Revised culture-based system for identification of Malassezia species

Forty-six strains of Malassezia spp. with atypical biochemical features were isolated from 366 fresh clinical isolates from human subjects and dogs. Isolates obtained in this study included 2 (4.7%) lipid-dependent M. pachydermatis isolates; 1 (2.4%) precipitate-producing and 6 (14.6%) non-polyethoxylated castor oil (Cremophor EL)-assimilating M. furfur isolates; and 37 (34.3%) M. slooffiae isolates that were esculin hydrolyzing, 17 (15.7%) that were non-tolerant of growth at 40 degrees C, and 2 (1.9%) that assimilated polyethoxylated castor oil. Although their colony morphologies and sizes were characteristic on CHROMagar Malassezia medium (CHROM), all strains of M. furfur developed large pale pink and wrinkled colonies, and all strains of M. slooffiae developed small (<1 mm) pale pink colonies on CHROM. These atypical strains were distinguishable by the appearance of their colonies grown on CHROM. Three clinically important Malassezia species, M. globosa, M. restricta, and M. furfur, were correctly identified by their biochemical characteristics and colony morphologies. The results presented here indicate that our proposed identification system will be useful as a routine tool for the identification of clinically important Malassezia species in clinical laboratories.     

Tween 40-based precipitate production observed on modified chromogenic agar and development of biological identification kit for Malassezia species.

We developed a simple identification kit for nine species of Malassezia (M. furfur, M. slooffiae, M. sympodialis, M. restricta, M. obtusa, M. globosa, M. pachydermatis, M. dermatis, and M. japonica) based on their biological features. This method utilizes Tween 40-based precipitate production on modified chromogenic agar (CHROMagar) Malassezia medium, growth on specific agars (Sabouraud's dextrose agar, Cremophor EL agar, Tween 60-esculin agar), and catalase reactions. This identification kit was verified with 11 type and reference strains of nine Malassezia species. An additional 26 clinical isolates were also successfully identified using the kit and the results were confirmed by molecular biological analysis.     

Species identification and strain typing of Malassezia species stock strains and clinical isolates based on the DNA sequences of nuclear ribosomal internal transcribed spacer 1 regions

This study demonstrated the application of internal transcribed spacer 1 (ITS1) ribosomal DNA sequences to the species identification and strain typing of 28 standard strains and 46 clinical isolates of the genus Malassezia. The size of ITS1 regions ranged from 162 to 266 bp. Members of the genus Malassezia (M. pachydermatis, M. furfur, M. sympodialis, M. globosa, M. obtusa, M. restricta and M. slooffiae) were classified into seven ITS1-homologous groups and 22 ITS1-identical, individual groups. The 46 clinical isolates of lipophilic Malassezia spp. were identified as belonging to just three ITS1-homologous groups, i.e., M. furfur (19 strains: 11 from pityriasis versicolor, 4 from seborrhoeic dermatitis and 4 from atopic dermatitis). M. sympodialis (22 strains: 7 from pityriasis versicolor, 3 from seborrhoeic dermatitis, 1 from atopic dermatitis and 11 from healthy controls) and M. slooffiae (five strains: three from chronic otitis media and two from healthy controls).     

Atopic dermatitis and Malassezia species: a study of antigenic components of Malassezia species for immunoglobulin E of patients with atopic dermatitis]

Antigenic components extracted by treatment with beta-mercaptoethanol from M. furfur, M. globosa, M. restricta, M. slooffiae, and M. sympodialis were studied for immunoglobulin E antibodies in sera of patients with atopic dermatitis. CBB staining and lectin blots of the extracts showed that each Malassezia species contained species-dependent components at the protein level. In a Western blot with the 2-ME extracts,IgE antibodies against the Malassezia species were found in sera of 83% (for M. globosa), 74% (for M. sympodialis), 65% (for M. furfur), 56% (for M. restricta) and 50% (for M. slooffiae) of the AD patients. In the Western blot inhibition test, the 2-ME extract of M. globosa partially inhibited the reaction of the antigenic components of other Malassezia species with the patients IgE antibodies. These results indicated that Malassezia species contained both species-specific and common antigenic components at the IgE antibody level. A major component of M. globosa was isolated from the 2-ME extract of this fungus by ion-exchange column chromatography and was referred to as Malg46b. Dot blot with the Malg46b containing fraction immunologically reacted with 69% of the sera of the patients, and with 83% of the sera of those who were positive for IgE antibodies to the 2-ME extract of M. globosa in Western blot. The intensities generated for each dot correlated well with the total intensities generated for the 2-ME extract of M. globosa in Western blot (r=0.763) The polyclonal antibody to Malg46b reacted strongly only with the 2-ME extract of M. globosa and reacted slightly with M. restricta. These results indicate that a glycoprotein, Malg46b of M. globosa, is dominantly expressed in this fungus and is a possible major antigen for IgE antibodies in patients with AD.     

A modified Christensen's urea and CLSI broth microdilution method for testing susceptibilities of six Malassezia species to voriconazole, itraconazole, and ketoconazole

Two supplemented broths (Christensen's urea with 0.1% Tween 80 and 0.5% Tween 40 and RPMI 1640 with 1% glycerol, 1% peptone, 1.8% glucose, and 0.05% Tween 80) were evaluated to determine voriconazole, itraconazole, and ketoconazole MICs for 200 Malassezia sp. isolates. Malassezia globosa and M. restricta were the least susceptible species (MICs at which 90% of the isolates tested were inhibited, 1 to >or=8 microg/ml versus 0.25 to 1 microg/ml).     

Examination of the causative agent of pityriasis versicolor]

Pityriasis versicolor (PV) is a superficial infection of the stratum corneum caused by Malassezia. Eleven species have been recognized within this genus, namely M. globosa, M. restricta, M. sympodiasis, M. furfur, M. obtusa, M. slooffiae, M. pachydermatis, M. dermatis, M. japonica, M. yamatoensis, M. nana. To examine the distribution of the microorganism in the skin of patients with PV, we detected Malassezia species without M. pachydermatis and M. nana using a non-culture-based method that consisted of nested PCR with specific primers. The most frequently isolated species were M. globosa and M. restricta (both 93.9%). M. globosa was detected in scales in which only the mycelial form (yeast cells, < 10/sample) were observed microscopically; M. restricta was not found. We suggest that M. globosa is the causative agent of PV.     

Improved detection of amphotericin B-resistant isolates of Candida lusitaniae by Etest

Both intrinsic and acquired resistance to amphotericin B have been documented for Candida lusitaniae. Amphotericin B remains the drug of choice for many critical fungal infections, and the detection of resistance is essential to monitor treatment effectively. The limitations of the National Committee for Clinical Laboratory Standards (NCCLS) reference methodology for detection of amphotericin B resistance are well documented, and several alternative methods have been proposed. Etest assays with RPMI and antibiotic medium 3 (AM3) agar were compared to the NCCLS M27-A broth macrodilution method using AM3 for amphotericin B resistance testing with 49 clinical isolates of C. lusitaniae. The panel included nine isolates with known or presumed resistance to amphotericin B on the basis of in vivo and/or in vitro data. The distribution of amphotericin B MICs by Etest with RPMI ranged from 0. 032 to 16 microg/ml and was bimodal. All of the putatively resistant isolates were inhibited by amphotericin B at >/=0.38 microg/ml and could be categorized as resistant using this breakpoint. Etest with AM3 yielded a broader amphotericin B MIC range (0.047 to 32 microg/ml), and there were six putatively resistant isolates for which MICs were >1 microg/ml. The separation of putatively susceptible and resistant isolates was less obvious. Broth macrodilution with AM3 generated a unimodal distribution of MICs (ranging from 0.032 to 2 microg/ml) and failed to discriminate most of the putatively resistant isolates at both 24 and 48 h. Etest using RPMI and, to a lesser extent, using AM3 provided better discrimination between amphotericin B-resistant and -susceptible isolates of C. lusitaniae.     

Taxonomy of genus Malassezia: progress and problems]

The genus Malassezia is composed of lipophilic basidiomycetous yeasts which were recently shown to consist of seven species, one lipid-independent species, M. pachydermatis and six lipid-dependent species, M. furfur, M. sympodialis, M. globosa, M. obtusa, M. restricta and M. slooffiae. Based on this classification, we will be able to analyze pathogenicity or relationship between Malassezia-related diseases and each species.     

Identification of Malassezia species isolated from patients with seborrhoeic dermatitis, atopic dermatitis, pityriasis versicolor and normal subjects.

We identified Malassezia species isolated from 42 patients with seborrhoeic dermatitis, 17 patients with atopic dermatitis, 22 patients with pityriasis versicolor, 35 normal subjects and 73 healthy medical students. Regarding the prevalence of Malassezia species in the 35 normal subjects, the frequency of isolation of Malassezia globosa was 22%, M. sympodialis 10% and M. furfur 3%. M. slooffiae, M. pachydermatis, M. restricta and M. obtusa were infrequently isolated from normal skin. Two different species were isolated coincidentally from seven samples. In the patients with atopic dermatitis, M. furfur was isolated more frequently from lesional skin (21%) than non-lesional skin (11%). However, there was no statistical significance. Therefore, this result, by itself, is insufficient to prove that M. furfur should be considered to be an exacerbating factor of atopic dermatitis. In seborrhoeic dermatitis, M. furfur (35%) and M. globosa (22%) were isolated from lesional skin on the face at significantly high rates in comparison with the normal subjects. Therefore, M. furfur and/or M. globosa may be pathogens of seborrhoeic dermatitis. M. globosa was isolated at a frequency of 55% from lesional skin of pityriasis versicolor, while all other species were below 10%. These data suggest that the pathogenic species of pityriasis versicolor is M. globosa.     

Molecular analysis of Malassezia microflora on the skin of atopic dermatitis patients and healthy subjects

Members of the genus Malassezia, lipophilic yeasts, are considered to be one of the exacerbating factors in atopic dermatitis (AD). We examined variation in cutaneous colonization by Malassezia species in AD patients and compared it with variation in healthy subjects. Samples were collected by applying transparent dressings to the skin lesions of AD patients. DNA was extracted directly from the dressings and amplified in a specific nested PCR assay. Malassezia-specific DNA was detected in all samples obtained from 32 AD patients. In particular, Malassezia globosa and M. restricta were detected in approximately 90% of the AD patients and M. furfur and M. sympodialis were detected in approximately 40% of the cases. The detection rate was not dependent on the type of skin lesion. In healthy subjects, Malassezia DNA was detected in 78% of the samples, among which M. globosa, M. restricta, and M. sympodialis were detected at frequencies ranging from 44 to 61%, with M. furfur at 11%. The diversity of Malassezia species found in AD patients was greater (2.7 species detected in each individual) than that found in healthy subjects (1.8 species per individual). Our results suggest that M. furfur, M. globosa, M. restricta, and M. sympodialis are common inhabitants of the skin of both AD patients and healthy subjects, while the skin microflora of AD patients shows more diversity than that of healthy subjects. To our knowledge, this is the first report of the use of a nested PCR as an alternative to fungal culture for analysis of the distribution of cutaneous Malassezia spp.     

Malassezia infectious diseases

Although Malassezia yeasts are a part of the normal microflora, under certain conditions they can cause superficial skin infection Pityriasis versicolor, Malassezia folliculitis. Lipophilic yeasts are being considered as major opportunistic pathogens for a very long time. Most of the yeasts show an absolute requirement for long fatty acid chains and specific procedures are required for their isolation, conservation and identification. To date, the genus is composed of one non lipid-dependent species M. pachydermatis and lipid-dependent species M. furfur, M. sympodialis, M. globosa, M. obtusa, M. restricta, M. slooffiae, M. dermatis, M. yamatoensis, M. japonica, M. nana, M. caprae, M. equina, M. cuniculi.     

Molecular analysis of malassezia microflora on the skin of atopic dermatitis patients and healthy subjects]

We compared cutaneous colonization levels of Malassezia species in patients with AD and healthy subjects using nested PCR. Malassezia-specific DNA was detected in all 32 of the patients with AD. M. globosa and M. restricta were detected in approximately 90% of these patients, with M. furfur and M. sympodialis being detected in approximately 40% of the cases. In healthy subjects, Malassezia DNA was detected in 78% of the samples, M. globosa, M. restricta and M. sympodialis were detected at frequencies ranging from 44 to 61%, and M. furfur was found in 11% of healthy subjects. Our results suggest that M. furfur, M. globosa, M. restricta and M. sympodialis are common inhabitants of the skin of both AD patients and healthy subjects, while the skin microflora of patients with AD shows more diversity than that of healthy subjects.     

Precipitate production by some Malassezia species on Dixon's agar.

The production of a precipitate by Malassezia species on Dixon's agar was observed. Malassezia furfur (n = 12), M. obtusa (n = 2) and M. slooffiae (n = 3) were precipitate negative, while M. sympodialis (n = 32) and M. globosa (n = 6) were precipitate positive. This test may be useful in differentiating Malassezia species.     

Identification of Malassezia species

Malassezia spp. are lipophilic unipolar yeasts recognized as commensals of skin that may be pathogenic under certain conditions. The genus Malassezia now comprises of seven species. This study was aimed at using a simple practical approach to speciate Malassezia yeasts from clinical material. Seventy skin scrapings from patients with pityriasis versicolor infection, positive in 10% potassium hydroxide (KOH), were cultured onto modified Dixon's agar (mDixon's agar) and Sabouraud dextrose agar (SDA) and incubated at 32 degrees C. Speciation was done on the basis of Gram stain morphology, catalase test, and utilization of Tweens. Out of 70 scrapings 48 (68.75%) showed growth on mDixon's agar. The commonest isolate was M. sympodialis (28, 58%) followed by M. globosa (19, 40%) and one isolate was (2%) of M. restricta. M. sympodialis was the commonest species affecting our population and there was no isolation of M. obtusa, M. slooffiae, M. pachydermatis and M. furfur.     

Identification of causative species in malassezia-associated dermatoses]

The Tween test was used to identify Malassezia species isolated from patients with pityriasis versicolor and seborrhoeic dermatitis, and suckling infants with seborrhoeic dermatitis. The most common species isolated from cases of pityriasis versicolor was M. globosa (55%), and this species was surmised to be the principal causative organism of this disease. In both adult and suckling infant cases of seborrhoeic dermatitis, M. globosa and M. furfur were isolated at high incidences compared with the healthy control subjects, indicating the possibility that one or both of these species are the causative organisms of these diseases. In addition, scales were collected from lesions of pityriasis versicolor for use as the template, and the involved Malassezia species were identified by the PCR method using the DNA sequences of nuclear ribosomal internal transcribed spacer l. The most commonly detected species was M. globosa, found in 97% of the cases, and this was followed in frequency by M. restricta (79%) and M. sympodialis (68%). It was also elucidated that multiple Malassezia species can be detected in the same specimen.     

Molecular analysis of Malassezia species isolated from three cases of Akatsuki disease (pomade crust)]

Malassezia spp. which normally colonize on the skin surface, are known as being either the cause or an exacerbating factor in a variety of skin conditions, including pityriasis versicolor, folliculitis, seborrheic dermatitis and atopic dermatitis. We report here three cases of Akatsuki disease (pomade crust). Scales and crusts were collected from the lesional skin and analyzed using a PCR-based non-culture method. Malassezia microflora in Akatsuki disease was compared to that of healthy subjects and atopic dermatitis patients. Samples were collected from upper and lower eyelids (Case 1), an operation scar (Case 2) and parietal scalp (Case 3). DNA was extracted from the scales and nested PCR was performed using specific primers for each species. Our analysis detected only M. obtusa and M. slooffiae in Cases 1 and 3 and only M. slooffiae in Case 2. Our previous data indicated that while M. globosa, M. restricta and M. sympodialis were common in healthy subjects, the two aforementioned species were rare, suggesting that the presence of M. obtusa and M. slooffiae in the subjects in the present study is correlated to the pathogenesis of Akatsuki disease.     

Antifungal activities of tacrolimus and azole agents against the eleven currently accepted Malassezia species

The lipophilic yeast Malassezia is an exacerbating factor in atopic dermatitis (AD) and colonizes the skin surface of patients with AD. With the goal of reducing the number of Malassezia cells, we investigated the antifungal activities of a therapeutic agent for AD, tacrolimus, and the azole agents itraconazole and ketoconazole against Malassezia species in vitro. We examined 125 strains of the 11 currently accepted Malassezia species by using the agar dilution method. All strains of the 11 Malassezia species were very susceptible to both azole agents, with MICs ranging from 0.016 to 0.25 mug/ml. Tacrolimus had antifungal activities against half of the strains, with MICs ranging from 16 to 32 mug/ml. Two of the major cutaneous floras, Malassezia globosa and Malassezia restricta, have several genotypes in the intergenic spacer region of the rRNA gene; the azole agents had slightly higher MICs for specific genotype strains of both microorganisms. A combination of azole agents and tacrolimus had a synergistic effect against Malassezia isolates, based on a fractional inhibitory index of 0.245 to 0.378. Our results provide the basis for testing these agents in future clinical trials to reduce the number of Malassezia cells colonizing the skin surface in patients with AD.     

Prevalence of Malassezia spp. in the ears of asymptomatic cattle and cattle with otitis in Brazil.

Yeasts of the genus Malassezia are lipophilic microorganisms that are saprophytes that can act as opportunistic pathogens in animals. Malassezia pachydermatis is commonly isolated from the ear canal and skin of healthy dogs, or in association with seborrheic dermatitis and otitis externa conditions. The objective of the present study was to determine the occurrence of Malassezia spp. in the ears of healthy bovines and bovines with otitis. Specimens (secretion or cerumen) were collected with sterile swabs, inoculated onto Mycosel medium, supplemented with olive oil, and incubated at 35 degrees C for 1 week. Yeasts were identified according to morphological characteristics, growth in Dixon medium at 32 degrees C and Sabouraud glucose medium modified by the addition of Tween 20, 40 or 80. The results showed that 54.7% of the cultures were positive in bovines with otitis (75) and 34.6% were positive in healthy bovines (378). Analysis of the positive cultures (41) from animals with otitis allowed presumptive identification of 24 strains corresponding to M. globosa (12), M. slooffiae (5), M. furfur (5) and M. sympodialis (2). Further studies on a larger number of animals may confirm the trend verified thus far, i.e. a higher frequency of isolation of Malassezia spp. from animals with otitis than from healthy animals (P<0.01) and a predominance of the species M. globosa.     

In vitro activity of nystatin compared with those of liposomal nystatin, amphotericin B, and fluconazole against clinical Candida isolates

We investigated the in vitro activity of nystatin and liposomal nystatin against 103 Candida isolates to determine the effect of both time and medium on MICs. We also compared the nystatin MICs with those of amphotericin B and fluconazole. Testing was performed in accordance with the National Committee for Clinical Laboratory Standards M27-A microdilution methodology with RPMI 1640, RPMI 1640 supplemented with glucose to 2% (RPMI-2), and antibiotic medium 3 supplemented with glucose to 2% (AM3). While nystatin MICs were similar to or slightly lower than liposomal nystatin MICs in RPMI 1640 and RPMI-2, they were markedly higher than liposomal nystatin MICs in AM3. Use of AM3 and determination of the MIC after 24 h of incubation provided a slightly wider range of liposomal nystatin MICs (0.06 to >16 microg/ml). Under these conditions, the MICs at which 90% of isolates were inhibited of nystatin and liposomal nystatin were 2 and 1 microg/ml, respectively. Nystatin and liposomal nystatin in general showed good activity against all Candida spp. tested. Although the MICs of nystatin and liposomal nystatin tended to rise in parallel with the amphotericin B MICs, nystatin and liposomal nystatin MICs of 1 to 2 and 0.5 to 1 microg/ml, respectively, were obtained for seven and six, respectively, of nine isolates for which amphotericin B MICs were >or=0.25 microg/ml. No correlation between fluconazole and nystatin or liposomal nystatin MICs was observed. As amphotericin B MICs of >or=0.25 microg/ml correlate with in vitro resistance, these results suggest that liposomal nystatin might have activity against some amphotericin B-resistant isolates. In vivo testing in animal models is required for clarification of this issue.