Antifungal Effect of Thymus Vulgaris Essential Oil and Chlorhexidine on Candida Albicans Colonies Accumulated on Removable Orthodontic Appliances: A Double- blinded Clinical Trial.

Objectives: To assess the ecacy of Thymus vulgaris (T. vulgaris) essential oil to decrease the count of Candida albicans (C. albicans) accumulated on removal orthodontic appliances. Methods: In this 2 arm parallel controlled clinical trial, Forty-four patients whose removable orthodontic appliances contaminated with C. albicans were randomly divided into two groups for use of T. vulgaris essential oil and 0.2% Chlorhexidine (CHX) by electronic random sampling. The C. albicans colony count was measured at baseline (T0) and at 2 (T1) and 5 (T2) days after the intervention. Microbial samples were collected by a sterile swab from 3 spots on the internal surface of orthodontic appliances. The Friedman test was used for within-group comparisons while the Mann Whitney test was applied to compare the ecacy of T. vulgaris essential oil and CHX. Results: No signicant difference was noted in the ecacy of 2% T. vulgaris essential oil and 0.2% CHX in decreasing the C. albicans colony count on removable orthodontic appliances. Conclusion: T. vulgaris essential oil has favorable antimicrobial activity against C. albicans. The medical and dental history of patients and their oral health, dental status, type of malocclusion, dental arrangement, and oral hygiene were evaluated before the study. Of 83 candidates for participation in the study, those with large diastema or periodontal problem, patients using antimicrobial mouthwashes, those who reported antibiotic intake past 3 and patients with very poor oral hygiene were excluded. According

Chlorhexidine (CHX) is the gold standard for chemical removal of microbial plaque, which is often prescribed as an adjunct to mechanical plaque removal methods. However, long-term use of chemical agents is not recommended [15][16][17][18].
Use of herbal essences for their antimicrobial properties has been previously evaluated [19][20][21]. Several medicinal plants such as thyme, cloves, lavender and tea leaves have been tested for disinfection of the oral cavity [22][23][24][25][26][27]. Given that medicinal plants are suitable alternatives to chemical agents, application of chemical agents can be limited to acute cases, and herbal products can be recommended for continuous long-term use by orthodontic patients during their treatment course [18].
Thymus (thyme) is an important medicinal herb that belongs to the Lamiaceae family. Thymus, Ziziphora, and Zataria are the three main genera of the Lamiaceae family. The thymus genus has 14 species in Iran, and Thymus vulgaris (T. vulgaris) is one of the most important species [33]. Thymol and carvacrol are the main constituents of T. vulgaris essence. Also, it contains tannin, avonoids, and saponins [34]. The T. vulgaris essential oil has several pharmaceutical and therapeutic applications [25]. It also has high chance of acceptance by patients due to its favorable taste and odor, insigni cant toxicity and side effects, and low cost [35].
Essential oil is the alcoholic extract with a volatile oil, which can be extracted by different techniques such as the cold-pressed extraction method, distillation, extraction by solvent, distillation under vacuum using a microwave, maceration, or extraction with absorbent oils obtained from herbal products [36].
Distillation is an important method of essential oil extraction, which is based on separating the components or substances in two phases [37]. Evidence shows that Zataria multi ora (Shirazi thyme) has suitable antimicrobial and antifungal properties for irrigation of root canals in endodontic treatment in vitro, compared with chemical irrigating solutions [38,39]. Also, Zataria multi ora essential oil is effective for disinfection of acrylic plates and orthodontic elastics in vitro [40,41]. Favorable antifungal activity of T. vulgaris essential oil, compared with CHX, has also been reported for disinfection of removable orthodontic appliances in vitro [42].

Speci c objectives and hypotheses
This study aimed to compare the effects of T. vulgaris essential oil and CHX on C. albicans accumulated on removable orthodontic appliances. The null hypothesis was that there was no difference in Antifungal e cacy of T. vulgaris essential oil and CHX.

Materials And Methods
Trial design and any changes after trial commencement This double-blind, single-center, parallel clinical trial with 1:1 ratio was approved by the ethics committee of Islamic Azad University, School of dentistry, Shiraz Branch (Reference: IR.IAU.SHIRAZ.REC.1397.004) and registered in the Iranian Registry of Clinical Trials (IRCT20180923041092N1). There were no changes to the trial after its commencement.
Participants, eligibility criteria and settings: Patients with removable orthodontic appliances with a midline expansion screw whose orthodontic treatment had commenced 6 to 8 months earlier in the Orthodontics Department of School of Dentistry of Islamic Azad University, Shiraz Branch were enrolled in this study. The medical and dental history of patients and their oral health, dental status, type of malocclusion, dental arrangement, and oral hygiene were evaluated before the study. Of 83 candidates for participation in the study, those with large diastema or periodontal problem, patients using antimicrobial mouthwashes, those who reported antibiotic intake in the past 3 months, and patients with very poor oral hygiene were excluded. According to the inclusion and exclusion criteria, 55eligible patients were selected. The participants were briefed about the study, and written informed consent was obtained from them.
Primary sampling to detect patients with C. albicans: One main inclusion criterion for the study was presence of C. albicans on removable orthodontic appliances of patients. Thus, a primary sampling was performed for assessment of the presence/absence of C. albicans on removable orthodontic appliances. For this purpose, a sterile swab dipped in sterile distilled water was used to collect samples from three spots on the internal surface of the removable appliance that was in contact with the oral mucosa. The three spots were (I) at the center of the appliance, (II) close to the gingival margin of teeth #4 and #5, or D and E in the right side, and (III) close to the gingival margin of teeth #4 and #5, or D and E in the left side. The collected sample was streak cultured on a speci c culture medium for C. albicans. The plates were coded and sent to a microbiology lab for the microbial culture. Each participant was allocated a code. Of 55 patients, 44 were positive for C. albicans and were enrolled in the study.
Microbial culture: The Sabouraud dexterous agar containing chloramphenicol is a speci c culture medium for C. albicans [26]. Thus, the collected microbial sample was streak cultured on this culture medium, and the plate was then incubated for 24-48 h. The proliferated colonies of C. albicans were observed on the culture medium after this time period.

Staining:
Lactophenol cotton blue dye, which is a strong dye for diagnostic staining of slides, was used for identi cation of C. albicans colonies, and their differentiation from other microorganisms.
Microorganisms absorb cationic chromatophores due to their slight negative charge, which reveals the morphology of microorganisms under a microscope. For staining, two drops of distilled water were applied at the center of a sterile slide and one drop of lactophenol cotton blue dye was added to distilled water. Next, some colonies were added and dissolved in the dye using a sterile loop such that a homogenous suspension was obtained. Next, the slide was inspected under a microscope at x4, x10, x40 and nally x100 magni cation to identify and study the microorganisms.

Intervention:
After nding the patients with orthodontic appliances contaminated with C. albicans, patients were randomly divided into two groups of 2% T. vulgaris essential oil and 0.2% CHX control group.
Intervention group: In the intervention group, the patients received T. vulgaris essential oil spray. In order to prepare the T. vulgaris essential oil, some research was conducted to nd T. vulgaris with higher concentrations of thymol and carvacrol. Thus, T. vulgaris was obtained from Aliabad Tang village in Beyza city of Fars Province, Iran, and T. vulgaris essential oil was extracted by distillation. Analysis of the essence was performed by gas chromatography/mass spectrometry (GC-MS; Agilent Technologies-5975C-MS,7890A-GC) to measure the concentrations of thymol and carvacrol. A clinical trial aiming to determine the minimum inhibitory concentration (MIC) of T. vulgaris essential oil against C. albicans reported that the MIC of T. vulgaris essential oil against C. albicans was 1.56% (equal to 15.6 µL/mL). 42 Thus, in this study, 2% concentration of T. vulgaris essential oil was formulated after taking into account the possibility of procedural errors and in order to increase the effect size.
In the formulation of T. vulgaris essential oil, distilled water was used as the aqueous phase, T. vulgaris essential oil was used as the organic phase, and Span 80 and Tween 80 were used as surfactants. In order to prepare 2% formulation, 2 wt% of a mixture of Tween 80 and Span 80 along with the essence were added to 96 wt% of distilled water. Regarding the order of mixing, rst Tween 80, Span 80, distilled water and essence were separately weighed. Next, distilled water was added to Tween 80, and the essence was added to Span 80. Eventually, the mixture of essence and Span 80 was added to the mixture of water and Tween 80. The nal solution was mixed for 1-3 min, and sonication was performed for 15 min at room temperature in order for the components to form a homogenous mixture. After preparing 2% T. vulgaris essential oil, it was packaged in the form of 50 mL sprays, which were lled with the solution using a funnel. The sprays were also coded. The patients received comprehensive instructions on how to use the sprays and also regarding daily hygienic measures. Also, comprehensive instructions for use were provided on the labels of the sprays. The patients were requested to spray the internal surface of their orthodontic appliance from 15 cm distance every night after washing their appliance and then rinse it again 15 min after spraying.

Control group:
The control group received 0.2% CHX Najo in the form of spray with the same packaging and labeling as those administered among the test group. Similarly, the control subjects were provided with thorough instructions on how to use the spray and perform daily hygienic measures. Also, comprehensive instructions for use were provided on the labels of the sprays. The sprays were then coded.
Outcomes (primary and secondary) and any changes after trial commencement The second microbial sampling was performed in both groups at baseline prior to the commencement of clinical trial (T0), and at 2 (T1) and 5 days (T2) after using the sprays. A sterile swab was used for sampling from three spots of the appliance as explained earlier for the rst sampling. The person preparing and providing the sprays to the participants and those responsible for culture and diagnosis of the slides were all blinded to the group allocation of samples. There were no changes to the trial after its commencement Sample size calculation: A total of 40 participants were required to achieve 85% power (instituted by G power, version 3.0.1; Franz Faul universitat, Kiel, Germany) and detect signi cant differences considering the effect size of 0.47 (P < 0.05). Considering the possible dropouts, the sample size was increased to 44.
Interim analyses and stopping guidelines Not applicable.

Randomization:
Before the trial commencement, randomization process, allocation concealment, and implementation were performed by a researcher who was not involved in the study. Random Allocation Software program was used for simple electronically-generated randomization.
Randomization ensured patients' allocation to both groups with 1:1 ratio. Allocation concealment was ensured by using numbered, sealed, opaque envelopes prepared before the trial commencement. The envelopes were sequentially opened only after the envelope had been irreversibly assigned to a participant Blinding: Double-blinding was ensured in this study. Only the chemist who cooperated with us was aware of the distribution of disinfectants. She assigned a number to each bottle that only she was aware of.

Statistical analysis:
Data were analyzed using SPSS version 22. Distribution of data was evaluated using the Kolmogorov-Smirnov test. Since data were not normally distributed, non-parametric tests were applied. The Friedman test was used to assess the e cacy of T. vulgaris essential oil and CHX within each group. The Mann Whitney test was applied to compare the e cacy of T. vulgaris essential oil and CHX.

Participant ow
Of 83 patients, 28 were excluded since they did not meet the eligibility criteria. Of the remaining 55, 44 had removable orthodontic appliances contaminated with C. albicans and were enrolled in the study. Recruitment began in April 2019 and was completed in August 2019. Two participants were later excluded since their orthodontic appliance broke and they did not cooperate well after the trial commencement; The CONSORT diagram is shown in Fig. 1.

Baseline data
The baseline characteristics for age, sex and the time point at which patients were recruited were similar in both groups ( Numbers analyzed for each outcome, estimation and precision, subgroup analysis For data analysis, the colony count was classi ed into ve groups of rare, few, moderate, many and numerous ( Table 2). The Friedman test revealed that continuous use of T. vulgaris essential oil or CHX signi cantly decreased the colony count (P < 0.05, Table 3). The Mann Whitney test showed that this reduction was not signi cantly different between the two groups of T. vulgaris essential oil and CHX (Table 3). Figure 2 shows the change in the mean rank of colony count (contamination rate) in the two groups. Although the mean difference in the rst and third samplings was the same and equal to 0.1, the 2% T. vulgaris essential oil graph was completely descending, while the 0.2% CHX graph reached a plateau from the second phase on.   Figure 3 shows the relative frequency distribution of different levels of colony count in the two groups at different time points. The relative frequency of rare class was 6.7% in both groups at baseline while the frequency of rare class was 53.3% in the 2% T. vulgaris essential oil and 26.7% in the CHX group in the nal sampling, which indicated higher e cacy of T. vulgaris essential oil than CHX. The relative frequency of numerous class was 80% in T. vulgaris essential oil group at baseline, which decreased to 40% in the nal sampling. The remaining 40% was distributed among different classes, with rare class having a higher frequency. This rate was 66.7% at baseline in the CHX group, which decreased to 33.3% in the nal sampling. The remaining percentage mainly belonged to the few class, which indicated the superiority of T. vulgaris essential oil to CHX.

Results of GC-MS:
The interpretation of GC-MS results revealed that the essential oil used in this study contained 43.7% thymol. Table 4 lists the 14 main constituents of this essential oil.

Harms
No harm was observed or reported from the participants in the study.

Discussion
Orthodontic appliances can impair the balance of microorganisms in the oral environment and may even lead to disease [10,11]. The acrylic base of removable orthodontic appliances is made of polymethyl methacrylate-based auto-polymerizing resin while the wires are made of stainless steel. The pathogenic microorganisms can lodge in resin porosities on the external and internal surfaces of acrylic components. Also, degradation of acrylic resin can cause bacterial bio lm accumulation [13].
The mechanical plaque removal methods such as tooth brushing are suitable for elimination of the accumulated bio lm. Nonetheless, mechanical methods alone are not highly effective in many individuals due to poor motivation or hand skills [43]. Thus, chemical plaque removal agents are always considered along with the mechanical methods. Many chemical and herbal products are used for elimination or reduction of microorganisms [44]. CHX is the gold standard for chemical removal of the microbial plaque. Nonetheless, CHX is a synthetic chemical agent, which can have many side effects following long-term application [18]. Thus, long-term use of chemical agents is not recommended [15]. In the recent years, the demand for alternative plaque removal methods has greatly increased. Herbal products are gaining increasing popularity due to their organic nature and fewer side effects [27]. The results of this clinical trial showed that T. vulgaris essential oil and CHX effectively decreased the C. albicans colonies accumulated on orthodontic appliances. In this study, the T. vulgaris plant was collected early in April, and the aerial part of the plant was used to ensure its high quality.
T. vulgaris is a medicinal plant with antimicrobial properties [45]. It has favorable taste and odor and low cytotoxicity [35]. Nonetheless, the in vivo effect of T. vulgaris essential oil on C. albicans isolated from removable orthodontic appliances has not been previously evaluated. According to a study by Kaviani et al, the MIC of T. vulgaris against C. albicans isolated from the surface of acrylic appliances was 1.56% (15.6 µL/mL) [42]. Thosar et al. reported the MIC of T. vulgaris essential oil to be 1.6% (16 µL/mL), with the difference that they used standard-strain C. albicans in vitro [24]. Moreover, Fani et al. showed that the MIC of T. vulgaris essential oil was 1.63% (16.3 µL/mL) [46]. In this clinical trial, 2% T. vulgaris essential oil was formulated to compensate for the possible procedural errors.
Although immersion of orthodontic appliances in disinfecting solutions for a certain period of time is the conventional method for disinfection of these appliances at home [47], immersions can lead to water sorption and subsequent adverse changes in the acrylic resin structure. In this study, the spraying technique was used instead of immersion to prevent possible changes in acrylic resin structure. Also, spraying is cost-effective and easier for patients [13,48].
The results of this clinical trial revealed that 2% T. vulgaris essential oil and 0.2% CHX had suitable antifungal effects on orthodontic appliances contaminated with C. albicans. Nonetheless, the reduction in colony count was not signi cantly different between the two groups.
The antimicrobial properties of medicinal plants mainly depend on the presence and concentration of phenolic compounds, saponins, and avonoids in their structure. These compounds can adversely affect the plasma membrane of microorganisms or inhibit their structural cell membrane enzymes and exert their antimicrobial effects as such [49].
Thymol and carvacrol are among the main constituents of T. vulgaris essential oil, and the antifungal properties of this plant can be attributed to these ingredients. Carvacrol is an iso-thymol that increases the activity of ATPase and inhibits the activity of the enzyme responsible for non-speci c permeability of the cell membrane of microorganisms such as bacteria, and consequently increases their sensitivity to external factors entering the cell [50]. Thus, difference in the MIC of T. vulgaris essential oil can be attributed to the variations in the percentage of its constituents.
Assessment of the mean rank-time graph revealed that continuous use of T. vulgaris essential oil during the study period caused a continuous reduction in C. albicans colony count. However, CHX caused a reduction in colony count to some extent and then reached a plateau. Also, assessment of the relative frequency of C. albicans colonies revealed that T. vulgaris essential oil was more effective than CHX for reduction of the relative frequency of C. albicans colonies.

Generalizability
The patients recruited for this study were 10 to 12 years old, this shows the age range for removable orthodontics appliance and allowing the results to be generalized across this age range. Limitation: Short intervention period: to evaluate the effect of prolonged use of T. vulgaris essential oil on oral mucosa and teeth, enlarging treatment duration is necessary. Further studies on the antimicrobial e cacy of T. vulgaris essential oil against other microorganisms accumulated on orthodontic appliances and dentures are required prior to extensive clinical use of this essential oil as an alternative to CHX.

Conclusion:
Although this study found no signi cant difference in antifungal e cacy of 2% T. vulgaris essential oil and 0.2% CHX against C. albicans isolated from removable orthodontic appliances, 2% T. vulgaris essential oil can be suggested as an alternative to CHX for elimination of fungal contamination of orthodontic appliances, considering its lower cytotoxicity, fewer side effects, and higher patient acceptance, compared with CHX. Availability of data and material: The datasets used and/or analyzed during the present study are available from the corresponding author.
Competing interests: Change in the mean rank of colony count in the two group s T0: rst day of intervention; T1: Two days after intervention; T2: Five days after intervention Supplementary Files