Investigation of Candidal Species among People Who Suffer from Oral Potentially Malignant Disorders and Oral Squamous Cell Carcinoma.

AIM AND
OBJECTIVE: The aim of the present study was to evaluate the candidal species among masses with oral potentially malignant disorders and oral squamous cell carcinoma.
MATERIALS AND METHODS: This prospective and observational study was conducted by the Department of Oral Pathology and Microbiology, S. B. Patil Dental College, Bidar, Karnataka, India, from February 2018 to January 2019. The study composed of total of 150 individuals, of which 50 individuals did not had any visible manifestations, 50 were analyzed with potentially malignant disorders (PMDs) in particular oral leukoplakia, oral lichen planus, and oral submucous fibrosis and last group of 50 individuals were suffering from oral squamous cell carcinoma (OSCC). First, the swab samples were elicited from culture technique after that incisional biopsy of the discernible investigated lesion was done for the purpose of justopathological verification. The swab samples were streak on Sabouraud dextrose agar (SDA) and HiCrome Candida Differential HiVeg agar/CHROMagar medium and incubation at 37°C for 24-48 h. Biopsy was done for all the samples.
RESULTS: The proportion of candidates as men and women in control was 45 (90%) and 5 (10%), in PMD was 30 (60%) and 20 (40%), and in OSCC was 45 (90%) and 5 (10%), correspondingly. On evaluation on SDA medium in controls, PMD and OSCC groups, Candida was founded in 14 (28%), 20 (40%), and 42 (84%) and not founded in 36 (72%), 30 (60%), and 8 (16%) folks, subsequently. Intragroup contrast illustrated exceedingly necessary distinction with P = 0.000 between both controls versus OSCC and PMD in comparison to OSCC. Nevertheless, controls versus PMD manifested insignificant, P = 0.119. Investigation on CHROM AGAR media among controls, PMD and OSCC groups, Candida species was seen in 11 (22%), 19 (38%), and 40 (80%) and absent in 39 (78%), 31 (62%), and 10 (20%) individuals, respectively. On statistical inspection, the variations noted were enormous, (P = 0.000). On speciation of Candida in CHROM agar among the controls, PMD and OSCC groups, Candida albicans species was present in 9 (18%), 16 (32%), and 6 (12%), Candida krusei in 3 (6%), 6 (12%), and 13 (26%), Candida glabrata in 0, 0, and 8 (16%), and Candida tropicalis in 0, 0, and 3 (6%) cases, respectively. Nonetheless, only OSCC group reveal amalgamation of species such as C. glabrata and C. krusei was present in 2 (4%) case, C. tropicalis and C. krusei in was present 3 (6%) cases, C. tropicalis and C. glabrata was present in 2 (4%) case, C. albicans and C. tropicalis was present in 2 (4%) cases, and C. krusei, C. glabrata with C. albicans was present in 1 (2%) case, respectively. All other types of fungi were regarded as infectious excluding Candida, on analysis on SDA medium, infestation in the form of fungal molds was seen in 18 (36%) in controls, 12 (24%) in PMD and 8 (16%) in OSCC groups.
CONCLUSION: We interpreted that the chief carrier of candidal species in PMDs and OSCC, yet more light is to be thrown on the topic that Candida has particular establishment in PMDs or in malignancy. Copyright:

INTRODUCTION

Clinically, apparent oral potentially malignant conditions are the most common precursors of oral squamous cell carcinoma (OSCC) potentially malignant disorder.[12] According to some findings, Candida spp. isolated from leukoplakia lesions can contain the carcinogen N-nitrosobenzylmethylamine.[3] and due to its ability to nitrosylate N-benzyl methylamine, candida species have been linked to the etiopathogenesis of OSCC and oral potentially malignant disorders.[345] There is a connection between epithelial dysplasia and histologically determined fungal infection of the oral mucosa.[6] It is suggested that Candida albicans tubular hyphal structure is needed because it allows for the entrance of precursors from saliva and the release of the nitrosamine agent into keratinocytes, possibly causing OSCC.[789] The ability of specific C. albicans strains to promote neoplastic transformation and the development of carcinogenic nitrosamines from saliva has revealed Candida's crucial role in malignant transformation.[1011] As compared to noncandidal leukoplakia, candidal leukoplakia is distinguished histologically by persistent (occasionally acute) intraepithelial inflammation with fungal hyphae entering the superficial layers of epithelium and has a significantly higher rate of malignant transformation.[1213]

MATERIALS AND METHODS

This prospective and observational study was done in the Unit Department of Oral Pathology and Microbiology, S. B. Patil Dental College, Bidar, Karnataka, India, from February 2018 to January 2019 after taking the approval of the protocol review committee and institutional ethics committee.

Methodology

Patients with PMDs and OSCC were also included in the report. Patients on oral or systemic corticosteroid medication, long-term broad-spectrum antibiotic therapy, a history of immune compromised illness, and those who had not received chemotherapy, radiotherapy, or antifungal medication were all excluded from the study. In all, 150 participants were studied, 50 of whom were healthy controls with no visible lesions, 50 of whom had PMDs such as oral leukoplakia, oral submucous fibrosis (OSMF), and oral lichen planus, and 50 of whom were OSCC patients. On a custom case history type, all of the study participants' socioeconomic statistics were registered. Using medical instruments, a thorough oral examination of all applicants is conducted. To begin, swab samples were obtained using the culture technique, and then, an incisional biopsy of the discernible examined lesion was performed for just pathological confirmation. Patients were often instructed to rinse their mouths with purified water to remove food particles, followed by salt water for microbiological culture. To collect samples from the buccal mucosa in disease-free controls and the lesion region in PMD and OS cases, sterile cotton swabs with a wooden stick placed on a blue capped high-density polyethylene tube sized 150 mm 12 mm were used. The swab samples were put back in the same polyethylene tube and held at 37°C until they were streaked on Sabouraud dextrose agar (SDA) medium and incubated for 24–48 h at 37°C. A sterile instrument, such as a cotton swab or an inoculation thread, was used to finish the streaking. To keep microbiological colonies alive and avoid contamination of the growth medium, aseptic techniques were used.[14] After that, the creamy/white pasty smooth colonies were inoculated again on HiCrome Candida Differential HiVeg agar/CHROMagar and incubated at 37°C for 24–48 h. According to HiMedia Laboratories' orders, microbial-colored colonies representing different species of Candida organisms were interpreted for color and unique colony characteristics.[15] After biopsy, the tissue sample was fixed in neutral-buffered formalin, then processed and embedded in paraffin wax as normal. The 4–5 mm thick sections were primed and stained with hematoxylin and eosin for histopathological confirmation using a binocular analysis microscope (Motic BA400) with a 5 MP camera for photomicrography (Moticam 2500, USB2.0).

Statistical methods

The whole set of statistics was then moved to a Microsoft Excel sheet. The Chi-square test was performed using the Statistical Package for the Social Sciences (SPSS edition 19.0, Chicago, IL, USA). P ≤ 0.05 was considered statistically significant and was often used in statistical studies.

RESULTS

There was age range for assortment of the participants in controls, PMD and OSCC of 15–58, 20–60 and 25–70 years, subsequently, and the mean age ± standard deviation was 30.92 ± 13.12, 43.1 ± 5.26 and 50.12 ± 12.96 years, correspondingly. The dispersal of the candidates between men and women in controls was 45 (90%) and 5 (10%), in PMD was 30 (60%) and 20 (40%) and in OSCC was 45 (90%) and 5 (10%), correspondingly. On evaluation on SDA medium in controls, PMD and OSCC groups, Candida was found in 14 (28%), 20 (40%) and 42 (84%) and not seen in 36 (72%), 30 (60%) and 8 (16%) people, respectively. On statistical investigations of intergroup comparison, huge difference (P = 0.000) was noted [Table 1]. As well as in intragroup comparison, there was colossal distinction with (P = 0.000) among both controls versus OSCC and PMD versus OSCC. However, controls versus PMD showed a nonsignificant, (P = 0.119) [Table 2]. On evaluation on CHROM agar media among controls, PMD and OSCC groups, Candida species was present in 11 (22%), 19 (38%), and 40 (80%) and absent in 39 (78%), 31 (62%) and 10 (20%) individuals distributively. On statistical analysis, an extremely significant difference was noted (P = 0.000) [Table 3]. On speciation of Candida in CHROM agar among the controls, PMD and OSCC groups, C. albicans species was present in 9 (18%), 16 (32%), and 6 (12%), Candida krusei in 3 (6%), 6 (12%), and 13 (26%), Candida glabrata in 0, 0, and 8 (16%) and Candida tropicalis in 0, 0, and 3 (6%) cases, respectively. Nonetheless, OSCC group solely showed union of species such as C. glabrata and C. krusei was present in 2 (4%) case, C. tropicalis and C. krusei in was present 3 (6%) cases, C. tropicalis and C. glabrata was present in 2 (4%) case, C. albicans and C. tropicalis was present in 2 (4%) cases, and C. krusei, C. glabrata with C. albicans was present in 1 (2%) case, respectively [Table 4]. All the types of the fungi excluding Candida were regarded as infection. On evaluation on SDA medium, contamination in the form of fungal molds was present in 18 (36%) in control, 12 (24%) in PMD, and 8 (16%) in OSCC groups [Table 5].

Table 1

Estimation of Candida in Sabouraud dextrose agar media among the study batches

Study groups	Candida in Sabouraud dextrose agar		P
	Present (%)	Absent (%)
Control	14 (28)	36 (72)	0.000
PMD	20 (40)	30 (60)
OSCC	42 (84)	8 (16)

OSCC: Oral squamous cell carcinoma, PMD: Potentially malignant disorders

Table 2

Intergroup contrast of Candida in Sabouraud dextrose agar media

Study groups	P
Control versus PMD	0.119 (NS)
Control versus OSCC	0.000 (HS)
PMD versus OSCC	0.000 (HS)

NS: Nonsignificant, HS: Highly significant, OSCC: Oral squamous cell carcinoma, PMD: Potentially malignant disorders

Table 3

Assessment of Candida in chrome agar media in the study group

Study group	Candida in chrome agar		P
	Present (%)	Absent (%)
Control	11 (22)	39 (78)	0.000 (HS)
PMD	19 (38)	31 (62)
OSCC	40 (80)	10 (20)

HS: Highly significant, OSCC: Oral squamous cell carcinoma, PMD: Potentially malignant disorders

Table 4

Recognition of different candidal species in chrom agar in the study groups

Candida species	Control (%)	PMD (%)	OSCC (%)
C. albicans	9 (18)	16 (32)	6 (12)
C. krusei	3 (6)	6 (12)	13 (26)
C. glabratta	Nil	Nil	8 (16)
C. tropicalis	Nil	Nil	3 (6)
C. krusei and C.tropicalis	Nil	Nil	3 (6)
C. krusei and C.glabratta	Nil	Nil	2 (4)
C.glabratta and C.tropicalis	Nil	Nil	2 (4)
C.albicans and C.tropicalis	Nil	Nil	2 (4)
C.albicans, C.krusei, and C.glabratta	Nil	Nil	1 (2)
Total	12 (24)	22 (44)	40 (80)

C. albicans: Candida albicans, C.krusei: Candida krusei, C. glabratta: Candida glabratta, C. tropicalis: Candida tropicalis, OSCC: Oral squamous cell carcinoma, PMD: Potentially malignant disorder

Table 5

Infestation in control, potentially malignant disorders, and oral squamous cell carcinoma groups

Contamination	Control (%)	PMD (%)	OSCC (%)
Molds	18 (36)	12 (24)	8 (16)

OSCC: Oral squamous cell carcinoma, PMD: Potentially malignant disorder

DISCUSSION

In oral Candida-based experiments, one of the key investment strategies is to approximate the occurrence of Candida and the species specificity for certain societal classes.[161718] OSCC has been shown to grown from PMDs such as leukoplakia, OSMF, and lichen planus in many ways.[1920] Any researchers believe that candidal inflammation increases the risk of premalignant lesions transforming into cancer. 22 Yeasts are usually found commensal organisms in masses, accounting for around 40% of the total, with C. albicans being the most common species isolated and its contribution to development of OSCC. C. albicans, C. tropicalis, and C. glabrata are the most commonly isolated Candida species, while Candida parapsilosis, Candida stellatoidea, Candida guilliermondii, Candida krusei, and Candida pseudotropicalis are the least often isolated Candida species from medical specimens.[2122232425]

The samples in this analysis were taken using a sterile swab. In contrast to Saigal et al.,[26] who used saliva instead of swab for their culture technique. The swab technique was simple to use and resulted in the isolation of viable cells from a specific location, while the saliva technique did not.[27] For speciation, the collected swab sample was first inoculated on SDA medium, then on CHROM agar (a differential medium). Nadeem et al.[28] and Odds and Bernaerts.[29] used the same mixture of culture media, while Saigal et al.[26] used corn meal agar as the main culture media, preceded by the germ tube examination, which only detects C. albicans and Candida dubliniensis and is unable to distinguish other Candida species.[26] Candida was found in 14 (28%) of the samples, 20 (40%) of the PMD patients, and 42 (84%) of the OSCC patients, respectively, and was absent in 36 (72%) of the controls, 30 (60%) of the PMD patients, and 8 (16%) of the OSCC patients. An extraordinarily large gap (P = 0.000) was found in the statistical study of intergroup comparison. Similarly, different researchers, such as Hongal et al.,[20] have shown that the findings of Vuckovic et al.,[31] Saigal et al.,[26] Anila et al.,[30] and Saigal et al.[26] in the present study, the most prevalent Candida in CHROM agar among the controls, PMD and OSCC groups, C. albicans species was present in 9 (18%), 16 (32%) and 6 (12%), C. krusei in 3 (6%), 6 (12%) and 13 (26%), C. glabrata in 0, 0, and 8 (16%) and Candida tropical is in 0, 0 and 3 (6%) cases, respectively. However, only OSCC group showed a combination of species such as C. glabrata and C. krusei was present in 2 (4%) case, C. tropicalis and C. krusei in was present 3 (6%) cases, C. tropicalis and C. glabrata was present in 2 (4%) case, C. albicans and C. tropicalis was present in 2 (4%) cases and C. krusei, C. glabrata with C. albicans was present in 1 (2%) case, respectively. This was in consistent to the study by Gall et al.,[15] He was the one who demonstrated that was shift in the dispersal of Candida species with PMD going through malignant transformation from C. albicans to nonalbicans species such as C. glabrata, C. tropicalis, C. krusei and C. parapsilosis.[30]

CONCLUSION

We summarized that the prime carriage of Candidal species in PMDs and OSCC, yet conclusion and more evidences are required to prove whether Candida has specific establishment in PMDs or in malignancy.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.