Cryogenic purification of Plasmodium parasites from blood

项目总结 疟疾的感染频率和死亡人数令人震惊，超过2亿例。 每年，有近100万人死亡，其中大部分是儿童。虽然疟疾的症状可能会相当 严重的、无症状的疟疾病例约占感染的50%-90%。无症状疟疾可持续 几个月，并有助于疾病的传播，因为这些人仍然对蚊子有感染力 向量。这些病例还使根除工作复杂化，因为在血液中循环的寄生虫数量 经常低于血液涂片的检测极限，这是疟疾诊断的黄金标准。这里, 我们的目标是开发一种从血液中冷冻浓缩寄生虫的方法，以提高对 显微镜观察，并能够识别无症状个体中的寄生虫。 低温生物学中的一个混杂因素是，每个独特的细胞系都需要自己独特的冷冻保存 协议。这对于非均质细胞和组织的冷冻保存来说尤其麻烦。这里, 我们的目标是利用低温生物学的这一原理来选择性地冷冻保存 疟疾寄生虫，同时破坏未感染的血细胞，从而实现“低温浓缩”。 加强对低寄生虫血症感染的显微镜检测。在整个提案中，重点是 采用低成本、简单和易于在现有基础设施中实施的方法，以确保 顺利过渡到护理点。 在具体目标1中，我们将优化低温浓缩方案，以提高形貌、回收率 和疟疾寄生虫的纯度。这将通过量化一系列 在纯化的血细胞(即未感染的红细胞、白细胞和疟疾寄生虫)中加入冷冻保护剂以识别 在寄生虫中出现最佳通透性(从而在超低温保存期间保护细胞)的条件， 但不适用于其他血细胞。在具体目标2中，我们将确定低温浓缩的可行性 现场实施的依据是：i)冷冻室的冷却要求；ii)可伸缩性和敏感性；iii) 与其他疟疾诊断方法的兼容性。我们的最终目标是开发一种简化的和 可持续低温浓缩方案，以提高显微镜在疟疾诊断中的灵敏度 世界各地的实验室和诊所都可以方便地访问。

PROJECT SUMMARY The frequency of infection and the number of deaths due to malaria are staggering with over 200 million cases each year, and nearly one million deaths, mostly among children. While the symptoms of malaria can be quite severe, asymptomatic malaria cases account for ~50-90% of infections. Asymptomatic malaria can last for months, and contributes to propagation of disease as these individuals remain infectious to the mosquito vector. These cases also complicate eradication efforts as the number of parasites circulating in the blood frequently falls below the limit of detection of blood smears, the gold standard for malaria diagnostics. Here, our goal is to develop a method to cryogenically enrich parasites from blood to enhance the sensitivity of microscopy and enable identification of parasites among asymptomatic individuals. One confounding factor in cryobiology is that each unique cell line requires its own unique cryopreservation protocol. This is particularly troublesome for the cryopreservation of non-homogenous cells and tissues. Here, our goal is to exploit this principle of low temperature biology in order to selectively cryopreserve malaria parasites while destroying uninfected blood cells, thereby achieving a `cryogenic enrichment' to enhance microscopic detection of low-parasitemia infections. Throughout this proposal, emphasis is given to methods that are low-cost, simple and easy to implement into the existing infrastructure to ensure smooth translation to point-of-care. In Specific Aim 1, we will optimize the cryogenic enrichment protocol to improve the morphology, recovery and purity of malaria parasites. This will be achieved by quantifying the transport properties of a range of cryoprotectants in purified blood cells (i.e. uninfected erythrocytes, leukocytes and malaria parasites) to identify conditions where optimal permeability (and hence cell protection during cryopreservation) occurs in parasites, but not for other blood cells. In Specific Aim 2, we will determine the feasibility of cryogenic enrichment for field implementation based upon i) cooling requirements for freezing cells, ii) scalability and sensitivity and iii) compatibility with alternative malaria diagnostic methods. Our ultimate goal is to develop a simplified and sustainable cryogenic enrichment protocol to enhance the sensitivity of microscopy for malaria diagnosis in a manner that is readily accessible to labs and clinics worldwide.