Development of a Single Nucleotide Polymorphism Barcode to Genotype Plasmodium vivax Infections

Abstract

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.

Higher Complexity of Infection and Genetic Diversity of Plasmodium vivax Than Plasmodium falciparum across all Malaria Transmission Zones of Papua New Guinea

“…Plasmodium vivax also has a wider geographical range due to its ability to develop within the mosquito vector at lower temperatures, 4 and at least in part due to a wider vector range in some endemic regions (though in PNG all local malaria vectors transmit both species). 85,86 Of particular note is relapse, which arises from triggering of the dormant liver stage to cause multiple sequential blood-stage infections in the absence of another mosquito bite. 58 These relapses not only cause further illness to the individual, they also provide more opportunities for multiple clones to be co-transmitted, resulting in outcrossing during sexual recombination in the mosquito midgut.…”

Malaria Genomics in the Era of Eradication

“…Statistical tools like COIL (complexity of infection using likelihood) can estimate the COI level of a malaria sample using only knowledge of SNP minor allele frequencies (MAFs) and a sample's genotype . SNP-based barcodes have also proven useful for distinguishing malaria parasites hailing from different geographic regions (Preston et al 2014;Baniecki et al 2015), suggesting they could be helpful in identifying the source of imported infections in pre-elimination settings. Amplicon-based sequencing approaches have also been useful in other intervention contexts, such as characterizing polymorphism in vaccine candidates (Juliano et al 2010;Bailey et al 2012;Gandhi et al 2012Gandhi et al , 2014Aragam et al 2013;Neafsey et al 2015;Mideo et al 2016).…”

Malaria Epidemiology at the Clone Level

“…25 P. vivax genomes had been available [8–12], studies published in 2016 have increased this number to over 500 [13–15]. Genome-wide P. vivax SNP assays as well as amplicon -based genotyping protocols were recently developed [16–18]. Table 1 gives an overview of the different types of genotyping markers, and their benefits and limitations.…”

“…Microsatellites [6, 39] as well as SNPs [3, 16, 40] have shown pronounced genetic differences between parasites from different continents, both for P. vivax and P. falciparum . It is thus possible to identify the origin of parasites imported from distant regions.…”