Valdez L M, Dang H, Okhuysen P C, Chappell C L
Center for Infectious Diseases, School of Public Health, The University of Texas Houston Health Science Center, 77030, USA.
J Clin Microbiol. 1997 Aug;35(8):2013-7. doi: 10.1128/jcm.35.8.2013-2017.1997.
Cryptosporidium parvum is an important pathogen that causes diarrhea in virtually all human populations. Improved diagnostic methods are needed to understand the risk factors, modes of transmission, and impact of cryptosporidiosis. In the present study, we fluorescently labeled and counted C. parvum oocysts by flow cytometry (FC) and developed a simple and efficient method of processing human stool samples for FC analysis. Formed stool (suspended in phosphate-buffered saline) from an asymptomatic, healthy individual was seeded with known concentrations of oocysts, and oocysts were labeled with a cell wall-specific monoclonal antibody and detected by FC. The method described herein resulted in a mean oocyst recovery rate of 45% +/- 16% (median, 42%), which consistently yielded a fourfold increase in sensitivity compared to direct fluorescent-antibody assay of seeded stool samples. However, in many instances, FC detected as few as 10(3) oocysts per ml. Thus, FC provides a reproducible and sensitive method for C. parvum oocyst detection.
微小隐孢子虫是一种重要的病原体，几乎可导致所有人群腹泻。需要改进诊断方法以了解隐孢子虫病的危险因素、传播方式及其影响。在本研究中，我们通过流式细胞术（FC）对微小隐孢子虫卵囊进行荧光标记和计数，并开发了一种简单高效的处理人类粪便样本以进行FC分析的方法。将已知浓度的卵囊接种到一名无症状健康个体的成形粪便（悬浮于磷酸盐缓冲盐溶液中）中，并用细胞壁特异性单克隆抗体标记卵囊，然后通过FC进行检测。本文所述方法的卵囊平均回收率为45%±16%（中位数为42%），与接种粪便样本的直接荧光抗体检测相比，灵敏度始终提高了四倍。然而，在许多情况下，FC每毫升能检测到低至10³个卵囊。因此，FC为微小隐孢子虫卵囊检测提供了一种可重复且灵敏的方法。
