PvGTSeq and PvCRiSP: Two amplicon-based targeted sequencing panels for Plasmodium vivax.

Plasmodium vivax is the main cause of malaria outside of sub-Saharan Africa, and in many settings it presents significant challenges to malaria elimination efforts. Despite some control successes in the Americas, regional annual case counts of malaria have increased by over 25% between 2014 and 2023, largely driven by P. vivax. Genomic surveillance can play a key role in understanding the extent to which disease persistence represents indigenous transmission as opposed to introduction of new strains through migration, and whether specific variants evade control measures. Efforts to make P. vivax genomic surveillance more cost-effective have led to the development of targeted sequencing-based methods, which strike a varying balance between assay sensitivity and breadth/informativeness. We introduce two new highly sensitive multiplexed amplicon sequencing panels for P. vivax: PvGTSeq and PvCRiSP. PvGTSeq requires selective whole-genome amplification (sWGA) and contains 249 amplicons-36 for antimalarial resistance and 213 for population structure-optimized for Latin America but applicable to all continents. PvCRiSP features four highly polymorphic amplicons that operate without sWGA and is designed to estimate complexity of infection (COI), identify instances of clonal transmission, and characterize recurrent episodes. Both panels use a single multiplex PCR with non-proprietary reagents, achieve ≥75% amplicon recovery at parasitemias as low as five parasites/μL, and PvCRiSP remains effective with low quality DNA. PvGTSeq showed high sequencing accuracy (error rate 3.85e-4% - 2.87e-3%), and both panels efficiently detected alleles from minority clones in simulated polyclonal infections. We validated both panels with samples from Colombia, Guyana, Honduras, Panama, and Venezuela, and performed in-silico assessments using data from 16 countries worldwide, confirming that these two panels have high power to discriminate samples and assign global geographic origin to imported cases. These panels will therefore be useful tools for P. vivax molecular surveillance in diverse geographic settings.