Analysis of Metabolically Active Bacteria After Adjunctive Steps for Disinfection of Teeth With Primary Endodontic Infections: RNA- and DNA-based Molecular Study
试验速览
- 阶段
- 不适用
- 状态
- 已完成
- 入组人数
- 45
- 试验地点
- 1
- 主要终点
- Metabolic Activity of Persistent Bacteria
研究概览
简要总结
This study aims to evaluate an endodontic treatment protocol using adjunctive steps for disinfection of teeth with primary endodontic infections.
详细描述
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.
研究设计
- 研究类型
- Interventional
- 分配方式
- Non Randomized
- 干预模型
- Single Group
- 主要目的
- Treatment
- 盲法
- None
入排标准
- 年龄范围
- 18 Years 至 65 Years（Adult, Older Adult）
- 性别
- All
- 接受健康志愿者
- 否
入选标准
- •Teeth with necrotic pulps and asymptomatic apical periodontitis
排除标准
- •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.
研究组 & 干预措施
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.
干预措施: Root canal preparation (Procedure)
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.
干预措施: Intracanal medication (Procedure)
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.
干预措施: Root canal preparation (Procedure)
结局指标
主要结局
Metabolic Activity of Persistent Bacteria
时间窗: 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.
次要结局
- Bacterial Community Diversity(After 14 day treatment period)
- Bacterial Levels(After 14 day treatment period)
研究者
Ericka Tavares Pinheiro
Professor
University of Sao Paulo