Kock N P, Petersen H, Fenner T, Sobottka I, Schmetz C, Deplazes P, Pieniazek N J, Albrecht H, Schottelius J
Section of Parasitology, Bernard Nocht Institute for Tropical Medicine, Hamburg, Germany.
Eur J Clin Microbiol Infect Dis. 1997 May;16(5):369-76. doi: 10.1007/BF01726365.
In view of the increasing number of cases of human microsporidiosis, simple and rapid methods for clear identification of microsporidian parasites to the species level are required. In the present study, the polymerase chain reaction (PCR) was used for species-specific detection of Encephalitozoon cuniculi. Encephalitozoon hellem, Encephalitozoon (Septata) intestinalis, and Enterocytozoon bieneusi in both tissue and stool. Using stool specimens and intestinal biopsies of patients infected with Enterocytozoon bieneusi (n = 9), Encephalitozoon spp. (n = 2), and Encephalitozoon intestinalis (n = 1) as well as stool spiked with spores of Encephalitozoon cuniculi and Encephalitozoon hellem and tissue cultures of Encephalitozoon cuniculi and Encephalitozoon hellem, three procedures were developed to produce PCR-ready DNA directly from the samples. Specific detection of microsporidian pathogens was achieved in the first PCR. The subsequent nested PCR permitted species determination and verified the first PCR products. Without exception, the PCR assay confirmed electron microscopic detection of Enterocytozoon bieneusi and Encephalitozoon intestinalis in stool specimens and their corresponding biopsies and in spiked stool samples and tissue cultures infected with Encephalitozoon cuniculi and Encephalitozoon hellem. Moreover, identification of Encephalitozoon spp. could be specified as Encephalitozoon intestinalis. Whereas standard methods such as light and transmission electron microscopy may lack sensitivity or require more time and special equipment, the PCR procedure described facilitates species-specific identification of microsporidian parasites in stool, biopsies, and, probably, other samples in about five hours.
鉴于人类微孢子虫病病例数量不断增加，需要简单快速的方法将微孢子虫寄生虫明确鉴定到种水平。在本研究中，聚合酶链反应（PCR）用于对兔脑炎微孢子虫、海伦脑炎微孢子虫、肠脑炎微孢子虫（Septata）和比氏肠细胞内微孢子虫在组织和粪便中的种特异性检测。使用感染比氏肠细胞内微孢子虫（n = 9）、脑炎微孢子虫属（n = 2）和肠脑炎微孢子虫（n = 1）患者的粪便标本和肠道活检组织，以及接种了兔脑炎微孢子虫和海伦脑炎微孢子虫孢子的粪便，还有兔脑炎微孢子虫和海伦脑炎微孢子虫的组织培养物，开发了三种程序以直接从样本中产生可用于PCR的DNA。在第一次PCR中实现了微孢子虫病原体的特异性检测。随后的巢式PCR允许进行种属鉴定并验证第一次PCR产物。无一例外，PCR检测证实了在粪便标本及其相应活检组织中，以及在接种了兔脑炎微孢子虫和海伦脑炎微孢子虫的粪便样本和组织培养物中，通过电子显微镜检测到的比氏肠细胞内微孢子虫和肠脑炎微孢子虫。此外，脑炎微孢子虫属可鉴定为肠脑炎微孢子虫。虽然标准方法如光学显微镜和透射电子显微镜可能缺乏敏感性，或需要更多时间和特殊设备，但所述的PCR程序有助于在约五小时内对粪便、活检组织以及可能的其他样本中的微孢子虫寄生虫进行种特异性鉴定。
