## 2 Materials and Methods  
### Study Design  
This is a pilot prospective study. Declarations of informed consent were signed by the parents of the children or their legal guardians. The study was approved by the Ethics Committee of the Medical University of Varna (protocol No. 82/28.03.2019). Participants in the study were recruited during their primary visit to the University Dental Medicine Center of the Medical University of Varna based on the inclusion and exclusion criteria. Inclusion criteria:  
1. Informed consent signed by the parent or guardian of each participant;
2. Age between 4-14 years;
3. No acute or chronic disease reported;
4. No medicine intake, including homeopathic remedies;
5. No known allergic reactions. Exclusion criteria:  
1. Children who are not collaborative during the examination and/or sample collection procedures;
2. Children with reported acute or chronic disease at the time of examination;
3. Children taking any treatment (anti-inflammatory, antibiotics, anti-allergy drugs, or homeopathic remedies);
4. Children with established allergic reactions;
5. Children displaying an open bite and mouth breathing;This pilot prospective study included 45 children (26 girls and 19 boys) with no acute or chronic disease reported (aged between 4-14 years). For all participants, the clinical indices of PLI and GI were recorded and applied for the evaluation of the accumulation of dental plaque (Silness-Loe plaque index--PLI), assessment of gingival conditions, and registration of the qualitative changes in gingival tissues (Loe-Silness gingival index--GI). The plaque index (PLI) is applied for the evaluation of dental plaque accumulation. This index is registered for Ramfjord teeth 16, 22, 24, 36, 42 and 44. In children with primary and mixed dentition, in the case of missing representative teeth, the PLI is registered for mesially situated teeth. The recording of the PLI index is conducted by an atraumatic periodontal probe on the vestibular, oral, mesial, and distal surfaces along the site of contact with the marginal gingiva. The degree of dental plaque accumulation is recorded by the scores 0, 1, 2, and 3. The criteria are as follows: 0--no dental plaque on the examined tooth surface; 1--low levels of dental plaque on the peak of the probe; 2--moderate levels of dental plaque scratched by the probe; 3--a considerable, macroscopically visible accumulation of dental plaque on the tooth surface. The sum of all of the registered figures is divided by the total number of examined surfaces. The values of PLI in the interval from 0.1 to 1.1 indicate a condition that is \"excellent with a tendency to have very good and good oral hygiene\". PLI values between 1.2 and 2.0 indicate a condition that is \"good, with a tendency to have satisfactory oral hygiene\". PLI values from 2.1 to 3.0 are descriptive of a condition that is \"satisfactory, with a tendency to have poor oral hygiene\". The GI is utilized to evaluate the severity of gingivitis. This index records the marginal and interproximal tissues separately on the bases of 0 to 3. The same Ramfjord teeth applied for the assessment for PLI are applied for the recording of GI, with the following criteria: 0--normal gingiva; 1--mild-inflammation hyperemia and slight edema, without bleeding on probing; 2--moderate-inflammation hyperemia, edema, and provoked bleeding; 3--severe-inflammation hyperemia, considerable edema, and spontaneous bleeding. The assessment of bleeding was applied by atraumatic sampling along the site of the soft tissue of the gingival sulcus. To calculate the GI, the values of the four walls of the gingival tissues encircling the tooth were summed up and divided by four. The sum of these values was divided by the total number of examined sites. By the application of the GI, the diagnostic scores were determined as follows: GI = 0--normal gingiva; GI levels from 0.1 to 1.0--mild degree of gingivitis; GI values from 1.1 to 2.0--moderate degree of gingivitis; GI values from 2.1 to 3.0--severe degree of gingivitis [21].