A Genome Wide Association Study of Plasmodium falciparum Susceptibility to 22 Antimalarial Drugs in Kenya

Abstract

BackgroundDrug resistance remains a chief concern for malaria control. In order to determine the genetic markers of drug resistant parasites, we tested the genome-wide associations (GWA) of sequence-based genotypes from 35 Kenyan P. falciparum parasites with the activities of 22 antimalarial drugs.Methods and Principal FindingsParasites isolated from children with acute febrile malaria were adapted to culture, and sensitivity was determined by in vitro growth in the presence of anti-malarial drugs. Parasites were genotyped using whole genome sequencing techniques. Associations between 6250 single nucleotide polymorphisms (SNPs) and resistance to individual anti-malarial agents were determined, with false discovery rate adjustment for multiple hypothesis testing. We identified expected associations in the pfcrt region with chloroquine (CQ) activity, and other novel loci associated with amodiaquine, quinazoline, and quinine activities. Signals for CQ and primaquine (PQ) overlap in and around pfcrt, and interestingly the phenotypes are inversely related for these two drugs. We catalog the variation in dhfr, dhps, mdr1, nhe, and crt, including novel SNPs, and confirm the presence of a dhfr-164L quadruple mutant in coastal Kenya. Mutations implicated in sulfadoxine-pyrimethamine resistance are at or near fixation in this sample set.Conclusions/SignificanceSequence-based GWA studies are powerful tools for phenotypic association tests. Using this approach on falciparum parasites from coastal Kenya we identified known and previously unreported genes associated with phenotypic resistance to anti-malarial drugs, and observe in high-resolution haplotype visualizations a possible signature of an inverse selective relationship between CQ and PQ.

Functional Comparison of 45 Naturally Occurring Isoforms of the Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT)

“…33 Bray et al showed that PQ potentiates CQ versus some CQR strains, 34 and recent genomewide association studies demonstrate increased sensitivity to PQ in some CQR isolates. 33 However, it is not clear whether this reciprocal relationship is due to PQ blocking CQ transport by PfCRT or perhaps to PQ transport by PfCRT in some of these strains. 34,36 …”

“…These data suggest inhibition of Dd2 PfCRT CQ transport by PQ is due to isoform-specific competition between the two drugs for transport, which is consistent with the recently observed reciprocal relationship between CQR status and PQ susceptibility for African P. falciparum isolates. 33 …”

From within host dynamics to the epidemiology of infectious disease: Scientific overview and challenges

“…The parasite undergoes continuous growth and development, throughout the different stages of its life cycle. With the chance of mutations, these may be associated with the parasite becoming resistant to drugs [48; 49; 50; 51]. The transfer and propagation of offspring within and between its hosts is requisite for its survival, and modeling efforts are of paramount importance to project and understand individual and population-based parasite loads, transmission and disease characteristics, and treatment regimens, as well as to devise new means for intervention [52; 53].…”

Overlap Extension Barcoding for the Next Generation Sequencing and Genotyping of Plasmodium falciparum in Individual Patients in Western Kenya

“…2a and b). This high analytical sensitivity and specificity is important because, in high transmission areas, most parasitemias comprise many unique parasite genomes6171840 and traditional genotyping approaches may either fail to generate analyzable data owing to complex templates or fail to capture minority variant subpopulations; these minority variant populations can harbor clinically-important genotypes41. The correlations between observed and expected allele frequencies in control DNA samples for pf-ama1 were lower than those reported in studies employing whole genome sequencing of polygenomic templates42.…”

“…In addition, the utility of these approaches is undermined by the polyclonality common to high transmission regions such as Sub-Saharan Africa due to diversity at the same nucleotides in multiple parasites within individual patients1416. More recent approaches use NGS platforms to reconstruct whole genomes, but this approach is complicated by the parasite’s genomic architecture, polyclonality of parasitemias, and the need for sophisticated genome-scale assembly and analytic tools61718. NGS platforms have also been employed in ecological studies to sequence amplicons generated from pools of parasites1920, but heretofore technical constraints have limited the ability to assign sequence reads to individual input parasites.…”