|Year : 2013 | Volume
| Issue : 2 | Page : 49-53
Characterisation of Malassezia species and their clinical correlation in a tertiary healthcare centre in South India
Ambujavalli Balakrishnan Thayikkannu1, Anupma Jyoti Kindo1, Mahalakshmi Veeraragahavan2
1 Department of Microbiology, Sri Ramachandra Medical College and Research Institute, Chennai, Tamil Nadu, India
2 Department of Dermatology, Sri Ramachandra Medical College and Research Institute, Chennai, Tamil Nadu, India
|Date of Web Publication||7-Jan-2014|
Anupma Jyoti Kindo
Department of Microbiology, Sri Ramachandra Medical College and Research Institute, Chennai, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Genus Malassezia consists of 14 species of yeast like fungi that commonly causes superficial mycoses. It is a topic of intense interest but its fastidious nature restricts its research. Speciation gives us better treatment strategies especially with global concern over high Minimum Inhibitory Concentrations (MICs) to anti-fungal agents and emerging resistance. Aim of the study was to speciate Malassezia using phenotypic methods and to analyse the risk factors and its clinical correlation. The study was conducted at our university teaching hospital in south India over a one year period after approval by the Institutional Ethics Committee. A total of 105 patients, who had skin lesions resembling diseases caused by Malassezia, were included. The skin scrapings were subjected to 10% KOH wet mount. Culture was put up on Sabouraud's dextrose agar (SDA), with and without olive oil overlay (SDA-O) and modified Dixon's agar. Gram's stain, catalase test, aesculin hydrolysis, Tween assimilation, temperature tolerance and Tween 40-based precipitate production were done to characterise Malassezia species. The most common age was 20-40 years, with a slight female predominance (58.1%). Among the study group (n = 105), 87 had the prototype disease - pityriasis versicolour (PV). Out of 105 patients, 62.9% were fresh cases and 37.1% came with recurrences. The most common site to be affected by PV was the back, followed by chest. M. globosa was the most common species in both the hypopigmented and hyper pigmented groups. In three patients, two species were isolated from the same lesion. A sequential use of these simple tests helps greatly in a financially constrained set up for speciation of Malassezia in the laboratory.
Keywords: Malassezia , pityriasis versicolour, superficial mycosis
|How to cite this article:|
Thayikkannu AB, Kindo AJ, Veeraragahavan M. Characterisation of Malassezia species and their clinical correlation in a tertiary healthcare centre in South India. J Acad Clin Microbiol 2013;15:49-53
|How to cite this URL:|
Thayikkannu AB, Kindo AJ, Veeraragahavan M. Characterisation of Malassezia species and their clinical correlation in a tertiary healthcare centre in South India. J Acad Clin Microbiol [serial online] 2013 [cited 2017 Apr 29];15:49-53. Available from: http://www.jacmjournal.org/text.asp?2013/15/2/49/124586
| Introduction|| |
Genus Malassezia consists of fastidious, saprophytic, opportunistic 'yeast like fungi'  characterised morphologically by small cells exhibiting unilateral, enteroblastic and repetitive, percurrent budding.  They form a part of the normal cutaneous biological flora of both animals and mammals. This group of yeast like fungi have lipophilic growth requirements (excepting Malassezia pachydermatis) and hence colonise the seborrhoeic parts of the skin where they utilise sebum for their growth and maintenance. Sebum consists of triglycerides and esters, which are broken down by microbes into diglycerides, monoglycerides and free fatty acids.  The hair infundibulum is the ecological niche of Malassezia yeasts in our body. 
The prevalence of Malassezia is high in tropical countries like India. It is responsible for causing varied illnesses from chronic recurring superficial mycosis like Tinea versicolour, which is aesthetically displeasing, to fatal fungaemia in pre-disposed patients, like those on total parenteral nutrition (TPN). 
Pityriasis versicolour (PV), also known as tinea versicolour is the prototype Malassezia infection.  It is a superficial chronically recurring infection of the stratum corneum, characterised by scaly, dyspigmented irregular macules most often occurring on the trunk and extremities.  It can be hypo- or hyper pigmented and are covered by fine scales (pityron, Greek for scale).  Some patients may experience pruritus, but most are asymptomatic. 
The other common clinical manifestations include the inflamed red papules and pustules of Pityrosporum folliculitis, and the erythematous, scaling facial and scalp skin seen in seborrhoeic dermatitis (SD). There is evidence that Malassezia even play a role in atopic dermatitis and psoriasis, especially in cases involving the scalp. 
There are presently 14 described species, namely, M. furfur, M. pachydermatis, M. sympodialis, M. globosa, M. obtusa, M. restricta, M. Slooffiae, M. equina, M. dermatis, M. japonica, M. nana, M. capre M. yamatoensis, and M. cuniculi. The fifteenth member has been isolated from hamsters. But there is no valid description and details on its physiological characteristics are not available. 
There is also rising global concern over the species dependent anti-fungal resistance to commonly used agents and high MICs. Hence early and easier species identification is required to study their clinical spectrum and pathogenic potential.
| Materials and Methods|| |
This study was conducted at a 1600 bedded university teaching hospital in south India over a one year period from October 2010 to November 2011.
The study was approved by the Institutional Ethics Committee (IEC-NI/10/Oct/19/38). All the patients gave written consent before samples were collected. A parent provided written informed consent on behalf of all child participants.
All patients who had skin lesions resembling diseases caused by Malassezia, like tinea versicolour (including hypo- and hyper pigmented scaling lesions), seborrhoeic dermatitis of scalp, ala of nose, folliculitis, confluent and reticulated pappillomatosis were included in this study.[Figure 1]a-c
A total of 105 patients were included in the study.
Risk factors such as increased sweating, working in a hot and humid environment, family history of similar complaints, sharing of linen with other members of the family/roommates, topical steroid therapy, malnutrition, conditions with increased cortisol level, history of TPN, diabetes, past history of similar lesions or any other lesion caused probably by Malassezia were documented including whether the patients had contact with animals.
The samples were processed at the Mycology section. KOH (10% for skin and 40% for nail) wet mount was prepared using a good representative part of the sample. Yeasts, hyphal forms or both were looked for and documented.
The samples were then plated immediately onto Sabouraud's dextrose agar (SDA), SDA with olive oil overlay (SDA-O) and modified Dixon's agar (mDA) containing Chloramphemicol and Cyclohexamide at a concentration of 0.05 μg and incubated at 32°C for up to 3 weeks. They require supplementation with long-chain (C 12 to C 24 ) fatty acids for growth in vitro.
Once the growth occurred, Gram's stain was performed and the colonies were subjected first to catalase test [Figure 2]b. The only catalase negative species is M. restricta.
|Figure 2: (a) KOH showing Spaghetti and meatball appearance (b) Catalase test + (c) growth and esculin hydrolysis seen in Tween 60 esculin agar slant|
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The β-glucosidase activity of different Malassezia species was assayed using the method described by Mayser et al.  A loopful of fresh yeast was inoculated deeply in the Tween 60 aesculin agar slant, consisting of Tween 60 (Sigma), ferric ammonium citrate, aesculin, and agar (Hi media). All slants were incubated for 5 days at 32°C. The splitting of aesculin is revealed by darkening of the medium [Figure 2]c. This test was used to distinguish M. furfur, M. slooffiae, M. cuneculi and M. sympodialis from other Malassezia species.
The ability to utilise individual Tween compounds like, Tween 20, 40, 60 and 80 (Sigma) was tested according to the method described by Guillot et al.  Sterile SDA (16 ml) was melted and allowed to cool to approximately 50°C. Malassezia yeast suspensions (2 ml) were mixed with Sabouraud's agar, and the mixtures were plated by pour plate method. Four wells were made in the agar by means of a 2 mm diameter punch, one in each quadrant, and filled with 5 μl Tween 20, 40, 60 and 80. The plates were incubated for one week at 32°C. Utilisation of Tween was assessed by the degree of growth and/or reaction (precipitation) of the lipophilic yeasts around individual wells [Figure 3]a.
|Figure 3: (a) Tween assimilation with Tween 20, 40, 60, 80 (b) SDA with Tween 40-precipitate production|
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Certain species have the ability to grow at temperatures higher than body temperature (40°C-45°C), for example M. slooffiae. Some do not grow well beyond 32°C, for example M. globosa, M. restricta and some grow at 37°C as well, for example M. furfur and M. pachydermatis.
Speciation was done according to the protocol of Cabanes et al., involving all the above mentioned tests. 
All the tests were performed with reference strains purchased from The Centraalbureau, Schimmelcultures (CBS), Netherlands, namely, M. furfur CBS 7019, M. globosa CBS 7966, M. restricta CBS 7222, M. sympodialis CBS 7877 and M. slooffiae CBS 7956.
Statistical analysis was done using simple percentages, chi-square test, Odd's ratio, logistic regression and SPSS software.
| Results|| |
In the 1-year study period, there were 105 patients aged from 10 to 58 years, 83% of whom were in the 20-40 years age group with a slight female predominance (58.1%).
Among the study group (n = 105), 87 had the prototype disease PV, 71 (81.6%) of whom had hypo pigmented lesions and 14 (16.1%) had the hyper pigmented chromic variety and 2 (2.3%) had both [Figure 1]a and b. Of the 105 cases, 66 (62.9%) were coming for the first time and 39 (37.1%) had recurrences.
The most common site of lesion was chest and back followed by back alone.
M. globosa was the most common species in both the hypo- and hyper pigmented groups and was also the most common in fresh and in recurrent cases.
Species isolated: [Figure 4]
In three patients, two species were isolated from the same lesion, namely
- M. pachydermatis and M. restricta were isolated from a 35-year-old male dog trainer with pityriasis versicolour.
- M. globosa and M. restricta were isolated from a 32-year-old male who had pityriasis versicolour, SD, dandruff and confluent reticulated papillomatosis (CRP).
- M. sympodialis and M. furfur were isolated from a 20-year-old female with recurrent PV in multiple sites who also had SD and Malassezia folliculitis.
Hammer and Riley in 2000,  reported the production of a precipitate on modified Dixon's agar by some Malassezia strains. M. furfur, M. obtusa and M. slooffiae were precipitate-negative strains, while M. sympodialis and M. globosa produced precipitate. The same was observed on 1% Tween 40 supplemented SDA [Figure 3]b.
| Discussion|| |
The present study was based in Chennai, south India, a tropical coastal city with high humidity that leads to excessive sweating. The most common age group was 20-40 years probably owing to the period of maximum sebum secretion. Malassezia sp lack the fatty acid synthase gene and are unable to synthesise their own lipids Hence they hydrolyse the sebum to satisfy this growth requirement.  Other reasons for increased prevalence in this age group are working in hot and humid environment, easy access to medical facilities, sharing clothes and probably owing to cosmetic concerns at this age. This study also suggests that it is only rarely found in the elderly.
In some studies in India, the disease has been recorded from 10 to 30 years of age while other studies have found that PV is uncommon in children. , In this study too, there were no patients in the 0-10 age group. Another study from Chandigarh (India), however, reported three neonatal cases of PV.  PV has also been documented in a 2-month-old child.  Several studies have been conducted among school-going children, establishing the presence of PV in this age group. ,,,
Similar to our results, on speciation, M. globosa was found to be the most common isolate (ranging from 53% to 97%) from PV patients, by several researchers. ,,, However, some studies found M. sympodialis to be the most common species.  Another study from central India, showed that 54% of isolates belonged to M. globosa and the next common was M. furfur (30%).  A study in Indonesia found M. furfur to be the most common (42.9%).  M. globosa's pathogenicity might be explained by its high esterase and lipolytic activity. 
M. pachydermatis is occasionally isolated from human skin, but its presence is transient and it is not a human commensal.  Isolating this species from PV can be significant in this case due to history of occupational exposure to dogs. This species did not grow from a swab taken from an unaffected site.
Different studies have shown variable male to female ratios. In this study there were marginally more women than men, similar to studies conducted in Bangkok. In contrast, most studies report a majority of males. This can be due to earlier reporting and easier access to hospitals. Another observation we made in our study is that, all our patients had the habit of applying oil to their hair and taking oil bath. When women plait their hair with oil and allow its contact with the skin of the back, exposed to sun by the traditional Indian attire, the sari, it could be a factor contributing to the development of PV, especially on the back.
We had varying clinical presentations of PV, from single lesions to multiple and involving different sites. Multiple lesions were more common, and so were multiple sites. The most common site to be affected by PV was the back, followed by chest and multiple sites many a time involving the back and chest.
Like most studies, the number of hypo pigmented achromic PVs outnumbered the hyper pigmented chromic type. Though the number of the chromic type was small, we observed that their culture recovery rate was higher and they were all fresh cases. In contrary, the hypochromic type had a lesser recovery rate and many experienced recurrences.
The total number of patients who suffered from dandruff was 29.5%. Those with a past history of untreated dandruff most often grew the same species in both lesions (scalp and the fresh lesions of PV).
Even in those patients who had recurrences, dandruff was a common complaint.
A few rarer presentations of Malassezia infections like CRP, folliculitis, psoriasis vulgaris, and tinea facei (post-steroid therapy) were seen during the study period. However, due to the small number, our study did not contribute much to the understanding of each of these special cases.
KOH wet mount is a valuable screening test confirmed by our study. We noticed both the yeast and mycelial form in PV lesions compared with yeast forms alone when seen as normal flora. In cases of SD, we found predominantly yeasts with no mycelial forms. We also had a smaller number of short hyphal forms. Among the two CRP patients, one had only the yeast form and the other had the yeast and the hyphal form (spaghetti and meatball) in KOH wet mount. In all pityrosporum folliculitis cases, only the yeast forms were seen. When a good representative sample is used for KOH it gives 100% correlation with growth.
There were 50 (47.6%) patients who gave history of contact with pets. There was one isolate of M. pachydermatis, which is commonly an animal pathogen from a patient who was a dog trainer.
There were a very small number of diabetics, all of whom were in good glycaemic control, and hence diabetes was not found to be a significant risk factor in this study. Many of the well-known risk factors were looked for, but they were not statistically significant, probably due to the small sample size.
| Conclusions|| |
A sequential use of KOH, Gram's stain, urease test, catalase test, aesculin hydrolysis, Tween assimilation test and temperature tolerance studies, helps greatly in early speciation of Malassezia in the laboratory.
A larger sample size is needed to come to a significant statistical conclusion in associating risk factors to disease.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]