Bialek Ralf, Feucht Antje, Aepinus Christian, Just-Nübling Gudrun, Robertson Valerie J, Knobloch Jürgen, Hohle Rolf
Institute for Tropical Medicine, University Hospital Tübingen, Keplerstrasse 15, 72074 Tübingen, Germany.
J Clin Microbiol. 2002 May;40(5):1644-7. doi: 10.1128/JCM.40.5.1644-1647.2002.
In order to evaluate the diagnostic relevance of two nested PCR assays for diagnosis of histoplasmosis in clinical specimens, 100 paraffin-embedded biopsy specimens were examined. Upon microscopy of tissue, 50 biopsy specimens were histoplasma positive and 50 were negative. Due to destruction by formalin fixation, successful extraction of amplifiable human DNA was limited to 29 and 33 samples, respectively. A product of the Histoplasma capsulatum nested PCR assay targeting the gene encoding the unique fungal 100-kDa-like protein was detected in 20 histopathologically positive biopsy specimens but in none of the microscopically negative samples. Sequencing revealed that all 20 products of 210 bp were identical to the sequence of H. capsulatum in the GenBank database. In contrast, the nested PCR assay targeting the fungal 18S rRNA genes amplified products in 26 histopathologically positive but also in 18 microscopically negative biopsy specimens. However, sequencing revealed that only 20 of these 44 PCR products (231 bp) were identical to the sequence of H. capsulatum. The remaining 24 sequences were homologous to those of several Euascomycetes. These PCR products were detected only in tissues possibly colonized by nonpathogenic fungi, possibly causing these nonspecific amplifications. The detection limit of both H. capsulatum nested PCR assays was 1 to 5 fungal cells per sample. The two assays were similarly sensitive in identifying H. capsulatum. In this preliminary study, the novel 100-kDa-like-protein gene nested PCR revealed a specificity of 100% without requiring sequencing, which was necessary for identification of the 18S ribosomal DNA nested PCR products in order to avoid a high rate of false-positive results.
为了评估两种巢式PCR检测方法对临床标本中组织胞浆菌病诊断的相关性，对100份石蜡包埋活检标本进行了检测。经组织显微镜检查，50份活检标本组织胞浆菌呈阳性，50份为阴性。由于福尔马林固定造成的破坏，分别仅有29份和33份样本成功提取出可扩增的人类DNA。针对编码独特真菌100 kDa样蛋白的基因进行荚膜组织胞浆菌巢式PCR检测，在20份组织病理学阳性活检标本中检测到产物，但在所有显微镜检查阴性样本中均未检测到。测序显示，所有20个210 bp的产物与GenBank数据库中荚膜组织胞浆菌的序列相同。相比之下，针对真菌18S rRNA基因的巢式PCR检测在26份组织病理学阳性活检标本中扩增出产物，但在18份显微镜检查阴性活检标本中也扩增出产物。然而，测序显示这44个PCR产物（231 bp）中只有20个与荚膜组织胞浆菌的序列相同。其余24个序列与几种真子囊菌的序列同源。这些PCR产物仅在可能被非致病性真菌定植的组织中检测到，可能导致了这些非特异性扩增。两种荚膜组织胞浆菌巢式PCR检测方法的检测限均为每个样本1至5个真菌细胞。两种检测方法在鉴定荚膜组织胞浆菌方面同样敏感。在这项初步研究中，新型100 kDa样蛋白基因巢式PCR显示特异性为100%，无需测序，而18S核糖体DNA巢式PCR产物的鉴定则需要测序以避免高假阳性率。
