Short communications & case reports

The use of Calcofluor white in the histopathologic diagnosis of oral candidiasis Denis P. Lynch, D.D.S., Ph.D..* and Deborah K. Gibson, H.T., H.T.L. (A.S.C.P.),** Houston. Texas

DEPARTMENT OF PATHOLOGY AND RADIOLOGY. THE UNIVERSITY OF TEXAS HEALTH SCIENCE CENTER AT HOUSTON. DENTAL BR.ANCH

Calcofluor white (CW) is a water-soluble, colorless dye that selectively binds to beta l-4 polysaccharides (cellulose) in fungal cell walls. The planar, transform of the molecule fluoresces light blue when exposed to UV light (345 to 365 nm). Recuts of all biopsy specimens, accessioned over a 6-month period, that had been stained with Grocott’s methenamine silver (GMS) were stained with CW and examined. Of the 34 cases from which specimens were reviewed, 17 had specimens that were positive for the presence of fungal organisms as indicated by both GMS and CW staining. Specimens from all of the 17 remaining cases were negative for the presence of fungal organisms, as staining with both GMS and CW indicated. Similar results were obtained with direct fungal smears. The CW technique has significant advantages over traditional methods, including rapidity, cost, sensitivity, simplicity, and absence of interference with permanent fungal stains. (ORAL SURG. ORAL MED. ORAL PATHOL. 1987;63:698-703)

A new technique for the rapid identification of fungi in tissues has been described by Monheit and co-workers’: the use of Calcofluor white (CW), a water-soluble, colorless dye used as an optical bright- ener in the textile and paper industries.7,‘6.‘7 CW selectively and permanently binds to beta 1-4 glyco- sidic linked polysaccharides (cellulose) in the cell walls of plants, algae, and fungi.?-’ Such polysaccha- rides are especially prominent in budding mycelia and septation sites, allowing an easy distinction from other possible fluorescing materials such as cotton fibers.8 The planar, trans-isomer fluoresces light blue upon exposure to UV light.4 Unbound molecules are photo-isomerized to the nonplanar, nonfluorescing cis form, further enhancing fungal staining (Fig. 1) .9 The characteristic fluorescence is particularly intense and easily discernible from stained back-

*Assistant Professor. **Chief Histology Technician

696

ground components such as collagen, elastin, and keratin, even in thick specimens.‘O CW has minimal binding to bacterial cell walls.’

We were interested in determining the applicabil- ity of CW as a nonspecific fluorochrome in the histopathologic diagnosis of oral fungal infections.

MATERIALS AND METHODS

All biopsy specimens accessioned over a 6-month period (n = 3 152) that had been stained for fungal organisms with Grocott’s methenamine silver (GMS) were retrieved from the departmental files. Of the 34 cases reviewed, 17 had specimens that were previously diagnosed as having fungal organisms present.

Additional 5 pm sections were cut from each block, deparaffinized, hydrated to water, and cover- slipped with a solution of 0.1% (w/v) CW (disodium salt of 4,4’-bis-[Canilino-bis-diethyl-amino-S-tri- azin-2-ylaminol-2,2’-stilbene-disulfonic acid, Calco- fluor, Polysciences, Inc., Warrington, Pennsylvania)

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I 300 320 340 360 380 400 420 440 460 480 500

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Fig. 1. CW solutions are photo-isomerized upon exposure to fluorescent room lights (320 to 380 nm) as

well as upon exposure to UV light, necessitating use of actinic (red) glassware for storage. More than 90% of potential photo-isomerization occurs within 2 hours of exposure.

Fig. 2. Both fungal hyphae and yeast forms are easily visible with CW staining. Budding mycelia and septation sites are especially prominent because of the increased amounts of beta l-4 polysaccharides present. (Original magnification, X50.)

in distilled water containing 0.05% Evans blue as a counterstain to suppress background fluorescence. Although the solution is relatively stable, it was kept in actinic (red) glassware to prevent photo-isomeri- zation and degradation. The procedure was perform- ed under fluorescent room lighting, and the slides were immediately placed in cardboard slide hold- ers.

All sections were examined with a Nikon Labo- phot microscope (Nikon, Nippon Kogaku K.K., Tokyo, Japan) equipped with plan achromatic lenses, episcopic-fluorescence attachment EF-D, a high- pressure mercury light source (HBO lOOw/2,

Osram, West Germany) and a UV filter cassette containing a 330 to 380 nm excitation filter, a 400 nm dichroic mirror, and a 420 nm absorption filter. Photomicrography was done with a Nikon Microflex UFX camera and Kodak Ektachrome daylight film (EL 135) (Eastman Kodak Company, Rochester, New York), ASA 400, exposed and developed at ASA 800.

RESULTS

There was complete concordance between positive GMS and CW diagnoses. There were no false- positives (Figs. 2 and 3).

700 Lynch and Gibson

Fig. 3. UV light photo-isomerizes CW. A, CW-stained section is shown after I5 seconds of exposure to UV light. B, Identical section is presented after 90 seconds of exposure. Not only is the staining of fungal hyphae enhanced. but background fluorescence is also suppressed. (Original magnilication. X50.)

DISCUSSION

Candida albicans is a common oral fungal orga- nism that can be cultured in 50% of clinically asymptomatic adults and 90% of patients with removable dentures.“, ” When the microbial balance is altered in some way, however, Candida albicans becomes opportunistically infective rather than sym- biotic. Contributing or predisposing factors to such opportunistic infection include antibiotic suppression of normal oral flora (either intentional or second- ary), immunodeficiency (either inherited or

acquired), diabetes mellitus, xerostomia brought about by radiation therapy, anticholinergic drugs or autoimmune sialadenitis, corticosteroid therapy, and chronic irritation (e.g., tobacco use).

Oral candidiasis has been classified into four general categories: (1) acute pseudomembranous, (2) acute atrophic, (3) chronic atrophic, and (4) chronic hyperplastic candidiasis.‘;

Acute pseudomembranous candidiasis or thrush is the most common form of the disease and is most often found in infants and the chronically debili-

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Diagnosis of oral candidiasis 701

60.

WAVELENGTH (nm,

Fig. 4. Absorbance range of trans-CW corresponds to spectral transmission wavelengths of UV light. Peak absorbance of CW parallels the peak transmission wavelengths of UV light with no overlap with violet and blue light wavelengths.

tated. It is characterized by soft white plaques that can be rubbed off, leaving a variably erythematous base. Acute atrophic candidiasis is manifested as a painful mucosal erythema without any appreciable white plaques which often follows antibiotic therapy.

Chronic atrophic candidiasis is variably symptom- atic and is usually found on denture-bearing mucosa. It has been historically referred to as denture sore mouth and angular cheilitis and is frequently related to poor oral hygiene. Chronic hyperplastic candidia- sis is a unique form of the disease that results in a reactive epithelial hyperplasia, which does not rub off. In addition, this form of candidiasis is thought by some investigators to represent a premalignant lesion becasuse of the prevalence of atypia noted in biopsy specimens from such lesions.lJ

The clinical diagnosis of oral candidiasis should be confirmed by culture and/or smear before the insti- tution of antifungal therapy. When the clinical presentation is classic and the patient is symptomat- ic, clinicians ofter prefer to empirically institute drug therapy before confirmatory laboratory tests. In many cases, however, other possible causes of the clinical signs and symptoms cannot be immediately excluded, and therapeutic intervention is delayed pending confirmatory laboratory results.

A number of procedures are currently employed in the diagnosis of candidiasis. When Candida albicans is suspected clinically, office procedures used to detect the organism include potassium hydroxide (KOH) preparations and Gram-stained smears. Potassium hydroxide preparations, although rapid, have inherent artifacts caused by protein denatur-

ation.15 Because of such problems, periodic acid- Schiff (PAS) stains are recommended for all KOH- negative preparations.” More time-consuming pro- cedures include PAS and Gomori’s or stains.” While some “rapid” silver stains have been introduced, they are comparatively slow, require frozen sections, and have an unacceptably high incidence of technical failure.’ The organism can also be cultured on Sabouraud’s medium, although with this procedure it often takes days to confirm fungal growth. While Candida albicans may be suspected in routine sec- tions of formalin-fixed, paraffin-embedded tissue stained with hematoxylin and eosin, the organisms are often obscure and the diagnosis must be con- firmed with special stains, such as PAS or GMS.’ Each technique has its own advantages and disad- vantages, such as cost, ease of technique, induction of artifacts, sensitivity, ease of diagnosis, rapidity of the procedure, and necessary equipment.

Other authors have reported that violet and blue light can also be used to induce fluorescence of CW.’ The maximum absorbance of the trans-isomer occurs at 340 to 400 nm (peak 345 to 365 nm), and longer-wavelength light is ineffective in achieving the desired amount of fluorescence (Fig. 4). The color of fluorescence of CW has also been reported as apple-green or blue-green.7.‘h In all of our experi- ments the fluorescence was uniformly light blue (400 to 440 nm). Occasional reddish orange background material was noted with the use of the 0.5% Evans blue counterstain.’ The use of CW did not interfere with the subsequent use of either GMS or PAS staining (Fig. 5).

702 Lynch and Gibson

Fig. 5. A, Routine section stained with CW is shown. B. Identical section is shoMn after rinsing with distilled water and staining with PAS. (Original magnification. X25.)

Similar sensitivity and specificity have been reported for frozen section and exfoliative cytology diagnoses of candidal infections.16, ” Deep fungal infections with Mucor species, Aspergillus fumigat- us, and Cryptococcus neoformans have also stained positively with CW, as have wet mount preparations of cultured organisms.” Body fluids and cellophane tape preparations reacted equally well.’ We have used similar techniques in our laboratory with direct

fungal smears and have experienced equally satisfy- ing results.

CONCLUSIONS

The CW technique has a number of advantages over traditional methods:

1. The technique is extremely rapid, requiring less than 30 seconds from preparation of hydrated speci- men to viewing of the slide.

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2. The technique is remarkably inexpensive, cost- ing less than $0.01 per slide. Questionable slides can be stained initially with CW and, if they are negative for fungal organisms, more expensive and time- consuming special stains need not be used for perma- nent filing of material.

3. No specific techniques are required other than routine histologic processing.

4. The stain is extremely sensitive. 5. CW staining does not disrupt cellular detail. 6. CW staining does not interfere with subsequent

GMS or PAS staining when required for permanent filing of material.

REFERENCES

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6.

7.

8.

Monheit JG, Cowan DF, Moore DG: Rapid detection of fungi in tissues using Calcofluor white and fluorescence microsco- py. Arch Pathol Lab Med 108:616-618,1984. Darken M: Applications of fluorescent brighteners in biolog- ical techniques. Science 133:1704-1705, 1961. Darken M: Absorption and transport of fluorescent brighten- ers by microorganisms. Appl Microbial 10~387-393, 1962. Data Sheet #265: Calcofluor white M2R, Purified, Poly- sciences, Inc., Warrington, Pa., April 1982. Harrington BJ, Raper KB: Use of a fluorescent brightener to demonstrate cellulose in the cellular slime molds. Appl Microbial 16:106-I 13, 1968. Hughes J, McCully ME: Use of an optical brightener in the study of plant structure. Stain Technol 50:3 19-329, 1975. Maeda H. Ishida N: Specificity of binding of hexopyranosyl polysaccharides with fluorescent brightener. J Biochem (To- kyo) 62~267-278, 1967. Hayashibe M, Katohda S: Initiation of budding and chitin- ring. J Gen Microbial 19:23-29, 1973.

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Lewis GN, Magel TT, Lipkin DL: Absorption and re- emission of light by cis- and trans-stilbenes and the efficiency of their photochemical isomerization. J Am Chem Sot 62: 2973-2980, 1940. Monheit JG, Brown G, Kott MM, Schmidt WA, Moore DG: Calcofluor white detection of fungi in cytopathology. Am J Clin Pathol 85~222-225, 1986. Arendorf TM, Walker DM: The prevalence and intraoral distribution of Can&da a&cans in man. Arch Oral Biol 25~1-7, 1980. Berdicevsky 1: Oral Candida of asymptomatic denture wear- ers. Int J Oral Surg 9:113-l 15, 1980. Lehner T: Classification and clinico-pathologic features of Candida infection in the mouth. In Winner HI. Hurley R (editors): Symposium on Candida infections, Edinburgh, 1966, E. & S. Livingstone, Ltd., p. 119. Cawson RA: Leukoplakia and oral cancer. Proc Roy Sot Med 62~610-614, 1969. Hageage GJ, Harrington BJ: Use of Calcofluor white in clinical mycology. Lab Med 15:109-l 12, 1984. Haley LD, Callaway CS: Laboratory methods in medical Mycology. U.S. Department of Health. Education and Wel- fare publication no. (CDC) 78-8361, Government Printing Office, 1978, p. 47. Luna LG: Manual of histologic staining methods of the Armed Forces Institute of Pathology, ed. 3, New York, 1968, McGraw-Hill Book Company, Inc., pp. 158-160 and 230- 232.

Reprint requests to: Dr. Denis P. Lynch Department of Pathology University of Texas Dental Branch P.O. Box 20068 Houston, TX 77225