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

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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) 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.