Assessment of Candidal carriage in patients with Type II Diabetes Mellitus

Correspondence: Dr. Ram Sudhan Lamichhane, MDS Department of Oral and Maxillofacial Pathology, Kist Medical College, Lalitpur, Nepal E-mail: drramsudhan@gmail.com Background: It is generally acknowledged that patients with diabetes mellitus are more susceptible to fungal infections, particularly with Candida albicans. Oral infection by Candida can result in a number of clinical lesions, including median rhomboid glossitis (central papillary atrophy), denture stomatitis, squamous cell carcinoma, Radiation therapy, immunocompromised status, etc. Different studies have shown that patients with diabetes mellitus have increased frequency of oral candidal carriage and increased risk of candidiasis, which is related to poor metabolic control, neutrophil dysfunction, reduced salivary flow, high glucose concentration in blood and saliva and in medications.

[3] The main complications associated with diabetes mellitus are retinopathy, neuropathy, nephropathy and macro/ microangiopathy.Several studies suggest a higher prevalence and severity of some pathologies in the oral tissues of diabetes mellitus patients, like gingivitis, periodontitis, dental caries, candidiasis, and other oral manifestations such as alteration of salivary flow and oral burning sensation.
It is generally acknowledged that patients with diabetes mellitus are more susceptible to fungal infections, particularly with Candida albicans. 4Oral infection by Candida can result in a number of clinical lesions, including median rhomboid glossitis (central papillary atrophy), denture stomatitis, squamous cell carcinoma, Radiation therapy, immunocompromised status (HIV), etc. 4 Different studies have shown that patients with diabetes mellitus have increased frequency of oral candidal carriage and increased risk of candidiasis, which is related to poor metabolic control, neutrophil dysfunction, reduced salivary flow, high glucose concentration in blood and saliva and in medications. 5thods to identify, quantify and establish the pathogenicity of Candida have included a number of techniques, such as smears and swabs, imprint or impression cultures, and estimates of the number of organisms by using colony counts from saliva and oral rinses. 4e present study aims to determine the candidal carriage in patients with type II diabetes mellitus as it is hypothesised that patients with type 2 DM may exhibit a certain degree of local immune suppression, thereby enabling the candidal population to proliferate beyond the levels that are normally seen when it is a commensal in the oral cavity.

MATERIALS AND METHODS
The present study was conducted in the Department of Oral Pathology and Microbiology, Manipal College of Dental Sciences, Mangalore over a period of 12 months from January 2010 to January 2011.The study involved the assessment of candidal carriage using oral rinse samples.

Study Sample
The study comprised of a total of 60 subjects divided into two groups: Study group: Consisting of thirty individuals above 40 years of age with a known history of type II diabetes mellitus for a minimum period of one year.The subjects with diabetes were selected from among the patients attending the dedicated Diabetic clinic of K. M. C. Hospital, Attavar Mangalore.Patients were included irrespective of whether they were under insulin or oral hypoglycaemic therapy for diabetes.
Control group: Consisted of thirty age and sex-matched healthy individuals without any history of diabetes mellitus (assessed by random blood sugar). 6

Exclusion criteria
Patients/ individuals with the following were excluded from the study:

Control group of volunteers having clinically healthy
oral mucosa and no clinical signs of systemic diseases and positive laboratory findings (absence of diabetes)

Methods of collection of Data:
Ethical clearance was obtained from the Institutional Ethics Committee (MCODS, Mangalore).Subjects of both the study and control groups were informed of the procedure and a written consent was obtained.All the subjects were clinically examined to assess the oral hygiene and to exclude the possibility of any other oral disease or systemic disease with oral manifestations.For study groups, haematological assessment for blood glucose/glycosylated haemoglobin levels was obtained from patients' medical records.

Assessment of candidal colonization:
Materials used: • Sterile physiological saline solution Oral Rinse Method: An overview of the procedures: Subjects of both the groups were given 10 ml of sterile normal saline and asked to rinse the mouth for one minute.The subjects were then asked to return the oral rinse in a sterile clean, broad-mouthed container which was capped, labeled and taken to the laboratory.The samples were then inoculated onto the culture medium (Sabouraud's dextrose agar with Chloramphenicol) with minimal delay (within 6-8 hours of collection of oral rinse).

Method of counting colonies:
Fifty μl of sample was taken using micropipette and streaked onto culture plates containing Sabouraud's Dextrose Agar with Chloramphenicol and incubated at 370 C for 48 hours.Number of colonies formed were counted and multiplied with a factor of 20 to get the colonies in 1 ml of a subject's sample. 8mber of colonies contained in 50 μl of saliva = n Therefore the number of colonies in 1000 μl (1 ml) = n x 1000/50 = n x 20

Statistical Analysis:
The data was analysed using SPSS software.(Version

RESULTS
The first table (Table .1) showed that the total colony forming units was higher in cases (1020) than in controls (270) (fig.1).
As the data was found to follow a skewed distribution pattern, nonparametric tests were used to study the differences.Thus, Mann Whitney U test (Table .2) was applied to assess the variations between cases and controls for each of the variables.The increase in CFU counts (median 1020) among cases was statistically significant (Z= -2.139, p=0.0324) (fig. 2 & fig.3).
Correlation of glucose level using linear regression (Table .3) showed no correlation.The colony forming units (0.096) showed no correlation.

DISCUSSION
Diabetes Mellitus (DM) is a disease affecting multiple organs and this combined with changes in the oral cavity such as xerostomia (dry mouth), alteration of taste and burning mouth sensation causes localised immune suppression thereby causing shift in homeostasis of the oral micro flora. 8andida albicans is a commensal yeast and 40-60% of the healthy adults harbour Candida in the oral cavity.5 Candida species especially Candida albicans have been frequently isolated from the oral cavity of patients with DM and it has been reported that upto 77% of insulin-treated diabetic patients harbour oral Candida.9 Various studies have been reported in support of [10][11][12] and against [13][14][15] the hypothesis that "poor glycaemic control predisposes to oral candidal infection in diabetic patients".
A study carried out by Aly et al 9 and Hill et al 12 found that oral carriage of yeasts was associated with increased plasma glucose levels (P<0.05) and concluded that poor glycaemic control was a significant contributory factor in palatal carriage of yeasts in patients with Type 2 DM (P <0.01).Also a study by Sashikumar R and Kannan R 11 found a significant correlation between the salivary glucose level and the blood glucose level in diabetic subjects and hence proposed that increased blood glucose level increases the salivary glucose level and this increase, likely contributes to increased yeast carriage and the potential for increased susceptibility to oral candidiasis.
Another study using an oral rinse technique for estimation of oral candidal carriage found no difference between the patients of Types 1 and 2 DM.Furthermore, the frequency of isolation and oral candidal load were not influenced either by duration or type of diabetes, glycated haemoglobin level or diabetic complications. 9ndidal carriage in DM is rendered non-comparable by the use of different methods like swabs, smears, imprint, oral rinse etc for the recovery of Candida from the oral cavity. 16,17he oral rinse technique using normal saline is known to be a sensitive technique mainly because, the entire oral cavity is sampled in a single rinse thereby ensuring coverage of greater surface area. 17 the present study, a comparison of oral candidal carriage between the study group of diabetics and the control group of non-diabetics showed significant increase (p=0.0324) in the colony forming units (CFUs) of Candida ranging from 0 to 8400 CFU/ml of saliva with the median of 1020 while in non-diabetics, the candidal counts ranged from 0 to 7740 CFU/ml with the median of 270.[18][19][20] However, Loiselle et al and Peter et al found no difference in the frequency of isolation of Candida organism from the oral cavities of diabetics and non-diabetics. 19e increased oral candidal carriage load may be attributed to hyposalivation, increased salivary glucose and/or altered neutrophil function (fig.4).The increased salivary glucose concentration may form chemically reversible glycosylation products with proteins in tissues during hyperglycaemic episodes.It is possible that accumulation of such glycosylation products on epithelial cells may increase the number of available receptors for Candida. 9bservations made by Darwazeh et al 21 where the adhesion of Candida albicans to Buccal Epithelial Cells (BEC) from 50 diabetic patients was 55% higher than the cells from 50 age-and sex-matched non-diabetic control subjects (P <0.001) supports this theory.These results agree with a study by Dorocka-Bobkowska et al 22   PEC for control subjects (P <0.001).
The other important manifestation in diabetics that could lead to high oral candidal carriage is hyposalivation. 8ehydration associated with elevated blood glucose increases osmotic gradients within the salivary glands, thereby limiting secretion. 10,23Autonomic neuropathies that diminish the ability of salivary glands to respond to a salivary stimulus or microvascular changes that compromise the ability of salivary glands to respond to neural or hormonal stimulation in diabetic patients could explain the significantly lower salivary flow rates in diabetics. 10,23Hyposalivation therefore compromises both the flushing and antimicrobial action of saliva.Also saliva possesses secretory immunoglobulin A (sIgA) and free secretory component (SC) which normally inhibit Candidal adhesion to oral epithelial cells.Thus, hyposalivation leads to imbalance of the normal homeostatic mechanism that otherwise ward off candidal infections. 9other host factor which may promote the oral carriage of Candida in diabetes is the possible defects in candidacidal activity of neutrophils, particularly in the presence of glucose.For instance, when the Candida killing ability was correlated with the production of superoxide, the polymorphonuclear leucocytes (PMN) from diabetic patients with candidiasis produced less free oxygen radicals and exhibited reduced phagocytosis and intracellular killing of Candida.Hence, decreased phagocytosis, intracellular killing, bactericidal activity and chemotaxis associated particularly with poorly controlled diabetes may render the diabetic patient more prone to candidal infection. 9ndidal infection in diabetics is thus a reflection of neural, hormonal and immunological status of an individual and the emphasis should be on a holistic approach to management of diabetes mellitus.The present study compared oral candidal carriage between the study group of diabetics and the control group of non-diabetics with results of a significant increase in the CFU count of diabetics.Therefore, our study, while limited in its sample size, suggests that oral candidal carriage can also serve as a surrogate marker of serum glucose levels, enabling non-invasive periodic evaluation of serum glucose levels.

CONCLUSION
Oral rinses obtained from the study and control groups showed significant increase in Colony Forming Units of Candida albicans among diabetics when compared to nondiabetics.
The increase in candidal colony counts in diabetics may be explained as: 1. Higher salivary glucose levels in diabetic patients favour yeast growth.
2. Accumulation of increased glycosylation products on oral epithelial cells of diabetic patients may increase the number of available receptors for Candida.
3. Hyposalivation secondary to neuropathy in diabetics may enhance candidal colonization as the normal anti-fungal properties of saliva stand diminished.
4. Possible defects in candidacidal activity of neutrophils, particularly in the presence of elevated levels of glucose.
The results indicate significant increase in colonization and carriage of candida in the oral cavity among diabetics when compared with non-diabetics.However, further research using larger samples is required which may lend credibility to the suggestion of increased candidal CFUs in diabetics serving as a surrogate marker of serum glucose levels.

Figure 1 :
Figure 1: Indicating the distribution of colony forming units (CFUs) among cases and control.

Figure 2 :
Figure 2: Smooth creamy while colonies of Candida growing on Sabouraud's Dextrose Agar (SDA) in control group.

Figure 3 :
Figure 3: Smooth creamy white colonies of Candida growing on Sabouraud's Dextrose Agar (SDA) in study group of diabetic patients.

Figure 4 :
Figure 4: Depicting interrelationship between DM and Oral Candidal Colonization

Table 3 : Pearson's correlation between the glucose levels and morphometric parameters
Lamichhane RS et al.
who reported that the mean candidal adherence to palatal epithelial cells (PEC) of diabetic patients was 34.5 yeasts/PEC versus 20.7 yeasts/