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ORIGINAL ARTICLE
Year : 2013  |  Volume : 26  |  Issue : 2  |  Page : 86-90

Malassezia species isolated from lesional and nonlesional skin in patients with pityriasis versicolor


1 Department of Dermatology, Venereology and Andrology, Menoufia University, Shebin Al Kawm, Egypt
2 Department of Microbiology, Menoufia University, Shebin Al Kawm, Egypt
3 Faculty of Medicine, Menoufia University, Shebin Al Kawm, Egypt

Date of Web Publication31-Jan-2014

Correspondence Address:
Ola A El-Kholy
MBBCH, 3rd Ibrahim Abukhaled, Military Hospital, Maadi, Cairo
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.126115

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  Abstract 

Objectives
This study aimed to identify Malassezia species found in pityriasis versicolor (PV) patients as well as to establish whether there is any association between the various species of Malassezia isolated from lesional and from nonlesional skin of those patients.
Background
PV is a common, benign, superficial cutaneous fungal infection caused by Malassezia species usually characterized by hypopigmented or hyperpigmented macules and patches on the chest and back. These lesions are scaly and their color varies from red, pale, yellow brown to dark brown.
Patients and methods
A total of 100 patients with PV were included in the study. The samples were obtained by scraping the skin surface, both from lesional and from nonlesional skin, and then incubated on Dixon's agar. The yeast isolated were identified using conventional biochemical tests and Tween assimilation.
Results
Skin lesions were 100% KOH positive for Malassezia species and 10% KOH positive in healthy skin. Growth from skin lesion was obtained on Dixon's agar in 83 patients (83%). The most common species was Malassezia furfur as it was detected in 47 samples (47%), followed by Malassezia globosa (23%) and Malassezia sympodialis (13%). From healthy skin of patients, growth of Malassezia was obtained from 19 specimens, M. furfur was detected in 13%, and M. globosa was detected in only three patients (3%) and M. sympodialis in three patients (3%).
Conclusion
M. furfur in its mycelial phase was the predominant species involved in the etiology of PV and M. globosa was the second most common species. The most frequently isolated species from clinically healthy skin was M. furfur and was recovered from healthy skin only in yeast form. We found no difference in the distribution of Malassezia species between lesional and nonlesional skin. Furthermore, no species predominance was observed whether with hyperpigmented or hypopigmented lesions as M. fufur was the most common species in both lesions in PV patients.

Keywords: Malassezia species, pityriasis versicolor, skin lesions


How to cite this article:
Shoeib MA, Gaber MA, Labeeb AZ, El-Kholy OA. Malassezia species isolated from lesional and nonlesional skin in patients with pityriasis versicolor. Menoufia Med J 2013;26:86-90

How to cite this URL:
Shoeib MA, Gaber MA, Labeeb AZ, El-Kholy OA. Malassezia species isolated from lesional and nonlesional skin in patients with pityriasis versicolor. Menoufia Med J [serial online] 2013 [cited 2020 Feb 27];26:86-90. Available from: http://www.mmj.eg.net/text.asp?2013/26/2/86/126115


  Introduction Top


Opportunistic yeasts of the genus Malassezia have been recognized as members of the microbiological flora of human skin and many other warm-blooded vertebrates but are also associated with a variety of diseases [1] . These lipophilic fungi are associated with different types of diseases, especially pityriasis versicolor (PV), a chronic, benign, and recurrent superficial infection that generally occurs in tropical regions [2] . PV affects predominantly young adults of both sexes and is characterized by circumscribed scaly maculae of various colors from ochre to brown, especially in sebum-rich areas of the skin. Until recently, classification of Malassezia species was a matter of debate. In the last reclassification by Guého et al. [3] , seven distinct species were recognized within this genus, namely, Malassezia furfur, Malassezia pachydermatis, Malassezia sympodialis, Malassezia globosa, Malassezia obtusa, Malassezia restricta, and Malassezia slooffia. More recently, new Malassezia species were defined: Malassezia dermatis, Malassezia equina, Malassezia japonica, Malassezia nana, Malassezia yamatoensis, Malassezia caprae, and Malassezia cunicoli; however, the first seven species have been well studied in relation to diseases in humans [4] . The different Malassezia species are distinguished on the basis of their morphology, growth characteristics, and enzyme activities [5] . Furthermore, in the last few years, molecular tools have been used in identification of the new species of Malassezia such as PCR [6] . In the last 10 years, studies have shown interesting geographical variations in the prevalence of different Malassezia species in PV. Several studies have reported M. globosa, M. sympodialis, and M. furfur to be the three dominant species isolated from PV lesions. Thus, in order to prevent recurrence and invasive PV, early laboratory diagnosis of the condition is required [2] . This study aimed to identify Malassezia species found in PV patients by means of morphological and physiological methods and their correlation to patients' clinical characteristics such as sex, age, number, and location of lesion and pigmentation and to compare the distribution of Malassezia species isolated from PV lesions and those isolated from healthy skin of the same patient.


  Patients and methods Top


Patients

This study included 100 patients (63 men and 37 women); their ages ranged from 18 to 49 years and they were randomly selected from those attending the dermatology outpatient clinic of Menoufia University hospitals. Patients had not received any topical treatment of PV within the previous 2 weeks.

Collection and culture of samples

Mycological examination was performed to confirm the diagnosis of PV. After explaining the procedure and obtaining patient consent, two specimens were collected from each patient; one sample was collected from skin lesion (scales were scraped by a scalpel and transported in a sterile filter paper to the laboratory for further processing) and another sample was taken from the same patient from the healthy skin away from the lesion. The scrapings in the PV lesions as well as normal samples were subjected to 20% potassium hydroxide (KOH) mount examination to detect hyphae and spores. Samples from both lesional and nonlesional skin were cultured on slant of Dixon's agar (prepared by 3.6% malt extract, 0.6% peptone, 2% desiccated ox-bile, 1% Tween 40, 0.2% glycerol, 1.2% agar, 50 mg chloramphenicol, and 500 mg cyclohexa-mide) and were incubated at 32°C for 10 days [7] .

Identification

Malassezia species were identified on the basis of morphological characteristics, catalase test, and growth in the presence of different types of individual Tween (20, 40, 60, 80) as unique lipid supplementation [Table 1] [8] .
Table 1: Scheme used for the identification of Malassezia species

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Tweens assimilation test [7]

The ability to utilize individual Tween compounds (Tween 20, 40, 60, and 80) was tested. Briefly, sterile Sabouraud's agar (16 ml) was melted and allowed to cool to ˜50°C. Malassezia yeast suspensions were mixed with Sabouraud's agar and the mixtures were plated. After solidification, four holes were made in the agar using a 2 mm diameter punch and filled with 5 μl Tween 20, 40, 60, and 80. The plates were incubated for one week at 32°C. Utilization of Tween was assessed by the degree of growth of Malassezia species around individual wells.

Statistical methods

Two types of statistical analysis were carried out:

  1. Descriptive statistics were expressed as number (N), percentage (%), mean (), and SD.
  2. Analytic statistics:

    1. Mann-Whitney U-test for comparison of two groups;
    2. Kruskal-Wallis test for comparison of three groups;
    3. Fisher's exact test was used;
    4. P value of less than 0.05 was considered statistically significant.



  Results Top


In the present study, of 100 patients with PV, 63 (63%) were men and 37 (37%) were women. On comparing the sex of the patients, site of the lesion, and type of Malassezia species isolated from PV patients with the clinical type of the lesion, we found no statistically significant difference with respect to the site of the lesion [Table 2]. In this study, the highest prevalence of PV was observed in the 20-30-year-old age groups. The most commonly isolated Malassezia species in our study were Malassezia furfur, followed by M. sympodialis and M. globosa. The primary diagnosis of PV in our study was based on microscopic examination of infected skin with 20% KOH in which hyphae were seen together with budding yeasts. On direct microscopic examination, PV lesions showed the presence of cylindrical bottle-shaped yeast cells M. globosa or ovoid-shaped cells M. furfur or M. sympodialis. In samples from normal skin, yeast cells were recorded in 10 patients (10%). The remaining 90 patients (90%) were negative, with a statistically highly significant difference (P<0.001; [Table 3]). From 100 lesion samples, the growth of Malassezia species on Dixon's media was obtained from 83 specimens (83%). Mixed cultures were not observed. The most common species was M. furfur (47%), followed by M. globosa (23%) and M. sympodialis (13%). In samples from healthy skin of our patients, the growth of Malassezia species was observed from 19 specimens that showed M. furfur (13%), M. globosa (3%), and M. sympodialis (3%). The percentage of negative cultures was 81% [Table 4] and [Table 5]. The majority of the patients (60%) had hyperpigmentation, whereas 40 patients (40%) had hypopigmentation. The predominant Malassezia isolate was M. furfur in both hypopigmented and hyperpigmented lesions, with no species predominance. We did not find any M. restricta isolate as the catalase reaction was positive for all of the isolates examined. The Tween test allowed distinction of Malassezia species in this study.
Table 2: Malassezia species, sex, and site of lesion in relation to clinical type of lesion in patients with PV

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Table 3: Comparison between the results of KOH examination among lesion and nonlesion skin samples

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Table 4: Results of culture of Malassezia species from lesion and nonlesion skin using Dixon's media

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Table 5: Distribution of Malassezia species among lesion and nonlesion skin in pityriasis versicolor patients

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  Discussion Top


The etiological role of Malassezia in PV is unquestioned. The disease is diagnosed on the basis of its clinical appearance and confirmed by mycological examination. In this study, the highest prevalence of PV was observed in the 20-30-year-old age groups. These data correlated with the results obtained from several previous studies that indicated that the disease generally appears in the late teens, with a peak in the 20 years, when the sebum production is at the highest level, and rarely found in elderly individuals [9] . Our study found no differences in the development of PV between both sexes. In the present study, the most affected areas of the body were the trunk and neck, in agreement with the majority of studies worldwide [10] . The back and the arms were the second most commonly affected sites. The lesions of PV can be hypopigmented, hyperpigmented, both, or erythematous. In the present study, 60% of cases corresponded to the hyperpigmented type. Our result was in agreement with the study by Prohic and Ozegovic [11] as they reported the same result. Crespo et al. [12] reported higher frequency of hypopigmented (51%) lesions than hyperpigmented lesions (43%). The diagnosis of PV is generally simple and is based on the clinical manifestations and microscopic examinations of the lesions. In our study, direct microscopy of PV scales from lesional skin showed 100% presence of yeast cells and short hyphae. Our result was similar to Taroozie et al. [13] and Prohic and Ozegovic [11] as they reported that direct microscopy yielded positive results in 100% of PV samples. In healthy skin, we found the presence of yeast in only 10% of the samples. Culture is necessary to distinguish the Malassezia species by morphological and physiological methods. In the present study, the recovery rate of Malassezia species from the PV lesions was 83%, which was comparable with a recent study by Nakabayashi et al. [14] , but our result was higher than previous studies. However, the higher recovery rate in our study may reflect the difference in culture media and sampling methods. In this survey, the most common isolated species in PV lesions was M. fufur (47%). Previous studies carried out in areas with tropical or subtropical climates showed a clear predominance of M furfur in PV lesions. De Quinzada [15] , in Panama, reported that M. furfur was the predominant species (42%). Also, Canteros et al. [16] and Giusiano et al. [17] found that M. furfur was the predominant species. However, Gupta et. al. [2] found that M. sympodialis was the predominant species isolated from PV lesion in temperate climate regions and M. globosa was the predominant agent in tropical regions. In contrast to our study, Rasi et. al. [18] found that M. globosa was the main agent of PV lesions in the temperate region of Iran. Hence, more studies are necessary to either confirm or reject this hypothesis [19] . In healthy skin, we found positive culture in 19% of samples, which was lower than the results reported by Crespo et al. [12] , and Prohic and Ozegovic [11] , who reported 60 and 90.1% positive culture from healthy skin. In this study, M. globosa was the second common species isolated from skin lesion and healthy skin, with rates of 23 and 3%, respectively. This result was in contrast to the observation of Ben Salah et al. [9] and Nakabayashi et al. [14] as they found that M. globosa was the most commonly detected species (97%). Although Malassezia species are considered as normal microflora of the human skin, these lipophilic yeasts are associated with PV. In healthy skin, we found Malassezia species by direct examination and culture with a frequency of 10 and 19%, respectively. It is widely believed that endogenous factors such as administration of corticosteroids, malnutrition, and increased plasma cortisol level mediate the development of PV. Besides, the role of high temperature and humidity is well established [19] . In this respect, in our study, no differences were observed in Malassezia species isolated from skin lesion and those isolated from healthy skin of PV patients.


  Conclusion Top


M. furfur in its mycelial phase was the predominant species involved in the etiology of PV and M. globosa was the second species. The most frequently isolated species from clinically healthy skin was M. furfur and was recovered from healthy skin only in yeast form. We found no difference in the distribution of Malassezia species between lesional and nonlesional skin. Furthermore, no species predominance was observed, whether with hyperpigmented or with hypopigmented lesions, as M. fufur was the most common species in both lesions in PV patients.


  Acknowledgements Top


Conflicts of interest

None declared.

 
  References Top

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4.Cabañes FJ, Vega S, Castellá G. Malassezia cuniculi sp. nov., a novel yeast species isolated from rabbit skin. Med Mycol 2011; 49 :40-48.  Back to cited text no. 4
    
5.Saadatzadeh MR, Ashbee HR, Cunliffe WJ, Ingham E. Cell-mediated immunity to mycelial phase of Malassezia spp. in patients with pityriasis versicolor and controls. Br J Dermatol 2001; 144 :77-84.  Back to cited text no. 5
    
6.Willimas JV, Eichenfield LF, Burke BL. Prevalence of scalp scaling in pre-pubertal children. Pediatrics 2005; 115 :1-6.  Back to cited text no. 6
    
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8.Chander J. Malassezia infections. In: Textbook of medical mycology. 3rd ed. India: Mehta Publishers; 2002. pp. 92-102.  Back to cited text no. 8
    
9.Ben Salah S, Affes M, Makni F. Les levures du genre Malassezia: identification mole´ culaire par nested-PCR des souches isole´ es a partir desdiffe´ rentes affections dermiques [abstract]. Medical Mycology. J Mycol Med 2005; 15 :175-180.  Back to cited text no. 9
    
10.Sugita T, Uto H, Unno T, Tsuboi R, Ogawa H, Shinoda T, et al. Molecular analysis of Malassezia on the skin of atopic dermatitis patients and healthy subjects. J Clin Microbial 2001; 39 :3486-3490.  Back to cited text no. 10
    
11.Prohic A, Ozegovic L. Malassezia species isolated from lesional and non-lesional skin in patients with pityriasis versicolor. Mycoses 2007; 50 :58-63.  Back to cited text no. 11
    
12.Crespo Erchiga V, Ojeda Martos A, Vera Casano A, Crespo Erchiga A, Sanches Fajardo F, Gueho E. Malassezia globosa as the causative agent of pityriasis versicolor. Br J Dermatol 2000; 143 :799-803.  Back to cited text no. 12
    
13.Tarazooie B, Kordbacheh P, Zaini F, Zaini B, Zomorodian F, Saadat 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.  Back to cited text no. 13
    
14.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 :337-341.  Back to cited text no. 14
    
15.De Quinzada M. Estudio de las especies de Malassezia relacionadas con la patología cutánea pitiriasis versicolor en Panamá. Panamá: Tesis Doctoral; 2005.  Back to cited text no. 15
    
16.Canteros CE, Soria M, Rivas C, Lee W, López Joffre MC, Rodero L, et al. Especies de Malassezzia aisladas de patologías de piel en un centro asistencial de la ciudad de Buenos Aires, Argentina. Rev Argent Microbiol 2003; 35 :156-161.  Back to cited text no. 16
    
17.Giusiano G, Sosa MA, Rojas F, Vanacore ST, Mangiaterra M. Prevalence of Malassezia species in pityriasis versicolor lesions in northeast Argentina. Rev Iberoam Micol 2010; 27 :71-74.  Back to cited text no. 17
    
18.Rasi A, Naderi R, Behzadi AH, Falahati M, Farehyar S, Honarbakhsh Y, et al. Malassezia yeast species isolated from Iranian patients with pityriasis versicolor in a prospective study. Mycoses 2010; 53 :350-355.  Back to cited text no. 18
    
19.Boekhout T, Guého-Kellerman E, Mayser P, Velegraki A. Malassezia and the skin science and clinical practice. Heidelberg/Dordrecht/London/New York: Springer; 2010.Malassezia species isolated from lesional and nonlesional skin in patients with pityriasis versicolor  Back to cited text no. 19
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]


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