Abstract
Genetic polymorphisms in P. falciparum can be used to indicate the parasite’s susceptibility to antimalarial drugs as well as its geographical origin. Both of these factors are key to monitoring development and spread of antimalarial drug resistance. In this study, we combine multiplex PCR, custom designed dual indexing and Miseq sequencing for high throughput SNP-profiling of 457 malaria infections from Guinea-Bissau, at the cost of 10 USD per sample. By amplifying and sequencing 15 genetic fragments, we cover 20 resistance-conferring SNPs occurring in pfcrt, pfmdr1, pfdhfr, pfdhps, as well as the entire length of pfK13, and the mitochondrial barcode for parasite origin. SNPs of interest were sequenced with an average depth of 2,043 reads, and bases were called for the various SNP-positions with a p-value below 0.05, for 89.8–100% of samples. The SNP data indicates that artemisinin resistance-conferring SNPs in pfK13 are absent from the studied area of Guinea-Bissau, while the pfmdr1 86 N allele is found at a high prevalence. The mitochondrial barcodes are unanimous and accommodate a West African origin of the parasites. With this method, very reliable high throughput surveillance of antimalarial drug resistance becomes more affordable than ever before.
Highlights
During the past fifteen years the global malaria case numbers are estimated to have decreased by 18% and overall death rates by 48%1
Among the P. falciparum genes hitherto implicated in conferring antimalarial resistance, the literature has focused on single nucleotide polymorphisms (SNPs) occurring in mainly five P. falciparum genes; P. falciparum chloroquine resistance transporter, P. falciparum multidrug resistance gene 1, P. falciparum dihydrofolate reductase, P. falciparum dihydropteroate synthase, and most recently P. falciparum putative kelch protein[5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20]
We present proof-of-concept of using custom dual indexing on the Illumina next-generation sequencing (NGS) (Miseq) platform, for targeted high throughput sequencing of genetic loci related to antimalarial drug resistance and mitochondrial barcoding of the infecting P. falciparum parasites
Summary
During the past fifteen years the global malaria case numbers are estimated to have decreased by 18% and overall death rates by 48%1. The current first-line treatments for P. falciparum malaria are the artemisinin-based combination therapies (ACTs). While specific and highly conservative SNPs have been identified globally in pfcrt, pfmdr[1], pfdhfr and pfdhps, studies indicate that tolerance/resistance towards artemisinins may rather be conferred by a variety of mutations in the propeller region of pfK1315, 21. Temporal surveillance of the prevalence of these SNPs provides crucial knowledge on development and geographic spread of antimalarial drug resistance in P. falciparum populations, which are important for antimalarial drug policy decisions for instance as shown regarding IPTp22. The geographic origin of the parasites is of major importance to a surveillance scheme aiming to monitor global emergence and spread of resistance, and perhaps thereby attempt to hinder or delay the spread of artemisinin resistance from South East Asia to SSA23–25. A surveillance scheme aiming to analyse all SNPs in question is a long process and requires sequencing
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