Determination of Hemolytic Activity and In Vitro Antifungal Susceptibility of Trichophyton rubrum Clinical Strains

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Trichophyton spp. which are among the agents of dermatophytosis with high morbidity, produce many virulence factors including hemolysins that exhibit toxic activity on immune system cells. Since relapses and chronicity are common problems related to dermatophytosis, prompt and appropriate treatment is of crucial importance. However, treatment is getting difficult due to the choice of inappropriate antifungals and increasing rates of cross-resistance among antifungal agents. The aims of this study were to investigate the hemolytic activities of Trichophyton rubrum strains isolated from patients with dermatophytosis and to detect the in vitro susceptibilities of those strains to ketoconazole, itraconazole, sulconazole, econazole and terbinaphine. Hair, skin and nail samples of patients were examined with direct microscopy using potassium hydroxide and cultivated on mycobiotic agar and Sabouraud dextrose agar. To determine hemolytic activities of T.rubrum strains, they were subcultured in Columbia Agar with 5% sheep blood and incubated for 7-14 days at 25 degrees C in aerobic conditions. Media which displayed hemolysis were further incubated for 1-5 days at 37 degrees C to increase hemolytic activity. Antifungal susceptibility testing was done with broth microdilution method guided by Clinical and Laboratory Standards Institute (CLSI) M38-A document. A total of 79 T.rubrum strains which exhibited negative urease and hair perforation tests, yielded pigmentation in potato-dextrose agar, were evaluated in the study. Hemolytic activity was detected in 71 strains (89.9%). Fifty strains showed incomplete (alpha) hemolysis and 21 strains showed complete (beta) hemolysis, whereas hemolysis was absent in eight of the isolates. Larger colonies created a larger zone of hemolysis and the smaller ones created a smaller zone. However, alpha-hemolysis did not turn to beta-hemolysis following further enlargement of the colony. According to antifungal susceptibility testing, the minimum inhibitory concentration (MIC) ranges, MIC50 and MIC90 values were found 0.0125-4 mu g/ml, 0.5 and 2 mu g/ml for ketoconazole; 0.0625-2 mu g/ml, 0.5 and 1 mu g/ml for itraconazole; 0.0313-4 mu g/ml, 0.25 and 1 mu g/ml for sulconazole; 0.0313-0.125 mu g/ml, 0.0313 and 0.0625 mu g/ml for econazole; 0.0313-0.0313 mu g/ml, 0.0313 and 0.0313 mu g/ml for terbinaphine, respectively. When the MIC values of hemolytic and non-hemolytic T.rubrum strains were compared, it was detected that hemolytic activity had no effect on MIC values. Our data have indicated that terbinaphine was the most effective antifungal agent against T.rubrum, while MIC values for itraconazole which is in common clinical use, were higher than expected and MIC values for econazole were lower than expected.


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Trichophyton rubrum, antifungal sensitivity, minimum inhibitory concentration, hemolytic activity



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