Ultrasensitive CRISPR-based diagnostic for field-applicable detection of Plasmodium species in symptomatic and asymptomatic malaria
Files
Supplementary information
Date
2020-10-13
Authors
Lee, Rose A.
Puig, Helena De
Nguyen, Peter Q.
Angenent-Mari, Nicolaas M.
Donghia, Nina M.
McGee, James P.
Dvorin, Jeffrey D.
Klapperich, Catherine M.
Pollock, Nira R.
Collins, James J.
Version
Published version
OA Version
Citation
R.A. Lee, H.D. Puig, P.Q. Nguyen, N.M. Angenent-Mari, N.M. Donghia, J.P. McGee, J.D. Dvorin, C.M. Klapperich, N.R. Pollock, J.J. Collins. 2020. "Ultrasensitive CRISPR-based diagnostic for field-applicable detection of Plasmodium species in symptomatic and asymptomatic malaria.." Proc Natl Acad Sci U S A, Volume 117, Issue 41, pp. 25722 - 25731. https://doi.org/10.1073/pnas.2010196117
Abstract
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.
Description
License
Copyright © 2020 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY).