SBIR Phase II: Recombinant Multi-epitope Mosaic Protein Design for Urine-based Diagnosis of Leptospirosis

该小型企业创新研究（SBIR）第二阶段项目旨在开发和验证一种非侵入性的多重尿液试纸，用于临床诊断由多种微生物感染引起的急性发热性疾病（AFI）。目标疾病包括钩端螺旋体病、伤寒、登革热以及恶性疟原虫和间日疟原虫疟疾，这些疾病每年在全世界造成约2.29亿例AFI和80万例死亡。目前的诊断工具是侵入性的，技术复杂，因此不适合这些疾病最流行的热带和亚热带国家，特别是在血液检测不切实际的农村和护理点环境中。 I期项目成功开发并验证了病原体特异性重组抗体试剂，证明了尿液诊断潜力。该II期项目设想了两个广泛的目标：（i）重组单克隆抗体试剂的详细表征，以验证其个体和集体诊断效用;以及（ii）开发和验证多重试纸检测装置，用于同时检测单个尿液样本中的一组急性发热的主要原因。交付成果将是一种简单的诊断设备，具有诊断恶性疟原虫、间日疟原虫和钩端螺旋体之一/两者的潜力，它结合了改善AFI特异性诊断的需要和解决急性发热替代原因的需要。将提供一种快速诊断工具，以应对资源匮乏、全球健康疾病负担沉重的人们所面临的一项重大挑战，即如何在没有快速鉴别诊断测试的情况下管理急性发热。虽然对每个潜在的发热原因进行单独的测试是有益的，但在这些环境中快速选择，运行和解释多个单独的测试通常是不可行或不切实际的。这种单独测试的成本影响也将是令人望而却步的。因此，迫切需要一种如本文所设想的多重快速装置来评价与病理学相关的发热性疾病。它将提供医疗和后勤两方面的好处，在一个单一的尿液样本中快速检测急性发热性疾病的主要原因。它将提供具有更广泛影响的单一答案多疾病诊断，并通过多路检测而不是单独检测提供鉴别诊断，最适合诊断实验室能力有限的资源有限环境。这种多重尿液试纸检测被设计为基础平台设备，可以反复添加其他急性发热诊断试剂以适应特定区域。

This Small Business Innovation Research (SBIR) Phase II project proposes to develop and validate a non-invasive, multiplex urine dipstick for clinical diagnosis of Acute Febrile Illness (AFI) caused by polymicrobial infections. The target diseases include leptospirosis, typhoid fever, dengue, as well as Plasmodium falciparum and P. vivax malaria, responsible for ~229M AFI's and 800,000 deaths a year worldwide. Current diagnostic tools are invasive, technically complex, and thus not suitable in tropical and subtropical countries where these diseases are most prevalent, especially in rural and point-of-care settings where blood testing is impractical. The Phase I project successfully developed and validated pathogen-specific recombinant antibody reagents that demonstrated diagnostic potential in urine. This Phase II project envisages two broad objectives: (i) detailed characterization of the recombinant monoclonal antibody reagents to validate their individual and collective diagnostic utility; and (ii) to develop and validate a multiplex dipstick test device for concurrent detection of a set of leading causes of acute fever in a single urine sample. The deliverable will be a simple diagnostic device with the potential to diagnose either/both Plasmodium falciparum, Plasmodium vivax and Leptospira, which combines the need to improve specific diagnosis of AFI with the need to address alternative causes of acute fever.The broader impact/commercial potential of this project, if successful, will be the delivery of a rapid diagnostic tool that addresses a major challenge faced by people in low resource settings with high burdens of global health diseases, i.e. how to manage acute fever without the benefit of rapid differential diagnostic tests. Although individual tests for each potential cause of fever would be beneficial, it is often not feasible or practical to rapidly select, run, and interpret multiple individual tests in these settings. The cost implications for such individual testing also will be prohibitive. Therefore, a multiplex rapid device as envisaged here that will evaluate groups of symptomatically related febrile diseases is urgently needed. It will offer both medical and logistical benefits for rapid testing of leading causes of acute febrile illness in a single urine sample. It will deliver a single answer multi-disease diagnostic with broader impact, and offer differential diagnosis through a multiplexed assay rather than individual testing, most suitable in resource limited settings where diagnostic laboratory capacities are limited. This multiplex urine dipstick test is designed to be a foundational platform device to which other diagnostic reagents for acute fever can be iteratively added to suit a particular region.