ETIOLOGY AND RISK FACTORS OF STOMATITIS AMONG YEMENI DENTURE WEARERS

Objectives: Denture stomatitis (DS) is a benign condition, usually asymptomatic, that can affect denture wearer’s patients. The aim of this study was to describe the etiology of DS among Yemeni denture wearers and the risk factors of DS. A cross sectional study was carried out at Prothodontics Departments at the Faculty of DentistrySana'a University, Sana’a city –Yemen. Methods: The study group consisted of 288 denture wearers' patients' contracting DS. The microbiological samples were collected by wiping sterile swabs on upper and lower denture fitting surfaces. Then, they were directly cultured for Candida in Chromomeric agar media and for bacteria in standard selective media, using standard bacteriological methods. Results: The most common microbial cause of DS was C. albicans (58.3%), followed by S. mutans (17%), while S. aureus (4.9%), lactobacillus (3.1%) and C. glabrata (4.2%) were less common. There was an association between male patients, older age group and longer period of denture wearers with high risk of contracting candidal DS and bacterial DS. Most of those patients were suffering from DS Type I and few from Type II, but no case of DS Type III was found. Ending, DS is a condition that commonly affects denture wearers, and should be treated even if asymptomatic. The condition requires a combined treatment approach from both patient and clinician, and the role of the patient must be stressed. Conclusion: Management of aetiological risk factors is key in order to prevent recurrence. Treatment modalities may include: treatment of any underlying systemic risk factors, improvement in the fit of existing dentures, replacement of existing dentures, improved denture hygiene and the use of antifungal agents. Whichever methods are employed, the main aim of treatment is to eradicate the biofilm from the patient’s dentures.


INTRODUCTION
Denture stomatitis (DS) is a chronic inflammatory reaction commonly seen in denture wearing patients. Where mild inflammation and redness of the oral mucous membrane occurs beneath a denture 1 . The etiology is considered as multi factorial, with the prosthesis is considered as the prime etiologic factor. Etiological factors include poor denture hygiene, continual and night time wearing of removable dentures, accumulation of denture plaque, age of the denture and dryness of the mouth. Poor-fitting dentures may increase mucosal trauma which play a vital role in type I denture stomatitis and least importance in other types of DS, plaque on the inner surface of the denture harbors micro-organisms causing inflammation of the mucosa, bacterial and yeast contamination of denture surface in which C. albicans has been shown to be highly implicated in the etiology of DS and may account for 90% of cases 2, 3 . C. albicans and S. aureus are microorganisms with an elevated adhesion capacity to the oral mucous. This adherence is enhanced in vitro when Candida is incubated simultaneously with Streptococcus mutans (S. mutans), Streptococcus sanguis (S. sanguis), Streptococcus salivairus (S. salivairus) or some other bacteria 3 . There are many predisposing factors for DS such as oral hygiene, old age, diabetes mellitus, trauma, xerostomia, high carbohydrate diet, use broad spectrum antibiotics and smoking tobacco 4-6 . Staging different classifications have been proposed, but the reference classification for DS is the one suggested by Newton in 1962, based exclusively on clinical criteria: According to Newton, it can be classified into 3 types: Type 1-Localized simple infection with pinpoint hyperemia and is usually trauma induced 7, 8 . Type 2-Erythematous type, covers the entire or a part of the denture covering area 7, 8 . Type 3-Granular type, involving the central part of the hard palate and the alveolar ridge 9 . The aims of the study were to determine the different microbial causative agents of denture stomatitis and also detect the risk factors contributing for the severe cases of denture stomatitis (type II and type III).

SUBJECTS AND METHODS
Cross sectional study was carried out in the Prosthodontic department at the Faculty of Dentistry-Sana'a University, from 11/2016 to 2/2017 on 288 Yemeni subjects (males and females), aged more than 35 years old. Data were collected by predesigned questionnaire. Samples were collected by wiping premoistened sterile cotton wool swabs on upper and lower denture fitting surfaces. Two swabs were collected for each patient one for mycological and the other for bacteriological investigation. For mycological investigations each sample was inoculated into both media: Sabouraud's dextrose agar and a differential and selective culture medium as Chrom agar Candida. Plates were incubated aerobically for 48 to 72 hours at 37 º C and were identified systematically by Colonial morphology, wet mount preparation, gram stain and germ tube. However for bacteriological investigations samples of S. mutans and S. aureus were cultured into Mitis Salivarius-Bacitrac in agar and blood agar plate media and incubated at 35-37°C for 48 hours with addition 1% Potassium Tellurite. Then, were identified and differentiated from each other by their colonial morphology, catalase test, gram staining and microscopically examine, mannitol and sorbitol fermentation tests, hemolysis and coagulase test. But for Lactobacillus species, it were cultured overnight in MRS agar plate and incubated at 37 º C and then it were identified by their colonial morphology, gram staining, growth at 15°C and growth at 45°C and catalase test. Data collection: Data including demographic data of the patients, clinical information, and potential risk factors of DS. The findings were recorded in a form with laboratory results.

Ethical approval
We obtained written consent from all cases. Assent was taken from participants before collecting the specimens. The study protocol was reviewed and approved by the Ethics Committee of Sana'a University, Faculty of Medicine and Health Sciences.

RESULTS
This study was conducted on a total of 288 patients contracting DS attending Prosthetic department at the Faculty of Dentistry-Sana'a University from 11/2016 to 2/2017. Male patients were predominant in which they counted 59.4%, while female patients were counted 40.6%. The patients' age ranged from 35-65 years, and most of the patients were at the age group of +65 years, (32.6%). The most common microbial causes of DS was C. albicans (58.3%) of the total isolates, while S. mutans was the most common bacterial cause of DS(17%), while S. aureus (4.9%), lactobacillus (3.1%) and C. glabrata (4.2%) were less common. There was a highly significant association between male and high risk of contracting Candida albicans DS (OR=2.33, CI=1.4 to 3.9, and p< 0.001).
There was a highly significant association between older age group (65+ years) and the high risk of contracting Candida albicans DS (OR=3.62 times, CI=2 to 6.6, with p <0.001).   When denture fitness was considered, there was a highly significant association between the poor fitness and the high risk of contracting bacterial DS (OR=10.22 times, CI=5.2 to 20.6, with p< 0.001). Also, there was a highly significant association between longer period of 18-over months and the high risk of contracting bacterial DS (the associated OR=2.5 times, CI=1.4 to 4.5 with p< 0.001). Most of patients were suffering from DS Type I in which it was counted 66.7%, while only 33.3% of patients were suffering from DS Type II, but no case of DS Type III occurred in those patients. Also, there was a significant association between good fitness and the high risk of developed DS type II (OR= 1.95 times, CI=1.01 to 3.7, with p= 0.03). When periods of having denture were considered, there was a highly significant association between longer period of 18-over months and the high risk of developed DS type II, (OR=10.2 times, CI=5.03 to 21.2 with p<0.001).

DISCUSSION
The most common microbial cause of DS in the present study was C. albicans (58.3%) of the total isolates. This result was similar to that reported by Salerno and Zomorodian 1, 10 in which the most common cause of DS was C. albicans. This occurrence of Candidal DS with high rate in denture wearers can be explained by the findings in which wearing denture was led to increase the chance of C. albicans oral colonization rate from 60 to 100% 9-12 . Oral colonization can be explained by the fact that dentures decrease the flow of oxygen and saliva to the underlying tissue producing a local acidic and anaerobic microenvironment that favours yeast overgrowth 13,14. In this study bacterial denture stomatitis among those patients counted about 29.1% of the total isolates. This result was similar to that reported by Prabha in which bacterial denture stomatitis was the second cause after Candida albicans DS 15 . These results can be explained by the hypothesis of Harold Marcott 16 , who assumed that bacteria present in the saliva may also cause ulcer when there is alteration in the salivary pH. Moreover, bacterial load in saliva increases due to caries, periodontal diseases and also other endocrine disorders like hypo-function of salivary glands which leads to decreased secretion and increased oral bacteria.  In this study, there was a highly significant association between males and the high risk of contracting Candida albicans DS (OR=2.33). The present study results supported the rejection of the null hypothesis which states that there would be no difference between male and female denture wearers in terms of the prevalence of Denture Related Stomatitis (DRS) and colonization by C. albicans of the inner surfaces of dentures and attachment surroundings. In a study by Gendreau and Loewy, a higher incidence has been reported in females 17 , but this is not always as in this case in which a higher incidence has been reported in males 18 . In the present study, there was a highly significant association between older age group and the high risk of contracting Candida albicans DS (OR=3.62). This study result was different from that reported from Philadelphia by Bouquot 19 , in which there was no different in the rate of DS with age, but similar to that reported from UK by Smaancyake et al., in which the highest rate occurred in older adult age groups 20 . In the current study, there was a highly significant association between longer period of 18-over months and the high risk of contracting Candida albicans DS. This result was similar to that reported by Barbeau in which the risk of developing Candida albicans DS increased with time duration 21 . Furthermore, there was a highly significant association between the poor fitness and the high risk of contracting bacterial DS (OR=10.22, and p< 0.001). This result was similar to that reported by Kulak in which poor-fitness was considered as a prime etiologic factor for contracting bacterial and/or Candida albicans DS 22 . This result explained by those poor-fitting dentures can increase mucosal trauma and ulcer formation. Most ulcers tend to have bacterial growth which sometimes might because of the non harmful normal flora, unless the count exceeds the normal and permissible levels which increase its ability to colonize both the denture and oral mucosal surfaces and cause stomatitis 22 . In the present study most of the patients were suffering from DS Type I which counted 66.7%, while only 33.3% of the patients were suffering from DS in Type II, but no case of sever DS in Type III. These results were different from that reported by Pinelli et al., 23 and Gendreau and Loewy et al., 24 in which Type II was the most common presentation followed by type III lesions. The high rate of type II and III in other studies might be explained by that poor denture hygiene in their patients which allows the increased growth of pathogenic micro-organisms within the dental plaque on the fitting surfaces of dentures 24 .

CONCLUSION
DS is a circumstance that commonly affects denture wearers, and should be treated even if asymptomatic. The condition requires a combined treatment method from both patient and clinician, and the role of the patient must be stressed. Management of aetiological risk factors is key in order to prevent recurrence. Treatment modalities may consist of: treatment of any underlying systemic risk factors, improvement in the fit of existing dentures, replacement of existing dentures, improved denture hygiene and the use of antifungal and antibacterial agents. Either methods are employed; the main aim of treatment is to eradicate the bio film from the patient's dentures. Regular review of patients suffering from DS is essential in order to ensure longterm successful treatment of the condition.