Prevalence of Candida spp. during radiographic examination in Diabetes mellitus patients

Introduction: It is suggested that individuals with diabetes are more susceptible to Candida infections than healthy people, especially if periodontal infection is associated. Objective: This study evaluated the prevalence of colonization by Candida spp. during radiographic examination in diabetic and non-diabetic patients. Material and methods: Twenty-six patients with type 2 diabetes mellitus and 20 patients without diabetes mellitus, presenting chronic periodontitis and presence of Candida  spp. in saliva were evaluated. During radiographic examination, samples of saliva were collected from: oral mucosa, conventional radiographic periapical film, digital x-ray sensor (CDR), and bite block of the receptor-positioning device. Colony forming units (cfu/mL) and identification of Candida yeasts were assessed. Result: Oral mucosa from both groups showed the highest colonization with Candida spp. if compared with others surfaces collected (p < 0.05). In diabetic patients, the mucosa of the upper left regions showed higher levels of colonization. In non-diabetic patients, the upper right molar region showed the highest level of colonization during the examination of the receptor-positioning device, the sensor and the nonsensitive film. Candida spp. levels in saliva were similar between diabetics (mean = 3.0 × 106) and non-diabetics (mean = 3.8 × 106). Conclusion: No difference in Candida spp. colonization (cfu/mL) in diabetics and non-diabetic patients was observed for the five collected surfaces and the simulated radiographic region. Candida albicans was the prevalent species of Candida spp. found on all the samples. Descriptors: Periodontal diseases; diabetes mellitus; dental digital radiography. 14 Volpato, Pires, Martinez et al. Rev Odontol UNESP. 2013; 42(1): 13-19

INTRODUCTION lnfection-control practices are designed to create and maintain a safe clinical environment to eliminate or minimize disease transmission during patient treatment 1 . There is high potential for cross-contamination of equipment and environmental surfaces with blood or saliva when taking dental radiographs 2 . Saliva has always been considered a potentially infectious material in dental infection control 2 . White, Glaze 3 found that dental healthcare workers can transfer oral microorganisms from the patient's oral cavity to radiographic equipment during routine intraoral radiography. These microorganisms remain viable on radiographic equipment for at least 48 hours.
Traditionally, intraoral radiographs are acquired using films. Since the introduction of digital radiography to dentistry, many dental schools and private practices have adopted digital imaging methods for acquiring radiographs. Digital radiology promises many advantages over traditional film-based techniques 4,5 . Digital radiography sensors come into contact with mucous membranes and are considered semicritical devices. They should be cleaned and ideally heat-sterilized or high-level disinfected between patients. However, there is a variety in the ability of digital radiographic sensors to be sterilized or high-level disinfected 2 . Semicritical items that cannot be reprocessed by heat sterilization or high-level disinfection should, at a minimum, be barrier protected by a plastic barrier sheaths to reduce gross contamination during use that is not guaranteed from contamination 2,6 .
Previous reports mentioned that individuals with diabetes present oral complications more frequently than healthy people 7 . Commonly, these oral complications are associated with fungi, for instance, Candida spp., which presents as white or white-yellow color, creamy, convex colonies, with a smooth and bright appearance, that are moist with a typical smell 8 . These oral complications are also associated with Candida species that have frequently been isolated from the oral cavities of patients with diabetes mellitus [9][10][11][12][13][14][15] . Besides, evidence indicates a strong correlation between the severity of periodontitis and diabetes, based on the fact that periodontal infection is associated with poor glycemic control in this particular group of patients 7,16,17 . Furthermore, it has been established that the higher occurrence rate of yeasts is associated with immunocompromised patients with periodontal diseases, such as patients with diabetes 12,13,18 .
Moreover, patients with long-standing, poorly controlled diabetes are at risk of developing oral candidiasis 19 . Although authors had related that 40% of the patients colonized with candidal species had no clinical signs of oral candidosis 20 . A number of candidal species were recovered from the oral cavity of insulin-treated diabetic patients. C. albicans was the most commonly recovered species, recovered from 85% of the diabetic patients. C. dubliniensis was the second most commonly occurring species 20 .
Considering the lack of dada about the prevalence of colonization by Candida spp. in diabetic and non-diabetic patients with periodontal disease during radiographic examination, this study was carried out in order to determine what surfaces would be contaminated. Therefore, the aim of this study was to determine the prevalence of colonization by Candida spp. in diabetic and non-diabetic patients during radiographic examination, on different surfaces.

Sample
This study was approved by the Ethics in Human Research Committee of the Dental School (protocol number 79/03). All volunteers were informed about the aims and methods of this study, and gave their written consent to participate. The sample size calculation was based on previous studies [21][22][23] . A post-hoc statistical power calculation test was performed based on cfu/mL for both groups and all evaluated surfaces, and the sample size was estimated in 20 patients per group, considering a power of 80%.
As inclusion criteria, all subjects that participated in this study must have been presenting chronic periodontitis and presenting Candida spp. in their saliva, regardless of gender, race, social group, age, oral hygiene, and nutrition habits. The following exclusion criteria were considered: history of antibiotic therapy within the previous 6 months and anti-inflammatory drugs within the previous 3 months; current history of immunosuppression; local or systemic use of antifungal drugs and use of mouthrinses.

Periodontal Analyses
Chronic periodontitis was established as probing pocket depth (PPD) and clinic attachment loss (CAL) ≥ 4 mm and bleeding on probing in more than three sites in non-adjacent teeth 24 .

Diabetes Analyses
The glycated hemoglobin exam (HbA1c; A1c) was requested for diabetic patients. The patient was classified as having adequate metabolic control when presenting results from the glycated hemoglobin test A1c < 7% 25 .

Saliva Analyses
The saliva collection was performed without stimulation, by asking the patients to retain their saliva and then deposit it in a funnel and bottles previously identified and sterilized, until obtaining 3 mL of saliva for microbiological analysis. The tubes containing saliva were dispersed by vortexing for 60 seconds (Vortex, Marconi Equipamentos para Laboratório Ltda., Piracicaba, SP, Brazil) and the saliva was diluted in a decimal series of 10 -1 to 10 -4 in phosphate-buffered saline. The saliva was then inoculated in Petri dishes with SDA and incubated at 37 °C for 48 hours, for posteriori microbiological analyses of presence and quantity (CFU/mL) of Candida spp.
Out of a total 153 periodontitis patients evaluated, 26 patients with diabetes mellitus and 20 patients without diabetes mellitus that presenting Candida spp. in their saliva were included in this study.

Radiographic Examination
For all participants who required full-mouth radiographic examination, a set of 14 radiographs was used. When the patient already had a radiographic examination, a simulation of the examination was performed with written consent from the participant. Then, 26 diabetic and 20 non-diabetic patients were submitted to radiographic simulation/examination. Before using the conventional periapical radiographic film (Kodak Insight, Eastman Kodak Company, Rochester, NY, USA) this material was placed in a plastic barrier that was sealed (Odontobrás, Ribeirão Preto, SP, Brazil) for 4s. The CDR sensor (Schick Technologies, Long Island City, NY, USA) was also protected with a plastic barrier. The conventional film and the CDR sensor were then disinfected by rubbing with sterile gauze dipped in 70% alcohol.
The intraoral radiographic examination (or the simulation) using conventional periapical radiographic film was taken with a Rinn film holder (XCP Instruments, Elgin, IL, USA). Initially radiographs were taken with conventional film from the regions solicited, after that, the CDR sensor was maintained in the mouth for 30

Microbiological Examination
Before the radiographic examination, saliva was collected from the oral mucosa of each patient with a sterile swab that was humidified in sterile phosphate buffered saline (PBS) solution. Each swab was then rubbed onto a culture plate of Sabouraud Dextrose Agar (SDA, Acumedia, Neogen, Lansing, MI, USA) 26 for posteriori microbiological analyses.
After the radiographic examination or simulation in each region, the conventional radiographic periapical film and the randomized regions of the sensor, still with the plastic barrier, were pressed into the SDA plate using sterile clinic tweezers; this procedure was carried out on both sides of the radiographic film and only on one side of the sensor. Only one side of the sensor is submitted to X-ray which was used because the other side presents a wire connection to the computer and this side would damage the procedure of pressure of the sensor into the DAS plate. Material was also collected from the surfaces of the RINN device, which remained in contact with the radiographic film, using a sterile swab, which was then maintained in sterilized saline solution that was then rubbed onto the culture medium on the plate. Samples were incubated at 37 °C for 48 hours, for posteriori microbiological analyses of quantity (cfu/mL) and identification of Candida species.

Identification of Candida spp.
After colony growth, the counting of the inoculum was performed in colony forming units per mL (cfu/mL). The characteristic colony morphology of the Candida yeasts was demonstrated using a stereoscopic microscope (Carl Zeiss do Brasil Ltda, Brazil) and different colonies were identified by Gram staining. A test in CHROMagar  Candida chromogenic medium (Difco, BD) was performed to identify the Candida species 27,28 and other identifying tests were also carried out according to Sandven 29 and Sullivan, Coleman 30 , i.e., germ tube formation, carbohydrate fermentation, carbohydrate assimilation, and thermotolerance assay 31 .

Statistical Analyses
The values of colony-forming units per mL (cfu/mL) for diabetic and non-diabetic patients were compared, considering independent variables of the study the surfaces collected (mucosa, both sides of the conventional radiographic periapical film, CDR sensor and bite block of the receptor-positioning device) and the simulated radiographic region. The non-parametric Kruskal-Wallis test was used for comparison of the surfaces of collection in diabetic and non-diabetic patients while the Wilcoxon test was used for comparison of the radiographic regions for each type of surface collected. ANOVA and t-test were used for comparison the diabetic and non-diabetic patients with CAL, using BioStat 4.0 software (Belém, PA, Brazil).
The total distribution of the concentration of glycated hemoglobin was similar in diabetic patients, considering that 25% male and 25% female presented less than 7% (good metabolic control) and 20% male and 30% female presented more than 8% (inadequate metabolic control).
Considering the percentage of patients with CAL ≥ 4 mm, it is possible to note that there was no statistical difference (ANOVA, t-test) between diabetic (male = 63.8%; female = 48.3%) and non-diabetic (male = 76.5%; female = 50.7%) patients.
Considering the presence and quantity of Candida species during radiographic examination, it is possible to note that the mucosa followed by the non-sensitive side of the conventional radiographic film showed the statistically highest cfu/mL mean if compared with the others surfaces collected, independently of diabetes presence (Table 1). Considering the different types of surfaces on simulating regions, it was observed that the upper right molar (Urm) region showed the highest level of colonization during the examination of the receptor-positioning device, the sensor and the non-sensitive film on non-diabetic patients. In diabetic patients, the mucosa of the upper left regions (Ulb and Ulm) showed higher levels of colonization (Table 1). Table 1 shows statistical differences for the cfu/mL mean, when the collection surface was the conventional radiographic film, on the non-sensitive side, when compared to the oral mucosa in the upper right molar region of diabetic patients. The lower left molars, lower right bicuspid and molar region also demonstrated statistical differences between the oral mucosa and the receptor-positioning device. There was a statistically significant difference between the colonization of the surface of the device and the sensor for the lower right molar (Krukal-Wallis, p < 0.005).
The mean cfu/mL of non-diabetic patients was statistically different in the oral mucosa and receptor-positioning device for the lower left molar region (Kruskal-Wallis, p < 0.05; Table 1). When a Wilcoxon test (p < 0.05) was applied to the data of Table 1, a statistical difference was observed in the cfu/mL mean of the upper right molar for the sensitive film surface.
Candida spp. in saliva did not differ significantly between diabetic (mean = 3.0 × 10 6 ) and non-diabetic patients    (mean = 3.8 × 10 6 ). Table 2 shows the high prevalence of Candida albicans for all collected surfaces from diabetic and non-diabetic patients, followed by Candida tropicalis. Tests for the identification of Candida dubliniensis were performed, but this specie was not found in any patients. The device surface was the surface that carried the least number of Candida colonies and the non-sensitive film for non-diabetics was the surface that presented the highest Candida albicans colonization.
Candida albicans was observed in 420 samples, C. tropicalis in 52, and C. parapsolsis in 21 diabetic and non-diabetic patients for all radiographic regions (Table 2). In this study, 1185 species of Candida spp. were identified in the simulated radiographic region for both groups ( Table 3). The lower left bicuspid region presented the highest Candida colonization (n = 108), whilst the upper central incisor region presented the lowest colonization (n = 67; Table 3).

DISCUSSION
This study did not confirm the hypothesis that the presence of Candida spp. is more frequent in the oral cavity of diabetic than non-diabetic patients, as has been observed in other studies 9,11,14,15 . This result may be due to the fact that half of the sample had good metabolic control. According to Hill et al. 32 , diabetes by itself does not put a patient at risk of developing fungal infections, unless his/her metabolic control is poor. It is speculated that there is a tendency towards a greater presence of Candida spp. in the oral mucosa of non-diabetic patients in this study. However, due to data analyses of statistical values and considering the extreme values shown by this data set, statistical differences were not observed, with the exception of some regions shown in Tables 2 and 3. This lack of difference suggests that studies about intraoral techniques should consider that the radiographic receptor or the receptorpositioning device may be infected by Candida spp. in a similar manner to the patient's oral mucosa, independently of whether the patient is diabetic or not. Often, students that are initiating their studies consider the oral cavity to be potentially infected, but handle the radiographic receptor as if it were not infected.
As regards the distribution of the participants in this study, measurements of glycated hemoglobin suggested that patients who presented glycated hemoglobin of higher than 8% did not have an increased risk of fungal infection. In contrast, a higher occurrence of fungal infections was shown by Hill et al. 32 in patients with concentrations of glycated hemoglobin of higher than 12%. The limitation of studies of small sample sizes of patients with diabetes should also be taken into consideration, since such sample sizes may prevent the analysis of risk factors for higher prevalence of fungal infections, such as metabolic control 33 .
C. albicans was the most commonly identified candidal species in this and other studies 12,20,34,35 .
According to Willis et al. 20 , forty per cent of patients colonized with candidal species had no signs of oral candidosis and a number of candidal species were recovered from the oral cavity of insulin-treated patients. C. albicans was the most commonly recovered species, being recovered from 85% of the diabetic patients. C. dubliniensis was the second most commonly occurring candidal species. The oral cavity reflects the state of systemic health more frequently than any other part of the body and increased susceptibility to general and oral superficial infections with yeasts has long been associated with diabetes mellitus.
Manfredi et al. 13 observed that diabetic patients with dentures had more species of Candida, with the exception of Candida albicans, isolated from their mouths than dentate diabetics. This study showed a higher prevalence of Candida albicans, followed by C. tropicalis and C. guillermondi. Similarly, a previous study reported a non-significant trend towards a prevalence of species other than C. albicans in non-diabetic patients compared to diabetic patients 13 .
In conclusion, there was no difference in colonization (cfu/mL) for the Candida spp. between diabetic and non-diabetic patients with periodontal disease, when considering the five collected surfaces and the simulated-radiographic regions studied. Candida albicans was the prevalent species of Candida spp. found on the collected surfaces and simulated radiographic regions, followed by Candida tropicalis.