Multiplex Screening ELISA for Giardia, Cryptosporidium and E. histolytica

描述（由申请人提供）：我们的快速STTR应用的具体目标是NIAID生物防御类别B肠道病原体贾第鞭毛虫，隐孢子虫属，和溶组织内阿米巴。这些肠道原生动物具有食物和水传播、感染剂量低和环境稳定的特点。E.溶组织菌感染可能是致命的，G.蓝氏隐孢子虫最常见，而隐孢子虫属（Cryptosporidium spp.）艾滋病患者慢性腹泻的一个重要原因是得不到有效治疗。目前缺乏低成本的肠道寄生虫粪便抗原筛查试验，这损害了腹泻患者的医疗保健，因为目前唯一可用的筛查试验是粪便O&P检查;这是一种众所周知的劳动密集型，非特异性和不敏感的程序。我们提出的诊断筛查（“Tri-Combo”ELISA）将通过从所有3种寄生虫均为阴性的进一步检测标本中消除，为临床实验室节省成本。Tri-Combo ELISA是一种常规的两步ELISA形式，使用HRP偶联的检测抗体进行比色显色。独特的多重性质的测试来与它的能力，同时筛选粪便标本的G。蓝氏隐孢子虫属（Cryptosporidium spp.）和E.使用单个测定孔进行溶组织试验。Tri-Combo ELISA可以使用结果的视觉解释进行，用于现场和发展中国家。与此同时，它能够转换为自动化平台，用于参考实验室和其他大规模监测方案;这将有助于军事和社区水源疫情。Tri-Combo ELISA的初步测试表明，与FDA批准的单个G.兰氏藻属，隐孢子虫属，和E.粪便中溶组织杆菌，通过RT-PCR分离。在这里，我们建议在研发实验室优化Tri-Combo ELISA，然后进行良好生产规范（GMP）验证和FDA产品批准。第一阶段将优化Tri-Combo ELISA，完成GMP程序，并准备临床研究中心进行评估。第二阶段将完成Tri-Combo ELISA的生产验证，在我们的联盟地点建立性能特征，并准备FDA 510（k）上市前许可申请文件。Tri-Combo ELISA的创新在于其能够以多重形式检测美国三种最常见的肠道原生动物寄生虫，并且其开发过程允许三种检测系统以单一测试形式组合在一起。在未来，该过程将用于开发其他多重病原体检测系统。 在美国，需要一种筛选试验来检测三种最常见的肠道原生动物寄生虫（贾第虫，隐孢子虫属，和溶组织内阿米巴）。它们存在于提交给参考实验室的所有粪便样本中的10%，它们是B类优先生物防御病原体，它们具有食物和水传播、低感染剂量和环境稳定性的特征。我们的目的是诊断性筛查贾第鞭毛虫、隐孢子虫和大肠杆菌。人粪便标本中的溶组织菌。

DESCRIPTION (provided by applicant): Our specific objective with this fast-track STTR application is a diagnostic screen for the NIAID Biodefense Category B intestinal pathogens Giardia lamblia, Cryptosporidium spp., and Entamoeba histolytica in stool. These enteric protozoa share the characteristics of food and water-borne transmission, low infectious dose, and environmental stability. E. histolytica infections may be lethal, G. lamblia is most common, while Cryptosporidium spp. stands out as an important cause of chronic diarrhea without effective treatment in AIDS patients. The current lack of a low-cost screening fecal antigen test for enteric parasites compromises the healthcare of individuals presenting with diarrhea, as the only screening test available today is the stool O&P exam; a notoriously labor-intensive, non-specific and insensitive procedure. Our proposed diagnostic screen (the "Tri-Combo" ELISA) will provide cost-savings for clinical laboratories via the elimination from further testing specimens that are negative for all 3 parasites. The Tri-Combo ELISA is a conventional two-step ELISA format with HRP-conjugated detecting antibodies for colorimetric development. The unique multiplex nature of the test comes with its ability to simultaneously screen stool specimens for G. lamblia, Cryptosporidium spp. and E. histolytica using a single assay well. The Tri-Combo ELISA can be performed using visual interpretation of results for use in the field and in developing countries. At the same time, it is capable of conversion to automated platforms for use in reference laboratories and other large-scale surveillance scenarios; this will be useful with military and community water source outbreaks. Preliminary testing with the Tri-Combo ELISA indicates a >97% correlation to individual FDA-cleared ELISAs specific for G. lamblia, Cryptosporidium spp., and E. histolytica in stool with resolution by RT-PCR. Here, we propose optimization of the Tri-Combo ELISA in the R&D laboratory, followed by Good Manufacturing Practice (GMP) validation and FDA product clearance. Phase I will optimize the Tri-Combo ELISA, finalize GMP procedures, and prepare the clinical sites for evaluation. Phase II will complete the Tri-Combo ELISA manufacturing validation, establish performance characteristics at our consortium sites, and prepare FDA 510(k) pre-market clearance application documents. Innovation of the Tri-Combo ELISA lies with its ability to detect the three most common enteric protozoan parasites in the United States in a multiplexed format, and with its development process that allows three detection systems to be combined together in a single testing format. In the future, the process will be utilized to develop other multiplexed pathogen detection systems. A screening test is needed to detect the three most common intestinal protozoan parasites in the US (Giardia lamblia, Cryptosporidium spp., and Entamoeba histolytica). They are present in up to 10% of all stool samples submitted to reference laboratories, they are Category B Priority Biodefense Pathogens, and they share the characteristics of food and water-borne transmission, a low infectious dose, and environmental stability. Our objective is a diagnostic screen for the Giardia, Cryptosporidium, and E. histolytica in human stool specimens.