Antifungal Susceptibility Testing of Dermatophytes by ABDD and E-Test, a Comparative Study

Aim: The objective of this study was to isolate, identify, and explore the in-vitro antifungal susceptibility pattern of dermatophytes isolated from clinically suspected cases of dermatophytosis (tinea infections) attending the Dermatology Department at J.S.S Hospital. Methods: This study was conducted at JSS Medical College and Hospital from December 2016 to December 2017. Clinical samples (e.g., skin scrapings and hair stumps) were collected under aseptic precautions. The identification of dermatophytes was performed through microscopic examination using 10%, 20% & 40% potassium hydroxide (KOH) and culture on Sabouraud dextrose agar (SDA), SDAac, PDA and Dermatophyte test medium (DTM). All dermatophytes isolates were subjected to antifungal susceptibility testing using the agar-based disk diffusion (ABDD) and E-test method against Terbinafine, Itraconazole, Fluconazole, and Griseofulvin. Data were analyzed by using Chi square test. Results: A 100 samples were studied, 46% tinea corporis, 2%tinea cruris, 9% tinea pedis, 5% tinea faciei. The dermatophytes isolated were Trichophyton rubrum 11 (35%), Trichophyton isolation of dermatophytes. The ABDD method appears to be a simple, cost-effective, and promising method for the evaluation of antifungal susceptibility of dermatophytes. E-test method is the most sensitive method due to the fact that quantitative MICs can be obtained directly from the E-strip. However, the E-test method is expensive and difficult in defining the precise borders of the inhibition zones in dermatophytes.


Introduction
Dermatophytes are a group of closely related filamentous fungi able to damage and utilize keratin found in the skin, hair and nails [1] [2]. These are classified into three genera: Microsporum, Trichophyton, and Epidermatophyton. Dermatophytoses is an infection produced by dermatophytic fungi in the keratinized tissues. Clinically, dermatophytoses can be classified depending on the site involved. These include Tinea capitis (scalp), Tinea corporis (non-hairy skin of the body), Tinea ungium (nail infection), tinea cruris (groin), Tinea pedis (athletes foot), and Tinea barabae (bearded areas of the face and neck) [1] [2]. World Health Organization (WHO) estimates dermatophytes affect about 25% of the world's population. It is also estimated that 30% -70% of adults are asymptomatic carriers of these pathogens, and that the incidence of this disease increases with age [1] [3]. The estimated life-time risk of acquiring dermatophytoses is between 10% -20%. The global prevalence of dermatophytoses is estimated to be 20% [2] [4]. The prevalence of dermatophytes has increased tremendously in the last few decades due to various factors like climatic changes, socio-economical and occupational situations [5] [6].
The diagnosis of a dermatophytic infection is mostly done clinically, but often confused with other skin infections due to the topical application of steroid ointments and creams, leading to further misdiagnosis and mismanagement [4] [7].
Hence, there arises the need for the correct, efficient, and rapid laboratory diagnosis of dermatophytes [8]. Another important point to consider is that resistance to antifungals has started appearing in dermatophytes [9]. The establishment of a reference antifungal susceptibility testing method may allow the clinician to select the appropriate therapy for the treatment of infections caused by dermatophytic fungi [10] [11].
The antifungal susceptibility test of dermatophytes has been well-studied in some developed countries, but few data are available in tropical countries [12].
Resistance to antifungal agents is also on the increase in dermatophytes seeks to improve knowledge of the molecular identification and the antifungal susceptibility test of dermatophytes [13]. The agar-based disk diffusion (ABDD) suscep-   Following growth, conidia were harvested in sterile saline and conidial suspension was adjusted to 10 × 10 6 using hemocytometer. Only two Antifungal discs were tested against the dermatophyte isolates. The discs used were Itraconazole (10 mg) and Fluconazole (25 mg). Muller Hinton Agar (MHA) plates were streaked evenly in three directions with a sterile cotton swab dipped into the standardized inoculums suspension. Plates were allowed to dry then antifungal discs were placed onto the medium. Each disc was pressed down to ensure complete contact with the agar surface and distributed evenly so that they are no closer than 24 mm from each other, center to center as illustrated by Figure 1 and

E-test
The E-test method was performed according to the manufacturer's protocol.
The isolated dermatophytes were sub-cultured on potato dextrose agar and incubated at 28˚C to enhance sporulation for one week. Four Antifungal agents were tested against the dermatophyte isolates. The antifungal agents were Itra-   Table 7.

Results
In the present study, a total of 100 clinically suspected cases of nail, skin and hair dermatophytosis were included in the study. Out of the 100 samples 85 (85%) were skin scrappings, 10 (10%) were hair and 5 (5%) were nail samples as illustrated in Table 1 Out of 100 samples 28 (28%) of them were KOH positive and as well as culture positive, 3 (3%) were KOH negative and culture positive, KOH positive and culture negative were 5 (5%). However, 64 (64%) samples were both KOH and culture negative as illustrated in Table 2. Table 3 illustrates that tinea corporis were the highest at 64% of the identified isolates while tinea unguium and tinea faceie made up 5% each. Table 4 shows the number of respective dermatophytes     Table 6 shows MICs criteria as provided by Himedia. The IZD were then carefully recorded in Table 7, Table 8 and Table 9 respectively.       [19]. Another important point to consider is that resistance to antifungals has started appearing in dermatophytes [20]. The establishment of a reference antifungal susceptibility testing method may allow the clinician to select the appropriate therapy for the treatment and also for studying mechanisms of Open Journal of Medical Microbiology drug resistance of dermatophytic fungi [21] [22].
In the present study a total of 100 specimens (skin scrapings, hair fragments and nail clippings) were collected from clinically suspected cases of dermatophytosis. 69% were from males and 31 (31%) were from female patients visiting the dermatology department. Out of the 100 clinical cases, tinea corporis accounted for 64%, tinea cruris accounted for 6%, tinea pedis 10%, tinea faciei 5%, tinea capitis 10% and tinea unguium accounted for 5% and. 5% samples were KOH positive and culture negative.
A comparison was made between the two antifungal agents used in both tests (E-test and ABDD). The MIC and IZD values produced by the organism were recorded in Table 4, Table 8 and Table 9. The MIC and the IZD values were found to be inversely proportional to each other [23].  [24].
The zones of inhibition were seen in all the strains except 1 strains of T. rubrum as could be seen in Figure 1 and Experience in determining MICs and careful attention to procedural details are critically important in conducting the E-test method because it is not as easy as it is with inhibition zone scale in disk diffusion method [2] [27]. The MICs and IZDs are inversely proportional to each other i.e. when the MIC for the drug is more; the IZD is smaller and vice versa. Successful treatment of fungal infections depends on the ability of a given antimycotic agent to eradicate the fungus from the tissue [28]. Though some in-vitro antifungal susceptibility tests are now available, no simple reference method has been standardized for testing the drug susceptibility of dermatophytes [28] [29].

Conclusions
The present study demonstrated a good correlation between MICs and IZDs of the drugs. It was observed that if the MIC value was low for a particular isolate a larger zone of inhibition was observed [30]. Furthermore, it was also observed that if MIC was higher, a smaller zone of inhibition was observed.
Disc diffusion method is simple, reproducible, cheap and easily adaptable.
Furthermore, it has potential for use in selection of appropriate antifungal agents Open Journal of Medical Microbiology once the conditions such as temperature and inoculums size are properly standardized [31] [32]. The inhibition zones for the disks were easy to measure with the inhibition zone scale compared to the reading of the E-strips. In addition to that, more than one antifungal can be tested in the same plate at the same time to conserve time and media [18] [33]. Antifungal susceptibility testing by disk diffusion can become an important method in treatment of patients with fungal infections.
Therefore, based on the results of the present study; it could be concluded that the method for antifungal susceptibility of dermatophytes is the disc diffusion method as is the simpler, cheap and reliable method in comparison with the E-test. However, when it comes to sensitivity the E-test supersedes the disc diffusion method. Previous studies have also shown that the E-test method is the most sensitive method due to the fact that quantitative MICs can be obtained directly from the E-strip [34] [35]. However, the E-test method is expensive and difficult in defining the precise borders of the inhibition zones in dermatophytes [36]. Despite that, the agar based disk diffusion method was found to be favorable in comparison to the E-test method. Future efforts must put more emphasis on establishing standard interpretive break-points for dermatophytes for licensed as well as newly introduced antifungals and correlating them to the clinical outcomes.