Mohammadpour Iraj, Bozorg-Ghalati Farzaneh, Motazedian Mohammad H
1Department of Medical Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences, Zand Blvd, Shiraz-7134845794, Iran
Department of Molecular Medicine, School of Advanced Technologies in Medicine, Shahid Beheshti University, Velenjak Street, Tehran 1956944413, Iran
Ann Parasitol. 2016;62(4):321-330. doi: 10.17420/ap6204.68.
Microsporidia and Cryptosporidium species are prominent agents of enteritis, capable of causing severe chronic diarrhoea in children, immunocompetent and immunocompromised individuals around the world. It is not possible to identify the parasites at species level solely on the basis of microscopy. The aim of the present study was to identify and characterize the species of Microsporidia and Cryptosporidium in immunocompetent humans with GI disturbances by nested PCR-RFLP, sequencing, and phylogenetic analysis. Fresh frozen and fresh paraffin-embedded biopsy specimens of the duodenum, jejunum, ileum and the cecum of 110 patients were examined. Genomic DNA was extracted from all bowel biopsies. Nested PCR targeting the 18S rRNA gene was performed by restriction endonuclease digestion of the PCR product followed by nucleotide sequencing and phylogenetic analysis. A total of three patients with chronic diarrhoea were positive for Microsporidia and Cryptosporidium spp. Species analysis showed the presence of C. parvum and E. bieneusi in two and one samples, respectively. This is the first PCR confirmation of the presence of E. bieneusi and C. parvum in a bowel biopsy of immunocompetent individuals in Iran. This study revealed that PCR, sequencing, and phylogenetic analysis are very powerful tools for the precise species identification of these pathogens.
微孢子虫和隐孢子虫是肠炎的主要病原体，能够在世界各地的儿童、免疫功能正常者和免疫功能低下者中引起严重的慢性腹泻。仅通过显微镜检查无法在种水平上鉴定这些寄生虫。本研究的目的是通过巢式PCR-RFLP、测序和系统发育分析，鉴定和表征免疫功能正常的胃肠道功能紊乱患者中的微孢子虫和隐孢子虫种类。对110例患者的十二指肠、空肠、回肠和盲肠的新鲜冷冻和新鲜石蜡包埋活检标本进行了检查。从所有肠道活检标本中提取基因组DNA。通过对PCR产物进行限制性内切酶消化，然后进行核苷酸测序和系统发育分析，对18S rRNA基因进行巢式PCR。共有3例慢性腹泻患者的微孢子虫和隐孢子虫属检测呈阳性。种类分析显示，分别在2份和1份样本中存在微小隐孢子虫和贝氏隐孢子虫。这是伊朗首次通过PCR证实免疫功能正常个体的肠道活检中存在贝氏隐孢子虫和微小隐孢子虫。本研究表明，PCR、测序和系统发育分析是精确鉴定这些病原体种类的非常强大的工具。
