Division of Microbiology, SydPath, St.Vincent's Hospital, Darlinghurst 2010, NSW, Australia.
J Clin Microbiol. 2011 Jan;49(1):257-62. doi: 10.1128/JCM.01796-10. Epub 2010 Nov 3.
The aim of this study was to describe the first development and evaluation of a multiplex tandem PCR (MT-PCR) assay for the detection and identification of 4 common pathogenic protozoan parasites, Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis, from human clinical samples. A total of 472 fecal samples submitted to the Department of Microbiology at St. Vincent's Hospital were included in the study. The MT-PCR assay was compared to four real-time PCR (RT-PCR) assays and microscopy by a traditional modified iron hematoxylin stain. The MT-PCR detected 28 G. intestinalis, 26 D. fragilis, 11 E. histolytica, and 9 Cryptosporidium sp. isolates. Detection and identification of the fecal protozoa by MT-PCR demonstrated 100% correlation with the RT-PCR results, and compared to RT-PCR, MT-PCR exhibited 100% sensitivity and specificity, while traditional microscopy of stained fixed fecal smears exhibited sensitivities and specificities of 56% and 100% for Cryptosporidium spp., 38% and 99% for D. fragilis, 47% and 97% for E. histolytica, and 50% and 100% for G. intestinalis. No cross-reactivity was detected in 100 stool samples containing various other bacterial, viral, and protozoan species. The MT-PCR assay was able to provide rapid, sensitive, and specific simultaneous detection and identification of the four most important diarrhea-causing protozoan parasites that infect humans. This study also highlights the lack of sensitivity demonstrated by microscopy, and thus, molecular methods such as MT-PCR must be considered the diagnostic methods of choice for enteric protozoan parasites.
本研究旨在描述一种用于检测和鉴定 4 种常见致病性原生动物寄生虫（隐孢子虫、脆弱双核阿米巴、溶组织内阿米巴和肠道贾第鞭毛虫）的多重串联 PCR（MT-PCR）检测方法的首次开发和评估。共纳入 472 份来自圣文森特医院微生物科的粪便样本。MT-PCR 检测方法与 4 种实时 PCR（RT-PCR）检测方法和传统改良铁苏木精染色显微镜检查进行了比较。MT-PCR 检测到 28 株肠道贾第鞭毛虫、26 株脆弱双核阿米巴、11 株溶组织内阿米巴和 9 株隐孢子虫。MT-PCR 对粪便原生动物的检测和鉴定与 RT-PCR 结果具有 100%的相关性，与 RT-PCR 相比，MT-PCR 显示出 100%的敏感性和特异性，而传统的染色固定粪便涂片显微镜检查对隐孢子虫的敏感性和特异性分别为 56%和 100%，脆弱双核阿米巴为 38%和 99%，溶组织内阿米巴为 47%和 97%，肠道贾第鞭毛虫为 50%和 100%。在包含各种其他细菌、病毒和原生动物物种的 100 份粪便样本中未检测到交叉反应。MT-PCR 检测方法能够快速、敏感、特异性地同时检测和鉴定感染人类的四种最重要的腹泻致病原生动物寄生虫。本研究还强调了显微镜检查显示出的敏感性不足，因此，必须将 MT-PCR 等分子方法视为肠道原生动物寄生虫的首选诊断方法。
