Gatei Wangeci, Greensill Julie, Ashford Richard W, Cuevas Luis E, Parry Christopher M, Cunliffe Nigel A, Beeching Nicholas J, Hart C Anthony
Liverpool School of Tropical Medicine, University of Liverpool, Liverpool, United Kingdom.
J Clin Microbiol. 2003 Apr;41(4):1458-62. doi: 10.1128/JCM.41.4.1458-1462.2003.
An 840-bp fragment of the 18S rRNA gene was used to identify Cryptosporidium spp. recovered from human immunodeficiency virus (HIV)-infected and -uninfected patients from Kenya, Malawi, Brazil, the United Kingdom, and Vietnam. Initial identification was by Ziehl-Neelsen acid-fast staining. Confirmation was by nested PCR, targeting the most polymorphic region of the 18S rRNA gene. Genotyping was by restriction endonuclease digestion of the PCR product followed by nucleotide sequencing. Among 63 isolates analyzed, four genotypes of Cryptosporidium were identified; 75% of the isolates were of the C. parvum human genotype, while the potentially zoonotic species were of the C. parvum bovine genotype (21.7%), the C. meleagridis genotype (1.6% [one isolate]), and the C. muris genotype (1.6% [one case]). HIV-infected individuals were more likely to have zoonotic genotypes than the HIV-uninfected individuals. Among the C. parvum group, strains clustered distinctly into either human or bovine genotypes regardless of the geographical origin, age, or HIV status of the patients. The intragenotypic variation observed in the C. parvum human genotype was extensive compared to that within the C. parvum bovine genotype group. The variation within genotypes was conserved in all geographical regions regardless of the patients' HIV status. The extensive diversity within genotypes at the 18S rRNA gene locus may limit its application to phylogenetic analyses.
利用18S rRNA基因的一个840碱基对片段,对从肯尼亚、马拉维、巴西、英国和越南的人类免疫缺陷病毒(HIV)感染及未感染患者中分离出的隐孢子虫属进行鉴定。初步鉴定采用齐-尼抗酸染色法。通过巢式PCR进行确认,该方法针对18S rRNA基因的最具多态性区域。基因分型通过对PCR产物进行限制性内切酶消化,然后进行核苷酸测序。在分析的63株分离株中,鉴定出4种隐孢子虫基因型;75%的分离株为微小隐孢子虫人基因型,而潜在的人畜共患病种为微小隐孢子虫牛基因型(21.7%)、火鸡隐孢子虫基因型(1.6%[一株分离株])和鼠隐孢子虫基因型(1.6%[一例病例])。与未感染HIV的个体相比,感染HIV的个体更有可能感染人畜共患病基因型。在微小隐孢子虫组中,无论患者的地理来源、年龄或HIV状态如何,菌株明显聚类为人或牛基因型。与微小隐孢子虫牛基因型组相比,微小隐孢子虫人基因型中观察到的基因型内变异更为广泛。无论患者的HIV状态如何,基因型内的变异在所有地理区域均保持一致。18S rRNA基因位点基因型内的广泛多样性可能会限制其在系统发育分析中的应用。