It was a cross-sectional, case control study conducted in a period of two years between 2013 to 2015, in which the study group consisted of 50 Indian patients suffering from oral cancer and undergoing RT and/or CT at Gujarat Cancer Research Institute, Ahmadabad. Permission from ethical committee of the institution was taken and patients consent was obtained prior to investigation. Patients with histological diagnosis of carcinoma of oral cavity e.g., carcinoma of lip, buccal mucosa, retro-molar triangle, lateral surface of tongue, base of tongue, maxillary alveolus and mandibular alveolus, undergoing RT and/or CT, were included in the study. The patients with carcinoma of vocal cord and oesophagus and who were undergoing antifungal medications were excluded from the study. The control group comprised of 50 healthy individuals of similar age group, not suffering from any major disease and were not undergoing any medication for any chronic illness. The demography, clinical and treatment characteristics of the study group were described in [Table/Fig-1]. After taking detailed history, intra-oral examination was done to note the presence and nature of oral Candidiasis. Only lesions that were clearly visible were noted.

In order to investigate possible presence of Candida and to differentiate between the carrier state and infective stage of the organism, imprint culture technique as described by Arendorf and Walker [9] was applied in all patients. In brief, a sterile square plastic foam pad dipped in sterile saline was held on the middle third of dorsal surface of the patient’s protruded tongue for 10 seconds. The pad was then pressed firmly on to a Sabouraud’s dextrose agar plate into which gentamycin 20μg/ml and chloremphenicol 50μg/ml had been incorporated to suppress bacterial growth. Cyclohexamide 0.5 mg/ml is also added to prevent growth of saprophytic fungi. The pad was left in situ for 60 minutes and was then removed. The plate was incubated aerobically at 37°C for 2 days. The candidal density was then determined by enumerating the colonies. The patient is considered as carrier if dentate patient have <30 colonies/cm², if denture wearer have <49 colonies/cm². The patient is considered as infective if dentate patient have > 30 colonies /cm², if denture wearer has >49 colonies / cm². Confluent growth obtained from a mucosal surface was highly suggestive of Candida infection [9].

The colony characteristics were recorded and compared with known colony characters of various candidal species. If no colonies were seen on Sabouraud’s agar after 48hrs incubation, culture plate was kept for seven days in incubator before being considered as negative.

The morphology of the organism was studied by preparing Wet mount. The yeast appeared as gram positive, round to oval bodies. The species of Candida was identified by sugar fermentation tests. Further identification of C. albicans species was done by germ tube test [Table/Fig-2] and chlamydospores formation [Table/Fig-3].

[Table/Fig-2]:

Photomicrograph showing tube like, out growth from yeast cell - germ tube, C. albicans (45 X).

[Table/Fig-3]:

Photomicrograph showing thick walled chlamydospores at the end of pseudo-hyphae on cornmeal agar plate. C. albicans (45 X).

Out of 50 healthy controls, 10 (20%) had shown positive Candida culture while out of 50 oral cancer patients undergoing RT and/ CT, 35 (70%) patients had shown positive Candida culture (p = 0.001, < 0.05) [Table/Fig-4]. Candida was cultured in 50% of the patients taking RT, 75% of the patients taking CT and 81.25% of the patients taking RT and CT both [Table/Fig-5].

All positive healthy controls had shown carrier growth pattern of Candida while out of 35 Candida positive oral cancer patients, 10 (28.57%) were in carrier and 25 (71.42%) were in infective state (p = 0.001, < 0.05) [Table/Fig-4]. The infective state of Candida was seen in 71.42% in patients undergoing RT, 80% in patients undergoing CT and 61.53% in patients on RT and CT both [Table/Fig-5].

In healthy controls, out of 10 positive controls, 7 (70%) were identified as C. albicans and 3 (30%) were C. Stellatoidea [Table/Fig-4]. In 35 Candida positive oral cancer patients undergoing RT &/CT, the most common candidal species identified were C. tropicalis, in 15 (42.85%) patients. followed by C. stellatoidea in 10(28.57%), C. albicans in 5 (14.28%), and C. Krusei in 5 (14.28%) patients [Table/Fig-5].

The relationship between prevalence and growth pattern of Candida and treatment modality of oral cancer patients: The oral cancer patients undergoing RT, showed 50% Candida positive culture with 71.42% were in infective state and 71.42% were non albicans species. The oral cancer patients undergoing CT, showed 75% Candida positive culture with 80% were in infective state and 80% were non albicans species. The oral cancer patients undergoing both RT and CT showed 81.25% Candida positive culture with 61.53% were in infective state and 100% were non albicans species [Table/Fig-5].

Oral candidiasis is one of the most common opportunistic infection of the oral cavity. It is caused by a yeast-like fungus, Candida, which is present in oral cavity as commensal and becomes pathogenic when the host defense is compromised. Oral candidiasis manifested as acute or chronic disease and either superficial or disseminated systemic mycosis [10]. There are many systemic conditions which leads to immune-compromised state e.g., diabetes mellitus, HIV, patients undergoing solid organ and bone marrow transplants and patients undergoing chemotherapy and radiotherapy targeted against carcinomas and sarcomas [4]. Oral candidiasis is common in patients with head and neck cancers, especially during chemotherapy and radiotherapy. It usually manifest as acute erythematous candidiasis, but the diagnosis may often be missed, as it may be mistaken for radiation mucositis [11].

In the present research, a total of 50 cases of oral cancer undergoing RT and CT, were screened and compared with a control group of 50, healthy controls of same age group, for oral Candidal isolation and species identification.

We observed significant increase in oral carriage of Candida in oral cancer patients taking RT &/ CT, it was 50% in patients taking RT, 75% in patients taking CT and 81.25% in patients taking RT v CT both, in comparison to 20% Candida prevalence in healthy controls. This is in accordance with findings of previous studies. Silverman et al., and Chen and Webster found 49% and 63% Candida carriage in patients taking RT in comparison to 22% and 29.1% in healthy controls respectively [12,13]. Samaranayake et al., and Main et al., found 70% and 75% Candida carriage in patients taking CT respectively [8,14]. The candidal carriage rate increased from 70% to 80% during the 3 months period of cytotoxic therapy [8]. Main et al., found that the saliva volume & salivary amylase activity decreased and candidal score increased after the cytotoxic therapy. The carriage of coliforms was also increased [14].

The significantly increased in Candida carriage may be due to compromised defence system of host because of underlaying disease and use of cytotoxic drugs. Moreover, RT causes mucositis and changes in salivary glands which lead to quantitative and qualitative changes in saliva [12]. Xerostomia and thick saliva makes the oral environment conducive for fungal colonization.

Apart from isolation of Candida, the growth pattern of Candida is also changed from carrier to infective state, in oral cancer patients undergoing RT and/or CT. In our observation all healthy controls (100%) showed carrier growth pattern while 71.42% oral cancer patients showed infective growth pattern. It shows significant increase in the infective state of Candida in oral cancer patients undergoing RT and/ CT (p = 0.001, < 0.05). According to Ueta et al., the virulence of Candida strain in chemotherapy and irradiated individuals is largely determined by activities of adherence, multiplication and release of a cytoplasmic antigen, enolase & Candida aspartic proteinase [15].

By far the most pathogenic as well as most common species of Candida occurring as a commensal in healthy man are C. albicans followed by C. glabrata, C. Krusei, C. tropicalis & C. parapsilosis [16]. In accordance we found C. albicans in 70% positive healthy controls. The pattern is different in the oral cancer patients undergoing RT and/or CT, non-albicans species of Candida are predominantly isolated out of which the most common candidal species identified was C. tropicalis, (42.85%). Our results were in congruence with that of Belazi et al., Redding et al., and Dahiya et al., who reported non-albicans species of Candida in 12% to 30% of irradiated patients [17–19]. Jham et al., also reported similar findings, 33% increase in non-albicans species colonization, which increased to 71.4% at the end of study [20]. Hence we could suggest that the prevalence of non albicans species of Candida were increased in oral cancer patient undergoing RT &/ CT. Candida species distribution, especially non-albicans, varies worldwide. Studies reveal that in the United State/Canada region, the most common non-albicans species is C. glabrata while in South America C. tropicalis and C. parapsilosis are most common [20]. In India, we found predominance of C. tropicalis in our study which should be further evaluated on a larger sample size.

In conclusion, we found significant increase in candidal colonization in oral cancer patients of Indian origin, undergoing RT and/or CT, with change in the growth pattern of candida, from carrier to infective state. There is a shift toward non albicans species of Candida, chiefly C. tropicalis which are more bizarre in presentation and resistant to conventional treatment. As the pattern of candidal species infection is changing, such studies are important for better diagnosis and treatment planning to gain good control over the disease. Since, a small sample size has been the chief limitation of this study, we suggest further studies on larger study groups should be conducted to confirming the above findings. It will contribute better towards the prevention and control of oral cancer related candidiasis.