1. Background
Malassezia yeasts are lipophilic fungi which are regarded as normal flora of the skin, and are recovered in 75–98% of healthy individuals (1-3). Malassezia yeasts may also have a role in pityriasis versicolor (4), seborrhoeic dermatitis (5, 6), dandruff (6), seborrhoeic blepharitis (7), Malassezia folliculitis (8), confluent, reticulate papillomatosis (9) and uncommon systemic infections. In the last category, Malassezia species are associated with catheter acquired sepsis, fungaemia and pulmonary infection in neonates; these infections can sometimes be life threatening (10-14) and have been reported as significant causes of fungal infections in patients with cancer (15, 16) and HIV positive individual (17).
The difficulty in culturing Malassezia spp. in vitro has delayed the determination the taxonomic status of these yeasts. Nowadays, morphological characteristics and physiological features, the use of different Tween compounds as well as molecular studies of Malassezia species have allowed their classification into 11 species; M. furfur, M. sympodialis, M. slloffiae, M. obtusa, M. globosa, M. restricta, M. pachydermatis, M. yamatoensis, M. nana, M. japonica and M. dermatis (18, 19). Since molecular methods are not cost effective and unavailable in clinical laboratories, thus the purpose of this study was to identify Malassezia species isolated from pityriasis versicolor patients in Sari, Iran, by conventional methods, based on morphological, physiological and biochemical techniques (20).
2. Materials and Methods
2.1. Sample Collection
This cross sectional study was conducted between Octobers 2011 to April 2012 and a total of 134 patients clinically suspected of pityriasis versicolor, attending the Department of Mycology, Boali Sina Hospital and Referral Laboratory of the Mazandaran University of Medical Sciences were consecutively included. Individuals who received local or systemic antifungal for at least 5 days or corticosteroid agents for 3 weeks before the mycological examination were excluded from the study.
Skin scrapings were taken from the scaliest sites and obtained by the use of sterile blades. The diagnosis of pityriasis versicolor was established by the presence of both hyphae and spores in direct microscopy with KoH 20% and methylene blue staining. The assessed samples were plated onto Sabouraud's dextrose agar (SDA, Difco)(1% w/v peptone, 1% w/v glucose, 0.2% w/v yeast extract, 0.8% desiccated ox bile, 0.1% v/v glycerol, 0.05% w/v glycerol monostearate, 0.5% v/v Tween 60, and 2% v/v oleic acid, 1% w/v agar in distilled water) and modified Leeming and Notman agar supplemented with cyclohexamide (0.5%) and chloramphenicol (0.05%)(21), as fungal general medium and incubated at 32ºC for two weeks in the dark. The study then proceeded as follows, to identify the Malassezia species. This research was approved by the ethics committee of Mazandaran University of Medical Science and written informed consent was obtained from the each participant prior to enrolment.
2.2. Microscopic and Physiological Characteristics
Among Malassezia species, only M. pachydermatis is capable of growing on Sabouraud’s agar. However, more tests are essential for identification of the remaining Malassezia species such as Tween assimilation test, catalase reaction and splitting of esculin. After one week of incubation on modified Leeming and Notman agar at 32º C, the yeast cells were examined morphologically by gram and methylen blue staining. The catalase reaction was performed by the application of a drop of hydrogen peroxide (30% solution) onto a culture smear on a glass slide. Production of gas bubbles was considered as a positive reaction. M. restricta is the only lipid- dependent species lacking catalase (22, 23).
2.3. Splitting of Esculin
According to the method described by Mayser et al. (24), the b-glucosidase activity of different Malassezia species was assayed. Briefly, the loop of fresh yeast was deeply inoculated in the esculin agar (Merck, Germany) tube and incubated for 5 days at 32 ºC. The splitting of esculin is indicated by darkening of the medium. This test was used to distinguish M. furfur, M. slooffiae and M. sympodialis from other Malassezia species.
2.4. Tween Assimilation Test
The ability to utilize individual Tween compounds (Tween 20, 40, 60 and 80) was tested according to Guillot et al. (20). Briefly; the sterile Sabouraud's agar (16 ml) was melted and allowed to cool to approximately 50˚C. Malassezia yeast suspensions (about 105 cfu/mL) were mixed with Sabouraud's agar, and the mixtures were plated. Five holes were made in the agar by the means of a 2 mm diameter punch and filled with 5 μl of Tween 20, 40, 60 and 80 (Merck, Germany). The plates were incubated for one week at 32º C. Utilization of Tween was assessed by the degree of growth and /or reaction (precipitation) of the lipophilic yeasts around individual wells.
2.5. Statistical Analysis
Data collected in checklists were analyses using SPSS version.16. The data were analyzed using the chi-squared test. A P value of < 0.05 was considered significant.
3. Results
Of the 134 skin scraping samples, 116 patients (86.5%) were confirmed with pityriasis versicolor, based on the presence of both hyphae and spores in direct microscopy with KoH 20% and methylene blue staining, however, the Malassezia spp. were isolated from100/116 (86.2%) or 100/134 (74.6%) patients in the culture media, who were the final enrolled subjects. Table 1 shows that the distribution of patients clinically suspected of pityriasis versicolor based on age/ sex, indicating that there was no significant difference between age and gender of patients with pityriasis versicolor.
Table 1.Distribution of Patients Clinically Suspected of pityriasis versicolor Based on Age/Sex
| Age, years | Male No. (%) | Female No. (%) | Total No. (%) |
|---|---|---|---|
| ≤ 5 | 4 (3) | 3 (2.2) | 7 (5.2) |
| 6 - 14 | 1 (9.7) | 15 (11.2) | 28 (20.9) |
| 15 - 24 | 20 (14.9) | 31 (23.1) | 51 (8.1) |
| 25 - 34 | 16 (11.9) | 21 (15.7) | 37 (27.6) |
| 35 - 44 | 3 (2.2) | 3 (2.2) | 6 (4.5) |
| ≥ 45 | 4 (3) | 1 (.7) | 5 (3.7) |
| Total | 60 (44.8) | 74 (55.2) | 134 (100) |
According to descending frequency for the presence of Malassezia species among patients, M. globosa was found in n = 54; 54% cases, M. furfur in n = 32; 32% cases, M. sympodialis in n = 6; 6% cases, M. restricta in n = 6; 6% cases and M. slooffiae in n = 2; 2% cases. In this survey M. pachydermatis and M. obtusa were not found in any of the cases. There was a significant association between age and Malassezia species frequency (χ² = 36.32 df = 20, P = 0.014) (Table 2). There was no significant association between gender of patients and Malassezia species frequency (χ² = 9.24 df = 4, P = 0.055) (Table 3). Table 4 has shown the frequency of isolated Malassezia species based on anatomical locations. There was no significant association between anatomical sites and Malassezia species frequency (χ² = 23.26 df = 20, P = 0.28) (Table 4).
Table 2.Malassezia Species Frequency According to Age of Patients (P = 0.014)
| Malassezia spp. | M. globosa % | M. furfur % | M. sympodialis % | M. restricta % | M. slooffiae % | Total, % |
|---|---|---|---|---|---|---|
| Age, y | ||||||
| ≤ 5 | 3 | 1 | 1 | - | - | 5 |
| 6 - 14 | 8 | 8 | 1 | 1 | 1 | 19 |
| 15 - 24 | 33 | 9 | 2 | 2 | - | 46 |
| 25 - 34 | 5 | 13 | 2 | 2 | - | 22 |
| 35 - 44 | 3 | - | - | 1 | - | 4 |
| ≥ 45 | 2 | 1 | - | - | 1 | 4 |
| Total | 54 | 32 | 6 | 6 | 2 | 100 |
Table 3.Frequency of Malassezia Species According to Gender (P = 0.055)
| M. globosa % | M. furfur % | M. sympodialis % | M. restricta % | M. slooffiae % | Total, % | |
|---|---|---|---|---|---|---|
| Male | 26 | 18 | 4 | - | 2 | 50 |
| Female | 28 | 14 | 2 | 6 | - | 50 |
| Total | 54 | 32 | 6 | 6 | 2 | 100 |
Table 4.Frequency of Malassezia Species According to the Lesion Site (P = 0.276)
| M. globosa % | M. furfur % | M. sympodialis % | M. restricta % | M. slooffiae % | Total ,% | |
|---|---|---|---|---|---|---|
| Chest | 12 | 8 | - | - | - | 20 |
| Back | 11 | 5 | 3 | 5 | 1 | 25 |
| Abdomen | 14 | 9 | 1 | - | 1 | 25 |
| Neck | 9 | 3 | 1 | - | - | 13 |
| Face | 7 | 4 | 1 | 1 | - | 13 |
| Arms | 1 | 3 | - | - | - | 4 |
| Total | 54 | 32 | 6 | 6 | 2 | 100 |
4. Discussion
Although, Malassezia yeasts are part of the normal flora of the skin and are discovered in 75–98% of healthy hosts, their association with various skin disorders have been established; many studies have been conducted on the distribution of these yeasts (1-3). A great number of epidemiological studies using culture or molecular-based methods, e.g. PCR-RFLP or nested-PCR, have been performed in different geographical regions in order to investigate Malassezia species in patients with pityriasis versicolor (23, 25-27), including culture-based studies carried out in Spain (25, 28, 29), Japan (26), Bosnia and Herzegovina (30) Tunisia (27), Iran (31, 32), Turkey (33) and India (34).
By using morphological and physiological characteristics particularly in tropical areas, M. globosa either alone or associated with other Malassezia spp. (e.g. M. sympodialis, M. slooffiae) was the most commonly isolated Malassezia species. This ratio that indicated the predominance of M. globosa was between 53.3% and 63.3 % (25-28, 30-35). In contrast, a study carried out by Gupta et al. (36) indicated that M. Sympodialis was the predominant pityriasis versicolor isolate (59.5%), followed by M. globosa (25.2%), M. furfur (10.8%), M. slooffiae (2.7%), M. restricta (0.9%) and M. obtusa (0.9%) in Canada. In the present study, the most predominant species observed was M. globosa (54%) followed by M. furfur (32%), M. sympodialis (6%), M. restricta (6%) and M. slooffiae (2%). Nevertheless, M. pachydermatitis and M. obtuse were not observed from pityriasis versicolor lesions.
Our findings for M. globosa were most compatible to that of other similar studies (25, 26, 28, 30-35). However, M. furfur and M. slooffiae seemed to occur more frequently than in other studies carried out in Canada (6) Spain (25, 28, 35), Japan (26) Iran (28,29), Tunusia (27) and Bosnia (25), in which a frequency between 0% and 25.3%, and 0% and 5.3% was reported , respectively. In this study, no significant differences were found in species isolated according to the clinical type or anatomical sites of lesions. In contrast, in Bosnia, Prohic et al. (30) and in Spain, Erchiga et al. (25) reported that no significant differences were found in the isolated species according to the clinical sign or anatomical sites of lesions. In this study, the recovery rate of Malassezia spp. from pityriasis versicolor lesions was 74.6 % (100/134). In similar studies, this ratio tended to vary between 43.8% and 91.3% (17, 25-28, 30-34, 37-40).
It was noted that detection of different ratios of Malassezia species in patients with pityriasis versicolor in different parts of the world could be due to the use of different culture media (Leeming–Notman and modified Dixon agar) (25), climatic regions (tropical, subtropical, etc) (41), different sampling methods (swabbing/scraping) (25) and characteristics of patients (41). Although pityriasis versicolor has worldwide occurrence, its frequency is variable and depends on different climatic, occupational and socio-economic conditions (42, 43). In high temperate areas, the disorder is common in young adults aged 17–24 years. In tropical climates, pityriasis versicolor is more common in all age groups, but most cases occur in individuals aged 10–19 years (39).
In agreement with other investigations (43, 44), the highest prevalence of pityriasis versicolor in this study was observed for those aged between 15 to 24 years old , suggesting that the peak of the infection coincided with age periods when the sebum production is atits highest level. The role of gender in propensity to development of pityriasis versicolor is still unclear (29). To date the data revealed that pityriasis versicolor affected male individuals more than females (45, 46). However, several studies indicated that the incidence was higher in women (47, 48). In this survey we did not find significant association between gender of infected patients and the frequency of Malassezia species, this may be attributable to the extra attention of both sexes to beauty and skin hygiene (22).
Lesions mostly were found in the neck, face, back, arms, abdomen, and chest and in this survey, the most affected areas were found to be the chest, back and abdomen, which is concordance with the majority of studies worldwide (17, 49). The distribution of Malassezia species on the back and chest is proportional to the density and activity of pilosebaceous glands in these areas. However, there are a few reports indicated that pityriasis versicolor lesions can occur in unusual locations such as the nipple, genital areas and groin (49, 50).
5. Conclusions
According to our data, M. globosa was the main species causing pityriasis versicolor, as it was isolated from 54% of the lesions; followed by M. furfur (32% of lesions), while M. pachydermatitis and M. obtuse were not found in any of the cases and in 13.8% (16/116) of patients the culture was negative. The results demonstrated a significant relationship between the frequency of Malassezia species and age, however, gender and anatomical site of lesions of pityriasis versicolor was not significant. However, more studies with larger samples have to be performed to clarify the investigated subject. Since Malassezia species show different responses to antifungal therapy, thus correct identification of Malassezia species and antifungal susceptibility tests could facilitate selection of appropriate antifungal agents.
