Barbosa Joana M M, Costa-de-Oliveira Sofia, Rodrigues Acácio G, Hanscheid Thomas, Shapiro Howard, Pina-Vaz Cidália
Department of Microbiology, School of Medicine, Porto, Portugal.
Cytometry A. 2008 Jan;73(1):44-7. doi: 10.1002/cyto.a.20502.
Cryptosporidium parvum is transmitted through water and can cause severe diarrhea. The diagnosis is usually based upon observer-dependent microscopic detection of oocysts, with rather low sensitivity and specificity. Our objective was to optimize a flow cytometric (FC) protocol for the detection of C. parvum. A specific monoclonal antibody conjugated with R-phycoerythrin was incubated with dead oocysts to determine the optimal antibody concentration. Serial concentrations of oocysts were stained with the optimized concentration and analyzed by FC. The lower detection limit was determined, and the possibility of cross-reaction was investigated using prokaryotic and eukaryotic microorganisms. A FC protocol was optimized to detect oocysts in spiked human stools. The optimal antibody concentration was found to be 3.0 mug/ml. The lowest number detectable was 2 x 10(3) oocysts/ml. Staining procedure was specific, as no cross-reactions were observed. This reliable and easy FC protocol allow the specific detection of Cryptosporidium oocysts, even at very low concentrations, which is important for public health and further studies of treatment efficacy.
微小隐孢子虫通过水传播，可导致严重腹泻。诊断通常基于依赖观察者的卵囊显微镜检测，其敏感性和特异性相当低。我们的目标是优化一种用于检测微小隐孢子虫的流式细胞术（FC）方案。将与藻红蛋白偶联的特异性单克隆抗体与死亡的卵囊孵育，以确定最佳抗体浓度。用优化后的浓度对系列浓度的卵囊进行染色，并通过流式细胞术进行分析。确定了最低检测限，并使用原核和真核微生物研究了交叉反应的可能性。优化了一种流式细胞术方案以检测加标的人粪便中的卵囊。发现最佳抗体浓度为3.0微克/毫升。最低可检测数量为2×10³个卵囊/毫升。染色程序具有特异性，因为未观察到交叉反应。这种可靠且简便的流式细胞术方案能够特异性检测微小隐孢子虫卵囊，即使在浓度非常低的情况下也是如此，这对于公共卫生和治疗效果的进一步研究很重要。
