Abstract: Objective To understand the prevalence of Cryptosporidium infection in diarrhea infants under 2 years old in Wuhan City， so as to provide the epidemiological evidence for the prevention and treatment of cryptosporidiosis. Methods The fecal samples from infants under 2 years old with diarrhea were collected in Hubei General Hospital and Central South Hospital in Wuhan City， Hubei Province from August 2014 to July 2015. The fecal samples were stored in 2.5% potassium dichromate at 4 ℃ after filtered. The DNA was extracted from the fecal pellets with the phenol?chloroform method. The Cryptosporidium species were detected by a nested PCR assay targeting the SSU rRNA gene of the parasite. All the positive PCR products were sequenced on ABI 3100 automated sequencer， and the amplified sequences were compared to homologous sequences in the NCBI database by using the Basic Local Alignment Search Tool （BLAST）. Phylogenetic analyses were performed by using the software MEGA （version 4.0） based on the Neighbour?Joining method. Results The human stool specimens （n = 298） were screened for the presence of Cryptosporidium by nested PCR. The infection rate of Cryptosporidium was 3.02% （9/298）. The infection rate of Cryptosporidium was 5.93% （7/118）in the infants between 1-2 years old， and the infection rate was 1.11% （2/180） in the infants under 1 year old， and there was a significant difference between the two groups （[χ2]= 4.13，P < 0.05）. The nine samples which were positive by nested PCR were successfully sequenced and compared with the reference sequences in GenBank. The results revealed the nine positive specimens were all infected with C. parvum， and two of them were co?infected with C. hominis. Neighbor?joining trees were constructed from the aligned partial SSU rRNA sequences of these nine isolates， and in the SSU rRNA locus， the nine isolates were grouped with C. parvum. Conclusion There exists Cryptosporidium infection in the infants under 2 years old with diarrhea in Wuhan City， and the main species of Cryptosporidium is C. parvum.

Key words: Cryptosporidium parvum； C. hominis； Infant； Diarrhea； Nested PCR； Molecular epidemiology； Wuhan City

摘要： 目的 了解武汉市2岁以下婴幼儿腹泻患者的隐孢子虫感染状况，为预防隐孢子虫感染提供分子流行病学依据。方法 采集2014年8月—2015年7月在湖北省人民医院和武汉大学中南医院就诊的2岁以下婴幼儿腹泻患者粪便，过滤沉淀后于2.5%重铬酸钾溶液中4 ℃保存；采用酚?氯仿法提取基因组DNA、18S rRNA巢式PCR（Nested?PCR）扩增技术检测隐孢子虫感染，并对阳性PCR产物测序；所获序列进行Blastn比对，应用MEGA软件采用邻接法构建系统进化树进行虫种鉴定。结果 共收集到298份婴幼儿腹泻患者的粪便样本。经巢式PCR扩增技术检测，298份样品中有9份扩增出隐孢子虫阳性条带，阳性率为3.02%（9/298），其中1～2岁腹泻幼儿阳性率为5.93%（7/118），< 1岁患儿的阳性率为1.11%（2/180），差异有统计学意义（[χ2]= 4.13，P < 0.05）。9份阳性PCR产物经测序后与GenBank中的参照序列进行比对，结果显示7份样本为微小隐孢子虫感染，2份样本为微小隐孢子虫与人隐孢子虫混合感染。绘制种系发育进化树发现，9条SSU rRNA基因序列与已发表的微小隐孢子虫（Cryptosporidium parvum）位于同一个进化支上。结论 武汉地区婴幼儿腹泻患者中存在隐孢子虫感染，主要感染虫种为微小隐孢子虫。

关键词: 微小隐孢子虫；人隐孢子虫；婴幼儿；腹泻；巢式PCR ；分子流行病学；武汉市