Mycotic infections have become a major cause of

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1 TECHNIQUES Detection of Candida by Calcofluor White Rashmi S. K. Bhavasar, M.D.S., Santosh Kumar Goje, M.D.S., Anant Arunrao Takalkar, M.D., Sindhu M. Ganvir, M.D.S., Vinay K. Hazarey, M.D.S., and Suchitra R. Gosavi, M.D.S. Objective To assess the efficacy of calcofluor white (CFW) is compared to Gram stain and periodic acid Schiff in detection of Candida in oral precancer and cancer. Study Design The study group consisted of patients with precancer (n = 45), cancer (n = 45) and control group (n=45). Presence of Candida was confirmed by culture inoculation along with a germ tube and carbohydrate fermentation test. The cytopathologic smears were analyzed by Papanicolaou-CFW and Gram staining, whereas, tissue sections were stained by periodic acid Schiff and CFW staining. Results Candida albicans was the predominant species identified. A highly significant association of Candida was seen more often in cancer than in precancer. Both in cytology and histopathology Candida detection by CFW was higher. In precancer it was 48.88% in smears and 40% in tissue sections, whereas, in cancer 60% in smears and 55.55% in histopathology. Conclusion Among the various diagnostic tools used in the present study, the use of CFW is seen to be a simple, effective, rapid CFW appears to enhance the detection of fungal elements due to its staining properties. and reliable method, both in cytopathology and histopathology. (Acta Cytol 2010;54: ) Mycotic infections have become a major cause of morbidity and mortality in clinically debilitated or immunocompromised patients. 1 The coexistence of Candida species within humans either as commensals or pathogens has been a subject of interest. 2 Also, the association of Candida due to its endogenous nitrosamine production has been reported to the extent that it is a causative agent with various precancer and cancerous lesions. 3,4 With the increasing importance of candidosis in various acute and chronic mucous membranes and deep seated infections, there is a need for a practical method for identification of the fungus. A new technique for the rapid identification of fungi with use of calcofluor white (CFW) in tissues has been described by Monheit and coworkers. 5,6 CFW is a disodium salt of 4,4 -bis-[4anilino-bisdiethyl-amino-s-tri-azin-2-ylamino]-2,2 -stilbenedisulfonic acid, a colorless dye that is used in the textile and paper industries as a whitening agent. 7 It has the ability to bind to β 1-3, β 1-4 polysaccharides (i.e., cellulose and chitin) and exhibits fluorescence of either an apple green or blue-white color when exposed to long-wavelength ultraviolet and short wavelength From the Departments of Oral Pathology and Microbiology and Orthodontics, Kamineni Institute of Dental Sciences, and Department of Social and Preventive Medicine, Kamineni Institute of Medical Sciences, Nalgonda, Andhra Pradesh; and Department of Oral Pathology and Microbiology, Government Dental College and Hospital, Nagpur, Maharashtra, India. Dr. Bhavasar is Assistant Professor, Department of Oral Pathology and Microbiology, Kamineni Insitute of Dental Sciences. Dr. Goje is Associate Professor, Department of Orthodontics, Kamineni Insitute of Dental Sciences. Dr. Takalkar is Assistant Professor, Department of Social and Preventive Medicine, Kamineni Insitute of Medical Sciences. Drs. Ganvir, Hazarey and Gosavi are Professors, Department of Oral Pathology and Microbiology, Government Dental College and Hospital. Address correspondence to: Rashmi S. K. Bhavasar, M.D.S., Department of Oral Pathology and Microbiology, Kamineni Institute of Dental Sciences, Sreepuram, Narketpally, Nalgonda, Andhra Pradesh , India (rashmi_bhavasar@rediffmail.com). Financial Disclosure: The author has no connection to any companies or products mentioned in this article. Received for publication July 6, Accepted for publication November 11, /10/ /$21.00/0 The International Academy of Cytology ACTA CYTOLOGICA 679

2 Bhavasar et al Table I Distribution of Study Group (90 Cases of Oral Precancer and Cancer, Diagnosed on the Basis of Clinical and Histopathologic Features) and Controls (45 Patients) Type of lesion No. Precancer Oral leukoplakia 15 Oral lichen planus 6 Oral submucous fibrosis 24 Oral cancer 45 Control 45 visible light. 8 It can be used on fresh, frozen, fixed, paraffin-embedded and clinical specimens, 7 and can be incorporated in the Papanicolaou (Pap) stain procedure to enhance the recognition of yeasts in smears. 6 The study was planned with the following aims and objectives to: determine the presence of Candida in oral precancer and cancer patients, using various laboratory tests, such as culture, germ tube (GT) test, and carbohydrate fermentation test; detect Candida in oral precancer and cancer lesions by using CFW staining in cytopathology and in histopathology under a fluorescence microscope; investigate the diagnostic efficacy of fluorescence microscopic evaluation of a CFW stain used in direct smears and tissue sections; and evaluate the sensitivity and specificity of CFW staining in cytopathology and histopathology in the detection of Candida as compared to Gram staining and periodic acid Schiff (PAS) staining. The sample for the present clinical study comprised a total of 135 patients of both sexes. The patients were selected from among those visiting the Outpatient Figure 2 C albicans showing GT production when incubated in serum-unstained slide seen under a light microscope ( 40). Department of Oral Pathology, Government Dental College and Hospital, Nagpur (Maharashtra, India) (Table I). Culture inoculation was done for each patient by the swab method. Smears were prepared by scrape cytology using a wooden stick moistened in normal saline. For histopathologic examination, a tissue was obtained by using a 7-mm punch. Swabs were inoculated immediately on the Sabouraud s slope. The growth was subcultured on a new slope of Sabouraud s agar, and the pure colony was subjected to the GT test and carbohydrate fermentation test (Figures 1 and 2). One of the two smears was Gram stained and observed under a light microscope for each patient (Figures 3 and 4). Another smear was wet fixed by dipping the slide in 95% ethyl alcohol for 1 hour and was subsequently stained with Pap-CFW staining (Table II). When observed under the fluorescence microscope, Candida hyphae appeared apple Figure 1 Candidal colonies appear as smooth, shiny, convex, creamy white on Sabouraud s dextrose agar when cultured at room temperature (37 C). (The colors are shown in the online version.) Figure 3 Yeast forms of Candida in cytopathology seen under a light microscope (Gram stain, 100). 680 ACTA CYTOLOGICA Volume 54 Number 5 September October 2010

3 Detection of Candida by CFW Figure 4 Dark blue hyphae and yeasts forms of Candida in cytopathology seen under a light microscope (Gram stain, 100). (The colors are shown in the online version.) green 6 (Figures 5 and 6). Punch biopsy tissues processed to obtain the paraffin-embedded sections both for PAS (Table III, Figure 7) and CFW staining (Table IV, Figure 8). The procedure was performed under fluorescent room lighting. All sections were examined with an Olympus fluorescent microscope (Olympus, Nagpur, Maharashtra, India) equipped with planachromatic lenses, episcopic-fluorescence attachment BX-FLA, a high-pressure mercury light source Table II Pap-CFW Staining for Cytopathology of Both Study and Control Groups of Patients Used for Detection of Candida Under Fluorescence Microscopy Agent Staining time Harris hematoxylin 6 minutes Aqueous HCl solution 6 dips Lithium carbonate 2 minutes 50% Ethanol 10 dips 95% Ethanol 6 8 dips 95% Ethanol 6 8 dips Orange G-6 1. Acid-eosin % Ethanol Rinse gently CFW (0.1%) 50% Ethanol Rinse gently 100% Ethanol 6 8 dips 100% Ethanol 6 8 dips Xylene 6 8 dips Xylene 6 8 dips Mount in DPX Figure 5 Yeast forms of Candida in cytopathology seen under a fluorescence microscope (Pap-CFW, 100). and an ultraviolet filter cassette containing a nm excitation filter, a 400-nm dichromic mirror and a 420-nm absorption filter. Sensitivity, specificity, positive predictive value and negative predictive value were calculated. Correlation between the staining procedures was determined by the κ test. 8 Distribution of males and females among the study and control groups are tabulated in Table V. Candida growth was observed from the primary culture medium of Sabouraud s dextrose agar, which is considered the gold standard (Figure 1). By using the carbohydrate fermentation test and GT test, the species identified was predominantly Candida albicans, whereas the remaining 6 cases were Candida tropicalis (Table VI). Evaluation of cytologic smears showed that among them Pap-CFW was more effective in detection of Candida as compared to Gram stain (Table VI). Similarly, evaluation of the histopathologic sections Figure 6 Hyphal forms of Candida as apple green in cytopathology seen under a fluorescence microscope (Pap-CFW, 100). (The colors are shown in the online version.) Volume 54 Number 5 September October 2010 ACTA CYTOLOGICA 681

4 Bhavasar et al Table III PAS Staining for Histopathology of Study Group (Precancer and Cancer) of Patients Used for Detection of Candida Under Light Microscope Agent Staining time Deparaffinize the sections with xylene Absolute alcohol Poured 0.2% PAS or 0.5% PAS 2 minutes Schiff s reagent solution 4 at 37 C Pouring of sulfurous vapors solution to remove excess Schiff s reagent Pouring of 0.2% solution of light green on the slide 5 10 minutes Washed with absolute alcohol Dehydrated and cleaned with xylene Solid mounted with DPX showed a higher detection rate of Candida by CFW as compared to PAS stain (Table VI). Pap-CFW staining showed high sensitivity (85.10%), with high specificity (88.63%) (Figure 9), a positive predictive value of 80% and negative predictive value of 91.76%. κ test (κ = ) suggested substantial agreement of Pap- CFW with cultures. Z test (Z = ) showed a highly significant probability of detection of Candida by the Pap-CFW procedure, and κ test (κ = ) suggested moderate agreement between the 2 staining methods, Gram and Pap-CFW. Sensitivity of CFW was high (82.22%), with specificity of 86.66% (Figure 10), positive predictive value of 86.04% and negative predictive value of 82.97%. κ Test (κ = ) suggested substantial agreement of Table IV CFW Staining for Histopathology of Study Group (Precancer and Cancer) of Patients Used for Detection of Candida Under Fluorescence Microscopy Commercially available CFW powder was dissolved in distilled water to produce 0.1% solution of CFW Sections of 5 μm were cut from each block Sections were deparaffinized with xylene and hydrated to water One or 2 drops of the working solution were placed on freshly prepared unstained paraffin-embedded sections and allowed to stand for Followed by a tap water rinse Counterstained for with a dilute solution of Evans blue to minimize background fluorescence Slides were mounted by DPX mounting medium and were followed by coverslip placement CFW with cultures. However, Z test (Z = ) suggested the equivalence of PAS and CFW procedures. κ test (κ = ) showed substantial agreement between both detection methods. χ 2 test showed a highly significant association of Candida in both oral precancer (χ 2 = , p < 0.001) and oral cancer (χ 2 = , p < 0.001). Discussion In recent years the incidence of fungal infections has been rising around the world. Mycoses of deep tissues require prompt recognition and early institution of treatment, which may include chemotherapy and surgical debridement. Thus, rapid intraoperative examination of tissues for fungi is important to control the spread of the infection. A variety of methods, including direct examination of smears, routine and special cultures, and cytopathologic and histopathologic evaluation are available for detection of Candida. The use Figure 7 Candidal colonization in superficial keratin as magenta hyphae penetrating the epithelium both vertically and horizontally, seen in a patient with oral leukoplakia under a light microscope (PAS, 20). (The colors are shown in the online version.) Figure 8 Candidal hyphae seen as green to yellow (arrow) against a dark orange background in superficial keratin seen in a patient with oral cancer under a fluorescence microscope (CFW, 20). (The colors are shown in the online version.) 682 ACTA CYTOLOGICA Volume 54 Number 5 September October 2010

5 Detection of Candida by CFW Table V Age and Gender Distribution for Study Group and Control Group of Patients Age (yr) Patients Mean ± SD Controls Males ± Females ± Total ± Oral precancer Males ± Females ± 17.8 Total ± Oral cancer Males ± Females ± Total ±11.33 of standard staining methods, such as hematoxylineosin (H-E), PAS and Gomori s methenamine silver (GMS) can detect Candida, but these organisms may be obscure on H-E; the latter two methods are slow. 5 Rapid identification of these candidal species may prevent unnecessary prescribing of commonly used antifungal agents to which they have become resistant. 9 This need of rapid identification of Candida was fulfilled by CFW. In the present study, the species identified predominantly was C albicans. The results were compatible with those of Krogh et al, 10 who found C albicans to be the dominant species in oral leukoplakia and lichen planus. Monheit et al 6 introduced the addition of CFW to Pap stain. The ability to use CFW in this manner allowed pathologists to confirm the presence of fungi without delay. In the present study, a higher percentage of Candida was detected with the use of Pap-CFW stain in oral precancer and oral cancer when compared with that of Gram stain. Barrett et al 11 recommended the use of PAS stain for the detection of fungi. In the present study, PAS showed high specificity (91.11%) and low sensitivity (66.66%) for the detection of Candida. Harrington 12 used CFW to demonstrate hyphae and yeasts in the paraffin-embedded tissues of mycotic lesions. In the present study, it was observed that CFW had a higher candidal detection capacity than PAS. Rajendran and Sivapathasundharam 13 reported that 60% of speckled leukoplakia showed Candida hyphae with cellular atypia. In the present study, although 12 cases of leukoplakia were positive by culture, Gram stain and Pap-CFW stain could detect only 7 and 8 cases, respectively, in cytopathology. Similarly, PAS and CFW identified 7 and 8 cases, respectively, in histopathology. In the present study, culture was positive in only 1 case of lichen planus for Candida, but Gram stain and PAS were negative for Candida in the same case. However, Pap-CFW and CFW staining of the same case showed the presence of Candida. Gram staining and Pap-CFW staining were positive for Candida in 8 (33.33%) and 13 (54.16%) cases, respectively. Similarly, in histopathology, 8 (33.33%) and 9 (37.5) cases were positive for Candida by PAS and CFW staining, respectively. The lack of published data on the occurrence of Candida in oral submucous fibrosis makes comparison of our data with other studies difficult. A statistically significant association has been found between Candida, precancer and cancer in the present study, in which 55.55% of oral cancer cases were culture positive, whereas cytopathology using Pap-CFW revealed the presence of Candida in 60% of oral cancer cases. In tissue microscopy, CFW and PAS revealed the presence of Candida in 55.55% and 42.22% of cases, respectively. Association of Candida with leukoplakia has been noted in earlier studies, but its association with oral submucous fibrosis has not been noted in many studies. The role of Candida in the causation of precancer and oral cancer needs to be determined. The use of CFW stain in candidal detection not only has myriad advantages, like rapid detection, higher sensitivity and higher specificity over Gram and PAS staining, but is also a unique stain that can detect Candida in both smears and tissues without disturbing cellular details under the fluorescence microscope. Table VI Results of Candidal Detection by Various Methods in Study and Control Groups Cytology Histopathology Culture GT test Gram stain Pap-CFW PAS CFW Total positive positive positive positive positive positive Oral precancer Leukoplakia Lichen planus Submucous fibrosis Oral cancer Control Total Volume 54 Number 5 September October 2010 ACTA CYTOLOGICA 683

6 Bhavasar et al Figure 9 Comparison of sensitivity and specificity of Gram and Pap-CFW staining procedures for Candida detection in cytopathology. Figure 10 Comparison of sensitivity and specificity of PAS and CFW staining procedures for Candida detection in histopathology. The question of which stain or procedure to use for the detection of fungal elements is one that can be resolved by personal preference and user experience. CFW has several distinct advantages for easy and rapid diagnosis of Candida in both cytopathology and histopathology in precancer and cancer, though it is slightly expensive and needs photo documentation. The technique is extremely rapid, requiring < 30 seconds from preparation of the hydrated specimen to viewing of the slide. No specific techniques are required other than routine histologic processing, and it does not disturb cellular details. CFW staining also does not interfere with the subsequent Gram or PAS staining when required. Certain cellular components or extraneous debris, which may interfere with the interpretation of GMS stain, do not interfere with fluorescence microscopic examination. 6 Also, distinct and clear fluorescence allows better cytologic and histologic recognition of Candida by fluorescence microscope when it is not easily distinguished from its surroundings by light microscopy. Thus, fluorescence microscopy with CFW, though slightly expensive (Fluostain I, 100 mg, Sigma Aldrich Foreign Holding Company, St. Louis, Missouri, U.S.A.), is superior to and quicker than other investigative techniques and enhances the sensitivity and specificity of cytologic and histologic investigation when a rapid and reliable identification of Candida is needed. Currently, laboratories must rely on the astute powers of observation and inquisitive minds of those performing the work. CFW appears to enhance the detection of fungal elements due to its staining properties. Laboratories will find that investing in a fluorescence microscope and appropriate filters will yield better patient care. However, further study is definitely required to grade the Candida in tissue sections and to determine the correlation between the presence of Candida and epithelial dysplasia in precancer and cancer cases and also to determine the nature of Candida: whether it is one of the causal factors in precancer and cancer. References 1. Wiesner SM, Bendel CM, Hess DJ, Erlandsen SL, Wells CL: Adherence of yeast and filamentour forms of Candida albicans to cultured enterocytes. Crit Care Med 2002;30: Samarnayake LP, MacFarlane TW: Oral Candidiosis. Butterworth, Domingues-Ferreira M, Grumach AS, Da Silva Duart AJ, De Moraes-Vasconcelos D: Esophageal cancer associated with chronic mucocutaneous candidiasis: Could chronic candidiasis lead to esophageal cancer? Med Mycol 2008;47: Krogh P, Hald B, Holmstrup P: Possible mycological etiology of oral mucosal cancer: Catalytic potential of infecting Candida albicans and other yeasts in production of N-nitrosobenzylmethylamine. Carcinogenesis 1987;8: Monheit JE, Cowan DF, Moore DG: Rapid detection of fungi in tissues using calcofluor white and fluorescence microscopy. Arch Pathol Lab Med 1984;108: Monheit JG, Brown G, Kott MM, Schmidt WA, Moore DG: Calcofluor white detection of fungi in cytopathology. Am J Clin Pathol 1986;85: Lynch DP, Gibson DK: The use of Calcofluor white in the histopathologic diagnosis of oral candidiasis. Oral Surg Oral Med Oral Pathol 1987;63: Mahajan BK: Methods in Biostatistics. Fourth edition. Smt Indu Mahajan Publications, Harrington BJ, Williams DL: Rapid, presumptive identification of Torulopsis (Candida) glabrata and Candida krusei using calcofluor white. Lab Med 2007;38: Krogh P, Homstrup P, Thorn JJ, Vedtofte P, Pindberg JJ: Yeast species and biotypes associated with oral leukoplakia and lichen planus. Oral Surg Oral Med Oral Pathol 1987;63: Barrett AW, Kingsmill VJ, Speight PM: The frequency of infection in biopsies of oral mucosal lesions. Oral Dis 1998;4: Harrington B: Use of calcofluor white in clinical mycology. Lab Med 1984;15: Rajendran R, Sivapathasundharam R (editors): Shafer s Textbook of Oral Pathology. Fifth edition. New Delhi, Elsevier, ACTA CYTOLOGICA Volume 54 Number 5 September October 2010