|Year : 2013 | Volume
| Issue : 3 | Page : 270-274
Study of the distribution of Malassezia species in patients with pityriasis versicolor and healthy individuals in Tertiary Care Hospital, Punjab
M Kaur1, T Narang2, M Bala1, S Gupte1, P Aggarwal1, A Manhas1
1 Department of Microbiology, Giansagar Medical College, Ramnagar, Patiala, Punjab, India
2 Department of Skin and VD, Giansagar Medical College, Ramnagar, Patiala, Punjab, India
|Date of Submission||11-Oct-2012|
|Date of Acceptance||11-Apr-2013|
|Date of Web Publication||25-Jul-2013|
Department of Microbiology, Giansagar Medical College, Ramnagar, Patiala, Punjab
Source of Support: None, Conflict of Interest: None
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 38 0 C. 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.
Keywords: Identification, Malassezia, pityriasis versicolor, skin
|How to cite this article:|
Kaur M, Narang T, Bala M, Gupte S, Aggarwal P, Manhas A. Study of the distribution of Malassezia species in patients with pityriasis versicolor and healthy individuals in Tertiary Care Hospital, Punjab. Indian J Med Microbiol 2013;31:270-4
|How to cite this URL:|
Kaur M, Narang T, Bala M, Gupte S, Aggarwal P, Manhas A. Study of the distribution of Malassezia species in patients with pityriasis versicolor and healthy individuals in Tertiary Care Hospital, Punjab. Indian J Med Microbiol [serial online] 2013 [cited 2021 Mar 7];31:270-4. Available from: https://www.ijmm.org/text.asp?2013/31/3/270/115636
| ~ Introduction|| |
The lipophilic yeast Malassezia furfur and related species are members of the normal human cutaneous flora of skin and produce clinical disease under conditions that permit massive growth of the fungus.  These yeasts are associated with clinical infections such as pityriasis versicolor (PV), folliculitis, seborrheic dermatitis, some forms of atopic dermatitis, confluent and reticulate papillomatosis and even systemic infections.  Pityriasis versicolor is the only cutaneous disease in which the involvement of Malassezia is undisputed whereas in the remaining dermatological disorders related to Malassezia, the role of these yeasts is controversial. 
PV is a superficial infection of the stratum corneum, characterized by hyperpigmented and hypopigmented scaly macules, primarily on the trunk and proximal extremities.  The fungal nature of PV was first recognized by Eichstedt in 1846.  It is present worldwide, but it is more common in tropical countries. The incidence is as high as 30-40% in tropical climates. 
Currently, Malassezia genus includes 11 species comprising Malassezia pachydermatis, M. furfur, Malassezia sympodialis, Malassezia slooffiae, Malassezia globosa, Malassezia obtusa, Malassezia restricta, Malassezia dermatis, Malassezia japonica, Malassezia nana and Malassezia yamotoensis. 
Only a few reports are available about the epidemiology of various Malassezia species in India. Therefore, the present study has been undertaken to identify the Malassezia species causing PV in north Indian population as well as to study Malassezia species microflora of healthy individuals.
| ~ Materials and Methods|| |
The study was conducted from July 2011 to February 2012. Patients of PV attending out-patient department of skin and sexually transmitted diseases of a tertiary care hospital were consecutively included. Observations were entered in detailed performa. Forty five clinically healthy subjects (without any dermatosis) were included as control.
Collection of specimen
Sampling from the PV lesions was made by scraping them with a sterile scalpel, taking care not to collect from healthy looking areas. In normal subjects and in cases where the scales were not sufficient, samples were taken by means of tape method. Site of sampling in healthy subjects was upper back.
Direct microscopy and culture
Direct microscopy with 20% KOH and methylene blue staining was carried out in the PV lesions as well as normal samples. All samples were inoculated on plates containing modified Dixon medium (MDA). The plates were incubated at 32°C for 2 weeks and examined at frequent intervals for developing colonies. Isolated colonies on modified Dixon agar were used for identification.
Identification of Malassezia species
Species were identified by following the scheme established by Guillot et al.,  completed by the use of Cremophor EL and esculin agar, as suggested by Mayser et al. 
Growth on Sabouraud dextrose agar
Among Malassezia species, only M. pachydermatitis is able to grow on Sabouraud agar as it is not an obligatory lipid dependent species. 
Presence of catalase was determined by using a drop of hydrogen peroxide (30% solution).
Tween assimilation test
Briefly, Malassezia yeast suspension (at least 10 7 cfu/ml) was made in 2 ml sterilized distilled water and mixed with Sabouraud agar at 45°C and the mixture was plated. After the solidification of medium, four holes were made in the agar by means of a 3 mm diameter punch and filled with 5 μl each of Tween 20, 40, 60 and 80 respectively. The agar plates were incubated at 32°C for 1 week and the growth was assessed around the individual wells.
Assimilation of Cremophor EL
Sabouraud agar was the medium used and procedure used was same as that for Tween assimilation test. Six mm wells were punched and filled with 50 μl of Cremophor EL. The agar plates were incubated at 32°C for 1 week and the growth was assessed around the individual wells.
Splitting of esculin
Briefly, a loop of fresh yeast was inoculated deeply in the esculin agar tube and incubated for 5 days at 32°C. The splitting of esculin was revealed by darkening of the medium. Significant brown staining of more than a third of the medium is considered demonstrative of M. sympodialis and M. obtusa. M. furfur causes weak staining. The other Malassezia species are negative. 
Growth at 38°C
The ability of the various Malassezia species to grow on modified Dixon agar at 38°C was studied.
Pearson's Chi-square test and Fisher's exact test were applied to analyze the difference in the distribution of Malassezia species in PV patients and healthy controls. A P < 0.05 was considered significant.
| ~ Results|| |
Of 65 patients, 54% of patients were male. The range and median age of patients was 8-50 years and 29 years respectively. The highest prevalence of Malassezia infection was seen in patients with 20-30 years of age.
Out of sixty five clinically diagnosed patients, seven patients turned negative on direct microscopy, while in 58 patients it demonstrated the characteristic "spaghetti and meatball" appearance [Figure 1]. Hypopigmented lesions were present in 51 patients and seven patients had hyperpigmented lesions. Recurrence of PV lesions was observed in 15 (26%) cases. Distribution of Malassezia species in PV patients on different body sites is shown in [Table 1].
|Figure 1: Typical globose yeasts and pseudohyphae in pityriasis versicolor skin scales (methylene blue staining (×400)|
Click here to view
|Table 1: Distribution of Malassezia species based on the body sites in pityriasis versicolor |
Click here to view
Out of fifty eight microscopically diagnosed cases, growth on MDA was obtained from 54 (93.10%) cases. Amongst the culture positive cases, 3 cultures yielded mixed growth and remaining 51 cultures showed single pure growth. All the three cultures with mixed growth yielded M. globosa and M. sympodialis. In PV lesions, the species most frequently isolated was M. globosa 28 (51.79%), followed by M. sympodialis 17 (31.42%) and M. furfur 10 (18.51%). The most frequent pattern was M. globosa as the sole species 25 (46.29%), although it was associated with M. sympodialis in 3 (5.5%) cases. M. sympodialis alone was seen in 14 (25.92%) cases. M. obtusa was isolated in just 2 (3.7%) cases. Other species like M. pachydermatis, M. restricta and M. slooffiae were not isolated from PV cases. Of the seven patients with hyperpigmented lesions, M. globosa was isolated in four, M. furfur in two and M. sympodialis in one case.
In case of healthy controls, out of 45 samples taken from upper back, culture was positive in 21 (46.66%) samples. M. sympodialis (47.61%) was the major isolate followed by M. obtusa and M. globosa, which made up 19.04% and 14. 20% respectively of the isolated Malassezia flora. One isolate of M. pachydermatis obtained from a farmer, was the only isolate which grew on SDA medium being lipid non-dependent. The distribution of various Malassezia species in controls and PV patients is shown in [Figure 2]. On statistical analysis, it was found that M. globosa was present in significantly more number in PV patients as compared to healthy controls (P = 0.01), which is not so in case of M. sympodialis and M. furfur (P > 0.05). On the other hand, M. obtusa appeared in significantly more number in healthy controls (P = 0.04), indicating its less pathogenicity.
|Figure 2: Distribution (%) of various malassezia species in pityriasis versicolor patients and healthy controls|
Click here to view
The Tween assimilation test allowed the differentiation of most Malassezia species. M. sympodialis assimilated all the four Tweens, but the growth was inhibited by high concentration of Tween 20. Few isolates of M. sympodialis also developed a ring of tiny colonies at some distance from the Cremophor source [Figure 3]. Isolates belonging to M. globosa and M. obtusa did not utilize any of the Tweens. Therefore, isolates of M. globosa and M. obtusa were further identified by use of esculin agar test. M. furfur assimilated all the four Tweens as well as Cremophor EL [Figure 4].
|Figure 3: Malassezia sympodialis showing assimilation of the four Tweens (right top is Tween 20 and then clockwise Tween 40, 60 and 80) and in the centre, a ring of tiny colonies around cremophor EL|
Click here to view
|Figure 4: Malassezia furfur showing assimilation of all the four tweens as well as cremophor EL (in the centre)|
Click here to view
| ~ Disscusion|| |
The highest prevalence of PV in the present study was observed in 20-30 year old group, which is similar to other investigators. , We found no difference in development of PV among both sexes as stated by other studies as well. ,
The most commonly affected area was trunk (70% of cases), which is concordant with many other studies. ,, Distribution of the lesions generally parallels the density of sebaceous gland distribution, with greater occurrence on the chest and back.  Moreover, areas which remain covered by clothing favours development of lesions, supporting the theory that the occlusion of glands plays a role in this disease.  The recurrence of PV lesions could be attributed to the idiosyncratic nature of the condition and the fact that local factors such as humidity and high temperature remain unchanged. 
In PV lesions, the most frequently isolated species was M. globosa, followed by M. sympodialis and M. furfur. Our result is concordant with the majority of studies worldwide which have identified M. globosa as the most common species in PV leisons. ,,, Amongst the Indian studies also, Dutta et al., and Chaudhary et al.,  have reported M. globosa to be the main species involved in PV in north central part of India constituting 54% and 57.5% respectively of the total isolates. , However, Kindo et al.,  showed that in south India, M. sympodialis is the commonest agent (58.3%), which goes well with the findings of Gupta et al., In the present study, M. sympodialis was the second most common agent involved, which matches with the findings of other studies. , This species can be easily recognized by its culture and biochemical features, mainly by its strong β-glucosidase activity. M. furfur is responsible for PV especially in tropical countries. Dutta et al., and Tarazooie et al., have reported M. furfur to be the second most frequent species isolated from PV lesions. However, in the present study, it is the third most common agent.
As the cases with hyperpigmented lesions of PV were only seven, no definitive conclusions could be drawn about the relationship of the species and the pigmentary changes. Azelaic acid and several tryptophan metabolites produced by Malassezia, which can interfere with melanization, are considered important in the skin pigmentation changes seen in PV. 
In healthy subjects, Malassezia colonies were recovered in nearly half of the cases (46.66%) which goes well with the study of Crespo et al. In this study, M. sympodialis was the main species isolated from the back of healthy individuals. Sandstrφm Falk et al., have also reported M. sympodialis as the predominant species in healthy skin, especially on the upper back.  Similar findings have been reported by other authors as well. ,,, However, various other studies have reported M. globosa to be main isolate from the trunk in normal individual followed by M. sympodialis. , These two species appear to be the main normal flora on the skin of the trunk. Although, in the present study, M. obtusa was second most common isolate obtained as normal flora, which goes well with the study of Sandstrφm Falk et al.
The differences between various studies could be explained not only by ethnic and geographical factors, but also by use of different sampling techniques and the different culture media (Leeming and Notman agar or modified Dixon agar).  There is still no single medium that is able to reliably recover and maintain all the species of Malassezia described so far.  Another problem associated with working on Malassezia is the difficulty of preserving isolates, with certain species being particularly fastidious. The most reliable method recommended for preservation of Malassezia is storage at − 80°C. 
Biomolecular techniques are considered as fast and more accurate methods to identify Malassezia species.  However, simple methods based on morphology and physiology evaluation, such as the ones employed in this study, are more accessible to every laboratory. Furthermore, strains of M. furfur, M. globosa and M. obtusa have been found to be more tolerant to terbinafine than the remaining Malassezia species,  which emphasize the need of studies related with identification as well as antifungal susceptibility testing of Malassezia isolates.
To conclude, M. globosa was the major pathogen in PV cases, but constituted significantly less number of isolates as normal flora from the back of healthy individuals. M. globosa has a better enzymatic endowment than the other species, especially in esterases and lipases enzymes closely related with skin pathogenicity.  Thus, probably the higher pathogenicity of M. globosa might be the cause of its predominance in PV lesions.
| ~ References|| |
|1.||Nakabayashi A, Sei Y, Guillot J. Identification of Malassezia species isolated from patients with seborrhoeic dermatitis, atopic dermatitis, pityriasis versicolor and normal subjects. Med Mycol 2000;38:337-41. |
|2.||Guého E, Boekhout T, Ashbee HR, Guillot J, Van Belkum A, Faergemann J. The role of Malassezia species in the ecology of human skin and as pathogens. Med Mycol 1998;36 Suppl 1:220-9. |
|3.||Crespo Erchiga V, Delgado Florencio V. Malassezia species in skin diseases. Curr Opin Infect Dis 2002;15:133-42. |
|4.||Aspiroz C, Ara M, Varea M, Rezusta A, Rubio C. Isolation of Malassezia globosa and M. sympodialis from patients with pityriasis versicolor in Spain. Mycopathologia 2002;154:111-7. |
|5.||Eichstedt E. Fungal structures in the pityriasis versicolor[in German]. Froriep Neue Notizen Naturkunde Heilkinde 1846;39:270. |
|6.||Ashbee HR. Update on the genus Malassezia. Med Mycol 2007;45:287-303. |
|7.||Guillot J, Gueho E, Lesourd M, Midgley G, Chevrier G, Dupont B. Identification of Malassezia species. A practical approach. J Mycol Med 1996;6:103-110. |
|8.||Mayser P, Haze P, Papavassilis C, Pickel M, Gruender K, Guého E. Differentiation of Malassezia species: Selectivity of cremophor EL, castor oil and ricinoleic acid for M. furfur. Br J Dermatol 1997;137:208-13. |
|9.||Midgley G. The lipophilic yeasts: State of the art and prospects. Med Mycol 2000;38 Suppl 1:9-16. |
|10.||Crespo EV, Ojeda MA, Vera CA, Crespo EA, Sanchez FF, Guého E. Mycology of pityriasis versicolor. J Mycol Med 1999;9:143-8. |
|11.||Tarazooie B, Kordbacheh P, Zaini F, Zomorodian K, Saadat F, Zeraati H, et al. Study of the distribution of Malassezia species in patients with pityriasis versicolor and healthy individuals in Tehran, Iran. BMC Dermatol 2004;4:5. |
|12.||Gupta AK, Kohli Y, Summerbell RC, Faergemann J. Quantitative culture of Malassezia species from different body sites of individuals with or without dermatoses. Med Mycol 2001;39:243-51. |
|13.||Gupta AK, Kohli Y, Faergemann J, Summerbell RC. Epidemiology of Malassezia yeasts associated with pityriasis versicolor in Ontario, Canada. Med Mycol 2001;39:199-206. |
|14.||Sunenshine PJ, Schwartz RA, Janniger CK. Tinea versicolor. Int J Dermatol 1998;37:648-55. |
|15.||Gupta AK, Bluhm R, Summerbell R. Pityriasis versicolor. J Eur Acad Dermatol Venereol 2002;16:19-33. |
|16.||Crespo Erchiga V, Ojeda Martos A, Vera Casaño A, Crespo Erchiga A, Sanchez Fajardo F. Malassezia globosa as the causative agent of pityriasis versicolor. Br J Dermatol 2000;143:799-803. |
|17.||Dutta S, Bajaj AK, Basu S, Dikshit A. Pityriasis versicolor: Socioeconomic and clinico-mycologic study in India. Int J Dermatol 2002;41:823-4. |
|18.||Chaudhary R, Singh S, Banerjee T, Tilak R. Prevalence of different Malassezia species in pityriasis versicolor in central India. Indian J Dermatol Venereol Leprol 2010;76:159-64. |
|19.||Leeming JP, Sansom JE, Burton JL. Susceptibility of Malassezia furfur subgroups to terbinafine. Br J Dermatol 1997;137:764-7. |
|20.||Gupta AK, Kohli Y, Li A, Faergemann J, Summerbell RC. In vitro susceptibility of the seven Malassezia species to ketoconazole, voriconazole, itraconazole and terbinafine. Br J Dermatol 2000;142:758-65. |
|21.||Kindo AJ, Sophia SK, Kalyani J, Anandan S. Identification of Malassezia species. Indian J Med Microbiol 2004;22:179-81. |
|22.||Sandström Falk MH, Tengvall Linder M, Johansson C, Bartosik J, Bäck O, Särnhult T, et al. The prevalence of Malassezia yeasts in patients with atopic dermatitis, seborrhoeic dermatitis and healthy controls. Acta Derm Venereol 2005;85:17-23. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
|This article has been cited by|
||Malassezia pachydermatis fungemia in a preterm neonate resistant to fluconazole and flucytosine
| ||Noura Al-Sweih,Suhail Ahmad,Leena Joseph,Seema Khan,Ziauddin Khan |
| ||Medical Mycology Case Reports. 2014; 5: 9 |
|[Pubmed] | [DOI]|