Fungal Species Detection in Onychomycosis by Culture and Direct Microscopy at Tertiary Care Hospital, Bahawalpur, Pakistan

Objective: This study aimed to detect the common organisms by culture and KOH mount microscopy of nail infections. Materials & Methods: It was a cross-sectional study. Three hundred sixty clinically diagnosed cases were collected from 1 st July 2018 to 31 st July 2021 from Dermatology OPD of Bahawal Victoria Hospital and clinics. The samples were processed in the Microbiology section of the Pathology department of Quaid-e-Azam Medical College Bahawalpur, Pakistan. The nail specimen was directly inoculated on SDA culture media and aerobically incubate at 25 Ć to 30 Ć for 3 to 4 weeks. The growth was identified in colony characteristics by using cotton blue stains. Whereas the nail is immersed in 20% KOH solution. The microscopic study revealed the hyphae or spores and that is considered a positive for the test. Result: out of three hundred sixty cases the culture positivity was 56.94% of a specimen while KOH mount was positive in 60.83% of specimens and the combination of Culture with KOH was 66.67%. Conclusion: The fungal culture and KOH mount microscopy combination are subtle laboratory methods for the detection of organisms causing onychomycosis. The species detection and precise usage of anti-mycological agents to prevent the complications raised public health considerably.


Introduction
Fungal nail infections are common, affecting mostly over 40 years of age and especially in the 20% elderly population. The Specified fungal treatment needs the precise isolation of fungal elements for the eradication of the disease. 1 Onychomycosis is a superficial cutaneous fungal infection that affected more than one nail. The identified causative agent as Dermatophytes (Trichophyton. Rubrum and Mentagrophytes) yeast or moulds (Fusarium, Chrysosporium, Aspergillus, etc.) on mycological culture. The Greek word "onyx" a nail and "mykes" known as onychomycosis a fungal infection. 2 it affects the toenail in 80% of cases which was identified on clinical grounds and confirmed on culture as Trichophyton rubrum a dermatophyte which is mostly occurring agents. It is counted as a public health problem and should be considered for decreasing complications like cellulitis in diabetics and peripheral vascular disease that affect the quality of life. 3 Onychomycosis is clinically remembered as superficial white onychomycosis (SWO), proximal subungual onychomycosis (PSO), distal and lateral subungual onychomycosis (DLSO), candidal onychomycosis (CO) and total dystrophic onychomycosis(TDO). 4 The alteration of immune and genetic makeup defects is the causation of onychomycosis. The large nail surface for invasion and slow nail growth was observed in adults. Keratin is a horn-like envelope that covers the dorsal aspect of the nail bed. The infection starts from the nail bed and progresses to dystrophic nature if not treated appropriately with anti-mycotic agents. 5 Onychomycosis is now considered no more a cosmetic problem The risk factors generally increased worldwide widely in fungal infection as in nail trauma, hyperhydrosis, compromised immunity, a metabolic disorder like diabetes, peripheral vascular diseases, poor hygiene and more water exposure of nails, especially in candidal onychomycosis. 6 Initially, onychomycosis starts as discoloration of the nail that may thicken, become rough and crumbly and separate its bed, and debris may accumulate under the nail. The thickening and dystrophy of nails result from the pressure erosions on the nail bed and Hyponychim. 7 In diabetes, the risk was observed 1.9 to 2.8 times higher in persons in the general population while in human immunodeficiency virus (HIV) infection that ranges from 15% to 40%. 8 The phenotypic characteristics of Onychomycosis varies with geographically and more dermatophytes cases are reported in a temperate zone while Aspergillus and Fusarium species are prevalent in tropical and subtropical countries. 9 Middle age adults' dermatophytes species Trichophyton rubrum while non-dermatophytes like Candida albicans are mostly identified species of onychomycosis. 10 Clinically identified cases of onychomycosis required laboratory confirmation and on KOH mount microscopy of nails that labeled as fungal detection but the recognition of the fungus species whereas culture specifies the genus that needed the drug for curing the disease. 11 The physical limitations are severe pain and discomfort in the nail-affected area. The dermatophytes are the main etiological agents and Trichophyton rubrum are common isolating pathogen. 12 Objective: The purpose of this study is to identify the causative agent, by mycological culture and with the combination of 20% KOH mount microscopy deceptive as the "gold standard" to diagnose onychomycosis. The appropriate drugs for a specified organism that cure the disease which limits the resistance against drugs.

Materials and Methods
Settings: Clinical diagnosis of 360 patients of onychomycosis included a detailed history of trauma, occupation, sharing of common facilities, personal habits such as smoking and drinking, personal hygiene, hyperhydrosis, and different predisposing diseases reported at the dermatology outpatient department of Bahawal Victoria Hospital Bahawalpur, a tertiary care hospital and performed at Microbiology section of pathology department Quaid-e-Azam Medical College Bahawalpur were included in this study.
Non-probability convenience sampling technique was applied. The study was carried out from 1 st July 2018 to 31 st July 2021. Inclusion Criteria: The specimens of severely affected nails diagnosed with Onychomycosis were selected for processing in the study. 13 Exclusion Criteria: Patients whose history of antifungal therapy were excluded from the study. 13 The variable of the study was age groups, genders were included with more than one affected nail and KOH mount, and culture positivity diagnostic techniques were adapted. 14 Direct Microscopy: The nail clipping specimen that immersed in 20% KOH and slightly warmed and examined under both low and high-power of microscopy. Fungal elements' presence may be hyphae, spores, budding cells, and pseudo-hyphae were noted. Mycological Culture: The detected fungal sample was placed on Sabouraud's dextrose agar and incubated at 25ºC to 30ºC. The growth with colony characteristics was observed periodically for 4 weeks follow-up and by using cotton blue reagent for the final identification of species. 15 Results Out of three hundred sixty patients, the distribution of 200 cases was male while 160 was female (Figure 1). The study age ranged between 10 to 70 years. The positivity of fungal infection was reported at 54%, 30%, and 7% in 21 to 30-year age, 31 to 50 years, and above 50 years of age respectively ( Figure 2 Table 2). In this study, Trichophyton rubrum was the higher most isolated organism while second was Candida albicans. The combination technique (mycological culture with KOH) positivity was 240 (66.67%). This study showed that fungal culture with KOH in combination is considerably effective either in single laboratory methods for revealing onychomycosis.

Discussion
The fungal nail is a public health problem that is difficult to treat without proper isolation of causative organisms and laboratory investigation. It is not a cosmetic problem and the prevalence of the disease is rising worldwide.
The factors contributing to the etiology of onychomycosis that was nail trauma, diabetes mellitus, peripheral vascular disease, immunecompromised, contaminated swimming pools, footwear, and genetic alteration. The microscopy screening for the presence of fungus or not whereas mycological culture reveals the isolation of pathogens at the species level for the proper selection of drugs. The combination of KOH and Culture was better to reach the fungal diagnosis that helps eradicate the cosmetic problem The finding of Shenoy M et al; 16 determined the KOH positivity was 53% and culture was 35%. That lower in consistence with our study results KOH was 60.83% and mycological culture was 56.94%. Our study concluded that KOH positivity was 60.83%, culture was positive in 56.94% of samples in lower concord with Agrawal A et al; 17 (KOH 89.23%, culture 84.62%). Moreover, our study revealed in a combination of KOH with culture was 66.67% and Dermatophytes was 56.58% is consistent (KOH and culture 65% and dermatophytes 57%) with Agrawal A et al. 17 Our results were lower compared with other authors Alkhayat et al 18  should be interpreted carefully in each individual case. In the majority, yeasts are likely to be a secondary infection and nondermatophyte moulds to be saprophytic in previously damaged nails Recommendation: Fungal culture is the gold standard and combination with KOH mount microscopy is a delicate laboratory method for onychomycosis diagnostic sensitivity. It is a routinely performed technique in pathology laboratory for improving the accuracy of diagnosis of onychomycosis. Limitation of Study: Anti-mycotic susceptibility is the stephead for further projects.