In Vitro Amphotericin B Susceptibility of Malassezia pachydermatis Determined by the CLSI Broth Microdilution Method and Etest Using Lipid-Enriched Media

Abstract

We determined the in vitro amphotericin B susceptibility of 60 Malassezia pachydermatis isolates by the CLSI broth microdilution method and the Etest using lipid-enriched media. All isolates were susceptible at MICs of <1 g/ml, confirming the high activity of amphotericin B against this yeast species. Overall, the essential agreement between the tested methods was high (80% and 96.7% after 48 h and 72 h, respectively), and all discrepancies were regarded as nonsubstantial. Malassezia pachydermatis is a lipophilic but non-lipid-dependent basidiomycetous yeast which thrives on the skin of most warm-blooded vertebrates (1-3). Although generally regarded as a commensal microorganism, M. pachydermatis can become pathogenic under the influence of diverse factors, leading to different clinical forms of dermatitis and/or otitis, mainly in small animals (1, 2, 4). Systemic infections caused by this yeast species are only rarely encountered, but some outbreaks of fungemia have been reported in intensive care nurseries (5, 6). Notably, in one such outbreak, the introduction of the yeast into a nursery was linked to its possible zoonotic transmission from colonized pet dogs to the health care workers' hands, with further spread of the microorganism in the unit through person-to-person contact (5).Antifungal susceptibility testing of Malassezia species remains a challenge, as their growth is not supported (or, in the case of M. pachydermatis, only poorly supported) on the standard lipid-free RPMI growth medium recommended for yeast testing by the CLSI and EUCAST (6-8). A variety of alternative procedures, most of which use lipid-enriched media, have been proposed, but published studies have mainly focused on M. pachydermatis susceptibility to ketoconazole, itraconazole, and other azole derivatives (e.g., 8-17). On the contrary, information on the in vitro susceptibility of M. pachydermatis isolates to the polyene amphotericin B, which is commonly used for treating bloodstream infections in human patients (5-7), is scarcer; the available studies were based on a single method and/or a low number of isolates (see Table 1), so the reliability of their results cannot be adequately assessed.The aim of this study was to expand the database of MIC values to amphotericin B for M. pachydermatis isolates, as determined by two different procedures using lipid-enriched media, (i) a modified broth microdilution (BMD) method based on CLSI guidelines (18) and (ii) the Etest, which is a commercial agar-based gradient technique that is widely used for antifungal susceptibility testing of yeasts.Isolates. A total of 60 Malassezia pachydermatis isolates obtained from clinical cases of canine otitis and primarily recovered in Sabouraud dextrose agar (SDA) containing 0.005% chloramphenicol (bioMérieux, Marcy l'Etoile, France) were included in this study. They were identified as M. pachydermatis on the basis of their macroscopic and microscopic morphologies and their ability to grow on media without lipid supplementation, such as SDA, at 32°C (19)...

Malassezia infections with systemic involvement: Figures and facts

“…Amphotericin B is among the preferred therapeutic options for the first‐line approach in patients who are usually under fluconazole prophylaxis . However, the variable results regarding the susceptibility profile testing should raise concern about treatment recommendation . Future comprehensive studies are warranted to elucidate which antifungal may be optimal for treatment of Malassezia systemic infections.…”

“…Susceptibility testing has not been standardized for these yeasts and therefore is not performed by routine in many clinical laboratories. The use of broth microdilution and E‐test methods has been reported, but both are still somewhat cumbersome . More recently, an optimized colorimetric broth microdilution method has been developed for faster and more efficient profiling of antifungal susceptibility of Malassezia isolates .…”

“…[36][37][38][39] More recently, an optimized colorimetric broth microdilution method has been developed for faster and more efficient profiling of antifungal susceptibility of Malassezia isolates. 7 According to available data, Malassezia organisms exhibited conflicting results regarding susceptibility testing to amphotericin B: M. pachydermatis being inhibited by low concentrations of amphotericin B (≤1 lg/mL); 39 and M. furfur and Malassezia globosa requiring high amphotericin B minimal inhibitory concentration (MIC) values. 36 More often, reports described high MIC and wide MIC ranges depending on the strains tested.…”

Susceptibility of Malassezia pachydermatis to aminoglycosides

“…Due to an increasing number of cases of fungal skin infections caused by Malassezia spp. reported in humans and animals, the in vitro susceptibility profiles to antifungal drugs has been recommended in order to control this yeast . Moreover, in vitro susceptibility testing for antifungal drugs has previously been described for clinical isolates that cause treatment‐resistant otitis externa and dermatitis …”

“…Each of these methods has previously been used for susceptibility testing with azoles . and amphotericin B to determine the in vitro susceptibility of M. pachydermatis .…”