic examination
Time frame: 1 year after endodontic treatment
Population: A subset of 30 patients had both intra-oral radiograph and cone beam computed tomography (CBCT) scans for analysis of the treatment outcome. After 1 year, 8 patients were lost during the follow-up period and 22 patients were analysed.
| Arm | Measure | Value (COUNT_OF_PARTICIPANTS) |
|---|---|---|
| Total Bacteria Analysis After 1rst- and 2nd-visit Procedures | Endodontic Success | 19 Participants |