Metagenomic Analysis and Post-operative Pain Assessment of Infected Root Canals Using Different Irrigation Protocols: A Randomized Controlled Clinical Trial
Phase: NA
Conditions: Dental Pulp Necrosis, Pulpitis
Sponsor: Suez Canal University

 
 
Suez Canal University 
Faculty of Dentistry 
 
 
 
1 
Official Title: 
Metagenomic Analysis and Post-operative Pain Assessment of Infected Root Canals 
Using Different Irrigation Protocols: A Randomized Controlled Clinical Trial 
 
NCT Number: 
Not yet assigned (Initial Submission) 
Document Date: 
December 25, 2025 
Study Facility: 
Faculty of Dentistry, Suez Canal University 
Ethical Approval No: 
REC 928/2024 
Researcher: 
Salma Mohamad Hassan Gohar 
 
 

 
 
Suez Canal University 
Faculty of Dentistry 
 
 
 
2 
Study Protocol 
1. Aim & Objectives 
The study compares bacterial reduction and postoperative pain in root canals using four 
irrigation protocols: ultrasonic activation, laser activation, negative pressure irrigation, 
and mechanical activation. It also evaluates bacterial changes with metagenomic analysis 
and patient-reported pain levels. 
2. Methodology 
This randomized clinical trial involves 40 adult patients divided into four groups. Pre- and 
post-irrigation bacterial samples are collected and analyzed using metagenomic 
sequencing to assess changes in microbial composition. 
Eligibility Criteria 
Inclusion Criteria: 
• 
1. Adult patients aged 18–60 years requiring root canal treatment for teeth with 
necrotic pulp and periapical radiolucency. 
• 
2. Patients in good general health. 
Exclusion Criteria: 
• 
1. Patients with open apices or severe periodontal disease. 
• 
2. Patients currently on antibiotics or allergic to materials used in the study. 
• 
3. Pregnant or breastfeeding women. 
3. Clinical Procedures 
Procedures include aseptic isolation with a rubber dam, access cavity preparation, and 
standard instrumentation with rotary NiTi files. Four irrigation protocols are used: 
Ultrasonic Activation (NaOCl activated by ultrasonic device), Laser Activation (Laser-
activated NaOCl with methylene blue), Easy Clean (Mechanical activation), and 
PulpSucker (Negative pressure suction). 
4. Evaluation 
Postoperative pain is evaluated using the Visual Analogue Scale (VAS) at 6 hours, 24 
hours, 48 hours, and 7 days. Bacterial reduction is assessed via Next-Generation 
Sequencing (NGS). 
5. Statistical Plan 
Sample size calculations  
Sample size calculation using G*Power software indicates a total sample of 48 (including 
20% dropout) to achieve 95% power. Data will be analyzed using two-way repeated 
measures ANOVA or corresponding nonparametric tests. 

 
 
Suez Canal University 
Faculty of Dentistry 
 
 
 
3 
Current research was performed to compare between four different treatment groups 
(A1, A2, A3, A4); Ultrasonic activation, Laser activation, Easy clean device, PulpSucker 
device at two different sampling time (S1, S2); Two-way repeated measure analysis of 
variance is proposed (ANOVA). A minimum total sample size of 32 samples will be 
sufficient to detect the effect size of 0.40, at a power (1-β=0.95) of 95% at a significance 
probability level of p<0.05, and a partial eta squared of 0.14. Applying a dropout of 20% 
to compensate for losses of samples, a total sample size of 48 will be applied in current 
study, according to sample size calculations each treatment group (A1, A2, A3, A4) would 
be represented by 6 samples as shown in Tables 1 and 2. (Santos Nogueira et al., 2021). 
The sample size was calculated according to G*Power software version 3.1.9.6 (Cohen, 
1988; Faul et al., 2007; 2013). 
Statistical analyses 
The statistical analysis will be performed for comparison between different four different 
treatment groups (A1, A2, A3, A4); Ultrasonic activation, Laser activation, Easy clean 
device, PulpSucker device at two different sampling time (S1, S2).  
The data will be collected, checked, revised, and organized in tables and figures using 
Microsoft Excel 2016. Data will be subjected to outliers’ detections and normality 
statistical test to detect whether the data are parametric or nonparametric.  
Data will be analyzed for descriptive statistically both graphical and numerical 
description. Inferential statistics for evaluating and comparing between four different 
treatment groups (A1, A2, A3, A4); Ultrasonic activation, Laser activation, Easy clean 
device, PulpSucker device at two different sampling time (S1, S2), will be performed by 
two-way repeated measures analysis of variance (ANOVA) or corresponding 
nonparametric analyses at significance levels of 0.05. ANOVA will be followed by Tukey’s 
HSD multiple range tests (DMRTs) to compare between treatment groups or 
corresponding posthoc for nonparametric data. Difference between four treatment 
groups at each sampling timepoint will be performed by one way analysis of variance 
(ANOVA) or Kruskal-Wallis for nonparametric data at 0.05 level. The difference between 
two sampling timepoint will be performed using Paired samples t-test. Data analyses will 
be carried out using computer software Statistical Package for Social Science SPSS (IBM-
SPSS ver. 30.0 for Mac OS) (Knapp, 2017).