Study Design
This was a randomised, single-blinded, placebo-controlled clinical study that was conducted at the Faculty of Dentistry, Universiti Sains Islam Malaysia (USIM). Ethical approval was obtained from the Research Ethics Committee, USIM (USIM/JKEP/2023-251). This clinical study followed the Consolidation Standards of Reporting Trials Statement and was registered on 24th January 2024 at ClinicalTrials. gov (No. NCT06223243).
Participants
Student volunteers from the Faculty of Dentistry, USIM, were invited to participate in the study. Written and informed consent were obtained from the participants. The sample size calculation for this pilot study was estimated based on Whitehead et al., 2016 to have 10 participants per arm. A total of ten participants per group were recruited for the study (Table 1). The inclusion and exclusion criteria are as follows:
The inclusion criteria:
1. Medically healthy dental students aged between 19 to 24
2. Healthy gingiva or localised gingivitis with the highest Basic Periodontal Examination (BPE) score of 2
3. Presence of at least 20 natural teeth
The exclusion criteria:
1. Patients with Type I or Type II Diabetes Mellitus
2. Any physical and/or mental disabilities that can impede the use of mouthrinses
3. The use of orthodontic or prosthodontic appliances
4. Undergoing antibiotic/antimicrobial therapy for the past 6 months
5. Previous or current smoker
6. Saliva secretion rate outside of normal ranges for the population
7. Pregnancy or taking any hormone therapy
8. Reported allergies to honey or bee stings
Randomisation
Participants were randomly allocated into Group A, Group B, or Group C based on the order of recruitment. Randomisation was done using Microsoft Excel software. Predetermined recruitment numbers (1-30) were randomly allocated into groups A, B, or C using computer-generated random numbers. The random allocation was generated and implemented by MHR and NSZ. Participants’ assignment and enrolment to interventions was performed by MHR and NSZ based on this randomization.
Blinding
This study was a single-blinded study where each participant and single clinician who took the plaque scores were blinded to the treatment assignment for the duration of the study. The clinician, NAAH and participants were not involved in the intervention assignment.
Mouthrinse Preparation
Three different mouthrinse formulations were prepared in this study: (i) Stingless bee honey (Bayu kelulut ®) mouthrinse containing honey diluted with distilled water to a concentration of 20% (Aina et al., 2021) (Figure 1), (ii) 0.12% chlorhexidine (Oradex Antibacterial Mouthwash, Malaysia. Reg no: MAL06011901XCZ), and (iii) placebo (negative control) containing 0.9% saline solution. These mouthrinse preparations were packed, sealed and coded in identical bottles as shown in Figure 2 before start of study. These formulations were mixed and kept in the refrigerator between 2 to 8°C.
Intervention
Pre-intervention, plaque score was taken at baseline by a single clinician (NAAH), a periodontist. This was to reduce any variation in plaque score recording between different clinicians. Both the participants and the clinician were blinded to the intervention given. After recording the plaque score, scaling and prophylaxis were performed (NAAH). This was to ensure a standardised level of hygiene among all the participants at the study commencement. 90 ml of mouthrinse in a sealed bottle was prescribed by two dental students following a randomised list.
Participants were instructed to refrain from all forms of tooth cleaning and rinse 15ml of mouthrinse twice daily for two minutes. This was done for three days. After three days, the plaque score was measured again by the same clinician (NAAH) and prophylaxis was given by NAAH, NHMB or MAS. Visual Analogue Scale (VAS) score for patient perception was taken for taste, halitosis, burning sensation and odour. The flow of study was summarized in Figure 3.
Outcome Measures
Patient Details
A self-reported questionnaire for patients were administered at Day 1 comprising of questions on sociodemographic information (age, gender, ethnicity), lifestyle habits, medical history and dental history.
Plaque Score
Plaque accumulation was measured using the Plaque Index (PI) - O’Leary et al. 1972 which measures the presence of supragingival plaque on four tooth surfaces. Plaque was scraped using a dental explorer. Plaque incidence in the oral cavity was expressed as an accurate percentage, and the presence (1) or absence (0) of plaque is noted in a simple chart.
Basic Periodontal Examination
Basic Periodontal Examination (BPE) of all sextants was performed before and after the interventions. BPE was performed based on the guidelines by the British Society of Periodontology (BSP) published in 2019. A Community Periodontal Index and Treatment Needs (CPITN) probe was used for all procedures by a single clinician. If any participants had any score of 3, a full mouth 6-point charting was carried out.
Visual Analogue Scale
Participants were asked on the acceptance or comfort towards mouthrinse in terms of taste, halitosis improvement and odour. The level of acceptance or comfort were measured using visual analogue (VAS) scale (Figure 4). The VAS scale consisted of one line where the far-left end scale (0) indicated worst taste while the far-right end (10) indicated best taste, improve halitosis and smell. Participant would choose any number between the two ends that best described the taste they experienced during intervention.
Data Analysis
Data obtained was analysed using IBM SPSS statistical program (Version 27). The normality of data was analysed using the Shapiro–Wilk test and was found to be normally distributed for all data set. Thus, the intragroup comparison of plaque score (before and after) was analysed using paired sample t test. One-way Analysis of Variance (ANOVA) followed by Tukey’s Post-Hoc test was used to compare plaque score between groups after the intervention and patient’s perception. All experimental results were presented as the mean ± standard deviation (SD). Mean values were considered statistically significant for p < 0.05.