Next-Generation Sequencing of Plasmodium vivax Patient Samples Shows Evidence of Direct Evolution in Drug-Resistance Genes

Abstract

Understanding the mechanisms of drug resistance in Plasmodium vivax, the parasite that causes the most widespread form of human malaria, is complicated by the lack of a suitable long-term cell culture system for this parasite. In contrast to P. falciparum, which can be more readily manipulated in the laboratory, insights about parasite biology need to be inferred from human studies. Here we analyze the genomes of parasites within 10 human P. vivax infections from the Peruvian Amazon. Using next-generation sequencing we show that some P. vivax infections analyzed from the region are likely polyclonal. Despite their polyclonality we observe limited parasite genetic diversity by showing that three or fewer haplotypes comprise 94% of the examined genomes, suggesting the recent introduction of parasites into this geographic region. In contrast we find more than three haplotypes in putative drug-resistance genes, including the gene encoding dihydrofolate reductase-thymidylate synthase and the P. vivax multidrug resistance associated transporter, suggesting that resistance mutations have arisen independently. Additionally, several drug-resistance genes are located in genomic regions with evidence of increased copy number. Our data suggest that whole genome sequencing of malaria parasites from patients may provide more insight about the evolution of drug resistance than genetic linkage or association studies, especially in geographical regions with limited parasite genetic diversity.

Genome-wide diversity and gene expression profiling of Babesia microti isolates identify polymorphic genes that mediate host-pathogen interactions

“…The availability of new sequencing technologies has made it possible to perform genome-wide profiling of genetic polymorphisms in a large number of species including several protozoan parasites2728293031. These analyses have significantly improved our understanding of the diversity and evolution of these pathogens, provided insights into their virulence, host modulation and the genetic basis of drug resistance phenotypes, and helped to develop novel approaches for disease control and prevention, and informed public health policy323334353637.…”

Whole genome sequencing of field isolates reveals extensive genetic diversity in Plasmodium vivax from Colombia

“…However, many of these apparent singletons have been recorded in other 285 studies. When we compare our SNVs to a catalogue of previously reported P. vivax 286 SNVs[18,19,31] only 6,854 (20.2%) are unique to this study. Our low-coverage samples 287 did not produce more singletons per callable-site than those with higher sequencing 288 coverage (Table 4), suggesting the remaining singletons are not simply artifacts…”

“…falciparum mrp proteins). There were no such variants in GTP cyclohydrase,341 Chloroquine resistance transporter ortholog, or Kelch 13, which are all considered 342 possible drug resistance genes[18].343…”

“…Using this procedure, we called a total of 34 SNVs from the >22 million 261 base pair reference genome, none of which were called as polymorphic alleles in our 262 patient-derived data. Repeating this procedure with a lower coverage (2x) dataset 263 generated fewer SNVs(18) including only one new putative variant. The small number 264 of variants called from the reference data confirms the conservative nature of our variant 265 calling procedure.In total, we identified 33,855 non-reference SNV alleles among the Colombian The first eight panels represent the distribution of the frequency of reference bases in sequencing reads produced from our samples across all sites in the P. vivax genome.…”

“…The majority of sites from all samples contain only non-reference bases; these sites were removed to allow clearer visualization of these distributions.populations, which contained 41,630 and 45,417 SNVs respectively in the genomic 272 regions included in our study (Fig. 3)[31]; however, it was three times the number 273 identified in the same genomic regions in a recent study using isolates from the Amazon 274 basin in Peru[18]. Approximately two-thirds of the Peruvian SNVs(7,232) are also 275 present in our Colombian samples.…”