This pilot randomized clinical study aims to compare bacterial profiles in symptomatic and asymptomatic primary apical periodontitis using 16S rRNA polymerase chain reaction sequencing. Twenty-four mandibular first molars requiring endodontic treatment will be selected from patients attending the Conservative Dentistry Clinic at the Dental and Oral Hospital of Hasanuddin University. The samples will be divided into two diagnostic groups: symptomatic primary apical periodontitis and asymptomatic primary apical periodontitis. Each diagnostic group will then be allocated into two irrigation groups using either 3% or 5.25% sodium hypochlorite.

Microbial samples will be collected from the distal root canal at three time points: before root canal treatment, after chemomechanical preparation and sodium hypochlorite irrigation with ultrasonic activation, and two weeks after intracanal calcium hydroxide medication. Root canal samples will be subjected to bacterial DNA extraction, 16S rRNA PCR amplification, sequencing, and taxonomic identification.

The primary outcome is the presence or absence of detectable bacterial DNA at each sampling stage. Secondary outcomes include the bacterial taxa identified in symptomatic and asymptomatic primary apical periodontitis and changes in bacterial profiles after sodium hypochlorite irrigation and calcium hydroxide medication. The study is designed to provide preliminary clinical-molecular evidence on bacterial profile changes during endodontic treatment of primary apical periodontitis.

rimary apical periodontitis is commonly associated with microbial infection of the root canal system. The bacterial community in primary endodontic infections is complex and may differ between symptomatic and asymptomatic cases. Persistent bacteria after chemomechanical preparation and intracanal medication may contribute to continuing periapical inflammation and unfavorable endodontic outcomes. Therefore, molecular identification of bacterial profiles during different stages of root canal treatment is important to better understand the microbial changes associated with endodontic disinfection.

Sodium hypochlorite is widely used as an endodontic irrigant because of its antimicrobial and tissue-dissolving properties. Different concentrations of sodium hypochlorite may influence bacterial reduction within the root canal system. In addition, ultrasonic activation may improve irrigant penetration and enhance disinfection by promoting acoustic streaming and disruption of bacterial biofilm. Calcium hydroxide is also commonly used as an intracanal medicament between appointments because of its high alkalinity and antibacterial activity.

This pilot randomized clinical study will evaluate bacterial profiles in mandibular first molars with primary apical periodontitis. A total of 24 mandibular first molars requiring endodontic treatment will be included from patients attending the Conservative Dentistry Clinic at the Dental and Oral Hospital of Hasanuddin University. The samples will be selected using purposive sampling based on predefined inclusion and exclusion criteria.

The samples will be divided into two diagnostic groups: 12 teeth with symptomatic primary apical periodontitis and 12 teeth with asymptomatic primary apical periodontitis. Each diagnostic group will then be divided according to the concentration of sodium hypochlorite irrigation, resulting in four treatment groups. Group 1 will consist of six mandibular first molars with symptomatic apical periodontitis irrigated with 3% sodium hypochlorite. Group 2 will consist of six mandibular first molars with symptomatic apical periodontitis irrigated with 5.25% sodium hypochlorite. Group 3 will consist of six mandibular first molars with asymptomatic apical periodontitis irrigated with 3% sodium hypochlorite. Group 4 will consist of six mandibular first molars with asymptomatic apical periodontitis irrigated with 5.25% sodium hypochlorite.

After communication, information, education, and informed consent, root canal treatment will be performed under aseptic conditions. The operative field will be isolated using a rubber dam, followed by disinfection of the working area. Access opening will be performed, and the mesial canal orifices will be isolated to allow sampling from the distal root canal. Working length will be determined using a K-file and apex locator, then confirmed radiographically. The first microbial sample will be collected from the distal root canal before chemomechanical preparation and labeled as S1. The sampling procedure and subsequent laboratory analysis are described in the uploaded research procedure, including collection using endodontic files and storage in DNA Shield before PCR examination.

Root canal preparation will be performed using rotary ProTaper Gold instruments up to F2. After instrumentation, the canals will be irrigated according to the assigned group using either 3% or 5.25% sodium hypochlorite. The irrigation protocol will include sodium hypochlorite, sterile distilled water, 17% EDTA, and sterile distilled water, followed by ultrasonic activation for 20 seconds per cycle for three cycles. The second microbial sample will be collected after chemomechanical preparation and irrigation, then labeled as S2.

After S2 sampling, intracanal medication with calcium hydroxide will be placed in the root canal. The tooth will be sealed with a cotton pellet and temporary restoration for 14 days. At the second visit, the tooth will be isolated and disinfected again, the temporary restoration will be removed, and calcium hydroxide will be cleaned from the canal using sterile distilled water. The third microbial sample will be collected from the distal root canal two weeks after calcium hydroxide medication and labeled as S3.

Bacterial identification will be performed using 16S rRNA polymerase chain reaction sequencing. DNA will be extracted from the samples, followed by PCR amplification using universal bacterial primers. The PCR process will include pre-denaturation, denaturation, annealing, extension, and final extension steps. PCR products will then be evaluated using gel electrophoresis. Positive amplified products will be further processed for sequencing and taxonomic identification by comparing DNA sequences with available sequence databases.

The primary outcome of this study is the presence or absence of detectable bacterial DNA in root canal samples at the three sampling stages: before treatment, after sodium hypochlorite irrigation, and after calcium hydroxide medication. Secondary outcomes include the identification of bacterial taxa in symptomatic and asymptomatic primary apical periodontitis, comparison of bacterial profiles between 3% and 5.25% sodium hypochlorite irrigation groups, and evaluation of bacterial profile changes from S1 to S2 and S3.

This study is designed as a pilot clinical-molecular investigation to generate preliminary evidence regarding bacterial profile changes during endodontic treatment of primary apical periodontitis. The findings are expected to provide useful information on the microbial characteristics of symptomatic and asymptomatic primary apical periodontitis and the effect of standard endodontic disinfection procedures on bacterial detection using 16S rRNA PCR sequencing.

Inclusion Criteria:

• Adult patients who are mentally and physically healthy, aged 18-40 years.

Mature permanent molar teeth diagnosed with symptomatic or asymptomatic apical periodontitis, determined based on:

Patient history; Intraoral clinical examination; For symptomatic apical periodontitis: negative pulp vitality test, negative palpation test, positive percussion test, with or without widening of the periodontal ligament space; For asymptomatic apical periodontitis: negative pulp vitality test, negative palpation test, percussion test findings, and radiographic assessment using a periapical index score of less than 5 based on periapical radiographs.

Teeth with straight root canals. Teeth that are restorable after root canal treatment. Patients who are willing to participate in the study and have signed the informed consent form after receiving an explanation of the study.

Exclusion Criteria:

• Participants will be excluded if the tooth or patient presents with any of the following conditions:

Patients who have taken antibiotics within 7 days before sample collection. Advanced tooth mobility greater than grade 2. Periodontal pockets greater than 4 mm. Teeth with previous endodontic treatment. Root fracture or fracture occurring during root canal treatment. Teeth showing internal or external resorption. Teeth with open apices. Teeth with calcified or obliterated root canals. Patients with systemically compromised conditions requiring antibiotic coverage for routine dental treatment or patients with systemic diseases.

The IPD will not be shared because the researchers have guaranteed the confidentiality of patient data

The allocation of sodium hypochlorite concentration will be performed randomly by a dental nurse and will not be known by the researcher during sample allocation. No participant masking is applied.