A Novel Contrast Stain for the Rapid Diagnosis of Dermatophytoses: A Cross-Sectional Comparative Study of Chicago Sky Blue 6B Stain, Potassium Hydroxide Mount and Culture.

BACKGROUND: The current upsurge of complicated dermatophytosis in India and other parts of the world has necessitated the development of rapid and accurate diagnostic techniques. Conventional methods such as potassium hydroxide (KOH) mount lack color contrast and require a trained eye, whereas fungal culture is laborious and costly. Chicago sky blue 6B (CSB) a new contrast stain promises to be a reliable and rapid diagnostic method. It contains 1% CSB stain and is used with 10% KOH as clearing agent. AIMS AND
OBJECTIVES: The objective of this study is to compare the efficacy of a novel contrast stain (CSB) and KOH mount in the diagnosis of dermatophytoses with culture as the reference method.
METHODS: The infected skin scrapings, nail clippings, and hair specimens from 100 patients, with clinically diagnosed dermatophytoses, were subjected to KOH mount, CSB stain, and culture on Sabouraud's dextrose agar. Using fungal culture as the reference standard, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of CSB stain and KOH mount were determined. An inter-rater reliability analysis was performed using the Cohen's κ statistic to determine agreement amongst the different diagnostic modalities.
RESULTS: Of the 100 cases, the CSB stain and KOH mount were positive in 85 (85%) and 70 (70%) cases, respectively. Cultures on Sabouraud's agar yielded growth in 59 (59%) cases. The sensitivity, specificity, PPV, NPV, and accuracy of CSB stain were 98%, 34%, 68%, 93%, and 72%, respectively. The same for KOH mount were 71%, 32%, 60%, 43%, and 55%, respectively.
CONCLUSION: CSB stain is a simple, rapid, sensitive, accurate, and inexpensive office-based method with qualitatively superior demonstration of dermatophytes compared to KOH mount.

Introduction

Dermatophytoses are the most common of the superficial fungal infections with an estimated lifetime risk of 10–20%.[1] Dermatophytic infections are usually diagnosed on clinical grounds based on the typical presentation.[2] However, due to the advent of immunosuppressive drugs and disease, and usage of over-the-counter steroid–antifungal preparations, atypical presentations are on the rise and it is increasingly becoming essential to seek laboratory aid to arrive at a confirmatory diagnosis.[3]

Traditional methods such as fungal culture and potassium hydroxide (KOH) mount are routinely used for diagnosing dermatomycoses.[4] The KOH preparation is simple, cheap and rapid, but it has 5–15% false-negative rate even in trained hands.[4] Fungal culture is expensive, time-consuming, and may take up to 4 weeks to obtain the results. Skin surface biopsy and nail biopsies followed by periodic acid–Schiff stain are complicated, time–consuming, and can be impractical. Molecular methods such as polymerase chain reaction are expensive and not available for general use.[56]

Many stains such as the Parker blue–black ink, Swartz–Lamkins stain, Chlorazole–KOH, KOH–acridine orange stain, and Calcofluor White with KOH mount, have been used to stain the fungal elements, but each of them has their own limitations.[78]

Chicago Sky Blue (CSB) is a novel contrast stain that highlights the fungal hyphae blue in color against a purplish background of cellular debris and it was found to be more sensitive than KOH mount in diagnosis of pityriasis versicolor and dermatomycoses.[5678] There is a paucity of Indian studies on the utility of this stain in fungal infections. Here, we compared the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of CSB stain and KOH mount in the diagnosis of dermatophytoses.

Aims and Objectives

We compared the performance of CSB stain with KOH mount in diagnosing dermatophytoses taking mycological culture as the reference standard.

The objective was to determine the sensitivity and specificity of KOH mount and CSB stain and to study the level of agreement between different diagnostic modalities.

Inclusion criteria

Clinically diagnosed cases of dermatophytosis of all ages and both genders who consented for the study were included.

Exclusion criteria

Treatment with oral or topical antifungals in the last 2 weeks

Patients with secondary infection.

Materials and Methods

This cross-sectional comparative study was conducted over 1 year at a tertiary care hospital attached to a medical college. After obtaining clearance from the institutional ethics committee, a total of 100 clinically diagnosed dermatophytoses were included after applying the aforementioned criteria and a written informed consent was obtained from all the patients.

Sample collection

The areas to be scraped were disinfected using 70% alcohol. Skin scrapings were taken from the actively growing peripheral margins of the lesions with the blunt edge of surgical blade no. 1. Hair specimens were collected by an epilating forceps in such a way that the base of the hair shaft was also included. Nail clippings along with the attached subungual debris were taken with a nail clipper.

The collected sample (skin scrapings, nail clippings, and hair specimens) was divided into three portions and subjected to (1) 10% KOH mount for skin scrapings, 20–40% KOH mount for nail clippings and hair specimens; (2) 1% CSB staining with 10% KOH as clearing agent; and (3) culture on Sabouraud's dextrose agar (SDA) with chloramphenicol (0.005%) and cycloheximide (0.05%).

Preparation of CSB stain

One percent solution was prepared by dissolving 1 g of CSB powder in 100 ml of distilled water.[79]

Light microscopy: CSB stain and KOH mount

Duplicate slides were made with the first two portions of the sample and labeled. One drop of 10% KOH was added to both the slides and one drop of CSB stain was added to the second. Both the slides were covered gently with cover slip and the excess stain was removed using a blotting paper. Microscopic examination was done after 20 min by a single untrained observer under scanner view (4×), low power (10×), high power (40×), and oil immersion (100×) objectives of an ordinary light microscope. Positive results with CSB were considered when blue and branched septate hyphae were noted. Similarly, the slides of KOH mount were also observed for hyphae and spores.

Culture

The third portion of the sample was collected onto a sterile black card paper, inoculated into plates of SDA with chloramphenicol (0.005%) and cycloheximide (0.05%), incubated at room temperature for 2 weeks, and examined daily for growth. If no growth was observed even after 4 weeks, the plates were considered negative for culture and discarded. The culture isolates were identified on the basis of phenotypic characteristics of the colonies and microscopic examination of lactophenol cotton blue wet mount.[10]

Statistical analysis

The sensitivity, specificity, PPV, NPV, and accuracy of CSB stain and KOH mount were calculated using culture as the reference method. Cohen's κ statistical analysis was performed to determine agreement amongst the different diagnostic modalities.

The P value less than 0.05 was taken as statistically significant.

Results

Out of the 100 patients included, 56 were male and 44 were female. The highest number of cases of dermatophytoses (27%) was found in young adult age group, that is, 21–30 years.

Microscopic examination with the CSB stain and the KOH mount revealed presence of hyphae in 85 (85%) and 70 (70%) cases, respectively. Mycological culture was positive in 59 (59%) cases.

Under KOH mount, the hyphae and spores appeared as individual refractile structures without color contrast [Figure 1]. On staining with CSB, dermatophytes appeared as long blue filaments against a pink to purplish background of cellular debris [Figure 2]. The color contrast made it easier to locate fungal elements under scanner view even when they were scanty. The branching and septate were better appreciated on higher magnification [Figure 2b]. Similar results were seen on hair mount with CSB stain [Figure 3].

Figure 1

(a) Microscopic examination of KOH mount of skin scrapings (×40) showing branched hyphae. There is however no contrast with surrounding keratinous debris. (b) Microscopic examination of KOH mount (×400): long-branching hyphae seen as refractile elements

Figure 2

(a) Microscopic examination of skin scrapings showing: multiple blue branching filaments seen contrasting against a light pink to purplish background (CSB stain, ×40). (b) Microscopic examination of skin scrapings showing long branched blue-colored hyphae with septae seen (CSB stain, ×100)

Figure 3

Microscopic examination of hair mount (CSB stain, ×400): multiple blue branching hyphae of dermatophytes seen within the hair shaft (Endothrix)

The sensitivity, specificity, PPV, NPV, and overall accuracy of CSB stain were all high when compared to KOH mount [Table 1]. Using the Cohen's κ statistical analysis, slight agreement was found between KOH mount and culture (55%, κ = 0.03, P = 0.826) [Table 2]. Statistically significant slight agreement was found between CSB stain and culture (72%, κ = 0.359, P < 0.001) [Table 3]. Statistically significant fair agreement was found between CSB stain and KOH mount (85%, κ = 0.583, P < 0.001) [Table 4].

Table 1

Comparison of CSB stain and KOH mount using culture as reference method

	KOH (%)	CSB (%)
Sensitivity	71	98
Specificity	32	34
PPV	60	68
NPV	43	93
Accuracy	55	72

PPV: Positive predictive value, NPV: negative predictive value

Table 2

Culture versus KOH mount in diagnosis of dermatophytoses (test for agreement: κ-statistic)

	Culture		Total
	Positive	Negative
KOH examination
Positive	42	28	70
	71.2%	68.3%	70.0%
Negative	17	13	30
	28.8%	31.7%	30.0%
Total	59	41	100
	100.0%	100.0%	100.0%

Overall agreement was 55% with κ=0.03 (P=0.826)

Table 3

Culture versus CSB stain in diagnosis of dermatophytoses (test for agreement: κ-statistic)

	Culture		Total
	Positive	Negative
CSB
Positive	58	27	85
	98.3%	65.9%	85.0%
Negative	1	14	15
	1.7%	34.1%	15.0%
Total	59	41	100
	100.0%	100.0%	100.0%

Overall agreement was 72% with κ=0.359 (P<0.001)

Table 4

CSB stain versus KOH in diagnosis of dermatophytoses (test for agreement: κ-statistic)

	CSB		Total
	Positive	Negative
KOH examination
Positive	70	0	70
	82.35%		70.0%
Negative	15	15	30
	17.64%	100%	30.0%
Total	85	15	100
	100.0%	100.0%	100.0%

Overall agreement was 85.0% with κ 0.583 (P<0.001)

Discussion

Dermatophytoses have always been among the commonest infective dermatoses in the outpatients clinics in India. The diagnosis although often made clinically, the recent changing profile of dermatophytoses both qualitatively and quantitatively has become a cause of great anxiety among treating dermatologists.[1112] Hence, the need of the hour is the development of improved diagnostic methods not only for accurate diagnosis and prompt treatment but also for posttherapeutic and preventive strategies. The standard KOH mount, although rapid and inexpensive, does not produce a color contrast and is expertise dependent. False-negative reports may occur even in experienced hands in case of sparse fungal elements.[13] Fungal culture is less sensitive and takes a long time.[8]

CSB 6B is a diazo dye compound. It is available as a dark blue powder and is water soluble. One percent solution once prepared can be stored in a brown dropper bottle at room temperature.[8] It is primarily used to dye cotton in the textile industry. It is also used in immunohistochemistry as a counterstain for background autofluorescence and in dye-assisted macular surgery. The stain has also been reported to be useful in the diagnosis of pityriasis versicolor and candidal infections. At low concentrations, as has been used in our study, it is unlikely to cause any significant problem, although it is a corrosive agent.[9] Nevertheless, protective gloves should be used while directly handling the stain, especially during preparation of the stain. A 30-ml bottle of CSB, enough for 500 smears, costs approximately Rs. 2500.[9]

The contrast staining provided by CSB makes it easy even for an untrained observer to locate the fungal elements under scanner view.[5] The superior highlighting of fungal elements compared to KOH decreases the screening time and gives quick results which can be very useful in busy outpatient departments and diagnostic laboratories with heavy work load.[1314] This simple time saving method is also of great help in cases of inflammatory tinea capitis where prompt and accurate treatment helps in preventing disfiguring cicatricial alopecia.[9] Instantaneous results in less than 5 min can be obtained by a slight modification of the staining technique by flame heating the CSB stained slide.[6]

Conclusion

CSB stain was found to be highly efficacious, less skill dependent, easy to perform and to interpret. It was more sensitive, more specific, and more accurate than the KOH preparation in diagnosis of dermatophytoses. Considering the statistically significant fair agreement found between CSB stain and KOH mount, high sensitivity of CSB stain and qualitatively superior contrasting appearance of dermatophytic hyphae, it can be adopted in the Out patient department (OPD) as a cost-effective alternative to KOH mount for the rapid diagnosis of dermatophytoses.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.