Candida Albicans and Its Correlation with Oral Epithelial Neoplasia

Int J Oral-Med Sci 10(3):140-148,2011 Original Article

Candida albicans and its Correlation with Oral Epithelial Neoplasia

Preeti Sharma,and Susmita Saxena

Department of Oral Pathology & Microbiology,Subharti Dental College,MEERUT (UP),250005,INDIA

Correspondence to: Preeti Sharma Objective: The present study aimed to compare and quantify the

E-mail: neepreeti 121＠yahoo.com presence of Candida albicans in precancerous and cancerous oral

mucosal lesions. Materials& Methods: A total of 135 cases were studied［Group I,45

cases of precancerous lesions; Group II,45 cases of malignant lesions; and Group III, 45 cases of precancerous conditions such as lichen

planus and oral submucous fibrosis (OSMF)］. Three cytological

smears were made from lesional tissue.One wet smear was made with

10% potassium hydroxide (KOH) solution and the two other smears

were stained with Gram’s stain and periodic acid Schiff(PAS) stain. Colonies were cultured on Sabouraoud’s dextrose agar(SDA)medium

followed by quantification on a colony counter.Germ tube test con-

firmed the species as C.albicans. Results: Using a two-way Anova test, a statistically significant

association was found between C.albicans and precancer(p＜0.05,p＜ 0.01).Similarly,the frequency of oral yeast carriage was significantly

greater (p＜0.05, p＜0.01) in the malignant lesions group than the

precancerous lesions as well as precancerous conditions.However,no

significant difference was observed between the two groups of precancerous lesions and precancerous conditions. Keywords: Conclusion : The present results suggested a close correlation of

Candida albicans, precancerous Candida infections to leukoplakias and squamous cell carcinoma lesions, precancerous conditions, (SCC),supporting an association between Candida species and oral malignant lesions neoplasia.

albicans. The dimorphic yeast Candida albicans has

Candida albicans, a diploid asexual fungus,is an been recognized as an increasingly important human opportunistic pathogen that commonly colonizes pathogen particularly in immunocompromised hosts. human mucosal epithelial cell surfaces.Colonization The other Candida species encountered in human may lead either to a sustained or transient sapro- infections are C.tropicalis, C.glabrata, C.parap- phytic association with the host or to localized infec- silosis,C.stellatoidea,C.krusei and C.kefyr (2). tions of mucosal surfaces. Infections are usually In recent years,many researchers have focused on confined to the mucosal epithelium that lines the studies supporting an association between Candida vaginal and oral cavities. C.albicans colonizes or species and oral neoplasia. Interest in the study of infects mucosal surfaces more often than any other oral candidiasis has markedly increased not only

Candida species because of its adherence capability because of its association with human im- (1). munodeficiency virus(HIV)infection,but also due to

The genus Candida includes characteristically its relation with precancerous lesions of the oral white asporogenous (imperfect) yeasts capable of mucosa.These lesions belong to the wide group of forming pseudohyphae. There are seven Candida leukoplakia (3). species of major medical importance, the most Candida leukoplakia, if untreated, has a signifi- important and most frequently isolated being C. cant rate of malignant transformation when compar- Int J Oral-Med Sci 10(3):140-148,2011 ed with non-candida leukoplakia.This high rate of malignant change in candida leukoplakia is strong The main objective of the present study was to evidence for the fungi being involved in neoplastic determine whether there is any significant associa- change. All forms of leukoplakia can be infected tion between C.albicans infection and precancerous with Candida species, but the non-homogeneous lesions/conditions and malignant lesions, and to types,particularly the speckled leukoplakias,have a evaluate the role of C.albicans in oral neoplasia.The predisposition for hyphal invasion and neoplastic study also aimed to quantify candidal carriage and change (3).In one immunohistochemical study Gao determine its correlation with the progression of

(4)elegantly demonstrated p53 expression in all 17 disease. candidal leukoplakia cases (100%) compared with

78% of the controls.They found that the number of p53-positive cells was significantly higher in lesions The present study was carried out to detect the exhibiting epithelial dysplasia than in those without presence of C.albicans in precancerous and cancer- dysplasia and concluded that p53 expression may be ous oral mucosal lesions under a binocular light considered as a biological marker for candidal leuko- microscope (Olympus CX-31),in the Department of plakia. Oral Pathology and Microbiology. The essential debate is whether the Candida species are secondary opportunistic pathogens acting on a pre-existing leukoplakia or, conversely, The study sample comprised 135 patients with whether the higher premalignant rate of change in precancerous and malignant oral mucosal lesions candida leukoplakias supports a role for this fungus diagnosed on the basis of clinical and histological in the development of neoplasia.Therefore,a study features. A working group of the World Health was planned to detect the presence of C.albicans in Organization proposed in 1978 that clinical presenta- precancerous lesions and conditions to explore and tions of the oral cavity that are recognized as precan- evaluate their association with oral neoplasia. cerous be classified into two broad groups: lesions

Table 1. Distribution of study sample groups with histopathological diagnosis

Study S. Clinical No.of Sample Histopathological Diagnosis No. Diagnosis Cases Group Hyperkeratosis with no dysplasia 06

Precancerous Hyperkeratosis with mild dysplasia 16 Leukoplakia 1. Hyperkeratosis with moderate dysplasia 12 lesions (45 cases) Hyperkeratosis with severe dysplasia 07 Erythroplakia Atrophic epithelium with moderate to severe dysplasia 04 Malignant Well-differentiated 21

2. lesions Oral SCC Moderately-differentiated 16

(45 cases) Poorly-differentiated 08 No dysplasia 19 Oral Lichen Mild dysplasia 04 Precancerous Planus Moderate to severe dysplasia 02 3. conditions No dysplasia 13 (45 cases) OSMF Mild dysplasia 05

Moderate to severe dysplasia 02 Int J Oral-Med Sci 10(3):140-148,2011

Table 2. Candidal detection in study sample groups by various methods

HISTO- Colony CYTOLOGY PATHOLOGY Count on Germ

Study Sample SDA tube Total Groups 10% medium test Gram PAS PAS positive KOH / ＞ positive positive (cfu ml positive positive 1000) Precancerous 45 23.3％ 73.3％ 71.1％ 71.1％ 84.4％ 55.5％

lesions Malignant 45 22.2％ 91.1％ 95.5％ 95.5％ 91.1％ 75.5％

lesions Pre-cancerous 45 11.1％ 40％ 42.2％ 42.2％ 44.4％ 31.1％

conditions Consider including the percentages of positive candidal detection for Table 2(example: PAS Positive for Group

I is 24%)given that percentages were discussed in the Results section. and conditions (5).The present study included precancerous lesions of the oral mucosa,mainly leukoplakia and erythroplakia cases, and precancerous conditions consisting of oral lichen planus and oral submucous fibrosis (OSMF) cases, while malignant lesions included oral squamous cell carcinoma patients only.Thorough clinical examinations were conducted for each patient. All relevant clinical details were entered in the case history proforma. Patients were informed about the procedure and prior consent was received(Institutional Ethics Com- mittee, Swami Vivekanand Subharti University, Meerut, India). The distribution of various study Fig.1. Stratified squamous epithelium showing invasion by groups are tabulated in Tables 1 and 2. Candida albicans in PAS-stained section.(×40). Section staining-For each biopsy,one histological section was stained using the hematoxylin and eosin (H&E)method (Medical Chemical Corp.,Torrance, Cytological smears- Prior to biopsy, three

CA, USA) and one section was stained using the cytological smears were made from the lesional periodic acid Schiff(PAS) method (Medical Chemi- tissue of each patient.One wet smear was made with cal Corp.). Tissues were processed to obtain the 10% KOH solution and the other two smears were paraffin-embedded sections both for PAS and H&E stained with Gram’s stain (Medical Chemical Corp.) staining.Sections of 5μm were cut from each block, and PAS stain. Budding yeasts and pseudohyphae deparaffinized, hydrated with water, stained with were identified as refractile structures under wet

PAS, and observed under a light microscope. Can- KOH mount (Fig.2).Candida were seen as dark blue dida could be seen in the superficial layer of the -colored hyphae and yeasts with Gram’s stain and epithelium as magenta-colored hyphae penetrating magenta-colored with PAS stain(Fig.3)when obser- the epithelium both vertically and horizontally(Fig. ved under a light microscope. 1). Colony counting and Germ tube test-Additionally, Int J Oral-Med Sci 10(3):140-148,2011

colony-forming units (cfu) of yeast present within

this pellet (and therefore present within the 10-ml

mouth rinse) was assessed. The colonies of C.albicans were cream-colored,pasty and smooth (Fig. 4).Generally,the rate of growth was rapid and the

colonies matured in 3 days.The germ tube(GT)test

confirmed the species as C.albicans (Fig.5).The GT

Fig.2. 10% KOH wet mount showing candidal hyphae with budding yeast forms (×40).

Fig.4. Colonies of Candida albicans showing greater than

1000 cfu/ml(on SDA medium).

Fig.3. PAS-stained smear showing presence of candidal hyphae with budding yeast forms (×100). patients were asked to rinse their mouth with 10 ml sterile saline for at least 1 min prior to expectora- tion. The mouth rinse sample was collected and centrifuged at 1700 rpm for 10 min and the pellet was plated onto Sabouraud’s dextrose agar (SDA) medium (Difco Laboratories, Detroit, MI, USA) containing amikacin (Aristo Pharma,Nani,Daman, Fig.5. Germ tube test confirming the species as Candida

India)and incubated for 48 h at 37°C.The number of albicans (×40). Int J Oral-Med Sci 10(3):140-148,2011 test provides a simple,reliable and economical proce- 0.01).However,no significant difference was obser- dure for the presumptive identification of C.albicans. ved for various parameters (p＞0.05,p＞0.01)when

About 95% of the clinical isolates produced GTs quantified and compared between the precancerous when incubated in serum at 35°C for 2.5 to 3 h. lesions and conditions.(Table 3). Results were expressed in terms of the presence or With the standard error of the mean, malignant absence of yeast in the oral mouth rinse,and the level lesions showed the highest average for all of colonization was expressed in terms of being cytological parameters. A highly significant differ- either above or below 1000 cfu/ml. ence was observed between precancerous conditions

and malignant lesions while the difference was least

significant between precancerous lesions and condi- A total of 135 cases of precancerous and cancerous tions.(Table 4). oral mucosal lesions were studied. Findings were Evaluation of cytological smears and colony count entered in the proforma prepared for record keeping. on SDA medium showed a higher percentage of

The results were then tabulated and subjected to candidal hyphae and colonies in the malignant statistical analysis using a two-way ANOVA (analy- lesions.The present study revealed the lowest per- sis of variance) test for correlation between the centage of candidal hyphae in cytological smears different groups and different parameters to evaluate and sections,as well as the fewest positive culture the relationship between C.albicans and neoplasia. samples,for the precancerous lesions and conditions. The presence of C.albicans was quantified and (comparable to those of healthy adults).However, compared between precancerous lesions and malig- candidal colonies were present in more than 75% of nant lesions as well as between precancerous condi- the cases of precancerous lesions and tested positive tions and malignant lesions.Two-way ANOVA test- for Candida in cytological smears. ing was applied for sections, smears and colony Of the 99 positive culture samples included in the counting to assess the associations between Candida study,73.7% tested positive using the GT test.The and oral precancer and between Candida and oral GT test identified the species as predominantly C. cancer. A significant difference was observed albicans. between the three groups as well as among different Elderly males comprised more of the affected parameters at a 1% level of significance(p＜0.05,p＜ patients in the precancer and cancer groups. Men

Table 3. Two-way ANOVA F table for significant difference in groups and different parameters

S.No. Source of variation d.f. S.S. M.S.S. F-ratio F 1. Between the groups 2 1174.78 (587.39)(38.00)F(２,10,0.01)＝(7.56) 2. Between different parameters 5 1287.09 (257.42)(16.65)F(５,10,0.01)＝(5.64) 3. Error 10 154.57 (15.457) －－

4. Total 17 2616.44 －－

d.f.:degree of freedom,S.S.: Sum of squares,M.S.S.: Mean sum of squares

Table 4. Standard error of mean

Mean±SD SEM

Precancerous lesions 28.5±9.5237 3.887 Malignant lesions 35.33±12.848 5.244 Pre-cancerous conditions 15.833±5.7067 2.329 Int J Oral-Med Sci 10(3):140-148,2011 represented a higher proportion of patients with microscopy of smears from cases of leukoplakia,

SCCs than women. A large number of cases were studies in countries such as Denmark,the UK,India seen to develop in the fourth and fifth decades of life. and Germany have revealed the presence of C.al- A larger number of patients in this study used smoke- bicans as the dominant species,making up 67-80% of less tobacco versus bidi or cigarettes. the isolated yeast flora (15).Similarly,the present

study reveals C.albicans as the predominant species

in as many as 55.5% of the cases of precancerous

C.albicans and other Candida species are oppor- lesions. tunistic commensals in the human oral cavity that However, our observations for smears revealed are detectable in 30% to 50% of adults and are candidal hyphae in about 73.3% of the cases,while usually asymptomatic.In recent years,oral patholo- colony counting on SDA medium showed more than gists have taken considerable interest in the possible 1000 cfu/ml in 84.4% of the cases of precancerous role of chronic oral candidosis in the induction of lesions.Several studies have asserted that the reli- oral (pre-)cancer,since C.albicans and other yeast- ability of the direct smear technique in the diagnosis like fungi have appeared histologically in dysplastic of candidal infection was found to be equal to that of lesions or in oral smears by cultivation(6,7,8,9,10). histologic examination of biopsy material from oral

Several authors have suggested that candidal infec- leukoplakias (3,16,17).It is noteworthy that in our tion may be a factor in the malignant transformation study the candidal colonization was greater than of leukoplakia (8, 11, 12, 13).After reviewing the 1000 cfu/ml in nonhomogeneous leukoplakias evidence for a role of Candida in oral epithelial (53.8%) as compared to homogeneous leukoplakias. neoplasia,Field et al. (3)postulated that nitrosamine In support of this,many studies have generally con- compounds produced by Candida species may direct- sidered that nonhomogeneous types of oral leukopla- ly,or in concert with other chemical carcinogens, kia have a higher probability than homogeneous activate specific proto-oncogenes and thus initiate types for malignant transformation (9,14,18).From the development of a malignant lesion. Certain previous histologic investigations,it became evident strains of C.albicans and other yeasts may therefore that malignant changes of the epithelium are more play a causal role in the development of oral cancer likely to occur in chronic candidal leukoplakias than by means of endogenous nitrosamine production. in non-candidal ones(3,8,10,12,13). Field et al.further suggested that the progression of In our study,out of 45 cases of SCC, nearly 95% the activated cell into a tumorigenic cell might be cases showed candidal hyphae in smears, while in linked to the amplification and over-expression of PAS-stained sections only 10 cases(22.2%)revealed oncogenes, a phenomenon observed in many other candidal hyphae.In 91% of cases,the colony count human malignancies. was found to be greater than 1000 cfu/ml.GT testing

The present study was undertaken to detect the confirmed the species as C.albicans in 75% of cases. presence of C.albicans in precancerous and malig- Thus,a statistically significant difference was obser- nant oral mucosal lesions and to evaluate the purpor- ved between the leukoplakia and SCC groups (p＜ ted role of C.albicans in oral carcinogenesis and 0.05,p＜0.01)regarding the presence of C.albicans. determine its correlation with progression of disease Our observations are in accordance with several process.Our investigation on biopsy specimens has other studies (8,11,17).Recently Kumar et al. (19) shown concurrent Candida in 24.4% of the cases of found a highly significant association of Candida in oral leukoplakia,which complements the findings of the cancer group as compared to the precancer a Scandinavian study(14).It is also roughly double group.A statistically significant positive association the 11% value found by another study on leukoplakia between fungal infection and moderate and severe

(12). Using histologic examination of biopsies or epithelial dysplasia was observed by various authors Int J Oral-Med Sci 10(3):140-148,2011

(14,17,20).Significantly more yeasts were isolated ities of patients with OSMF using the same oral rinse from patients with more advanced levels of dysplasia technique as performed in this study.Our results also as categorized histopathologically in our study.Sith- confirmed this finding. As the oral mucosa is com- eeque and Samarnayake (21) reported a positive promised in OSMF, it can be argued that the pres- association between fungal infection and mild,mod- ence of Candida may predispose the individual to erate,and severe epithelial dysplasia,squamous cell candidal infection and invasion,although our biopsy carcinoma and hyperkeratosis.Their results did not samples refuted this.Admittedly,the cohort chosen show a causal relation between Candida species and for our study is relatively small.Further studies with dysplastic lesions and carcinomas,but confirmed the a larger pool of patients are required to confirm or higher presence of the microorganism in those refute our findings and ascertain factors that may lesions. influence oral candidal carriage in patients with

Out of 45 cases of precancerous conditions in our OSMF.The lack of published data on the occurrence study,candidal hyphae were detected only in 10 out of Candida with OSMF makes the comparison of our of 25 cases of lichen planus.The frequency of yeasts data with other studies difficult. in lichen planus patients was about the same as in Interestingly, lesions like leukoplakia and oral healthy persons (30% to 50%),whereas the isolation squamous cell carcinoma were more prevalent in of yeasts in leukoplakia patients was slightly higher elderly males.Similarly,OSMF was more prevalent and was far above the expected frequency among in males because of gutkha chewing habit in this healthy dentate adults when quantified in oral cancer region of India.Arendorf and Walker(7)suggested patients.Our observations are in accordance with the that cigarette smoke contains a soluble “can- conclusions drawn by some past studies(9,22).Oral didacidal factor”that is inactivated by saliva and lichen planus has considerably less susceptibility hence does not interfere with the colonization by than oral leukoplakia to invasion by C.albicans (22). Candida of mucosa changed by tobacco smoking. Another study revealed that yeasts were present in The findings of Bastiaan and Reade (25),however, the lesions of 82% of leukoplakia patients,compared differ from the foregoing in that their results showed to 37% of lichen planus patients, a frequency of that smoking tobacco did not influence the preva- yeasts corresponding to that in healthy adults (9). lence of Candida, nor was the prevalence of this

However,contradictory results have been reported organism related to the presence of oral mucous by Thankun et al. (23). Results from their study membrane keratoses shown to be an irritant. A indicated the significant finding of oral Candida similar study in a female Cambodian cohort did not infection in oral lichen planus patients.There are no indicate that betel quid chewing had a significant data indicating that yeast infection is involved in the effect on oral colonization by Candida species(26). etiology of oral lichen planus.Yeasts may,however, The present study supports previous evidence that be involved in the malignant transformation in some there may be a role for yeast in the development of cases of oral lichen planus (9, 22).When quantifica- oral epithelial dysplasia/neoplasia (3,10,13,14,15, tion of C.albicans was compared between patients 17).However, although the present study provides with lichen planus and oral squamous cell car- supporting evidence for a role of oral yeast in epith- cinoma, a statistically significant association was elial carcinogenesis,it may also be possible that the observed in our study(p＜0.05,p＜0.01). increased colonization and prevalence of oral yeast

When quantification of oral yeast carriage was in these lesions is entirely coincidental and merely determined in OSMF patients,our results confirmed reflects a changed local environment that allows for the findings of the study by Ariyawardana et al.(24). the proliferation of these common oral commensals. Although not statistically significant, their study The probable role of yeast in oral carcinogenesis is demonstrated higher candidal carriage in oral cav- still unclear. It could be argued that the increased Int J Oral-Med Sci 10(3):140-148,2011 colonization and infestation associated with ・The frequency of oral yeast carriage was signifi- dysplasia reflects a change in the environmental cantly greater(p＜0.05,p＜0.01)in the malignant conditions conducive to the proliferation of these lesion than the precancerous lesions and condi- ubiquitous commensals,and is therefore not a cause tions.In the precancerous conditions,no signifi- of carcinogenesis.Jobbins et al. (27)identified sub- cant correlation was found with yeast carriage. stantial changes in the oral flora of patients with ・On comparing the study groups,a high positive advanced cancer and reported a reduction in the correlation was observed between precancerous normal colonization resistance of the oral mucosa, lesions and malignant lesions. A statistically with a high level of candidal carriage and candidosis. significant association was also found between

Xerostomia is a common problem in advanced can- precancerous conditions and malignant lesions. cer patients and is another factor that reduces coloni- However,no significant difference was observed zation resistance of the oral mucosa. between precancerous lesions and precancerous

Cawson (12)suggested that the presence of Can- conditions. dida can be regarded as coincidental－that is, an ・Quantification of candidal carriage helps in infection of an already diseased surface with little determining an interaction between oral car- relevance to the pathologic nature of the lesion as a riage of yeast and oral epithelial dysplasia. whole.Lynch(6)explained that C.albicans presents ・C.albicans was the predominant species something of an enigma.The simple presence of the identified. organism does not represent disease.Nevertheless, The present study yields strong evidence for a oral candidiasis may serve as a useful clinical close correlation between Candida infections and marker for the presence of significant predisposing chronic and, in particular, precancerous leukopla- conditions such as immunosuppression and endocrine kias.This may be interpreted as a sign of prognostic dysfunction.In respect to the association between C. importance implying local conditions that allow albicans and leukoplakia, it seems reasonable to opportunistic Candida species to invade leukoplakic suppose that candidal infection is at least a co-causa- lesions. From the remarkable positive correlation tive factor in some cases of human oral leukoplakia. between leukoplakia and squamous cell carcinoma, The essential debate is whether the Candida species we do not immediately conclude that there is a true are secondary opportunistic pathogens acting on a etiologic connection between both conditions.How- pre-existing leukoplakia or,conversely,whether the ever, the debate remains open as to whether can- higher premalignant rate of change in candida leuko- didosis may be causal to,or only symptomatic for, plakias may support a role for this fungus in the the malignant transformation of the lesions. The development of neoplasia. The relationship of question of the role of the Candida in the production precancer,candidiasis and malignant transformation of these leukoplakias and its possible carcinogenic remains controversial. action is an important one.

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