This case-control study will be conducted between January and June 2026 and will include 148 participants aged over 30 years to investigate differences between individuals with periodontitis (n = 111) and those with periodontal health (n = 37). Participants will be selected based on strict inclusion and exclusion criteria to minimize confounding factors, and ethical approval along with informed consent will be obtained prior to participation. Periodontitis will be diagnosed according to the 2017 classification criteria, requiring clinical attachment loss and periodontal pocketing, while periodontal health will be defined by minimal bleeding on probing, shallow pocket depths, and absence of attachment loss. The sample size will be determined using G*Power to ensure adequate statistical power.

Clinical periodontal parameters-including plaque index, gingival bleeding index, probing pocket depth, and clinical attachment loss-will be recorded using standardized methods at multiple sites per tooth by calibrated examiners to ensure reliability. Following clinical assessment, subgingival biofilm samples will be collected using sterile paper points under controlled conditions and stored at -80°C for subsequent real-time PCR analysis. Calibration procedures, including inter- and intra-examiner agreement assessed by intraclass correlation coefficients, will be implemented to enhance measurement consistency. Overall, the study will combine rigorous clinical evaluation with molecular techniques to enable a comprehensive assessment of periodontal status and associated microbiological factors.

This case-control study will be conducted between January and June 2026 and will include a total of 148 participants aged over 30 years. The study population will consist of 111 subjects with periodontitis and 37 subjects with periodontal health. Ethical approval will be obtained from the Institutional Ethics Review Board (Certificate No. 2015-16/1118), and written informed consent will be secured from all participants prior to examination. Eligible individuals will be selected based on predefined inclusion and exclusion criteria and will be categorized into a healthy group and a periodontitis group according to clinical periodontal parameters. Inclusion criteria will require participants to be over 30 years old and to have at least 20 teeth. Exclusion criteria will include individuals with active caries, those undergoing orthodontic treatment, recent antibiotic use within three months, history of valve replacement requiring prophylactic antibiotics, recent use of non-steroidal anti-inflammatory drugs, pregnancy or lactation, alcohol use, and receipt of periodontal therapy within the last six months.

Participants in the periodontitis group will be diagnosed based on the 2017 classification, defined by interdental clinical attachment loss (CAL) at ≥2 non-adjacent teeth or buccal/oral CAL ≥3 mm with probing pocket depth (PPD) >3 mm at ≥2 teeth. Cases will be further characterized as generalized periodontitis affecting more than 30% of teeth, with unstable disease indicated by sites exhibiting PPD ≥5 mm or PPD ≥4 mm with bleeding on probing (BOP), and staged from I to IV. The control group will include individuals with periodontal health, defined by BOP <10%, PPD ≤3 mm, and absence of attachment loss. The sample size will be calculated using G*Power software (version 3.1.9.7) based on an expected prevalence of 25% in healthy individuals and 50% in periodontitis patients, with 80% power and a significance level of 0.05, resulting in a required total of 148 participants.

Clinical periodontal parameters will be assessed following participant selection. Plaque Index (PI) will be recorded at four sites per tooth using a disclosing agent and a dichotomous scoring system. Gingival Bleeding Index (GBI) will be evaluated at the same sites using a UNC-15 periodontal probe, with bleeding recorded 30 seconds after probing. Probing pocket depth (PPD) and clinical attachment loss (CAL) will be measured at six sites per tooth using a manual periodontal probe. To ensure measurement reliability, inter-examiner calibration will be performed between two examiners on selected quadrants and sites, while intra-examiner calibration will be conducted by repeating measurements in two sessions. Agreement will be analyzed using the intraclass correlation coefficient (ICC) in SPSS.

Following clinical examination, subgingival biofilm samples will be collected from selected sites. The sampling area will be isolated with cotton rolls to prevent contamination, and sterile paper points will be inserted into the base of the periodontal pocket for 30 seconds. Samples will then be transferred into sterile tubes containing preservative solution, properly labeled, and stored at -80°C to maintain DNA integrity. These samples will subsequently be analyzed using real-time polymerase chain reaction (RT-PCR) to identify and quantify bacterial presence.

Inclusion Criteria:

• (1) Participants aged > 30 years old. (2) Participants who have at least 20 teeth

Exclusion Criteria:

• 1) Participants who have active caries. (2) Participants who are undergoing orthodontic treatment. (3) Participants with a history of antibiotic use within the last three months will not be eligible.

  (4) Participants with a history of valve replacement which requires prophylactic antibiotic coverage. (5) Participants who require systemic or topical non-steroidal anti-inflammatory drugs within the past three months. Participants who are pregnant or lactating women. (6) Participants with a history of alcohol use. (7) Participants who had periodontal therapy within the last six months

This study is an observational case-control study. Patients with generalized unstable periodontitis, at any grade, with (>4 mm) probing pocket depths (PPD), as well as periodontal health participants, will be recruited from the Department of Periodontics, College of Dentistry, University of Babylon. A written consent forms will be obtained from all participants. Ethical approval will be obtained.

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