Characterisation of Non-Dermatophyte Fungus on The Toes of a Patient Diagnosed with Tinea Pedis

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INTRODUCTION
There is a considerable variety of irresistible sicknesses in Indonesia, whether Athlete's foot infects about 42% of men, compared to 1.7% of women, and its pervasiveness increases with age, although it can also infect children.The signs and side effects of competitor's foot (athlete's foot) are a layered rash that causes chills and irritation (7).Based on previous research, the infection with species from nondermatophyta groups can also exacerbate dermatocytosis in humans.Therefore, the purpose of this study was to characterise the type of fungi that infects the toes of tinea pedis in butchers.

MATERIALS AND METHODS
The materials used in this study were Sabouraud Dextrose Agar (SDA) media

RESULTS
Seven

Fungus Pure Isolate 1
The macroscopic results of pure fungi isolate 1 are shown in the Figure 1.These results are taken through a magnifying glass (lup).The characteristics obtained from the macroscopic observations as shown in Figure 1, are presented in detail in Table 1.Similary, the microscopic result of pure fungus isolates 1 are documented in Figure 1, acquired thorugh a 400x magnification microscope.
The characteristics obrained from the microscopic observations of fungus isolates 1 are outlined in Table 1.
Based on the results of macroscopic and microscopic observations of pure fungal isolate 1, it has the characteristics of white colonies with a mixture of yellow inside, the reverse color is yellowish white with smooth colonies.Hyphae are not septate.
Conidiophores are colorless (hyaline), smooth and unbranched.Phialide grows on metulae with a round shape with a brownish color.Presence of round brownish vesicles with transparent conidia of rough round shape.The macroscopic and microscopic observations of fungus isolate 2 revealed green colonies that were a mixture of yellowish-white and yellowish-white, with the colonies' shape resembling that of a rather  The pure isolates obtained were The presence of round brownish vesicles with conidia dark brown, rough round shape (21).
Based on these characteristics, fungal isolates   Macroscopic observation of pure fungus isolates 5 obtained the characteristics as shown in Table 5, based on Figure 5. Based on microscopic observations made on fungal isolates 5, the characteristics shown in Table 5 were obtained, as shown in Figure 5.The pure isolates obtained were identified and observed macroscopically and microscopically.
From macroscopic observations of fungus isolates 6, the characteristics shown in Table 6 were obtained, according to those in Figure 6.
Based on microscopic observations made on fungus isolates 6, the characteristics shown in Table 6 were obtained, according to Figure 6.
caused by fungal pathogens, bacteria, or viruses.A mycotic disease, particularly good superficial mycoses that occur in the nails (onychomycosis) or on the skin, is one of the diseases that are frequently found in tropical environments such as Indonesia.Personal hygiene and the environment are two of the most important factors in determining whether a disease is caused by a particular pathogen or others (1).Most skin and nail diseases are caused by dermatophytes, a group of pathogenic fungi.Many dermatophyte fungi have a stratum corneum and keratin (such as skin, scalp, and nails), accordingly, they can easily grow and infect those areas of the human body (2).Additionally, non-dermatophyte companions of the fungal group such as Candida spp.and Aspergillus spp., are frequently associated with and exacerbated by dermatophytecaused pathogenic fungal infections (3).Indonesia is a nation characterised by a climate of high humidity and heat.Thereby making it easier for pathogenic fungal spores that cause superficial mycosis (onychomycosis) to germinate and grow.Pathogenic fungal infections are also increasing, according to recent reports, notably among people with compromised immunity.In addition, Indonesia has a high rate of deep mycosis.The superficial site of deep mycosis attacks the body's surface, infects internal organs, and eventually spreads to the circulatory system (4-6).As noted by the Gorontalo City Health Office in 2020 in its data report on the level of morbidity at the a local puskesmas, the prevalence of skin diseases caused by fungal infections reached 4.76% (476) cases, one of which was tinea pedis (56 cases).One of the variables causing athlete's foot is a workplace and type(s) of work that fail to satisfy basic work guidelines.Athlete's foot, also called competitor's foot, is an infection brought about by a contagious dermatophyte disease that contaminates the skin on the toes, bottoms, and parallel pieces of the feet.Athlete's foot represents 20.4% of all dermatomycosis cases.The reason for this illness is the entire lot of dermatophytes, notably Trichophyton rubrum and Trichophyton mentagrophytes.

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625 gr), 10% Eosin solution (50 mL), Aquades (225 mL), and 70% alcohol.The samples used were skin scrapings of tinea pedis found on butchers' toes.The samples were extracted from a total of 15 patients.The research encompassed multiple stages.Initially, the respondents ere prepared for sampling, which involved a systematic 148 procedure: the skin were cleansing with 70% alcohol.Samples were obtained using a knife, and placed in a plastic clip bag with tweezers.Each sample was meticulously labeled with the corresponding patient's name and age.Subsequently, the samples were prepared for transportion to the Microbiology Laboratory at Bina Mandiri University, Gorontalo, for comprehensive analysis.The subsequent phase involving crafting the growth medium for cultivating the fungi and embedding samples within SDA.The SDA was dissolved in distilled and heated on a hotplate to achieve homogenity.Following this, the SDA media underwent sterilization in an autoclave at 121°C for 15 minutes (8-10).The next step was inoculation of the sample on SDA media.It was imperative to utilize gloves and a mask to avoid contamination.The wire loop was heated over a spirit lamp, and a skin scraping was taken in a sterile container using a ring loop.The skin scrapping sample was promptly inoculated into specific areas of the SDA media.Subsequently, the petri dish was closed, and the edges were heated over the spirit flame.The media placed in an incubator set at a temperature of 25-28ºC for seven days, during which daily observations were conducted.In the event of a mixed cultire, the cultivation process was continued to obtain a pure isolate.Each colony on a petri dish was carried out by inoculating a sample of the suspected colony with straight loops, then inoculating it in the center of sterile SDA media.The media was appropriately labeled with the pertinent details, including the name, date, and sample number.As previously noted, the media was then incubated at room temperature (25-28ºC) for seven days.After the incubation period, the fungal culture examination process was initiated.The third stage involves assessment of growth patterns of the fungal culture daily, both with the naked eye (macroscopic examination) and under a microscope (microscopic examination).The macroscopic analaysis encompasses the observation of various aspects, such as colony surface texture (grains, velvet, fine threads like cotton, floccose), colony color, the absence of growth zones, concentration lines or circles, distinctive odor and exudate droplets.Meanwhile, the microscopic examination employs a direct technique.A drop of eosin solution is applied to the object glass, and fungal colonies are carefully collected using a sterile loop, placed on the slide and made like a smear, then covered with a cover glass.The preparations were observed under a 10× or 40× magnification microscope.This microscopic examination focuses on the structure or arrangement of hyphae and fungal spores (9-20).The final step involves to enforce the results of the inspection.A positive result (+) signifies the presence of on-dermatophyte fungi within the fungal culture.Conversely, a negative result (-) is assigned when the examination detects nondermatophyte fungi.

Figure 2 .
Figure 2. Colony morphology of pure fungus isolates 2 on SDA isolated from skin in this study -Isolate top view (A).Microscopic characteristics of pure fungus isolates 1 at 400 x magnification (B) 1. Conidiophore; 2. Conidia.
identified and observed macroscopically and microscopically.Macroscopic observation of pure fungal isolates 4 showed the characteristics shown in Figure 4. Based on microscopic observations made on fungal isolates 4, the characteristics as shown in Table 44 were obtained.For more details, microscopic observations on the isolation media can be seen in the Figure 4.From macroscopic and microscopic observations (Figure 4), the isolates 4 fungus has the characteristics of a bright green slightly brown colony, the reverse color is yellowish white with smooth colonies.Hyphae are not septate.Conidiophores are colorless (hyaline), smooth and unbranched.

Figure 5 .
Figure 5. (A) Colony morphology of pure fungus isolates 5 on SDA isolated from skin in this study -Top view isolate.(B) Microscopic characteristics of pure fungal isolate 5 at 400 x magnification. 1. Blastospore; 2. Chlamydospore

DISCUSSION
pure isolate of fungus 6 reveal that it exhibits characteristics of white to gray colonies, with a brownish-white reverse color.The colony structure appears cottony in nature.It possesses hyphae that form rhizoids to attach to the substrate, and has coenocytic hyphae that lack septa or have septa.Stolons spread over the substrate, accompanied by sporangiophores with upwards-growing stalks containing numerous spores, as well as large, dark brown, smooth-walled sporangia (20).Based on these characteristics, fungus isolate 6 belongs to the Rhizopus sp.species, which can be classified as follows: In view of the results of observing tests of skin scrapings from meat dealers in small butchers' shops in Gorontalo city, which were undertaken utilising the way of life strategy on Saboraud Dextrose Agar (SDA) media, non-dermatophyte parasites were found, notably Aspergillus sp, Aspergillus niger, Aspergillus terreus, Rhizopus sp, Candida albicans and different species, for example, Metarhizium anisopliaea.Fifteen examples of foot skin scrapings which were analysed visibly and minutely from meat venders in customary business sectors in Gorontalo City, subsequent to being consulted 12 respondents were found to not constantly wear shoes or other foot coverings when moving about and selling meat.Of these 12, three only occasionally wore shoes.The average type of fungus obtained from 12 respondents determined that the most isolates detected were the fungus Aspergillus sp.(13).Microscopically, the fungus Aspergillus sp. has hyphae that branch and are partitioned, while the conidiophores do not have branching but some have partitions and do not have partitions depending on the type of species.Free-living and widespread contaminant fungi include Aspergillus sp., Aspergillus niger, and Aspergillus terreus (14).The air is where the fungus Aspergillus sp. can spread, and it can contaminate in unfavorable conditions such as a humid environment.Aspergillus sp. is hazardous as it can cause contaminations in the human body such as sensitivities, and in addition it can cause diseases in the human lungs (13).The way of life aftereffects of skin scratching tests additionally found growths of the family Rhizopus in samples of foot skin scrapings.One of the pathogenic fungi that can infect the human body and cause inflammation or infection is the fungus Rhizopus sp.Humidity has an impact on the likelihood of detecting the fungus Rhizopus sp. in samples.For fungi to grow, humidity is all-important.For example, the fungus Rhizopus sp.needs a low-level environment with 90% humidity.As well, the Rhizopus sp.In small skin wounds, dermatophytosis fungi can also spread by direct contact(15).Furthermore, analysts additionally foundCandida albicans organism.This is a type of fungus that infects the skin and thrives in warm, humid environments(16).The chance of the development of the Candida albicans parasite can occur when there are inclining variables, for example, neatness of the skin of the feet, the propensity for absorbing the feet water for a long time, causing maceration which makes it easier for the development of the Candida albicans organism, and warm and moist environments cause expanded sweat.Candida albicans is considered as the most pathogenic species and the most common cause of candidiasis, a fungal disease that attacks the skin, hair, nails, mucous membranes and internal organs (10).The species C. albicans is among the most important and most frequently isolated from healthy and diseased persons and is considered a commensal organism and, at the same time, a pathogen causing health complications.C. albicans represents over 80% of isolates from all forms of human candidosis (22), (23).This makes it workable for the parasite Candida albicans to be present in the foot skin scratching test.The fungus Metarhizium anisopliae was also discovered in this study.Metarhizium anisopliae is one of the endopathogenic parasites that has the potential for bother control, which can lessen bug populaces in a rural region contrasted with different organic entities.It is conceivable that the organism Metarhizium anisopliae was found in the butcher's foot scratching test on the grounds that at the time preceding examining, the respondent was taking creature feed from the ranch region.The growth Metarhizium anisopliae ordinarily occurs in plants like corn, rice, sugar cane and is typically found in soil.The fungus Metarhizium anisopliae is parasitic and saprophytic in the soil by surviving on plant debris.Metarhizium anisopliae is widely used to treat insect larvae and plant-disturbing caterpillars (17).Some of the respondents complained of tingling between the toes and also of moistness.As per Nigam and Saleh (24), the development of Athlete's foot on the feet is brought about by the state of the feet which are wet and hot when wearing foot wear.This can cause the development of Non dermatophyta growths, and taint between the butcher's toes, causing athlete's foot.CONCLUSIONS Based on the findings from macroscopic and microscopic examinations conducted among meat sellers with tinea pedis patient in Gorontalo City, various groups of nondermatophyte fungi were identified.These groups included species such as Aspergillus sp., Aspergillus niger, Aspergillus terreus, Rhizopus sp., Candida albicans, as well as others like Metarhizium anisopliaea.AUTHOR CONTRIBUTIONS Yolan Dunggio: Planning and designing research, processing research results, interpreting results, and compiling research manuscripts.Ismail Mukusibu: Took samples at the location, prepared tools and materials, and looked for references related to the research.ACKNOWLEDGEMENTS The author would like to thank the laboratory assistants and several related parties who have provided direction and support in this study.Much obliged to you also, the Chancellor of Bina Mandiri University, Gorontalo.Microbiology

Table 1 .
Results of Macroscopic and Microscopic Characteristics of Pure Fungal Isolates 1

Table 2
were obtained, based on microscopic observations in

Table 2 .
Results of Microscopic Characteristics of Pure Fungal Isolates 2

Table 3 .
Results of Macroscopic Characteristics of Fungal Isolate 3

Table 4 .
Results of Macroscopic Characteristics of Fungal Isolate 4

Table 5 .
Results of Macroscopic Characteristics of Fungal Isolate 5

Table 6
Results of Macroscopic Characteristics of Fungal Isolate 6