Université de Montréal Phase-I clinical trial on the effect of palatal brushing on denture stomatitis par Marla Kabawat Département de dentisterie de restauration Faculté de médecine dentaire Université de Montréal Mémoire présenté à la Faculté des études supérieures en vue de l’obtention du grade de Maîtrise ès Sciences (M.Sc.) En sciences buccodentaires Août 2013 © Marla Kabawat, 2013 Université de Montréal Faculté des études supérieures Ce mémoire intitulé: Phase-I clinical trial on the effect of palatal brushing on denture stomatitis Présenté par: Marla Kabawat A été évalué par un jury composé des personnes suivantes: Dr Benoît Lalonde, président-rapporteur Dre Elham Emami, directrice de recherche Dr Louis de Koninck, co-directeur de recherche Dr Jean Barbeau, co-directeur de recherche Dr Gilles Gauthier, membre du jury ii RÉSUMÉ Introduction: La stomatite prothétique est une condition inflammatoire chronique de la muqueuse buccale recouverte par une prothèse. Cette maladie est considérée comme la lésion buccale la plus fréquente chez les porteurs de prothèses amovibles. Des études récentes sur l'étiologie de la stomatite prothétique suggèrent que des traitements basés sur la réduction de l'inflammation seraient efficaces dans le traitement de cette maladie. Objectifs: Évaluer l'efficacité du brossage du palais dans le traitement de la stomatite prothétique. Méthodes: Quarante-huit participants (âge moyen : 66,0 ± 11,2 ans) avec un diagnostic de stomatite prothétique, ont été sélectionnés à partir d’un examen préalable de 143 individus, afin de participer à cet essai clinique de phase I à deux centres, réalisé selon un devis de type pré-test/post-test à un seul groupe. L'intervention a consisté en un brossage du palais avec une brosse manuelle après chaque repas et avant le coucher. Des examens cliniques et microbiologiques ont été effectués avant le traitement, et à 1 mois et 3 mois de suivi. Des données supplémentaires ont été obtenues par l'utilisation d'un questionnaire validé. Les résultats primaires et secondaires étaient, respectivement, la rémission de stomatite prothétique et la diminution du nombre de colonies de Candida. Des tests statistiques descriptifs et non paramétriques ont été menés pour analyser les données. Résultats: À 3 mois de suivi, 10,4 % des participants ont été guéris et 70,8 % ont eu une amélioration clinique de la stomatite prothétique grâce au brossage du palais. Une iii réduction statistiquement significative de la surface et de l’intensité de l’inflammation après 3 mois de brossage du palais a été démontrée (p < 0,0001). L’ampleur de l’effet a varié d’un effet modéré à important (0,34 à 0,54) selon la classification utilisée pour le diagnostique de la stomatite prothétique. De plus, le nombre de colonies de Candida, recueillies par sonication des prothèses et par échantillonnage du palais, a diminué de manière statistiquement significative après 3 mois de brossage (p ≤ 0,05). Conclusion: Les résultats de cette étude suggèrent que le brossage du palais est efficace comme traitement de la stomatite prothétique. Mots-clés: Stomatite prothétique, brossage du palais, prothèse complète, Candida, étude de phase I. iv ABSTRACT Introduction: Denture-related erythematous stomatitis (denture stomatitis) is a chronic inflammation of the oral mucosa covered by a removable prosthesis. This disease is considered the most prevalent mucosal lesion associated with prosthesis use. Recent research on the etiology of denture stomatitis suggests that treatments based on the reduction of the inflammation are effective in the management of this disease. Objectives: To assess the efficacy of palatal brushing in the treatment of denture stomatitis. Methods: After screening 143 individuals with a potential diagnosis of denture stomatitis, 48 (mean age: 66.0 ± 11.2 years) were enrolled in a phase-I two-center clinical trial with one-group pre-test/post-test design. The intervention of interest was manual palatal brushing after each meal and before bedtime. Clinical and microbiological examinations were performed at baseline, 1 month and 3 months postintervention. Additional data were obtained by the use of a validated questionnaire. The primary and secondary outcomes were the remission of denture stomatitis and the diminution of Candida Colony-Forming Units (CFUs), respectively. Descriptive and non-parametric statistical tests were conducted to analyze the data. Results: At 3-month follow-up, denture stomatitis was completely cured in 10.4 % of the study participants, and 70.8 % of denture wearers showed improvement in the clinical signs of denture stomatitis. There was a significant reduction in the area and severity of the palatal inflammation at 3-month follow-up (p < 0.0001). The effect size v ranged from medium to large (0.34 to 0.54), depending on the classification used for the diagnosis of denture stomatitis. Furthermore, a significant reduction in the number of Candida CFUs isolated from the palatal mucosa and dentures was observed (p ≤ 0.05). Conclusion: The results of this study suggest that palatal brushing is effective in the treatment of denture stomatitis. Keywords: Denture stomatitis, palatal brushing, complete denture, Candida, phase I trial vi TABLE OF CONTENTS RÉSUMÉ ...................................................................................................................................... iii ABSTRACT .................................................................................................................................. v TABLE OF CONTENTS ............................................................................................................ vii LIST OF TABLES ........................................................................................................................ x LIST OF FIGURES ...................................................................................................................... xi LIST OF SYMBOLS AND ABBREVIATIONS ........................................................................ xii DEDICATION ........................................................................................................................... xiii ACKNOWLEDGMENTS .......................................................................................................... xiv CHAPTER I................................................................................................................................... 1 LITERATURE REVIEW .............................................................................................................. 1 1.1 INTRODUCTION .............................................................................................................. 1 1.2 ORAL INFLAMMATION ................................................................................................. 2 1.2.1 Definition of oral inflammation....................................................................................... 2 1.2.2 Physiology of oral inflammation ..................................................................................... 3 1.2.3 Types and etiology of oral inflammation ........................................................................ 6 1.3 DENTURE-RELATED ERYTHEMATOUS STOMATITIS ............................................ 7 1.3.1 Definition, diagnosis and classification ........................................................................... 7 1.3.2 Epidemiology of denture stomatitis............................................................................... 11 1.3.3 Histopathology and physiopathology of denture stomatitis .......................................... 16 1.3.4 Etiology of denture stomatitis ....................................................................................... 18 1.3.5 Systemic effects of denture stomatitis ........................................................................... 20 1.4 TREATMENT OF DENTURE-RELATED ERYTHEMATOUS STOMATITIS .......... 21 1.4.1 The conservative methods ............................................................................................. 21 1.4.1. I Oral hygiene measures ....................................................................................... 22 1.4.1. II Prosthodontic measures .................................................................................... 28 1.4.2 Antifungal medications ................................................................................................. 29 CHAPTER II ............................................................................................................................... 32 METHODOLOGY ...................................................................................................................... 32 2.1 PROBLEMATIC, HYPOTHESIS, OBJECTIVES .......................................................... 32 2.1.1 Objectives ...................................................................................................................... 33 vii 2.1.2 Hypotheses .................................................................................................................... 33 2.2 RESEARCH METHODOLOGY ..................................................................................... 34 2.2.1 Study design .................................................................................................................. 34 2.2.2 Study participants and inclusion criteria ....................................................................... 34 2.2.3 Experimental procedure................................................................................................. 35 2.2.4 Data collection and measurement instruments .............................................................. 36 2.2.4. I Clinical investigation ......................................................................................... 36 2.2.4. II Microbiological investigation ........................................................................... 39 2.2.5 Outcome and explanatory variables .............................................................................. 40 2.2.6 Statistical analysis ......................................................................................................... 41 2.2.7 Ethical considerations.................................................................................................... 42 2.3 STUDY RELEVANCE .................................................................................................... 42 2.4 CANDIDATE’S ROLE IN THE PROJECT .................................................................... 43 2.5 KNOWLEDGE TRANSFER ........................................................................................... 43 CHAPTER III .............................................................................................................................. 45 RESULTS .................................................................................................................................... 45 3.1 MANUSCRIPT ................................................................................................................ 45 CHAPTER IV .............................................................................................................................. 72 DISCUSSION.............................................................................................................................. 72 4.1 PALATAL BRUSHING EFFECTS ................................................................................. 72 4.2 MECHANISM OF ACTION OF PALATAL BRUSHING ............................................. 75 4.3 THE ETIOLOGY OF DENTURE-RELATED ERYTHEMATOUS STOMATITIS . 78 4.4 MODIFICATION TO THE CLASSIFICATION OF DENTURE-RELATED ERYTHEMATOUS STOMATITIS ................................................................................. 79 4.5 ORAL HEALTH KNOWLEDGE .................................................................................... 79 4.6 THE CHOICE OF STUDY DESIGN AND LESSON LEARNED FROM INTERNATIONAL COLLABORATION....................................................................... 81 4.7 STUDY LIMITATIONS .................................................................................................. 82 4.8 FUTURE RESEARCH..................................................................................................... 83 CHAPTER V ............................................................................................................................... 85 CONCLUSIONS ......................................................................................................................... 85 BIBLIOGRAPHY ....................................................................................................................... 86 APPENDICES ................................................................................................................................ i viii APPENDIX I: CONSENT FORM ............................................................................................ i APPENDIX II : QUESTIONNAIRE ...................................................................................... ix APPENDIX III: CLINICAL FORM ...................................................................................... xx APPENDIX IV: MICROBIOLOGICAL FORM ............................................................... xxvii APPENDIX V: INSTRUCTION FORM ............................................................................ xxix ix LIST OF TABLES Table 1.1: Denture stomatitis prevalence in the last decade (2003-2013)......................13 Table 3.1: Assessments of denture stomatitis ………………………………...….........66 Table 3.2: Socio-demographic characteristics of the participants at baseline (T0) by study center......................................................................................................................67 Table 3.3: Study participants’ profiles by study center at baseline (T0) according to denture stomatitis risk factors..........................................................................................68 Table 3.4: Effect of the intervention at 3-month follow-up............................................69 Table 3.5: Effect of the intervention on the number of Candida colonies at 3-month follow-up.........................................................................................................................69 x LIST OF FIGURES Figure 1.1: The inflammatory process.............................................................................5 Figure 1.2: Clinical features of denture-related erythematous stomatitis......................10 Figure 3.1: Flow chart of the study................................................................................65 Figure 3.2: Treatment effect at 3-month follow-up (T2)................................................70 Figure 3.3: Palatal mucosa of patients at T0 and T2.…………………………..............71 Figure 4.1: Palatal brushing effects……………………………………………….…...77 xi LIST OF SYMBOLS AND ABBREVIATIONS CFUs % OR CI F WHO NHANES RCT IL TNF C. RR PDT VAS Colony-Forming Units Percentage Odds Ratio Confidence Interval Female World health Organization National Health and Nutrition Examination Survey Randomized Controlled Trial Interleukin Tumor Necrosis Factor Candida Relative Risk Photodynamic Therapy Visual Analogue Scale xii DEDICATION To my beloved parents and sister, To the love of my life, Thank you Rizkallah, Lina, Nai and Nidal for being a source of love and encouragement, and for believing in me. xiii ACKNOWLEDGMENTS There are a number of people without whom this thesis might not have been written, and to whom I am deeply grateful. First of all, I would like to sincerely thank my supervisor, Dr. Elham Emami who was a role model for me throughout my master’s program. Thank you Dr. Emami for your tremendous kindness and patience, and for your invaluable support. Secondly, I would like to thank my co-directors, Dr. Louis de Koninck and Dr. Jean Barbeau, for their support and helpful comments which guided me over the past two years. I would also like to acknowledge Dr. Raphael de Souza for making our collaboration so simple and pleasant. I am also thankful for M. Pierre Rompré for his help with the statistical analyses. Of equal importance I would like to thank my colleague Dr. Amal Idrissi Janati for the two years we spent together. I would like to express my gratitude to Mme Annie Leduc for her kindness, support and expert assistance with microbiologic techniques, as well as Mme Jacynthe Séguin and M. Daniel Chartrand for welcoming me in the lab. I would also like to thank Dr. Benoît Lalonde for agreeing to be the president of my committee and Dr. Gilles Gauthier for the time spent reviewing this thesis. Finally, I am thankful for my parents, my sister and my fiancé for their endless love and support and for giving me the opportunity to pursue my studies. xiv CHAPTER I LITERATURE REVIEW 1.1 INTRODUCTION Over the last few decades, there has been substantial progress in understanding the role of oral inflammation in systemic health. Oral inflammation is a protective, non-specific response of the immune system to a pathogenic or traumatic injury 1-3 . Although this defensive inflammatory mechanism is necessary for the protection of the oral cavity, it can be potentially deleterious when it becomes long-lasting and persistent. Denture-related erythematous stomatitis (denture stomatitis) is the most common mucosal disease associated with the wear of removable prostheses, and is characterized by a chronic inflammation of the palatal mucosa 4-6 . The treatment of this recurrent condition has challenged clinicians because of its resistant and refractory nature. Although its recalcitrance has been related to Candida biofilms and their attachment to prosthetic surfaces, the direct role of Candida in the injury to the adjacent mucosa has been questioned. The new evidence suggests that trauma initiates the inflammatory reaction in denture stomatitis 7. Subsequently, the complex structured microbiological communities play an intermediary role in the process of denture stomatitis 7. Based on this hypothesis, the treatment of denture stomatitis should focus on the resolution of the inflammation. 1 The first chapter of this thesis will review the literature to discover the rationale behind this hypothesis. 1.2 ORAL INFLAMMATION 1.2.1 Definition of oral inflammation The oral cavity is a very complex system that is vulnerable to inflammatory diseases due to the exposure of the oral environment to different pathogenic stimuli. Although the primary role of an inflammatory response is to protect oral tissues from a deleterious injury 1, 3 , this defensive mechanism could affect different sites of the oral cavity and manifest as a primary sign of oral diseases such as periodontitis, pulpitis, mucositis, and stomatitis 8. Inflammation is defined as a nonspecific response of the body in reaction to a mechanical, chemical or microbial stimulus 9. Its etymology comes from the Latin inflammare, “to set on fire”. Cornelius Celsus 10, a Roman encyclopaedist, was the first person who introduced the cardinal signs of inflammation. These include redness, swelling, heat and pain, and represent the clinical manifestation of increased local vascularity, exudation of tissue fluid, increased blood flow and the release of inflammatory mediators, as well as the stimulation of pain receptors 1, 3. In 1858, Rudolf Virchow added loss of function to the four signs of inflammation. Loss of function is caused by the combination of pathophysiological events that occur during inflammation 11 . 2 1.2.2 Physiology of oral inflammation An oral inflammatory process is similar to any other inflammation in the human body and consists of complex interactions between inflammatory mediators and different types of cells 1, 3 . Inflammatory mediators are soluble molecules that represent the physiological messengers of the inflammation and can positively or negatively influence the inflammatory process 7, 12 . Their role is to induce and maintain a host response. Inflammatory mediators include vasoactive amines (histamine, serotonin), arachadonic acids (prostaglandins, leukotrienes) and cytokines (tumor necrosis factor, interleukins, interferons, and colony stimulating factors). Those mediators are released from different types of cells, including mast cells, dendritic cells, platelets, neutrophils and monocytes 1, 9 . mediators initiate numerous physiological processes including Inflammatory vasodilatation, increased microvascular permeability, cellular activation, cellular adhesion, and coagulation. Vasodilatation and increased microvascular permeability at the site of injury increase the available oxygen and nutrients, generate heat, and provoke tissue oedema. This increased permeability leads to the infiltration of plasma proteins and leucocytes from the circulation. The leucocytes, which consist principally of polymorphonuclear leukocytes (neutrophils) and macrophages, are attracted by inflammatory mediators, become activated, and aggregate at the site of the injury. They also become engaged in the production of cytokines and other inflammatory mediators 3, 13 . Endothelial cells also become active and secrete additional cytokines and secondary 3 inflammatory mediators. These processes result in the activation of coagulation cascades and lead to local thrombosis and isolating of the inflamed areas 9. 4 STIMULI MECHANICAL, CHEMICAL OR MICROBIOBIAL INFLAMMATORY RESPONSE RELEASE OF INFLAMMATORY MEDIATORS Vasodilatation and ↑ microvascular permeability Activation of endothelial cells ↑ Oxygen, nutrients, heat and oedema Chemical attraction and infiltration of plasma proteins and leucocytes Secretion of additional mediators and activation of coagulation cascade  local thrombosis Activation and aggregation of leucocytes at the site of injury + production of additional inflammatory mediators OUTCOMES Resolution of the inflammation Abscess formation Chronic inflammation Fibrosis Figure 1.1: The inflammatory process 5 1.2.3 Types and etiology of oral inflammation An oral inflammatory response can be classified into two main types: acute and chronic inflammation. An acute inflammatory response is a process characterized by a rapid onset, usually appearing within a few minutes or hours after the injury. This prompt response is also of short-term duration and ceases after the removal of the stimulus 3. Acute inflammation is marked by the exudation of fluid and plasma proteins and by the migration of leukocytes, most particularly neutrophils, to the site of injury 1. Conversely, a chronic inflammatory response is of prolonged duration and is characterized by the presence of lymphocytes and macrophages. Chronic inflammation could result from failure to eliminate the injurious stimulus, an autoimmune response, or from a chronic low-intensity irritant that persists for an extensive amount of time. A low-intensity irritation could cause deleterious changes in tissues, such as fibrosis and necrosis 1, 14. Oral inflammation can also be described as being localized or generalized/systemic. Localized inflammation is generally confined to the site of injury. In contrast, when the local control of the inflammation is lost, an exaggerated response with systemic activation of the inflammatory response occurs 9. The etiology of oral inflammatory reactions is multifactorial. Oral inflammation could be caused by infections (bacterial, viral, fungal), oral biofilms chemotherapy and radiation therapy systemic conditions 1. 15 14 , trauma, neoplasia, 16 , as well as immune-mediated disorders and 6 1.3 DENTURE-RELATED ERYTHEMATOUS STOMATITIS 1.3.1 Definition, diagnosis and classification Denture-related erythematous stomatitis, or denture stomatitis, was first described in the medical literature in 1936 by Cahn 17 under the name “denture sore mouth”. Denture stomatitis is a localized chronic inflammation of the oral mucosa covered by a removable prosthesis 4-6 . It usually affects the palatal mucosa under a complete upper prosthesis. The mandibular mucosa is rarely involved as a result of the lesser amount of tissue coverage and the continuous contact between the saliva and the lower alveolar mucosa 6, 18. This pathology is often diagnosed clinically by an oral healthcare professional during routine examination of denture wearers since denture stomatitis is often asymptomatic 6, 19 . However, symptoms like mucosal bleeding, burning sensation of the palate and tongue, tenderness, halitosis, xerostomia, unpleasant taste and dysphagia, could occasionally be displayed 20-22. In research and academic settings, various methods have been used to classify denturerelated erythematous stomatitis. Generally, the classifications are based on three essential criteria: the distribution and the stages of the inflammation 21, 23, the intensity of the inflammation 24 and the extent of the inflammation 25, 26. The first classification of denture stomatitis was introduced in 1958 by Östlund 27 . He distinguished three types based on the distribution of inflammation: I. Localized inflammation; II. Diffused erythema limited by the denture margins; III. Granular 7 reaction. Four years later, Newton 23 presented a new classification based on Östlund’s. This classification, which has been widely used in clinical practice and research, has three types: Newton Type I: Petechia: pinpoint hyperaemia around the orifices of the ducts of the palatal mucous glands; Newton Type II: Diffuse hyperaemia: a generalised inflammation of the denturebearing area; Newton Type III: Inflammatory papillary hyperplasia. In 1970, Budtz-Jørgensen and Bertram 21 removed the pinpoint hyperaemic lesions of Newton’s classification and used the terms: “simple localized inflammation”, “simple diffuse inflammation” and “granular inflammation”. A decade later, Bergendal and Isacsson 24 classified denture stomatitis according to the intensity of the inflammation: Grade 0 : Normal pink, pale mucosa; Grade 1 : Slightly erythematous mucosa; Grade 2 : Moderately erythematous mucosa; and Grade 3: Pronouncedly erythematous mucosa. Schwartz et al. 26 added the concept of the extent of the inflammation: 0 1 2 No inflammation Inflammation of the palate extending up to 20 mm on denture-bearing tissue Inflammation of the palate extending more than 20 mm on denture-bearing tissue 3 Inflammation covering more than 50 % of the palatal denture-bearing tissue 25 Finally in 2003, Barbeau et al. modified the Newton classification to provide a more accurate evaluation of the extent of the lesion. Newton types II and III were 8 subclassified as A, if inflammation was present in 1 to 2 quadrants, or as B if inflammation was present in 3 to 4 quadrants. In this master’s research project, in order to capture the difference between petechiae and localized inflammation in regard to denture stomatitis treatment, we added two subtypes to Newton's Type I denture stomatitis: Type IA: Petechiae around the orifices of the ducts of the palatal minor salivary glands; Type IB: Localized area of inflammation in denture-bearing area (Figure 1.2). 9 Type IA Type IB Type II Type III Figure 1.2: Clinical features of denture-related erythematous stomatitis 10 1.3.2 Epidemiology of denture stomatitis Denture-related erythematous stomatitis is considered the most prevalent mucosal lesion associated with prosthesis use 4, 5, 28. In a recent review of the literature by Gendreau and Loewy 29 , the global prevalence of this disease has been reported to be between 15 % and 77.5 %. Table 1.1 presents the worldwide prevalence of denture stomatitis in the last decade. In Quebec, the prevalence of denture stomatitis in complete denture wearers has been reported to be up to 77.5 % 7, 30. However, it should be mentioned that these studies were mainly university-based and their populations were not representative of Quebec’s general population. Although denture stomatitis is generally seen in complete denture wearers 6, 28 , a recent systematic review 31 revealed that denture stomatitis has a prevalence of 1.1 % to 36 % in partial denture wearers. Regarding the relationship between gender and denture stomatitis, many studies found that this pathology is more prevalent in women 17, 32, 33, whereas some others did not find any gender difference 34, 35 . It could be argued that females may be more susceptible to this disease as they are more likely to wear their prosthesis continuously to avoid an unesthetic appearance 36. It has been reported that the prevalence of denture stomatitis increases with age 34, 37 . This could be explained by long-term denture use, inadequate oral hygiene due to the lack of dexterity, presence of systemic diseases and medication use, as well as a decrease in host immunity in the elderly 38-42 . Paradoxically, several studies did not demonstrate any association between aging and denture stomatitis 7, 28, 43. 11 Several risk and predisposing factors have been related to the occurrence of denture stomatitis, including continuous and nocturnal wear of the prosthesis 7; nutritional deficiencies in proteins, vitamins A and B, and iron 6; several systemic diseases such as diabetes mellitus and immunosuppressive diseases and therapies 44, 45 ; medications such as antibiotics, corticosteroids, xerogenic agents and hormone supplementation therapy 46, 47 ; as well as smoking 6, 24. Although the literature shows contradictory results in regard to the association between these risk factors and denture stomatitis 6, 7, 35, 48, 49 , there is some evidence that highlights the dominant role of nocturnal wear of the prosthesis in this disease 34, 50, 51. In the cross-sectional study by Barbeau et al. 25 , participants with extensive palatal inflammation were five times more likely to wear their prosthesis at night than healthy denture wearers (OR = 5.00; 95 % CI 1.35 to 18.55; p = 0.03). Nocturnal and continuous prosthesis wear could reduce the protective effect of saliva, decrease the cleaning effect of the tongue, prevent proper oxygenation of the palatal mucosa, and finally, increase local trauma to the mucosa. These effects make denture wearers more prone to mucosal mechanical and microbiological injuries, and therefore increase the risk of denture stomatitis in this population 7, 30, 51-54. The noticeable variations between studies in regard to the epidemiology of denture stomatitis is justified by the inconsistency in methodological aspects of the studies, especially in the diagnostic criteria of denture stomatitis, data collection, the choice of the study population and its underlying spatial, socio-demographic and lifestyle characteristics 6, 17, 34, 45, 55. 12 Table 1.1: Denture stomatitis prevalence in the last decade (2003-2013) Study/year of study/Country Study design and study population Sample size (n) and population characteristics  n = 889  62 % F  ≥ 65 years old Denture stomatitis predisposing factors Diagnosis of denture stomatitis Epidemiology guide for the diagnosis of oral mucosal diseases (WHO) Modified version of Newton’s classification. Prevalence of denture stomatitis (%) 34 % Espinoza et al. 56 , 2003, Chile Barbeau et al. 25 , 2003, Canada  Cross-sectional study  Random sample of individuals from public and private health systems  Cross-sectional study  Convenience samples of 2 cohorts:  Cohort 1: universitybased study  Cohort 2: Individuals living or working in a nursing home  Cross-sectional university-based study  Convenience sample of completely edentate individuals  Cross-sectional study  Edentate individuals from the NHANES III (1988–1994) survey  Cross-sectional study  Random sample of completely edentate institutionalized individuals  Cross-sectional university-based study  Completely edentate individuals Not available Cohort 1:  n = 47  66 % F  Mean age 63.7 ± 11.6 years Cohort 2:  n = 21  76.2 % F  Mean age 56.3 ± 11.0 years  n = 236  75 % F  Mean age: 62 ± 12.8 years  n = 3450  42.3 % F  Mean age: 59.2 ± 0.50 years  n = 765,  49 % F  Mean age: 35.6 ± 26.6 years  n = 40,  77.8 % F  Mean age: 64.5 years  Nocturnal denture wear  Smoking Cohort 1: 76.6 % Cohort 2: 57.1% Combined: 70.6 % Marchini et al. 19 , 2004, Brazil  Poor oral hygiene Not available 42.4 % Shulman et al. 6 , 2005, United States  Vitamin A deficiency  Nocturnal denture wear  Smoking ≥15 cigarettes per day Not available Classification of Newton 27.9 % Mumcu et al. 57, 2005, Turkey Classification of Newton 20.5 % Emami et al. 30, 2007, Canada  Nocturnal denture wear Classification of Newton 77.5 % 13 Emami et al. 7, 2008 , Canada  1-year follow-up of a previous RCT.  University-based study  Completely edentate individuals  n = 173  53.8 % F  Mean age: 72.13 ± 4.39 years  Nocturnal denture wear  Type of the prosthesis (implantsupported overdenture or conventional)  Poor denture hygiene habits Classification of Newton 63.6 % Baran et al. 58, 2009, Turkey  Cross-sectional university-based study  Completely edentate individuals  Cross-sectional university-based study  Completely edentate individuals  Cross-sectional study  Random sample of completely edentate individuals from nursing homes  Cross-sectional university-based study  Convenience sample of partially or completely edentate individuals  Cross-sectional study  Convenience sample of completely edentate individuals from a hospital dental clinic  n = 310  49 % F  Mean age: 65.74 ± 2.73 years  n = 873  49.45 % F  Mean age: 72.0 ± 5 years  n = 335  73.1 % F  ≥ 60 years old Classification of Newton 35.8 % Divaris et al. 51, 2010, Greece  Nocturnal denture wear Classification of Newton 6% Ferreira et al. 5, 2010, Brazil Not available Jainkittivong et al. 4, 2010, Thailand  n = 380  59.2 % F  Mean age 65.2 ± 9.1 years Not available Epidemiology guide for the diagnosis of oral mucosal diseases (WHO) Epidemiology guide for the diagnosis of oral mucosal diseases (WHO) Not available 15.2 % 18.1 % Mandali et al. 37 , 2011, Turkey  n = 153  50.3 % F  Mean age: 61.8 ± 9.8 years  Age  Continuous use of the dentures 35.3 % Evren et al. 34, 2011, Turkey  Cross-sectional  n = 269 study  55.8 % F  Convenience sample  ≥ 65 years old of institutionalized completely edentate individuals  Age  Education  Nocturnal denture wear  Denture hygiene Classification of Newton 44 % 14 Kossioni 35, 2011, Greece  Cross-sectional university-based study  Convenience sample of completely edentate individuals  Cross-sectional study  Convenience sample of institutionalized of partially of completely edentate household farmers  Cross-sectional study  Convenience sample of institutionalized elderly individuals  Cross-sectional study  Random sample of completely edentate individuals from a list of patients attending medical routine consultations in a polyclinic.  Cross-sectional study  Convenience sample of institutionalized elderly individuals  n = 106  64.2 % F  Mean age: 67.7 ± 9.9 years  Continuous denture use Classification of Newton 39.6 % Da Silva et al. 49 , 2011, Brazil  n = 102  82 % F  Mean age: 49 years  Female  Length of denture use Classification of Newton 48.2 % Mozafari et al. 59 , 2012, Iran  n = 202  91.1 % F  Mean age: 79.59 ± 8.88  n = 126  75 % F  Mean age: 70.2 years Not available Not available 54.6 % Cueto et al. 60, 2012, Chile  Nocturnal denture wear  poor denture hygiene  Not well-fitted prosthesis Not available 37.1 % Sakar et al. 54, 2013, Turkey  n = 365  66 % F  Mean age: 70.5 ± 13.2 years  Age of maxillary denture  Nocturnal denture wear  Reduced Vertical dimension of occlusion  Prosthesis quality Classification of Newton 46.3 % Pesee et al. 43, 2013, Thailand  Cross-sectional study  Convenience sample of partially or completely edentate individuals from a dental clinic located in a hospital.  n = 128  67.2 % F  Mean age: 57.11 years Classification of Newton 52.3 % 15 1.3.3 Histopathology and physiopathology of denture stomatitis The chronic inflammatory reaction of the palatal mucosa in denture-related erythematous stomatitis is characterized by histopathological and physiopathological alterations similar to any other inflammatory reaction 61. The epithelial changes that occur in denture stomatitis include reduced thickness of the epithelium, with areas of epithelial hyperplasia or atrophy, as well as accelerated cellular turnover of the epithelium 61-63 . Another important change that was demonstrated in several studies was the parakeratinization or absence of keratinization of the surface layers of the palatal mucosa 61-64. Regarding connective tissue changes, Le Bars et al. 65 62, compared biopsies of the palatal mucosa of healthy patients wearing complete upper dentures with those affected by Type II denture stomatitis. They found a moderate inflammation in the connective tissue with the presence of polymorphonuclear leucocytes and lymphocytes and the display of thin collagen and a disorganized lamina densa. Furthermore, they demonstrated disruptions in the basement membrane. Other cytological studies of palatal mucosal smears also showed higher inflammatory infiltrates in patients with denture stomatitis than healthy patients 63, 66. Recently, many research groups highlighted the role of salivary inflammatory mediators in denture-related erythematous stomatitis. In a case-control study by Barros et al. 67 , high levels of salivary cytokines such as IL-1β and IL-8 were found in patients with Type II and III denture stomatitis. Gasparoto et al. 68 assessed patients with Candida- associated denture stomatitis and found that they had higher salivary levels of IL-4 and IL-10. Pesee et al. 43 found no association between the occurrence of denture stomatitis 16 and the salivary levels of IL-6, IL-8, IL-10, IL-17, and TNF-α. However, in this study 43 , the majority of the study participants (65.7 %) had Type I denture stomatitis. With regard to denture stomatitis and the systemic levels of inflammatory markers, the literature is still inconclusive. The findings of the longitudinal cohort study by Ajwani et al. 69 in completely edentate individuals demonstrated higher systemic levels of C67 reactive protein in patients affected by denture stomatitis. In contrast, Barros et al. found that the amount of C-reactive protein was similar in patients with denture stomatitis and healthy edentate, suggesting a lack of systemic inflammation associated with denture stomatitis. Pietruski et al. 70 found that denture wearers had higher serum levels of IL-6 and TNF-α, regardless of the presence of denture stomatitis, compared to dentate individuals. Recently, Matsumoto et al. 71 evaluated the cytogenetical damage induced by denture stomatitis by means of micronucleus assay in exfoliated cells of the palatal mucosa. The findings of this study showed a lack of malignancy risk in denture stomatitis patients. However, this chronic inflammation had cytotoxic effects on the cells of the oral mucosa by inducing nuclear alterations such as nucleus pyknosis, karyorrhexis and karyolysis 71. Finally, Kaplan et al. 72 compared palatal biopsies of patients with normal mucosa and patients with papillary hyperplasia or Type III denture stomatitis. They found no evidence of dysplasia in denture stomatitis patients. Similarly, Flanagan and Porter reported no histological premalignant changes in Type III denture stomatitis. 73 17 1.3.4 Etiology of denture stomatitis The etiology of denture-related erythematous stomatitis remains poorly understood and controversial 25, 30 . Three etiologic factors have been reported to play a major role in 32, 74 denture stomatitis including fungal infections (particularly Candida) biofilm 75 and trauma 7, 52. Fungal Infection: , denture In 1936, Cahn 17 was the first to propose Candida infection as a main etiologic factor for denture stomatitis. Since then, a relationship between denture stomatitis and fungal infection has been widely reported in the literature 18, 74, 76. However, several studies did not prove any statistically significant association between the presence of denture stomatitis, the severity of the inflammation, and the number of Candida ColonyForming Units (CFUs) isolated from the prosthesis and the palate of patients affected by denture stomatitis 22, 25, 30, 67, 77. Furthermore, high recurrence rates of the clinical signs of denture stomatitis and recolonization of Candida after the antifungal therapy have been widely reported 78-80. Denture Biofilm: Denture biofilm is a surface-attached, dense and complex layer consisting of microbial communities and their metabolites, which are embedded in an extracellular polysaccharide matrix 81 . It has been reported that denture biofilm contains 1011 82 microorganisms per gram amoebae 83 , including aerobic and anaerobic bacteria, yeasts, and . Poor hygienic habits such as continuous and nocturnal wear of the 18 prosthesis, in addition to inadequate denture cleaning, promote the formation and accumulation of denture biofilm. This biofilm harbours a wide array of pathogenic microorganisms which produce toxins and metabolic waste. These metabolites in turn could initiate the inflammatory process in denture stomatitis 6, 32, 34, 48, 83, 84. Furthermore, biofilms provide a protective niche for these microorganisms and allow them to become resistant to antimicrobials 83, 85, 86. Trauma: The role of trauma in the onset of denture stomatitis was suggested for the first time in 1929 by Wright 87 and since then has been reported in several studies 20, 21, 52 . Recently, many studies have assessed the plausibility of this hypothesis. In the study by Pesee et al. 43 , there was a statistically significantly relationship between the prevalence of denture stomatitis and the absence of adequate hygiene, fit, retention and stability of the prosthesis. Furthermore, the results of a randomized controlled trial by Emami et al. 7 suggest that a lack of stability of the lower denture can cause trauma to the palatal mucosa following the displacement of the upper denture and thus, promote the development of denture stomatitis. In fact, in this study, the risk of denture stomatitis was 4.5 times greater in patients wearing mandibular conventional dentures than in those wearing more stable and less traumatic two-implant overdentures (Adjusted OR = 4.54, 95 % CI 2.20 to 9.40). 19 1.3.5 Systemic effects of denture stomatitis 88, 89 There is overwhelming evidence of the link between oral and general health . The oral mucosa constitutes the lining of ports of entry to several systems of the human body. When the mucosal defences are breached, the systemic health could be affected 90. Although the association between denture stomatitis and systemic disease such as diabetes mellitus has been the focus of several studies 45, 91, this investigation has always been unidirectional, and the direct and indirect impact of denture stomatitis has not been examined yet mainly because of the silent nature of this disease. We can hypothesize that this disease could affect the general health through two pathways: local infection and local inflammation. Evidence regarding the role of local inflammation and infection in the development of several diseases supports this hypothesis. Denture stomatitis favours the colonization of infectious pathogens which in turn could lead to serious disease such as oral candidiasis, bacterial endocarditis and aspiration pneumonia, especially in individuals with a compromised immune system, hospitalized patients, and elders with cognitive impairments and dementia 46, 92-98. There is also persuasive scientific evidence on the consequences of chronic inflammation 99, 100 . The evidence of the presence of inflammatory markers such as C- reactive protein in the saliva and serum of patients affected by denture stomatitis suggests that this chronic inflammation may be involved in many seemingly unrelated diseases. As we get older, chronic inflammation can have pathological consequences throughout the body. As supported by Ajwani et al. 69 , among the edentulous, chronic inflammatory lesions like denture stomatitis are important determinants of inflammatory 20 markers, comparable to periodontal disease in the dentate individuals. This evidence shows the need for future and novel studies on this topic. 1.4 TREATMENT OF DENTURE-RELATED ERYTHEMATOUS STOMATITIS The history of the treatment of denture-related erythematous stomatitis demonstrates the recognition of this disease as being multifactorial. Therefore, denture stomatitis has not received a unique treatment, and a variety of different therapies have been used to treat this chronic inflammation 101-103. The first trace of denture stomatitis treatment dates back to 1929. At that time, the etiology of this disease was considered to be trauma and treatments based on the reduction of traumatic injuries were introduced. Since 1935, the hypotheses on the role of fungal infections in denture stomatitis have convinced most of the clinicians to prescribe different kinds of antifungal medications to treat this disease. In 1952, Fisher and Rashid 104 reported that denture stomatitis is caused by a lack of denture hygiene, and recommended improving the oral hygiene of patients. In general, we can classify denture stomatitis treatments into two major categories: the conservative methods and the use of antifungal medications 94. 1.4.1 The conservative methods The conservative approach includes methods that improve the oral and prosthesis hygiene and that reduce the trauma to the underlying palatal mucosa. 21 1.4.1. I Oral hygiene measures Oral hygiene measures include discontinuous use of the denture, use of mouthwash, palatal brushing, laser and photodynamic therapy, prosthesis hygiene, microwave disinfection of the prosthesis, and finally the use of phytomedicines. 1. Discontinuous use of dentures As previously mentioned in section 1.3.2, continuous wear of the prosthesis represents a risk factor for denture-related erythematous stomatitis 28, 51 . Several studies show a 6, relationship between nocturnal denture wear and the prevalence of denture stomatitis 25, 34 , and clinicians usually suggest to their patients to remove their prosthesis at night to 51 prevent or improve this disease. A study by Divaris et al. demonstrated that removing the prostheses during the night was associated with a decreased risk of having denture stomatitis (OR = 0.63, 95 % CI 0.44 to 0.90). 2. Mouthwash use Mouthwashes such as chlorhexidine gluconate (Peridex™, Cordosyl™) dioxide (CloySYS™) 106 105 , chlorine , and lawsone methyl ether mouthwash 105 107 , have been widely used to remove palatal biofilm. In a clinical study by Lal et al. , 0.12 % chlorhexidine gluconate (Peridex™) was effective in elimination of C. albicans colonies isolated from the denture biofilm and led to a decrease in palatal inflammation. In a randomized controlled trial, Koray et al. 108 demonstrated that hexetidine mouthrinse was as effective as fluconazole capsules in reducing the number of Candida albicans Colony-Forming Units (CFUs). Similarly, in a clinical study by Uludamar et al. 22 106 , chlorhexidine gluconate and chlorine dioxide mouthwashes decreased the number of C. albicans colonies. However, the recurrence of denture stomatitis after cessation of the use of these kinds of mouthwash has been reported 105 . In addition, mouthwashes containing chlorhexidine could cause side effects such as staining of the teeth and oral mucosa 109. 3. Palatal brushing Oral health-care providers often suggest oral and prosthesis hygiene instructions such as palatal brushing to complete denture wearers. In a recent observational study 110 , the chance of remission of denture stomatitis was 3.9 times higher in participants who brushed their palate (RR = 3.9, CI 1.0 to 15.9, p = 0.04). In addition, an association between the lack of palatal brushing and the occurrence of C. albicans was reported (OR = 1.8, 95 % CI 1.3 to 2.4, p = 0.03) 30. However, there is still a paucity of research on this topic; and to our knowledge, no interventional study has examined this mode of oral hygiene. 4. Laser and photodynamic therapy Photodynamic therapy (PDT) and Diode laser irradiation have been successfully used in the treatment of denture stomatitis 111-114. Photodynamic therapy consists of activation of a photosensitizing agent by means of oxygen and a light source (Light-Emitting Diode) with specific wavelengths. This results in the formation of cytotoxic oxgen molecules that damage the cells of the microorganisms 115 . In a randomized controlled trial, Mima 23 et al. 113 evaluated the efficacy of PDT and found this therapy as effective as nystatin in the treatment of denture stomatitis. The mechanism underlying the diode laser (low-power laser) therapy is not well understood. One hypothesis suggests that laser irradiation produces reactive oxygen, which reduces the proliferation of the microorganisms trial, Maver-biscanin et al. 112 116 . In a randomized controlled compared the effects of diode laser irradiation with antifungal treatment regarding the reduction of Candida CFUs. They found no difference between the laser treatment and the antifungal medication regarding the remission of denture stomatitis. The disadvantages of PDT and diode laser irradiation include the need for special equipment and difficulty in implementation. In addition, recurrence of denture stomatitis was noted for both treatments 112, 113. 5. Prosthesis hygiene Chemical disinfection and mechanical cleaning of the prosthesis can be used to clean the dentures. The prostheses can be chemically disinfected by immersing them in cleaning solutions containing alkaline peroxide (Polident®, Efferdent®) chlorhexidine gluconate (Peridex™) 105 92, 106, 117 , 0.12 % , or sodium hypochlorite 0.05 % (10 ml hypochlorite 1 % in 200ml water for 10 minutes) 118. Sodium hypochlorite is considered an effective, accessible and inexpensive disinfecting agent 119, especially when used as a Milton™ solution (2 % aqueous solution of sodium hypochlorite with 16.5 % salt). This solution causes less damage to the dentures when compared to household bleach 120, 121. 24 Many studies have been conducted to test the effectiveness of chemical methods in denture biofilm removal 122, 123 . In a randomized crossover trial, Gornitsky et al. 123 assessed the efficacy of three commercial denture cleaners (Denture Brite®, Polident® and Efferdent®) in the removal of denture biofilm and in the reduction of the number of microorganisms. They did not find any difference between the three brands after 3 weeks of use. In an in-vitro study, Glass et al. 92 found that soaking of the dentures in Polident® was more effective in reducing microorganisms’ load than microwaving. Some other findings showed that the use of denture cleaners containing chlorine dioxide and 0.2 % chlorhexidine gluconate were effective in the elimination of the biofilm 122 . Furthermore, the combination of a denture disinfectant such as chlorhexidine with an antifungal treatment was shown to improve the efficacy of the antifungals 124 . However, this combination could increase the resistance of Candida species over time 125. Several studies compared the efficacy of the chemical and mechanical methods of denture cleaning in the treatment of denture stomatitis. Silva-Lovato et al. 126 compared the cleaning capacity of brushing the prosthesis to soaking in a denture cleaner containing sodium lauryl sulphate (NitrAdine™). They found that the chemical method was more effective in terms of biofilm removal (p < 0.001) and reduction of Candida colonies (p < 0.05) than cleaning the prosthesis with a denture brush. Paranhos et al. 117 conducted a longitudinal study to compare the cleaning capacity of alkaline peroxide solution (Bonyplus®), mechanical brushing with toothpaste, and the combination of both methods. The findings of this study suggest that brushing the denture was more effective in biofilm removal than the use of a sodium peroxide 25 soaking solution. However, the most effective method for denture hygiene was the combination of mechanical and chemical methods 117, 127. It should be noted that denture cleansing agents could be corrosive and detrimental to the prostheses in long-term use and could have some side effects such as altering the taste of denture wearers 108, 126. 6. Microwave disinfection Microwaves have been used for disinfection of the prosthesis. The mechanism of action of microwaving is not well understood and is attributed to the thermal or non-thermal effect 128. According to thermal theory, heat generated by the vibration of the molecules could result in the disintegration of the microorganisms. The disinfecting effect of microwaving could also be the result of non-thermal interactions between the molecules of the cell wall of microorganisms and the microwave electromagnetic field 128-130. Although there is still no definitive guideline for microwave use 131 , according to the literature, the prosthesis should be disinfected once a week by immersion in a 600 ml container with 200 ml of water, and irradiated at 650 W for 3 minutes 132, 133. According to several studies, microwaving is as efficient as other alternatives such as sodium hypochlorite soaking 121 , and miconazole and nystatin medications 80, 128, 132 , in the treatment of denture stomatitis. This method is simple and user-friendly, especially for the elderly with decreased dexterity 131 . However, in most of these studies, high recurrence rates of denture stomatitis after cessation of the microwaving were demonstrated 80, 128 . In addition, microwave disinfection could lead to the shrinkage of the denture bases 134 or a decrease in the hardness of the denture teeth 135. 26 7. Phytomedicines Lately, there has been a tendency toward the use of herbal and natural remedies (phytomedicines). The recent narrative review by Casaroto and Lara several studies 78, 137, 138 136 , in addition to , reported that phytomedicines are effective in the treatment of denture stomatitis. In a recent randomized double-blind clinical trial, Bakhshi et al. 137 compared the effect of nystatin and garlic aqueous extract on denture stomatitis. Both treatments produced a statistically significant decrease in the extent of the erythema caused by denture stomatitis. Although a faster effect was noted with the use of nystatin, the garlic extract had fewer side effects than the antifungal. In another randomized controlled trial, the efficacy of the essential oil of the Z. multiflora herb was compared to 2 % miconazole gel 78 . Z. multiflora exhibits antimicrobial activity and can inhibit the activity of 139 inflammatory mediators , and proved to be as effective as the miconazole gel in the treatment of denture stomatitis. The extract of Punica granatum Linné (pomegranate) 140 and propolis gel 138, 141 (a resinous substance collected by bees from plants), were also as effective as miconazole gel in the treatment of denture stomatitis. Other natural substances that were examined and were effective in the treatment of denture stomatitis include vinegar alternifolia extracts (tea tree oil) 143 142 , Melaleuca , Satureja hortensis essential oil 144 and Ricinus communis 145. Nonetheless, it should be noted that phytomedicines can have side effects such as bad taste, in addition to recurrence and relapse after cessation of their use 78, 137. 27 1.4.1. II Prosthodontic measures Several prosthodontic procedures with the aim of improving the fit and the stability of the prosthesis can be performed in the cycle of treatments of denture stomatitis. These include the use of tissue conditioners, prosthesis adjustments, and the renewal of the prosthesis 38. Tissue conditioners are used to manage ill-fitting dentures and inflamed tissue under the prostheses. They have a cushioning effect that improves distribution of the occlusal forces and decreases trauma to the underlying tissues 146 . Tissue conditioners have been shown to be successful in reducing palatal inflammation associated with denture stomatitis 147, 148 . However, they were not effective in the treatment of the candidiasis 102 associated with this disease . Furthermore, the resilient quality of tissue conditioners decreases with time and they become hard, stained and porous. This time-related deterioration of the material contributes to the microorganisms’ colonization 149, 150. A number of studies examined the efficacy of incorporating antifungals in denture lining materials 143, 150-152 . Geerts et al. 150 evaluated the benefit of adding an antifungal in a short-term denture liner. Their findings showed that antifungals inhibit the colonization of Candida species in the relining material. Hard autopolymerising reline materials have also been examined in the treatment of denture stomatitis. These hard liners are long lasting, produce a low polymerising exothermic reaction and can be used directly in the mouth. In a randomized controlled trial by Marin Zuluaga et al. 153 , treatments with hard and soft tissue conditioners were found to be equally effective in the management of denture stomatitis. However, the 28 time needed for remission of inflammation was longer for the soft tissue conditioners compared to the harder one. In a one-group pre-test/post-test study, Pires et al. 18 evaluated the effect of new well- fitted complete dentures on denture stomatitis. They found a 30 % decrease in the frequency of this disease. However, there was no change in the number of Candida colonies. In accordance with several studies 103, 154 , Arikan et al. 124 found that the making a new prosthesis for patients with denture stomatitis will improve the localized inflammation but will have no effect on generalized inflammation and colonization of Candida. 1.4.2 Antifungal medications Antifungal medications are used to eliminate different fungal species, particularly Candida species. The antifungals used in the treatment of denture stomatitis can be applied topically or used systemically. Topical antifungals include the polyenes (nystatin or amphotericin B) used as lozenges or suspensions and the imidazoles derivatives (such as clotrimazole and miconazole), used as gel or lacquers. Ketoconazole and the triazole derivatives (fluconazole and itraconazole) could be prescribed as tablets or capsules for systemic use 21. Recently, other triazole antifungal agents with broader spectrum activity like voriconazole, ravuconazole, and posaconazole have been used to treat oral candidiasis associated with denture stomatitis, especially in immunocompromised patients 155, 156. 29 Nystatin is the most widely used antifungal in the treatment of oral candidiasis and denture stomatitis. Dentures can be soaked in a solution of nystatin (100,000 U/ml). Nystatin cream (100,000 U/g) could also be applied to the inner surface of the prosthesis. Treatment should continue for a minimum of 4 weeks along with meticulous oral hygiene maintenance 157. The use of antifungal medications to treat denture stomatitis has been studied by several research groups 24, 101, 158. In a randomized controlled trial by Cross et al. 79, itraconazole cyclodextrin solution and itraconazole capsules were found to be similar in terms of reduction of clinical signs of denture stomatitis and reducing the colonization of the yeast. However, recolonization of Candida species was seen at 6-month follow-up. Similar results were found in studies comparing the systemic and topical application of antifungals such as ketoconazole, fluconazole and amphotericin 101, 159. Some attempts have been conducted to produce a topical antifungal with long-lasting effect to avoid the side effects of systemic antifungals. For example, miconazole has been incorporated in lacquer agents. This treatment proved to have a short-term effect and recolonization of Candida was observed after treatment 160, 161. As previously mentioned in section 1.4.1, several studies 26, 78, 108, 113, 128 compared antifungal treatments with different conservative methods. No significant differences between the antifungal and the conservative methods were found. Antifungal medications have multiple disadvantages such as side effects, the emergence of resistance, and recurrence. 30 The excessive use of antifungals may produce some side effects, such as bitter taste, nausea, gastrointestinal disturbance, hypersensitivity, renal and liver toxicity, and interaction with other medicines 46, 47, 50. Side effects are especially seen with the use of systemic antifungals 79, 101, 137, 162. Furthermore, resistance, which is defined as non-resolution of an infection despite the use of an antimicrobial agent, have been demonstrated with antifungal medications 163 156, . High recurrence rates of denture stomatitis have also been reported after cessation of 32, 78, 101, 150, 164 the antifungal treatment . The emergence of resistance may explain this extensive recurrence of denture stomatitis 86, 125, 165, 166. 31 CHAPTER II METHODOLOGY 2.1 PROBLEMATIC, HYPOTHESIS, OBJECTIVES Denture stomatitis is the most prevalent oral disease in denture wearers 35, 60 . The treatment of this disease consists of two different approaches: the use of antifungal medications and the conservative approach 94. Antifungal therapy is the main choice of oral healthcare professionals for the treatment of this disease, based on some evidence that Candida is the main etiological factor in the onset of denture stomatitis. However, a direct cause-and-effect relationship has never been shown, and high recurrence rates of denture stomatitis clinical signs and recolonization of Candida have been reported after cessation of the antifungal treatment 78-80 . Recent research on the etiology of denture stomatitis suggests that treatments based on the reduction of the inflammation are effective in the management of this disease 7, 25. In this regard, some new findings suggest palatal brushing as a curative method 110 . However, to our knowledge, there is still no interventional study that has evaluated the efficacy of such an alternative approach. 32 2.1.1 Objectives The objectives of this master’s research project were: 1. Primary objective: To assess the efficacy of palatal brushing in the treatment of denture stomatitis. 2. Secondary objectives: a) To assess the efficacy of palatal brushing in controlling the colonization of Candida species. b) To standardize the study procedures and to produce preliminary data for a phase-II clinical trial. 2.1.2 Hypotheses We tested the following null hypotheses: 1. There is no difference in the extent of palatal inflammation in individuals with denture stomatitis before and 3 months after palatal brushing. 2. There is no difference in the number of Colony-Forming Units (CFUs) of Candida isolated from the palate and denture of patients with denture stomatitis before and 3 months after palatal brushing. 33 2.2 RESEARCH METHODOLOGY 2.2.1 Study design This study was a phase-I clinical trial with a one-group pre-test/post-test design. It has been registered in clinicaltrials.gov and received the identification number NCT01643876. The project was conducted at two Faculties of Dentistry of the Université de Montréal in Canada and the University of São Paulo (Ribeirão Preto) in Brazil. 2.2.2 Study participants and inclusion criteria Study participants were recruited from the general population of the area of Metropolitan Montreal and Ribeirão Preto via advertisements in local newspapers, through flyers placed within dental clinics of the two dental schools, or by clinicians during clinical examination at diagnostic clinics of these faculties. All potential participants were informed about the study, and those who were willing to participate in the study were invited to a screening clinical session. During this session, the master’s students explained all aspects of the study, and examined candidates for suitability for inclusion in the study. Candidates were considered for inclusion in this study if they: 1) were 18 years old or older; 2) were wearing a complete upper denture; 3) had a clinical diagnosis of denture stomatitis; 34 4) had the ability to understand and sign an informed consent form. Candidates were excluded from the study if they: 1) had any conditions known to promote Candida carriage such as uncontrolled diabetes, anemia, xerostomia or immunosupression; 2) received a treatment with an antibiotic, an antifungal or corticosteroids in the last four weeks prior to the study or were being treated with chemotherapy or radiotherapy; 3) included palatal brushing in their routine oral hygiene; 4) were planning to change their existing prosthesis during the trial. Each individual meeting the eligibility criteria were invited to read and sign the consent form (Appendix I). They were given an opportunity to ask questions and to take the research consent form home for further consideration. Finally, forty-eight consecutive participants (men = 16, women = 32) from the two study centers (Canada, n = 22; Brazil, n = 26) participated in the clinical trial. 2.2.3 Experimental procedure The experimental procedure (intervention) consisted in brushing the palate with a softbristle manual brush (Oral-B® CrossAction® Pro-Health™) after each meal and before sleeping, for a period of 3 months. In order to standardize the experimental technique, the participants were asked to keep to their usual oral and denture hygiene routine during the trial. A cast model was used to show the participants how to brush the palate. The study participants also received a 35 written instruction sheet (Appendix V) to remind them of the study’s experimental procedure. 2.2.4 Data collection and measurement instruments The data collection was conducted at the postgraduate prosthodontic clinics of the Faculties of Dentistry of the Université de Montréal and the University of São Paulo at baseline (T0), 1 month (T1), and 3 months (T2) of the intervention, during the period extending from May 2012 to January 2013. One master’s student in each center (MK and MB) was responsible for the data collection. Data were collected by means of a self-administered questionnaire (Appendix II), a clinical examination (Appendix III) and a microbiological investigation (Appendix IV). All procedures were standardized between the two centers during research team meetings before data collection. 2.2.4. I Clinical investigation In each center, the diagnosis of denture stomatitis was conducted by three trained, calibrated dentists (interobserver reliability 0.6 to 0.84) using a front-surface mirror and probe (XP23/QW, HuFreidy). Furthermore, the photographs of palate were taken with a Nikon D90 camera (105mm f/2.8 D; macro flash SB-21). These photographs were used to obtain a diagnostic consensus from research team members during a workshop held at the faculty of Dentistry of the Université de Montréal from November 19th to December 7th, 2012. 36 Denture stomatitis was diagnosed according to the area and severity of inflammation indices 26 and a modified classification of Newton 23, as detailed below: I. Inflammation area index: 0: No inflammation 1: Inflammation of the palate extending up to 25 % of the palatal denture-bearing tissue 2: Inflammation of the palate extending between 25 % and 50 % of the palatal denture-bearing tissue 3: Inflammation covering more than 50 % of the palatal denture-bearing tissue. II. Inflammation severity index: 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) A score between 0 and 6 for total inflammation was then given, which equals the area + intensity of inflammation 26. III. The modified classification of Newton: 0: Healthy mucosa 37 Type IA: Petechiae in a normal palatal tissue, which are usually found around the orifices of the ducts of the palatal mucous glands Type IB: Localized area of inflammation of the denture-bearing area Type II: Generalized area of inflammation of the denture-bearing area Type III: Hyperplasic palatal surface with inflammation of the denture-bearing area. The other assessed variables included denture cleanliness, which was evaluated by the modified Hoad-Reddick classification 167, as clean (without any soft/hard debris or stain) or dirty (with soft and hard debris or stain after washing under water). The upper prostheses were also evaluated regarding their stability and retention. The stability was evaluated by determining the movement of the prosthesis over the supporting tissues and its resistance to rotation. The presence or absence of a noticeable rocking motion was also noted 168, 169 . The retention was evaluated by asking the participant to touch the vermillion border of their upper lip with their tongue and by opening their mouth to the maximum. If the denture dropped, it was considered nonretentive. The upper prosthesis was grasped by the thumb and index finger and a downward force was applied. The absence or presence of adequate resistance to dislodgment was noted 168, 169. Other explanatory variables included the clinical signs of parafunctional habits, salivary flow, and the resorption and tissue resilience of the upper residual ridge addition to the vertical dimension of occlusion 168, 169. 170-173 , in 38 Information about socio-demographic variables (age, sex, education, medical and dental histories, medication profiles), years of edentulism and age of the dentures, hygienic habits (cleaning frequency, nocturnal wear, mouthwash use) and smoking, were obtained from a validated questionnaire 30. The level of the reported oral hygiene was estimated through questions with categorized answers (How many times per day do you clean your dentures? How do you clean your dentures?). The answers were binary summarized as cleaning of the dentures less than two times/day or 2 times and more/day; and brushing the dentures or washing without brushing. A 100 mm visual analogue scale (VAS), with anchor words of “not at all satisfied” and “extremely satisfied” 7, was also used to assess the general satisfaction with oral condition. Furthermore, symptoms and side effects of palatal brushing, if present, were reassessed after 1 and 3 months of the intervention. 2.2.4. II Microbiological investigation In order to evaluate the colonization of Candida species, microbiological investigations on the upper denture and palatal plaques were conducted. With this aim, the sonication technique 121, 174 and swabs were used to collect the upper denture biofilm and palatal biofilm, respectively. In brief, the upper prosthesis was rinsed under tap water and placed in a Ziploc™ plastic bag with 30 ml of sterile saline water (0.85 % sodium chloride). The first bag was then put in a second bag and sonicated for 5 minutes at room temperature in an 39 ultrasonic bath containing distilled water (Cole Parmer 08890-21, 50/60 Hz, 1,3 Amp). The recovered plaque (sonicate) was transferred to a 50 ml sterile tube. A sample of palatal plaque from 1 cm² of the central surface of the palate was collected by the use of a sterile swab. The swab was then placed in a sterile tube with 5ml saline and sonicated for 2 minutes. Both denture and palatal plaque sonicates were placed on ice until the microbiological examination. Sonicates were mixed by vortex for one minute and diluted 10-fold serially with saline (dilution factors: 100, 10-1 and 10-2). A volume of 100 μL of each dilution was spreadplated in duplicate on Sabouraud-Dextrose 4 % Agar (Difco™, Becton, Dickinson and Company, USA). All cultures were incubated at 37o C for 48 hours. Colony-Forming Units (CFUs) were counted and expressed as a number of CFUs/ml, after correction for volume and dilution factor. When a growth was observed, an imprint of colonies was obtained onto a sterile filter paper which was transferred on a Candida selective growth medium (CHROMagar Candida, Paris, France) and incubated under the same conditions. This chromogenic selective medium allows identification of the different Candida species 175. 2.2.5 Outcome and explanatory variables The remission of denture stomatitis was considered as a primary outcome variable. The secondary outcome variable was the diminution of the Candida CFUs. The explanatory variables included socio-demographic characteristics and well-known denture stomatitis risk factors 30. 40 2.2.6 Statistical analysis Assuming that the minimal practically important pre/post difference in the mean change score is 20 % and the standard deviation of the distribution of the change in score is 0.8 (based on estimates from a previous study 110 ), a sample size of 44 participants is required to ensure a power of 90 % of rejecting the null hypothesis if it is indeed false. An additional number of individuals were considered to prevent the effect of potential 10 % drop-outs on the study results. Inter-rater agreement for the diagnosis of denture stomatitis was evaluated by using Cohen’s kappa (k) 176 , with k value > 0.75 representing excellent agreement, 0.4–0.75 fair to good agreement and < 0.40 poor agreement. In order to obtain frequency counts, percentages and univariate means, and to test for normality, the data was first subjected to descriptive statistical tests. Non-parametrical analyses were applied because of the deviation from a normal distribution. Between-center differences in regard to baseline characteristics of the participants and treatment effects were analyzed by means of the Fisher's exact test, two-sample t-test and the Mann–Whitney U test. The Wilcoxon Signed Rank Test was used to analyze the change in the classifications level, in the total score, and in the number of Candida colonies between T0 and T2. McNemar’s test was used to determine if there were differences in the participants’ reported symptoms between T0 and T2. The Budtz-Jorgensen 177 index was used to categorize the clinical effects of treatment:  Large effect: inflammation resolved 41    Moderate effect: inflammation reduced No effect: no change in inflammation Negative effect: increased inflammation The effect size r was calculated from the Z obtained from the Wilcoxon Signed Rank Test (r = Z/√N, where N is the number of observations over the two time points) and was defined as 0.1 = small effect, 0.3 = medium effect and 0.5 = large effect 178. Differences were considered statistically significant at p ≤ 0.05. All statistical analyses were performed by using SPSS version 20 (SPSS Inc., Chicago, IL, USA) and SAS 9.1 software. 2.2.7 Ethical considerations The ethical approval for this master’s research project was obtained from the Université de Montréal Research Ethics Board (CERES, certificate number 12-019-CERES-D) and the Institutional Review Board of the Ribeirão Preto Dental School (certificate number 00625912.6000.5419). 2.3 STUDY RELEVANCE To our knowledge, this study is the first clinical trial on the effect of palatal brushing on denture stomatitis. The results of this study will provide clinicians with scientific evidence on a simple, conservative, cost/effective therapeutic and preventive approach that will contribute to the improvement of oral health in edentate individuals wearing complete dentures. The findings of this clinical trial will also assist clinicians in their 42 decision-making in regard to the treatment of denture stomatitis and will avoid the over-prescription of antifungal medications. Finally, this phase-I clinical trial will allow the elaboration of a randomized clinical trial on the efficacy of palatal brushing in comparison with the use of antifungal medications. 2.4 CANDIDATE’S ROLE IN THE PROJECT During this master’s project, the candidate has fulfilled several roles. First of all, she helped in the research protocol development. She was responsible for the recruitment of the study participants, data collection and data analyses. She was in charge of the coordination of the study between the Canadian and the Brazilian centers. She also participated actively in the knowledge-transfer phase of this research project. 2.5 KNOWLEDGE TRANSFER The candidate presented the results of this research project during several scientific meetings and conferences: 1) Oral presentation: “Séminaires de recherche en médecine dentaire” (SAB6604A11) at the Université de Montréal; 2) Poster presentation : i. Kabawat M, De Souza RF, De Koninck L, Barbeau J, Rompré P, Emami E. Phase-I clinical trial on the effect of palatal brushing on denture stomatitis. Journées Dentaires Internationales du Québec (JDIQ), Montreal, Canada, May 24th to 28th 2013. 43 ii. Kabawat M, De Souza RF, De Koninck L, Barbeau J, Rompré P, Emami E. Phase-I clinical trial on the effect of palatal brushing on denture stomatitis. Canadian dental research student workshop, Ontario, Canada, June 10th to 11th 2013. The candidate was also co-author of 3 articles on the topics related to the master’s project: 1) Emami E, De Souza RF, Kabawat M, Feine JS. The impact of edentulism on oral and general health. International Journal of Dentistry; 2013:498305. doi: 10.1155/2013/498305. Epub 2013 May 8. 2) Emami E, Kabawat M, De Koninck L, Gauthier G, de Grandmont P, Barbeau J. La stomatite prothétique: nouvelle perspective. Journal de l’Ordre des Dentistes du Québec 2013; 50(4):7-12. 3) Emami E, Kabawat M, Rompré P, Feine JS. Linking evidence to treatment for denture stomatitis: a meta-analysis. Journal of Dentistry 2013; Submitted. The article included in chapter III of this master’s thesis was also submitted for publication in The International Journal of Prosthodontics. Finally, the candidate will present the results of this study during The International Association for Dental Research (IADR) General Session in 2014. 44 CHAPTER III RESULTS 3.1 MANUSCRIPT Phase-I clinical trial on the effect of palatal brushing on denture stomatitis Marla Kabawat1, Raphael F. de Souza2, Maurício M. Badaró2, Louis de Koninck1, Jean Barbeau3, Pierre Rompré3, Elham Emami1 1 Département de Dentisterie de Restauration, Faculty of Dentistry, Université de Montréal, Montreal, Quebec, Canada. 2 Department of Dental Materials and Prosthodontics, Ribeirão Preto Dental School, University of São Paulo, Ribeirao Preto, SP, Brazil. 3 Département de stomatologie, Faculty of Dentistry, Université de Montréal, Montreal, Quebec, Canada. Corresponding author: Dr Elham Emami, DDS, MSc, PhD Associate professor and Director of Oral Health and Rehabilitation Research Unit Faculté de médecine dentaire, Université de Montréal C.P. 6128, succ. Centre-ville Montréal (QC), Canada H3C 3J7 Keywords: Denture stomatitis, palatal brushing, complete denture, Candida, therapy. 45 Phase-I clinical trial on the effect of palatal brushing on denture stomatitis ABSTRACT Objectives: To assess the efficacy of palatal brushing in the treatment of denture stomatitis. Methods: After screening 143 individuals with a potential diagnosis of denture stomatitis, 48 (mean age: 66.0 ± 11.2 years) were enrolled in a phase-I two-center clinical trial with one-group pre-test/post-test design. The intervention of interest was manual palatal brushing after each meal and before bedtime. Clinical and microbiological examinations were performed at baseline, 1 month and 3 months postintervention. Additional data were obtained by the use of a validated questionnaire. The primary and secondary outcomes were the remission of denture stomatitis and the diminution of Candida Colony-Forming Units (CFUs), respectively. Descriptive and non-parametric statistical tests were conducted to analyze the data. Results: At 3-month follow-up, denture stomatitis was completely cured in 10.4 % of the study participants, and 70.8 % of denture wearers showed improvement in the clinical signs of denture stomatitis. There was a significant reduction in the area and severity of the palatal inflammation at 3-month follow-up (p < 0.0001). The effect size ranged from medium to large (0.34 to 0.54), depending on the classification used for the diagnosis of denture stomatitis. Furthermore, a significant reduction in the number of Candida CFUs isolated from the palatal mucosa and dentures (p ≤ 0.05) was observed. 46 Conclusion: The results of this study suggest that palatal brushing is effective in the treatment of denture stomatitis. 47 INTRODUCTION Denture stomatitis is a chronic inflammation of the oral mucosa covered by a removable denture. It is considered the most common mucosal lesion associated with denture use 2 1, , affecting one in every three complete denture wearers 3. Several risk factors have been reported to be associated with denture stomatitis, including trauma 4, denture biofilm 5, bacterial and fungal infections, particularly by Candida albicans 6. However, the etiology of this pathological condition remains multifactorial and controversial 7, 8 . A variety of treatments reflecting the multifactorial etiology of denture stomatitis have been used in dental practice. These treatments can be basically classified into two categories: the conservative approach and the use of antifungal medications. Nowadays, antifungal medications are prescribed routinely by oral healthcare professionals for the treatment of denture stomatitis, based on the hypothesis that an infection by Candida is the main etiological factor of this disease 9-11 . However, a convincing cause-and-effect relationship between the presence of denture stomatitis and Candida has never been demonstrated 7, 8, 12 . Furthermore, high recurrence rates of denture stomatitis and recolonization of Candida have been reported after the cessation of antifungal treatment 9, 13, 14. Recent research findings suggest that trauma from unstable dentures induces a local inflammation and creates an environment favourable to the proliferation of microorganisms 4. Consecutively, Candida colonization becomes a secondary stage in the pathogenesis of denture stomatitis 7, 15 . This suggests that treatments that enable the remission of inflammation could be effective in the treatment of this disease. 48 Palatal brushing is a simple procedure that could reduce the extent of inflammation by different mechanisms such as the removal of denture plaque and the stimulation of the mucosal circulation and salivary flow. However, no previous clinical trial has evaluated palatal brushing as a treatment modality for denture stomatitis. Therefore, the objective of this study was to assess the efficacy of palatal brushing in the treatment of denture stomatitis. We tested the null hypotheses that, in individuals with denture stomatitis, there are no difference in the extent of palatal inflammation and in the number of Candida Colony-Forming Units (CFUs), before and 3 months after palatal brushing. MATERIALS AND METHODS Study Design and Study Participants A one-group pre-test/post-test research design was used to conduct a two-center phase-I clinical trial (clinicaltrials.gov ID # NCT01643876), at two Faculties of Dentistry of the Université de Montréal (Canada) and the University of São Paulo (Ribeirão Preto, Brazil). Participants were recruited from the general population of the area of Metropolitan Montreal and Ribeirão Preto via advertisements in local newspapers, through flyers placed within dental clinics of the two dental schools, or by clinicians during examination at diagnostic clinics of these faculties. The inclusion criteria of the study were: a) being 18 years old or older, b) wearing a complete upper denture, and c) having a clinical diagnosis of denture stomatitis. Patients were excluded if they: a) had any conditions known to promote Candida carriage such as uncontrolled diabetes, anemia, xerostomia or immunosuppression; b) received a 49 treatment with an antibiotic, an antifungal or a corticosteroid or if they were under chemotherapy or radiotherapy in the last four weeks prior to the enrollment in the study; c) used palatal brushing as a routine oral hygiene procedure; and d) if they changed their existing prosthesis during the trial. The study was approved by the Université de Montréal Research Ethics Board and the Institutional Review Board of the Ribeirão Preto Dental School. Written informed consent was obtained from the participants prior to their participation in the clinical trial. Experimental Procedures Data collection was conducted at baseline (T0), 1-month (T1) and 3-month (T2) after the intervention, by means of a self-administered questionnaire, clinical examination and microbiological investigation. 1. Intervention The intervention consisted of brushing the palate with a soft-bristle manual toothbrush (Oral-B® CrossAction® Pro-Health™, Procter & Gamble, Iowa, IA, USA) after each meal and before sleeping, for a period of 3 months. Participants were instructed to brush their palate using horizontal, vertical and vibration movements. They were also asked to keep their usual oral and denture hygiene habits during the trial. 2. Clinical investigation Denture stomatitis was assessed according to the modified Newton classification 16, and by means of the area and severity of the inflammation indices 17 (Table 3.1). The clinical assessment was conducted by two trained, calibrated dentists using a front surface mirror and probe (XP23/QW, Hu-Friedy, Chicago, IL, USA). Photographs of 50 palate were taken with a Nikon D90 camera (105mm f/2.8 D; macro flash SB-21, Nikon Co., Tokyo, Japan). These photographs were used to obtain a diagnostic consensus from research team members. A good to excellent interobserver reliability was obtained (κ = 0.6 to 0.84). 3. Microbiological investigation Collection of the upper denture biofilm was carried out by the sonication technique, according the protocol described by Emami et al. 8. A sample of palatal biofilm was also collected by the use of a sterile swab 18 , placed in a tube with 5ml saline and sonicated for 2 minutes. Both denture and palatal biofilm sonicates were subsequently mixed by vortex for one minute and diluted 10 fold serially with saline (dilution factors: 10 0, 10-1 and 10-2). A volume of 100 μL of each dilution was spread -plated in duplicate on Sabouraud-Dextrose 4 % Agar (Difco™, Becton Dickinson Co., Detroit, MI, USA). All cultures were incubated at 37oC for 48 hours. Colony-Forming Units (CFUs) were counted and expressed as a number of CFUs/ml, after correction for volume and dilution factor. When a growth was observed, an imprint of colonies was obtained onto a sterile filter paper, which was transferred on a Candida selective growth medium (CHROMagar™, Candida, Paris, France) and incubated under the same conditions. This chromogenic selective medium allows identification of the Candida species 19 4. Outcome measures and explanatory variables The remission of denture stomatitis was considered as a primary outcome variable, and it was defined as decrease in the level of the modified Newton classification, decrease in 51 the area and the severity of the inflammation, as well as decrease in the total score of inflammation (inflammation area + severity) 17. The Budtz-Jorgensen index 20 was used to evaluate the magnitude of the treatment effect (based on the total score of inflammation): Large effect: inflammation resolved; Moderate effect: inflammation reduced; No effect: no change in inflammation; Negative effect: increased inflammation. The secondary outcome variable was the reduction in the number of CFUs of Candida. Explanatory variables included sociodemographic variables (age, sex, education, medical and dental histories, and medication profiles), years of edentulism and age of the upper dentures, hygienic habits (cleaning frequency, nocturnal wear, mouthwash use) as well as smoking. This information was obtained from a self-administrated questionnaire 8. General satisfaction with oral condition was assessed by a 100 mm visual analogue scale (VAS) 4. Other independent variables included denture cleanliness, evaluated by means of the modified Hoad-Reddick classification and retention of upper prostheses 22, 23 8, 21 , and the stability . Denture stability was evaluated by determining the movement of the prosthesis over the supporting tissues and its resistance to rotational movement. The upper prosthesis was grasped in the premolar region with the thumb and index finger and a rotational force was applied in the occlusal plane. A displacement of 5 mm or more was considered as prosthesis instability. The prosthesis was considered retentive if there was a resistance to downward force when grasping the prosthesis by the thumb and index finger 22, 23. 52 The wear of the denture teeth, the salivary flow, the resorption and the resilience of the upper residual ridge, as well as the vertical dimension of occlusion evaluated. Furthermore, symptoms of denture stomatitis and side effects of palatal brushing, if any, were documented. Statistical Analyses Assuming that the minimal practically important pre/post difference in the mean change score is 20 percent and the standard deviation of the distribution of the change in score is 0.8 28, a sample size of 44 participants is required to ensure a power of 90 % of rejecting the null hypothesis if it is indeed false. An additional number of individuals were considered to address potential 10 % drop-outs on the study results. Cohen’s kappa (κ) coefficient 29 22-27 , were also was used to evaluate the inter-rater agreement for the diagnosis of denture stomatitis, with κ value > 0.75 representing excellent agreement, 0.4–0.75 fair to good agreement and < 0.40 poor agreement. In order to obtain frequency counts, percentages, univariate means and to test for normality, the data was first subject to descriptive statistical tests. Non-parametrical tests were applied because of the non-normal distribution of data. Between-center differences in regard to baseline characteristics of the participants and treatment effects were analyzed by means of the Fisher's exact test, two-sample t-test and the Mann–Whitney U test. The Wilcoxon Signed Rank test was used to compare the level of the modified Newton classification, the area and the severity of the inflammation, the total score of 53 inflammation as well as the number of Candida colonies between baseline and followups. McNemar’s test was used to compare nominal data on patient-reported symptoms at baseline and follow-ups. The level of significance was set at 0.05. The Fisher's exact test and two-sample t-test were used to examine the significance of association between the explanatory variables and the treatment effect. Logistic regression was not conducted because of non-significant association (p > 0.10) in bivariate analyses. Statistical analyses were performed using SPSS version 20 (SPSS Inc., Chicago, IL, USA) and SAS 9.1 software (SAS Institute, Cary, NC, USA). RESULTS From a total of 143 individuals who participated in the screening sessions, 48 patients (16 men, 32 women; mean age: 66.0 ± 11.2 years) were enrolled in this study. Figure 3.1 illustrates the flow of participants throughout the study, indicating that there were no dropouts. Tables 3.2 and 3.3 present the socio-demographic characteristics of the participants and their profiles according to denture stomatitis risk factors at baseline, by study center. The mean year of edentulism in the maxillary arch was 37.2 ± 14.7 years and the mean age of the current upper prostheses was 15.3 ± 13.7 years. Signs of wear facets on denture teeth were observed in 77.1 % of the participants. The majority of the patients brushed their prostheses with toothpaste (79.2 %), and 31.3 % used a denture-cleaning agent. However, only the Canadian center participants used the latter. Twenty percent of the participants had previously received denture hygiene instructions. Only one 54 participant used denture adhesive on his upper denture. At baseline, the most reported symptoms of denture stomatitis were halitosis (52.1 %) and a dry mouth sensation (66.7 %). The salivary flow was adequate in 83.3 % of the participants, and 91.7 % had a wellrounded ridge with sufficient height and width (Class III resorption 27 ). Finally, 39.6 % of the patients had resilient tissue covering their residual ridge. There was no statistically significant difference between the two study centers regarding the demographic characteristics and risk factors associated with denture stomatitis, except for gender, income, educational level, denture cleanliness, and the mean age of the current prostheses. The Brazilian participants had more women enrolled in the trial, less income, lower education, and finally, older and less clean upper prostheses than the Canadian participants (Tables 3.2 and 3.3). At T0, 6.3 % of the participants had petechiae (Type IA), 16.7 % had localized inflammation (Type IB), 39.6 % had generalized palatal inflammation (Newton Type II) and finally, 37.5 % had hyperplasic inflammation (Type III). Also, 16.7 % of the participants had an inflammation extending up to 25 % of the palate, 33.3 % had an inflammation covering between 25 % and 50 % of the palate and 50 % of the participants had an inflammation covering more than 50 % of the palatal denture-bearing area (area of inflammation index). In addition, 25 % of the participants had a mild inflammation, 43.8 % had moderate inflammation and 31.3 % had a severe inflammation (severity of the inflammation index). The microbiological analysis of the denture sonicates at T0 revealed that 39 participants were Candida carriers. Candida albicans was the most frequent species isolated (59 % of 55 the cultures from the denture sonicates). Other species included C. glabrata, C. krusei, C. tropicalis and C. parapsilosis. The cultures were negative in 9 patients (18.8 %) including patients classified as Type IA, Type IB and Type II. Cultures from the palatal swab were negative in 77 % of the cultures. There was no statistically significant difference between the two research centers according to the frequency of different types of denture stomatitis, and Candida carriage at baseline. There were no statistically significant changes in results at 1-month follow-up (T1). At 3-month follow-up (T2), denture stomatitis was cured in 10.4 % of the participants, and 70.8 % showed substantial improvement in the clinical signs of denture stomatitis. There was a worsening of the clinical signs of denture stomatitis in only one participant (Figure 3.2). There was a significant inflammation decrease according to the modified Newton classification (p = 0.001), and the area (p < 0.0001) and severity (p < 0.0001) of inflammation indices at 3-month follow-up. The reduction in the total score value of the inflammation was also significant (p < 0.0001) (Table 3.4 and Figure 3.3). Subgroup analyses showed that there was no change in the inflammation indices for patients with Type IA. Patients with Type II and III denture stomatitis showed a significant decrease in the total inflammation score (p < 0.0001). However, the hyperplasic tissue remained in all patients affected by Type III denture stomatitis. The effect size ranged from medium to large (0.34 to 0.54), depending on the classification used (Table 3.4). In addition, statistically significant improvements in the perceived oral condition of the participants (p = 0.003) and palatal burning sensation (p = 56 0.008) were found at 3-month follow-up. Overall, 40 % of the participants reported minor side effects of palatal brushing such as mild pain and some bleeding during the first days of treatment. There was no statistically significant difference between the centers regarding the treatment effect. However, there was a statistically significant difference between the two centers regarding the reported side effects of palatal brushing (p < 0.0001), with the majority of reported side effects occurring in the Brazilian center. Microbiological analyses showed a significant reduction in the number of CFUs of Candida isolated from denture plaque sonicates (p = 0.05) and from the palatal swabs (p = 0.048) at the 3-month follow-up (Table 3.5). Bivariate analyses did not reveal any statistically significant association between explanatory variables and treatment effect. DISCUSSION To our knowledge, this is the first phase-I clinical trial which provided data on the efficacy of the palatal brushing on denture stomatitis. The results of this study confirmed our previous reports on the positive effect of palatal brushing. In a recent observational study 28, we demonstrated that the chance of the remission of denture stomatitis was 3.9 times higher in participants who brushed their palate (RR = 3.9, 95 % CI 1.0 to 15.9, p = 0.04). Furthermore, we have shown that there is an association between the lack of palatal brushing and the occurrence of C. albicans (OR = 1.8, 95 % CI 1.3 to 2.4, p = 0.03) 8. 57 Several mechanisms could explain the effect of palatal brushing, including mechanical stimulation and oral biofilm removal 30 . An oral biofilm is a protective niche that may harbor a wide array of pathogenic microorganisms encased in extracellular polysaccharide matrix, including aerobic and anaerobic bacteria, and yeasts 31, 32 . There is overwhelming evidence that denture and palate biofilms are important risk factors for denture stomatitis 5, 33-35. Palatal brushing could eliminate this reservoir of pathogens and the source of irritation. This could justify the statistically significant decrease in Candida CFUs that was demonstrated in our study. The results of several studies demonstrated that mechanical stimulation encourages keratinisation, reduces the infiltration of inflammatory cells and enhances the proliferation of fibroblasts and collagen synthesis 30, 36-38 . It has been shown that even in the presence of oral biofilm, brushing stimulation with techniques such as vibration motion can improve the tissue microcirculation 39 . Consequently, the effects of mechanical stimulation could counteract the effects of the inflammatory process in denture stomatitis. This resolution could lead to the re-establishment of an undamaged epithelium and basement membrane within the palatal mucosa. This healthy mucosa then serve as a mechanical barrier against microbiological colonization 40, 41. Impaired salivary flow and xerostomia have been considered as predisposing factors in denture stomatitis 42, 43 . Palatal brushing could increase salivary flow by mechanical stimulation of the minor salivary glands of the palate. The stimulation of the salivary glands could have a mechanical cleansing effect and thus eliminate the denture biofilm 32, 40, 44 . In addition, saliva acts as an immune defence mechanism against 58 microorganisms 45 . In this study, patient-reported dry mouth was the most commonly 46, reported side effect of palatal brushing. However, in accordance with several studies 47 , the patients’ assessment did not correlate with the findings of the clinical examinations. Further research should include a more accurate assessment of unstimulated salivary flow to confirm these results 47. There are conflicting hypotheses on whether the inflammation in denture stomatitis is associated with trauma from unstable prostheses or if it results from fungal biofilm 4, 7, 41, 46, 48 . However, there is considerable evidence demonstrating the lack of a direct cause7, 8, 12 and-effect relationship between the presence of denture stomatitis and Candida . Furthermore, several studies on the efficacy of antifungal medications in the treatment of denture stomatitis demonstrated a high recurrence rate of clinical signs of denture stomatitis and the re-colonization of the Candida after cessation of the antifungal treatment 13, 14 . Our study results support the previous hypothesis that trauma is a primary etiologic factor in denture stomatitis. Thus, we encourage oral healthcare professionals to use conservative approaches such as oral hygiene instructions, palatal brushing, and prosthesis adjustments rather than antifungal medications in the treatment of denture stomatitis. In this study, we used a modified version of the Newton classification 16 . This modification introduced two subtypes for Newton's Type I denture stomatitis and allowed us to differentiate palatal petechiae (Type IA) from localized inflammation (Type IB). Our findings demonstrated a difference between these two different clinical manifestations. The microbiologic analyses of the dentures and palatal swabs of the 59 participants with Type IA denture stomatitis were negative for the presence of Candida. Furthermore, petechiae remained after palatal brushing. These results suggest that petechiae are merely the widening of the minor salivary glands ducts, which could be considered as a variation of the normal anatomy caused by trauma from the denture, and not a pathological sign of denture stomatitis 4, 49. We also found that the use of the area and severity of the inflammation indices 17, 50 permit a better classification of the clinical signs of denture stomatitis than the Newton classification. We recommend the use of these indices in clinical and research training to ensure the standardization of the methods and comparisons between trials. The results of this study should be interpreted with caution since a one-group pretest/post-test design was used, and the study did not include any control group. The encouraging results of this study should be confirmed by a phase-II clinical trial. CONCLUSION In conclusion, the results of this study suggest that palatal brushing is effective in the treatment of denture stomatitis. We recommend the use of palatal brushing as a crucial adjunct to the routine management of this condition. AKNOWLEDGMENTS This study was supported by the research funds of the Faculty of Dentistry of Université de Montréal and received financial support from the São Paulo Research Foundation (FAPESP, grant 12/04234-3), as well as the Ministère du Développement économique, de l’Innovation et de l’Exportation (MDEIE). Dr Elham Emami is supported by 60 Clinician-scientist Canadian Institutes of Health Research Salary Award. Marla Kabawat received a financial support from the International Health Unit (IHU) of the University of Montreal Hospitals’ Research Centre. The authors would like to acknowledge the expertise of Mrs. Annie Leduc regarding the microbiological investigations. 61 REFERENCES 1. Jainkittivong A, Aneksuk V, Langlais RP. Oral mucosal lesions in denture wearers. Gerodontology 2010;27:26-32. 2. Cueto A, Martinez R, Niklander S, Deichler J, Barraza A, Esguep A. Prevalence of oral mucosal lesions in an elderly population in the city of Valparaiso, Chile. Gerodontology 2012. 3. Zissis A, Yannikakis S, Harrison A. Comparison of denture stomatitis prevalence in 2 population groups. Int J Prosthodont 2006;19:621-625. 4. Emami E, de Grandmont P, Rompre PH, Barbeau J, Pan S, Feine JS. Favoring trauma as an etiological factor in denture stomatitis. J Dent Res 2008;87:440-444. 5. 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Development of Candida-associated denture stomatitis: new insights. J Appl Oral Sci 2008;16:86-94. 43. Campisi G, Panzarella V, Matranga D, Calvino F, Pizzo G, Lo Muzio L, et al. Risk factors of oral candidosis: a twofold approach of study by fuzzy logic and traditional statistic. Arch Oral Biol 2008;53:388-397. 44. Mese H, Matsuo R. Salivary secretion, taste and hyposalivation. J Oral Rehabil 2007;34:711-723. 45. Dodds MW, Johnson DA, Yeh CK. Health benefits of saliva: a review. J Dent 2005;33:223-233. 46. Altarawneh S, Bencharit S, Mendoza L, Curran A, Barrow D, Barros S, et al. Clinical and histological findings of denture stomatitis as related to intraoral colonization patterns of Candida albicans, salivary flow, and dry mouth. J Prosthodont 2013;22:13-22. 47. Wiener RC, Wu B, Crout R, Wiener M, Plassman B, Kao E, et al. Hyposalivation and xerostomia in dentate older adults. J Am Dent Assoc 2010;141:279284. 48. Ramage G, Tomsett K, Wickes BL, Lopez-Ribot JL, Redding SW. Denture stomatitis: a role for Candida biofilms. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;98:53-59. 49. Arendorf TM, Walker DM. Denture stomatitis: a review. J Oral Rehabil 1987;14:217-227. 50. Bergendal T, Isacsson G. Effect of nystatin in the treatment of denture stomatitis. Scand J Dent Res 1980;88:446-454. 64 List of Figures and Tables Screening Total n=143 Canada n=50 Brazil n=93 N( Recruitment and Signature of consent form Total n=48 Canada n=22 Brazil n=26 n=95 individuals excluded because failed to meet the inclusion criteria: - No denture stomatitis (n=69) - Partial edentulism (n=5) - Use of antiobiotics/corticosteroids/ radiotherapy (n=8) - Uncontrolled diabetes mellitus (n=6) - Oral lesions (n=3) - Unavailable for follow-up appointments (n=4) Pre-treatment data collection (T0) Total n=48 Canada n=22 Brazil n=26 Post-treatment data collection: one-month follow-up (T1) Total n=48 Canada n= 22 Brazil n=26 Post-treatment data collection: 3-month follow-up (T2) Total n=48 Canada n= 22 Brazil n=26 Figure 3.1: Flow chart of the study 65 Table 3.1: Assessments of denture stomatitis Modified Newton classification 18 0: Healthy mucosa Type IA: Petechiae in a normal palatal tissue, which are usually found around the orifices of the ducts of the palatal mucous glands Type IB: Localized area of inflammation of the denture-bearing area Type II: Generalised area of inflammation of the denture-bearing area Type III: Hyperplasic palatal surface with inflammation of the denture-bearing area Inflammation area index 19 0: No inflammation 1: Inflammation of the palate extending up to 25 % of the palatal denture-bearing tissue 2: Inflammation of the palate extending between 25 % and 50 % of the palatal denture-bearing tissue 3: Inflammation covering more than 50 % of the palatal denture-bearing tissue Inflammation severity index 19 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) Total score for inflammation = area+intensity (range 0 to 6) 19 66 Table 3.2: Socio-demographic characteristics of the participants at baseline (T0) by study center Variables Combined Canada Brazil p value Mean Age (years)† N Gender  Male  Female Marital status  Single/Separated/Divorced/ Widowed  Married/Partnered Living arrangements  Alone  With family or other adults Education  High school or less  College and higher Yearly income  Less than $10,000  $10,000 -$30,000  $30,000 or more † 66.0 (±11.2) % 33.3 66.7 47.9 52.1 16.7 83.3 81.3 18.7 64.6 (±12.3) N 12 10 9 13 5 17 13 9 % 54.5 45.5 41 59 22.7 77.3 59 41 N 4 67.3 (±10.2) % 15.4 84.6 53.8 46.2 11.5 88.5 100 0 0.407 16 32 23 25 8 40 39 9 0.006 22 14 12 3 23 26 0 0.401 0.442 <0.0001 14 25 9 29.2 52.1 18.8 0 14 8 0 63.6 36.4 14 11 1 53.8 42.3 3.8 <0.0001 Mean (standard deviation) 67 Table 3.3: Study participants’ profiles by study center at baseline (T0) according to denture stomatitis risk factors Variables Combined Canada Brazil p value Mean years of edentulism† Mean age of current prosthesis (years) Presence of systemic diseases Medications use Unacceptable VDO Inadequate upper retention Unstable upper denture Inadequate denture hygiene (reported by the patient) Dirty denture (reported by the clinician) No mouthwash use Nocturnal wear (upper denture) Smoking † 37.2 (±14.7) † 40.55 (±15.92) 9.93 (±9.53) N 12 17 13 9 12 4 13 13 12 6 % 54.5 77. 3 59 41 54.5 18.2 59 59 54.5 27.3 34.3 (±13.3) 19.8 (±15.1) N 19 29 17 8 10 1 26 22 16 7 % 73.1 73.1 65.4 30.8 38.5 3.8 100 84.6 61.5 26.9 0.146 0.011 0.232 1.000 0.558 0.551 0.384 0.165 <0,0001 0.059 0.770 1.000 15.3 (±13.7) N 31 36 30 17 22 5 39 35 28 13 % 64.6 75.0 62.5 35.4 45.8 10.4 81.3 72.9 58.3 27.1 Mean (standard deviation) 68 Table 3.4: Effect of the intervention at 3-month follow-up Diagnosis worse unchanged improved/cured p value (T0-T2) N Modified Newton classification Inflammation area index Inflammation intensity index Total inflammation (area+intensity) † Effect size† (T0-T2) % 2.1 2.1 2.1 2.1 N 32 18 15 8 % 66.6 37.5 31.2 16.7 N 15 29 32 39 % 31.3 60.4 66.7 81.2 0.001 <0.0001 <0.0001 <0.0001 0.34 0.49 0.52 0.54 1 1 1 1 Effect size: 0.1= small effect, 0.3=medium effect and 0.5=large effect Table 3.5: Effect of the intervention on the number of Candida colonies at 3-month follow-up Candida colonies counts increased unchanged decreased p value (T0-T2) N Number of CFUs‡ from the prosthesis Number of CFUs from the palate ‡ † Effect size† (T0-T2) % 27.1 8.3 N 7 34 % 14.6 70.8 N 28 10 % 58.3 20.9 0.050 0.048 0.19 0.20 13 4 ‡ Colony-forming units (CFUs) Effect size: 0.1= small effect, 0.3=medium effect and 0.5=large effect 69 2.1% 10.4% 16.7% Large effect: inflammation resolved Moderate effect: inflammation reduced No effect: no change in inflammation 70.8% Negative effect: increased inflammation % participants Figure 3.2: Treatment effect at 3-month follow-up (T2) 70 Figure 3.3: Palatal mucosa of patients at T0 and T2 71 CHAPTER IV DISCUSSION The results of this clinical trial demonstrated that palatal brushing can: 1. Reduce the extent of palatal inflammation in individuals affected by denture stomatitis. 2. Reduce the Candida colonization in the denture plaque and palate of individuals affected by denture stomatitis. 4.1 PALATAL BRUSHING EFFECTS This master’s research project is the first clinical study specifically designed to provide evidence on the efficacy of palatal brushing in the treatment of denture stomatitis. Research on palatal brushing and its association with denture stomatitis is scarce. The only two observational studies that aimed to provide evidence on this simple hygienic measure are the previous studies of our research group consisted of a university-based cohort study 110 30, 110 . One of these studies that assessed the evolution of denture stomatitis in term of severity and frequency and its association with potential risk factors. 135 edentate elders wearing a set of complete dentures (either with mandibular denture or mandibular implant-retained overdenture) were followed over two years. The results showed that edentate elders suffering from type II or type III denture stomatitis who brushed their palate had approximately 6 times more chance to have a decrease in 72 the severity of their condition (OR = 5.88, 95 % CI 1.1 to 32.2, p = 0.04). Another study by Emami et al. 30 showed a statistically significant relationship between the lack of palatal brushing and the occurrence of oral candidiasis (OR = 1.8, 95 % CI 1.3 to 2.4, p = 0.03). The findings of this master’s research project confirmed the results of these previous studies. In the present study, after 3 months of palatal brushing, denture stomatitis was cured in 10.4 % of the participants, and 70.8 % showed substantial improvement in the clinical signs of denture stomatitis. Furthermore, there was a statistically significant reduction in the number of Colony-Forming Units (CFUs) of Candida isolated from the palatal mucosa and from the denture biofilm. However, the effect size obtained for the microbiological data was reported to be small to medium (r = 0.19 to 0.2), which was less important than the clinical effect size (r = 0.34 to 0.54). These promising data showed a potential success of palatal brushing in the treatment of denture stomatitis. However, in our study, one participant showed an increase in palatal inflammation. Similarly, in participants classified with Type IA denture stomatitis (the presence of petechiae in a healthy mucosa), there was no change in the level of the modified Newton classification neither in the level of area and severity of the inflammation indices. Furthermore, there was no change in the Newton classification for the patients with Type III denture stomatitis. These negative results can be explained by three hypotheses: 1. The non-compliance to intervention. This hypothesis can be specifically attributed to the only patient with increased inflammation after palatal brushing. 73 2. The misconception about Type I denture stomatitis. The findings of the present study suggest that petechiae should not be considered as a type of denture stomatitis since it could be simply a variation in the normal anatomy of the minor salivary gland openings. This hypothesis can be supported by the fact that in our study, the microbiological analyses of the dentures and palatal swabs of individuals with petechiae did not show any trace of Candida colonization. 3. The shortcoming of conservative methods in the management of papillary hyperplasia of the palate, or Type III denture stomatitis. Hyperplasia is a reactive tissue overgrowth characterized by an inflamed mucosa with a nodular or papillary appearance in response to chronic irritation 72, 179 . The results of the present study support findings in the literature suggesting that treatment of Type III denture stomatitis should consist of the excision of the hyperplasic tissue 181 180, . However, our findings showed that there was a statistically significant decrease in the extent and the severity of the inflammation in the individuals with Type III denture stomatitis, even though the hyperplasic tissue remained. This finding confirms the efficacy of palatal brushing in reducing the inflammation associated with denture stomatitis. 74 4.2 MECHANISM OF ACTION OF PALATAL BRUSHING Palatal brushing can have two separate effects on the palatal mucosa: the effect of the mechanical stimulation and the effect of biofilm removal, both of which can lead to a reduction in the inflammation (Figure 4.1) 182. Mechanical stimulation improves the microcirculation of an inflamed tissue by dilation of the vasculature. This action leads to an increased blood flow and oxygenation of the tissue 183 . Moreover, mechanical stimulation encourages keratinisation, reduces the 182 infiltration of polymorphonuclear leukocytes and enhances the proliferation and collagen synthesis of fibroblasts 184-186. The process of action of the mechanical stimulus will decrease or inhibit the inflammatory reaction and will lead to the resolution of the inflammation. This resolution allows the re-establishment of undisrupted stratified squamous epithelium and basement membrane that provide an impervious mechanical barrier against microbiological colonization and permit the restitution of a healthy palatal mucosa under a removable denture 62, 65, 187, 188. The mechanical stimulus can also increase the salivary flow by stimulating the minor salivary glands of the palate. This stimulation could have a mechanical cleansing effect and thus, contribute to the elimination of the biofilm immune defence mechanism against microorganisms 190. Palatal brushing also eliminates of the oral biofilm, which is considered as an etiologic factor for denture stomatitis 6, 32, 34, 48. As previously stated in Chapter I, oral biofilm acts as a protective niche that harbours a wide array of pathogenic microorganisms 75 83, 85 85, 187, 189 . Saliva also has an . Those microorganisms produce toxins and metabolic waste, which can instigate the inflammatory process in denture stomatitis 6, 32, 34, 48, 83, 84. Palatal brushing can eliminate this source of irritation and this reservoir of pathogens. This could explain the statistically significant decrease in Candida CFUs isolated from the palate and the dentures after 3 months of palatal brushing. 76 PALATAL BRUSHING Mechanical stimulation Elimination of oral biofilm ↑ Keratinisation ↓ Leukocyte infiltration ↑ Proliferation of fibroblasts ↑ Microcirculation and oxygenation ↑ Salivary flow ↓ Source of irritation ↓ Reservoir of pathogens Resolution of the inflammation Re-establishment of a mechanical barrier resistant to microbiological colonization Healthy palatal mucosa Figure 4.1: Palatal brushing effects 77 4.3 THE ETIOLOGY OF DENTURE-RELATED ERYTHEMATOUS STOMATITIS The etiology of denture-related erythematous stomatitis is still controversial; the literature offers conflicting results on whether the inflammation in denture stomatitis is associated with trauma from unstable prostheses or if it results from the fungal biofilm 7, 25, 65, 191 . The lack of a direct cause-and-effect relationship between the presence of denture stomatitis and Candida has been demonstrated in many studies 7, 18, 30 . In addition, no difference between antifungal medications and alternative treatments of denture stomatitis were found 26, 78, 108. Moreover, high recurrence rates of denture stomatitis and the re-establishment of the Candida colonization after cessation of the antifungal treatment have been frequently reported 78, 80. There is considerable evidence supporting the hypothesis of trauma as a primary etiologic factor in denture stomatitis 7, 13, 26, 27, 29 . The histopathological changes that occur in denture stomatitis, such as incomplete or absent keratinisation, and modifications in components and structure of the epithelium and connective tissue, in addition to dissociations in the basement membrane, suggest that traumatogenic stress from unstable dentures results in an inflammatory reaction. This reaction will increase the susceptibility of the palatal mucosa to microbiological colonization 62, 63, 65. Based on this concept we could hypothesize that Candida is not a primary etiologic factor of denture stomatitis 7, 18, 25 and that fungal infection contributes to the maintenance of the inflammatory reaction rather than its initiation 25, 63, 65. 78 4.4 MODIFICATION TO THE CLASSIFICATION OF DENTURE-RELATED ERYTHEMATOUS STOMATITIS In the present study, we modified the Newton classification by adding two subtypes for Newton's Type I denture stomatitis. This modification allowed the differentiation between petechiae (Type IA) and localized inflammation (Type IB) in terms of the effect of treatment (as mentioned in section 4.1). Newton classification is the most frequently used classification for the diagnosis of denture stomatitis in research and clinical settings. However, this classification does not allow an accurate representation of the clinical signs of denture stomatitis. In this clinical trial, we used the area and severity of the inflammation indices to account for this limitation. We found that this classification, which was introduced by Schwartz et al. 26, is easy to use in clinical settings and is sensitive enough to measure the effects of treatment. The inter-rater reliability results (kappa measurements) were higher with the use of those indices (κ = 0.71 to 0.86) when compared to the classification of Newton (κ = 0.60 to 0.84). 4.5 ORAL HEALTH KNOWLEDGE Hygienic measures are essential to ensure a healthy mucosa under removable dentures. Those measures include: daily cleaning of the dentures by brushing after every meal, using a mouthwash, soaking the prosthesis in a denture-cleaning agent, and avoiding nocturnal wear of the dentures 192 . However, only one study included palatal brushing 79 among other hygienic instructions given to the participants assessing the effects of palatal brushing as an intervention. 108 and there was no study In the present study, only 18.7 % of the participants had a clean prosthesis. Only 20 % of participants reported having previously received oral and denture hygiene instructions from their oral health care provider. Furthermore, half of the participants stated that they never consult a dental health care professional. There was a significant difference (p < 0.0001) between the two centers regarding the prosthesis hygiene of the participants. This result could be influenced by the lower level of income and education of the Brazilian participants when compared to the Canadian participants. In fact, none of the Brazilian participants were using mouthwash as a hygienic method. However, although these differences between the two centers were statistically significant, they did not have any influence on the treatment effects. In agreement with the literature 32, 193, these findings suggest that edentate elders lack the necessary education about oral and denture hygiene. Oral health knowledge deficiency could play an important role in the prevalence of oral diseases such as denture stomatitis. It is the responsibility of the clinicians to raise awareness about oral hygiene and the necessity of periodic follow-up visits. The importance of oral health knowledge is more evident when taking into account the increase in the life expectancy and worldwide growth of the elderly population in the next decade 48, 194, 195 . This growing geriatric population is more susceptible to denture-related oral mucosal lesions due to the longterm use of removable prostheses, especially elders with systemic diseases and those who use multiple medications 38-42. 80 Palatal brushing could be an effective preventive measure against the development of denture stomatitis. However, it should be noted that this mode of prevention or treatment would be difficult for elderly patients with lack of manual dexterity 32, 117, 196. Alternative hygienic methods that could be considered for this population could be the use of denture cleaners, oral mouthwashes, phytomedicines, and the microwave disinfection of the prosthesis 106, 128, 137, 197. 4.6 THE CHOICE OF STUDY DESIGN AND LESSON LEARNED FROM INTERNATIONAL COLLABORATION Clinical trials are conducted in a series of phases and each phase is designed to achieve different objectives. Phase-I trials are conducted to test a first-time intervention or treatment in a small group of individuals in order to 198-200: 1. Standardize the study procedures; 2. Assess the safety of the intervention; 3. Assess the recruitment strategies; 4. Collect preliminary data on the treatment effect for sample size calculations; 5. Assess the practicability of an international collaboration; 6. Guide the planning of a large-scale trial. The evaluation of the feasibility of the collaboration between the two study centers was an important aspect of this master research project. In order to coordinate all the methodological aspects of the study and to interpret the results, three meetings between the research groups were organized, two in Brazil and one in Canada. 81 During the course of this study, we found that the standardization of the methodology is a critical step in multi-center trials. This ensures the adequacy of data for combined statistical analyses. 4.7 STUDY LIMITATIONS The results of this phase-I clinical trial should be interpreted with caution because of certain study limitations. The primary threat to the internal validity of this research project was its design as a single group pre-test/post-test quasi-experimental study. This design comprises a variety of biases: 1. History bias: the possibility that events other than the treatment could have happened between the pre-test and the post-test and could have affected the outcome; 2. Maturation bias: the natural process that leads participants to change as a function of the passage of time. History and maturation biases occur primarily because of the absence of a control group and lack of randomization 201. 3. Pre-test effects: the information that participants acquire during the pre-test information session could influence the outcome of the study 202 . During this project, the pre-test clinical examination could have sensitized the participants to the presence of a pathologic lesion in their oral cavity. As a result, although they were asked to keep to their routine hygiene regimen, they could have altered their behavior by improving their oral hygiene because they are taking part in an 82 experiment. This effect, also called the Hawthorne effect, could have led to an overestimation of the study results 201-204. Furthermore, although this project was a two-center study with a diverse population in term of socioeconomic and educational level, and denture characteristics (denture cleanliness, the mean age of the current prostheses), the study was limited in terms of external validity and the results cannot be generalized to other populations. In addition, we cannot generalize the study results across time because of the duration of the follow-up. In our trial, the follow-up data was collected only after 1 month and 3 months. Therefore, we couldn’t assess the long-term effect of palatal brushing and the potential for the recurrence of denture stomatitis. 4.8 FUTURE RESEARCH In order to develop clinical practice guidelines in regard to the treatment of denture stomatitis, high-quality randomized controlled trials are needed 205 . The encouraging results of this master research project will help in the development of a randomized controlled trial with valid and generalizable results. Also, as the etiology of denture stomatitis is still subject to debate, we need welldesigned long-term cohort studies to shed light on the main causal factor of this disease. Inferences about cause-effect relationships may be valid or generalized to other populations only in the presence of a strong and direct relationship between the etiological factor and denture stomatitis, based on experimental evidence 206 . 83 Furthermore, diagnostic studies should focus on investigating the salivary or systemic biomarkers involved in denture stomatitis. As recent studies suggest that denture-related erythematous stomatitis has cytotoxic effects on the cells of the oral mucosa and induce nuclear alterations 71, further research should be conducted to assess the cytotoxic and premalignant changes occurring in denture stomatitis. 84 CHAPTER V CONCLUSIONS The results of this phase-I clinical trial suggest that: 1. Palatal brushing is an effective method for the treatment of denture stomatitis. 2. Palatal brushing reduces the extent and severity of the palatal inflammation in individuals affected by denture stomatitis. 3. 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Lancet 2002;359(9302):248-52. 98 APPENDICES APPENDIX I: CONSENT FORM i ii iii iv v vi vii viii APPENDIX II : QUESTIONNAIRE ix x xi xii xiii xiv xv xvi xvii xviii xix APPENDIX III: CLINICAL FORM xx xxi xxii xxiii xxiv xxv xxvi APPENDIX IV: MICROBIOLOGICAL FORM xxvii xxviii APPENDIX V: INSTRUCTION FORM xxix