ASSESSMENT OF Candida SPP . PROLIFERATION IN OCCLUSAL AND PALATAL SPLINTS AVALIAÇÃO

: Candida species inhabit the oral cavity of all individuals who wear complete denture and whose material is the same as that used in splints. Assess the growth of C. albicans in occlusal and palatal splints used for treatment of TMD so that the potential risks of oral microbiota can be assessed. The growth of Candida spp . was assessed in the saliva of 27 individuals wearing splints for treatment of TMD. They were divided into two groups: G1 (n = 14), individuals wearing occlusal splint; and G2 (n = 13), individuals wearing palatal splint. Saliva samples were collected during placement of the splints (T1) and after 4 months (T2), being stored in PBS (10 mL) after 60-second rinses. It was observed that patients wearing occlusal splints (G1) had an increase of 0.648 CFU/mL (Log 10), with statistically significant differences ( P = 0.043) for C. albicans (42.33%), C. glabrata (5.52%), C. krusei (41.72%) and C. tropicalis (10.43%). In the group of patients wearing palatal splints (G2), there was a decrease of 0.101 CFU/mL (Log 10), was observed with ( P = 0.964) only the presence of C. albicans . The results suggest that growth of Candida species was greater in patients wearing occlusal splints compared to those wearing palatal ones as the presence of different yeast species was found in the former.


INTRODUCTION
Temporo-mandibular joint dysfunction (TMD) is a complex set of symptoms involving structures surrounding the temporo-mandibular joint (TMJ) and masticatory muscle, or both (DE LEEUW, 2008), in association with pain (BENOLIEL;SHARAV, 2008).TMD can be classified as muscular, articular or mixed (DWORKIN, 2011), being the most prevalent type of chronic orofacial pain and the third most prevalent type of chronic pain, only behind common headaches and backaches.(DWORKIN, 2011).Although several types of treatment have been suggested in the literature (MINAGI et al., 2001;TSUKIYAMA et al., 2001), such as occlusal splint therapy, drug therapy, surgical therapy, occlusal adjustment, physical therapy and psychological counselling, it is clear that the use of occlusal splints is consecrated for treating muscle TMD.However, palatal splints are an alternative to the occlusal ones as the former have the distinction to adjust to the hard palate without interfering occlusion semiadjustable articulator mounting or splint adjustment (MINAGI et al., 2001;HASEGAWA et al., 2007).Although the patients are guided to wear the splints for several hours consecutively on a daily basis, it is important to emphasise that these dental appliances are colonised by pathogens which have the potential to cause risks.
Candida yeasts are micro-organisms which are widely spread in the nature, with some species living as saprophytes or parasites in human beings and other animal species.Associated with human beings and other warm-blooded animals, Candida albicans usually lives in the oropharynx, mouth, skin folds, bronchial secretion, vagina, urine and faeces.The occurrence of this species in water and soil is relatively rare and is related to the contamination of these environments by human beings and animals (LIM et al., 2012).
Considered the most prevalent pathogenic fungus in humans, C. albicans can be commonly isolated from the oral cavity of adults and children, often resulting in systemic or superficial infections (THEIN et al., 2006;RAMAGE et al., 2006), such as candidiasis (RAMAGE et al., 2007;VIDAL et al., 2011), and even in death in the cases of immunocompromised individuals (LIM et al., 2012).Because C. albicans is a component of the oral microbiota (WILLIAMS; LEWIS, 2011), this microorganism can be easily isolated from approximately 60% of healthy individuals (PEREIRA et al., 2008;JAROSZ et al., 2009).
Among the most important virulence factors of Candid spp., one can observe that this microorganism has the ability to adhere to surfaces by producing filamentous growth and releasing hydrolytic enzymes capable of damaging the host cells, in addition to forming biofilms, thus reducing the susceptibility to antimicrobial agents (WILLIAMS;LEWIS, 2011;SILVA et al., 2012).In general, tongue, oral mucosa, tooth surface, biofilm and saliva are areas of preference for colonisation by this micro-organism.Local factors such as poor oral hygiene, poorly-adjusted dentures, decreased salivary flow, presence of caries as well as systemic factors such as nutritional deficiency, malignant tumours, use of broad-spectrum antibiotics, smoking, diabetes, salivary gland disorders and immune response deficiency can favour the proliferation of Candida species in the oral cavity (PEREIRA et al., 2008;ROWAN et al., 2009).Some cases of candidiasis are associated with several implantable medical devices and appliances made of acrylic resin (NIKAWA et al., 1994;PEREIRA et al., 2007), such as dental prostheses and others, whose surfaces are susceptible to biofilm formation and which, in turn, tend to impede the immune response and other defence mechanisms (RIZK; FALKLER; MEILLER, 2004; SAMARANAYAKE; WU; SAMARANAYAKE; SO M; YUEN, 1994).Candida species are found in the oral cavity of 60-100% of the individuals wearing complete denture (DAĞISTAN; AKTAS; CAGLAYAN; AYYILDIZ; BILGE, 2009), whose material is the same as that used in the manufacture of splints.Therefore, as there is a shortage of studies on the colonisation of these plates by Candida species, it is important and necessary to assess the growth of this micro-organism in splints.The objective of the present study was to assess the growth of Candida species in occlusal and palatal splints used for treatment of TMD in order to verify potential risks to the oral microbiota.

Volunteers
The volunteers of this study comprised a convenience sample, as we have assessed 14 occlusal (G1) (Figure 1) and 13 palatal splints (G2) (Figure 2) in individuals undergoing treatment for TMD at the Centre for Occlusion and Temporomandibular Joint Treatment (COAT) of the Institute of Sciences and Technology, São José dos Campos Dental School (UNESP).For inclusion, patients had to be between 18 and 44 years old, have myofascial pain and have complete permanent dentition up to second molars.Those excluded were individuals in perimenopause; those with complete, uni-or bi-maxillary, or removable dentures; those with uni-or bi-lateral lack of posterior support; those who made continual use of any drugs or medication.
The patients were guided to wear the splints during sleeping for 8-10 hours for 4 months.They were also guided to clean their splints by using dentifrice and toothbrush only after removing them in the morning.Microbial samples were collected twice, that is, immediately before placement of the splint (T1) and after four months of use (T2) according to protocol already validated in the literature (24).

Sample Collection
The patient rinsed the mouth with 10 mL of phosphate buffered saline solution for 60 seconds before spitting out the rinse material into the same container.

Centrifugation
The samples underwent centrifugation at 8,000xg for 10 minutes and the supernatant was disregarded.The resulting pellet was resuspended in 2.5 mL of PBS solution and homogenised for 30 seconds by means of a shaker.

Culture
One-hundred mL of the Falcon tube content were seeded onto CHROMagar Candida agar plates and the inoculums were spread out by using a Drigalski spatula.Next, the agar plates were incubated at 37 o C for 48 hours.

Bacterial Counting
Colony-forming units per millimeter (CFUs/mL) were counted per agar plate.Presumptive identification of Candida species was performed by observing the morphology and colour of the colonies.

Splints Protocol of use
All the patients were given the same instructions regarding the use and cleaning of the splints.They were also guided to wear the splint during sleeping at night and to keep it in water when not in use in order to avoid dimensional changes.
It was also recommended that the splints should be thoroughly cleaned by using toothbrush and fluoride dentifrice.

Statistical Analysis
For statistical analysis, we used the GraphPad Prism 5 software.For verification of the adherence to normal distribution, the mean and standard deviation (SD) of the results obtained from each experimental group were analyzed with the Shapiro-Wilk test.After analysis it was verified that the data does not have the normal distribution, and then nonparametric Wilcoxon and Mann-Whitney tests were used.In all tests, a 5% significance level was used.
The literature shows that when certain types of dental materials are exposed to oral milieu, they are immediately covered by salivary components and then by micro-organisms in the oral cavity.Oral biofilms can contribute to the occurrence of diseases, such as caries, periodontitis or prosthetic stomatitis (HAHNEL et al., 2009).Candida yeast has been identified as a major agent of these infections (RAMAGE; MARTINEZ; LOPEZ, 2006; HAHNEL; ROSENTRITT; HANDEL; BÜRGERS, 2009;OLIVEIRA et al., 2007), also being found to be adhered to numerous dental materials such as acrylic resins (e.g.prosthesis) and silicone (e.g.overlay), as reported by in vitro and in vivo studies (NIKAWA et al., 1994;PEREIRA et al., 2007;RADFORD;CHALLACOMBE;WALTER, 1999;NEVZATOĞLU et al., 2007).
As the present study has assessed the growth of Candida species in a convenience sample of patients wearing splints at COAT, maybe the results shouldn't be generalizable to all Brazialian individuals with TMD.However, as the inclusion and exclusion criteria guarantee no usage of medications or drugs even presence of any illness that could interfere in the oral microbiota, our results are important to identify the amount of Candida associated to the use of mouth splints and according to the results obtained, a greater microbial growth was observed in occlusal ones.A number of factors may contribute to both growth and replication of micro-organisms, and possibly one of the main factors is the habitat formation, which would enable conditions for development and survival of Candida species.
Candida yeasts can grow as a biofilm on several human tissues as well as on several types of biomaterials, including acrylic prostheses, thus causing morbidity (MEGHA; NOBILE, 2016;PIERCE et al., 2017).Oral candidiasis, in association with biological materials, is the most common fungal infection in wearers of dental prostheses (WILLIAMS; LEWIS, 2011).
Based on our results, we have observed the presence of different Candida species in the oral cavity of patients wearing occlusal splints, whereas C. albicans was predominant in individuals wearing palatal splints.As Candida species are oral commensal micro-organisms present in 50-60% of the population, they can be transitory or permanent (KADIR et al., 2002).C. albicans are the most opportunistic pathogenic fungus.
Factors determining the virulence of Candida species can be unchained by local environmental stimuli (RAMACHANDRA et al., 2014), including pH, nutritional availability and gaseous compensation (KADIR; GÜMRÜ; UYGUN, 2007).In addition, studies have already demonstrated that the properties of colonised surfaces (e.g.surface roughness and load) and the composition of salivary pellicle increase the development of biofilm (CUSTODIO et al., 2015;DA SILVA et al., 2014).
Earlier studies showed that the surface properties of bio-materials play a significant role in the development of micro-organisms and formation of biofilm (DA SILVA et al., 2014), mainly due to the increased microbial colonisation on rough surfaces (PEREIRA et al., 2007;DA SILVA et al., 2014).
Surface roughness is extremely important during the adhesion phase of C. albicans (HAHNEL; ROSENTRITT; HANDEL; BÜRGERS, 2009;RADFORD;CHALLACOMBE;WALTER, 1999).Some in vitro studies have concluded that surface roughness accounts for the C. albicans adhesion to dental bio-materials (PEREIRA et al., 2007).These studies on dental development and protein plaques have similarities regarding biofilm formation in different types of surfaces in the oral cavity.Rough surfaces tend to promote in vivo biofilm formation as these surfaces are more likely to have micro-organisms.In addition, rough surfaces provide larger areas and a safer environment for development of micro-organisms (RADFORD; CHALLACOMBE; WALTER, 1999).
Adhesion of micro-organisms to the splints may also be related to salivary flow, both reducing the volume of saliva and hindering it to reach areas of the oral cavity covered by the splint.Such a finding was also observed in the study conducted by BARBEAU et al. (2003), who assessed a case of prosthetic stomatitis and found evidence of reduced salivary flow, including lack of saliva on acrylic surface, which contributes to formation and accumulation of biofilm (WILLIAMS et al., 2013).
Although the precise mechanisms by which the adhesion of Candida species to acrylic surfaces occurs are not known, many factors affecting this process have already been described, such as surface roughness, salivary pellicle, and hydrophobic and electrostatic interactions (RADFORD; CHALLACOMBE; WALTER, 1999).SANCHEZ et al. (2013) reported the presence of other Candida species in the oral cavity by means of a longitudinal study to quantify biofilms in oral clinical isolates from adult individuals presenting local and systemic predisposing factors for candidiasis.A total of 69 Candida yeasts isolated from 63 Mexican patients were evaluated.These isolated (39 C. albicans, 15 Candida tropicalis, 7 Candida glabrata, 4 C. krusei, 1 Candida lusitaniae, 1 Candida kefyr, 1 Candida Guilliermondii and 1 Candida pulcherrima) were obtained from two clinical origins: 62.3% (n = 43) from oral mucosa of fully or partially edentulous individuals and 37.7% (n = 26) from oral mucosa of diabetic individuals.Standard strains of Candida were used as controls in each experiment.The authors concluded that biofilm formation depends on both Candida species and clinical origin, reporting that oral isolates of C. glabrata are strong producers of biofilm, whereas C. albicans and C. tropicalis are moderate ones.However, C. albicans was the most common species.
These results corroborate our study regarding the diversity of other Candida species, despite the fact that C. albicans prevailed.THEIN et al. (2007) evaluated the interactions between C. albicans and C. krusei in acrylic prostheses, concluding that quantitative and qualitative characteristics of a given Candida species can change the other's physiology during co-habitation in the biofilm.In addition, human saliva can modulate this process depending on the nature and number of species in the habitat.Therefore, competing interactions between fungal species are important in the formation of biofilms on acrylic surfaces, with human saliva modulating this process.
ARIAS et al. ( 2009) described the isolation of C. dubliniensis from patients with prosthetic stomatitis and compared the presence of yeasts in the oral cavity of prosthesis wearers, with the authors reporting the predominance of C. albicans other than non-albicans species such as C. tropicalis, C. guilliermondii, C. glabrata and C. parapsilosis.These results are in accordance with ours regarding patients wearing occlusal splints, demonstrating that C. albicans was present in the oral cavity in 100 percent of the cases.
In view of the impossibility to treat fungal infection by using antifungal therapy, we have emphasised the importance of treatment methods for reducing the initial fungal adhesion since colonisation and consequently the C. albicans adhesion to oral surfaces, including mucosa and prosthetic surfaces (VERRAN; MARYAN, 1997;NIKAWA et al., 1997;ZAMPERINI et al., 2010;PIERCE et al., 2017).
Other important factor in determining the microbial colonisation of the oral cavity in patients wearing splints was the hygienisation of both appliance and mouth, that is, frequency of tooth brushing and cleaning of the splints according to the guidance received.
In sum, it is impossible to separate the set of factors which determine the conditions for microbial development, growth and colonisation of the oral cavity.Saliva composition, salivary flow rate, predisposition to caries, immune response, hygiene habits (i.e.manner and frequency of tooth brushing), time length of the splint within the mouth, cleaning of the splint, and type of material and structure of the splint are factors which may predispose the patient to a greater number of micro-organisms in the oral cavity, thus allowing for the development of candidiasis.Therefore, our results are consistent with the findings reported elsewhere, but not all patients responded to treatment in a similar way.

CONCLUSION
Patients wearing occlusal splints showed a greater growth of Candida species compared to those wearing palatal ones as different species of yeasts were observed.

Figure 1 .
Figure1.Mean and standard deviation of CFU/mL (Log 10) obtained in occlusal (G1) and palatal splints (G2) immediately before placement of the splint (T1) and after four months of use (T2).

Table 1 .
Percentage of microorganisms found in the splints.