The Global Technical Strategy for Malaria 2016-2030 calls for the transformation of malaria surveillance into a core intervention. Molecular tools can quantify malaria importation risk, characterize changing transmission intensity and guide malaria control by distinguishing imported vs. indigenous cases and sources of reintroduction. SNP barcodes for Plasmodium vivax (Pv) were frequently designed at a global level with insufficient resolution at smaller geographical scales to serve national malaria control programs (NMCPs).
We have designed novel within-country SNP-barcodes for Vietnam and Peru using whole genome data. SNPs were selected based on allele frequency, neutrality and contribution to geographical spread. These SNP barcodes were genotyped using a highly multiplexed Pv AmpliSeq NGS assay, including amplicons targeting 11 drug resistance genes, ama1 and a previously reported 33-SNP global barcode (vivaxGEO).
Allele frequencies at barcode positions were evaluated in Pv genomes (N=604; 23 countries in 4 WHO regions). Barcode performance for different use cases was evaluated in the Pv AmpliSeq assay applied to 1) isolates from travelers from our clinic at ITM Antwerp (N=137; 26 countries in 5 WHO regions), 2) routine collections at sentinel sites in Vietnam (N=286), and 3) retrospective samples from Peru (N=345). The spatial specificity of the vivaxGEO-barcode performs very well for between-country prediction of origin, while our barcodes have higher diversity and increased resolution for within-country spatio-temporal surveillance.
As molecular surveillance platforms transition to incorporation into NMCPs, simple standardized laboratory protocols feasible on benchtop sequencers and automated analysis are necessary to generate reproducible results. We prioritize putting this tool in the hands of researchers and control programs in endemic countries to increase ownership and ensure data usage for decision-making and policy. Our approach performs well in geographically differentiating isolates at different levels, detecting variants in putative resistance markers, and can be easily adjusted to suit the needs for other settings.