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Denture stomatitis (DS) is the most common form of oral candidiasis with an overall incidence of 11–65% in complete denture wearers. This recurring disease is characterized by different degrees of inflammation of the mucosa under the maxillary denture, ranging from petechiae to generalized inflammation with papillary hyperplasia . The aetiology of this problem is multifactorial: decreased salivary flow, medication, endocrinopathies, immunosuppression, metabolic and nutritional factors, smoking, increased age of denture, denture trauma, continuous denture wearing, and poor denture hygiene have been implicated . Nonetheless, the denture–palatal interface offers a unique ecological niche for microorganism colonization because of the relatively anaerobic and acidic environment favouring yeast proliferation without any other predisposing factor present.
Candida albicans is the yeast species most frequently isolated in significant quantities from subjects with DS [1–8]. This oral fungal pathogen is able to grow in a variety of morphological forms, ranging from blastospores to hyphae. The filamentous growth can promote tissue penetration during the early stages of infection . Moreover, on soft and hard surfaces within the oral cavity, C. albicans grows as a biofilm, which consists of a complex community of cells embedded in a matrix of extracellular polysaccharide. When cells exist in a biofilm they exhibit phenotypic properties that are distinct from those of planktonic cells and they have increased resistance to antimicrobial agents . Although C. albicans is the most prevalent and virulent species of the genus Candida, other non-C. albicans species are often isolated from acrylic surfaces and the palatal mucosa, such as: C. glabrata, C. tropicalis, C. parapsilosis, C. pseudotropicalis, C. krusei and C. guilliermondii. The emergence of other Candida species is important because they may exhibit higher denture surface adherence, and species such as C. glabrata, C. krusei and C. lusitaniae show inherent resistance or intrinsic reduced susceptibility to antifungal agents .
Antifungal agents are commonly used to treat DS, but improvement in denture hygiene, discontinuation of nocturnal denture wearing, and eventually relining or replacing the denture are also required. Topical agents such as nystatin and miconazole have been used effectively [12,13]. However, the diluent effect of saliva and the cleansing action of the oral musculature tend to reduce the concentration of these agents to sub-therapeutic levels. Hence, treatment regimens tend to be prolonged and recurrence rates are high. Systemic antifungal agents such as amphotericin B and fluconazole are also effective, but they do not eradicate the microorganisms that colonize the denture . Nonetheless, the major problem associated with the prolonged or recurrent use of antifungal drugs is the development of resistant species [9,11,13].
This makes it necessary to seek new therapeutic approaches. A promising modality is Photodynamic Therapy (PDT), which uses a photosensitizing agent and light of appropriate wavelength. The interaction between the photosensitizer and light in the presence of oxygen produces reactive species, such as singlet oxygen and free radicals, which cause cell damage and death [14,15]. As a consequence of these non-specific oxidizing agents, organisms resistant to conventional antifungal agents could be successfully killed by PDT, and it seems unlikely that they will develop resistance to such a therapy. PDT is effective against oral species and may not promote damage to host cells and tissues [16,17].
Investigations have shown that Candida spp. are susceptible to photoinactivation [14–16,18], including resistant strains [19,20]. Previous studies have shown that PDT is effective in reducing C. albicans counts in a murine model of oral candidiasis  and for denture disinfection [21,22] when a porphyrin was associated with light-emitting diode (LED) light. In a recent case report, five patients with clinical and microbiological diagnoses of DS were successfully treated with PDT . Nonetheless, the clinical effectiveness of PDT in comparison with conventional antifungal therapy in the treatment of DS is not yet known. Hence, the aims of the present randomized clinical trial were to compare the efficacy of PDT with that of topical nystatin in the treatment of DS and to identify the prevalence of Candida species.
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The present investigation compared the clinical and microbiological efficacy of PDT with that of conventional antifungal therapy in patients with DS. The microbiological results showed no significant difference between PDT and conventional antifungal therapy in the inactivation of Candida spp. At the end of both treatments (day 15), a significant reduction in CFU/mL recovered from dentures and palate was found in comparison with baseline (day 0). This result is in agreement with a previous case report in which reduction of CFU/mL of five patients with DS was observed after six sessions of PDT . Interestingly, on day 30 of the follow-up period (15 days after the end of both treatments), the mean values of CFU/mL were still significantly lower than those at baseline. This could be explained by the patients’ compliance with denture and oral hygiene advice given during this research. Although not sufficient to treat DS [24,25], denture and oral hygiene are essential to maintain a low level of microorganisms on dentures, as well as oral health.
Although several in vitro investigations have confirmed the potential of PDT to inactivate Candida spp. [14–16,18–22], this is the first clinical trial to evaluate the effect of PDT on the treatment of Candida infection. Although Teichert et al.  stated that mice with oral candidiasis were treated using PDT, they only observed a dose-dependent eradication of C. albicans from the oral cavity using methylene blue and laser irradiation. However, the regression of lesions, which is an important clinical sign of treatment, was not evaluated by these authors. Furthermore, the association of Photogem with LED light showed a significant reduction in the CFU/mL of C. albicans recovered from the tongues of mice with oral candidiasis and no adverse effects were observed on the tissues of the tongue . Nevertheless, complete eradication of yeast was not achieved. A previous in vivo study also showed that PDT, using approximately the same parameters as this investigation, was not toxic to the rat palatal mucosa when 500 mg/L of Photogem was associated with 144 J/cm2 of blue LED light . Hence, potential toxic or adverse effects of PDT on palatal tissue may seem unlikely. In fact, it is difficult to compare the results of this clinical trial with those of in vitro studies and animal models because the oral environment of humans is different, and factors such as microbiota and biofilm composition, salivary flux, oral hygiene and food habits might influence the response to PDT. Nonetheless, the outcome of the present investigation suggests that PDT may be an alternative method for inactivation of Candida spp. in patients with DS.
During the follow-up period (days 30, 60 and 90), the mean values of CFU/mL recovered from dentures were significantly higher than those found at the end of the treatment (day 15). This result was expected because progressive recolonization of dentures starts after the end of the treatment. On the other hand, only on day 60 of the follow-up period was the mean value of CFU/mL recovered from the palate significantly higher than that at the end of treatment (day 15). The results obtained during the follow-up period may be explained by the high affinity of Candida spp. to adhere to and colonize acrylic surfaces. Virulence factors of Candida spp., such as cell-surface hydrophobicity  and ability to form biofilm , increase its adherence to acrylic surfaces. Other investigations have also demonstrated that Candida spp. are primarily found on the tissue surface of the denture rather than on the palatal mucosa of patients with or without DS [5,25,28]. This result corroborates that of the present study, because the palatal cultures evidenced lower CFU/mL mean values than the denture cultures for all the time intervals evaluated. The low level of CFU/mL recovered can be partially attributed to the sample technique (swab sampling) employed in the present investigation. An in vitro study showed that C. albicans was able to invade a reconstituted human oral epithelium over a period of 48 h through hyphal penetration into the superficial epithelium together with features of cellular internalization of yeasts . Therefore, a delicate cotton swab used in this study for recovering yeast may underestimate the real burden of Candida spp. present on the palatal mucosa and denture surface. Hence, another sampling technique, such as an oral rinse method using saline or sterile water, would have been valuable to monitor the overall Candida burden in the oral cavity of the patients at baseline and at subsequent evaluations.
In the present study, a higher percentage of clinical success was verified in the NYT group (53%) than in the PDT group (45%). Nystatin would have also reduced Candida cells on the tongue and buccal mucosa in patients from the NYT group, which could justify the higher percentage of success in this group. On the other hand, no antimicrobial treatment of the dentures was performed in this group, which could have contributed to the rate of clinical failure obtained (47%). The high rate of clinical failure observed in the PDT group (55%) might be attributed to the recolonization of the palatal mucosa after PDT by Candida cells from other sites of the mouth, such as tongue, particularly mid-dorsum, and buccal mucosa. In addition, the clinical conditions in which PDT was performed, such as treatment time (three times a week for 15 days, a total of six sessions) and the parameters used (only one type and concentration of photosensitizer and light fluence) might also explain the rate of clinical failure observed in this group. A longer treatment period might achieve a better resolution of palatal inflammation. Further trials are necessary to evaluate the effect of additional parameters of PDT, such as other types and concentrations of photosensitizer and light fluences, in an endeavour to find a higher clinical success rate.
In the present investigation, the clinical failure rates are not in agreement with the microbiological results obtained, i.e. a reduction in CFU/mL values was observed even in patients who showed no improvement in palatal inflammation. This corroborates the findings of Barbeau et al. , and Zomorodian et al.  who found no significant relationship between DS and number of yeast colonies. Furthermore, other reports have demonstrated that healthy denture wearers were also Candida carriers [3,6,22,28]. Although both treatments resulted in a significant reduction in values of CFU/mL from dentures and palates in this study, and given that the aetiology of DS is multifactorial, a more effective treatment should be directed towards all aetiological factors involved in this pathology.
This investigation demonstrated that among the patients who showed clinical success, a high rate of recurrence was found in the follow-up period (75% for NYT group and 78% for PDT group). Recurrence of infection after the conclusion of treatment has often been reported [4,5,12], because the tissue surface of the acrylic resin denture acts as a reservoir that harbours microorganisms, and is therefore, a potential source of re-infection of patients. Replacement of dentures is also necessary for complete resolution of DS , especially when dentures are very old. The present study also showed that the age of dentures was the only predisposing factor significantly associated with the degree of inflammation of the palate. The age of maxillary denture of 52.5% of patients was over 12 years, which could have contributed to the proliferation of yeasts on denture surfaces at follow-up. This result is in agreement with that of Neppelenbroek et al. , Zomorodian et al.  and Figueiral et al. , who found that the time of denture use was related to DS. From this standpoint, the provision of new dentures should also be considered during the management of DS.
The outcomes of the present study showed that C. albicans was the predominant species isolated from the dentures and palates of patients from the NYT and PDT groups (63.3% and 54%, respectively). This finding is in agreement with previous reports [2,3,6–8,22,28]. The next most common species found was C. tropicalis followed by C. glabrata in both groups. Investigations have also observed that C. tropicalis was the second most prevalent species isolated from the oral cavity [6,7]. However, other studies have found that C. glabrata was the most common yeast after C. albicans [2–4,8,22,28]. The presence of non-C. albicans species is a cause for concern because they are able to cause infection and are frequently resistant to antifungal agents. In the present study, considerable rates of reduction in the number of isolates of C. albicans, C. tropicalis and C. glabrata, as well as other yeasts species, were verified at the end of the treatments. This result demonstrated that these species were susceptible to nystatin and PDT, except C. tropicalis, which showed higher susceptibility to nystatin (90% of reduction) than PDT (45.5% of reduction). Nonetheless, the increased number of these isolates observed in the follow-up period when compared with the end of the treatments may be attributed to recolonization of the mucosa and denture.
During all the time intervals of this investigation, 60% and 70% of the patients from NYT and PDT groups showed mixed-species populations. In other studies, more than one yeast species was found in 27.27% , 14.4%  and 48.5%  of subjects without DS, and 38.5%  and 32.5%  of individuals with DS. The complex interactions among yeasts in synergistic relationships are not well known, but may be involved in the enhanced pathogenic potential of these associations. In this investigation, mixed-species were reduced by 50% and 54% in the NYT and PDT groups, respectively, at the end of the treatments. This suggests that both treatments were effective in reducing more complex biofilms.
In conclusion, despite the limitations of this first clinical investigation, such as the low number of patients evaluated, the results demonstrated that PDT was as effective as topical nystatin in reducing the CFU/mL of Candida spp. from the dentures and palates of patients with DS, but a higher number of patients showed decrease in palatal inflammation after nystatin. In addition, a high rate of recurrence was observed, and C. albicans was the most prevalent species identified. Further studies are required to determine more effective clinical parameters for a better response to PDT in patients with DS, and more patients should be assessed to draw firmer conclusions. The principal advantage of PDT is that, unlike antifungal agents, development of resistance to phototherapy seems to be improbable because of its mechanism of action, and potential toxic or adverse effects of PDT on palatal tissue are unlikely. Moreover, according to the results of this investigation, fewer sessions of PDT are necessary to achieve the same clinical outcome as nystatin, which, on the other hand, requires multiple daily doses, which can lower the patient’s compliance. As PDT is performed in the dental office, dentists can also monitor the therapeutic sessions and the patient’s response gradually. Therefore, PDT seems to be a promising method for the treatment of DS.