Gaitanis G, Velegraki A, Frangoulis E, Mitroussia A, Tsigonia A, Tzimogianni A, Katsambas A, Legakis N J
Department of Microbiology, Mycology Reference Laboratory, Medical School, University of Athens, Greece.
Clin Microbiol Infect. 2002 Mar;8(3):162-73. doi: 10.1046/j.1469-0691.2002.00383.x.
This study was aimed at the development of a DNA-based procedure directly applicable to pathological skin scales and at the assessment of its value in rapid laboratory confirmation and identification of each of the seven Malassezia species. These lipophilic basidiomycetous yeasts in predisposed individuals are involved in pityriasis versicolor, seborrheic dermatitis, blepharitis, folliculitis, atopic dermatitis and fungemia. Standard identification procedures to species level are available, but so far no system for direct detection and characterization of Malassezia species in clinical specimens is available.
Malassezia DNA was extracted from pathological skin scales by a modified hexadecyltrimethylammonium bromide (CTAB) method and amplified by single and nested polymerase chain reaction (PCR), assays using the general fungal ITS 1/4 and 3/4 primers for amplification of sequences from the Malassezia major ribosomal DNA complex. Restriction fragment length polymorphism (RFLP) analysis of PCR products was used in subsequent species identification. DNA extracted from culture-positive skin scales was also tested by PCR and the RFLP patterns obtained were analyzed.
A total of 36 isolates were tested. Distinct pure culture and skin-scale ITS 3/4 HinfI and AluI restriction patterns differentially identified M. furfur, M. globosa, M. restricta, M. sympodialis, M. pachydermatis, M. obtusa and M. slooffiae. Malassezia DNA was extracted from pathological skin scales and RFLP identified solitary and multiple Malassezia species in the same specimen. Molecular identification was confirmed by cultures and biochemical tests. Concurrent detection and identification of Candida and Yarrowia species was also feasible from skin scales.
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.
本研究旨在开发一种直接适用于病理性皮肤鳞屑的基于DNA的方法，并评估其在快速实验室确认和鉴定七种马拉色菌属菌种中的价值。这些亲脂性担子菌酵母在易感个体中可引发花斑癣、脂溢性皮炎、睑缘炎、毛囊炎、特应性皮炎和真菌血症。目前已有针对菌种水平的标准鉴定程序，但到目前为止，尚无用于直接检测和鉴定临床标本中马拉色菌属菌种的系统。
采用改良的十六烷基三甲基溴化铵（CTAB）法从病理性皮肤鳞屑中提取马拉色菌DNA，并通过单重和巢式聚合酶链反应（PCR）进行扩增，使用通用真菌ITS 1/4和3/4引物扩增马拉色菌主要核糖体DNA复合体的序列。PCR产物的限制性片段长度多态性（RFLP）分析用于后续菌种鉴定。从培养阳性的皮肤鳞屑中提取的DNA也通过PCR进行检测，并对获得的RFLP模式进行分析。
共检测了36株分离株。不同的纯培养物以及皮肤鳞屑ITS 3/4 HinfI和AluI限制性模式可分别鉴定糠秕马拉色菌、球形马拉色菌、限制马拉色菌、合轴马拉色菌、厚皮马拉色菌、钝形马拉色菌和斯洛菲马拉色菌。从病理性皮肤鳞屑中提取了马拉色菌DNA，RFLP鉴定出同一标本中存在单一和多种马拉色菌属菌种。通过培养和生化试验证实了分子鉴定结果。同时从皮肤鳞屑中检测和鉴定念珠菌属和耶氏酵母属菌种也是可行的。
首次描述的该方法可为马拉色菌属菌种提供一种灵敏、快速的检测和鉴定系统，可应用于流行病学调查和常规实践。
