Study Design
Fifty middle-aged male completely edentulous patients wearing complete dentures visiting the Excellence Medical Centre, National Research Centre, Cairo, Egypt were screened for the possibility of having any accompanied denture stomatitis (DS) and/or Candida albicans (C. albicans). Patients were all randomly distributed into two groups. Group I patients were treated with chlorhexidine mouthwash and oral hygiene measures (OHM), while Group II patients received ozonated water as a mouthwash and OHM. Evaluation of inflammation, pain grade, and oral Candidiasis count took place for all patients in all visits; initiating from the initial day of treatment and then after 7, 14, and 30 days.
Ethical Approval and Clinical Trial Registration
The current contemplate has been applied to the Code of Ethics of the World Medical Association, following the ethics stated in the Declaration of Helsinki in 1975. This research has been permitted by the “Medical Research Ethical Committee of the National Research Centre, Cairo, Egypt” with agreement number 4447082022. All patients were up-to-date with the existing study’s practical phases and contracted the printed consent. The research was registered on ClinicalTrials.gov with the identifier: NCT06021847.
Sample Size Calculation
The Sample size was estimated liable on preceding research as a reference (Aoun et al 2015). Accordingly, when the mean ± standard deviation of Candida albicans count in the chlorhexidine group was (8 ± 18.807), while the estimated mean difference with the other group was (17) the minimally accepted sample size was 20 per group when the power was 80 % and type I error probability was 0.05. The total sample size was enlarged to 25 per group to reward 20 % drop out. The independent t-test was employed to estimate sample size by using P.S. power version 3.1.6.
Inclusion and Exclusion Criteria
The inclusion criteria were completely edentulous male patients aged from 45-50 years wearing heat-cured acrylic resin complete dentures and suffering from denture stomatitis. Though exclusion criteria were smokers, uncooperative patients together with those having systemically immunosuppressive diseases, Temporomandibular joint (TMJ) disorders, parafunctional habits, and history of radiotherapy or chemotherapy.
Methods
Preparation of Ozonated Water
Ozonated water as a mouthwash was prepared utilizing Ozone of (2-4 mg/l) for 1 min. in a 25 mg of double distilled water 37oC employing an Ozone generator (Ozone generator type N 1888A, China.)(Celakil et al 2017).
Patient's Instructions and Follow-Up during the Healing Process
All patients in this contemplate were properly diagnosed and instructed to have strict oral and denture hygienic measures together with sticking to their frequent follow-up visits to regularly check their healing progress. Furthermore, proper elimination of all the reasons for their inflammation and pain from their prosthesis took place. Group I patients were treated with chlorhexidine mouthwash, while group II patients received ozonated water as a mouthwash.
Assessment of Inflammation and Pain Grade
Inflammation was estimated by utilizing the Schwartz et al 2016. Inflammation index comprises two color-coded sections; the yellow one represents the area where inflammation is located intraorally and the other green one symbolizes its severity. The yellow area section provides a choice of 4 answers, and each is given a score (0 = No inflammation; 1 = Inflammation of the palate extending up to 25% of the palatal denture-bearing tissue; 2 = Inflammation of the palate covering between 25% and 50% of the palatal denture-bearing tissue; 3 = Inflammation covering more than 50% of the palatal denture-bearing tissue). The green severity section provides a choice of 4 answers, and each is given a score (0 = Normal tissue; 1 = Mild inflammation (slight redness, no swelling or edema); 2 = Moderate inflammation (redness with some edema); 3 = Severe inflammation (acutely inflamed redness, edema).
Whereas, the pain scale was estimated by employing the Granick et al 1998 pain index which comprises 5 grades of pain providing a choice of 5 answers, and each is given a score (Grade 0 = No symptoms; Grade 1 = Mild discomfort and capable of eating; Grade 2 = Moderate discomfort but still capable of eating; Grade 3 = Sever discomfort and unable to eat; Grade 4 = Tolerated pain and unable to eat). The overall score was estimated by gathering the replies to all the questions. The lowest score revealed a lack of inflammation with normal tissues and asymptomatic pain, consequently a satisfactory perception of an individual’s oral condition; as displayed in Table (1).
Table (1): Schwartz et al 2016 inflammation index and pain scale by Granick et al 1998 for determination of signs of inflammation and pain grade for patients of both groups in all visits
Schwartz et al 2016 Inflammation index
|
Area
|
Severity
|
0
|
1
|
2
|
3
|
0
|
1
|
2
|
3
|
No inflammation
|
Inflammation of the palate extending up to 25% of the palatal denture-bearing tissue
|
Inflammation of the palate covering between 25% and 50% of the palatal denture-bearing tissue
|
Inflammation covering more than 50% of the palatal denture-bearing tissue
|
Normal tissue
|
Mild inflammation (slight redness, no swelling or edema)
|
Moderate inflammation (redness with some edema)
|
Severe inflammation (acutely inflamed redness, edema)
|
Granick et al 1998 Pain scale
|
Grade 0
|
Grade 1
|
Grade 2
|
Grade 3
|
Grade 4
|
No symptoms
|
Mild discomfort and capable of eating
|
Moderate discomfort but still capable of eating
|
Sever discomfort and unable to eat
|
Tolerated pain and unable to eat
|
Assessment of Oral Candidiasis
Oral Candidiasis was assessed employing the swab technique, as swabs were obtained for all patients from both the palate and maxillary dentures’ fitting surfaces to detect the existence or absence of C. albicans and its counting if present.
a. Preparation of Chromogenic Agar Media for Identification of Candida Albicans
A suspension was fabricated comprising 47.9 gm of the medium properly mixed in one liter of distilled water and dissolved by heating with regular whisking. Then accompanied by boiling for one minute till the entire dissolution and followed by dispersion into Petri dishes. Then a homogeneous, free-flowing, clear amber and slightly opalescent medium was ready and stored at 8-15°C.
b. Isolation of Candida Albicans
Swabs were attained by dynamic rubbing of both palate and the entire maxillary complete dentures’ fitting surfaces by employing a sterile cotton-tipped wooden swab for 30 sec/ patient. Then these cotton swabs were inoculated separately in 10.0 mL sterile screw-capped tubes, where each tube comprised 2.5 mL nutrient broth medium then incubated at 37 ºC for 48-hour incubation period in a peculiar incubator (*VWR Shel-Labs 1530 Lab Incubator-China). Afterward, during the proper incubation period, dishes were removed from the incubator and detected to check the incidence of light green colonies with the naked eye resembling C. albicans colonies as displayed in figure (1).
Statistical Analysis
All numerical data were offered as mean and standard deviation values. Normality exploration of the given data was accomplished utilizing the Shapiro Wike and Kolmogorov test which revealed area and degree of inflammation as well as degree of pain demonstrated non-parametric data. Accordingly, a comparison between different follow-up visits was performed by employing Friedman`s test accompanied by Dunn's multiple comparisons test, while a comparison among the two dissimilar groups was performed by utilizing Mann Whiteny`s test. On the other hand, Candida albicans growth revealed normal data distribution. Consequently, comparison between different follow-up visits was performed by employing Repetitive One-Way ANOVA test accompanied by Tukey`s Post Hoc test for multiple comparisons, while comparison between different surfaces of both groups was accomplished via One-Way ANOVA test followed by Tukey`s Post Hoc test for multiple comparisons.