Diagnostic utility of Chicago sky blue stain in superficial dermatophytosis

INTRODUCTION

Fungal skin infections are categorized into superficial and deep. Superficial infections are defined as those limited to the stratum corneum of the epidermis, or the hair and nails. The three most common types of superficial mycoses are dermatophytosis, diseases caused by Malassezia and superficial candidiasis.¹ The prevalence of dermatophytosis varies according to geographical location and climatic conditions. In the world, 20-25% of people are infected by dermatophytosis, while in India it ranges from 36.6-78.4%.^2,3 Although the majority of the studies are hospital-based, an increasing trend of dermatophytosis is evident in published literature. Dermatophytosis infection most commonly presents as blackish macules with scaling associated with itching and irritation. Though the infection is non-invasive and curable, its widespread nature and therapeutic costs are a major public health problem. The most economic and easiest method for diagnosis of dermatophytosis is by direct examinations of samples using a KOH preparation.⁴ However, the gold standard for diagnosis of the infection is culture, which is rarely used as it is time-consuming and expensive. Also, the standard KOH preparation wet mount lacks a colour contrast and depends on the skills of the observer for the detection of fungal elements. CSB 6B stain is a new promising contrast stain that can stain the fungal hyphae and spores with a blue contrast against a light purplish background of cellular debris making them easily identifiable under a light microscope. Only a few reports have been published on using CSB as a diagnostic modality for dermatophytosis, despite it being easily available. Thus, the current study was planned to explore the feasibility and reliability of CSB as a diagnostic modality for the diagnosis of superficial dermatophytosis.

METHODS

A cross-sectional, observational study was conducted in the dermatology OPD of a tertiary care hospital. Permission for conducting the study was obtained from the institutional ethics committee. A total of 352 suspected cases of superficial dermatophytosis were included in the study over 9 months. After obtaining informed consent for participation in the study, sociodemographic details of all patients were noted. Thereafter relevant laboratory investigations were carried out.

Scraping method

After cleaning the affected area with 70% isopropyl alcohol, scraping of skin was taken with the blunt edge of a sterile scalpel from the edge of the lesion and was placed on two clean microscopic slides. The 10% KOH was added to both microscopic slides. CSB was added to one of the slides and both were examined microscopically after 30 minutes. The slides were visualized under low power (10× magnification) as well as high power (40× magnification). The presence of hyphae and arthroconidia was considered positive for the presence of dermatophytes in both KOH and CSB stains.

Culture

The samples were collected in a sterile container. Scales were inoculated on SDA and dermatophyte test medium (DTM). Culture medium incubated at 300ºC in a BOD incubator. Growth of dermatophytes was identified by colony morphology and other standard mycological tests.

Data were entered using Microsoft excel and analysis was done using SPSS version 22. Taking culture method as reference method for diagnosis, sensitivity, specificity, PPV, NPV of KOH and CSB staining method calculated. Kappa statistics were used to determine the degree of congruence between the various diagnostic methods.

RESULTS

A total of 352 patients were included in the study. KOH mount showed 227 (64.5%) of the samples to be positive for fungal elements while others were negative. When CSB staining was added to KOH mount 251 (71.3%) samples were found to be positive for fungal elements. While culture showed growth of fungal species in 292 (82.9%) of the specimens. The distribution of species of dermatophytes identified in culture is shown in Table 1. The sensitivity, specificity, PPV, NPV of the KOH mount and CSB staining method taking mycological culture as the reference method is enumerated in Table 2. Cohens Kappa was used to see the agreement between KOH mount and culture which showed negligible agreement (Kappa=0.09) which was statistically significant (p<0.05) at 95% of CI (Table 3). KOH with CSB staining and culture (Kappa=0.09) also showed negligible agreement (Kappa=0.09) which was statistically significant (p<0.05) (Table 4). KOH with CSB staining and KOH mount showed substantial agreement (Kappa-0.8) which was statistically significant (p<0.05) (Table 5).

*P<0.05=significant at 95% CI. #Kappa interpretation: <0: poor agreement, 0.0-0.20: slight agreement, 0.21-0.40: fair agreement, 0.41-0.60: moderate agreement, 0.61-0.80: substantial agreement, 0.81-1.00: almost perfect agreement.

*P<0.05=Significant at 95% CI, #Kappa interpretation: <0: poor agreement, 0.0-0.20: slight agreement, 0.21-0.40: fair agreement, 0.41-0.60: moderate agreement, 0.61-0.80: substantial agreement, 0.81-1.00: almost perfect agreement.

*P<0.05=Significant at 95% CI, #Kappa interpretation: <0: poor agreement, 0.0-0.20: slight agreement, 0.21-0.40: fair agreement, 0.41-0.60: moderate agreement, 0.61-0.80: substantial agreement, 0.81-1.00: almost perfect agreement.

DISCUSSION

We observed that T. mentagrophytes is the most commonly identified species followed by T. rubrum. The least commonly identified species was Aspergillus niger. Nenoff et al similarly in their study reported T. mentagrophytes and T. rubrum as the most commonly identified species of dermatophytosis.⁹ Also, Verma et al in their systemic review stressed the increasing prevalence of T. mentagrophytes and T. rubrum from studies around the world.⁶ Our study found that although combining CSB staining with KOH mount increases the sensitivity of diagnosis of superficial dermatophytosis as compared to only KOH, also the specificity, PPV and NPV increases. The diagnostic accuracy of KOH with CSB increased to 98.32% compared to only 69.57% in the case of culture. In congruence with our findings, Lodha et al demonstrated 100% sensitivity, high specificity, PPV and NPV for KOH plus CSB in the diagnosis of superficial dermatophytes.⁴ Also, Lim et al and Tambiosis et al reported a better sensitivity and specificity for the diagnosis of superficial dermatophytosis on using CSB stain as a contrast.^10,11 We observed negligible agreement between KOH wet mount and culture while a very high agreement was observed between KOH wet mount and KOH with CSB staining in the diagnosis of superficial dermatophytosis. This was a unique finding in our study as very few studies in the open domain have tried to establish an agreement between the new diagnostic methods with an already established method of diagnosis for superficial dermatophytosis.

CONCLUSION

In conclusion, the addition of CSB stain to KOH provides a colour contrast that highlights the fungal elements well, making interpretation easy. This increases the overall diagnostic accuracy of it as compared to established methods of diagnosis. Also, CSB stain is inexpensive and requires an ordinary light microscope for reading as opposed to other methods which are technically demanding and expensive. Further population-based studies involving a larger sample size should be undertaken to mainstream KOH plus CSB as a method of diagnosis of dermatophytosis which would help in early diagnosis and treatment initiation. This would go a long way in halting the silent epidemic of dermatophytosis in the world.

Funding: No funding sources

Conflict of interest: None declared

Ethical approval: The study was approved by the institutional ethics committee

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