Asymptomatic carriers of Plasmodium parasites hamper malaria control and eradication. Achieving malaria eradication requires ultrasensitive diagnostics for low parasite density infections (<100 parasites per microliter blood) that work in resource-limited settings (RLS). Sensitive point-of-care diagnostics are also lacking for nonfalciparum malaria, which is characterized by lower density infections and may require additional therapy for radical cure. Molecular methods, such as PCR, have high sensitivity and specificity, but remain high-complexity technologies impractical for RLS. Here we describe a CRISPR-based diagnostic for ultrasensitive detection and differentiation of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae, using the nucleic acid detection platform SHERLOCK (specific high-sensitivity enzymatic reporter unlocking). We present a streamlined, field-applicable, diagnostic comprised of a 10-min SHERLOCK parasite rapid extraction protocol, followed by SHERLOCK for 60 min for Plasmodium species-specific detection via fluorescent or lateral flow strip readout. We optimized one-pot, lyophilized, isothermal assays with a simplified sample preparation method independent of nucleic acid extraction, and showed that these assays are capable of detection below two parasites per microliter blood, a limit of detection suggested by the World Health Organization. Our P. falciparum and P. vivax assays exhibited 100% sensitivity and specificity on clinical samples (5 P. falciparum and 10 P. vivax samples). This work establishes a field-applicable diagnostic for ultrasensitive detection of asymptomatic carriers as well as a rapid point-of-care clinical diagnostic for nonfalciparum malaria species and low parasite density P. falciparum infections.

疟原虫无症状携带者阻碍了疟疾的控制和根除。实现消灭疟疾需要对在资源有限的环境（RLS）中起作用的低寄生虫密度感染（每微升血液中＜100个寄生虫）进行超灵敏诊断。非恶性疟疾也缺乏灵敏的即时诊断，其特征在于感染密度较低，可能需要进行其他治疗才能根治。分子方法（例如PCR）具有很高的灵敏度和特异性，但对于RLS仍然不可行。这里介绍一个诊断为超灵敏检测和分化基于CRISPR-恶性疟原虫，间日疟原虫，卵形疟原虫和疟疾疟原虫，使用核酸检测平台SHERLOCK（特定的高灵敏度酶促报告分子解锁）。我们提出了一种简化的，现场适用的诊断方法，该诊断方法包括10分钟的SHERLOCK寄生虫快速提取方案，然后通过荧光或侧流条读出的SHERLOCK进行60分钟的疟原虫物种特异性检测。我们使用简化的样品制备方法（与核酸提取无关）优化了单罐，冻干，等温测定，并表明这些测定能够检测到每微升血液中两个寄生虫以下的检出限，这是世界卫生组织建议的检出限。我们的恶性疟原虫和间日疟原虫检测对临床样品（恶性疟原虫5个和间日疟原虫10个）表现出100％的敏感性和特异性。这项工作为无症状携带者的超灵敏检测建立了现场适用的诊断方法，并为非恶性疟疾和低寄生虫密度恶性疟原虫感染提供了快速的即时临床诊断。