Malassezia spp. are part of the resident skin flora of humans and other warm-blooded vertebrates that are discovered in 75–80% of healthy individuals 1. These yeasts are associated with various superficial skin diseases, including pityriasis versicolor (PV), seborrheic dermatitis (SD), dandruff, and folliculitis, and can exacerbate atopic dermatitis (AD) 2. Malassezia spp. are also thought to be involved in psoriasis and acne vulgaris, although the detailed role of the microorganisms is unknown 3, as well as nosocomial bloodstream infection in pediatric care units 4.
The genus Malassezia has undergone several taxonomic revisions based on morphology, ultrastructure, physiology, and molecular biology. In the reclassification by Guého et al. 5, seven distinct species were identified within this genus (Malassezia furfur, Malassezia sympodialis, Malassezia pachydermates, Malassezia globosa, Malassezia obtusa, Malassezia restricta, and Malassezia slooffiae). Recently, on the basis of DNA relatedness, new species have been included: Malassezia dermatis, Malassezia nana, Malassezia japonica, Malassezia yamatoensis6–8, Malassezia caprae, Malassezia equina, and Malassezia cuniculi, and thus Malassezia spp. yeasts are classified into 14 species at present 9,10.
The study on Malassezia spp. has been postponed for many years owing to its nutritional requirements and its morphological variability. Molecular biology methods led to the taxonomic revision of this genus and to a new interest for its clinical importance. Molecular methods are an accurate tool in the identification and they lead to a better knowledge of the ecology and epidemiology of this genus 11.
The aim of this study was to elucidate the pathogenic role of Malassezia spp. in Malassezia spp.-related skin diseases, and to evaluate PCR, which is a reliable and rapid molecular diagnostic tool in identification compared with the conventional methods.
Patients and methods
The study included four groups of patients (groups I, II, III, and IV, which are PV, SD, dandruff, and AD, respectively) and a control group (V). They were randomly selected from those attending the Dermatology Outpatient Clinic at Misr University for Science and Technology (MUST) Hospital (Table 1).
Patients who received any treatment within 2 weeks from sample collection or had any dermatological and/or systemic diseases that could affect the outcome of the study were excluded.
All patients and normal candidates included in the study were subjected to history taking, general examination, dermatological examination including distribution and morphology of the lesions, and assessment of disease severity.
Collection of samples
Specimens were taken by scraping the lesions with a scalpel. Moreover, in normal individuals and in cases with few scales, samples were taken by means of sellotape. In groups I and V, specimens were collected from the face, neck, upper trunk, and upper limb; in group II from the face and chest; in group III from the scalp; and in group IV from two body sites, the face and flexures. Direct microscopy with KOH 20% was carried out on all samples.
Culture of skin scrapings
A total of 245 isolates were cultured on Sabouraud dextrose agar, Dixon, Modified Dixon medium, Mycosel agar, and Potato Dextrose agar. In the last two media, drops of olive oil were added after inoculation. All the media were incubated at 31°C for 2 weeks and examined at frequent intervals for yeast growth.
Identification of the isolates
Conventional (culture-based) identification of Malassezia spp.
The isolates were identified based on morphological characteristics, growth on Dixon’s medium at different temperatures (32, 37, and 40°C), catalase test, tryptophan utilization, tween assimilation (20, 40, 60, and 80), castor oil assimilation, and splitting of esculin 5,12,13.
Molecular identification of Malassezia spp. using 26S rDNA PCR-restriction fragment polymorphism
- DNA extraction: The DNA extraction was carried out using a glass bead phenol chloroform method according to Yamada et al.14.
- PCR amplification (Thermal cycler; Biometra; Analytik Jena company, Germany): two primers: forward, 5′-TAACAAGGATTCCCCTAGTA-3′ and reverse, 5′-ATTACGCCAGCATCCTAAG-3′ were used to amplify the internal transcribed spacer (ITS) region in the rDNA gene in all Malassezia spp. PCR amplification was carried out in a final volume of 50 µl. Each reaction contained 1 µl of template DNA, 0.5 µmol/l of each primer, 0.20 mmol/l of each deoxynucleoside triphosphate, 5 µl of 10× PCR buffer, and 1.25 U of Taq polymerase. An initial denaturation step at 94°C for 5 min was followed by 30 cycles of denaturation at 94°C for 45 s, annealing at 55°C for 45 s, and extension at 72°C for 1 min, with a final extension step of 72°C for 7 min. Amplified products were visualized by 1.5% (w/v) agarose gel electrophoresis in TBE buffer, stained with ethidium bromide (0.5 µg/ml), and photographed under UV transillumination, to show successful isolation of the Malassezia genus 15.
- Fragmentation of PCR products by restriction enzymes: The PCR products were electrophoresed in 1.5% agarose gel and digestion was performed by incubating a 21.5 µl aliquot of PCR product with 10 U of the enzyme in a final reaction volume of 25 µl at 37°C for 3 h, followed by 2% agarose gel electrophoresis in TBE buffer and staining with ethidium bromide. The enzymes CfoI (Roche Diagnostics, Manheim, Germany) and BstF51 (SibEnzyme, Novosibirsk, Russia) were selected according to Mirhendi et al.15. The restriction fragments were analyzed according to the size and number of DNA fragments seen under the UV transilluminator (dual-intensity UVP) using Uvisoft image acquisition and analysis software (http://www.uvitec.co.UK).
The statistical analysis of data was performed using statistical package for the social sciences (SPSS) (IBM Company, Armonk, New York, USA), version 16 on Windows XP. The description of data was done as frequency and proportion for qualitative data and mean±SD for quantitative data. Pearson’s χ2-test and Z-test to compare significance between two proportions were conducted. Pearson’s χ2-test was conducted to compare Malassezia spp. isolated from healthy and Malassezia spp.-associated dermatoses, and to statistically examine the difference in the frequency and distribution of Malassezia spp. by different ages, sex, and body sites. Z-value was carried out to compare identification rate between Malassezia spp.-associated dermatoses and healthy individuals. P-value less than 0.05 was considered significant.
The study included 245 individuals (Table 1), direct microscopy revealed positive hyphae and spores in 114 skin scrapings, showing characteristic ‘spaghetti and meatball’ appearance in patients complaining of PV (Fig. 1), whereas the remaining 131 specimens were positive only for yeast cells. Culture for the 245 specimens collected from patients and controls revealed isolation of 160 Malassezia spp. isolates with a total recovery rate of 65.3%. The highest recovery rate was observed in patients with PV (100/114, 87.71%) followed by SD patients (15/28, 53.57%), dandruff cases (15/31, 48.38%), AD patients (15/32, 46.87%), and the least recovery rate was observed in normal individuals (15/40, 37.5%).
A significant difference between isolation rates of Malassezia spp.-associated dermatoses (145/205, 70.7%) and healthy individuals (15/40, 37.5%), being higher in Malassezia spp.-associated dermatoses, was found. (Z=3.071, P<0.05).
According to phenotypic identification methods, three species were identified, the most commonly isolated species was M. furfur (112/160, 70%) followed by M. globosa (46/160, 28.75%) and M. sympodialis (2/160, 1.25%). Only one Malassezia spp. was isolated from each specimen.
Isolated M. furfur was characterized by smooth colonies, grew on Dixon’s medium at 32, 37, and 41°C, and microscopically showed oval elongated cells with broad base buds. It gave catalase, tweens, castor oil, and tryptophan test–positive reactions (Figs 2–4).
M. globosa was characterized by creamy buff and rough colonies, did not grow at 37 or 41°C, and microscopically showed spherical yeast cells with narrow base buds (Fig. 5). It gave catalase positive but did not assimilate tweens or castor oil and showed esculin splitting and tryptophan test-negative.
M. sympodialis was characterized by creamy-soft and smooth colonies, grew at 32, 37, and 41°C on Dixon’s medium, and microscopically showed sympodial conidiation (Fig. 6). It gave catalase-positive reaction, assimilated tweens 40, 60, and 80, and hydrolyzed esculin, but did not assimilate tween 20 or castor oil.
A significant difference (χ2=14.9, P=0.021) between species isolated from healthy and Malassezia spp.-associated dermatoses was found, as M. sympodialis was only isolated from the PV patients.
In patients with PV, three species were identified: 76 (76%) isolates were M. furfur, 22 (22%) were M. globosa, and 2 (2%) were M. sympodialis. Analysis of the data obtained showed that PV occur maximum in the age group 20–30 years (55%), minimum at extreme ages 0–10 years (2%), and at a rate of 11% in more than 40 years, with no statistical significance in relation of species to age (χ2=11.7, P=0.166). The occurrence of Malassezia spp. is verified in both the sexes, but the prevalence of PV is observed more frequently in male patients (79%). Comparing species isolated in male and in female patients showed no statistical significance (χ2=1.21, P=0.57). In addition, hyperpigmentation and hypopigmentation seen in the lesions and their body sites was shown to be caused by different species, with no statistical difference observed (χ2=2.94, P=0.230 and χ2=3.48, P=0.747, respectively) (Table 2).
In patients with SD, two species were identified, 7 (46.6%) isolates were M. furfur and 8 (53.3%) were M. globosa. Analysis of the data showed that SD was observed more in male patients (14/15, 93.3%) than female patients, more in the age group of 20–30years (8/15, 53.3%), with no statistical significance in relation of species to age, sex, body site, and severity score (Table 3).
In patients with dandruff, two species were identified: 12 (80%) isolates were M. furfur and 3 (20%) were M. globosa. Statistical analysis to investigate the relation of isolated species to age, sex, or severity assessment showed no significance (P>0.05) (Table 4).
In AD patients, two species were identified: 6 (40%) isolates were M. furfur and 9 (60%) were M. globosa. Statistical analysis of data showed that most of the cases are within the age group of 0–10 years (14/15, 93.3%), AD occurs more in male patients (12/15, 80%), with no statistical relation of species to age, sex, site, or severity of the disease (Table 5).
In normal controls, two species were identified: 11(73.3%) isolates were M. furfur and 4 (26.6%) were M. globosa. Statistical analysis of the data obtained revealed no relation of species identified to age, sex, or site of isolation (Table 6).
A total of 61 identified species by conventional methods were randomly chosen from healthy and Malassezia spp. disease-related dermatoses to reconfirm the identification using PCR.
The primers successfully amplified the target part of 26S rDNA from all Malassezia spp. tested, providing a single PCR product of the expected size (∼580 bp) (Fig. 7).
On analyzing the PCR-restriction fragment polymorphism (PCR-RFLP) of the 26S rDNA using restriction enzymes Bsft51 and CfOI, three different restriction patterns were found by gel electrophoresis (Fig. 8).
Analysis of the bands using Uvisoft image acquisition and analysis software, three species (M. furfur, M. globosa, and M. sympodialis) were identified (Fig. 9). M. furfur showed two bands, 311 and 429 bp; M. globosa shows two bands, 306 and 361 bp; and M. sympodialis shows three bands, 306, 373, and 429 bp.
The identification of all the 61 isolates by molecular methods showed 100% concordance to that of the conventional methods.
Unlike many bacteria and other fungi, Malassezia spp. yeasts are rarely found in the environment. Malassezia spp. abounds in the skin of subjects from tropical and subtropical climates 16.
In clarifying how normal lipophilic Malassezia spp. yeast could lead to disease, the results indicate that the difference of Malassezia spp. is not the cause but rather the increased colonies and overgrowth of Malassezia spp. yeasts, owing to the destruction of skin barriers and to abnormalities of the immune system in predisposed individuals 17.
PV is the skin infection where Malassezia spp. plays a definite causative role. The occurrence of clinical disease by Malassezia spp. depends on the factors permitting conversion of the saprophytic yeast phase of the organism to the mycelia phase 18. Direct microscopic examination revealed hyphae and yeast cells among the scales collected from the lesions of our PV patients, whereas hyphae was not found in the other Malassezia spp.-related diseases. Therefore, the presence of hyphae is important in elucidating the causative agent of PV, also after antifungal treatment, and no hyphae but only yeast cells are detected in PV scales 19.
In cases of dandruff, SD, and AD, Malassezia spp. could increase the inflammatory response by three main mechanisms: (a) altering skin barrier function through the production of lipases and phospholipases; (b) increasing the local immune response through the production of bioactive metabolites (indoles) and stimulating keratinocytes to produce an array of interleukins; and (c) sensitization to cross-reactive allergens produced by Malassezia spp. yeasts through the development of specific antibodies 10.
Similar to other investigations 20–22, the highest prevalence of Malassezia spp.-associated dermatoses (except AD) in the present study was observed in 20–30-year-old patients, suggesting that the peak of infection coincides with age when the sebum production is in the highest level.
The Malassezia spp. distribution as normal flora is related to the sebaceous gland density and activity; thus, the scalp, face, central chest, and back bears the highest number of fungi 17. In this survey, the most affected areas were the trunk and neck, which is concordant with the majority of studies worldwide 2,19,21.
The role of sex in propensity to the development of Malassezia spp. dermatoses is still unclear. Some studies found that diseases were more common in men than women, 23,24 as in our PV and SD patients. However, others indicated that the incidence of this is higher in women, 25 as in our dandruff patients, which may be because of the extra attention of women to beauty and skin hygiene. However, many reports 2,21 found no differences in the development of Malassezia spp. dermatoses among both sexes.
In this survey, the most common isolated species in the lesions was M. furfur, which is in concordance with the study by Makimura et al.26 and others 27,28. This was contrary to the observation of other studies 22,29,30 that isolated M. globosa as the predominant species. M. sympodialis was the main isolated species in other investigations 21,25,26,31. Gemmer et al.32 in cases of dandruff isolated M. restricta (50%), then M. globosa (33%), and least was M. sympodialis (8%).
The differences between the studies may be a result of geographic variation in species prevalence 26. M. globosa is the predominant species found in temperate climates. The yeast has been isolated from the soil in Brazil and central European forests and has been described to show favored growth at temperatures below 38°C. On the contrary, M. furfur is the predominant species found in tropical and subtropical zones 27. The predominance of M. furfur in this study may be because of the higher temperature and humidity that may play a role in its pathogenicity. Different Malassezia spp. may need different temperature requirements to undergo yeast mycelial transformation. A gene encoding a secreted lipase of M. furfur possibly associated with both its growth, and pathogenicity was cloned and characterized, and it was found that this gene is most active at higher temperature of 40°C 33.
Similar to previous studies, 19 no significant correlation was found regarding different Malassezia spp. identified and the age, sex, body sites (neck, trunk, and upper limb), clinical picture (hypopigmentation or hyperpigmentation in PV), and severity in the studied Malassezia spp.-associated diseases.
Accurate and reproducible methods of species identification are essential for epidemiological purposes. Most studies have been focusing on morphological and biochemical analysis, which are time-consuming and subject to controversy. Several conventional diagnostic tests must be used as culture-based methods that can be biased because of different growth rates and culture requirements of different species 34. In addition, the metabolic traits that conventional identification methods use do not have enough variables to differentiate this number of species 10. Species differentiation has to be complemented by molecular analysis of genes encoding subunits of the ribosomal RNA (rRNA) 35. Therefore, molecular methods are species-specific, less time-consuming, and more cost-effective. Identification of Malassezia spp. isolates by PCR-RFLP of ITS1–1TS2 regions has been applied recently, 36–38 albeit for a restricted number of species.
The 26S rDNA, which was targeted in this study, contains highly conserved base sequences and enough sequence variation that can serve as markers for species-specific restriction enzyme analyses and species identification. In addition, it is compatible with morphological methods and appropriate for identification of the currently known Malassezia spp. In agreement with the new taxonomic classification, it requires only two restriction enzymes, CfoI and BstF51, and has been proven to be technically easier to perform than other molecular techniques 39.
In the present study, conventional identification was in complete concordance with the PCR results, and the same three Malassezia spp. were identified. In both cases, the method proved accurate and simple.
The possibility to carry out Malassezia spp. detection and identification directly on the clinical sample, without nucleic acid extraction and eliminating the time for culture, together with the reduction of costs, represent some of the main goals of microbiological clinical diagnostics.
In conclusion, PCR-RFLP method that was used in this study enabled us to examine genetic variations through cleaving the amplified DNA with restriction enzymes and analyzing the patterns of the fragments. It confirmed the conventional identification of Malassezia spp. in Malassezia spp.-associated dermatoses in our study. The method has many advantages in that it is less time-consuming, more accurate as it is species-specific, and more cost-effective, and therefore can be utilized in rapid diagnosis and epidemiological studies.
In clarifying the relation between Malassezia spp.-related dermatoses and Malassezia spp. yeasts, further studies are needed. Species-specific and strain-specific research may help elucidate the role of the individual Malassezia spp. in various diseases. It is not only necessary to compare the qualitative difference of the Malassezia spp. but also to etiologically analyze the quantitative difference.
We are all ‘carriers’ of this fungus and it remains in equilibrium with the human skin (commensal/symbiotic status). What disturbs this equilibrium and turns Malassezia spp. yeasts to pathogens remains to be elucidated.
Conflicts of interest
There are no conflicts of interest.
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