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Year : 2018  |  Volume : 5  |  Issue : 1  |  Page : 39-41

A simple but yet novel stain in the diagnosis of onychomycosis

Department of Dermatology, Venereology and Leprosy, Yenepoya Medical College, Yenepoya University, Mangalore, Karnataka, India

Date of Web Publication22-May-2018

Correspondence Address:
Dr. Thansiha Nargis
Department of Dermatology, Yenepoya Medical College, Yenepoya University, Deralakatte, Mangalore - 575 018, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijdpdd.ijdpdd_19_17

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Background: Onychomycosis can resemble other nail dystrophies and hence the clinical diagnosis should be supplemented with a laboratory diagnosis. Rapid diagnosis of onychomycosis is the necessary to initiate early therapy. In this study, the efficacy of Chicago Sky Blue (CSB) stain in comparison with that of routine potassium hydroxide (KOH) preparation in the diagnosis of onychomycosis was evaluated. Methods: A prospective study of 42 nails with a clinical diagnosis of distal lateral subungual onychomycosis. KOH mount followed by direct microscopic examination of the subungual debris was performed. This was followed by microscopic examination after addition of 1% CSB. Results were interpreted by two experienced dermatologists. Results: 18 (42.9%) nails showed KOH positivity and 37 (88.1%) were positive for CSB stain. Conclusions: CSB staining was more sensitive and was more accurate in the rapid diagnosis of onychomycosis. Colour contrast provided by the CSB made it easier identification of fungal elements.

Keywords: Chicago sky blue stain, onychomycosis, potassium hydroxide mount

How to cite this article:
Nargis T, Shenoy MM, Bejai V, Gopal V. A simple but yet novel stain in the diagnosis of onychomycosis. Indian J Dermatopathol Diagn Dermatol 2018;5:39-41

How to cite this URL:
Nargis T, Shenoy MM, Bejai V, Gopal V. A simple but yet novel stain in the diagnosis of onychomycosis. Indian J Dermatopathol Diagn Dermatol [serial online] 2018 [cited 2023 Mar 27];5:39-41. Available from: https://www.ijdpdd.com/text.asp?2018/5/1/39/232950

  Introduction Top

Onychomycosis has to be differentiated from other causes of nail dystrophy before treating them with antifungals. Detecting fungal elements in the nail is time-consuming and often difficult to visualize using conventional methods such as potassium hydroxide (KOH) mount. Many stains such as the Parker blue–black ink, Swartz-Lamkins stain, Chlorazole-KOH, KOH-acridine orange stain, and Calcofluor White with KOH stains both the dermatophytes and the Malassezia species have been described.[1],[2],[3],[4] The Chicago Sky Blue (CSB) 6B (Direct Blue 1) stain along with KOH was helpful in the rapid detection and also improving the accuracy of nail fungal elements in routine dermatophytosis.[5] Very few studies have been conducted to explore the utility of CSB staining in the diagnosis of onychomycosis.

  Materials and Methods Top

A total of 42 nails from 36 patients with a clinical diagnosis of distal lateral subungual onychomycosis (DLSO) were selected for the study. Subungual debris was collected from the under the surface of the infected nails as proximal as possible after cleaning with 70% alcohol. A drop of 20% KOH was added and the slides were left at room temperature for 30 min. Examination of the mount was done under microscope for the fungal elements by two experienced dermatologists. Slides showing branching hyphae were considered as positive. A drop of 1% CSB stain was added to the mount and examined under microscope after 10 min by the same dermatologists. CSB stain was prepared by dissolving 1 g of the CSB dye in 100 ml of distilled water forming approximately 1% solution.[5],[6] Positive results were considered when blue filaments were noted.

  Results Top

Out of the 42 nails with clinical distal lateral onychomycosis, we detected fungal elements from 18 nails (42.9%) [Table 1] using KOH preparation [Figure 1]. All these positive slides were also positive with CSB staining. An addition of nails which were negative by KOH preparation [Figure 2] were detected to be positive by CSB staining making a total 37 positive results (88.1%) [Figure 3] and [Figure 4]. Among the 42 nails, fungal elements could not be detected in 5 nails both by KOH preparation and CSB staining. Although we did not notice the color contrast mentioned by the other articles, we noted that the fungal hyphae stained deeply to give a bluish color when left for a longer time. Fungal elements which were not visualized on KOH preparation due to incompletely lysed keratin [Figure 2] was visualized when CSB staining was performed [Figure 3].
Table 1: Percentage of nails positive for fungal elements (n=34)

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Figure 1: Microscopic examination of potassium hydroxide preparation of subungual debris of onychomycosis showing fungal hyphae (×10)

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Figure 2: Microscopic examination of potassium hydroxide preparation of subungual debris that was negative for fungal elements (×10)

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Figure 3: Branching fungal filaments identified in the slide that was negative for potassium hydroxide preparation after adding 1% Chicago Sky Blue (×10)

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Figure 4: Chicago Sky Blue stain (×40): Morphology of the fungal elements clearly visible with branching filaments and septations

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  Discussion Top

Diagnosing onychomycosis is a challenge as well as time-consuming. Mycological confirmation requires weeks to isolate the agent on culture and often the yields on culture are poor.[7],[8] The initiation of antifungal therapy is often based on the interpretation of direct examination such as KOH preparation. The interpretation of KOH mount often requires skills, and at times, it has been reported as false negative even by experienced persons.[9],[10] The detection of fungal elements by KOH mount from a nail clipping takes a longer time for it to be lysed.[11] In this study, CSB staining could confirm significantly more cases of onychomycosis compared to KOH preparation (88.1% v/s 42.9%). Time taken for the CSB staining is a 10 min addition to the KOH preparation totaling to about 40 min; hence, it is a rapid test for the diagnosis. The percentage of cases with KOH positivity would have increased if the nails were left longer for a period of 2 h or more.[8] Utilizing CSB staining abolishes this waiting period and also enhances the ease of identifying the fungus. We need a rapid method for the diagnosis as well as a simple staining technique since most laboratories in our country are equipped only with basic infrastructure and limited qualified workforce. We found a number of staining procedures which were helpful for the rapid detection of fungal elements, but each had its own drawbacks.[1],[2],[3] Panasiti et al. compared chlorazol-KOH and KOH-acridine orange in diagnosing onychomycosis and noticed to have a sensitivity of 55% and 39%, and specificity of 74% and 85%, respectively, but it needed a fluorescent microscope.[2] Calcofluor-white has shown to be 87% sensitive and 89% specific for the diagnosis of onychomycosis, and it also requires a fluorescent microscope for the analysis.[2] Swartz-Lamkins stain was difficult to prepare and was not effective in thicker specimens like nail.[7] Periodic acid–Schiff stain has 90% sensitivity in identifying fungal elements, but the staining is used in the histopathological sections.[8]

CSB is a dye used in the textile industry, inks, and paints and also as a counterstain in immunohistochemistry. Studies by Lim and Lim et al. showed that the CSB is more sensitive and more specific than the KOH preparation. CSB provides a color contrast that makes the easier location of fungal elements even when they were scanty. It also highlighted the morphologic characteristics of both spores and hyphae and enabled oil-immersion examination.[3],[4] In this study, we observed that though the nail scrapings did not lyse, the fungal hyphae and spores had taken up the stain making the identification of the fungal elements. The staining could be observed in a simple microscope, could be easily performed in a basic office setting and by a person not highly trained in the laboratory. The stain occasionally has been seen to have taken up by artefacts, but a careful observation can distinguish them from the fungus. The limitations of this were small study group and comparison with fungal culture or nail histopathology as a gold standard method would have increased the accuracy of the results.

  Conclusion Top

CSB stain is an inexpensive, rapid office procedure that requires less skill than the routine.

KOH preparation. It can be used as a routine test in the diagnosis of superficial fungal infections, especially onychomycosis.

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Conflicts of interest

There are no conflicts of interest.

  References Top

Swartz JH, Lamkins BE. A rapid, simple stain for fungi in skin, nail scrapings, and hairs. Arch Dermatol 1964;89:89-94.  Back to cited text no. 1
Panasiti V, Borroni RG, Devirgiliis V, Rossi M, Fabbrizio L, Masciangelo R, et al. Comparison of diagnostic methods in the diagnosis of dermatomycoses and onychomycoses. Mycoses 2006;49:26-9.  Back to cited text no. 2
Abdelrahman T, LetscherBru 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;16:87-91.  Back to cited text no. 3
Lim SL, Lim CS. New contrast stain for the rapid diagnosis of pityriasis versicolor. Arch Dermatol 2008;144:1058-9.  Back to cited text no. 4
Lim CS, Lim SL. New contrast stain for the rapid diagnosis of onychomycosis. Arch Dermatol 2011;147:981-2.  Back to cited text no. 5
Lim CS, Lim SL. Practical tip: Chicago sky blue (CSB) stain can be added to the routine potassium hydroxide (KOH) wet-mount to provide a color contrast and facilitate the diagnosis of dermatomycoses. Dermatol Online J 2011;17:11.  Back to cited text no. 6
Burke WA, Jones BE. A simple stain for rapid office diagnosis of fungus infections of the skin. Arch Dermatol 1984;120:1519-20.  Back to cited text no. 7
Shenoy MM, Teerthanath S, Karnaker VK, Girisha BS, Krishna Prasad MS, Pinto J, et al. Comparison of potassium hydroxide mount and mycological culture with histopathologic examination using periodic acid-Schiff staining of the nail clippings in the diagnosis of onychomycosis. Indian J Dermatol Venereol Leprol 2008;74:226-9.  Back to cited text no. 8
[PUBMED]  [Full text]  
Weitzman I, Summerbell RC. The dermatophytes. Clin Microbiol Rev 1995;8:240-59.  Back to cited text no. 9
Elewski BE. Large-scale epidemiological study of the causal agents of onychomycosis: Mycological findings from the multicenter onychomycosis study of terbinafine. Arch Dermatol 1997;133:1317-8.  Back to cited text no. 10
Shenoy MM, Shenoy MS. Superficial fungal infections. In: Sacchidanand S, Oberoi C, Inamdar AC, editors. IADVL Textbook of Dermatology. 8th ed. Mumbai: Bhalani Publishing House; 2015. p. 1489-501.  Back to cited text no. 11


  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

  [Table 1]

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