Portable and cost-effective genetic detection and characterization of Plasmodium falciparum hrp2 using the MinION sequencer

Abstract

AbstractThe prevalence ofPlasmodium falciparum hrp2(pfhrp2)-deleted parasites threatens the efficacy of the most used and sensitive malaria rapid diagnostic tests and highlights the need for continued surveillance for this gene deletion. While PCR methods are generally adequate for determiningpfhrp2presence or absence, they offer a limited view of its genetic diversity. Here, we present a portable sequencing method using the MinION.Pfhrp2amplicons were generated from individual samples, barcoded, and pooled for sequencing. To overcome potential crosstalk between barcodes, we implemented a coverage-based threshold forpfhrp2deletion confirmation. Amino acid repeat types were then counted and visualized with custom Python scripts followingde novoassembly. We evaluated this assay using well-characterized reference strains and 152 field isolates with and withoutpfhrp2deletions, of which 38 were also sequenced on the PacBio platform to provide a standard for comparison. Of 152 field samples, 93 surpassed the positivity threshold, and of those samples, 62/93 had a dominantpfhrp2repeat type. PacBio-sequenced samples with a dominant repeat-type profile from the MinION sequencing data matched the PacBio profile. This field-deployable assay may supplement the World Health Organization’s existing protocol for surveillingpfhrp2deletions and facilitate timely implementation of diagnostic policy change when needed.