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Figure 1.
Contrast stain of short bluish hyphae and flask-shaped yeasts of Malassezia furfur (oil immersion, original magnification ×1000). Malassezia furfur cell walls stain blue whereas their cytoplasm stain purplish. Hyphae were sometimes observed arranged end to end, falsely giving the appearance of septate filaments. Flask-shaped budding yeasts with septa at the neck can be seen. The inset depicts the contrast stain at an original magnification of ×400.

Contrast stain of short bluish hyphae and flask-shaped yeasts of Malassezia furfur (oil immersion, original magnification ×1000). Malassezia furfur cell walls stain blue whereas their cytoplasm stain purplish. Hyphae were sometimes observed arranged end to end, falsely giving the appearance of septate filaments. Flask-shaped budding yeasts with septa at the neck can be seen. The inset depicts the contrast stain at an original magnification of ×400.

Figure 2.
Parker–potassium hydroxide stain of bluish short hyphae and spores against orange cellular debris (a) and bluish precipitate (b) (original magnification ×400).

Parker–potassium hydroxide stain of bluish short hyphae and spores against orange cellular debris (a) and bluish precipitate (b) (original magnification ×400).

1.
Cohen  MM Simple procedure for staining tinea versicolor (M. furfur) with fountain ink. J Invest Dermatol 1954;22 (1) 9- 10
PubMedArticle
2.
Crespo-Erchiga  VFlorencio  VD Malassezia yeasts and pityriasis versicolor. Curr Opin Infect Dis 2006;19 (2) 139- 147
PubMedArticle
3.
Haldane  DJRobart  E A comparison of calcofluor white, potassium hydroxide, and culture for the laboratory diagnosis of superficial fungal infection. Diagn Microbiol Infect Dis 1990;13 (4) 337- 339
PubMedArticle
Research Letter
August 18, 2008

New Contrast Stain for the Rapid Diagnosis of Pityriasis Versicolor

Arch Dermatol. 2008;144(8):1058-1059. doi:10.1001/archderm.144.8.1058

Successful treatment of pityriasis versicolor requires an accurate diagnosis. Culture is not useful because its etiologic agent Malassezia furfur is part of the normal skin flora. Hence, diagnosis is usually based on direct microscopic examination of skin scrapings. The standard potassium hydroxide (KOH) wet mount suffers from a lack of color contrast. Parker blue-black ink has been added to potassium hydroxide to highlight fungal hyphae and spores against the surrounding cellular debris.1,2 The fluorescent brightener calcofluor white specifically binds to chitin, but this method incurs the additional cost of a fluorescence microscope.3 In this study, we compared a new contrast stain with the Parker-KOH stain in patients with a clinical diagnosis of pityriasis versicolor.

Methods

The study population comprised male and female patients who were diagnosed as having pityriasis versicolor by the dermatologist Kah-Beng Lim, MRCP(UK) (K.B.L.).

Areas of skin to be scraped were first cleaned with an alcohol swab to remove traces of creams and reduce surface bacteria. Skin scrapings were taken with a number 15 scalpel blade, placed on a clean microscope slide, and covered by a second microscope slide. The 2 slides were then bound at the ends with microporous tape, labeled with the patient's name, and stored in a screw-capped plastic bottle for microscopic examination.

Duplicate slides were made of each patient's skin scrapings for microscopic examination and coded by K.B.L. One slide was stained with the contrast stain and the other with the Parker-KOH stain containing 1 part Parker blue-black ink and 1 part 20% KOH. Both investigators collaborated in the reading of the slides but were not aware of all other test results when they read the slides. The Parker-KOH stain was used as the reference method for calculating the sensitivity and specificity of the contrast stain.

Contrast Stain

The new contrast stain contains 1% Chicago sky blue 6B and 8% KOH as the clearing agent. A drop of the contrast stain was mixed into the specimen on a clean microscope slide and left to sit in a humidifying chamber (covered plastic container lined by moist paper towel) for about 20 minutes at room temperature. A coverslip was applied over the specimen and gently pressed to remove air bubbles. Excess stain was blotted off with a paper towel. The slide was first scanned at an original magnification of ×10 using an Olympus CH-2 microscope (Olympus, Tokyo, Japan). Fungal elements stained blue against a purplish background of cellular debris. Malassezia furfur was confirmed at an original magnification of ×40 on the finding of short angular hyphae and spherical spores. Oil immersion (original magnification ×100) was used for a more detailed study of fungal morphology.

Parker-KOH Stain

Briefly, a drop of the stain was mixed into the specimen, covered with a coverslip, and left to sit at room temperature for 20 minutes. The coverslip was then pressed gently to remove air bubbles and excess solution was blotted off with a paper towel. Slides were examined at original magnifications of ×10 and ×40 with the Olympus CH-2 microscope. Malassezia furfur was confirmed by the presence of blue staining hyphae and spherical spores.

The sensitivity and specificity of the new contrast stain were calculated using 2 × 2 contingency tables with the Parker-KOH stain as the reference method.

Results

A total of 24 specimens were examined, 22 (92%) of which were positive for M furfur by both methods. The new contrast stain had a sensitivity and specificity of 100%, using the Parker-KOH stain as the reference method. Malassezia furfur cell walls stained blue against the purplish background of cellular debris. Hyphae were short and angular, and there were often clusters of spherical or flask-shaped yeasts, giving the appearance of spaghetti and meatballs (Figure 1). Darker staining was observed when the slide was left to sit longer. Hyphae and spores appeared blue against a light orange background of cellular debris with the Parker-KOH stain, and bluish precipitation was noted (Figure 2).

Comment

Both investigators are novices to microscopic examination of skin scrapings. Hence, it was decided that they could confer in the interpretation of the slides. The new contrast stain achieved a sensitivity and specificity of 100% when compared with the Parker-KOH stain as the reference method. A bluish precipitate was commonly observed with the Parker-KOH stain, whereas none occurred with the new contrast stain. In addition, the new stain is better at highlighting the morphologic features of both spores and hyphae compared with the Parker-KOH stain. Budding M furfur yeasts with septa at the neck can be clearly seen under oil illumination. The new contrast stain is therefore a useful alternative to the Parker-KOH stain. Unpublished results (January 6, 2008) from an ongoing study by the authors suggest that this stain may also be very useful for the diagnosis of dermatophytosis.

The new contrast stain contains KOH as the clearing agent. Laboratory overalls and gloves should be worn to protect clothes and hands. The staining procedure is simple and rapid to perform and requires only an ordinary light microscope.

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Article Information

Correspondence: Dr S.-L. Lim, Department of Pediatrics, Wollongong Hospital, Loftus St, Wollongong, NSW 2500, Australia (iota@tpg.com.au).

Author Contributions: Drs S.-L. Lim and C. S.-H. Lim had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Both authors contributed equally to the study. Study concept and design: S.-L. Lim and C. S.-H. Lim. Acquisition of data: S.-L. Lim and C. S.-H. Lim. Analysis and interpretation of data: S.-L. Lim and C. S.-H. Lim. Drafting of the manuscript: S.-L. Lim and C. S.-H. Lim. Critical revision of the manuscript for important intellectual content: S.-L. Lim and C. S.-H. Lim. Administrative, technical, and material support: S.-L. Lim and C. S.-H. 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.

References
1.
Cohen  MM Simple procedure for staining tinea versicolor (M. furfur) with fountain ink. J Invest Dermatol 1954;22 (1) 9- 10
PubMedArticle
2.
Crespo-Erchiga  VFlorencio  VD Malassezia yeasts and pityriasis versicolor. Curr Opin Infect Dis 2006;19 (2) 139- 147
PubMedArticle
3.
Haldane  DJRobart  E A comparison of calcofluor white, potassium hydroxide, and culture for the laboratory diagnosis of superficial fungal infection. Diagn Microbiol Infect Dis 1990;13 (4) 337- 339
PubMedArticle
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