IDENTIFICATION OF MALASSEZIA SPECIES

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IDENTIFICATION OF MALASSEZIA SPECIES

AJ Kindo, SKC Sophia, *J Kalyani, S Anandan

Abstract

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 Dixons agar (mDixons agar) and Sabouraud dextrose agar (SDA) and incubated at 32C. 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 mDixons agar. The commonest isolate was M. sympodialis (28, 58%) followed

byM. globosa (19, 40%) and one isolate was (2%) ofM. restricta. M. sympodialis was the commonest species

affecting our population and there was no isolation ofM. obtusa,M. slooffiae,M. pachydermatis andM. furfur.

Key words:Malassezia spp., pityriasis versicolor, lipophilic yeasts, tween assimilation

*Corresponding author

Department of Microbiology (AJK, JK), and Department

of Dermatology and STD (SKCS, SA), Sri Ramachandra

Medical College and Research Institute, Porur, Chennai

- 600 116, Tamil Nadu, India.

Received : 28-04-2003

Accepted : 19-09-2003

Indian Journal of Medical Microbiology, (2004) 22 (3):179-181

morphological criteria,Malassezia yeasts are primarily

differentiated by their ability to assimilate various

polyoxyetheylene sorbitan esters (Tween) following the

methodology of Guillot et al.6 The present study was

aimed to identify theMalassezia spp. causing pityriasis

versicolor in a south Indian population.

Materials and Methods

This study was conducted from August 2001 to

October 2002.The samples were collected from patients

with pityriasis versicolor in the out-patient departmentof skin and STD and processed in the mycology

laboratory of the department of microbiology.

Entry of observations in a detailed proforma, clinical

evaluation, Woods lamp examination and mycological

evaluation by microscopic examination of KOH treated

skin scrapings were done.

Culture

Only samples which were KOH positive (n=70) were

cultured. The scales were inoculated into modified

Dixons agar (mDixons agar) as described by Guillot et al6

and into Sabouraud dextrose agar containing 0.05%

chloramphenicol and 0.05% cycloheximide (SDA). The

tubes were incubated at 32C for 3-4 days.

Microscopic features

The morphology of the yeast cells was studied by

making Gram stained smears of the isolates from

mDixons agar after one week incubation at 32C.

Physiological characteristics

The catalase reaction was determined by application

of a drop of hydrogen peroxide (10 vol.) onto a portion

For more than a century, yeasts of the genus

Malassezia have been known to be a part of the normal

flora of human skin and other warm-blooded animals.1,2

Being lipid dependent, they are normally found in areas

that are rich in sebaceous glands.3 Once the lipophilic

nature of these yeasts was recognized and cultures were

made possible, different workers observed spontaneous

changes from one morphological type to the other which

led to the conclusion thatP.orbiculare,P. ovale andM.

furfurwere only variants of the same species.4

However, apart from their lipid dependence, little is

known about the metabolism and nutritional

requirements ofMalassezia species.2 A first approach,

recently presented by Gueho et al5 has been translated

into a practice oriented identification system by Guillot

et al.6 There was considerable confusion regarding the

classification ofMalassezia, with different groups

tending to favour their own classification scheme.

Following the reclassification of the genus Malassezia

and the definition of four new species9 much of this

confusion has been resolved.

There are seven proposed species in the genusMalassezia based on molecular, morphological and

biochemical profiles: six lipid dependent species namely

M.furfur, M.sympodialis, M. globosa , M.obtusa , M.

restricta and M.slooffiae and one lipid independent

species, Ma la ss ez ia pachyder mati s. Apart from

Brief Communication


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July, 2004 Kindo et al- Identification of Malassezia species 180

the narrow base (Fig.3a, 3b). The catalase reaction was

positive for all except one isolate ofM. restricta, whichis the only lipid dependent species ofMalassezia to

consistently lack catalase.

The Tween diffusion test allowed the differentiation

of mostMalassezia species in our study population. The

growth of 28 isolates was inhibited by high

of a colony on a glass slide, or directly on colonies on

the culture media. The production of gas bubbles

indicated a positive reaction. For each isolate, the ability

to utilize individual Tweens was tested by the following

procedure. Sterile SDA (16 mL) was melted and allowed

to cool to about 50C. The yeast being identified were

added (2 mL) to the medium. The suspension was

obtained by inoculating 5 mL of sterile distilled water

with a loopful of actively growing yeasts and the

concentration was adjusted to about 105 cell/mL. The

seeded agar was then vigorously mixed and poured into

a 9 cm diameter petri dish. Once the medium had

solidified, four wells were made by means of a 2 mm

diameter punch and filled with 5 L of Tween 20, 40, 60

and 80 respectively. Since these reagents are water-

soluble, a concentration gradient formed around each

well. The plates were systematically incubated for oneweek at 32C. Utilization of Tweens was assessed by the

degree of growth and/or reaction (precipitation) of the

lipophilic yeasts around individual wells.

Results

All the 70 skin scrapings showed hyphae and spores

exhibiting the characteristic spaghetti and meatball

appearance in the KOH preparation (Fig. 1a). Growth was

obtained on mDixons agar from 48 (68.57%) of the 70

skin scrapings (Fig. 1b). Twenty-eight (58%) of the

isolates belonged toM. sympodialis, 19 (40%) isolates

belonged toM. globosa and 1(2%) case belonged toM.restricta. There was no growth in SDA, ruling out the

presence of M. pa chyder mati s the only lipid-

independent species. Other species namelyM.furfur,M.

slooffiae, and M. obtusa, were not isolated from our

population.

M.sympodialis had small ovoid cells with sympodial

budding, which is a characteristic feature (Fig.2). M.

globosa had stable spherical cells. Buds were formed on

Figure 1a : Microscopic examination of KOH treated skin

scrapings showing the hyphae and spores- characteristic

spaghetti and meatball appearance (KOH, x 400).

Figure 1b : Smooth, cream coloured colonies ofMalassezia

spp. in modified Dixons agar.

Figure 2 : Malassezia sympodialis with small ovoid and

characteristic sympodial budding (Gram stain, x 1000).

Figure 3a : Malassezia globosa with stable, spherical cells

with a broad base (Gram stain, x 1000).


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of the Tweens (M. globosa or M. restricta or M.

obtusa). Based on the catalase reaction and the

microscopic morphology in Gram stained smears, these

isolates were speciated asM.globosa.

Discussion

The few reports that have appeared since the

taxonomic revision of the genus Malassezia in 1996,

suggest thatM.sympodialis is the predominant species

of human skin, in health or disease, and is mainly found

on the trunk,M.globosa is found in scales of pitryiasis

versicolor and in healthy skin, and M. restricta seems

to be associated with pityriasis capitis.7

In our study, out of the 70 specimens that were

inoculated, 68.75% yielded growth ofMalassezia in

culture. Out of this, the most frequently isolated specieswas Malassezia sympodialis (58.33%), followed by

M.globosa (39.58%) andM.restricta (2.08%).

In an earlier study, Crespo et al9 reported that

M.globosa was recovered from 97% of their patients,

alone in 60% of them and associated withM.sympodialis

in 29% andM.slooffiae in 7%. These authors concluded

that M.globosa in its mycelial phase is the causative

agent of pityriasis versicolor.8

Our results suggest that M. sympodialis in its

mycelial phase, is the most common Malassezia spp.

associated with pityriasis versicolor in the population

attending our hospital in south India. The presence of

this species, in its yeast phase in diseased and even in

healthy skin, indicates that local factors (humidity, sweat,

heat) together with some degree of idiosyncratic

individual predisposition, are responsible for the

transformation to the mycelial form and development of

clinical lesions.

concentrations of Tween 20. This phenomenon resulted

in a characteristic ring of tiny colonies around the

corresponding well (Fig. 4). 19 isolates did not utilize any

References

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Figure 3b : Single budding cell ofMalassezia globosa (Gram

stain, x 1000).

Figure 4 : Tween assimilation test- Malassezia sympodialis:

growth around all Tweens but inhibited by high concentration

of Tween 20.

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IDENTIFICATION OF MALASSEZIA SPECIES