Multiplex Diagnostic for Enteric Category B Pathogens and Intestinal Biomarkers

描述(由申请人提供)：我们的目标是开发一种多重免疫诊断小组，专门用于检测粪便中产生志贺氏菌和大肠杆菌的贾第鞭毛虫、隐孢子虫、溶组织肠杆菌和志贺氏菌毒素。额外的努力将量化粪便生物标志物乳铁蛋白和血红蛋白，这是肠道炎症和出血的指标。全面的多重专家小组将提供对腹泻/痢疾的主要B类肠道病原体的快速诊断，并描述疾病的严重程度。该检测采用了敏感和特异的基于Luminex的免疫荧光技术。Luminex允许使用一组唯一可检测的珠组对单个样品中的分析物进行多路检测。分析物特异性抗体是从现有的FDA批准的免疫分析中挑选出来的，为其性能和稳定性提供了保证。初步数据表明，Luminex对肠道原虫的敏感性超过了ELISA法，与RT-PCR相当，具有纯化的寄生虫曲线。粪便检测确定了108例寄生虫FDA清除的ELISA阳性反应中的108例，但仍需完全验证临床敏感性。Luminex对乳铁蛋白和血红蛋白的检测与商业ELISA检测相关，并提供了更大的定量范围，提高了粪便分析的效率。目的#1详细介绍了贾第虫、隐孢子虫和溶组织性肠炎原虫的肠道寄生虫群。目的#2扩大腹泻/痢疾的检测范围，包括志贺毒素、志贺样毒素(STX1)和志贺样毒素2(STX2)的检测。目的#3增加乳铁蛋白和血红蛋白的检测作为腹泻/痢疾严重程度和患者病情的生物标志物。这些研究的成功完成将提供一个原型诊断，能够同时检测B类肠道病原体和描述感染严重程度的肠道生物标记物。多重检测将在第二阶段NIH小企业资金完成制造转移和FDA 510(K)许可验证方面具有竞争力。基于Luminex的多重检测是一个很好的商业化候选者，它允许未来包括其他肠道病原体和肠道功能生物标记物。 公共卫生相关性：本研究提出了一个诊断小组，以鉴定腹泻和痢疾患者的B类生物防御性肠道病原体：贾第虫、隐孢子虫、溶组织志贺菌和志贺氏菌毒素产生的志贺氏菌和大肠杆菌。该小组还将量化粪便生物标记物，以描述肠道炎症和出血，提供有关疾病严重程度和患者状况的新信息。

DESCRIPTION (provided by applicant): Our goal is development of a multiplex immunodiagnostic panel specific for Giardia, Cryptosporidium, E. histolytica, and shiga toxin-producing Shigella and E. coli detection in stool. Additional effort will quantify the fecal biomarkers lactoferrin and hemoglobin, indicators of intestinal inflammation and hemorrhage. The full multiplex panel will provide rapid diagnosis of major Category B enteropathogenic agents of diarrhea/dysentery and describe severity of disease. The assay utilizes sensitive and specific immunofluorescence-based Luminex technology. The Luminex allows multiplexed detection of analytes in a single sample using an array of uniquely detectable bead sets. Analyte-specific antibodies are selected from existing FDA-cleared immunoassays, providing assurance of their performance and stability. Preliminary data indicate Luminex sensitivity for the enteric protozoa exceeds that of ELISA, and is comparable to RT-PCR, with purified parasite curves. Detection in stool identified 108 of 108 parasite FDA-cleared ELISA positive reactions, but still requires complete validation of clinical sensitivity. Luminex detection of lactoferrin and hemoglobin correlate with commercial ELISA detection and provide an expanded range of quantification, increasing efficiency of fecal analysis over ELISA. Aim #1 details an enteric protozoan parasite panel for Giardia, Cryptosporidium, and E. histolytica. Aim #2 expands this diarrhea/dysentery panel to include detection of shiga toxin, shiga-like toxin (Stx1) and shiga-like toxin 2 (Stx2). Aim #3 adds detection of lactoferrin and hemoglobin as biomarkers of diarrhea/dysentery severity and patient condition. Successful completion of these studies will provide a prototype diagnostic capable of the simultaneous detection of Category B enteropathogens and intestinal biomarkers that describe severity of infection. The multiplex assay will be competitive for Phase II NIH small business funding to complete manufacturing transfer and FDA 510(k) clearance validations. The Luminex-based multiplex detection is an excellent candidate for commercialization, and it allows for future inclusion of other enteric pathogens and gut function biomarkers. PUBLIC HEALTH RELEVANCE: This study proposes a diagnostic panel to identify the Category B Biodefense intestinal pathogens Giardia, Cryptosporidium, E. histolytica, and shiga toxin-producing Shigella and E. coli in patients with diarrhea and dysentery. The panel will also quantify fecal biomarkers to describe intestinal inflammation and hemorrhage, providing novel information regarding disease severity and patient condition.