This study was conducted at the Department of Pedodontics and Preventive Dentistry, Manipal College of Dental Sciences, Manipal, in collaboration with the Department of Microbiology, Kasturba Medical College, MAHE. Approval from the institutional Ethics Committee was obtained before initiating the study.
A total of 1431 students in the age group 8–12 years, belonging to classes forth to seventh, were the subjects for this study.
Materials used for recording indices
Mouth mirror, explorer, periodontal probe, tweezers, and chip syringe.
Materials used for the determination of salivary Streptococcus mutans and lactobacilli
- Sterile penicillin bottles for collection of stimulated saliva.
- Agars to be used: Mitis salivarius agar (with bacitracin)
- Lactobacillus MRS agar
- A standard loop
Obtaining informed consent
Before the commencement of this study, an informed consent from the principal of the school and the parents of the students participating in this study was obtained. Children also gave assent to participate.
Selection of the students
The subjects were allocated to the specific treatment by block randomization. Children with similar socioeconomic status, dietary habits, oral hygiene methods, oral hygiene status, and KAP status were included. Further, only children who had a minimum of one to two established carious lesions were considered. The subjects were selected from residential schools.
Assessment of knowledge, attitude and practice
The Knowledge, Attitude and Practice (KAP) of the subjects was determined using a questionnaire.
The subjects were divided into three groups:
Group I (n = 457): using Triphala mouthwash (0.6%)
Group II (n = 440): using Chlorhexidine mouthwash (0.1%) (positive control)
Group III (n = 412): using distilled water (negative control).
The schools were distributed in such a manner so that there was no intermingling within the students of different groups. It was a double-blind clinical trial.
Plaque scores were recorded using the methodology given by Silness and Loe[3
] The gingivitis index was calculated according to the method given by Loe and Silness[4
and lactobacilli count was done in stimulated saliva. The subjects were asked to simulate chewing action with sterilized cotton rolls for 4 min. At the end of 4 min, the students were made to expectorate into sterile penicillin bottles. The stimulated saliva was then transported to the microbiology department within 30 min. A semi-quantitative that is four-quadrant streaking method was adopted (Sitges-Serra and Linares)[5
] Using a standard loop, the saliva was streaked on Mitis salivarius agar with bacitracin (for Streptococcus mutans
) and Lactobacillus
MRS agar (for lactobacilli).
The growth in all the four quadrants was recorded. The colonies were identified based on colony morphology and gram staining. Growth in each quadrant was accorded the scores in CFU/ml. Thus,
<10,000 CFU/ml: three primary streaks in one quadrant;
25–50,000 CFU/ml: growth in one complete quadrant;
50–75,000 CFU/ml: growth in two complete quadrants;
75–100,000 CFU/ml: growth in three complete quadrants;
>100,000 CFU/ml: growth in four complete quadrants.
Preparation of mouthwashes
Triphala mouthwash was prepared in the pharmacy manufacturing center, Manipal, in the concentration of 0.6%, and it was then dispensed in 1-liter cans and delivered for use. Chlorhexidine gluconate mouthwash (Proprietary name: Clohex, concentration 0.2%) was procured from the market and given to the pharmacy manufacturing center. It was then diluted and the final concentration of Chlorhexidine gluconate was 0.1%. This was dispensed in the 1-liter cans.
Both solutions were made of identical colors to eliminate bias. The bottles were then coded and then at the end of the study, the decoding was done.
For the Streptococcus mutans and Lactobacillus counts, Wilcoxon and Mann-Whitney tests were applied for intragroup and intergroup comparison, respectively. All the tests were carried out in the SPSS software.
Administration of mouthwash
The teachers were educated and trained in the use of mouthwash so that the children, under the supervision of the teachers, could use the mouthwash. Each of the groups used the respective mouthwash, as a daily, supervised rinse after lunch in the afternoon. The children were advised not to eat or rinse for the next 30 min. They were instructed to carry home the mouthwash bottles on weekends and during vacations.
The Chlorhexidine mouthwash was used in concentration of 0.1% such that 10 ml was dispensed at one time. The mouthwash was swished in all quadrants of the mouth for a period of 2 min. An equal quantity of Triphala mouthwash and placebo (distilled water) was dispensed.
Plaque, gingivitis scores, and microbiological analysis were recorded at baseline 3, 6, and 9 months after baseline.
The collected data were subjected to statistical analysis. For intragroup comparison of plaque and gingival scores, the paired sample t-test was applied, while for the intergroup comparison Tukey's test was applied.