Agar-Based Disk Diffusion Assay for Susceptibility Testing of Dermatophytes

Abstract

Currently, no agar-based susceptibility testing method has been standardized for testing dermatophytes. We describe a newly developed agar-based method employing disk diffusion assay to test the susceptibility of 47 isolates of dermatophytes against 8 antifungals. Our results show that the method is reproducible, is simple, and could be used to determine the antifungal susceptibility of dermatophytes.The incidence of dermatophytosis (caused by Trichophyton, Epidermophyton, or Microsporum spp.[23]) has increased considerably, especially among immunocompromised patients (2,20). Relapse reported for some dermatophyte species and primary resistance of Trichophyton rubrum strains to terbinafine (18) underscore the need for determination of their in vitro antifungal susceptibilities. A reference microdilution method (M38-A2) is approved by the Clinical and Laboratory Standards Institute (CLSI) for antifungal susceptibility testing of molds and dermatophytes (7,19). However, no agar-based susceptibility testing method has been standardized for the testing of dermatophytes. Advantages of a standardized disk diffusion-based assay for evaluating the antifungal susceptibility of dermatophytes include the ease of use, reproducibility, accuracy, and low cost (1,3,10,15,16,22).In the current study, we optimized an agar-based disk diffusion method to determine the susceptibility of dermatophytes to various antifungals. We tested 47 clinical isolates (Trichophyton tonsurans [ Commercially available discs (9 mm diameter) preloaded with ciclopirox (50 g/disk), fluconazole (25 g/disk), itraconazole (8 g/disk), ketoconazole (15 g/disk), miconazole (10 g/disk), and voriconazole (1 g/disk) were used (Rosco NeoSensitabs; Key Scientific, TX). Discs containing griseofulvin (10 g/disk) and terbinafine (1 g/disk) were not commercially available and were prepared in our laboratory as part of this study. The concentrations of the drugs to be loaded in griseofulvin and terbinafine disks were determined by first performing preliminary experiments to determine the optimal concentration that produced inhibition zones which can be conveniently measured on the 100-by 15-mm plate (Fisher Scientific Co., KY). For these two antifungals, the drugs terbinafine (Novartis, NJ) and griseofulvin (Acros Organics, NJ) were obtained in powdered form. A stock solution of each drug was prepared using dimethyl sulfoxide, as follows: griseofulvin, 1.25 mg/ml; terbinafine, 50 g/ml. Blank paper discs (6 mm diameter) were loaded with 20 l of the prepared stock solutions to obtain the desired drug concentration per disk (1 g and 25 g for terbinafine and griseofulvin, respectively) and allowed to air dry at room temperature. The air-dried disks were stored at 4°C in a refrigerator.Organisms were subcultured on potato dextrose agar (PDA) or oatmeal agar (for T. rubrum) at 30°C for 4 to 15 days. Following growth, conidia were harvested in sterile saline, and using a hemacytometer, the conidial suspension was adjusted to 1.0 ϫ 10 6 conidia/ml. Mueller-Hinton (MH) agar (...

Antifungal Resistance Pattern among Dermatophytes in Western Rajasthan

“…Resistance against miconazole was not observed (0%) in our study which is similar to other studies conducted by few authors (Nweze et al, 2010). Terbinafine which is used systemically as well as topically, resistance against it was also very high (48%) and have also been reported by other authors like our study (Mukherjee et al, 2003).…”

“…Different dermatophyte strains may have different antifungal susceptibility patterns which may vary geographically also. Some strains of dermatophytes resistant to particular antifungal agent have been reported (Nweze et al, 2010). Resistance against fluconazole and terbinafine is found very high, this may be due to easy availability and indiscriminate topical as well as systemic use of these antifungal drugs.…”

“…Plates were allowed to dry then antifungal discs were applied over MHA plates. Plates were inverted and incubated at 28˚C and inhibition zone diameters were measured after 3-7 days for fungal growth (Nweze et al, 2010). Inhibition zone diameter was measured in millimeters and recorded as sensitive, intermediate or resistant (Table 1).…”

“…The Agar based disc diffusion method was performed as described by Nweze et al, (2010) [14] . All dermatophytes were subcultured on potato dextrose agar and incubated at 28˚C to enhance sporulation for one week.…”

A overview: non-steroidal anti-inflammatory drugs and mechanisms

“…Various scientific studies revealed that Sida acuta Burm. f. not only used as traditional medicine but also contain various useful activities such as Antibacterial [9,10], Anticancer [11], Antiplasmodial [12], Antioxidant [13], Analgesic [14,15], Antimalarial [16], Hepatoprotective [17], Demulcent, Diuretic [4], Larvicidal and Repellent activities [18] which are scientifically validated.…”