The Effect of Probiotics on Oral Health

Thirty consenting patients aged 20-24 years who will meet the inclusion criteria will be recruited in the study. At the preparatory phase patients will be recruited and randomized, their oral hygiene protocols will be standardized, researchers will be calibrated and trained. The main phase will include a double-blind, randomized, two-arm parallel group, placebo-controlled study of probiotics effects, and wash-out period. Patients will be randomly divided into 2 groups (group 1 - probiotics, group 2 - placebo). Clinical examination will be performed at baseline, after 4 weeks (probiotic/placebo intake) and 6 weeks (wash-out), and will include the assessment of DMFT index, oral hygiene level (the Turesky Modification of the Quigley-Hein Plaque Index (TMQHPI)), gingival inflammation (PMA index), salivation rate (unstimulated salivary flow), and salivary concentration of secretory immunoglobulins A.

The study is estimated to complete enrollment within 4 weeks from study initiation; however, enrollment will remain open until the study goal is met. The duration of this study for each subject will be a maximum of eight (10) weeks: 4 weeks - preparatory period, 4 weeks - intervention period, and wash out period (2 weeks).

Screening (First) visit

• Review the study with the subject and obtain written informed consent
• Assign the subject a unique screening/enrollment number
• Review and record medical history, and medication history to determine eligibility based on inclusion/exclusion criteria
• Record demographics (age, gender)
• Document vitals
• Document all current medications, including medications over-the-counter and herbal medications
• Perform physical examination
• Determine DMFT index
• Determine oral hygiene level (TQHPI)
• Provide training in standardised brushing techniques.

The allocation concealment will be performed by the use of containers numbered by a "third party" (person, who will not participate in the study). The tablets in the bottles without titles will be placed in the containers. The tablets in different groups will look similar. The patient on enrollment will receive a container with tablets. Neither patients nor researchers will be aware of the type of a tablets received by each patient (double blinding).

Baseline (Second) visit (4 weeks from the Screening visit) - all groups

• Perform physical examination
• Determine DMFT index
• Determine oral hygiene level (TQHPI)
• Determine gingival tissue condition (PMA index)
• Determine unstimulated salivary flow rate
• Determine a concentration of secretory immunoglobulins A in saliva (ELISA)

Third visit (4 weeks from the Baseline visit) - all groups

• Perform physical examination
• Determine DMFT index
• Determine oral hygiene level (TQHPI))
• Determine gingival tissue condition (PMA index)
• Determine unstimulated salivary flow rate
• Determine a concentration of secretory immunoglobulins A in saliva (ELISA)
• Assess for adverse events

Fourth visit (2 weeks from the Third visit) = Final Study Visit - all groups

• Perform physical examination • Determine DMFT index
• Determine oral hygiene level (TQHPI))
• Determine gingival tissue condition (PMA index)
• Determine unstimulated salivary flow rate
• Determine a concentration of secretory immunoglobulins A in saliva (ELISA)
• Assess for adverse events

Early Termination visit

• Assess for adverse events
• Assess for complications following treatments
• Document all current medications, including medications over-the-counter and herbal medications

Preparatory phase of the study Patient training Oral hygiene instructions will be given and the compliance with the recommendations will be controlled with weekly visits to the dental office. The participants will be familiarized with the assessment techniques and trial procedures Qualification and calibration of the observers The clinical examinations will be performed by two dentists who are the members of Therapeutic Dentistry Department, Sechenov University, Moscow, Russia. The examiners will be trained and calibrated to obtain an intra- and inter-examiner agreement of 90%, using Kappa statistics.

Randomization Thirty people aged 20-24 years will be examined and randomly assigned to one of the study groups: Group 1 will use tablets containing probiotic (Streptococcus salivarius K12). Group 2 will use tablets containing placebo. The allocation concealment will be performed by the use of containers numbered by a "third party" (person, who will not participate in the study). The tablets in bottles without any titles will be placed in the containers. The tablets in both groups will look similar. The patient on enrolment will receive a container of tablets. Neither patients nor researchers will be aware of the type of tablets used.

Treatment protocol The patients will use the prescribed tablets ("Bactoblis", registration number: AM.01.06.01.003.Е.000024.07.18; 20.07.18, MEDICO DOMUS, d.d.o. (18116, Nis, Serbia)) for 4 weeks 1 tablet per day. The end of the treatment course will be followed by a two-week wash-out period. During the wash-out period, patients will have to stop taking their prescribed tablets. A wash-out period is necessary to assess the stability of the results achieved. Control examinations will be carried out in the following periods: 4 weeks and 6 weeks.

Physical examination

The examination will include determination of:

DMFT index, oral hygiene level (TQHPI), gingival tissue condition (PMA index), unstimulated salivary flow rate, and a concentration of secretory immunoglobulins A in saliva (ELISA).

Data collection and reporting. Data will be collected at the following points: at baseline, and after 4 and 6 weeks of the study.

Data from the study will be maintained for two (2) years after the date the investigation is completed, terminated or until the records are no longer required to support the protocol, whichever date is later. Patient records and data are eligible for inspection and/or copying by applicable regulatory authorities.

Data handling and record keeping Data entry will be completed in the RedCap database (which will cover all the created CRFs). The data will be exported into CSV file format, which will then be used for data analysis. Only de-identified data will be used for data analysis. All hard copy documents will be shredded within five years after completion of the study upon Sponsor approval. Collected de-identified data will be sent to a biostatistician for statistical analysis.

Adverse reactions. There is no expectation of any adverse outcomes or reactions due to a patient using probiotics or placebo. However, hypersensitivity reactions to some of the tablets' components are still possible. All participants will be given access to contact info of the investigators. Any adverse reactions should be reported immediately to the investigators.

Reasons for Withdrawal or Termination

The following is a list of possible reasons for study treatment discontinuation:

• Screening Failure
• Subject withdrawal of consent
• Subject is not compliant with study procedures
• Adverse Event that in the opinion of the Investigator would be in the best interest of the subject to discontinue study participation
• Protocol violation requiring discontinuation
• Lost to follow-up All subjects are free to withdraw from participation at any time, for any reason, specified or unspecified, and without prejudice.

Statistical analysis plan All analyses will be performed using per-protocol population: all subjects will be analyzed who did not substantially deviate from the protocol as to be determined on a per-subject basis by the study principal investigator immediately before database lock.

Data will be presented as means and standard deviations with 95% confidence intervals, medians and 25 and 75 percentiles, and percentages depending on the type of variables. The normality and sphericity of distribution of continuous variables will be assessed with Shapiro-Wilk and Leven's tests, respectively. If the assumptions of normality and sphericity are satisfied, repeated measures mixed ANOVA model will be performed followed by post-hoc Tukey test with adjustment for multiple comparisons. If the aforementioned assumptions are not satisfied, the differences between groups will be assessed using Mann-Witney U-test and the difference within the groups at different study timepoints will be assessed with Friedman test with post-hoc comparisons. The same non-parametric tests will be used for the analyses of categorical and ordinal variables. Fisher's exact test will be used to access the frequencies of categorical variables in the groups. Correlation between Salivation rate, salivary sIgA concentration, TQHPI, DMFT, Decay, and PMA will be assessed using Spearman correlation coefficient.

All summary tables will be structured with a column for each timepoint and rows for each assessed outcome and will be annotated with the total population size relevant to that table/treatment, including any missing observations.