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

Abstract

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), ...

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

“…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 .…”

Fungi on the Skin: Dermatophytes and Malassezia

“…The next-generation sequencing technology used in this study resulted in more than five million sequences, whereas approximately 140 isolates were recovered from the culturing method (Findley et al 2013). Sequence analysis revealed that Malassezia is the most abundant fungus on many human skin sites, as previously shown for the scalp (Gemmer et al 2002). These authors cultured three of the human-associated Malassezia species: M. globosa, M. restricta, and M. sympodialis.…”

“…The number of scalp Malassezia has been reported to be higher in patients with seborrheic dermatitis or dandruff than in normal controls (McGinley et al 1975), but the evidence is controversial and contradicted by other reports ). The differences are likely caused by the difficulty in accurately enumerating Malassezia from human skin (Kligman 1979;Weiss et al 1991;Gupta et al 2001b;Gemmer et al 2002;Sugita et al 2002). D/SD is most commonly treated by shampoos containing antifungal ingredients, indicating the role of fungi.…”

“…Species can be recognized using ribosomal DNA (rDNA) sequence analysis, especially that of the ITS 1 þ 2 regions and the D1/D2 domains of the large subunit (LSU) rDNA (Guillot and Guého 1995;Gemmer et al 2002;Gupta et al 2004). Based on this molecular recognition of species, it became possible to differentiate them by phenotypic means to some extent (Guého et al 1996(Guého et al , 1998Guillot et al 1996;Mayser et al 1997;Crespo Erchiga and Guého 2005;Guého-Kellermann et al 2011).…”

Detection of Malassezia Species Isolated From Patients With Pityriasis Versicolor and Seborrheic Dermatitis Using Nested-PCR

“…In 1889, Baillon suggested that the genus of Malassezia was the cause pityriasis versicolor (5,6). In 1996 Gemmer et al based on 28S rRNA gene sequences classified Malassezia genus to seven species (7). Fourteen species of Malassezia genus have been identified based on phenotypic features and molecular methods (8 …”