IN VITRO CULTURE AND SUSCEPTIBILITY TESTING OF MICROSPORIDIA

在过去的几年中，已经发表了相当多的病例报告，主要描述了获得性免疫缺陷综合征患者微孢子虫感染的胃肠道、泌尿生殖和眼部表现。虽然文献中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。在人类病原体微孢子虫的主要种类中，除了bienusenterocytozoon （E. bienusi）外，所有的微孢子虫都在细胞培养中繁殖。我们将继续寻找增强微孢子虫体外繁殖的方法。目前的细胞培养系统效率低下。我们已经评估了许多细胞系和培养条件，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的繁殖。一旦达到这些目标，我们打算筛选潜在有用的抗菌药物，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。化合物还将检查其防止细胞培养单层被微孢子虫感染的能力。将使用各种实验室方法来评估抗孢剂的有效性。从已建立的培养中释放孢子的定量将提供有关药剂对微孢子复制和孢子产生影响的信息。Nash博士（NIAID， LPD）生产的单克隆抗体将用于染色和计数感染细胞，以确定抗菌药物对感染的抑制作用。我们已经开发了一种可靠的定量分析，使用聚合酶链反应和铕标记的DNA探针。微孢子DNA的定量将提供有关药物阻止宿主细胞内微孢子复制的能力和药物阻止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.