Prevalence and identification of oral candida species in patients with type 2 diabetes in Yogyakarta

Patients with diabetes are prone to recurring and even resistant Candidiasis, making treatment challenging. Many hypotheses proposed related to susceptibility of diabetic patients to Candida . The prevalence and species of Candida in a particular diabetic community might be different compared to other diabetic community. This study aimed to determine the prevalence of Candida colony and its species in the oral cavity of diabetic patients included in the CDM (Chronic Disease Management) program in Yogyakarta. One hundred patients with type 2 diabetes (n= 100) were recruited as the subjects of this study. The subjects were classified into controlled and uncontrolled Diabetes mellitus (DM). Samples of oral rinse solution were collected to determine the species of Candida and number of Candida colonies using CHROMagar Candida medium. There were 47 and 53 of subjects with controlled and uncontrolled DM, respectively. The mean number of Candida colony in the subjects with controlled diabetes (1003.13) was higher than that in the subjects with uncontrolled diabetes (478.43). The Candida colony most commonly identified in the subjects with controlled and uncontrolled diabetes were C. albicans and C. glabrata, respectively. Female patients had higher mean number of Candida colony (859.51) compared to male (299.21). The Candida colony most often identified in both genders was C. albicans. In addition, the subjects of this study consisted of 83 geriatric subjects and 17 non geriatric subjects, in which the mean number of Candida colony in the geriatric subjects (761.77) was higher than that in the non-geriatric subjects (545.71). The Candida colony most often identified in the geriatric subjects and non-geriatric subjects was C. albicans and C. glabrata, respectively. The Mann Whitney test demonstrated that there was a significant difference (p = 0.009) of the mean number of Candida colony between male and female. However, there was no significant difference of the mean number of Candida colony between ages (p = 0.060) and diabetic status (p=0.175). It can be concluded that the Candida species most commonly identified in all the subjects was Candida albicans with the mean colony number of 349.96, followed by C. glabrata (225.97), C. krusei (144.91), C. tropicalis (3.67), and other species (2.02).


INTRODUCTION
Diabetes mellitus (DM) is a disease caused by a chronic metabolic disorder of carbohydrate, lipid, and protein due to absolute or relative reduction in insulin secretion or activation that is characterized by increasing blood glucose. 1 Indonesia is the fourth most populated country, with the seventh highest prevalence of diabetic patients in 2012 worldwide. 2The Ministry of Health of the Republic of Indonesia reported in 2018 that around 1.5% (1.017.290)people of Indonesia suffered from diabetes. 3There are two main types of DM, i.e. type 1 and type 2, and the majority of diabetic patients (±90%) had type 2 DM and the rest had type 1 DM. 2 Many oral findings are attributed to uncontrolled blood glucose in diabetic patients such as xerostomia, hyposalivation, burning mouth sensation, periodontal disease, dysgeusia, and oral candidiasis.Candida itself is normal commensal of oral flora.However, if immune status of host is altered such as in patients with HIV-infection, malignant disease, nutritional deficiency, and metabolic disorder, Candida becomes pathogenic and manifests as oral candidiasis.There are around more than 150 species of Candida, but only ten species are more responsible for infection in human. 4Out of the ten species, Candida albicans is the most common species isolated from the oral cavity.6][7][8] Principally, many hypotheses have been proposed related to the susceptibility of diabetic patients to Candida, i.e. (i) high salivary glucose; (ii) hyposalivation; (iii) chemotaxis disorder; (iv) phagocytosis defect especially due to polymorphonuclear leukocyte deficiency. 9,10Several risk factors such as age, gender, nutrition, oral hygiene, smoking, denture usage, salivary pH, and xerostomia are contributed to the susceptibility of diabetic patients to oral candidiasis. 11,12Clinically, oral candidiasis manifests as white lesion, erythematous lesion, or hyperplastic lesion accompanied by pain or burning sensation.Candida colonies could be isolated from patients with oral candidiasis through oral rinse, saliva, or swab technique.However, it is also possible that there is no clinical evidence of oral candidiasis although Candida colonies can be isolated from laboratory examination -it is called Candida carriage. 13Previous studies showed that the prevalence of yeast carriage in uncontrolled diabetic patients reached up to 90% 10 and in controlled diabetic patients up to 63.3% of the subjects, 14 and Candida albicans was responsible for 51% of the isolates. 15n diabetic patients, oral candidiasis is difficult to cure, it is prone to be recurrent even resistant. 13ain and burning sensation in diabetic patients with oral candidiasis deteriorate oral functions which, in turn, will decrease oral health-related quality of life (OHRQoL).][18][19] The prevalence of Candida and its species in particular a diabetic community might be different compared to other diabetic communities, as the disease is closely related to diabetic conditions, systemic health, medications, oral microbiome and health, hyposalivation, lifestyles and habits, gender, ages, and denture usage.][22] Occurrence of oral candidiasis might also be affected by various diseases, such as HIV infection. 23It was also reported that diet could also affect the disease progression, as fructose was found to be able to slow down Candida growth rate. 24The prevalence, especially on chronic lesions, might also be affected by antifungal susceptibility profile of the dominating species and the antifungal drug prescribed. 25Consequently, determination of treatment planning for diabetic patients with oral candidiasis varies, depending on the etiology, risk factors, and the nature of the dominant species in each patient.
The aim of this study was to determine the prevalence of Candida colony and its species in the oral cavity of Chronic Disease Management (CDM) diabetic patients in Yogyakarta.By knowing the prevalence and species of Candida in the oral cavity of diabetic patients, the results can be used as a reference to plan the adequate management for diabetic patients, since every Candida species has its own susceptibility to antifungal medication.By minimizing the oral discomfort faced by diabetic patients with oral candidiasis, it can finally increase the OHRQoL of diabetic patients.

MATERIALS AND METHODS
This was a laboratory observational communitybased cross-sectional study.A total of 100 patients with type 2 diabetes in Yogyakarta ( The subjects were gathered and given explanation about the goal, significance, and course of the study.The subjects who were willing to participate in the study were asked to sign a written informed consent.The subjects were asked to fill in an identity form.Then, samples of oral rinse solution were collected in a sterile container by asking the subjects to rinse the mouth with 10 mL sterile saline (Otsuka TM , Japan), which was held in the mouth for 15 seconds.The concentrated rinse solution was prepared by centrifuging it at 2000 x g for 10 min.After the supernatant was removed, the cell pellet was resuspended in 500 µL, which was then inoculated onto CHROMagar Candida medium (CHROMagar TM , Paris, France) in 200 µL aliquots.Candida colonies were counted after incubation at 37 °C for 48 hours.
For the statistical analysis, the subjects were initially classified according to the diabetic status based on three variables i.e., age, gender, and presentation of Candida colony.Then, the mean number of Candida colony within the variable groups was counted based on four Candida species (C.albicans, C. glabrata, C. krusei, and C. tropicalis), others, and total Candida.Mann Whitney test for nonparametric data was conducted to know whether there was a significant difference in the mean number of Candida colony within the variable groups.

RESULTS
Type 2 DM was the only type of DM detected in this study (hereinafter referred to as DM).There were 47 and 53 of the subjects with controlled DM (M: 11, F: 36) and uncontrolled DM (M: 13, F: 40), respectively (Table 1).The subject age ranged from 45-89 years.Meanwhile, the duration of having DM in all the subjects was between <1 year up to 30 years.
Table 2 shows that the Candida colony most commonly identified in the subjects with controlled DM was Candida albicans, followed by C. krusei, C. glabrata, C. tropicalis, and other species of Candida, respectively.On the other hand, the Candida colony most often identified in the subjects with uncontrolled DM was Candida glabrata, followed by Candida albicans, C. krusei, C. tropicalis, and other species of Candida, respectively.The mean number of Candida colony in the subjects with controlled DM (1003.13)was higher than that in the subjects with uncontrolled DM (478.43).Females had higher mean number of Candida colony (859.51)compared to males (299.21).The Candida colony most commonly identified in both genders was C. albicans.This study consisted of 83 geriatric subjects and 17 non geriatric subjects, in which the mean number of Candida colony in the geriatric subjects (761.77) was higher than that in the non-geriatric subjects (545.71).The Candida colonies most often identified in the geriatric subjects and non-geriatric subjects were C. albicans and C. glabrata, respectively.It can be said that the Candida species most frequently identified in all the subjects was Candida albicans with the mean number of Candida colony of 349.96, followed by C. glabrata (225.97),C. krusei (144.91),C. tropicalis (3.67), and other species (2.02).
The results from the Mann Whitney test (Table 3) demonstrated that there was a significant difference (p = 0.009) in the mean number of Candida colony between the male and female subjects, in which females had higher mean number of Candida colony than male.However, there was no significant difference in the mean number of Candida colony between the other two variables (age and diabetes status).

DISCUSSION
In this study, the number of geriatric female subjects was higher than that of non-geriatric male subjects.This is understandable because in recent decades almost all countries in the world, including Indonesia, have experienced a rapid increase in the elderly population.Based on the population censuses in Indonesia in 1971, 1990 and 2010, there was an increase in the percentage of the elderly population by 4.5%, 6.3%, and 7.6%, respectively.The increase in the elderly population will continue, so it is estimated that by 2035 the number of elderly people in Indonesia will reach 15.8% of the total population. 26This is in line with the increase in the proportion of the elderly population globally from 6.1% to 8.8% between 1990 and 2017, which is expected to be 16% by 2050. 27The increase in the elderly population is partly due to an increase in the life expectancy of the population and improved health services.Based on the distribution of elderly population by province, Daerah Istimewa Yogyakarta had the highest percentage of elderly people in Indonesia. 28n the other hand, the proportion of women in Indonesia continues to increase in higher age groups.The proportion of women at the age of 60-64 years reaches >50%, and at higher ages the proportion of women becomes >65%.This indicates that elderly women have a higher life expectancy.Globally, the proportion of elderly women is also projected to increase, from 336 million in 2000 to more than one billion in 2050. 29he presence of Candida in the oral cavities of diabetic patients has been investigated in several places in the world, with quite diverse results.The prevalence of Candida in this study was 89%.Based on the results above, the number of colony in each Candida species between the controlled and uncontrolled DM groups varied.Previous studies reported their findings on the positive correlation and influence of blood glucose level on oral candidiasis and Candida colonization. 30,31owever, our study did not reveal statistically significant differences between the controlled and uncontrolled subjects.This might corroborate a previous study that reported independency of Candida carriage prevalence from HbA1c level. 4his shows that other risk factors might play bigger roles in the disease progression in diabetic patients.
Several risk factors such as age, gender, nutrition, level of oral hygiene, smoking habits, denture usage, salivary pH, and xerostomia are associated with the susceptibility of DM patients to Candidiasis. 12,17,32,33However, in this study not all these factors were revealed.A study by Lynne et al. showed that Candida could be detected in 74.8% of 107 subjects with DM, namely 36 of 52 controlled subjects (69.23%) and 44 of 55 uncontrolled subjects (80%). 4In this study, Candida was detected in 44 of 47 controlled subjects (93.61%) and 45 of 53 uncontrolled subjects (84.91%).A study by Mohammadi et al. showed a significant difference in Candida frequency among people with well-controlled, moderately-controlled, and poorly-controlled diabetes, as well as healthy people (p = 0.045), with the highest frequency found in people with poorly controlled diabetes. 12he results of this study indicated that the geriatric subjects had a higher mean number of Candida than the non-geriatric subjects.One of characteristics of elderly is a decline in immune system, so the elderly are more susceptible to diseases, especially chronic degenerative diseases (such as DM, malignant disease) and infections.Candida itself is a normal flora in the oral cavity.However, if the body's immune system decreases, Candida becomes pathogenic and can infect the oral cavity.There is a tendency that when someone is getting older, there is a higher possibility of Candida infection, especially related to deterioration of oral hygiene and use of dentures.In this study, there were only 6 subjects with a removable denture, so the possibility of association between Candida colonies and denture usage was less significant.The varying frequency of Candida in DM patients is due to the different abilities of fungi to attach to epithelial cells and genetic susceptibility of each patient with DM to infection.The increase in Candida colonies in diabetic subjects may be related to increased salivary glucose, oral microenvironmental changes such as reduced salivary flow or altered salivary composition, decreased immunity, 34 and disruption of oral microbial balance. 35Another reason for the association between geriatric subjects and high number of Candida colony is related to polymedication and polypathology in older people.The elderly tend to suffer from some diseases, so they have to take more medications, such as antibiotics and immunosuppressive drugs which tend to make someone more easily develop Candidiasis in the oral cavity.Unfortunately, this study did not reveal anamnesis regarding longterm medication with antibiotics and immunosuppressive drugs.
Another result of this study also demonstrated that the female subjects had a higher mean number of Candida than the male subjects.In addition, the Mann Whitney test proved that there was a significant difference in the mean number of Candida between the female and male subjects.Several reasons can be put forward regarding this; one of which is because women have estrogen.Estrogen can increase the fungal virulence of C. albicans, so they are able to avoid the innate immune system. 36Estrogen can also increase glycogen production in vaginal mucosa.Glycogen is a nutrient required for the growth of C. albicans. 36,37Possibly, the same thing also takes place in the oral mucosa.The results of this study are supported by other findings, including Brazil's Center for Diagnosis of Oral Diseases, which revealed that women's risk of developing Chronic Atrophic Candidiasis is four times higher than that of men. 35In addition, according to Loster et al., there was a significant relationship (p = 0.01) between intensity of fungal growth and gender, where infection-free individuals were more in males than females. 38ifferent works indicated C. albicans as isolated Candida with the highest frequency in the oral cavities of diabetic patients. 4,12,39,40n this research, this species was the most prevalent and it was found more frequently in the controlled subjects, while C. Glabrata was found more frequently in the uncontrolled sub jects.This is in line with previous findings that reported the same result, in which C. glabrata was found to be more prevalent in the uncontrolled diabetic patients compared to the controlled patients. 14This is because of the nature of C. glabrata that is more aggressive and produces higher amount of biofilm than C. albicans.This result showed possible specific pathogenicity and colonization of each Candida species in different microenvironment.Uncontrolled blood glucose and microenvironment might promote the aggressivity and colonization of C. glabrata in the oral cavity. 11The aggressivity will lead to enhanced pathogenicity and tissue destruction.
Diabetes mellitus could promote secreted aspartyl proteinases, thus leading to enhanced colonization, biofilm formation, and tissue invasion.Biofilm formation could impact antifungal treatment; each species has its own biofilm forming capacity.Biofilms formed by low biofilm former (LBF) were found to contain sparse yeast cells and pseudo hyphae, whereas high biofilm former (HBF) could produce denser hyphae.HBFs are known to need a specific consideration of drug choice and dose. 41Treatment to each species might differ, as some species have been reported to be resistant to particular antifungal drugs, but still in the same principle of antifungal, etiological elimination and blood glucose control. 11,42One of previous studies found that HBF needs antifungal dose of 8-fold higher than LBF. 43Some studies reported that C. parapsilosis and C. tropicalis generate great heterogenic biomass, whereas C. parapsilosis and C.glabrata secret biofilm matrix containing more carbohydrates than C.tropicalis. 41The specific nature of each species and its complexity in biofilm formation could affect treatment choice of patients with oral candidiasis because of potential resistance.
In other previous studies involving diabetic subjects, C. albicans was also the most commonly detected fungal species in the oral cavity. 12,34,44. albicans is known to predominantly exist in the oral cavity with its commensal nature 8,42 Abundant C. albicans on the mucosal surface might be caused by its pathogenicity and ability to adapt to oral microenvironment.C. albicans could infect diverse niches because it could change its morphological form, express adhesin and invasin, form biofilm to protect itself, and switch its phenotype.This led to rapid adaptation to microenvironment and pH to acquire nutrient and survive. 45 Calbicans were reported to produce lower amount of biofilm than nonalbicans species, thus leading to consideration of emerging pathogens of non-albicans species.However, the lower capacity of biofilm formation was also reported to not particularly influence clinical manifestation of the infection.41 This shows that various factors and co-infection might play roles in the disease progression.
The complexity of the relation between Candida spp.infection and DM causes the difficulty of concluding which species is the most prevalent in the oral cavity.We reported that the most prevalent species in the controlled patients was C. albicans, followed by C. krusei, C. glabrata, C. Tropicalis, and other Candida species.Meanwhile, the most prevalent species in the uncontrolled patients was C. glabrata, followed by Candida albicans, C. krusei, C. Tropicalis, and other Candida species.One of previous studies reported that the most prevalent species was C. albicans, followed by C. parapsilosis, C.dubliniensis, and C. tropicalis. 4 Meanwhile, another study reported that the most frequently identified Candida species were C. albicans, C. krusei, C. glabrata, C. kefyr, and C. tropicalis. 12Another study also reported the most prevalent species were C. glabrata, C. albicans, and C. tropicalis in both uncontrolled and controlled diabetic patients. 14These differences are significantly affected by the complex and diverse risk factors that play roles in the disease progression; these could also be affected by environmental factors surrounding the subjects.
Many risk factors that might influence Candida colonization in the oral cavity in the subjects with DM such as xerostomia, smoking, and salivary pH were not revealed in this study.In principle, these risk factors will affect the ecology of the oral cavity, making it more susceptible to oral candidiasis.Therefore, future studies on these risk factors can be carried out more comprehensively, so preventive measures to minimize the occurrence of oral candidiasis in people with DM can be taken.

CONCLUSION
It can be concluded that the Candida species most commonly identified in all the subjects with type 2 DM in this study was Candida albicans with the mean of Candida colony number of 349.96, followed by C. glabrata (225.97),C. krusei (144.91),C. tropicalis (3.67) and other species (2.02).
The subjects were classified into controlled DM if Fasting Blood Glucose (FBG) was < 126 mg/dL or 2 h Post Prandial Blood Glucose (2 pp BG) was < 200 mg/dL around the last few months.Meanwhile, the subjects were classified into uncontrolled DM if FBG was ≥126 mg/dL or 2 h Post Prandial Blood Glucose (2 pp BG) was ≥ 200 mg/dL around the last few months.The protocol of the study was approved by the Research Ethic Committee of Faculty of Dentistry, Universitas Gadjah Mada with approval number: 00140/KKEP/FKG-UGM/EC/2019.

Table 2 .
Means of Candida colony number within variable groups

Table 1 .
Diabetic status of subjects within variable groups

Table 3 .
Mann Whitney test of means of total Candida colony number within variable groups