This prospective observational study aims to investigate the prevalence and clinical significance of two oral protozoa, Entamoeba gingivalis and Trichomonas tenax, among patients attending the Bolu Abant İzzet Baysal University Faculty of Dentistry, Department of Periodontology. While the oral microbiome typically maintains a delicate balance, disruptions in this ecosystem are thought to trigger periodontal diseases. Recent evidence suggests that these parasites may contribute to increased inflammation and tissue destruction, potentially playing a role in the etiology of gingivitis and periodontitis.

The study will include 120 participants aged 18 and older who meet the inclusion criteria. Following the collection of demographic data and oral hygiene habits via a questionnaire, a single calibrated examiner will perform comprehensive clinical periodontal examinations. Measurements will include plaque index (PI), gingival index (GI), bleeding on probing (BOP), probing pocket depth (PPD), clinical attachment loss (CAL), and gingival recession (GR) based on the 2017 World Workshop Classification of Periodontal and Peri-Implant Diseases and Conditions.

To detect the presence of parasites, unstimulated whole saliva samples and subgingival plaque samples from the deepest periodontal pockets will be collected from each participant. These samples will be analyzed immediately at the Parasitology Laboratory using light microscopy (10X and 40X magnification) to identify live trophozoites. By evaluating the relationship between parasite prevalence and periodontal status, this research aims to contribute to the limited literature on oral protozoa in Turkey and increase clinical awareness regarding their impact on oral health.

The primary objective of this single-center, cross-sectional, prospective study is to evaluate the frequency of Entamoeba gingivalis and Trichomonas tenax in patients with different periodontal health statuses. Despite being the first protozoa identified in the oral cavity, the role of these parasites in the pathogenesis of periodontal destruction remains a critical area of investigation. This study seeks to correlate the presence of these protozoa with clinical periodontal parameters and patient-related factors.

1. Patient Selection and Recruitment:

   A total of 120 patients who apply to the Bolu Abant İzzet Baysal University Faculty of Dentistry, Department of Periodontology, will be screened. Following written informed consent, a detailed questionnaire will be administered to collect data on:

   Demographics (Age, sex, education, and economic status)

   Medical history (Systemic diseases, smoking, and pregnancy status)

   Oral hygiene habits and recent use of antibiotics or antiseptic mouthwashes.

2. Clinical Periodontal Examination:

   Periodontal diagnosis will be performed according to the "2017 World Workshop Classification of Periodontal and Peri-Implant Diseases and Conditions." All clinical measurements will be conducted by a single calibrated examiner using a Williams periodontal probe. The following parameters will be recorded:

   Plaque index (PI): Evaluated at 4 sites per tooth (0-3 scale).

   Gingival index (GI): Assessment of gingival inflammation (0-3 scale).

   Bleeding on probing (BOP): Presence/absence of bleeding within 10 seconds of probing, expressed as a percentage.

   Probing pocket depth (PPD): Distance from the gingival margin to the base of the pocket (6 sites per tooth).

   Clinical attachment loss (CAL): Distance from the cemento-enamel junction to the base of the pocket.

   Gingival recession: Distance from the cemento-enamel junction to the gingival margin.

3. Sample Collection and Laboratory Analysis:

   Two types of biological samples will be collected from each participant:

   Subgingival plaque: After isolating the area with cotton rolls and removing supragingival plaque, samples will be collected from the 4 deepest periodontal pockets using sterile curettes and transferred into tubes containing 0.9% NaCl.

   Unstimulated saliva: Approximately 2 mL of whole saliva will be collected after at least 30 minutes of no food intake or mouth rinsing.

   Samples will be transported immediately to the Parasitology Laboratory at Bolu Abant İzzet Baysal University Faculty of Medicine. Three slide preparations per sample will be examined under a light microscope (10X and 40X magnification) to identify the presence of live trophozoites.

4. Sample Size and Power Analysis:

The sample size of 120 was determined based on similar prevalence studies in the literature.

Inclusion Criteria:

• Participants aged 18 years or older.
• Having at least 10 natural teeth.
• Willingness to undergo periodontal examination and providing written informed consent.
• Includes both periodontally healthy individuals and patients with gingivitis or periodontitis.

Exclusion Criteria:

• Use of systemic antibiotics within the last 3 months.
• Use of chlorhexidine or similar antiseptic mouthwashes within the last month.
• Having received any periodontal treatment within the last 3 months.
• Receiving immunosuppressive therapy or having a serious systemic immune deficiency.
• Pregnancy or breastfeeding.

Patients aged 18 and older who applied to the Bolu Abant İzzet Baysal University Faculty of Dentistry, Department of Periodontology, and met the inclusion criteria.