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Christopher Seng-Hong Lim, MBBS; Siew-Lin Lim, MBBS

Arch Dermatol. 2008;144(9):1228-1229.

Rapid diagnosis of superficial dermatomycoses is important so that treatment can be initiated without delay. We compared a new contrast stain with the routine test of potassium hydroxide (KOH) wet mount and culture for the diagnosis of dermatophytic and candidal dermatomycoses.

Methods
Skin scrapings were taken from outpatients with superficial dermatomycoses and cultured in Sabouraud dextrose agar with cycloheximide. They were then evaluated under a microscope using both the KOH wet mount technique and the new contrast stain. Two investigators read the slides together but were blinded to all other test results.

The new contrast stain contains 1% Chicago sky blue 6B and 8% KOH as the clearing agent. Staining was performed as previously described.¹ Slides were scanned at original magnification x10 to locate blue-staining fungal hyphae. Dermatophytes were confirmed at original magnification x40 on the finding of septate filaments. Candidal yeasts were detected as refractile oval budding yeast cells and/or pseudohyphae after lowering the condenser. Candidal slides were returned to the humidifying chamber and read again the following day to detect any change in staining intensity.

A 20% KOH solution was used for the KOH wet mount, and microscopic examination was performed in the usual manner.

Results
The findings in 36 of 59 specimens were culture positive (61%)—27 dermatophytes (24 Trichophyton species and 3 Microsporon species) and 9 candidiasis (8 Candida albicans and 1 Candida guilliermondii).

Overall sensitivities were 86% for the contrast stain and 75% for the KOH wet mount (P = .23). Overall specificities were 96% and 83%, respectively (P = .16). Respective false-positive rates were 4% and 14%, and respective false-negative rates were 17% and 25%. The stain detected 26 of 27 dermatophytic infections (96%); KOH wet mount detected 22 of 27 infections (81%) (P = .08). They were both equally sensitive in detecting 5 of 9 candidiasis cases (56%).

Dermatophytes appeared as blue septate filaments against purplish cellular debris (Figure 1). Candida species stained poorly at 20 minutes and were confirmed, with the condenser lowered, as refractile oval budding yeast cells and/or pseudohyphae (Figure 2). Reexamination the following day confirmed that Candida species took up enough stain to be directly visualized without lowering the condenser.

View larger version (105K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. In both the main photograph (original magnification x400) and the inset (original magnification x1000), new contrast stain shows bluish, long, septate filaments of Trichophyton species against a background of purplish cellular debris.

View larger version (138K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. New contrast stain shows refractile budding yeasts and pseudohyphae of Candida albicans with the condenser slightly lowered (original magnification x400).

Comment
The KOH method does not produce a color contrast and requires experience and skill to perform and interpret. The Parker-KOH stain works better for Malassezia furfur than for dermatophytes.² The procedure of obtaining skin surface biopsy specimens followed by staining³ is more complicated and less suitable for obtaining samples from interdigital webs because of the anatomy. Chlorazol-KOH testing is 60% sensitive and 71.9% specific,⁴ but the compound may be a carcinogen. Potassium hydroxide–acridine orange staining has a sensitivity of 61.7% and specificity of 67.2%,⁴ and testing with calcofluor white with KOH is 92% sensitive and 95% specific,⁵ but both require a fluorescent microscope.

Analysis with the new contrast stain was more sensitive and specific than the KOH wet mount, although the difference was not statistically significant. It made locating dermatophytes particularly easy. We also found in an earlier study¹ that this stain was as good as the Parker-KOH stain for confirming the diagnosis of pityriasis versicolor.

The new contrast staining method described herein is rapid and simple, requiring only an ordinary light microscope. We recommend further evaluation of this staining technique by experienced investigators in a larger study.

AUTHOR INFORMATION
Correspondence: Dr C. Lim, Department of Surgery, St George Hospital, Gray Street, Kogarah, NSW 2217, Australia (fluppy888{at}yahoo.com).

Author Contributions: Drs C. Lim and S.-L. Lim contributed equally to this study. Study concept and design: C. Lim and S.-L. Lim. Acquisition of data: C. Lim and S.-L. Lim. Analysis and interpretation of data: C. Lim and S.-L. Lim. Drafting of the manuscript: C. Lim and S.-L. Lim. Critical revision of the manuscript for important intellectual content: C. Lim and S.-L. Lim. Administrative, technical, and material support: C. Lim and S.-L. Lim.

Financial Disclosure: None reported.

Additional Contributions: Kah-Beng Lim, MRCP(UK), who provided the new contrast stain used in this study, is the father of the investigators. He collected and coded the specimens and provided advice on the study design but was not involved in its evaluation. Yazhong Deng, MD, MBA, Gleneagles Clinical Research Center, helped with statistical analysis.

REFERENCES
1. Lim S-L, Lim CS-H. New contrast stain for the rapid diagnosis of pityriasis versicolor. Arch Dermatol. 2008;144(8):1058-1059. FREE FULL TEXT 2. Crespo-Erchiga V, Florencio VD. Malassezia yeasts and pityriasis versicolor. Curr Opin Infect Dis. 2006;19(2):139-147. ISI | PUBMED 3. Lousbergh D, Buntinx F, Pierard G. Diagnosing dermatomycosis in general practice. Fam Pract. 1999;16(6):611-615. FREE FULL TEXT 4. Panasiti V, Borroni RG, Devirgillis V; et al. Comparison of diagnostic methods in the diagnosis of dermatomycoses and onychomycoses. Mycoses. 2006;49(1):26-29. FULL TEXT | ISI | PUBMED 5. Haldane DJ, Robart E. A comparison of calcofluor white, potassium hydroxide and culture for the laboratory diagnosis of superficial fungal infections. Diagn Microbiol Infect Dis. 1990;13(4):337-339. FULL TEXT | ISI | PUBMED

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