Lasker Brent A, Butler Geraldine, Lott Timothy J
Mycotic Diseases Branch, Division of Bacterial and Mycotic Diseases, National Centers for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, Mailstop G-11, Atlanta, GA 30333.
J Clin Microbiol. 2006 Mar;44(3):750-9. doi: 10.1128/JCM.44.3.750-759.2006.
Candida parapsilosis, a pathogenic yeast, is composed of three newly designated genomic species that are physiologically and morphologically indistinguishable. Nosocomial infections caused by group I C. parapsilosis are often associated with the breakdown of infection control practices and the contamination of medical devices, solutions, and indwelling catheters. Due to the low levels of nucleotide sequence variation that are observed, an investigation of the size polymorphisms in loci harboring microsatellite repeat sequences was applied for the typing of C. parapsilosis group I isolates. PCR primer sets that flank the microsatellite repeats for seven loci were designed. Following amplification by PCR, the size of each amplification product was determined automatically by capillary electrophoresis. A total of 42 C. parapsilosis group I isolates were typed by microsatellite analysis, and their profiles were compared to the hybridization profiles obtained by use of the Cp3-13 DNA probe. A high degree of discrimination (discriminatory power = 0.971) was observed by microsatellite analysis. The number of different alleles per locus ranged from 14 for locus B to 5 for locus C. Microsatellite analysis detected 30 different microsatellite genotypes, with 24 genotypes represented by a single isolate. Comparison of the genotypes obtained by microsatellite analysis and those obtained by analysis of the Cp3-13 hybridization profiles showed that they were similar, and these methods were able to identify related and unrelated isolates. Some discrepancies were observed between the methods and may be due to higher mutation rates and/or homoplasy by microsatellite markers. Identical results were observed between microsatellite analysis and Cp3-13 DNA hybridization profile analysis for C. parapsilosis isolates obtained from two patients, demonstrating the reproducibilities of the methods in vivo. Identical microsatellite profiles were observed for isolates displaying different phenotypic switching morphologies. Indistinguishable Cp3-13 DNA hybridization profiles were observed for six epidemiologically related isolates; however, only three of six primary isolates had identical microsatellite profiles. Size variation at a single locus was observed for three of six isolates obtained either after the outbreak period or from a different body site, suggesting the potential of the method to detect microevolutionary events. Interestingly, for most loci a single allele per strain was observed; in contrast, two alleles per locus were observed for some strains, and consistent with the findings for natural isolates, some isolates may be aneuploid. Due to the potential for high throughput, reproducibility, and discrimination, microsatellite analysis may provide a robust and efficient method for the genotyping of large numbers of C. parapsilosis group I isolates.
近平滑念珠菌是一种致病性酵母，由三个新指定的基因组种组成，它们在生理和形态上无法区分。I组近平滑念珠菌引起的医院感染通常与感染控制措施的破坏以及医疗器械、溶液和留置导管的污染有关。由于观察到的核苷酸序列变异水平较低，因此对含有微卫星重复序列的基因座中的大小多态性进行了研究，以用于I组近平滑念珠菌分离株的分型。设计了侧翼位于七个基因座微卫星重复序列的PCR引物组。通过PCR扩增后，每个扩增产物的大小通过毛细管电泳自动测定。通过微卫星分析对总共42株I组近平滑念珠菌分离株进行了分型，并将它们的图谱与使用Cp3 - 13 DNA探针获得的杂交图谱进行了比较。微卫星分析显示出高度的区分能力（鉴别力 = 0.971）。每个基因座的不同等位基因数量范围从基因座B的14个到基因座C的5个。微卫星分析检测到30种不同的微卫星基因型，其中24种基因型仅由单个分离株代表。通过微卫星分析获得的基因型与通过Cp3 - 13杂交图谱分析获得的基因型的比较表明它们相似，并且这些方法能够识别相关和不相关的分离株。在这些方法之间观察到了一些差异，这可能是由于微卫星标记的较高突变率和/或平行进化。对于从两名患者获得的近平滑念珠菌分离株，微卫星分析和Cp3 - 13 DNA杂交图谱分析观察到相同的结果，证明了这些方法在体内的可重复性。对于表现出不同表型转换形态的分离株观察到相同的微卫星图谱。对于六个流行病学相关的分离株观察到无法区分的Cp3 - 13 DNA杂交图谱；然而，六个主要分离株中只有三个具有相同的微卫星图谱。在暴发期之后或从不同身体部位获得的六个分离株中有三个在单个基因座上观察到大小变异，这表明该方法具有检测微进化事件的潜力。有趣的是，对于大多数基因座，每个菌株观察到一个等位基因；相反，对于一些菌株，每个基因座观察到两个等位基因，并且与自然分离株的发现一致，一些分离株可能是非整倍体。由于具有高通量、可重复性和区分能力的潜力，微卫星分析可能为大量I组近平滑念珠菌分离株的基因分型提供一种强大而有效的方法。
