In Vitro Culture and Susceptibility Testing of Microsporidia

在过去的几年里，有相当多的案件 主要描述胃肠道、泌尿生殖道和 获得性微孢子虫感染的眼部表现 免疫缺陷综合征患者的资料。 虽然有传闻表明， 文献中出现了许多抗微生物剂， 很少或没有体外数据来证实这些声明。的 微孢子虫是人类病原体的主要种类， 比氏肠胞虫（Enterocytozoonbienusi）; bienusi）已经在细胞中繁殖 文化。我们继续寻找方法，以加强传播 微孢子虫体外培养。目前的细胞培养系统是 效率低下。我们已经评估了一些细胞系和文化 在我们的测定中优化微孢子虫生长的条件。努力 将继续寻找一种细胞培养系统，用于E. 谢谢一旦实现这些目标，我们打算筛选 潜在有用的抗微生物剂，如果有的话， 对微孢子虫的复制有有害影响，如果这 抑制是物种特异性的。还将检查化合物的 它们通过以下途径防止细胞培养单层感染的能力 微孢子虫。各种实验室方法将用于评估 抗微孢子虫药物的有效性。定量 从已建立的培养物中释放出的孢子 关于药剂对微孢子虫复制的影响， 孢子生产纳什博士生产的单克隆抗体 （NIAID，LPD）将用于染色和计数感染细胞， 确定抗菌剂对感染的抑制作用。我们有 开发了一种可靠的定量分析方法， 链反应和铕标记的DNA探针。定量 微孢子虫DNA将提供关于微孢子虫的能力的信息。 防止微孢子虫在宿主细胞内复制的试剂， 试剂防止细胞宿主细胞感染的能力。

In the past few years, a considerable number of case reports describing predominantly gastrointestinal, urinogenital, and ophthalmic manifestations of microsporidial infections in acquired immune deficiency syndrome patients have been published. Although anecdotal evidence of the therapeutic efficacy of a number of antimicrobial agents has appeared in the literature, there has been little or no in vitro data to substantiate these claims. Of the principal species of microsporidia that are human pathogens, all but Enterocytozoon bienusi (E. bienusi) have been propagated in cell culture.We continue to look for ways to enhance the propagation of microsporidia in vitro. The current cell culture systems are inefficient. We have evaluated a number of cell lines and culture conditions to optimize growth of microsporidia in our assay. Efforts will continue to find a cell culture system for the propagation of E. bienusi. Once these objectives are achieved, we intend to screen potentially useful antimicrobial agents to determine which, if any, have deleterious effects on microsporidial replication, and if this inhibition is species specific. Compounds will also be examined for their ability to prevent infection of cell culture monolayers by microsporidia.Various laboratory methods will be used to evaluate the effectiveness of antimicrosporidial agents. Quantitation of released spores from established cultures will provide information concerning the effect of agents on microsporidial replication and spore production. A monoclonal antibody produced by Dr. Nash (NIAID, LPD) will be used to stain and count infected cells to determine inhibition of infection by antimicrobial agents. We have developed a reliable assay for quantitation that uses polymerase chain reaction and a europium-labeled DNA probe. Quantitation of microsporidial DNA will provide information about the ability of agents to prevent microsporidial replication within the host cell and the ability of agents to prevent infection of cell host cells.