|Year : 2015 | Volume
| Issue : 1 | Page : 63-67
Frequency of fungal infection in biopsies of oral mucosal lesions: A prospective hospital-based study
Thimmarasa Venkappa Bhovi1, Swarnasmita Pathak2, Manas Gupta1, Parvathi Devi Munishwar3, Devarshi Nandi4, Ankur Nandi5
1 Department of Oral Medicine and Radiology, Rishiraj College of Dental Sciences, Bhopal, Madhya Pradesh, India
2 Department of Oral Medicine and Radiology, Regional Dental College, Guwahati, Assam, India
3 Department of Oral Medicine and Radiology, Teerthankar Dental College, Moradabad, Uttar Pradesh, India
4 Department of Oral Medicine and Radiology, Hazaribag College of Dental Sciences, Hazaribag, Jharkhand, India
5 Department of Orthodontics, Hazaribag College of Dental Sciences, Hazaribag, Jharkhand, India
|Date of Submission||10-Apr-2015|
|Date of Acceptance||30-Jun-2015|
|Date of Web Publication||12-Oct-2015|
Thimmarasa Venkappa Bhovi
Department of Oral Medicine and Radiology, Rishiraj College of Dental Sciences, Bhopal - 462 036, Madhya Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aims and Objectives: To determine the frequency and common site of fungal infection in biopsies of oral mucosal lesions and also to detect the lesions most likely to be infected with fungal infection. Materials and Methods: A total of 100 patients with oral mucosal lesions were advised routine hematological examination followed by incisional biopsy under local anesthesia. The specimen were fixed in 10% neutral buffered formalin and processed. One section from the specimen was stained with hematoxylin and eosin staining for histopathological diagnosis of the lesion and a second section was stained with Periodic acid-Schiff (PAS) stain for detection of fungal infection. Results: Out of the 100 patients, the most common mucosal lesion encountered was carcinoma (56%) followed by lesions with dysplastic changes (28%), benign lesions (9%), squamous papilloma (2%) and oral submucous fibrosis (5%). The most common anatomic location affected by the mucosal lesions were buccal mucosa, followed by the tongue, gingiva, maxillary tuberosity and floor of the mouth with values of 73%, 16%, 6%, 4% and 1%, respectively. Squamous papilloma had the highest positive association with fungal infection (100%) followed by lesions with dysplastic changes (17.9%) and carcinoma (8.9%). The maximum fungal positive association was encountered in the mucosal lesions over the tongue (18.7%) followed by the buccal mucosa (12.3%). Conclusion: There is statistically significant association of fungal infection with dysplastic lesions and papilloma with the tongue and buccal mucosa as the most common sites. Hence a PAS stain should be performed whenever epithelial dysplasia on the tongue and buccal mucosa is diagnosed.
Keywords: Epithelial dysplasia, mycoses, oral candidiasis
|How to cite this article:|
Bhovi TV, Pathak S, Gupta M, Munishwar PD, Nandi D, Nandi A. Frequency of fungal infection in biopsies of oral mucosal lesions: A prospective hospital-based study. J Indian Acad Oral Med Radiol 2015;27:63-7
|How to cite this URL:|
Bhovi TV, Pathak S, Gupta M, Munishwar PD, Nandi D, Nandi A. Frequency of fungal infection in biopsies of oral mucosal lesions: A prospective hospital-based study. J Indian Acad Oral Med Radiol [serial online] 2015 [cited 2019 Dec 8];27:63-7. Available from: http://www.jiaomr.in/text.asp?2015/27/1/63/167083
| Introduction|| |
Optimal patient care requires the ability to recognize and diagnose clinically suspicious oral findings. At one time, oral fungal infections were rare findings in dental clinics. They were more commonly seen in hospitalized and severely debilitated patients. However, with enhanced medical and pharmaceutical technology, increasing numbers of ambulatory immunosuppressed individuals with oral fungal infections are seeking out dentists for diagnosis and treatment of this lesion. 
Candida albicans may exist as yeast or in a hyphal form and is the most common commensal and pathological fungus of the oral cavity. The overgrowth of Candida species causes oral candidiasis which is a common opportunistic infection of the oral cavity. The consensus view is that yeast represents the morphotype in health, whereas the hyphal form is pathogenic.  This reflects the ability of the yeast to colonize different oral surfaces and the variety of factors which predispose the host to Candida colonization and subsequent infection.  The pathological significance of fungal, particularly candidal infection of the oral mucosa remains unknown; but, candidal leukoplakia is considered a potentially malignant lesion and infection may play an important role in the etiology of oral epithelial neoplasia. Although the hyphae do not always breach the epithelium, penetration of the cell membrane and occupation of the cytoplasm may either produce clinical entities which are readily recognizable or subclinical infection which is only detected after biopsy and microscopic examination. 
Histological evaluation of tissues is a quick and easy way to identify fungal organisms, and a strong adjunct to microbiologic culture for diagnosis of fungal infections. Hematoxylin and eosin (H and E) is a versatile stain which can not only be used to evaluate the host response, but can also be used to confirm the presence of naturally pigmented fungi and demonstration of the nuclei of yeast-like cells. However, one of the drawbacks in using just the H and E stain for fungal diagnosis, is the difficulty to distinguish poorly stained fungi from tissue components, even at higher magnifications.  Most fungi can be readily demonstrated with the common special stains such as Gomori's methenamine silver (GMS), Gridley's fungus (GF), and periodic acid-Schiff (PAS), also referred to as "broad spectrum" fungal stains. Gomori's methenamine silver and PAS are the two most common stains used to look for fungi in tissues and in cytology specimens. Fungal morphology is better demonstrated by the PAS stain than the silver stains. Degenerated fungi that may not be visible on H and E stain can be stained by the PAS stain. 
Although epithelial hyperplasia, hyperparakeratosis, superficial microabscess formation and chronic inflammation of lamina propria are the histopathological features suggestive of candidal infection, the thin, hematoxyphilic hyphae, can be much more easily visualized with the use of PAS stain, which reacts with the mannan, glucan and other polysaccharide moieties in the fungal hyphal unit. Fungal infection, especially that attributable to C. albicans, has been extensively researched in individual lesions, but the aim of this study was to determine how frequently fungal hyphae are detected by the PAS stain in biopsies of oral mucosal lesions, and to note which oral mucosal lesions are most likely to be infected.
| Materials and Methods|| |
This prospective study was carried out in the Department of Oral Medicine and Radiology, Rama Dental College Hospital and Research Centre and Health Square-Gian Pathology Lab, Kanpur after obtaining Institutional Ethical Committee clearance and informed written consent from the subjects. The inclusion criteria for the study group were patients with clinically suspected oral mucosal lesions in the 2 nd to 7 th decade of life who were reporting for the first time with no history of any treatment. A total of 100 patients with oral mucosal lesions visiting the dental out-patient department during January 2009 to March 2010 with simple random sampling were selected for the study. An incisional biopsy was performed under local anesthesia. All specimens were fixed in 10% neutral-buffered formalin and processed. One section from the specimen were stained with H and E staining for histopathological diagnosis of the lesion and a second section was stained with PAS stain for detection of fungal infection. No attempt was made to identify the fungus, which was assumed to be a species of Candida, most likely Candida albicans. All the cases were distributed according to the histopathological diagnosis as shown in the [Table 1].
|Table 1: Distribution of specimen according to the histopathological diagnosis|
Click here to view
The statistical analysis was done using Statistical Package for Social Sciences (SPSS) version 15.0 statistical analysis software. To seek an association between different variables and fungal positivity Chi-square test was used. The level of significance was assessed by calculating P-value (probability of chance error). The confidence level of the study was kept at 95%, hence a P-value less than 0.05 was considered to be significant statistically.
| Results|| |
Out of the 100 patients included in our study, the most common age group involved was 40-50 years (49%) followed by 30-40 years (21%), 50-60 years (20%), 20-30 years (%) and >60 years (5%). The most common age group affected by oral mucosal lesions among males (46.5%) and females (64.3%) was 40-50 years. The most common mucosal lesion encountered was carcinoma (56%) followed by lesions with dysplastic changes (28%), benign lesions (9%), squamous papilloma (2%) and oral submucous fibrosis (5%). The most common anatomic location affected by the mucosal lesions was buccal mucosa, tongue, gingiva, maxillary tuberosity and floor of the mouth with values of 73%, 16%, 6%, 4% and 1%, respectively.
The incidence of fungal positivity in biopsies of mucosal lesions through PAS staining was observed to be 12% [Graph 1 [Additional file 1] ]. Squamous papilloma had the highest positive association with fungal infection (100%) followed by lesions with dysplastic changes (17.9%) and carcinoma (8.9%) with P-value <0.001 [Table 2] and [Graph 2 [Additional file 2] ]. The maximum fungal positivity was encountered in the mucosal lesions over the tongue (18.7%) followed by buccal mucosa (12.3%) with P-value >0.05 [Table 3] and [Graph 3 [Additional file 3] ]. Among the 12 positive cases with fungal infection, six cases were found positive with fungal infection in the age group of 40-50 years, three cases were found positive in the age group of 50-60 years, two cases were found positive in the age group of 30-40 years and one case was found positive in the age group of >60 years. Similarly, among the 12 positive cases with fungal infection, nine cases were found positive among males, whereas three cases were found positive in females with P-value >0.05.
|Table 2: Type of oral mucosal lesions and fungal infection (Percentages have been calculated row wise)|
Click here to view
|Table 3: Site of oral mucosal lesions and fungal infection (Percentages have been calculated row wise)|
Click here to view
| Discussion|| |
Periodic acid-Schiff stain, also referred to as a 'broad spectrum' fungal stain, is more frequently employed for detection of fungal infections. , Therefore, in this study H and E stain was used for establishing the diagnosis of the mucosal lesions whereas PAS stain was used to determine the presence of fungal infection. In our study the accuracy of PAS stain in diagnosing various mucosal lesions infected by fungal infection was 12% [Graph 1]. Our finding is consistent with the study conducted by Spolidorio et al.  where in a detection rate of 12.2% was found for fungal infection in oral mucosal biopsies using PAS stain. Similar studies were conducted by Barrett et al.  where a detection rate of 5% was found and Daftary et al.  who did PAS staining only for oral leukoplakia and found a detection rate of 6.8%.
Considering specific oral mucosal lesions and its association with fungal infection, we have observed a significant association of fungal infection with squamous papilloma and dysplastic lesions followed by carcinoma. The fungal positivity for squamous papilloma in our study was 100% (2/2). In case of dysplastic lesions the detection rate was found to be 17.9% (5/28) whereas in cases of carcinoma it had a detection rate of 8.9% (5/56). Statistically significant association between type of lesion and fungal positivity was observed at 95% significance level (P < 0.001) [Table 2]. Similar results were observed in the study done by Barrett et al.  in which significant association of fungal infection was found with dysplastic lesions.
The finding of positive association in squamous papilloma and less association in carcinoma is somewhat anomalous. A possible explanation for the former is the transformation into hyphae by blastospores which are protected within the environment of a folded environment. The less association in carcinoma might be accounted for by the destruction of infected epithelium as a result of ulceration and invasion. Fungal species may have a causal role in the malignant development of oral cancer and that malignant development of oral pre-cancer may be elicited by particular biotypes of C. albicans. It has been suggested that certain strains of C. albicans and yeasts play an etiologic role in the development of oral cancer by means of endogenous nitrosamine production. , Although present literature 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 fungal infection in some potentially malignant lesions in comparison with inflammatory lesions with no malignant potential.
A number of studies have been conducted in the past to evaluate the correlation of fungal infection with oral mucosal lesions like leukoplakia and oral lichen planus. Vidas et al.  conducted a study to evaluate the association of fungal infection with oral leukoplakia with an accuracy of 46.67% (28/60). Lipperheide et al.  in 1996 conducted a study in a similar study group comprising 35 oral leukoplakia and 34 oral lichen planus patients and found an accuracy of 76% for leukoplakia and 88.2% for lichen planus. Later, Dorko et al.  and Vuckovic et al.  in similar studies found positive association of fungal infection in oral leukoplakia and oral lichen planus with positivity of 35.9% (23/64) and 40% (12/30), respectively. In the present study, the percentage positivity of dysplastic lesions was 17.9% (5/28) involving oral leukoplakia and lichen planus [Table 2] and [Graph 2]. The accuracy of detection rate was less compared to the previous studies, but on statistical analysis we found significant association with dysplastic lesions (P < 0.001). In our study we encountered benign lesions which included pyogenic granuloma, fibroma and fibrous epulis. None of the cases were positive for determining the frequency of fungal infection with PAS stain. A similar study was conducted by Barrett et al.  with an accuracy of 10.8% (24/223) in benign fibrous overgrowths and 1.3% (3/223) in pyogenic granuloma. Ariyawardana et al.  in their study on patients with oral submucous fibrosis (OSMF) found no significant association between fungal infection and OSMF. Similar results were present in our study while evaluating OSMF. Five cases were seen and none stained positive for fungal infection. On the other hand, in a study conducted by Barrett et al.,  they came across one case of OSMF positive with fungal infection out of 19 cases.
As reported by Arendorf and Walker, the tongue, particularly the dorsal surface and the buccolabial mucosa are the sites most vulnerable to infection.  In our study also the maximum percentage of fungal positivity was observed for the tongue (n = 3, 18.7%) and buccolabial mucosa (12.3%) [Table 3] and Graph 3]. On statistical evaluation no significant association between the site of involvement and fungal positivity was observed (P > 0.05). This is in accordance with the study conducted by Barrett et al.  who found maximum percentage of fungal positivity for the tongue (17.3%) and buccolabial mucosa (17.9%). The reason for increased hyphal colonization or positivity on the dorsum of the tongue may be explained by the physical protection afforded by a papillary epithelium, as noted above with respect to squamous papilloma.
| Conclusion|| |
Oral fungal infections have emerged as a major cause of human illness. The result of this study concluded that histological methods identify fungal infection where it may not be evident clinically and are the quickest, most reliable means to determine whether there is fungal invasion. In our study statistically significant association with fungal infection in dysplastic lesions and papilloma was observed with the tongue and buccal mucosa as the most common sites. Hence a PAS stain should be performed whenever epithelial dysplasia on the tongue and buccal mucosa is diagnosed. Furthermore, on histological confirmation of dysplasia, antifungal therapy should also be considered in the management of these lesions.
The authors acknowledge the services of Health Square-Gian Pathology Lab, Kanpur for assisting in carrying out the specimen processing and staining procedure.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Muzyka BC, Glick M. A review of oral fungal infections and appropriate therapy. J Am Dent Assoc 1995;126:63-72.
Akpan A, Morgan R. Oral candidiasis. Postgrad Med J 2002;78:455-9.
Cannon RD, Holmes AR, Mason AB, Monk BC. Oral candida: Clearance, colonization, or candidiasis? J Dent Res 1995;74: 1152-61.
Barrett AW, Kingsmill VJ, Speight PM. The frequency of fungal infection in biopsies of oral mucosal lesions. Oral Dis 1998;4:26-31.
Haque A. Special stains use in fungal infections. Connection 2010;187-94.
Treagan L. Candida and its role in opportunistic mycoses. California Association for Medical Laboratory Technology (CAMLT). Distance Learning Program. Course DL-986. California: California Association for Medical Laboratory Technology (CAMLT); 2008. p. 1-19.
Spolidorio LC, Geraldo VR, Norgueira RD, Spolidorio DM. The frequency of Candida sp. in biopsies of oral mucosal lesions. Pesqui Odontol Bras 2003;17:89-93.
Daftary DK, Mehta FS, Gupta PC, Pindborg JJ. The presence of Candida in 723 oral leukoplakias among Indian villagers. Scand J Dent Res 1972;80:75-9.
Mehdipour M, Taghavi Zenouz A, Hekmatfar S, Adibpour M, Bahramian A, Khorshidi R. Prevalence of Candida species in erosive oral lichen planus. J Dent Res Dent Clin Dent Prospects 2010;4:14-6.
Krogh P, Hald B, Holmstrup P. Possible mycological etiology of oral cancer: Catalytic potential of infecting Candida albicans
and other yeasts in production of N-nitrosobenzylmethylamine. Carcinogenesis 1987;8:1543-8.
Vidas I, Temmer K, Zuziæ P, Palaversiæ M. Candida albicans
in leukokeratotic lesions of oral mucosa. Acta Stomatol Croat 1988;22:311-7.
Lipperheide V, Quindós G, Jiménez Y, Pontón J, Bagán-Sebastián JV, Aguirre JM. Candida biotypes in patients with oral leukoplakia and lichen planus. Candida biotypes in leukoplakia and lichen planus. Mycopathologia 1996;134:75-82.
Dorka E, Zibrín M, Jenca A, Pilipcinec E, Danko J, Tkáciková L. The histopathological characterization of oral Candida leukoplakias. Folia Microbiol (Praha) 2001;46:447-51.
Vuckovic N, Bokor-Bratic M, Vuèkovic D, Picuric I. Presence of Candida albicans
in potentially malignant oral mucosal lesions. Arch Oncol 2004;12:51-4.
Ariyawardana A, Panagoda GJ, Fernando HN, Ellepola AN, Tilakaratne WM, Samaranayake LP. Oral submucous fibrosis and oral yeast carriage - A case control study in Sri Lankan patients. Mycoses 2007;50:116-20.
Arendorf TM, Walker DM. The prevalence and intra-oral distribution of Candida albicans in man. Arch Oral Biol 1980;25: 1-10.
[Table 1], [Table 2], [Table 3]