Description

Study Aim The overall aim of the study is to characterize the occurrence of small intestinal bacterial overgrowth (SIBO) in patients with severe (stage III/IV) periodontitis and to explore its potential association with periodontal clinical status, gastrointestinal symptoms, and biological markers related to intestinal barrier function and systemic exposure to bacterial products. A secondary longitudinal aim is to observe whether changes in periodontal clinical status following routine, clinically indicated periodontal therapy are accompanied by changes in SIBO status and intestinal barrier-related biomarkers.

Working Hypothesis The working hypothesis is that patients with severe periodontitis may present with SIBO and that SIBO may coexist with alterations in intestinal barrier-related biomarkers and systemic exposure to bacterial products. Periodontal inflammation may represent an additional source of systemic inflammatory burden and may contribute to alterations of the oral-gut microbial interface. Conversely, intestinal microbial alterations and barrier dysfunction may influence host inflammatory responses relevant to periodontal disease. These relationships are considered bidirectional and exploratory; the present study is not designed to establish causality.

Specific Objectives

1. To estimate the proportion of patients with Stage III/IV periodontitis who present with SIBO, as assessed by a standardized lactulose/glucose breath test.
2. To explore the association between SIBO status and the clinical characteristics and severity of Stage III periodontitis, based on standardized periodontal parameters.
3. To assess intestinal barrier-related biomarkers and explore their association with SIBO status and periodontal clinical parameters.
4. To explore the associations between SIBO, intestinal barrier-related biomarkers, and systemic exposure to bacterial products in patients with Stage III/IV periodontitis.
5. To prospectively observe whether changes in periodontal clinical status following routine periodontal therapy are accompanied by changes in SIBO status and intestinal barrier-related biomarkers.
6. To assess gastrointestinal symptoms and selected potential confounding factors, including relevant systemic diseases, medication use, smoking, and lifestyle/dietary characteristics, in relation to SIBO and periodontal status.

Study Design This is a prospective, longitudinal, observational cohort study conducted in a single cohort of at least 20 adults with Stage III/IV periodontitis, Grade B or C. No healthy control or comparison group will be included.

All participants will receive periodontal treatment according to their individual clinical indication and routine periodontal care. The periodontal treatment is not assigned experimentally by the research protocol. The research component consists of observing clinical and biological changes before and after routine periodontal therapy.

The study will include a baseline assessment (T0) before or at the beginning of periodontal treatment and a follow-up assessment (T1), planned approximately 6-8 weeks after completion of the etiological periodontal treatment.

Study Population and Sample Size The study will include at least 20 adult participants with a diagnosis of Stage III/IV periodontitis, Grade B or C, according to the 2017 World Workshop classification adopted by the EFP/AAP framework.

The sample size is based on the feasibility of the doctoral research project and the available resources for SIBO breath testing and biological assays. The study is exploratory and is intended to provide preliminary estimates and longitudinal observations that may inform the design and sample-size calculation of future controlled studies.

Eligibility Criteria

1. Inclusion criteria
   • Adults aged ≥18 years.
   • Diagnosis of Stage III/IV periodontitis, Grade B or C, according to the applicable periodontal classification criteria.
   • Presence of sufficient dentition to permit a meaningful full-mouth periodontal examination.
   • Ability and willingness to perform the breath test and provide blood samples and questionnaire data.
   • Written informed consent.
   • Patients with stable chronic systemic conditions may be included, provided that the condition is clinically controlled and does not meet any exclusion criterion. Relevant systemic diseases and concomitant medications will be recorded and considered as potential confounding factors.
2. Exclusion criteria
   • Uncontrolled or severe systemic disease likely to substantially affect periodontal inflammation, intestinal microbiota, intestinal barrier function, systemic inflammatory status, or the safety of periodontal treatment.
   • Antibiotic, systemic antifungal, or probiotic therapy within the preceding 4 weeks.
   • Recent periodontal treatment that could materially influence baseline periodontal or biological measurements.
   • Diagnosed inflammatory bowel disease, celiac disease, major intestinal surgery, or another major gastrointestinal condition that would substantially interfere with interpretation of SIBO or intestinal barrier-related outcomes.
   • Current pregnancy or breastfeeding.
   • Other condition or treatment judged by the investigator to materially interfere with the study outcomes or procedures.

Clinical and Biological Assessments A complete periodontal examination will be performed using standardized clinical procedures. Periodontal parameters will include probing pocket depth (PPD), clinical attachment level (CAL), bleeding on probing (BOP), plaque-related parameters, and other clinically relevant variables required for severe-stage periodontal characterization.

SIBO will be assessed using a standardized lactulose and/or glucose breath test, according to the finalized validated test protocol and manufacturer's instructions. Breath-test interpretation will follow the predefined diagnostic criteria specified in the final study protocol and registration.

Gastrointestinal symptoms will be assessed using the Gastrointestinal Symptom Rating Scale (GSRS), only if a validated version is finalized, or using the Visual Analogue Scale for Irritable Bowel Syndrome (VAS-IBS).

Approximately 5 mL of venous blood will be collected under standardized conditions. The biological assays will evaluate intestinal barrier-related and/or systemic bacterial-exposure biomarkers selected before database lock. If zonulin and LBP are the finalized assays, both will be prespecified in the statistical analysis plan. The laboratory matrix (serum or plasma), collection tube, processing procedure, storage temperature, and assay characteristics will be standardized across participants.

Systemic Conditions and Potential Confounders Because patients with Stage III/IVperiodontitis frequently present with chronic systemic conditions, controlled comorbidities will not be used as automatic exclusion criteria. Diabetes mellitus, hypertension, hypothyroidism, dyslipidemia, obesity/BMI, smoking, relevant gastrointestinal conditions, and concomitant medications will be recorded when present.

Diabetes mellitus will be characterized by type and, where available, recent glycemic control (e.g., HbA1c). Relevant medications and recent antibiotic, probiotic, or other microbiota-modifying exposures will be recorded. Given the sample size of 20 participants, comorbidities will primarily be described and considered as potential confounders rather than analyzed as independently powered subgroups.

Study Procedures and Timeline At T0, participants will undergo periodontal examination, assessment of gastrointestinal symptoms, SIBO breath testing, collection of relevant medical and lifestyle information, and blood sampling for the prespecified biological biomarkers.

Participants will then receive routine, clinically indicated periodontal etiological therapy according to their individualized periodontal treatment plan.

At T1, approximately 6-8 weeks after completion of etiological periodontal treatment, periodontal clinical parameters, SIBO status, gastrointestinal symptoms, and the prespecified biological biomarkers will be reassessed, where applicable.

Outcomes Primary outcome: the proportion of study participants with a positive SIBO breath test at baseline.

Secondary outcomes: (1) associations between SIBO status and periodontal clinical parameters; (2) associations between SIBO status and intestinal barrier-related biomarkers; (3) associations between periodontal parameters and intestinal barrier-related/systemic bacterial-exposure biomarkers; (4) gastrointestinal symptom scores in relation to SIBO status; and (5) within-participant changes in periodontal parameters and biological biomarkers between T0 and T1.

Statistical Analysis Descriptive statistics will be used to characterize the study population and estimate the proportion of participants with SIBO. Continuous variables will be summarized using appropriate measures of central tendency and dispersion, and categorical variables using frequencies and percentages.

Associations between SIBO status and periodontal, gastrointestinal, and biological variables will be explored using statistical methods appropriate to the distribution and measurement scale of each variable. Longitudinal changes between T0 and T1 will be assessed using paired statistical methods or appropriate regression models.

Potential confounders, including age, sex, smoking, BMI, diabetes, relevant systemic diseases, and medication use, will be considered where feasible. Because the sample size is limited, multivariable analyses will be parsimonious and interpreted as exploratory. No subgroup analysis will be considered adequately powered solely on the basis of the planned sample size of 20 participants.

Ethical Considerations The study will be conducted in accordance with the Declaration of Helsinki, applicable national regulations, institutional requirements, and the approved ethics protocol. Written informed consent will be obtained from all participants before study-specific procedures.

The periodontal treatment provided to participants is clinically indicated routine care and is not experimentally assigned by the study. The research procedures include periodontal examination, breath testing, questionnaires, and venous blood collection.

Data Management and Confidentiality Participant data will be coded using study identification numbers. Identifying information will be stored separately from research data and accessed only by authorized study personnel. Biological samples will be labeled using coded identifiers. Data will be stored and analyzed in accordance with institutional requirements and applicable data-protection regulations.

Scientific Significance The study is designed to provide preliminary clinical evidence regarding the occurrence of SIBO among patients with Stage III periodontitis and to characterize the relationship between SIBO, periodontal inflammation, intestinal barrier-related biomarkers, and systemic exposure to bacterial products.

The study may also help determine whether routine periodontal treatment is accompanied by measurable changes in gastrointestinal microbial status or intestinal barrier-related biomarkers. Such findings would not establish causality but could identify clinically relevant associations and generate hypotheses for larger controlled longitudinal studies.

An additional potential contribution is to highlight the importance of considering gastrointestinal symptoms and possible SIBO in the broader systemic assessment of selected patients with advanced periodontitis, particularly when clinically relevant gastrointestinal or systemic inflammatory features are present. Any diagnostic or therapeutic decisions regarding SIBO will remain within appropriate medical/gastroenterological care and will not be dictated by the research protocol.