Susceptibility testing of Malassezia furfur, Malassezia globosa and Malassezia sympodialis. Comparison of disk diffusion and broth microdilution methods

ROJAS F; ZALAZAR L; FERNÁNDEZ M; SOSA MA; CATTANA ME; ALEGRE L; CARRILLO MUÑOZ AJ; GIUSIANO G

Lisboa

Congreso; 7th Trends in Medical Mycology; 2015

European Confederation of Medical Mycology (ECMM)

Objectives: The aims of this study were to evaluate the antifungal susceptibility of three Malassezia species against fluconazole, voriconazole, ketoconazole, itraconazole, miconazole, amphotericin B and terbinafine using both broth microdilution and agar diffusion methods, and to compare bothmethodologies using a model of linear regression analysis and categorical agreement.Methods: A total of 50 Malassezia isolates, including 30 M. furfur, 10 M. globosa and 10 M. sympodialis, were studied. Yeasts were identified by PCR‐RFLP. The broth microdilution method was performed as described in CLSI M27‐A3 reference document and disk diffusion test was performed on Mueller‐Hinton agar using tablets and disks. To support optimal growth of these yeasts, culture media were supplemented. In order to correlate both methods, linear regression analysis was performed and categorical agreement was determined.Results: Both supplemented media allowed optimal growth of Malassezia yeasts and the results were read at 72 h of incubation at 32ºC. Results of broth microdilution and agar diffusion testsobteined for the 50 isolates studied are summarized in Table 1. Malassezia yeasts were highly susceptible to itraconazole, ketoconazole and voriconazole. In contrast, a significant dispersion of values was observed for fluconazole, miconazol and terbinafine.The strongest linear association was observed for FLC. The relationship between variables for KTZ and ITZ was not linear. For the other drugs, coefficients obtained were not enough to conclude that a linear relationship exists between MIC and inhibition zone diameters variables. The highest categorical agreement between both methods was observed for itraconazole and voriconazole and the lowest for amphotericin and fluconazole. Table 2 indicates results of linear correlation analysis and categorical agreements when comparing both methodologies.Conclusions: Susceptibility profiles of Malassezia yeasts against FLC, AMB and TRB were variable and this is very important considering that are commonly used drugs. On the other hand, isolates were inhibited at low concentrations of ITZ, KTZ and VCZ, showing to be highly effective drugs. Although modifications made to both methodologies allowed obtaining susceptibility data for Malassezia yeasts, both methods cannot be associated through linear correlation model. When analysis was applied, the determination coefficient values obtained were very low for all drugs test, indicating that based on the diameter of the inhibition zone there is a low probability of minimal concentration inhibitory prediction using a linear model.The agar diffusion method is a low‐cost, fast and simple technique that is easily applicable in routine clinical laboratories. The diameters of the inhibition zones were clearly defined when modifications were made in agar diffusion method for Malassezia susceptibility study. However, not a goodcorrelation between this technique and the CLSI reference method was obtained for evaluating antifungal susceptibility of Malassezia yeasts. This highlights the need for to develop a reference protocol for Malassezia susceptibility testing in order to obtain comparable results by reproducible methods.