Clinico-Cytological Study of Fungal Elements in the Buccal Smears of Prediabetic, Diabetic, and Nondiabetic Individuals in Association with Burning Sensation.

INTRODUCTION: Diabetes mellitus is a metabolic disorder that may have profound effects on oral mucosa being prone to infections, particularly candidiasis. In healthy individuals, the candida is a commensal, but in diabetic patients, it forms severe colonization, even in the absence of any clinically evident lesion and may cause a burning sensation of the mouth. This study aims to identify the presence or absence of fungal elements in buccal smears of nondiabetic, prediabetic, and diabetic individuals and to correlate the presence or absence of burning sensation in the oral cavity.
MATERIALS AND METHODS: A total of ninety individuals were selected for the study and divided into Group 1 - thirty cases of diabetics, Group 2 - thirty cases of prediabetic patients, and Group 3 - thirty cases of nondiabetic individuals. Detailed case history and thorough intra and extraoral examination were recorded. The buccal smears were cultured in Sabouraud dextrose agar, subcultured in HiCrome™ agar, observed for the colonies of fungal elements, and were analyzed for species differentiation.
RESULTS: There was a positive correlation between the presence of fungal elements and burning sensation in diabetic individuals, whereas it was not the case in the nondiabetic and prediabetic individuals.
CONCLUSION: Candida colonization is one among a factor in diabetic individuals related with burning sensation of the mouth. Copyright:

INTRODUCTION

Diabetes mellitus is a chronic metabolic disorder which manifests as hyperglycemia and alteration in insulin production or action or both.[1] Diabetes mellitus is epidemic, and there is a high carriage of Candida in the oral cavity of the diabetic patients.[2] Candida albicans is the most common species found in them and may or may not be symptomatic.[3] Burning sensation of the oral mucosa is a common manifestation that is usually multifactorial and characterized by epithelial atrophy induced by fungal colonization, especially C. albicans.[4] The factors that contribute to the candidal infections or carriage, in the oral mucosa of diabetic patients, are xerostomia,[5] smoking,[6] poor oral hygiene,[7] long-standing denture wearers,[8] increasing age,[9] nutritional deficiency, salivary pH disorder, presence of complications,[10] etc., These factors thus contribute in the transition of Candida species from commensal to pathogen, which in turn causes clinical infection.[11] From the literature search, it is evident that fungal colonization in the oral mucosa of diabetic individuals is one of the reasons for burning sensation irrespective of the demonstrable clinical lesions. However, the presence of subclinical Candidal colonization and burning sensation in the oral mucosa of diabetic individuals is inconclusive.[12]

Aim

The aims were to study the presence or absence of fungal elements in the oral mucosa of the diabetic, prediabetic, and nondiabetic individuals without having a demonstrable clinical lesion and to correlate with the presence of burning sensation.

MATERIALS AND METHODS

This cross-sectional study was carried out between June 2019 and February 2020 after obtaining ethical clearance and written informed consent from the patients. Ninety individuals were selected for the study and were divided into Group 1 consisting of thirty known diabetics, Group 2 consisting of thirty prediabetics, and Group 3 consisting of thirty nondiabetic individuals.

Group 1 – (known diabetics) individuals with past medical history of diabetes mellitus (confirmed by glucose tolerance test and under medications) and having normal or increased random blood sugar level which is more than 140 mmHg and glycated hemoglobin (HbA1c) more than 7.5 mmol.[911] Group 2 – (prediabetics) having either normal or increased random sugar but having HbA1c <7%.[91314] Group 3 – (nondiabetics) having blood sugar and HbA1c within the normal limits which is in between 120 and 140 mmHg and HbA1c below 6.5 mmol. Exclusion criteria: Patients with anemia, ulcers, sharp teeth, loss of vertical dimension, prosthesis, smoking, attrition, and mucosal diseases such as leukoplakia and lichen planus were excluded from the study.

A detailed case history was recorded and a thorough extra and intra-oral examination was made. Presence or absence of burning sensation was recorded. The samples were collected by scrapping of the buccal mucosa with sterile cotton swab and inoculated in Sabouraud dextrose agar (SDA) plates and were incubated at 37°C for 48 h. The inoculated plates were observed for colony formation. The colonies formed in the SDA growth plate were subcultured into the HiCrom™e agar (HiMedia, Mumbai, Maharashtra, India) for the isolation, presumptive identification, and differentiation of candida species. The plates were incubated and pure colonies were identified after incubation at 25°C for 72 h.

Statistical analysis

The information collected regarding all the selected cases was recorded in a Master Chart. Data analysis was done with the help of a computer by using SPSS 16 Software developed by IBM Software. Using this software, percentages, means, standard deviations, and “P” values were calculated through one-way ANOVA test for raw data and Chi-square test for consolidated data to test the significance of difference between variables. “P” value <0.05 was taken to denote statistically significant relationship.

RESULT AND INTERPRETATION

Among 90 individuals, 49 were male and 41 were female, with the age range between 20 years and 50 years. On clinical examination, 14 presented with a burning sensation and 18 did show positivity for candida colonization [Table 1]. The smear study upon incubation in SDA agar in 48 h, showed candida growth, which was identified by the colonies with the smooth creamy, white-colored buttery formation on the culture plates [Figure 1a and b]. The presence of creamy white colonies in SDA agar media indicates colonization of both C. albicans and nonalbicans [Figure 1a and b]. All the 18-Candida-positive samples isolated from SDA agar were inoculated in HiCrome agar plates. The growth of various species of candida in HiCrome agar based on the variable colored colonies was identified [Figure 1c–f]. Out of 18 Candida-positive samples, 12 C. albicans (14.6%) [Figure 1c], 4 Candida tropicalis (4.8%) [Figure 1d], 1 Candida glabrata (1.2%) [Figure 1e], and 1 Candida krusei (1.2%) [Figure 1f] were found.

Table 1

Correlation of presence of Candida species with the presence of burning sensation in all the three groups

Study groups	Presence of Candida species	Presence of burning sensation
Group 1 - Diabetic	14	12
Group 2 - Prediabetic	4	2
Group 3 - Nondiabetic	Nil	Nil
P	<0.001 significant

Figure 1

(a and b) Creamy white colonies indicates C. Albicans in SDA agar Candida species color in HiCrome agar. (c) Light green colony color indicates Candida albicans. (d) Steel blue colonies indicate Candida tropicalis. (e) Large glossy white colonies indicate Candida glabrata. (f) Large fuzzy rose color colony indicates Candida krusei

DISCUSSION

Diabetes mellitus is a condition, characterized by increased blood glucose level, leading to the accumulation of glycosylation products on the surface of oral mucosa and colonization of Candida species.[15] In our study, 14 individuals reported burning sensation and all of them are diabetic and prediabetic individuals, which are in consistence with the previous study by Sun et al.[16] In our study, we derived the correlation between diabetes mellitus and the presence of burning sensation by clinical, as well as culturing methods and thereby determined the relationship between diabetes mellitus, burning sensation, and fungal elements in the oral mucosa. Hence, we suggest that the presence of burning sensation in the diabetics and prediabetics may be due to the development of candida in the oral cavity of the individuals. The SDA agar is a commonly used cultural method to identify the Candida species.[10] Baradkar et al.'s study portrayed that Chrome agar (HiCrome agar, HiMedia, Mumbai, Maharashtra, India) can distinguish all types of Candida in clinical specimens.[17] For the purpose of specific identification, we used SDA agar and chrome agar as culturing media for candida identification.[18] Sharma A et al. suggested that C. albicans was the most frequent isolated candidal species followed by C. glabrata in patients with diabetes mellitus.[19] In our study, we observed that C. albicans is the common isolate to colonize in patients with diabetes mellitus, and we observed that in the 18-positive candida species, about 12 of them were C. albicans, 4 were Candida tropicalis, 1 was Candida Krusei, and 1 was C. glabrata. In our study, we have observed that out of the 14 Candida-positive diabetic individuals, 12 of them had burning sensation, which was in correlation with a study by Indurkar et al.[20] and suggest that there is a strong correlation between burning sensation and candida colonization in patients with diabetic mellitus. In our study, the exclusion criteria played a major role in differentiating the present study from previous studies as well as in correlating burning sensation with the fungal elements.

Limitations

We focused only on the aim of this study, so we did not correlate the other clinical symptoms related to diabetes and candida infection. In addition, the duration of the diabetes mellitus and medications taken has not been recorded in our study that may or may not influence the development of candidiasis.

CONCLUSION

Increased candida colonization is evident in diabetic patients than prediabetics and normal control groups. Presence of fungal elements in the oral mucosa of diabetics may be one of the reasons for burning sensation and should be considered by clinicians to provide appropriate treatment. Furthermore, large-scale study is necessary to confirm the findings in our study.

Financial support and sponsorship

Nil.

Conflicts of interest

All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Divyadharshini. The first draft of the manuscript was written by C. Divyadharshini, Dr. R. Sathya, and Dr. N. Gururaj, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. All the authors agree to be accountable for all aspects of the work in ensuring that question related to accuracy or integrity of any part of the work are appropriately investigated and resolved.