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Journal of Infection and Public Health (2013) xxx, xxxxxx

The prevalence of oral Candida infections in periodontitis patients with type 2 diabetes mellitus
Sultan Al Mubarak a,, Asirvatham Alwin Robert b, Jagan Kumar Baskaradoss c, Khalid Al-Zoman a, Abdulaziz Al Sohail d, Abdulaziz Alsuwyed e, Sebastian Ciancio f
a

King Faisal Specialist Hospital & Research Center, Riyadh, Saudi Arabia Research Center, Sultan Bin Abdulaziz Humanitarian City, Riyadh, Saudi Arabia c Department of Dental Public Health, College of Dentistry, King Saud Bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia d Prince Abdulrahman Bin Abdulaziz Institute for Higher Dental Studies, Riyadh, Saudi Arabia e Dental Department, King Abdulaziz Medical City, Riyadh, Saudi Arabia f Department of Periodontics and Endodontics, School of Dental Medicine, State University of New York at Buffalo, NY, USA
b

Received 28 June 2012 ; received in revised form 24 December 2012; accepted 28 December 2012

KEYWORDS
Diabetes; Infection; Candidiasis; Saudi Arabia

Summary Objectives: The purpose of this study was to determine the prevalence of Candida spp. in periodontitis patients with type 2 diabetes mellitus. Methods: This cross-sectional study included 42 diabetic patients with periodontitis (aged 2170 years; 18 males and 24 females). Clinical measurements included probing pocket depth (PPD), clinical attachment level (CAL) and hemoglobin A1c (HbA1c) levels. Sub-gingival samples were collected from the mesio-buccal aspect of 3 teeth for fungal analysis. Candida species, including Candida albicans, Candida dubliniensis, Candida tropicalis and Candida glabrata, were identied using Gram staining, the germ tube test, CHROMagar, Staib agar and API 20C AUX. Results: The overall prevalence of Candida in diabetic patients with periodontitis observed in our study was 52%. The most common spp. of Candida identied were C. albicans (38%), followed by C. dubliniensis (9.5%), C. tropicalis (4.7%) and C. glabrata (4.7%). Compared to females, male patients were characterized by increased levels of Candida infections. Our results also indicate that individuals over the age of 40 had increased levels of Candida infections compared to patients younger than 40. Candida infections were higher among subjects with elevated

Corresponding author at: King Faisal Specialist Hospital & Research Center, P.O. Box 3354, Riyadh 11211, Saudi Arabia. Tel.: +966 1 4424238; fax: +966 1 4427894. E-mail addresses: smubark@kacst.edu.sa, salmubarak@kfshrc.edu.sa (S. Al Mubarak).
1876-0341/$ see front matter 2013 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.jiph.2012.12.007

Please cite this article in press as: Al Mubarak S, et al. The prevalence of oral Candida infections in periodontitis patients with type 2 diabetes mellitus. J Infect Public Health (2013), http://dx.doi.org/10.1016/j.jiph.2012.12.007

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blood sugar levels (HbA1c > 9) compared to individuals with well-controlled blood sugar levels (HbA1c < 6). Patients with PPDs 5 had an increased risk of Candida infection compared to patients with PPDs between 3 and 4. Conclusion: This study indicates that the frequency of C. albicans is higher than the frequencies of C. dubliniensis, C. tropicalis and C. glabrata in diabetic patients with periodontitis. Candida infections were observed at increased frequencies among subjects with high blood sugar levels and PPDs 5. 2013 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.

Introduction
The worldwide prevalence of adult-onset diabetes is approximately 171 million, and the World Health Organization (WHO) and International Diabetes Federation (IDF) have predicted that this prevalence will increase to approximately 366 million by 2030 [1]. Loe reported that diabetes is a risk factor for periodontitis and that periodontal disease is the sixth-leading complication of diabetes [2]. Hyperglycemia appears to trigger a series of events that lead to an increased risk of infection. Indeed, a signicant association between diabetes and increased susceptibility to oral infections, including periodontal disease, has been reported [3]. Candida albicans is the periodontal species that has been most commonly associated with oral lesions, but other Candida spp. have also been isolated [4] from the saliva of subjects with and without oral candidiasis. The isolation of Candida from the oral cavity does not imply the presence of disease [5]. However, fungal organisms commonly colonize the tongue, palate and buccal mucosa, and such colonization may occur in sub-gingival plaques of adults with periodontitis [6]. The frequent occurrence of Candida infections in patients with diabetes mellitus has been recognized for many years, and oral candidiasis in particular is thought to be more prevalent among these individuals [7]. Different Candida spp. have been frequently isolated from the oral cavities of patients with diabetes. In addition to C. albicans, which has been commonly isolated from diabetic patients [8], Candida glabrata, Candida tropicalis, Candida dubliniensis, Candida kuusei, Candida colliculosa, Candida fomata, Candida parapsilosis, Candida guillermondii and Candida rugosa have all been recovered from diabetic patients [911]. The aim of this study was to investigate the prevalence of Candida spp. in periodontitis patients with type 2 diabetes mellitus.

Materials and methods

This cross-sectional study included 42 diabetic patients with periodontitis (18 males and 24 females; aged 2170 years) who were registered at Sultan Bin Abdulaziz Humanitarian City (SBAHC), Riyadh, Kingdom of Saudi Arabia, from March 2008 to August 2009. The subjects were recruited during their routine dental follow-up appointments at SBAHC. The International Diabetes Federation (IDF) and the American College of Endocrinology (ACE) recommend maintaining hemoglobin A1c (HbA1c) values below 6.5%, while the American Diabetes Association (ADA) recommends that HbA1c levels be below 7.0% for most patients [12]. In this study, fasting venous blood samples for the laboratory analysis of glycated HbA1c levels in all subjects were obtained from the antecubital vein between 8:00 a.m. and 10:30 a.m. by venipuncture using a 27-G buttery needle and the DCA VantageTM Analyzer (Siemens Health Care Diagnostics, New York, United States). Poor glycemic control was dened as having levels of HbA1c 7%. Clinical measurements included probing pocket depth (PPD) and clinical attachment level (CAL). Periodontitis was dened as a disease state characterized by the active destruction of supporting periodontal tissue, which manifests as the presence of PPDs 3 mm and 3 mm periodontal attachment loss at the same site [13]. Subjects who were willing to participate in the research were required to sign an informed consent agreement. The following inclusion criteria were used: age range between 21 and 70 years; a diagnosis of type 2 diabetes that had been made at least 1 year prior to enrollment; a minimum of 18 remaining natural teeth without crown restorations; and good physical condition with no serious medical conditions or transmittable diseases, such as malignant disease and active hepatitis. The exclusion criteria included the use of fungal treatment or periodontal treatment within 3 months prior to

Please cite this article in press as: Al Mubarak S, et al. The prevalence of oral Candida infections in periodontitis patients with type 2 diabetes mellitus. J Infect Public Health (2013), http://dx.doi.org/10.1016/j.jiph.2012.12.007

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Table 1 The demographic and socio economic data of the study population. Variables Gender Male Female Age <30 3040 4150 >50 Marital status Married Unmarried Education Primary Intermediate High school University Employment status Employed Unemployed Smoking Smoking Non-smoking Number of patients 18 (42.8%) 24 (57.2%) 4 (9.5%) 11 (26.2%) 15 (35.7%) 12 (28.6%) 35 (83.3%) 7 (16.7%) 9 (21.4%) 10 (23.8%) 12 (28.6%) 11 (26.2%) 17 (40.5%) 25 (59.5%) 9 (21.4%) 33 (78.5%)

Candidiasis in diabetic patients with periodontitis the study. Female patients who were pregnant or nursing were also excluded from the study. All participants were duly notied of the nature of the investigation, and this study was approved by the Research and Ethics Committee of SBAHC. Sub-gingival samples were collected from 3 of the deepest pockets in the oral cavity, and the samples were sent for fungal analysis at the Research Center of SBAHC. The sub-gingival samples were plated on special media, CHROMagar [14], to determine the presence of Candida. Cells were either pipetted onto CHROMagar Candida plates (CHROMagar, Paris, France) as 10 l drops of a cell suspension containing 2 107 yeast cells/ml or streaked-out to form single colonies from the suspension. Cultures on the plate medium were incubated at 37 C and examined after 2 days [15]. Suspected isolates were then incubated [16] at 42 C to differentiate between C. albicans and C. dubliniensis. All isolates were analyzed using the API 20C AUX system (bioMeriux, Marcy-lE toile, France) to identify them denitively based on their carbohydrate assimilation. Suspended yeast cells in PBS, which were equivalent to McFarland Standard No. 0.5, were spread on a yeast nitrogen base agar plate (Difco) that did not contain carbohydrates. Paper disks soaked in 1% (w/v) solutions of various carbohydrates were placed on the plate, and the results were read after incubating the plate at 25 C for 23 days [17].

Statistical analysis
All of the available data from these subjects were analyzed, and no imputations were performed for missing data. Data analysis was conducted using Microsoft Excel 2002 (Microsoft Corporation, Seattle, WA, USA) and the Statistical Package for Social Sciences, version 16 (SPSS Inc., Chicago, IL, USA).

dubliniensis, C. tropicalis and C. glabrata. Fig. 2 indicates that patients over the age of 40 had increased levels of C. albicans, C. dubliniensis, C. tropicalis and C. glabrata compared to individuals under the age of 40. The inuence of diabetes severity on Candida infections is shown in Fig. 3. Candida infections were more prevalent among subjects with high blood sugar levels (HbA1c > 9) compared to those with well-controlled blood sugar levels (HbA1c < 6). The inuence of PPD on Candida is shown in Fig. 4. Patients with PPDs 5 mm had

Results
The socio-demographic data of the study population are shown in Table 1. The mean age of the study participants was 47.3 14.4 (mean SD) years. Eighteen participants were male (42.8%), and 24 (57.2%) were female. The overall prevalence of Candida in diabetic patients was 52%, and the identied species included C. albicans (38%), C. dubliniensis (9.5%), C. tropicalis (4.7%) and C. glabrata (4.7%). Fig. 1 shows the gender-specic prevalence of Candida in diabetic patients with periodontitis. Compared to female patients, males exhibited increased levels of C. albicans, C.

Figure 1 Prevalance of oral candidiasis in diabetic patients with periodontitis.

Please cite this article in press as: Al Mubarak S, et al. The prevalence of oral Candida infections in periodontitis patients with type 2 diabetes mellitus. J Infect Public Health (2013), http://dx.doi.org/10.1016/j.jiph.2012.12.007

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S. Al Mubarak et al. in the microbial colonization that contributes to the progression of oral diseases [18,19]. Species of Candida, mainly C. albicans, have been recovered from periodontal pockets in 7.119.6% of patients with chronic periodontitis [6,20]. Both C. albicans and C. dubliniensis were capable of colonizing periodontal pockets in patients with chronic periodontitis [21]. Published reports have also indicated that C. albicans is the predominant species of yeast isolated from gingival crevicular uid and the periodontal pockets of periodontal patients and healthy subjects, but the presence of C. glabrata and C. tropicalis has also been reported infrequently [19,22]. In present study, we report that the prevalence of Candida infection is higher in diabetic patients. In addition, we found that males exhibit increased levels of Candida infections compared to females. Our results also indicate that individuals over the age of 40 exhibit increased levels of Candida infections compared to younger patients. Polymerase chain reaction (PCR) experiments revealed that the quantities of several Candida spp. were higher in diabetic patients with chronic periodontal disease than in patients without diabetes [23]. Among diabetic patients, C. albicans, C. dubliniensis, C. tropicalis and C. glabrata were observed in 57%, 75%, 16% and 5% of the periodontal pockets, respectively. Among non-diabetic patients, C. albicans and C. dubliniensis were present in 20% and 14% of the periodontal sites, respectively. In contrast, no evidence of C. tropicalis or C. glabrata colonization was found in the periodontal sites [23]. In the present study, we found that periodontitis patients with PPDs 5 mm had a higher risk of Candida infection than patients with PPDs between 3 and 4 mm. Previous studies have reported that diabetic patients colonized with Candida have higher salivary glucose levels than non-colonized patients [24]. In fact, patients whose diabetes is well controlled have fewer Candida infections than individuals with poorly controlled diabetes. Good diabetic control is the best protection against Candida infection, and studies have shown that controlling blood sugar levels lowers the risk of Candida infection [2527]. In the present study, we found that Candida infection levels were increased in uncontrolled diabetics compared to the wellcontrolled subjects. Indeed, Candida infections occurred less frequently in subjects who had well-controlled blood sugar levels (HbA1c < 6) compared to subjects without proper diabetic control (HbA1c > 9). The major limitations of this study were the relatively small number of patients and the limited

Figure 2 Age wise prevalance of oral candidiasis in diabetic patients with periodontitis.

Figure 3 Prevalance of Candida in patients with different HbA1c level.

an increased risk of Candida infection compared to patients with PPDs between 3 and 4 mm.

Discussion
The virulence factors in Candida species facilitate colonization and proliferation in the periodontal pockets. The Candida spp. can co-aggregate with bacteria in dental biolms and adhere to epithelial cells. These interactions may be important

Figure 4 Prevalance of Candida with probing pocket depth (mm).

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Candidiasis in diabetic patients with periodontitis number of risk factors examined. Further research is required to address these limitations. However, despite the limitations, this study provides valuable data related to the Saudi population. In addition, this study highlights the need for extensive research in this area in Saudi Arabia, which could facilitate the planning and design of appropriate strategies and interventions. In conclusion, our ndings indicate that the frequency of C. albicans is higher than the frequencies of C. dubliniensis, C. tropicalis and C. glabrata in diabetic patients with periodontitis. In addition, Candida infections within this patient population were found to be more prevalent among subjects with high blood sugar levels and PPDs 5 mm. However, further studies in different clinical settings will be required to provide a more comprehensive picture of Candida infections among periodontitis patients with diabetes mellitus.

Conict of interest
Funding: No funding sources. Competing interests: None declared.

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Please cite this article in press as: Al Mubarak S, et al. The prevalence of oral Candida infections in periodontitis patients with type 2 diabetes mellitus. J Infect Public Health (2013), http://dx.doi.org/10.1016/j.jiph.2012.12.007