Identification of Malassezia species from patient skin scales by PCR-RFLP

Gaitanis, George; Velegraki, Aristea; Frangoulis, E.; Mitroussia, Ageliki; Tsigonia, A.; Tzimogianni, Aleka; Katsambas, Andreas; Legakis, Nicholas J.

doi:10.1046/j.1469-0691.2002.00383.x

Cited by 99 publications

(81 citation statements)

References 26 publications

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“…Several findings from this study are consistent with those previously reported by Sugita et al (45) and Gaitanis et al (19). The results of these studies are expected to be free of potential culture bias.…”

Section: Discussionsupporting

confidence: 93%

“…In addition, all of the methods mentioned above (except denaturing gradient gel electrophoresis) require cultivation to enhance sensitivity, thereby increasing both the potential for culture bias and the turnaround time for analysis. Two methods have been reported to differentiate complex Malassezia communities from skin without cultivation (19,27,45), but these methods require either separate amplification with specific primer sets for each species or restriction digestion. The terminal fragment length polymorphism (tFLP) method uses only three different primer sets, minimizing the potential bias related to amplification efficiency.…”

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confidence: 99%

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Fast, Noninvasive Method for Molecular Detection and Differentiation of Malassezia Yeast Species on Human Skin and Application of the Method to Dandruff Microbiology

et al. 2002

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Malassezia fungi have been the suspected cause of dandruff for more than a century. Previously referred to as Pityrosporum ovale, Pityrosporum orbiculare, or Malassezia, these fungi are now known to consist of at least seven Malassezia species. Each species has a specific ecological niche, as well as specific biochemical and genetic characteristics. Malassezia yeasts have fastidious culture conditions and exceedingly different growth rates. Therefore, the results of surveys of Malassezia based on culture methods can be difficult to interpret. We developed a molecular technique, terminal fragment length polymorphism analysis, to more accurately survey the ecology of Malassezia yeasts without bias from culture. This technique involves fluorescent nested PCR of the intergenic transcribed spacer (ITS) ITS I and ITS II region ribosomal gene clusters. All known Malassezia species can be differentiated by unique ITS fragment lengths. We have used this technique to directly analyze scalp samples from subjects enrolled in a demographic scalp health study. Results for subjects assigned composite adherent scalp flaking scores (ASFS) <10 were compared to those for subjects assigned composite ASFS >24. Malassezia restricta and M. globosa were found to be the predominant Malassezia species present in both groups. Importantly, we found no evidence of M. furfur in either group, indicating that M. furfur can be eliminated as the causal organism for dandruff. Both groups also showed the presence of non-Malassezia fungi. This method, particularly when it is used in combination with existing fungal ITS databases, is expected to be useful in the diagnosis of multiple other fungal infections.Recently, members of the genus Malassezia have become viewed as opportunistic yeasts of increasing importance (1, 2, 31, 35, 42). They are lipophilic or lipid-dependent yeasts, and at least some belong to the normal cutaneous microflora. Some Malassezia species may act as pathogens when exposed to certain changes in the skin microclimate. For decades the genus Malassezia remained limited to two species, namely, the lipiddependent Malassezia furfur and the lipophilic M. pachydermatis. In 1995, 28S rRNA gene sequences revealed seven distinct genetic entities (25), which are now accepted as species (M. furfur, M. obtusa, M. globosa, M. slooffiae, M. sympodialis, M. pachydermatis, and M. restricta) (22). Malassezia species are exceptionally difficult to cultivate, so additional species may be discovered as DNA-based differentiation techniques are refined and applied to multiple ecosystems.While several of the seven described Malassezia species have been associated with human infection, the pathological role of each species is not fully understood. For example, M. furfur infections have been observed in hospitalized neonates with very low birth weights receiving intravenous lipid emulsions (5,7,15,23,41,46). M. globosa, which corresponds to the original description of Pityrosporum orbiculare and correlates to the former serovar B of M. furfur (14), ...

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Section: Discussionsupporting

confidence: 93%

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confidence: 99%

Fast, Noninvasive Method for Molecular Detection and Differentiation of Malassezia Yeast Species on Human Skin and Application of the Method to Dandruff Microbiology

et al. 2002

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“…To overcome the limits of morphological assessment, recent studies have used a variety of molecular methods such as the nested polymerase chain reaction (PCR) 12 , real-time PCR 13 , pulsed field gel electrophoresis (PFGE) 14 , amplified fragment length polymorphism (AFLP) 15,16 , denaturing gradient gel electrophoresis (DGGE) 16 , random amplification of polymorphic DNA (RAPD) 16,17 , single strand conformation polymorphism (SSCP) 18 , terminal fragment length polymorphism (tFLP) 19 , restriction fragment length polymorphism (RFLP) [20][21][22][23] , and sequencing analysis 24 .…”

Section: Introductionmentioning

confidence: 99%

Comparison of Nested PCR and RFLP for Identification and Classification of Malassezia Yeasts from Healthy Human Skin

Song

Lee

et al. 2009

Ann Dermatol

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Background: Malassezia yeasts are normal flora of the skin found in 75∼98% of healthy subjects. The accurate identification of the Malassezia species is important for determining the pathogenesis of the Malassezia yeasts with regard to various skin diseases such as Malassezia folliculitis, seborrheic dermatitis, and atopic dermatitis. Objective: This research was conducted to determine a more accurate and rapid molecular test for the identification and classification of Malassezia yeasts. Methods: We compared the accuracy and efficacy of restriction fragment length polymorphism (RFLP) and the nested polymerase chain reaction (PCR) for the identification of Malassezia yeasts. Results: Although both methods demonstrated rapid and reliable results with regard to identification, the nested PCR method was faster. However, 7 different Malassezia species (1.2%) were identified by the nested PCR compared to the RFLP method. Conclusion: Our results show that RFLP method was relatively more accurate and reliable for the detection of various Malassezia species compared to the nested PCR. But, in the aspect of simplicity and time saving, the latter method has its own advantages. In addition, the 26S rDNA, which was targeted in this study, contains highly conserved base sequences and enough sequence variation for inter-species identification of Malassezia yeasts. (Ann Dermatol 21(4) 352

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“…This phenotypic system has been used as the conventional method of identification, though in practice, the test results are not always easy to read. Therefore, several research groups have explored the use of molecular techniques, such as pulsed-field gel electrophoresis (3, 4, 36), randomly amplified polymorphic DNA analysis (1, 3), amplified fragment length polymorphism (AFLP) analysis (40), denaturing gradient gel electrophoresis (40), multilocus enzyme electrophoresis (30), sequencing analysis (37), restriction analysis of PCR amplicons of ribosomal sequences (8,15,16,20,28), and chitin synthase gene sequence analysis (1,4,8,37), for the identification of Malassezia species. Recently, Gemmer et al (9) devised a remarkably efficient, novel technique, terminal fragment length polymorphism analysis, for the rapid and reliable identification of Malassezia species.…”

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Identification and Typing of Malassezia Species by Amplified Fragment Length Polymorphism and Sequence Analyses of the Internal Transcribed Spacer and Large-Subunit Regions of Ribosomal DNA

et al. 2004

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Malassezia yeasts are associated with several dermatological disorders. The conventional identification of Malassezia species by phenotypic methods is complicated and time-consuming, and the results based on culture methods are difficult to interpret. A comparative molecular approach based on the use of three molecular techniques, namely, amplified fragment length polymorphism (AFLP) analysis, sequencing of the internal transcribed spacer, and sequencing of the D1 and D2 domains of the large-subunit ribosomal DNA region, was applied for the identification of Malassezia species. All species could be correctly identified by means of these methods. The results of AFLP analysis and sequencing were in complete agreement with each other. However, some discrepancies were noted when the molecular methods were compared with the phenotypic method of identification. Specific genotypes were distinguished within a collection of Malassezia furfur isolates from Canadian sources. AFLP analysis revealed significant geographical differences between the North American and European M. furfur strains.The genus Malassezia has received considerable attention in recent years from dermatologists and other clinicians. This group of basidiomycetous yeasts, long known to be the causal agents of pityriasis (tinea) versicolor, is also increasingly being associated with the causation of folliculitis, papillomatosis, and invasive human infections, as well as potential immunogenic triggering of atopic dermatitis, seborrheic dermatitis, and dandruff (6,9,11,14,18,25,26,29). Malassezia species are listed among the new and emerging yeast pathogens (22,24).The genus Malassezia, until recently, was characterized on the basis of rRNA sequences as consisting of seven species, including the lipid-dependent species M. furfur, M. sympodialis, M. globosa, M. obtusa, M. restricta, and M. slooffiae and the lipophilic species M. pachydermatis (12, 13). Recently, two new species have been identified: M. dermatis (39), which was isolated from atopic dermatitis patients, and M. equi (unpublished), a species that was isolated from the skin of horses (32). The latter species has only tentatively been named and still awaits formal description; the present study deals only with the eight described species.Guillot et al. (17) introduced a physiological system based on lipid assimilation and other phenotypic characteristics for identifying the various Malassezia species. This phenotypic system has been used as the conventional method of identification, though in practice, the test results are not always easy to read. Therefore, several research groups have explored the use of molecular techniques, such as pulsed-field gel electrophoresis (3, 4, 36), randomly amplified polymorphic DNA analysis (1, 3), amplified fragment length polymorphism (AFLP) analysis (40), denaturing gradient gel electrophoresis (40), multilocus enzyme electrophoresis (30), sequencing analysis (37), restriction analysis of PCR amplicons of ribosomal sequences (8,15,16,20,28), and chitin synthase ...

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Identification of Malassezia species from patient skin scales by PCR-RFLP

Abstract: The proposed method, described for the first time, could provide a sensitive and rapid detection and identification system for Malassezia species, which may be applied to epidemiological surveys and routine practice.

Cited by 99 publications

References 26 publications

Fast, Noninvasive Method for Molecular Detection and Differentiation of Malassezia Yeast Species on Human Skin and Application of the Method to Dandruff Microbiology

Fast, Noninvasive Method for Molecular Detection and Differentiation of Malassezia Yeast Species on Human Skin and Application of the Method to Dandruff Microbiology

Comparison of Nested PCR and RFLP for Identification and Classification of Malassezia Yeasts from Healthy Human Skin

Identification and Typing of Malassezia Species by Amplified Fragment Length Polymorphism and Sequence Analyses of the Internal Transcribed Spacer and Large-Subunit Regions of Ribosomal DNA

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