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Background: Sri Lanka was certified as a malaria-free nation in 2016; however, imported malaria cases continue to be reported. Evidence-based information on the genetic structure/diversity of the parasite populations is useful to understand the population history, assess the trends in transmission patterns, as well as to predict threatening phenotypes that may be introduced and spread in parasite populations disrupting elimination programmes. This study used a previously developed Plasmodium vivax single nucleotide polymorphism (SNP) barcode to evaluate the population dynamics of P. vivax parasite isolates from Sri Lanka and to assess the ability of the SNP barcode for tracking the parasites to its origin., Methods: A total of 51 P. vivax samples collected during 2005-2011, mainly from three provinces of the country, were genotyped for 40 previously identified P. vivax SNPs using a high-resolution melting (HRM), single-nucleotide barcode method. Minor allele frequencies, linkage disequilibrium, pair-wise F ST values, and complexity of infection (COI) were evaluated to determine the genetic diversity. Structure analysis was carried out using STRUCTURE software (Version 2.3.4) and SNP barcode was used to identify the genetic diversity of the local parasite populations collected from different years. Principal component analysis (PCA) was used to determine the clustering according to global geographic regions., Results: The proportion of multi-clone infections was significantly higher in isolates collected during an infection outbreak in year 2007. The minor allele frequencies of the SNPs changed dramatically from year to year. Significant linkage was observed in sample sub-sets from years 2005 and 2007. The majority of the isolates from 2007 consisted of at least two genetically distinct parasite strains. The overall percentage of multi-clone infections for the entire parasite sample was 39.21%. Analysis using STRUCTURE software (Version 2.3.4) revealed the high genetic diversity of the sample sub-set from year 2007. In-silico analysis of these data with those available from other global geographical regions using PCA showed distinct clustering of parasite isolates according to geography, demonstrating the usefulness of the barcode in determining an isolate to be indigenous., Conclusions: Plasmodium vivax parasite isolates collected during a disease outbreak in year 2007 were more genetically diverse compared to those collected from other years. In-silico analysis using the 40 SNP barcode is a useful tool to track the origin of an isolate of uncertain origin, especially to differentiate indigenous from imported cases. However, an extended barcode with more SNPs may be needed to distinguish highly clonal populations within the country.

Plasmodium vivax, one of the five species of Plasmodium parasites that cause human malaria, is responsible for 25-40% of malaria cases worldwide. Malaria global elimination efforts will benefit from accurate and effective genotyping tools that will provide insight into the population genetics and diversity of this parasite. The recent sequencing of P. vivax isolates from South America, Africa, and Asia presents a new opportunity by uncovering thousands of novel single nucleotide polymorphisms (SNPs). Genotyping a selection of these SNPs provides a robust, low-cost method of identifying parasite infections through their unique genetic signature or barcode. Based on our experience in generating a SNP barcode for P. falciparum using High Resolution Melting (HRM), we have developed a similar tool for P. vivax. We selected globally polymorphic SNPs from available P. vivax genome sequence data that were located in putatively selectively neutral sites (i.e., intergenic, intronic, or 4-fold degenerate coding). From these candidate SNPs we defined a barcode consisting of 42 SNPs. We analyzed the performance of the 42-SNP barcode on 87 P. vivax clinical samples from parasite populations in South America (Brazil, French Guiana), Africa (Ethiopia) and Asia (Sri Lanka). We found that the P. vivax barcode is robust, as it requires only a small quantity of DNA (limit of detection 0.3 ng/μl) to yield reproducible genotype calls, and detects polymorphic genotypes with high sensitivity. The markers are informative across all clinical samples evaluated (average minor allele frequency > 0.1). Population genetic and statistical analyses show the barcode captures high degrees of population diversity and differentiates geographically distinct populations. Our 42-SNP barcode provides a robust, informative, and standardized genetic marker set that accurately identifies a genomic signature for P. vivax infections.