Molecular Detection and Identification of Zygomycetes Species from Paraffin-Embedded Tissues in a Murine Model of Disseminated Zygomycosis: a Collaborative European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Fungal Infection Study Group (EFISG) Evaluation
Molecular Detection and Identification of Zygomycetes Species from Paraffin-Embedded Tissues in a Murine Model of Disseminated Zygomycosis: a Collaborative European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Fungal Infection Study Group (EFISG) Evaluation
复制标题
DOI：
10.1128/jcm.02319-09
复制
发表时间：
2010-06-01
影响因子：
9.4
通讯作者：
Lortholary, Olivier
中科院分区：
文献类型：
--
作者：
Dannaoui, Eric;Schwarz, Patrick;Lortholary, Olivier
本研究旨在评估实验感染小鼠经福尔马林固定石蜡包埋的肾脏和脑组织中接合菌的分子检测和鉴定的实验室间可重复性。动物感染5种真菌（米根霉、小孢子根霉、冠孢衣螨、圆柱根霉和环形毛霉）中的一种。从每个石蜡块中制备1、10或30个组织切片的样品，对样品身份进行盲法分析，并将样品邮寄到七个实验室中的每个实验室进行敏感性评估。提供了一份描述提取方法和PCR扩增程序的协议。用真菌通用引物ITS1和ITS2扩增内部转录间隔1 （ITS1）区，并对其进行测序。由于93%的组织标本呈阴性，因此将该物种的数据排除在分析之外。切片30次、10次、1次的PCR阳性检出率分别为93%（52/56）、89%（50/56）、27%（15/56）。根据器官组织、真菌种类和实验室的不同，存在细微的差异。100%（30刀）、98%（10刀）和93%（1刀）的病例能正确识别物种。在本研究中使用的方案下，当有足够的材料可用时，用于鉴定福尔马林固定石蜡包埋组织中主要接合菌的ITS测序的实验室间可重复性可达100%。
The present study was performed to assess the interlaboratory reproducibility of the molecular detection and identification of species of Zygomycetes from formalin-fixed paraffin-embedded kidney and brain tissues obtained from experimentally infected mice. Animals were infected with one of five species (Rhizopus oryzae, Rhizopus microsporus, Lichtheimia corymbifera, Rhizomucor pusillus, and Mucor circinelloides). Samples with 1, 10, or 30 slide cuts of the tissues were prepared from each paraffin block, the sample identities were blinded for analysis, and the samples were mailed to each of seven laboratories for the assessment of sensitivity. A protocol describing the extraction method and the PCR amplification procedure was provided. The internal transcribed spacer 1 (ITS1) region was amplified by PCR with the fungal universal primers ITS1 and ITS2 and sequenced. As negative results were obtained for 93% of the tissue specimens infected by M. circinelloides, the data for this species were excluded from the analysis. Positive PCR results were obtained for 93% (52/56), 89% (50/56), and 27% (15/56) of the samples with 30, 10, and 1 slide cuts, respectively. There were minor differences, depending on the organ tissue, fungal species, and laboratory. Correct species identification was possible for 100% (30 cuts), 98% (10 cuts), and 93% (1 cut) of the cases. With the protocol used in the present study, the interlaboratory reproducibility of ITS sequencing for the identification of major Zygomycetes species from formalin-fixed paraffin-embedded tissues can reach 100%, when enough material is available.