Superficial mycoses include superficial fungal infections of the skin, hair, and nail (onychomycosis). It is caused by dermatophytes, yeasts (e.g., Candida, Malassezia), and non-dermatophyte molds. Amongst these, dermatophytes are responsible for the highest proportion of cases.[1] Dermatophytes are classified under three asexual genera, namely Trichophyton, Epidermophyton, and Microsporum.[1] The prevalence of superficial mycoses has recently increased by many folds in India resulting in an epidemic-like situation.[2,3] Various factors have been implicated, such as hot and humid climatic conditions, overcrowding, poor hygiene, occlusive tight garments and footwear, low compliance to treatment, and irrational use of topical corticosteroids.[4] Furthermore, there is a changing trend in the disease presentation, severity, treatment response, and relapse rate.[1] This has a significant impact on the quality of life of the patients[5] and poses a huge psychosocial and financial burden.[6] Laboratory diagnosis of fungal infections depends on direct microscopic examination of fungal elements and mycological culture of the particular fungal species in the clinical sample. Every diagnostic laboratory must be able to differentiate between pathogens and contaminants in the culture plate. The microscopic examination is generally done by KOH and calcofluor white fluorescent stain (Rippon, 1988; Miller et al., 1993).7 Direct microscopic examination of specimens in suspected cases of superficial mycoses provides early detection when compared to culture, which may take days or weeks. KOH examination is very simple, fast and most cost-effective technique used for the diagnosis of fungal infections but, an inexperienced observer may misdiagnose certain artifacts as hyphae and it does not allow species identification (Lilly et al., 2006; Kaur et al., 2008).8 The calcofluor white stain is very sensitive technique which helps in the identification of fungal elements, even in small quantities and less experience of the observer, but it is an expensive technique which requires purchasing a fluorescence microscope.7 OBJECTIVES: 1. To compare the sensitivity and specificity of direct microscopic examination by KOH, CFW stain and fungal culture. 2. To study the microbiological profile causing superficial mycosis.

PLACE OF STUDY: Department of Microbiology, Konaseema Institute of Medical Sciences and Research Foundation, Amalapuram DURATION OF STUDY: October 2024 to November 2025 INCLUSION CRITERIA: All patients with superficial mycosis are included SAMPLE SIZE - 100 STUDY DESIGN: Observational laboratory diagnosis METHOD OF COLLECTION OF DATA: The samples which are received to the microbiology laboratory are processed. Skin scrapings, hair, Nail clippings and nail scrapings from subungual debris were collected in black paper and divided into two parts; one part was used for direct microscopy (KOH and CFW) and the other for culture. METHODOLOGY: KOH mount Specimen was placed on a slide, and a drop of 20% KOH was added. Incubation was done for 2 hours or more (up to 48 hours) until softening or digestion of the specimen occurred. Slides were evaluated for the presence of branching thread-like structures (hyphae). Samples with the presence of hyphae was considered to be a positive. Calcofluor White (CFW) : Two drops of calcofluor white stain (comprising 1g/L calcofluor white and Evans blue) was added to the specimen on the slide. The slide was then left to stand for 10 minutes and was examined under fluorescence microscopy using blue light excitation (300-400 nm for the emission wavelength with excitation at around 355 nm) and looked under 10X and 40X of fluorescent microscope for 7 apple green filaments Calcofluor white staining (CFW) Fungal culture Culture was done using Sabouraud’s dextrose agar (SDA) with and without antibiotics (chloramphenicol and cycloheximide). The plates were examined daily during the first week and twice weekly during the next two weeks. The isolates were identified by standard laboratory procedures (LPCB) and in case of any doubt in morphology, was confirmed by slide culture. If KOH/CFW showed spaghetti and meat ball appearance, an additional two tubes of SDA were inoculated, one of them with overlay of olive oil. STASTISTICAL ANALYSIS: The collected data was compiled, tabulated, presented in graphs and is being statistically analysed using Statistical Package for Social Sciences software (SPSS), 21st version.

Total 100 samples were processed and among them 76% are positive with KOH, 84% positive with CFW and 78% positive with fungal culture. Among the fungal cultures 44% are dermatophytes which includes Trychophyton rubrum 30.7%, Trychophyton mentagrophytes 23%, Microsaporum gypseum 2.5%. Non dermatophytes include Aspergillus 19.2%, Penicillium spp 5.1%, Fusarium 2.5%. Yeasts include Candida albicans 8.9% and Candida nonalbicans 7.6%. CFW study showed high sensitivity and specificity with positivity 84%, fungal culture showed 78% positivity and KOH showed 76% positivity. Test No.of positive cases No of negative cases Percentage of positive cases KOH 76 24 76% Calcofluor white staining (CFW) 84 16 84% Fungal culture 78 22 78% S.No Fungal group Species Number Percentage 1 Dermatophytes Trichophyton rubrum 24 30.7 Trichophyton mentagrophytes 18 23.0 Microsporum gypseum 2 2.5 2 Non Dermatophytes Aspergillus spp 15 19.2 Penicillium spp 4 5.1 Fusarium 2 2.5 3 Yeast Candida albicans 7 8.9 Candida non-albicans 6 7.6

Identification of fungi causing superficial mycosis is necessary, to find out the source of infection, for treatment and to exclude other skin disorder which mimic superficial fungal infections making laboratory diagnosis essential.9 Commonest dermatophyte was T.rubrum (30.7%) similar to study done by Vijaya D et al9 showing T.rubrum (64.06%) and . This species might have developed increased virulence and better adaption to hard keratin 8 of skin, hair an Rahmath AG et al10 nail leading to its increased prevalence. Other organisms isolated are T.mentagrophytes which is also isolated by Rahmath AG et al10 , Shakthi R et al study11 and Dass M et al.12 Non dermatophytes isolated in this study are Aspergillus, Penicillium, Fusarium and Candida spp. In Dass M et al12 studies also the same were isolated except for Penicillium. Commonest dermatophyte isolated in this study is T.rubrum (30.7%) similar to study done by Vijaya D et9 al showing T.rubrum (64.06%) and Rahmath AG et al10(54.7%). CFW study showed high sensitivity and specificity with positivity 84%, fungal culture showed 78% positivity and KOH showed 76% positivity. Which is also similar to the studies done by Vijaya D et al9 Rahmath AG et al10 , Shakthi R et al study11 and Dass M et al.12

Superficial mycosis is the commonest fungal infection occurring more commonly in diabetes and immunocompromised patients. It is necessary to have early and appropriate diagnosis in treatment of fungal infection. CFW is showing a higher positivity rate compared to fungal culture and KOH mount and also more easy to find out fungal elements if we have fluorescent microscopy and stains.

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