Original article. The use of essential oils based antiseptic solution in the treatment of denture stomatitis

Summary Introduction Local therapy of denture stomatitis (DS) associated with Candida species fungi infection usually involves the application of nystatin and miconazole. Due to the fact that these drugs may be less efficient against biofilm and possible resistance development, a new approach in the treatment includes the use of antiseptic agents. The aim of the study was to compare clinical and microbiological therapeutic outcomes of antiseptic solution Listerine® and Daktanol® antifungal oral gel in the treatment of DS associated with Candida species fungi. Material and Methods The study included 30 patients affected by DS, divided into the two treatment groups, control group (n=15) treated by Daktanol® gel and experimental group (n=15) treated by the antiseptic solution Listerine®. Successful treatment was evaluated based on palatal mucosa inflammation reduction classified according to the Newton classification and the difference in the number of fungal colony- forming units (CFU) isolated by smears before and after the treatment that lasted 14 days. Results Reduction in inflammation intensity and fungal CFU number on palatal mucosa (p<0,01) as well as on denture base (p<0,01) were observed in both groups of subjects after the treatment. Conclusion Antiseptic solution Listerine® and Daktanol® antifungal gel both reduced palatal mucosal inflammation and CFU number of fungi in mouth without significant differences among them. CFU number of fungi isolated from denture base was significantly lower after the treatment with Listerine® (p<0.05).


INTRODUCTION
Denture stomatitis (DS) is an inflammation of oral mucosa covered by the denture base with reported incidence of 15-70% [1]. It is linked to a number of non-infectious aetiologies such as poor quality of dentures, poor hygiene and nocturnal denture wearing as well as infectious etiological factor, Candida species fungi [2]. Number of fungal cells in the saliva and on dentures is leading etiological factor for DS development [3]. The degree of Candida albicans (C.albicans) denture base contamination is directly correlated to inflammation intensity [4]. This disease is characterized by erythema of oral mucosa covered by the denture base; it is usually asymptomatic and often associated with angular cheilitis [5]. Its intensity is clinically evaluated according to the Newton classification criteria [6,7]. DS treatment most frequently includes local and systemic administration of antifungals, reduction and eradication of biofilm, change of bad habits related to nocturnal denture wearing and poor denture hygiene as well as replacement of inadequate dentures. The most frequently used local antifungal drugs in dental practice are nystatin and miconazole [8,9]. Although effectively act against fungi, these drugs have no effect on biofilm matrix, therefore well-protected pathogens can survive in extracellular matrix. Also, their use comes with the risk of resistance development [10,11]. Commercially available oral antiseptic Listerine® acts directly against fungal cell, chemically, causing damage to the cell wall structure and membrane permeability. Also, it disrupts metabolic processes dependant on microorganism membrane enzymes, and, as other phenolic products, exerts anti-inflammatory effect [12]. Listerine® acts against free unbound Candida cells as well as against formed biofilm and it is shown to be more efficient than the azole antifungal drugs [13]. This Listerine® solution property has not been studied in a clinical trial yet.
The aim of this study was to compare clinical and microbiological outcomes of antiseptic agent Listerine® and antifungal Daktanol® gel in the treatment of DS associated with Candida species fungi.

MATERIAL AND METHODS
This prospective clinical study was conducted at the Department of Dental Medicine in Foca and the Microbiological laboratory of the University Hospital Foca. The research was conducted in accordance with the principles of the Helsinki Declaration of 2008. Prior to inclusion in the study subjects were informed about the aim and the protocol of research and gave their consent to participation. The study included 30 patients, maxillary acrylic dentures wearers, affected by DS. Criteria for inclusion of patients in the study were good general health, or chronic well-controlled disease and dental acrylic dentures wearing for at least one year. Also, swabs of palatal mucosa and denture basal surface had to be positive on Candida species fungi. Exclusion criteria were as follows: the use of corticosteroids and immunosuppressive therapy, tumours of the maxillofacial region and radiotherapy in the head and neck area, chemotherapy in the last year, blood disorders, local surgical therapy in the last 3 months, local or systemic use of antibiotics and antifungal drugs in the last 3 months, the use of hormones for therapeutic purposes, the existence of other diseases in the oral cavity and also functionally, prophylactically and cosmetically inadequate dentures that are indicated for replacement with new dentures.
By the means of random numbers table, participants who joined the study under even-number were classified into the control group and received standard treatment with Daktanol® oral gel (2% miconazole, Galenika a.d. Belgrade, Serbia). The participants included in the study under odd-number were classified into the experimental group and were treated with antiseptic agent Listerine® cool mint TM (Johnson&Johnson, S.p.A. Rome, Italy). Each group consisted of 15 patients, and therapy was administered according to the following protocol: 1. Control group of patients (n = 15) received standard local antifungal therapy in the form of Daktanol® gel containing 2% miconazole, used at the dose of 1-2 teaspoons, 4 times per day for 14 days. Basal surface of dentures were coated with gel overnight and rinsed with water in the morning prior to use.
2. Experimental group of patients (n = 15) used an antiseptic agent Listerine® also for 14 days as follows: 4 times a day 1 minute long mouth swish with antiseptic solution that was spitted afterward. Dentures were immersed in this solution overnight with base facing upward in a glass container, and completely overflowed by the Listerine® solution. In the morning dentures were rinsed with water.
Parameters used for the evaluation of inflammation and denture contamination, as well as the presence of expected improvement were clinical and microbiological. Clinical improvement implied palatal mucosa inflammation intensity reduction after the treatment compared to the inflammation intensity prior to the therapy.

Clinical parameters
The intensity of palatal mucosa inflammation was assessed by Newton classification which distinguishes three clinical types of denture stomatitis [6,7]: type Newton I: dotted hyperaemic shape and size of a pinhead lesion (pin-point) that present localized areas of poorly expressed inflammation; type Newton II: diffuse erythema of palatal mucosa covered by the denture base -general-ized inflammation; type Newton III: granular surface of the mucosa-inflammatory papillary hyperplasia.

Microbiological parameters
Swabs of palatal mucosa surfaces and denture bases were taken without prior mouth and dentures rinsing, in the morning, before any food intake. Sabouraud dextrose agar was used to grow fungi and it was incubated at 37 ° C, for 48 hours. The number of fungal colonies (CFUcolony forming units) was counted after 48 hours. The following criteria were used for CFU quantification: <10 colonies present after incubation -smear is negative; 10-25 colonies present after incubation -fungi present in small numbers; >25 colonies present after incubationfungi are present in large numbers [14]. Two days after the treatments, control examinations were conducted and swabs for samples cultivation were taken again.
The obtained data were statistically analyzed in the SPSS software (SPSS for Windows, version 11.5, Chicago, Ill.). Description of the sample was carried out by descriptive statistics methods. Therapeutic results within a group (paired samples) were evaluated by the Wilcoxon Signed Rank test. The effect of the therapy on the clinical improvement was assessed using the Fisher's Exact Test, and therapeutic results between observed groups were compared using χ² test. The relationship between certain characteristics and inflammation intensity as well as therapy outcomes were estimated using χ² test and Fisher's Exact Test. Results are presented in tables.
In both groups, significant reduction in the CFU number at the palate (p <0.01) as well as at the denture base was observed after the treatments (p <0.01) ( Table 1).
Inflammation intensity reduction was observed in most patients, but significant difference after treatments between the two applied therapeutic modalities was not observed ( Table 2). There was no significant difference in palate smear CFU number reduction in relation to the applied therapy, but significant difference (p <0.05) was observed in denture smear CFU number reduction in patients treated with antiseptic agent Listerine® compared to patients treated by Daktanol® oral gel (Table 3).
Gender, age of patients or age of dentures, had no statistically significant effect on clinical improvement after the treatment, as well as on the reduction in the CFU numbers isolated from palatal mucosa and denture basal surface after the treatment (

DISCUSSION
The present study evaluated the effect of oral antiseptic Listerine® on palatal mucosa inflammation and Candida species CFU number isolated from palatal mucosa and denture base among denture wearers affected with DS.
Literature review couldn't identify other similar clinical studies. Yet, there are indirect evidences of Listerine® antifungal properties that justify its use in DS treatment. Meiller et al. conducted an in vitro study in which they observed the effect of Listerine® on clinically isolated Candida species, British and American strains of the same species. Fungal cells were incorporated into an experimental biofilm. Authors reported that after 60 seconds of experimental biofilm exposure to this antiseptic, no living fungal cells were observed in the sample [15]. In a study conducted with clinically isolated Candida species, Lis-terine® showed very good antimicrobial activity in laboratory testing. After 60 minutes, there was no living cell of Candida species in the sample [16]. Listerine® was also efficient against experimental biofilm composed of one laboratory and 34 clinically isolated C. albicans strains where it reduced metabolical activity of fungi for 75-80% [13]. The effect of Listerine® against C.albicans clinical isolates was confirmed in a recent study where Listerine® reduced C.albicans growth on Sabouraud dextrose agar [17]. The results presented in this study clinically confirmed findings of previous in vitro studies.
In the present study, Listerine® showed better efficacy in reducing the number of CFU isolated from denture basis compared to the Daktanol® gel. This finding could be the result of different viscosity of used agents. Miconazole was applied in a form of gel, what might hinder its denture base coating. However, due to its physical caracteristics, Listerine® solution, could reach rugged, porous acrylic surface more easily. Beside therapy, improved hygiene and avoiding nocturnal dentures wearing could have positive impact on palatal mucosa inflammation reduction.

CONCLUSION
Therapeutic outcomes after the use of antiseptic agent Listerine® in DS treatment are similar to the therapeutic outcomes obtained by standard Daktanol® oral gel therapy. Therefore, Listerine® can be used in the treatment of DS associated with Candida species.