Dermatophytes and bacterial superinfections in tinea pedis patients at Haji Adam Malik Central Hospital, Medan-Indonesia

Background: Tinea pedis is a dermatophyte infection of the feet, especially the toes and soles of the feet. The clinical features of tinea pedis consist of four forms. They are interdigital, chronic hyperkeratotic, vesiculobullous, acute ulcerative or a combination. Tinea pedis mainly interdigital type often involved bacterial superinfection, initially Gram-positive bacteria, but Gram-negative bacteria may also be involved. In a state of severe infection, the fungus is getting hard to find. Otherwise, the bacteria were easier to find, so it is necessary to do fungal and bacterial culture with antibiotic sensitivity tests. Objective: To determine dermatophyte species and bacterial superinfection in tinea pedis at H. Adam Malik General Hospital Medan. Methods: This study is a descriptive observational with cross sectional design. The results of the study are presented in the form of frequency distribution table. These research involved 45 subjects with tinea pedis who met the inclusion and exclusion criteria. We conducted anamnesis, dermatologic examination, and collection of samples that were skin scrapings and smears on the lesion. The samples then examined on microscopic directly by using KOH. When KOH result was positive, it followed by fungal and bacterial culture. Results: The number of subjects who suffer from tinea pedis is not much difference between men (48.9%) and women (51.1%).The highest prevalence was in the age group 37-46 years (28,9%), mostly graduated from elementary school (40%) and mostly housekeepers (22.5%), with the most prevalent type was interdigital of 40 cases (88.9%). Dermatophytes T. mentagrophytes was most found by 44.4%, followed by T. rubrum, E. floccosum, and T. violaceum. Bacterial superinfection was mostly involved S.aureus by 30,8%, but B. subtilis, S.epidermidis, K.oxytoca, K.pneumoniae, E. coli, P. vulgaris were


INTRODUCTION
Tinea pedis is a dermatophyte infection of the feet especially between the toes and the soles of the feet. 1 This disease is estimated to affect 10% of the world's population. 1 The cause of tinea pedis is dermatophyte. The most common dermatophyte in tinea pedis lesions are Trichophyton rubrum (T. rubrum), Trichophyton mentagrophytes var interdigitale (T.mentagrophytes var interdigitale) and Epidermophyton floccosum (E.floccosum). 1,2,3, 4 Normal microflora on the sidelines of the toes include Micrococcae (staph), aerobic coryneform and some Gram-negative bacteria can proliferate when dermatophytes damage the stratum corneum barrier. It manifests of the development from uncomplicated superficial fungal infections becoming more aggressive with erosion and maceration on the sidelines of the toes. 5, 6 Tinea pedis especially the interdigital type is often involving of bacterial superinfection which causes the treatment to be difficult. 7,8,9 also, untreated tinea pedis or improper treatment may lead to secondary infection by bacteria. 3 In severe infections, the fungus was harder to found. Otherwise, bacteria are more easily to be found, so it required the examination of fungal culture and also bacterial culture accompanied by antibiotic sensitivity test. 1, 6 This study aims to determine dermatophyte species and bacterial superinfection in tinea pedis at H. Adam Malik General Hospital Medan.

Research Design
This study is a descriptive observational with cross sectional design. This study conducted at Mycology Division department of dermatology and venereology department of H. Adam Malik Hospital Medan from January to November 2016. This study aims to determine dermatophyte species and bacterial superinfection in tinea pedis at H. Adam Malik General Hospital Medan.

Research Sample
The sample of the study were patients with tinea pedis who fulfilled the inclusion and exclusion criteria. Inclusion criteria were patients aged above 17 years with tinea pedis based on clinical examination with positive KOH examination and willing to participate in research by signing informed consent. Exclusion criteria are being treated with topical antifungals in the past week, oral antifungals in the past month and oral and topical antibiotics in the past week. 45 samples were taken using consecutive sampling method.

Sampling Technique
Sampling for KOH examination and fungal cultures were selected in lesions with active edges. First, the area is cleaned with 70% cotton alcohol, then awaited dry. Scrambled with the blunt side of sterile scalpels in the areas of active edges. For lesions in the form of vesicles, bullae or pustules, done by opening the roof of the lesion using a sterile scalpel and then scrape the base of the lesion. The specimens were inserted into 2 specimen containers (envelopes) and labeled with patient's identity. Taking a sample of specimens for bacterial culture, swabbed using sterile lid cotton, then inserted into the transport medium swabs and labeled with patient's identity.

Microscopic Examination with KOH
The specimens were taken sufficiently, placed on object glass, dropped with 10% KOH solution then covered with a tumbler glass. The specimens are Flown over a small fire and left for 5 minutes. The specimen was examined under a microscope with a magnification of 10 x 40 to see the presence or absence of hyphae and arthroconidia. When examination results are found hyphae and/or arthroconidia, the investigation is continued to fungal culture and bacterial culture.

Fungal Culture
The specimens were applied to the surface of Sabaroud's dextrose agar medium with added cycloheximide (0.5 g / l) and chloramphenicol (0.05 g / l), then incubated at room temperature (26°C). Observations were made until there was fungal growth (maximum until the fourth week), then identified in macroscopic and microscopic. The macroscopic identification was made by observing the morphology (surface color, base color, surface texture, shape, and edges) of growing fungal colonies. Microscopic identification was performed using lactophenol cotton blue solution, observed hyphae, and conidia (macroconidia and microconidia).

Bacterial Culture
If in the Gram staining examination looks like a chain, signifying a Gram-positive cocci bacteria (streptococcus), so the bacterial culture was grown on blood agar medium only. The specimens were removed from the transport medium swab, applied to the blood agar medium and Mac Conkey agar medium, incubated at 37°C for 24 hours. If in the Gram staining examination looks like grapes, it is suspected the other Gram-positive cocci bacteria (staphylococcus), so the bacterial culture was grown on mannitol salt agar medium, incubated at 37°C for 24 hours (the color of S. aureus colonies are yellow gold, S.epidermidis colonies are white). Whereas, in the Gram staining examination suspected Gram-negative bacilli bacteria (Enterobacteriaceae), identified by biochemical reactions.
The result of the study showed the most bacteria species found from bacterial cultures of 35 specimens of dermatophyte was S. aureus a gram-positive coccus (30.8%), followed by 6 specimens (15.4%) of
The result of the study showed T. mentagrophytes (43.6%) was the most species of dermatophyte found in the interdigital type of tinea pedis, followed by T. rubrum with 13 cases (33.3%). T. rubrum was found in 1 case with the vesiculobullous type of tinea pedis. 1 case of both T. mentagrophytes (2.6%) and T. rubrum (2.6%) were found in mixed interdigital and vesiculobullous types. T. mentagrophytes was found in 2 cases of mixed interdigital and hyperkeratotic types of tinea pedis from the fungal culture examination ( Table 3).
The result of bacterial cultures in the interdigital type of tinea pedis showed S. aureus (28.2%) was the most species of bacteria, followed by K. oxytoca with 5 cases (12.8%), Mixed infections of S. aureus and K. pneumoniae with 1 case (2.6%) and 3 cases (7.7%) of no bacterial growth. K. pneumoniae was found in 1 case with the vesiculobullous type of tinea pedis. 1 case of both E. coli and S. Aureus species was found in mixed interdigital and vesiculobullous types. K.oxytoca found in 1 case of mixed the interdigital and hyperkeratotic (Table 4).

DISCUSSION
The result in his study showed the number of female subjects was almost equal to the number of male subjects. In general, tinea pedis is most commonly found in males than females, possibly due to greater activity and exposure to trauma and humid/hot conditions. 3 The largest age group in this study was 37-46 years old (28.9%), in accordance with the literature that mentions the most prevalence of tinea pedis was in adulthood and increases with age. 3 In this age group of 37-46 years old is mainly active in work, so they are more exposed to the causes of disease including trauma and humid/hot conditions. Most subject occupations are housemaids (22.2%) who in their daily work are often in wet and humid conditions that facilitate the occurrence of fungal infections.
The most dermatophyte species in this study were T. mentagrophytes (44.4%), followed by T. rubrum (33.3%), T. violaceum (5.1%) and E. floccosum (5.1%) ( Table 1). It accordance with the previous research by Tarigan in North Sumatra, Singapore, and Italy, The primary causal organisms of tinea pedis are T. rubrum, followed by T.mentagrophytes and E. floccosum. [11][12][13] The Difference results in species of fungi that have been found showed the differences of a geographical, climatical, and environmental factor. 10 The most common bacterial culture results in this study were S. aureus a gram-positive coccus (30.8%), followed by K. oxytoca (15.4%), and E. coli (12.8%) ( Table 2). Mainiadi in his study also found some bacterial species as secondary causes of infection in dermatophytosis with S. aureus was most commonly found (72.5%) followed by S. epidermidis, Proteus, and E. coli. 14 According to previous studies, either normal or pathological toe are often colonized by significant amounts of bacteria, including the Micrococccaceae family (staphylococci and micrococci), aerobic diphtheroid bacteria (primarily lipophilic strains) and Gram-negative bacteria. In complex dermatophytosis, the fungus grew less frequently between macerated toes, but the prevalence of S. aureus, another Gram-positive bacteria, and Gramnegative bacteria, is significantly increased. In the condition of damaged stratum corneum, the pathogenic bacterial species will be induced inflammation and maceration process. 6 In this study, the most common clinical features of tinea pedis encountered were interdigital types (88.9%). It accordance with many studies worldwide. 1,2,3,10 Interdigital types of tinea pedis often involve bacterial superinfection in the progression of the disease. 5-9 The result in this study showed T. mentagrophytes (43.6%) was the most species of dermatophyte found in the interdigital type of  The result of bacterial cultures examination in interdigital type of tinea pedis showed S. aureus (28.2%) was the most species of bacteria were grown, followed by K. oxytoca (12.8%), mixed infections of S. aureus and K. pneumoniae (2.6%) and 7.7% of no bacterial growth (Table 4). It suggests, there is a role of Gram-positive bacteria in producing of clinical manifestations in the interdigital type of tinea pedis, called complex dermatophytosis. But there are 7.7% cases with no bacterial growth, so in this condition of tinea pedis without bacterial involvement was called simplex dermatophytosis. According to Leyden et al., Simplex dermatophytosis is a pure fungal infection. When the toe area is excessively hydrated, microflora bacteria such as coccus and diphtheroid will overgrow manifested as a complex dermatophytosis (a collaboration between dermatophyte and bacterial infections). 5, 6 In the condition of severely complex dermatophytosis, Gram-negative organisms will be entered and aggravated the clinical manifestation of tinea pedis. 5,6,15

CONCLUSION
In this study, the most dermatophyte species found in tinea pedis were T. mentagrophytes followed by T. rubrum, T. violaceum and E. floccosum and the most common bacterial species were S. aureus followed by K. oxytoca, S. epidermidis, E. coli, B. subtilis, K. pneumoniae and Proteus sp. The role of dermatophyte and bacteria in clinical manifestation of tinea pedis have already known.