Onychomycosis is an intriguing and common nail infection caused by nail pathogens like dermatophytes, yeasts, and non-dermatophyte moulds. It accounts for up to 20-40% of nail diseases and 30% of cutaneous mycotic infections.^[1] Onychomycosis leads to pain, discomfort, impaired tactile functions and also poses cosmetic problems.^[1,2] It commonly affects elderly population, immunocompromised individuals and diabetics.

Based on the clinical presentation and route of invasion, onychomycosis is clinically classified into distal lateral subungual onychomycosis (DLSO), proximal subungual onychomycosis (PSO), endonyx subungual, superficial onychomycosis (SO), mixed, and secondary onychomycosis.^[3,1]

The prevalence of etiological agents causing onychomycosis show a vast difference in distribution related to climate, socio-economic status, and individual predisposing factors.^[4] Onychomycosis is not easily cured as nail serves as reservoir of fungi which leads to chronicity, eventually leading to total dystrophic onychomycosis.^[5] Dermatophytes are the leading pathogens followed by non-dermatophytic fungi and yeast.^[1,6,7] Identifying the causative pathogen of clinically suspicious onychomycosis is a challenge to the treating physician as well as the Microbiologist. It is essential to establish the diagnosis of Onychomycosis before starting treatment so that specific therapeutic modalities can be monitored during the course of treatment.^[8]

Our study aims at comparing the efficacy of direct microscopy and culture with periodic acid Schiff stain (PAS) to get an insight into the fungi causing onychomycosis in our region.

OBJECTIVES

1. To isolate the etiological fungi in clinically suspected cases of onychomycosis.
2. To compare different laboratory techniques in diagnosing onychomycosis.

A prospective study was conducted in the Department of Microbiology, Srinivas Institute of Medical Sciences and Research Centre, Central Diagnostic laboratory, Mukka for a period of six months from December 2024 to May 2025. A total of 125 nail clippings from clinically suspicious cases of onychomycosis were collected under strict aseptic precautions. The nail clippings were subjected for culture on two tubes of Sabouraud Dextrose Agar (SDA) and incubated at 25°C and 37°C respectively for 2 to 4 weeks and was observed periodically for growth.^[9,10] The fungal growth on SDA was identified by colony characteristics, pigmentation, growth rate, slide culture, microscopic examination by Lactophenol cotton blue (LPCB) stain and urease test.^[6,10,11] Slide culture is used to study undisturbed morphological features of fungi.

Rest of the sample was subjected to various concentrations of Potassium Hydroxide (KOH) like 10%, 20% and 40%;^[6,9] calcofluor white stain^[10,12,13] and periodic acid Schiff stain (PAS).^[6,13,14]

On KOH mount, the fungal elements appear as highly refractile, hyaline septate branching filaments.^[12,15]

Calcofluor white stain a bleaching agent binds to chitin and cellulose in fungal cell wall and upon excitation by long wavelength ultraviolet rays or short wavelength, visible light produces a chalk white or apple green fluorescence. ^[6,13]

The polysaccharide of fungi is oxidised by periodic acid to form aldehyde groups which gives magenta-coloured compounds with Schiff’s fuchsin sulphite. The protein and nucleic acid remain unstained. The nuclei stains blue, fungi magenta or deep pink.^[6,14]

In the present study involving 125 clinically suspicious cases of onychomycosis, 61(48.8 %) were males and 64(51.2%) were females. Majority 34(27.2%) of the study population belonged to the age group of 31-40 years. In this age group, females outnumbered males.

Of the 125 samples, subjected for culture on SDA 56(44.8%) revealed fungal growth. Out of the growth, dermatophytes accounted for only 5(8.9%) and rest 51 (91.1%) were non-dermatophytic fungi.

Comparing the microscopic diagnostic tools for onychomycosis, it was found that PAS 60(48%) showed better results in showing the fungal elements.

Onychomycosis is a non-fatal disease of the nail plate, but it has gained relevance due to the inconvenience caused to an individual because of the chronicity and cosmetic disfiguration of the involved nails, and the financial burden involved in the long treatment course. Of the 125 clinically suspicious cases of onychomycosis in this study, age group commonly involved was 31-40 years, 34(27.2%) which was in accordance with Gopi A et al.,^[16] Veer P et al.^[17] The highest incidence in young adults may be due to increased physical activity and increased opportunity for exposure. Females 24 (70.6%) were majority in number compared to males 10(29.4%) in this age group. This could be attributed to the constant contact with water.

In the present study, culture positivity was shown in 56(44.8%) of nail samples cultured on SDA. These similar findings were observed in N Patwardhan et al.,^[18] Sweetha N et al.^[14] Other studies had higher rates of positivity (66.3%) Kannan P et al.,^[19] and it could be due to bacterial contamination of the samples as well as non-availability of repeat sample from the patient. Majority of the isolates were non-dermatophytic fungi, Aspergillus species 35/56(62.5%) being the commonest. Garg A et al.,^[20] reported Aspergillus as the most common causative organism of onychomycosis in his study.

In this study out of 125 samples collected, 37(29.6%) were positive for fungal elements in KOH mount. Lesser percentage of KOH sensitivity may be due to thicker nail samples. Calcofluor white stain was found to be positive in 42 (33.6%) and this can be used as an alternative staining technique in the diagnosis of fungal elements. Calcofluor white stain picked up 5 more samples positive for fungal elements which were negative by KOH mount. Presence of collagen fibre and artifacts can pose difficulty in differentiating it from fungal elements. In such situations, calcofluor white stain has found to be more superior.

Sixty (48%) out of 125 samples were positive by PAS stain. Higher rate of positivity by PAS makes it the most reliable microscopic staining technique especially in onychomycosis. Shenoy MM et al.,^[2] Alkhayat H et al.,^[21] reported similar results.

In this study of 125 clinically suspicious cases of onychomycosis, PAS 60(48%) was superior than culture on SDA 56(44.8%) in establishing diagnosis of onychomycosis.

Although PAS is not a substitute for culture, it can give almost equal accuracy in detecting fungal elements in onychomycosis.

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