Enhancing detection and mobile monitoring of schistosomiasis with urine-based analyte pre-concentration technology

摘要 血吸虫病是一种使人衰弱的寄生虫病，每年感染2.3亿多人，造成28万人死亡。 对全球疾病负担的新估计将血吸虫病列为第三大最具影响力的传染病，仅次于 艾滋病毒/艾滋病和疟疾。因此，在过去十年中，全球卫生组织重新对 控制和消灭血吸虫病。减少与自闭症相关的发病率的努力主要涉及大规模的 药物给药（MDA）和阻断传播的措施（例如，通过改善获得安全饮用水和蜗牛 （载体）对照）。这两种疾病控制策略的关键是检测感染个体的能力;然而， 高度流行的地区往往缺乏基本的实验室基础设施，因此必须在实地或 床旁检测（POC），以满足大量需要检测的研究中心和个人的需求。 目前的疾病监测在很大程度上依赖于显微镜技术来识别/量化零星脱落的卵 粪便或尿液中蠕虫。虽然高度特异性，但显微镜方法费力、耗时、不一致， 并且通常具有低于5%的临床灵敏度。显微镜检查的替代方法包括免疫测定法， 来自人的特异性抗原，例如循环阴极抗原（CCA）或循环阳极抗原（CAA）， 尿POC CCA尿液检测已商业化并用于监测和绘图研究，但不敏感 足以检测低强度感染，并且仅由六种常见血吸虫之一可靠地产生， 这限制了其在某些地区的使用。相反，CAA由所有血吸虫属物种产生，与显微镜不同， 与监测治疗反应的总蠕虫负荷直接相关。因此，预期CAA免疫测定可 显着提高血吸虫病诊断监测，但没有商业上可用的POC-CAA测定存在。 为了满足这一未满足的需求，我们将整合两种互补技术，实现超灵敏的POC现场测试 血吸虫病的尿液样本我们的合作者Paul Corstjens博士是莱顿全球知名的血吸虫病专家 大学医学中心开发了一种超灵敏的上转换磷光体侧流测定（UCP-LFA），用于 CAA检测。该检测方法在几项研究和临床试验中显示出巨大的前景，但仍然需要时间- 为了达到检测最低强度所需的灵敏度， 感染.与此同时，Salus Discovery开发了一种名为FLOW™的新技术， LFA的操作概念，可在检测前将20 mL尿液中的分析物预浓缩至100 µL 一个LFA。最近，Salus和Corstjens博士的小组开发了第一个集成FLOW尿液的设备原型 使用UCP-LFA读数（FLOW-S）进行预浓缩。FLOW-S原型初步评估了一套 30个临床样本，其灵敏度达到79%，特异性达到100%，证明了其作为一种全新的， 血吸虫病的POC友好，超灵敏的基于CAA的检测。在本SBIR第1阶段提案中，我们将基于 通过优化FLOW-S设备，使检测限达到0.1 pg/mL，即使是最低浓度的 强度（即，单蠕虫）感染（目标1），并进行了临床研究与新鲜尿液样本（目标2）。

ABSTRACT Schistosomiasis is a debilitating parasitic disease that infects over 230 million people and causes 280,000 deaths per year. New estimates of global disease burden rank schistosomiasis as the third most impactful infectious disease behind only HIV/AIDS and malaria. Therefore, over the past decade, global health organizations have renewed interest in schistosomiasis control and elimination. Efforts to reduce schistosomiasis-associated morbidities primarily involve mass drug administration (MDA) and measures for interrupting transmission (e.g., via improved access to safe water and snail (vector) control). Critical to both disease control strategies is the ability to detect infected individuals; however, because highly endemic areas often lack basic laboratory infrastructure, it is vital that monitoring be performed in the field or at the point-of-care (POC) to address the vast numbers of sites and individuals needing testing. Current disease monitoring largely depends upon microscopy techniques to identify/quantify eggs that are sporadically shed by adult worms in stool or urine. While highly specific, microscopy methods are laborious, time-consuming, inconsistent, and often have clinical sensitivities under 5%. Alternatives to microscopy include immunoassays that detect schistosome- specific antigens, such as the circulating cathodic antigen (CCA), or the circulating anodic antigen (CAA), from human urine. A POC CCA urine assay has been commercialized and used in surveillance and mapping studies, but is not sensitive enough to detect low-intensity infections, and is only reliably produced by one of the six common Schistosoma species, which limits its use to certain regions. In contrast, CAA is produced by all Schistosoma species and, unlike microscopy, is directly correlated to total worm burden for monitoring therapy response. Therefore, a CAA immunoassay is expected to significantly enhance diagnostic monitoring of schistosomiasis, but no commercially available POC-CAA assay exists. To address this unmet need, we will integrate two complementary technologies that enable ultra-sensitive POC field-testing for schistosomiasis from urine. Our collaborator, Dr. Paul Corstjens, a globally recognized schistosomiasis expert at Leiden University Medical Center, has developed an ultrasensitive Up-Converting Phosphor Lateral Flow Assay (UCP-LFA) for CAA detection. The assay has shown tremendous promise in several studies and clinical trials but still requires time- consuming and resource-intensive sample pre-concentration to reach the sensitivity required to detect the lowest-intensity infections. In parallel, Salus Discovery has developed a new technology, termed FLOW™, that expands upon the operational concepts of LFAs by enabling pre-concentration of analytes from 20 mL of urine into 100 µL prior to detection on an LFA. Recently, Salus and Dr. Corstjens' group developed the first prototype of a device that integrates FLOW urine pre-concentration with a UCP-LFA readout (FLOW-S). The FLOW-S prototype was preliminarily evaluated with a set of 30 clinical samples where it achieved 79% sensitivity and 100% specificity, demonstrating its use as a fundamentally new, POC-friendly, ultra-sensitive CAA-based assay for schistosomiasis. In this SBIR Phase 1 proposal we will build upon our success by optimizing the FLOW-S device to achieve a detection limit of 0.1 pg/mL, enabling detection of even the lowest intensity (i.e., single-worm) infections (Aim 1), and performing a clinical study with fresh urine samples (Aim 2).