Epidemiology of Pityriasis versicolor in Tunisia: Clinical features and characterization of Malassezia species

Malassezia (M.) genus includes commensal yeasts of increasing medical importance, as they result in many diseases, ranging from pityriasis versicolor (PV) to systemic infections. Previous studies reported geographical variations in distribution of Malassezia species in PV lesions. The aims of the current study were to define the clinico-demographic features of PV in Tunisia, to characterize Malassezia isolates using phenotypic and molecular techniques and to find out any association between species and clinico-demographic parameters.In total, 120 PV patients were enrolled in this study. Skin scrapings were collected and inoculated on Sabouraud agar and modified Dixon medium. Malassezia species were identified using conventional phenotypic methods and 26 s rDNA PCR-RFLP. The highest prevalence of PV was observed among young adults’ group. The most affected body areas were the back and neck. In overall, 50.8% and 35% of PV cases had pruritus and history of recurrence respectively. The overall concordance between phenotypic and molecular methods was high (80.95%). The discordant results are rather due to the presence of multiple species in a single culture than true misidentification. Using PCR-RFLP, M. furfur was the most isolated species (38.7%) followed by M. globosa (37.7%), M. restricta and M. sympodialis. No statistically significant association was noted between Malassezia spp. and clinico-demographic characteristics. Unlike many reports from temperate climate countries, M. furfur and M. globosa along together were the most frequently isolated species in Tunisian PV patients. Although phenotypic methods remain simple and cost-effective, molecular techniques are considered as fast and accurate methods for diagnosis purposes.     

Study of the distribution of Malassezia species in patients with pityriasis versicolor and healthy individuals in Tertiary Care Hospital,Punjab

Purpose: Pityriasis versicolor (PV) is a chronic superficial fungal disease caused by Malassezia species. Our aim was to identify Malassezia species from PV patients and healthy individuals in Punjab. Materials and Methods: Modified Dixon agar was used as isolation culture medium. Identification was based on morphological observation and biochemical evaluation. The biochemical evaluation consisted of culture onto Sabouraud dextrose agar, catalase reaction, Tween assimilation, Cremophor EL assimilation, splitting of esculin and growth at 38⁰C. Results: Out of 58 microscopically diagnosed cases of PV, growth was obtained from 54 (93.10%) cases. The most frequently isolated species were M. globosa, M. sympodialis and M. furfur which made up 51.79%, 31.42% and 18.51% of the isolated etiological agents respectively. However, the major isolate from the back of healthy individuals was M. sympodialis (47.61%), followed by M.obtusa (19.04%), M. globosa (14.20%), M. furfur (9.52%), M. pachydermatis (4.76%) and M. slooffiae (4.76%). Conclusions: M. globosa in its mycelial phase was the main etiological agent, but as normal flora from the back of healthy subjects, it was found in significantly less number (P = 0.01), suggesting that the higher pathogenicity of M. globosa in terms of enzymatic endowment, might be the cause of its predominance in PV lesions.     

MALDI-TOF MS identification of Malassezia species isolated from patients with pityriasis versicolor at the seafarers’ medical service in Dakar,Senegal

Pityriasis versicolor (PV) is a superficial mycosis caused by yeast of the genus Malassezia. The most common isolated Malassezia species in PV lesions differ among M. furfur, M. globosa and M. sympodialis. We purpose to determine the distribution of Malassezia species in PV patients at the seafarers’ medical service in Dakar, Senegal and to examine whether any association between identified Malassezia species and patients’ profile. From May 2017 to August 2017, first a questionnaire was filled to get informative data before collection of skin scrapings taken from most scaly site using sterile scalpel blade and application of scotch^® for direct examination (DE). At the laboratory, DE, culture and identification by MALDI-TOF MS were done. One hundred patients with PV – all men – were included with a mean age of 34 years. Among seafarers, 81% were sailors. Clinical prevalence of PV was highest in aged adults patients with ages of 31 to 60 years (56%). Seafarers with high level of education were less representative with only 2%. The mean duration of the PV was 26.83 months. 20% of subjects suffered lesions in more than one location. The chest was the most affected anatomical site. Furthermore, possible predisposing factors associated with PV were also detected. DE was positive in 95% but culture growth only in 46%. MALDI-TOF MS analysis of the positive cultures could be performed in 84.8% (39/46). Only M. furfur was identified in 100% (39/39). In definitive, M. furfur is the only causative agent of PV in Dakar.     

Distribution of Malassezia species on the skin of patients with psoriasis

IntroductionMalassezia species can induce the expression of interleukin-17 (IL-17), which plays an important role in the inflammatory and immune response in psoriasis (PS). The purpose of this study was to investigate the Malassezia species composition in patients with PS and healthy individuals and explore the role of Malassezia species in the pathogenesis of PS.Materials and methodsA total of 28 patients with PS and 10 age- and sex-matched healthy individuals participated in this study. Specimens collected from the lesional and non-lesional skin of patients with PS and the skin of healthy individuals were analyzed by using nested PCR.ResultsThe relative abundance of Malassezia species was 84.96% in healthy subjects, more than twice that in patients with PS (P < 0.01). M. restricta (43.09%) and M. globosa (41.38%) were the main Malassezia species in patients with PS followed by M. furfur (4.84%) and M. sympodialis (2.49%). M. sympodialis accounted for 18. 81% of the Malassezia species in healthy subjects, which was nearly eight times higher than in patients with PS (P < 0.01). Further, M. furfur was detected both on lesional and non-lesional psoriatic skin, but it was not found on the skin of healthy individuals.ConclusionsThe Malassezia species composition in patients with PS differed from that of healthy individuals. M. restricta and M. globosa were the main Malassezia species in patients with PS.     

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.     

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.     

Physiological and molecular characterization of atypical isolates of Malassezia furfur

The species constituting the genus Malassezia are considered to be emergent opportunistic yeasts of great importance. Characterized as lipophilic yeasts, they are found in normal human skin flora and sometimes are associated with different dermatological pathologies. We have isolated seven Malassezia species strains that have a different Tween assimilation pattern from the one typically used to differentiate M. furfur, M. sympodialis, and M. slooffiae from other Malassezia species. In order to characterize these isolates of Malassezia spp., we studied their physiological features and conducted morphological and molecular characterization by PCR-restriction fragment length polymorphism and sequencing of the 26S and 5.8S ribosomal DNA-internal transcribed spacer 2 regions in three strains from healthy individuals, four clinical strains, and eight reference strains. The sequence analysis of the ribosomal region was based on the Blastn algorithm and revealed that the sequences of our isolates were homologous to M. furfur sequences. To support these findings, we carried out phylogenetic analyses to establish the relationship of the isolates to M. furfur and other reported species. All of our results confirm that all seven strains are M. furfur; the atypical assimilation of Tween 80 was found to be a new physiological pattern characteristic of some strains isolated in Colombia.     

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.     

Quantitative culture of Malassezia species from different body sites of individuals with or without dermatoses.

Quantitative cultures were obtained using contact plates to determine whether the quantity and composition of Malassezia species at a given anatomic site in normal individuals differs from that of patients with various cutaneous dermatoses. The sample included 20 clinically healthy individuals (without any dermatosis) and 110 patients with dermatoses (including 31 with atopic dermatitis [AD], 28 with psoriasis [PS], 28 with seborrheic dermatitis [SD] and 23 with pityriasis versicolor [PV]). Contact plates filled with special culture medium were used to obtain a quantitative culture from five body sites (scalp, forehead, arm, trunk and leg) of every individual. The number of cfu were recorded for every plate that grew Malassezia yeasts, and 3-5 colonies were isolated for identification to species level using microscopic, physiological and molecular characteristics. The mean cfu counts observed among patients with AD, PS and SD was significantly lower than normal control subjects (P < 0.05). The mean cfu counts from PV patients was not different from that of healthy control subjects. Overall, for all conditions considered together, the mean cfu counts in lesional sites were significantly lower than in non-lesional sites (P <0.05). Furthermore, the mean cfu counts from lesional sites in patients with AD and PS were significantly lower than the corresponding value in patients with PV (P <0.05). Six Malassezia species were recovered from the different dermatoses. Malassezia sympodialis was the most common species associated with AD and PV patients and healthy control subjects, while M. globosa was most frequently isolated from PS and SD patients. More than one Malassezia species was recovered at any given anatomic site from both controls as well as individuals with dermatoses. M. globosa was equally likely to be recovered from scalp, forehead and trunk, but less likely to derive from arms and legs. M. restricta and M. slooffiae were recovered more frequently from the upper body (scalp and forehead) than from the lower body. Among normal individuals and for patients with AD and PV, M. sympodialis was significantly more likely to affect the forehead than the legs.     

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.     

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.     

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.     

Efficacy of modified Leeming-Notman media in a resazurin microtiter assay in the evaluation of in-vitro activity of fluconazole against Malassezia furfur ATCC 14521

Background

Malassezia furfur is lipodependent yeast like fungus that causes superficial mycoses such as pityriasis versicolor and dandruff. Nevertheless, there are no standard reference methods to perform susceptibility test of Malassezia species yet.

Antagonistic Activity of <Emphasis Type="Italic">Malassezia Spp.</Emphasis> towards Other Clinically Signifi cant Yeast Genera

Antagonistic activity of Malassezia yeast towards clinically signifi cant yeast species was studied. Ten Malassezia strains exhibited this activity. M. furfur strain exhibited maximum activity and the least sensitivity to “foreign” metabolites. M. globosa proved to be the most sensitive and the least active. M. furfur metabolites exhibited pronounced activity towards 6 Basidiomycetes strains. This effect was signifi cantly higher in comparison with antagonistic activity towards 13 Ascomycetes species. Studies of a complex of M. furfur antagonistic metabolites showed that it has at least two components: thermolabile proteins with molecular weights of 33 and 35 kDa and a thermostable one, proteinase-resistant. In contrast to metabolites of many other yeast species, this substance is more effective against related Basidiomycetes microorganisms (Cryptococcus albicans), while antagonistic proteins are active mainly towards Ascomycetes, such as Candida albicans. It was found that mycocin-like activity of Malassezia is encoded by chromosomes, but not plasmids.     

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.     

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.     

Epidemiology of Malassezia yeasts associated with pityriasis versicolor in Ontario, Canada.

The genus Malassezia was recently revised to include seven species, but the clinical significance of each of these species is not clearly understood. To obtain a better understanding of the contribution of individual Malassezia species to the epidemiology of pityriasis (tinea) versicolor, we used Leeming-Notman medium to culture patient skin specimens showing positive evidence of Malassezia infection in direct microscopy. Isolates were identified on the basis of recently published morphological and physiological tests for distinction of the new species. Identification using recently developed molecular criteria was also carried out for the ambiguous cases. Malassezia species were cultured from 111 cases of pityriasis versicolor in the Canadian province of Ontario. The most frequently isolated species were Malassezia sympodialis, M. globosa and M. furfur which respectively made up 59.4%, 25.2% and 10.8% of the isolated etiological agents. M. globosa was commonly isolated from a small number of pityriasis versicolor specimens obtained from investigators outside Canada. A large number of additional Ontario specimens with positive direct microscopy failed to yield a culture; however, it is suggested that this is consistent with the standard sampling practice of scraping the older portions of pityriasis lesions rather than the margins, where viable fungal cells are most likely to occur.     

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.     

Occurrence of Malassezia spp. in the external ear canals of dogs and cats with and without otitis externa.

We studied the lipophilic microbiota of the external ear canals of 332 animals (264 dogs and 68 cats), with and without otitis externa, over an 11-year period from 1988 to 1999. Malassezia pachydermatis was isolated from 62.2% and 50% of dogs with and without otitis externa, respectively, and from 41.2% and 17.6% of cats with and without otitis externa, respectively. In the group of animals studied for lipid-dependent species, these yeasts were isolated from 4.5% of dogs with otitis externa and from 23.1% and 8.9% of cats with and without otitis externa, respectively. M. sympodialis and M. furfur were isolated from cats and M. furfur and M. obtusa from dogs. Our findings show that lipid-dependent Malassezia species may contribute to the etiology of otitis externa in dogs and cats.