European Journal of Cardiovascular Medicine

Oro‑pharyngeal cancer is a significant component in the global burden of cancer. Globally More than 2 lakh cases of head and neck cancers are diagnosed each year.India contributes up to 7.8% of the global cancer burden and 8.33% of global cancer deaths. A considerable proportion of oral squamous carcinomas develop from precancerous lesions and conditions of the oral cavity. [1] WHO collaborating centre for oral precancerous lesions and conditions in 2007 recommend the term “potentially malignant disorders,”(PMD) as it conveys that not all lesions and conditions described under this term may transform to cancer . Among them frequently associated lesions with Candida is leukoplakia , erythroplakia, OSMF, and lichen planus [2]. Etiology of PMD is multifactorial with Tobacco and alcohol being the major risk factors .[3]

Candida albicans (C.albicans) is frequently found in histologic sections of Potentially malignant disorders . Certain strains have been shown to produce nitrosamines, chemicals that have been implicated in carcinogenesis. Some candidal strains may have the potential to promote the development of oral cancer [4]. Candidal leukoplakia is regarded as a potentially malignant lesion and infection may be a pivotal factor in the etiology of oral epithelial dysplasia. The most commonly encountered commensal and pathological fungus of the oral cavity is C. albicans which may exist as yeast or in a hyphal form [5].

Techniques available for the isolation of Candida within the oral cavity include the use of a smear, a plain swab, an imprint culture, collection of whole saliva, the concentrated oral rinse, and mucosal biopsy [6]. Fungal morphology is better demonstrated by the PAS stain than the silver stains [7].

Compared with the PAS stain   culture method has a markedly increased upper limit of detection in quantifying yeast carriage . Sabouraud's dextrose agar medium is the suitable medium for the identification of fungus specially candida species [8].

The present study  was conducted to evaluate  the association of candida species in oral potentially malignant disorders by  microbiological , cytological and  histological  methods.

Aims and Objectives:

 The aims of the study were to investigate the presence of Candidal organisms in potentially Malignant disorders, by using two different techniques that is by studying PAS stained cytosmear and salivary sampling. The  second aim of the study was investigate the degree of dysplasia in histopathological sections of potentially maligant disorders.

The objective of the study was to correlate the presence of Candidal organisms if any, with the degree of dysplasia of oral potentially malignant disorders.

A total of 100 patients of either sex with clinically suspected potentially malignant oral mucosal lesions will be selected from the outpatient clinic of the Department of Oral Medicine and Radiology.An informed consent was obtained from the subjects to participate in the study and to undergo clinical investigation and biopsy procedure.

Co-operative patients with clinically suspected potentially malignant lesions affecting oral cavity are included in the study. Previously treated cases of oral PMDs , Patients having systemic disorders and contributing local factors influencing the Candida such as xerostomia, under medications, taking high carbohydrate diet, patients wearing dentures  were  excluded from the study group.

Collection of sample done by The oral rinse technique described by Samaranayake¹ et al was used to collect samples. All Subjects  were asked to rinse their mouth with 10 ml of phosphate buffered saline (PBS) for 2 minutes and expectorate into a sterile container and  transported to microbiology laboratory for culture then cytosmears were taken, under normal conditions with the wooden spatula and clean glass slide  allowed to dry  and transported to pathology laboratory for PAS staining  . Finally biopsy specimen was sent for histopathological examination to Department of Oral Pathology,. The slides were stained with Haemotoxylin and Eosin Stain to evaluate the presence of dysplastic changes.the clinical presentation diagnosis of the lesion and the result of laboratory investigations and the histopathological features were noted in the proforma.

The results obtained was statistically analyzed by chi square test and Student unparity test. Statistical analysis was carried out with the help of SPSS software 17.0. . p-value<0.05 was considered statistically significant.

Out of the 100 patients , there were 53 males and 47 females with mean ages of 41.9 and 49.1 years of age respectively participated in the study with commonly involved lesion was OSMF.

( Table -1)

Table 1 AGE AND LESION DISTRIBUTION

On Sabourad’s dextrose agar Culture medium :   Out of 100 individuals 72 cases showed round colonies in SDA medium(Fig-1) with the percentage of 71.6%, 28 cases were negative with the percentage of 28%. On PAS staining Out of 100 individuals  82 cases showed presence of candidal pseudo hyphae   in cytosmear ( Fig -2)with the percentage of 82%, 18 cases were negative with the percentage of 18%.

Fig 1- Photograph showing Candida colonies in Sabouraud’s Dextrose Agar

Fig 2- Photograph showing PAS Stained Candidal Hyphae Embedded in the Oral Mucosa

On Histopathological examination Out of 100 individuals 72 cases shows presence of dysplasia (Fig-3)with percentage of 71.5% and 28 cases were negative with the percentage of 27.6%. (Table 2)

Figure 3 -Histopathologic picture of Leukoplakia showing epithelial dysplaia (40x)

TABLE 2: Presence and association of Candida in Culture , PAS and Dysplasia

Association of culture with dysplasia Out of 100 patients Both culture and dysplasia positive in 60 cases and negative in 16 cases. So there is a correlation of candida and epithelial dysplasia in 76 cases . statistical analysis shows based on Chi square test , it was significant as P value 0.000. (Statistically significant)  (Table 3) .

TABLE 3: Culture Vs Dysplasia

Statistical Analysis: Chi-square test. Statistically significant if P<0.05

Association of PAS with dysplasia  Out of 100 patients Both PAS and dysplasia positive in 60 cases and negative in 6 cases. So there is a correlation of candida and epithelial dysplasia in only 66 cases . statistical analysis shows based on Chi square test , it was Non significant as P value 0.578 (Statistically Non significant)  (Table 4) .

TABLE 4: PAS Vs Dysplasia

Statistical Analysis: Chi-square test  Statistically significant if P<0.05

Association of PAS and  Culture  Both Culture and PAS  positive in 72 cases and negative in 18 cases. So there is a correlation of candida and PAS  in 90 cases . statistical analysis shows based on Chi square test , it was significant as P value 0.000. (Statistically significant) (Table 5) .

TABLE 5: PAS Vs Culture

Statistical Analysis: Chi-square test Statistically significant if P<0.05

Association of candida in different grades of dysplasia of Oral PMDs: (Association with Culture) Among 70  positive cases of  dysplasia candida association seen in  60 cases,   Out of which, 23 cases of mild dysplasia , 15 cases of moderate dysplasia, 13 cases of mild to moderate dysplasia, 7 cases of moderate-severe dysplasia and 2 cases of severe dysplasia were all associated with candida, shows statistically significant. (Table 6)

Table 6 Association of Candida in different grades of dysplasia of oral PMDS ( In Culture)

Statistical Analysis: Chi-square test  Statistically significant if P<0.05

The probable role of yeast in oral carcinogenesis remains the subject of considerable debate [9]. Studies in this field are fraught with difficulty as Candida organisms are commensals in the oral cavity; thus, establishing their role in carcinogenesis is challenging.

In vitro and animal studies have suggested that Candida organisms catalyze the production of nitrosamine, which is considered to be carcinogenic.

Previous studies by various investigators have demonstrated a significant correlation between epithelial dysplasia and fungal invasion, but the mechanisms responsible remain unclear [10].

Presence of Candida in the mouth together with epithelial changes may predispose to candidal infection. Candidal infection together with other co-factors may also induce epithelial atypia and dysplasia leading to malignant change  [11]

The present study evaluated the association of C. albicans with patients who are having potentially malignant disorders  for which we used oral rinse method for saliva culture  , cytosmear for PAS staining and took biopsy from the suspected lesion. However,  other authors suggested different sampling and identification methods for Candida spp. That would also certainly influence the results, because of the uneven distribution of C. albicans throughout the oral cavity; swab samples can yield false-negative culture more often than oral rinse samples or imprint culture [12,13] , Compared with the imprint method the rinse culture method has a markedly increased upper limit of detection in quantifying yeast carriage which is particularly useful when dealing with highly infested individuals [7].

The results of the present study confirm previous evidence on the association between oral epithelial dysplasia and the presence of yeast species in the oral cavity. Other studies also supports that there may be an association of candida and its role in malignant transformation of PMDs [14,15,16,17,18,19] .It may well be that certain strains of  C. albicans  possess properties  that are important in the development of pathological conditions and malignant changes.

Till now so many studies has undertaken to know the candidal presence and association with epithelial dysplasia by using only one technique either cytosmear (PAS) or culture. In the present  study to evaluate the correlation between presence of Candida and epithelial dysplasia in oral PMDs by using 2 different techniques simultaneously  ( Using PAS staining and Salivary culture) .There is abundant evidence that values of fungal infection as assessed by PAS staining are lower than those obtained by culture  [20] . So in the present study to reduce the disharmony in the study results as we have done culture.By doing these 2 techniques we trying to increase the sensitivity and specificity of the study finally we can eliminate the false positive results.

Microbiological analysis is a reliable method to assess the presence of Candida species. Salivary sampling techniques certainly influence the results, because of uneven distribution of Candida species throughout the oral cavity. Samaranayake et al (1986) compared oral rinse technique to collect samples and had a markedly increased upper limit of detection in quantifying yeast carriage which was particularly useful when dealing with highly infested individuals. Swab samples can yield false negative culture more often than oral rinse samples or imprint culture [21]. In our study 71.6% of cases showed positive results i. e out of 100 cases 71.6% cases showed presence of candida  as compared to the study done by  Horstein et al(1979) [22]. In the study conducted by Preethi Sharma and Sushmita Saxena(2011) the results showed 91.1% statistically significant association between C.albicans and precancer [23], 

the results of Saigal et al(2011) , showed 60% which is statistically significant association between C.albicans and precancer [21].In the present study under  PAS staining candida was diagnosed in 82% of cases out of 100 cytosmears. The study done by Anjali guptha et al 2014 shows the non significant result  [20] and in the study done by Thimmarasa et al 2015 shows the accuracy of PAS stain in diagnosing various mucosal lesions infected by fungal infection was 12% [24].

Summarizing the results of the present study, dysplasia was present in 83.3% cases of culture positive cases and also dysplasia present in 73.17% of PAS positive cases, which means there was a correlation exists between presence of candida and epithelial dysplasia based on culture technique. Epithelial Dysplasia is more associated with culture than PAS , that  means culture was more specific test to identify candida.In this study we compared two different techniques for the rapid production of candida spieces . we found the significant result .  out of 100 cases both culture and PAS are positive in 72 cases and negative in 18 cases , so there is correlation of culture and PAS in 90 cases shows statistically significant.

This is the first of its kind of study to know the association of candida and epithelial dysplasia by using 2 techniques and we found the statistically significant result.Based on the result we proved that culture is more specific test to ruled out the candidal association with dysplasia.

Although present study does not clarify whether pathogenic fungi cause epithelial dysplasia or the altered tissue is infected by the fungi, it is confirmed that there is increased frequency of fungus in patients who are having  potentially malignant disorders and the association between presence of fungus and epithelial  dysplasia showed statistically significant.

The present study highlights the potential role of Candida in the malignant transformation of oral potentially malignant disorders (PMDs). A significant association was observed between candidal presence and epithelial dysplasia, with higher isolation rates in patients with PMDs. Salivary culture was identified as a specific test for candidal detection, while the PAS technique demonstrated greater sensitivity. Interestingly, Candida was more frequently observed in PMDs with mild dysplasia than in those with moderate or no dysplasia, suggesting its possible role in the early stages of transformation. These findings emphasize the need for further research and support the potential importance of antifungal therapy and meticulous oral hygiene in management strategies.

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