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A Novel CRISPR-Based Malaria Diagnostic Capable of Plasmodium Detection, Species Differentiation, and Drug-Resistance Genotyping

Citation

Cunningham, Clark H.; Hennelly, Christopher M.; Lin, Jessica T.; Ubalee, Ratawan; Boyce, Ross M.; Mulogo, Edgar M.; Hathaway, Nicholas; Thwai, Kyaw L.; Phanzu, Fernandine; & Kalonji, Albert, et al. (2021). A Novel CRISPR-Based Malaria Diagnostic Capable of Plasmodium Detection, Species Differentiation, and Drug-Resistance Genotyping. EBioMedicine, 68, 103415. PMCID: PMC8213918

Abstract

BACKGROUND: CRISPR-based diagnostics are a new class of highly sensitive and specific assays with multiple applications in infectious disease diagnosis. SHERLOCK, or Specific High-Sensitivity Enzymatic Reporter UnLOCKing, is one such CRISPR-based diagnostic that combines recombinase polymerase pre-amplification, CRISPR-RNA base-pairing, and LwCas13a activity for nucleic acid detection.
METHODS: We developed SHERLOCK assays capable of detecting all Plasmodium species known to cause human malaria and species-specific detection of P. vivax and P. falciparum, the species responsible for the majority of malaria cases worldwide. We further tested these assays using a diverse panel of clinical samples from the Democratic Republic of the Congo, Uganda, and Thailand and pools of Anopheles mosquitoes from Thailand. In addition, we developed a prototype SHERLOCK assay capable of detecting the dihydropteroate synthetase (dhps) single nucleotide variant A581G associated with P. falciparum sulfadoxine resistance.
FINDINGS: The suite of Plasmodium assays achieved analytical sensitivities ranging from 2•5-18•8 parasites per reaction when tested against laboratory strain genomic DNA. When compared to real-time PCR, the P. falciparum assay achieved 94% sensitivity and 94% specificity during testing of 123 clinical samples. Compared to amplicon-based deep sequencing, the dhps SHERLOCK assay achieved 73% sensitivity and 100% specificity when applied to a panel of 43 clinical samples, with false-negative calls only at lower parasite densities.
INTERPRETATION: These novel SHERLOCK assays demonstrate the versatility of CRISPR-based diagnostics and their potential as a new generation of molecular tools for malaria diagnosis and surveillance.

URL

http://dx.doi.org/10.1016/j.ebiom.2021.103415

Reference Type

Journal Article

Year Published

2021

Journal Title

EBioMedicine

Author(s)

Cunningham, Clark H.
Hennelly, Christopher M.
Lin, Jessica T.
Ubalee, Ratawan
Boyce, Ross M.
Mulogo, Edgar M.
Hathaway, Nicholas
Thwai, Kyaw L.
Phanzu, Fernandine
Kalonji, Albert
Mwandagalirwa, Kashamuka
Tshefu, Antoinette
Juliano, Jonathan J.
Parr, Jonathan B.

Article Type

Regular

PMCID

PMC8213918

ORCiD

Boyce, R - 0000-0002-9489-6324