IN VITRO CULTURE AND SUSCEPTIBILITY TESTING OF MICROSPORIDIA

在过去的几年里，已经发表了相当数量的病例报告，描述了获得性免疫缺陷综合征患者微孢子虫感染的主要胃肠道、泌尿生殖和眼部表现。尽管文献中已经出现了一些抗菌剂治疗效果的轶事证据，但很少或根本没有体外数据来证实这些说法。在作为人类病原体的微孢子虫的主要种类中，除贝努斯肠殖子(E.bienusi)外，所有的微孢子虫都已在细胞培养中繁殖。我们继续寻找促进微孢子虫体外繁殖的方法。目前的细胞培养系统效率低下。在我们的实验中，我们评估了一些细胞系和培养条件以优化微孢子虫的生长。将继续努力寻找一种细胞培养系统来繁殖E.bienusi。一旦这些目标实现，我们打算筛选潜在有用的抗菌剂，以确定哪些(如果有的话)对微孢子虫复制有有害影响，以及这种抑制是否具有物种特异性。还将检查化合物防止细胞培养单层感染微孢子虫的能力。将使用各种实验室方法来评估抗微孢子虫剂的有效性。从已建立的培养物中释放的孢子的定量将提供有关药剂对微孢子虫复制和孢子产生的影响的信息。Nash博士生产的一种单抗(NIAID，LPD)将用于对感染细胞进行染色和计数，以确定抗菌剂对感染的抑制作用。我们已经开发出一种可靠的定量方法，它使用聚合酶链式反应和Eu标记的DNA探针。微孢子虫DNA的定量将提供有关试剂防止微孢子虫在宿主细胞内复制的能力以及试剂防止细胞宿主细胞感染的能力的信息。

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.