Rapid diagnosis of onchocerciasis using urinary biomarkers

盘尾丝虫病或河盲症是一种影响撒哈拉以南非洲1700万人的寄生虫病。它会导致视觉障碍，有时会导致不可逆的失明，以及皮肤的强烈瘙痒。这种疾病的症状可以通过每年使用伊维菌素治疗来控制，伊维菌素可以杀死皮肤中的幼虫，但成虫不能被消灭，可以存活10年以上。几十年来，该病的诊断一直依赖于幼虫期皮肤剪伤的显微镜检查。这是一个痛苦的过程，不足为奇的是，在多年来反复采样的社区中，依从性降低了。在这个建议中，我们的目标是通过使用患者的尿液样本检测成虫的存在，从根本上改善盘尾丝虫病的诊断。我们已经发现，蠕虫释放蛋白质和小RNA分子到尿液中，可以通过质谱仪和测序技术检测到。这些分子与称为细胞外囊泡的小“包”有关。我们将从尿液样本中分离出这些囊泡，以确定疾病的最佳标记物。为了促进这一进程，我们还将检测非洲牛的尿液样本，这些牛感染了一种与导致人类盘尾丝虫病的寄生虫非常密切相关的寄生虫。通过用杀死成年寄生虫的药物治疗牛，我们将测试尿液中释放的分子是否能很好地指示活的寄生虫是否留在宿主体内。这在目前尤为重要，因为几种对成虫具有潜在活性的药物正在人体临床试验中进行测试，目前唯一知道蠕虫是否被杀死的方法是对志愿者进行手术。在提案的最后阶段，我们将研究从尿液中有效捕获寄生虫分子的方法。从依赖昂贵的实验室设备的诊断测试转向最终可以在非洲农村使用的试剂盒，这将是必要的。

Onchocerciasis or river blindness is a parasitic disease affecting 17 million in sub-Saharan Africa. It causes visual impairment, sometimes leading to irreversible blindness, and intense itching of the skin. The symptoms of the disease can be controlled by annual treatment with the drug ivermectin, which kills the larval stages in the skin, but the adult worms are not eliminated and can survive for more than 10 years.Diagnosis of the disease has been dependent for many decades on microscopic examination of skin snips for the larval stage. This is a painful procedure that, not surprisingly, is being met with reduced compliance in communities that have been sampled repeatedly over many years.In this proposal, we aim to radically improve the diagnosis of onchocerciasis by testing for the presence of adult worms using a patient's urine sample. We have discovered that the worms release proteins and small RNA molecules into urine that can be detected by mass spectrometers and sequencing technology. The molecules are associated with small "packets" called extracellular vesicles. We will isolate these vesicles from urine samples to identify the best markers for the disease. To facilitate the process, we will also test urine samples from African cattle, which are infected with a parasite very closely related to that which causes human onchocerciasis. By treating the cattle with drugs that kill the adult parasites, we will test whether the molecules released in urine are a good indicator of whether live worms remain in the host's body. This is a particularly important at the present time, as several drugs with potential activity against adult worms are being tested in clinical trials in humans, and currently the only way to know whether the worms have been killed is to perform surgery on the volunteers.In the final stages of the proposal, we will investigate methods to capture parasite molecules from urine in an efficient manner. This will be necessary to move away from a diagnostic test that is dependent on expensive laboratory equipment to a kit that ultimately could be used in rural Africa.