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Figure 1. Bluish septate hyphae of Trichophyton species (Chicago Sky Blue stain, original magnification  ×400). The inset shows Trichophyton species under oil immersion (Chicago Sky Blue stain, original magnification  ×1000).

Figure 1. Bluish septate hyphae of Trichophyton species (Chicago Sky Blue stain, original magnification ×400). The inset shows Trichophyton species under oil immersion (Chicago Sky Blue stain, original magnification ×1000).

Figure 2. Bluish septate branching hyphae of Fusarium species (Chicago Sky Blue stain, original magnification  ×400). The inset shows septate hyphae and conidia of Fusarium species under oil immersion (Chicago Sky Blue stain, original magnification  ×1000).

Figure 2. Bluish septate branching hyphae of Fusarium species (Chicago Sky Blue stain, original magnification ×400). The inset shows septate hyphae and conidia of Fusarium species under oil immersion (Chicago Sky Blue stain, original magnification ×1000).

Figure 3. Bluish septate hyphae of Aspergillus species showing dichotomous branching (Chicago Sky Blue stain, original magnification  ×400).

Figure 3. Bluish septate hyphae of Aspergillus species showing dichotomous branching (Chicago Sky Blue stain, original magnification ×400).

Table. Fungi Isolated From 38 Nail Specimens
Table. Fungi Isolated From 38 Nail Specimens
1.
Panasiti V, Borroni RG, Devirgiliis V,  et al.  Comparison of diagnostic methods in the diagnosis of dermatomycoses and onychomycoses.  Mycoses. 2006;49(1):26-29PubMedArticle
2.
Abdelrahman T, Letscher Bru V, Waller J, Naocco G, Candolfi E. Dermatomycosis: comparison of the performance of calcofluor and potassium hydroxide 30% for the direct examination of skin scrapings and nails.  J Mycol Med. 2006;16(2):87-91Article
3.
Weinberg JM, Koestenblatt EK, Tutrone WD, Tishler HR, Najarian L. Comparison of diagnostic methods in the evaluation of onychomycosis.  J Am Acad Dermatol. 2003;49(2):193-197PubMedArticle
4.
Lim SL, Lim CSH. New contrast stain for the rapid diagnosis of pityriasis versicolor.  Arch Dermatol. 2008;144(8):1058-1059PubMedArticle
5.
Lim CSH, Lim SL. New contrast stain for the rapid diagnosis of dermatophytic and candidal dermatomycoses.  Arch Dermatol. 2008;144(9):1228-1229PubMedArticle
Research Letter
Aug 2011

New Contrast Stain for the Rapid Diagnosis of Onychomycosis

Author Affiliations

Author Affiliations: Medical Classification Center, Central Manpower Base, Ministry of Defense, Singapore (Dr C. S.-H. Lim); Department of Pediatrics, Wollongong Hospital, Wollongong, New South Wales, Australia (Dr S.-L. Lim).

Arch Dermatol. 2011;147(8):981-982. doi:10.1001/archdermatol.2011.208

Rapid confirmation of onychomycosis is desirable to differentiate it from nonfungal causes of nail dystrophy and to initiate appropriate therapy. In this study, we compared the performance of Chicago Sky Blue (CSB) stain with that of routine potassium hydroxide (KOH) preparation, using culture as the standard, in the diagnosis of onychomycosis.

Methods

Nail specimens from patients with a clinical diagnosis of onychomycosis were analyzed with CSB stain, KOH preparation, and Sabouraud dextrose agar culture with added antibiotics. Patients were selected on a random sequential basis. Scrapings were taken from the affected nail surface with a blunt scalpel, and subungual debris was obtained with an eye curette. Nail clippings were taken to include subungual debris as far proximal as was tolerable, and scrapings were taken from their undersurface for tests.

Slides for CSB stain were fixed with acetone and left to dry. A drop of 20% KOH was added, and the slides were left to sit at room temperature for 20 to 30 minutes before adding a drop of CSB stain. The slides were examined 10 minutes later with an Olympus BX41 microscope (Olympus, Center Valley, Pennsylvania) at ×10, ×40 and where necessary, under oil immersion at ×100 magnification. The 20% KOH prepared slides were examined after 20 minutes at ×10 and ×40 magnifications with the condenser lowered.

Results

Thirty-eight of 63 nail specimens were culture positive (60%) (Table). Overall sensitivities were 63% for CSB stain and 39% for the KOH wet mount (P  =  .09). Overall specificities were 96% and 92%, respectively (P  =  .67). Positive and negative predictive values were 96% and 63%, respectively, for CSB stain and 88% and 49%, respectively, for the KOH preparation.

Fungal elements stained blue against a pink background of cellular debris with CSB stain. Dermatophytes appeared as blue septate filaments (Figure 1); yeasts appeared as budding or nonbudding yeast cells; and nondermatophytes appeared as septate branching hyphae, which were more contorted and wider and of more variable diameters than dermatophytes (Figure 2 and Figure 3).

Comment

The number of nondermatophyte molds isolated in the present study is unusually high. This could be related to the fact that the dermatology practice is located in a private tertiary hospital where many of the patients are referred by their general practitioners or specialist physicians and may have had prior treatments or underlying medical problems.

The KOH preparation is inexpensive but does not produce a color contrast and requires considerable skill to interpret. Even in experienced hands, KOH has been reported to have a false-negative rate of 5% to 15%. We analyzed data provided by Panasiti et al1 and calculated chlorazol-KOH and KOH-acridine orange to have a sensitivity of 55% and 39%, respectively, and specificity of 74% and 85%, respectively, for onychomycosis.1

Calcofluor white with KOH, which is known to be very sensitive and specific for skin scrapings, has been shown to be 87% sensitive and 89% specific for the diagnosis of onychomycosis,2 but analysis requires a fluorescent microscope. Histologic evaluation of nail plate clippings stained with periodic acid –Schiff reagent is the most sensitive test,3 but it is not practical as an office procedure.

Polymerase chain reaction methods offer the possibility of rapid species identification, but such molecular approaches are still developing and are not available for general use. Although we used culture as the gold standard for this study, it should be remembered that false negatives might arise when the specimen does not contain an adequate number of organisms or contains only dead or nonviable fungi.

The CSB stain was both more sensitive and more specific than the KOH preparation, although the difference was not statistically significant. The CSB provided the color contrast that made it easier to locate fungal elements even when they were scanty. It also did a superior job of highlighting the morphologic characteristics of both spores and hyphae and enabled oil-immersion examination. Oil-immersion analysis might be useful for differentiating between dermatophytes, yeasts, and nondermatophyte molds, and we suggest that this is an interesting area for further research by experienced mycologists.

In conclusion, the present study confirms that CSB stain is useful for the rapid diagnosis of onychomycosis as well as pityriasis versicolor4 and dermatophytic infections.5 Staining with CSB is an inexpensive, rapid office procedure that is less skill dependent than the routine KOH preparation and requires only an ordinary light microscope. The cost for a 12-mL bottle of CSB, which stains about 200 specimens, is only $25.00. We recommend it as an alternative to calcofluor white and periodic acid –Schiff stain.

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Article Information

Correspondence: Dr Christopher Seng-Hong Lim, Medical Officer, Central Manpower Base, Ministry of Defense, Depot Road, Singapore 109680 (fluppy888@yahoo.com).

Accepted for Publication: December 7, 2011.

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

Financial Disclosure: None reported.

Additional Contributions: Kah-Beng Lim, MRCP(UK), the father of both investigators, developed CSB stain and supplied the stain used in the study. He also collected and coded the specimens and provided advice on the study design; he was not involved in study evaluation.

References
1.
Panasiti V, Borroni RG, Devirgiliis V,  et al.  Comparison of diagnostic methods in the diagnosis of dermatomycoses and onychomycoses.  Mycoses. 2006;49(1):26-29PubMedArticle
2.
Abdelrahman T, Letscher Bru V, Waller J, Naocco G, Candolfi E. Dermatomycosis: comparison of the performance of calcofluor and potassium hydroxide 30% for the direct examination of skin scrapings and nails.  J Mycol Med. 2006;16(2):87-91Article
3.
Weinberg JM, Koestenblatt EK, Tutrone WD, Tishler HR, Najarian L. Comparison of diagnostic methods in the evaluation of onychomycosis.  J Am Acad Dermatol. 2003;49(2):193-197PubMedArticle
4.
Lim SL, Lim CSH. New contrast stain for the rapid diagnosis of pityriasis versicolor.  Arch Dermatol. 2008;144(8):1058-1059PubMedArticle
5.
Lim CSH, Lim SL. New contrast stain for the rapid diagnosis of dermatophytic and candidal dermatomycoses.  Arch Dermatol. 2008;144(9):1228-1229PubMedArticle
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