Cryogenic purification of Plasmodium parasites from blood

血液中疟原虫寄生虫的低温纯化

• 批准号: 9973147
• 负责人: Rebecca Sandlin
• 金额: $ 21万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-05 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9973147
• 关键词: Biology; Blood; Blood Cells; Blood Stains; Blood specimen; Caring; Cell Line; Cell Volumes; Cells; Cessation of life; Child; Clinic; Community Surveys; Consumption; Coupled; Cryopreservation; Cryoprotective Agents; Cytoprotection; Data; Detection; Devices; Diagnosis; Diagnostic; Diagnostic Procedure; Diagnostic Sensitivity; Diagnostic tests; Disease; Disease Reservoirs; Engineering; Ensure; Erythrocytes; Exhibits; Freezing; Frequencies; Giemsa stain; Goals; Gold; Individual; Infection; Infrastructure; Laboratories; Lead; Leukocytes; Liquid substance; Malaria; Methods; Microscopic; Microscopy; Morphology; Nitrogen; Parasitemia; Parasites; Patients; Permeability; Plasmodium; Property; Protocols documentation; Public Health; Readiness; Recovery; Resources; Stains; Symptoms; Time; Tissues; Translating; Translations; Validation; Whole Blood; base; cold temperature; cost; cryobiology; cryogenics; density; disease transmission; falls; improved; infection rate; malaria infection; malaria transmission; point of care; preservation; rapid diagnosis; remote location; vector mosquito

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.

项目摘要 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，其中大多数是儿童。虽然疟疾的症状可能相当 严重的无症状疟疾病例占感染病例的约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具有传染性 vector.这些病例也使根除工作复杂化，因为血液中循环的寄生虫数量 血涂片是疟疾诊断的黄金标准。在这里， 我们的目标是开发一种从血液中低温富集寄生虫的方法， 显微镜检查，并使无症状个体中的寄生虫识别。 冷冻生物学中的一个混淆因素是，每个独特的细胞系都需要其独特的冷冻保存 议定书这对于非同质细胞和组织的冷冻保存特别麻烦。在这里， 我们的目标是利用低温生物学的原理， 疟疾寄生虫，同时破坏未受感染的血细胞，从而实现“低温浓缩” 以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 考虑到低成本、简单和易于在现有基础设施中实施的方法， 顺利地转移到护理点。 在具体目标1中，我们将优化低温浓缩方案， 和疟疾寄生虫的纯度。这将通过量化一系列的传输特性来实现， 纯化血细胞（即未感染的红细胞、白细胞和疟疾寄生虫）中的冷冻保护剂， 在寄生虫中出现最佳渗透性（以及因此在冷冻保存期间的细胞保护）的条件， 而不是其他血细胞。在具体目标2中，我们将确定低温浓缩的可行性， 现场实施基于i）冷冻细胞的冷却要求，ii）可扩展性和灵敏度，以及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个简化的， 可持续的低温浓缩协议，以提高疟疾诊断显微镜的灵敏度， 这种方式在世界各地的实验室和诊所都很容易使用。