Identification of Malassezia Species Isolated from Iranian Seborrhoeic Dermatitis Patients

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European Review for Medical and Pharmacological Sciences 2010; 14: 63-68 Identification of Malassezia species isolated from Iranian seborrhoeic dermatitis patients

M.T. HEDAYATI, Z. HAJHEYDARI*, F. HAJJAR**, A. EHSANI§, T. SHOKOHI#, R. MOHAMMADPOUR‡

Department of Medical Mycology and Parasitology; *Department of Dermatology; **Department of Medical Mycology and Parasitology, School of Medicine, Sari (Iran) §Department of Dermatology, School of Medicine, Tehran University of Medical Sciences, Tehran (Iran) #Department of Medical Mycology and Parasitology, School of Medicine; ‡Department of Statistics, School of Health, Mazandaran University of Medical Sciences, Sari (Iran)

Abstract. – Background and Objectives: Key Words: In recent years, the genus Malassezia has come Seborrhoeic dermatitis, Malassezia species, Tween to be considered important in the etiology of se- assimilation. borrhoeic dermatitis (SED). The aim of present study was identification of Malassezia species on the lesions of Iranian SED patients. Methods: 100 patients with SED were enrolled in the study. The patients were evaluated both clinically for the severity of SED and microscopically for the presence of the yeast Malassezia. Diagnosis of Malassezia was made Introduction after the yeast Malassezia was microscopically observed on skin scales stained with methylene Yeasts of the genus Malassezia, formerly blue. All samples were also cultivated on Leem- known as Pityrosporum, are ubiquitous skin resi- ing and Notman and Sabouraud’s dextrose agar culture media. The agar plates were incubated at dents of humans and other warm-blooded animals 32°C for 2 weeks and evaluated for the existence and also associated with several dermatological of growth every day for one week. Identification disorders. In recent years, the genus Malassezia of isolated yeast was based on morphological has come to be considered important in the etiolo- and physiological characteristics. gy of seborrhoeic dermatitis (SED)1-3. The im- Results: From 100 patients with SED, 60% provement of SED (and the related condition, were female. The age range was 12-65 years with dandruff) by treatments directed against median 27.3 years. The highest prevalence of SED was seen in 20-29 years age group. 59% and 41% Malassezia supports the role of the yeasts in of patients had local and generalized lesions, re- these conditions, although other factors such as spectively. 58% of patients showed lesion on an impaired barrier function are also thought to scalp. Microscopic examination of skin scales be important4. So the epidemiology and the ecol- was positive in 100% of SED lesions. 96% of pa- ogy of Malassezia species in SED has been con- tients showed more than 1-3 yeasts in each mi- sidered by different investigators1-3,5-8. Epidemio- croscopic field whereas only 4% patients showed 1-3 yeasts in whole slide. Totally, 77% of the spec- logical data suggest geographical variations in imens yielded Malassezia in culture. Malassezia the rate of the isolated Malassezia species from globosa was the most commonly isolated SED1-3,5-8. Malassezia species (55.8%). Malassezia globosa The taxonomy of Malassezia has undergone had also most frequencies on scalp and face le- extensive revisions in the last 10 years and is still sions. Malassezia furfur had most frequency on in a state of flux. Guillot and Gueho described trunk lesions. and named 7 species of Malassezia (M.): M. fur- Conclusion: The results of our study showed high recovery rate of Malassezia species on le- fur, M. sympodialis, M. obtusa, M. globosa, M. 9 sions of patients with SED. So it might be play- restricta, M. slooffiae and M. pachydermatis . ing a causative role in the etiology of this dis- Subsequently, other species have been described, ease. including M. dermatis10, M. japonica11, M.

Corresponding Author: Mohammad T Hedayati, PhD; e-mail: [email protected] 63 M.T. Hedayati, Z. Hajheydari, F. Hajjar, A. Ehsani, T. Shokohi, R. Mohammadpour nana12, M. yamotoensis13, and M. equi, although Species Identification the latter has not been formally recognized as a We identified Malassezia species according to species14. Guillot et al.17. Isolates from the primary cultures In Iran, only a few culture studies have been were used for identification. M. pachydermatis is made using the new classification of the able to grow on Sabouraud’s agar. Tween assimila- Malassezia yeasts15,16 but clinical studies are tion and catalase test were applied for identification lacking. Therefore, it is important to look at the of other Malassezia species. Briefly, Malassezia distribution of the various Malassezia species on yeast suspensions were prepared in sterile distilled the skin of patients with Malassezia associated water, which were adjusted to McFarland No. 2 tur- skin diseases including SED patients. In our bidity, mixed with Mycobiotic agar with cyclohexa- study we present some informations about the mide and chloramphenicol, and poured into the distribution of Malassezia species on the lesions plates. Four holes were made in the agar by means of Iranian SED patients. of a 3-mm diameter punch and filled with 30 μl each of tween 20, 40, 60 and 80, respectively. The agar plates, incubated at 32°C for 1 week, were examined each day for the existence of any growth Materials and Methods around the wells that contained tween compounds.

Patients Catalase Test 100 patients with SED from the Department of One drop of hydrogen peroxide solution was Dermatology, of Boali Hospitals (Sari City) and applied onto a yeast colony, which was on a glass from the Department of Dermatology, Hospital slide. Production of gas bubbles was considered Razi, Tehran, were enrolled in the study. The pa- a positive catalase reaction. tients filled out the consent form to participate in research which was approved by the Ethical Statistical Analyses Committee of Mazandaran University of Medical Chi-square test was performed using SPSS Sciences. The severity of the SED lesions was software (version 13.0) and differences were evaluated using a semiquantitative scale of 0 to 3 considered significant at p<0.05. for each of the symptoms (erythema, scaling, itching and seborrhoea), where a value of 0 cor- responded to the absence of symptoms, 1 to mild symptoms, 2 to moderate symptoms and 3 to se- Results vere symptoms. The sum of these values is re- garded as score of SED. 60% of the patients with SED were female. The age range was 12-65 yrs with median and Sampling and Microbiological Evaluation SDF yrs of 27.3 and 10.2, respectively. The high- Sampling was performed from the lesions af- est prevalence of SED was seen in patients with ter diagnosis of SED. Skin scales were scraped 20-29 yrs old. 59% and 41% of patients had local off with a sterile blade and transferred to the lab- and generalized lesions, respectively. 58% of pa- oratory. Diagnosis of Malassezia was made after tients showed lesion on scalp (Table I). budding yeast cells that were microscopically observed on skin scales stained with methylene blue. To counting of yeast cells, each slide was Table I. Distribution of SED patients on the basis of age examined under high power field (hpf) of micro- and gender. scope and it was recorded as number of observed yeast cells per hpf. All samples were also culti- Total Male Female Age vated on Leeming and Notman medium and n (%) n (%) n (%) Yrs Sabouraud’s dextrose agar culture media. The 24 (24) 11 (28.9) 13 (21) 10-19 agar plates were incubated at 32 °C for 2 weeks 44 (44) 14 (36.8) 30 (48.4) 20-29 and evaluated for the existence of growth every 16 (16) 6 (15.8) 10 (16.1) 30-39 day for one week. Identification of isolated yeast 13 (13) 7 (18.4) 6 (9.7) 40-49 was based on morphologic and physiologic tests, 3 (3) 0 (0) 3 (4.8) 50≤ namely tween assimilation profiles and catalase 100 (100) 38 (100) 62 (100) Total reaction.

64 Identification of Malassezia species isolated from Iranian seborrhoeic dermatitis patients

A: 1-3 yeast cells in whole slide B: 1-3 yeast cells per hpf C: 4-7 yeast cells per hpf D: > 7 yeast cells per hpf

Figure 1. Distribution of observed yeast cells number in direct microscopic examination (DME) on lesions of patients with SD.

Direct microscopic examination (DME) of Table IV shows the frequency of observed specimens was positive in 100% of SED lesions. yeasts number in DME on lesions of patients Figure 1 shows the observed yeast cells number with SED based on age groups. 48% and 49% of in DME on lesions of patients with SED. 96% patients in 20-29 years age group showed 4-7 (96/100) of patients showed more than 1-3 yeast and >7 yeast cells per hpf, respectively. There cells per hpf whereas only 4% (4/100) patients was significant difference between observed showed 1-3 yeast cells in whole slide. yeasts number in DME on lesions of patients Totally, 77% of the specimens yielded with SED and age groups (p=0.003). Malassezia in culture. The most commonly iso- Out of 100 patients with SED, 9, 74 and 17 pa- lated Malassezia species was M. globosa tients showed mild, moderate and severe SED, re- (55.8%), followed by M. furfur (32.5%), M. respectively (Table V). With respect to the observed stricta (9.1%), M. sympodialis (1.3%) and M. yeasts number in DME on lesions, 22.2%, 56.7% japonica (1.3%). M. globosa was most common and 41% of patients with mild, moderate and se- isolated species from face (17/27, 63%) and vere SED had a density of >7 yeast cells per hpf, scalp (25/45, 55.5%). M. furfur had most fre- respectively (Table V). There was not significant quency in trunk (4/5, 80%) (Table II). There was difference between the observed yeasts number in no statistically significant difference between DME and severity of SED (p=0.176). cultured Malassezia species and body sites (p=0.613). Table III shows the culture results from differ- Discussion ent areas of the body in SED patients. There was not significant difference between culture result In our study the most frequency of SED was and body sites (p=0.935). observed in women and the age group of 20-29

Table II. Distribution of various Malassezia species on lesions of patients with SED.

Body site species Scalp n (%) Face n (%) Trunk n (%) Total n (%)

M. globosa 25 (55.5) 17 (63) 1 (20) 43 (55.8) M. furfur 13 (28.8) 8 (29.6) 4 (80) 25 (32.5) M. restricta 5 (11.1) 2 (7.4) 0 (0) 7 (9.1) M. sympodialis 1 (2.2) 0 (0) 0 (0) 1 (1.3) M. japonica 1 (2.2) 0 (0) 0 (0) 1 (1.3) Total 45 (100) 27 (100) 5 (100) 77 (100)

65 M.T. Hedayati, Z. Hajheydari, F. Hajjar, A. Ehsani, T. Shokohi, R. Mohammadpour

Table III. Frequency of culture results based on body sites in patients with SED.

Body site culture result Scalp n (%) Face n (%) Trunk n (%) Total n (%)

Positive 45 (77.6) 27 (77.1) 5 (71.4) 77 (77) Negative 13 (22.4) 8 (22.9) 2 (28.6) 23 (23) Total 58 (100) 35 (100) 7 (100) 100 (100)

yrs old. Although it has suggested that SED can that was only sporadically identified in previous appear at any age, the highest prevalence is ob- reports7,15. Regarding to the most commonly iso- served in individuals aged 30 to 60 years18 and it lated species, there were differences between pa- is more common in males than in females19, in tients with various skin diseases and also the re- contrast to the results of our study. ported results from different countries. This dif- In DME 100% of samples yielded a positive ference may be attributable to the sampling and result which is the same reported by Crespo Er- culture techniques as well as to geographical dif- chiga et al20 in pityriasis versicolor. Moreover, ferences and it could be elucidated by worldwide 96% of our patients showed more than 1-3 yeast epidemiological studies. cells per hpf. This is a further evidence that our In the present research there was not a signifi- patients with SED harbour a higher number of cant difference between the positive culture re- Malassezia yeasts. One reason may be the in- sults and the various body sites. However, M. creased amount of lipids in the skin of seborrhoic globosa was most common isolated species on patients21. Some Authors have also shown a scalp and face lesions and M. furfur had most greater number of Malassezia cells in lesional frequency on trunk lesions. This result in part skin of patients with SED22,23 or a greater de- agrees with Gupta et al.25 that used the contact tectable incidence in affected subjects24. Howev- plates method to isolate and identify Malassezia er, others have reported a decrease in the density species from the different body sites of individu- of recovered yeasts from lesional skin25,26. It has als with or without dermatoses including SED also been suggested that seborrhoeic dermatitis is patients. It has also been suggested that different not caused by an overgrowth of Malassezia cells, Malassezia species tend to be found on different but by an abnormal host response to the yeasts on body sites25,28 in both normal and diseased skin. the skin21. We have isolated single separated colony from In the present study, 77% of the specimens each sample, as suggested by Tarazooie et al15 in yielded Malassezia in culture which is nearly patients with pityriasis versicolor. However, in concordant to other studies7,23,27, and M. globosa many studies, more than one species have been and M. furfur were the most commonly isolated recovered from each sample21,25,27. It should be Malassezia species. According to earlier reports, underlined that providing a pure culture and the M. sympodialis, M. globosa, M. restricta and M. separation of a species from a mixed sample is furfur were the most commonly isolated too difficult. This might be due to this point that Malassezia species in SED patients5-8. Converse- fast growing species usually cover other species ly, Swedish SED patients’ skin lesions are more in the culture. Besides, because of hydrophobic frequently colonized with M. obtusa27, a species characteristic of Malassezia yeast, preparing ho-

Table IV. Frequency of observed yeast cells number in DME on lesions of patients with SED based on age group.

Age groups 10-19 20-29 30-39 40-49 ≥50 Total Number of observed yeast cells in DME n (%) n (%) n (%) n (%) n (%) n (%)

1-3 yeasts in whole slide 1 (25) 1 (25) 0 (0) 0 (0) 2 (50) 4 (100) 1-3 yeast cells per hpf 4 (20) 6 (30) 6 (30) 4 (20) 0 (0) 20 (100) 4-7 yeast cells per hpf 5 (20) 12 (48) 4 (16) 3 (12) 1 (4) 25 (100) >7 yeast cells per hpf 14 (27.4) 25 (49) 6 (11.8) 6 (11.8) 0 (0) 51 (100)

66 Identification of Malassezia species isolated from Iranian seborrhoeic dermatitis patients

Table V. Frequency of observed yeast cells number in DME on lesions based on severity of SED.

Severity of SED Mild Moderate Severe Number of observed yeast cells in DME n (%) n (%) n (%)

1-3 yeasts in whole slide 0 2 (2.7) 2 (11.8) 1-3 yeast cells per hpf 4 (44.4) 13 (17.6) 3 (17.6) 4-7 yeast cells per hpf 3 (33.3) 17 (23) 5 (29.4) >7 yeast cells per hpf 2 (22.2) 42 (56.7) 7 (41.2) Total 9 (100) 74 (100) 17 (100) mogenous suspension is very difficult to separate References them by culture29. Moreover, some Malassezia species may loss their viability after several sub- 1) GUPTA AK, BATRA R, BLUHM R, BOEKHOUT T, D AWSON cultures9. TL Jr. Skin diseases associated with Malassezia species. J Am Acad Dermatol 2004; 51: 785- Our data showed the density of Malassezia 798. yeast cells on the lesions of patients in the age 2) CRESPO ERCHIGA V, D ELGADO FLORENCIO V. group of 20-29 yrs is significantly higher in com- Malassezia species in skin diseases. Curr Opin parison with other age groups. However, the rela- Infect Dis 2002; 15:133-142. tionship between colonization patterns and age 3) DEANGELIS YM, GEMMER CM, KACZVINSKY JR, KENNEAL- group is less clear. Some studies indicate that LY DC, SCHWARTZ JR, DAWSON TL, Jr. Three etiologic there is a difference in the colonization pattern of facets of dandruff and seborrhoeic dermatitis: Malassezia species in different age groups25,30. Malassezia fungi, sebaceous lipids, and individual Bergbrant et al.31 also suggested that the colony sensitivity. J Invest Dermatol Symp Proc 2005; counts generally are higher in adults than in chil- 10: 295-297. dren and it decreases again in elderly individuals. 4) HARDING CR, MOORE AE, ROGERS JS, MELDRUM H, This condition may due to high sebum produc- SCOTT AE, MCGLONE FP. Dandruff: a condition char- acterized by decreased levels of intercellular tion in above mentioned aged groups. lipids in scalp stratum corneum and impaired bar- In the present study there was not a significant rier function. Arch Dermatol Res 2002; 294: 221- difference between the observed yeasts number 230. in DME and severity of SED. The Malassezia 5) GAITANIS G, VELEGRAKI A, ALEXOPOULOS EC, CHASAPI V, species density in DME was higher in patients TSIGONIA A, KATSAMBAS A. Distribution of Malassezia with moderate and severe form in comparison species in pityriasis versicolor and seborrhoeic with patients with mild SED. The existing litera- dermatitis in Greece. Typing of the major pityriasis versicolor isolate M. globosa. Br J Dermatol ture generally disagrees whether or not the SED 2006; 154: 854-859. lesions are associated with an excess numbers of 6) TAJIMA M, SUGITA T, N ISHIKAWA A, TSUBOI R. Molecular Malassezia cells, perhaps due to a lack of stan- analysis of Malassezia microflora in seborrheic 32 dardization in the techniques used. Zaidi et al , dermatitis patients: comparison with other dis- using direct microscopy, found that Malassezia eases and healthy subjects. J Invest Dermatol increases in proportion with the severity of SED. 2008; 128: 345-351. These findings are also similar to those of other 7) NAKABAYASHI A, SEI Y, G UILLOT J. Identification of groups, who used different sampling and culture Malassezia species isolated from patients with methods to enumerate yeast cells present in SED seborrhoeic dermatitis, atopic dermatitis, pityria- 24,26,30,33,34 sis versicolor and normal subjects. Med Mycol or dandruff . 2000; 38: 337-341. In conclusion, we found an high recovery rate of Malassezia species on the lesions of patients with 8) RINCÓN S, CELIS A, SOPÓ L, MOTTA A, CEPERO DE GAR- CÍA MC. Malassezia yeast species isolated from SED. M. globosa was the most commonly isolated patients with dermatologic lesions. Biomedica Malassezia species on the SED lesions. In addi- 2005; 25: 189-195. tion, the density of Malassezia species on lesions 9) GUEHO E, MIDGLEY G, GUILLOT J. The genus was higher in patients with moderate and severe Malassezia with description of four new species. SED in comparison with patients with mild SED. Antonie van Leeuwenhoek 1996; 69: 337-355. Therefore, we think that Malassezia might be play- 10) SUGITA T, TAKASHIMA M, SHINODA T, S UTO H, UNN T, ing a causative role in the etiology of this disease. TSUBOI R, OGAWA H, NISHIKAWA A. New yeast

67 M.T. Hedayati, Z. Hajheydari, F. Hajjar, A. Ehsani, T. Shokohi, R. Mohammadpour

species, Malassezia dermatitis, isolated from pa- 24) GEMMER CM, DEANGELIS YM, THEELEN B, BOEKHOUT T, tients with atopic dermatitis. J Clin Microbiol DAWSON TL. Fast, noninvasive method for molecu- 2002; 40: 1363-1367. lar detection and differentiation of Malassezia yeast species on human skin and application of 11) SUGITA T, TAKASHIMA M, KODAMA M, TSUBOI R, the method to dandruff microbiology. J Clin Micro- NISHIKAWA A. Description of a new yeast species, Malassezia japonica, and its detection in patients biol 2002 40; 3350-3357. with atopic dermatitis and healthy subjects. J Clin 25) GUPTA AK, KOHLI Y, S UMMERBELL RC, FAERGEMANN J. Microbiol 2003; 41: 4695-4699. Quantitative culture of Malassezia species from different body sites of individuals with or without 12) HIRAI A, KANO R, MAKIMURA K, DUARTE ER, HAMDAN dermatoses. Med Mycol 2001; 39; 243-251. JS, LACHANCE MA, YAMAGUCHI H, HASEGAWA A. Malassezia nana sp. nov., a novel lipid-dependent 26) PECHÈRE M, KRISCHER J, REMONDAT C, BERTRAND C, yeast species isolated from animals. Int J Syst TRELLU L, SAURAT JH. Malassezia spp carriage in Evol Microbiol 2004; 54: 623-627. patients with seborrheic dermatitis. J Dermatol 1999; 26: 558-561. 13) SUGITA T, TAJIMA M, TAKASHIMA M, AMAYA M, SAITO M, TSUBOI R, NISHIKAWA A. A new yeast, Malassezia 27) SANDSTRÖM FALK MH, TENGVALL LINDER M, JOHANSSON yamotoensis, isolated from a patient with sebor- C, BARTOSIK J, BÄCK O, SÄRNHULT T, WAHLGREN CF, rhoeic dermatitis, and its distribution in patients SCHEYNIUS A, FAERGEMANN J. The prevalence of and healthy subjects. Microbiol Immunol 2004; Malassezia yeasts in patients with atopic derma- 48: 579-583. tis, seborrheic dermatitis and healthy controls. Ac- ta Derm Venereol (Stockh) 2005; 85: 17-23. 14) NELL A, JAMES SA, BOND CJ, HUNT B, HERRTAGE ME. Identification and distribution of a novel 28) KIM SC, KIM HU. The distribution of Malassezia Malassezia species yeast on normal equine skin. species on the normal human skin according to Vet Rec 2002; 150: 395-398. body region. Korean J Med Mycol 2000; 5; 120- 128. 15) TARAZOOIE B, KORDBACHEN P, Z AINI F, Z OMORODIAN K, SAADAT F, Z ERAATI H, HALLAJI Z, REZAIE1 S. Study of 29) MIDGLEY G, GUEHO E, GUILLOT J. Disease caused by the distribution of Malassezia species in patients Malassezia species. In Topley and Wilson’s Mi- with pityriasis versicolor and healthy individuals in crobiology and microbial infections Volume 4. 9th Tehran, Iran. BMC Dermatol 2004; 4:5. edition. Edited by: Ajello L, Hay RJ. London: 16) ZOMORODIAN K, MIRHENDI H, TARAZOOIE B, ZERAATI H, Arnold; 1998: 201-211. HALLAJI Z, BALIGHI K. Distribution of Malassezia 30) FAERGEMANN J. The use of contact plates for quan- species in patients with psoriasis and healthy in- titative culture of Pityrosporum orbiculare. Mykos- dividuals in Tehran, Iran. J Cutan Pathol 2008; 35: es 1987; 30: 298-304. 1027-1031. 31) BERGBRANT IM, IGREUD A, NORDIN P. An improved 17) GUILLOT J, GUEHO E, LESOURD M, MIDGLEY G, CHE´ method for quantitative culture of Malassezia fur- VRIER G, DUPONT B. Identification of Malassezia fur. Res Microbiol 1992; 143: 731-735. species: a practical approach. J Mycol Med 1996; 6: 103-110. 32) ZAIDI Z, WAHID Z, COCHINWALA R, SOOMRO M, QUREISHI A. Correlation of the density of yeast 18) SCHWARTZ RA, JANUSZ CA, JANNIGER CK. Seborrheic Malassezia with the clinical severity of seborrhoe- dermatitis: an overview. Am Fam Physician 2006; ic dermatitis. J Pak Med Assoc 2002; 52: 504- 74: 125-130. 506.

19) GUPTA AK, BLUHM R, COOPER EA, Summerbell RC, 33) LEE YW, KANG HJ, AHN KJ. Malassezia species cul- Batra R. Seborrheic dermatitis. Dermatol Clin. tured from lesions of seborrhoeic dermatitis. Ko- 2003; 21: 401-412. rean J Med Mycol 2001; 6: 70-76.

20) CRESPO ERCHIGA V, O JEDA MARTOS A, VERA CASAÑO A, 34) HENG MCY, HENDERSON CL, BARKER DC, HABERFELDE CRESPO ERCHIGA A, SANCHEZ FAJARDO F. Malassezia G. Correlation of Pityrosporum ovale density with globosa as the causative agent of pityriasis versi- clinical severity of seborrheic dermatitis as as- color. Br J Dermatol 2000; 143: 799-803. sessed by a simplified technique. J Am Acad Der- 21) BERGBRANT IM, FAERGEMANN J. Seborrhoeic dermati- matol 1990; 23: 82-86. tis and Pityrosporum ovale: a cultural and im- munological study. Acta Derm Venereol 1989; 69: 332-335. 22) SUGITA T, N ISHIKAWA A. Molecular and quantitative –––––––––––––––––––––––– analysis of Malassezia microflora on the skin of Acknowledgements atopic dermatitis and genotyping of Malassezia globosa DNA. JPN J Med Mycol. 2003 44; 61- 64. This work was supported by grants from the Pharmaceu- 23) MIRZA SH, REHMAN H, KHAN MA. Role of tical Sciences Research Center of Mazandaran University Malassezia yeast (Pityrosporum) in seborrhoeic of Medical Sciences, Sari, Iran. We thank the participat- dermatitis (SD). J Coll Physicians Surg Pak 2005; ing patients for their kind co-operation, which has been 15: 771-773. essential for the completion of the study.