A novel CRISPR-based malaria diagnostic capable of <i>Plasmodium</i> detection, speciation, and drug-resistance genotyping

ABSTRACT 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. We developed SHERLOCK assays for malaria capable of detecting all Plasmodium species known to cause malaria in humans and species-specific detection of P. vivax and P. falciparum , the species responsible for the majority of malaria cases worldwide. We validated these assays against parasite genomic DNA and achieved analytical sensitivities ranging from 2.5-18.8 parasites per reaction. We further tested these assays using a diverse panel of 123 clinical samples from the Democratic Republic of the Congo, Uganda, and Thailand and pools of Anopheles mosquitoes from Thailand. When compared to real-time PCR, the P. falciparum assay achieved 94% sensitivity and 94% specificity in clinical samples. In addition, we developed a SHERLOCK assay capable of detecting the dihydropteroate synthetase ( dhps ) single nucleotide variant A581G associated with P. falciparum sulfadoxine-pyrimethamine resistance. 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. These novel SHERLOCK assays have potential to spawn a new generation of molecular diagnostics for malaria and demonstrate the versatility of CRISPR-based diagnostic approaches. One-sentence summary Novel malaria SHERLOCK assays enabled robust detection, speciation, and genotyping of Plasmodium spp . in diverse samples collected in Africa and Asia.