Oral Candidal carriage and associated risk indicators among adults in Sakaka, Saudi Arabia

Background Candida is a ubiquitous organism in nature which inhabits the oral cavity as part of the normal microbial flora. The oral carriage of Candida is perpetuated by several predisposing factors. Methods This cross-sectional study was designed to investigate the carriage rate of Candida among 104 voluntary adults at the college of medicine - Jouf University. The concentrated oral rinse technique using Sabouraud Dextrose agar medium supplemented with 0.05% Chloramphenicol was used to isolate Candida. The relative factors affecting the colonization of Candida and the concentration of each type were also determined. Results Candida species were isolated from the oral cavity of 45 (43.4%) subjects. Of these 55.6% were identifies as C. albicans as determined by the Vitek 2 compact system. Other Candida species were represented by C. glabrata (11.1%), C. krusei (11.1%), C. dubliniensis (8.9%), C. parapsilosis (6.7%), C. tropicalis (4.4%), and C. famata (2.2%). Subjects with very poor plaque status, severe gingivitis and diabetes had significantly (P = 0.001) high concentration of Candida spp. Conclusion Plague, severe gingivitis, and diabetes were found to be significantly associated with higher Candida colonization.


Background
The term Candida originates from the Latin word "candid," which means white. Following the emergence of the Human Immunodeficiency Virus (HIV) and the widespread usage of immunosuppressive therapy, there has been a wide interest among scientists in these white fungi [1,2]. Of the over 200 known species of Candida only 40 are capable of causing disease. Candida albicans is the most prevalent isolated species in both symptomatic and asymptomatic candidiasis cases. In addition, C. parapsilosis, C. krusei, C. tropicalis, C. glabrata, C. dubliniensis, C. lusitaniae, and C. kefyr, which are collectively referred to as non-albicans, are increasingly involved in Candida cases and have been recognized as important pathogens [3,4].
Candida are considered to be normal commensal organisms of the mouth that present most frequently in the posterior part of the dorsum of the tongue and can also be found on other body surfaces in the vagina and digestive tract [5,6]. However, Candida are known to elicit disease only if there is debilitation to an individual's immune status either locally or systemically [7]. In such cases, these commensals invade and penetrate the mucosal surfaces and form an essential step in the development of candidiasis [8]. Since candidiasis is often caused through endogenous infection by the Candida species, it is important to study and investigate the normal commensal flora of asymptomatic hosts at the population level.
The oral carriage rate of these organisms among the general population fluctuates between 3 and 75% without the appearance of any symptoms [9]. The disparity that has been noticed in the oral carriage rates of Candida might be influenced by multiple factors including demographic status, oral hygiene, and inconsistencies in the criteria of sample selection as well as in the analytical and sampling techniques used [6,9,10]. In addition, the carriage rates of oral Candida that have been reported to be more frequently associated with other factors are female gender, systemic diseases (e.g. diabetes mellitus), different blood groupings, tobacco smoking, and poor oral hygiene [11][12][13][14]. Additional factors such as geographical variations have also been suggested as affecting oral Candida carriage [9].
According to the information obtained from the indexed literature, data on asymptomatic oral Candida carriage in Saudi Arabia is limited to just one study [15] in which some aspects of oral Candida carriage and its relationship to poor oral hygiene were described in healthy Saudi subjects. Therefore, the objective of the present investigation was to study the carriage of Candida species (spp.) among adult individuals at Jouf University's Medical College, in Saudi Arabia.

Methods
This is a cross-sectional study that was carried out by performing clinical checkups, filling a questionnaire, and conducting a culture based microbiological investigation. Adult volunteers participated from both genders attending the college of medicine Jouf University. All candidates were given sufficient information about the study before being asked to formally consent to participate. The study protocol was also approved by the Medical Ethics Committee of the college of medicine, Jouf University. The population study was made of 104 adult individuals based on the calculation of a sample size measurement. The questionnaire contained questions on antibiotic/antifungal intake, and other health measurements and dental status.

Collection of samples and identification of Candida isolates
The concentrated oral rinse technique as described by Samaranayake et al. [16] for oral Candidal sampling was used in the present study. In brief, each participant was requested to rinse his/her mouth thoroughly for 60 s with 10 ml sterile saline. The rinse was recollected into a sterile container, and was centrifuged for 15 min at 1700 g. The supernatant was discarded, and the pellet was dissolved into l ml sterile saline. After thorough mixing for 30 s, 100 mL of each sample was transferred onto two agar plates of Sabouraud Dextrose medium (SDA, Oxoid -Basingstoke, U.K) supplemented with 0.05 μg chloramphenicol and incubated for 48-72 h at 35°C in air. All isolates that resembled Candida were gram stained and tested for Germ tube formation. Further identification of the yeast isolates was performed using the Vitek 2 compact 60 system (Biomerieux, France) according to the manufactures instructions.

Oral assessment
A visual examination using a probe and dental mirror was employed to perform the dental health status of the participants by the same researcher throughout the study. The dental health status and DMFT index (decayed, missing, and filled teeth) were assessed as described by Schuller and Holst [17]. The plaque thickness at the gingival area, and the plaque index (PI) were measured using the Silness and Loe method [18]. While, the gingival index (GI), and the gingival condition were evaluated using the Loe and Silness method [19].

Self-reported data
Information regarding age, gender, education or work status, oral health condition (history of dental caries), frequency of teeth brushing, and frequency of daily usage of mouth washes, smoking (current smoker, non-smoker) were all tabulated in a self-explanatory questionnaire. The body mass index (BMI, kg/m 2 ), blood grouping (ABO, A, B, and O), presence of diabetes and hypertension were also recorded for all the participants.

Statistical analysis
The results were statistically analyzed using the Statistical Package of Social Sciences (SPSS) version 22.0. The Student's t-test or Mann-Whitney test (for non-normally distributed data) was used to compare the difference between the means; while the Chi-squared test was used to analyze the differences between frequencies in the different groups. The one-way ANOVA analysis was used to determine the correlation between the density of Candida and the plaque/gingival status. A value of P < 0.05 was considered significant.

Clinical characteristics and demographic status
Oral rinses were obtained from 104 adult subjects, of which 79 were males (76%) and 25 were females (24%). As shown in Table 1, the mean age of the study population was (25 years, SD ± 10). Blood group O was the most common (44.2% among the study participants followed by groups A; AB and B). Only 12.5% of the study populations were smokers, 6.7% hypertensive and 5.8% were diabetics ( Table 1).
Factors associated with colonization of Candida spp.
The oral Candidal carriage and concentration (density) among both males and females regardless of their ages (Table 3) was found to be not significantly different (P = 0.35). Other factors such as, BMI (obese vs. non-obese), hypertension, and smoking all had no significant effect on either carriage rate or concentration of Candida among the study group. Although, the prevalence of oral carriage rate of Candida was comparable between diabetic participants and non-diabetic participants [4/45 (8.9%) versus 41/45 (91.1%) respectively; P = 0.23], nevertheless, the mean carriage concentration of Candida was significantly higher among diabetic participants than their non-diabetic counterparts [4492 (SD ± 5629) versus 293 (SD ± 634) P ≤ 0.00; Table 3). Subjects with Blood group O were found to be associated with a higher frequency of oral Candidal carriage, while those with blood group A showed a higher amount (density) of Candida growth, however neither was significant ( Table 2).
Correlation between oral/dental hygiene and Candidal carriage As shown in Table 2, the oral carriage rate and concentration of Candida spp. among those who brushed or used mouth wash on a regular basis was comparable, but not significantly different. Similarly, dental caries though was associated with a higher rate of Candida carriage; however, it was not statistically significantly different from those reporting no dental caries. Additionally, no significant differences in the oral Candidal carriage (P = 0.85) among the subjects with the different plaque status was revealed. There were also no significant differences regarding the prevalence of oral Candida between subjects with no gingival inflammation, mild, moderate or severe gingival inflammation status (P = 0.11). Nevertheless, the mean concentration of Candida concentration was increased significantly as a result of poor gingival status, and likewise due to a poor plaque status (P = 0.01, and P = 0.01 respectively) as revealed by ANOVA analysis.

Discussion
Based on the very limited information regarding the oral carriage of Candida in our region [9] and to the knowledge obtained from the indexed literature, only one study has investigated the asymptomatic oral carriage of Candida [14]. Other studies have focused on Candida infections of the bloodstream, called Candidemia, or exclusively on diabetic groups or infants [19][20][21].
However, several studies [12][13][14] have shown wider variations of oral Candida carriage among healthy individuals. Also, Odds et al. [7] reported that the rate of Candida colonization found in the saliva or mouth washes of normal subjects ranges from 25 to 71.3%. In a healthy Saudi Arabian population, Darwazeh et al. [14] reported a 52% prevalence rate for total Candida, of which 80.8% was C. albicans. These rates are comparable to our present findings (43.3% total Candida and 55.6% C. albicans) and consistent with the prevalence rate of 53% reported by Patil et al. [22] among Indian adults. In addition, our results confirm that C. albicans is the most prevalent species in the oral cavity in comparison to other non-Candida albicans. Some of the factors that were found to be associated with oral Candida colonization include age, gender, poor oral hygiene, smoking, and the presence of systemic diseases [2, 10-15, 22, 23]. Loster et al. [24] reported that oral Candida colonization was influenced by both age and gender; however, the subjects in this study were mainly denture-wearers.
A correlation between body mass indexes (BMI) and oral conditions has also been observed [25]. BMI was classified into the following three categories in the World Health Organization (WHO)'s World Health Report 1995: overweight (BMI ≧ 25.1), acceptable weight (BMI > 18. , and underweight (BMI ≦ 18.4). We attempted to analyze the relationship between BMI and oral Candida carriage, but find no correlation. It was observed that smoking can affect Candida colonization [26] by indirectly influencing the level of salivary glucose, which in turn leads to increased Candida growth and colonization [26]. Nonetheless, our study did not demonstrate any clear link regarding this phenomenon, nor did it affect the status of the mean number of Candida colonization. Our results were found to be consistent with previous studies [11,12] as  they found that tobacco smoking on its own influenced neither the carriage rate of oral Candida nor the number of colonizing Candida.
Although several studies that have been conducted focus on the association between A, B, and O blood groups and Candida carriage [13,27], contradictory observations have been reported, and other results lacked significant findings [28]. Therefore, the relationship between Candida species and ABO blood grouping remains inconclusive. Bamford-Mason et al. [13] showed that there is a significant relationship between the blood group O and Candida colonization, and our results were consistent with this, as we found that individuals with blood group O had higher carriage rates than those with other blood groups. Although Shin et al. [29] showed that most oral Candida carriers had blood group A, but there was no significant differences between the different blood groups.
The role of Candida species as a precursor of disease among diabetic patients was first described by Odds et al. [23], who observed a higher rate of oral candidiasis in patients with diabetes mellitus (DM) than in healthy individuals [30,31]. However, another study has contradicted this relationship between DM and oral colonization by Candida [32]. Our results showed a significantly (P = 0.00) higher Candida count (CFU/ml) among diabetics than non-diabetics, as shown in Table 3. Although, these findings are consistent with some previous investigations [31][32][33], other studies did not show any significant differences between DM and the number of colonizing Candida [34].
Investigations into whether there is a relationship between hypertension and Candida colonization have also been conducted over the past 5 years [25]. Our study found that subjects who had mild hypertension had a prevalence rate of 71.4% for oral Candida carriage. There was also a higher mean number of colonies associated with the mild hypertensive subjects than their counterparts; however, neither of these findings was proven to be statistically significant. The relationship between hypertension and Candida colonization has yet to be clarified and might in fact be due to unhealthy lifestyles.
Several clinical investigations were performed to study the presence of a possible relationship between Candida colonization status and oral hygiene [12,35]. In these studies, researchers observed an inversely relationship between better oral hygiene and reduced carriage rate of the number of colonizing cells of Candida spp. A recent study showed that a lower frequency of tooth-brushing was suggested to be associated with metabolic syndromes [36]. Other studies reported using mouth wash and aqueous extract of green tea led to reduction in the number of Candida cells [37]. On the contrary, our prospective study couldn't demonstrate any association between frequencies of teeth brushing on a daily basis or the frequencies of usage of mouth wash on daily basis and Candida colonization.
In this study, we found no correlation between the plaque index status and the rate of Candida colonization. However, we found the concentration of Candida cells was significantly increased as a result of poor plaque index status (P = 0.001). We also couldn't demonstrate any significant differences between oral Candidal carriage and gingival status. On the other hand, a significant increase in the density of the number of Candida colonies among subjects diagnosed with severe gingivitis (P = 0.001) was observed. These findings are in agreement with previous studies that reported a significant relationship between the presence of oral candidiasis and gingival erythema in patients diagnosed with HIV infection [38]. Other studies have reported no significant relationship between Candida, plaque and gingival status [12]. We didn't found any significant correlation between caries history or poor DMFT scores and Candida colonization. Our findings regarding the Data represent mean CFU/ml of colonizing Candida, (±SD) and P-values increased density of Candida cells in subjects with very poor plaque status and/or severe gingivitis might substantiate other hypothesis which links Candida reluctance to preferably reside in subjects whose dental plaque is associated with poor oral hygiene, high level of plaque status, and presence of high level of gingival inflammation [38].
Limitation of study Using of self-reported data such as information on comorbidities (hypertension and diabetes) was based on recall basis.

Conclusion
Of all the studied indicators none seem to affect the rate of oral carriage of Candida among our subjects. However, very poor plague status, severe gingival inflammation, and diabetes were significantly associated with higher density of colonizing Candida spp.