Multiplex Screening ELISA for Giardia, Cryptosporidium and E. histolytica

描述(由申请人提供)：我们的快速跟踪STTR应用程序的具体目标是对NIAID生物防御B类肠道病原体进行诊断筛查，包括粪便中的蓝氏贾第鞭毛虫、隐孢子虫和溶组织内阿米巴。这些肠道原虫具有食物和水传播、低感染剂量和环境稳定的特点。溶组织性肠杆菌感染可能是致死性的，兰氏革兰氏菌是最常见的，而隐孢子虫属。突出地成为艾滋病患者慢性腹泻得不到有效治疗的重要原因。目前缺乏一种针对肠道寄生虫的低成本粪便抗原筛查测试，这损害了出现腹泻症状的个人的医疗保健，因为目前唯一可用的筛查测试是粪便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。我们建议的诊断筛查(“三联”酶联免疫吸附试验)将为临床实验室提供成本节约，通过消除进一步的测试样本，所有3种寄生虫都是阴性的。Tri-Combo酶联免疫吸附试验是一种传统的两步酶联免疫吸附试验，用于比色显色法检测抗体。这项检测的独特的多重性质是能够同时筛查粪便样本中的革兰氏菌和隐孢子虫。和组织裂解埃希氏菌的单项检测结果良好。可通过目视解释结果进行三组合酶联免疫吸附试验，以供现场和发展中国家使用。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情景；这将对军事和社区水源暴发有用。用Tri-Combo ELISA法进行的初步检测表明，RT-PCR法与粪便中针对革兰氏菌、隐孢子虫和溶组织性肠杆菌的单个FDA清除的ELISA有97%的相关性。在这里，我们建议在研发实验室中优化三组合酶联免疫吸附试验，然后进行良好制造规范(GMP)验证和FDA产品批准。第一阶段将优化Tri-Combo ELISA法，最终确定GMP程序，并准备临床站点进行评估。第二阶段将完成Tri-Combo ELISA制造验证，在我们的联合体站点建立性能特征，并准备FDA 510(K)上市前许可申请文件。Tri-Combo ELISA的创新之处在于它能够以多路复用的形式检测美国最常见的三种肠道原虫寄生虫，以及它的开发过程，允许三个检测系统在单一检测格式中组合在一起。未来，该技术将被用于开发其他多路病原体检测系统。 需要进行筛查试验来检测美国最常见的三种肠道原虫寄生虫(蓝氏贾第鞭毛虫、隐孢子虫和溶组织内阿米巴)。在提交给参考实验室的所有粪便样本中，它们的存在比例高达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、低感染量和环境稳定的特点。我们的目标是对人类粪便标本中的贾第虫、隐孢子虫和溶组织性肠杆菌进行诊断筛查。

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.