Plasmodium evolution and drug resistance in vitro and in populations using whole genome technologies

Malaria remains a significant worldwide cause of morbidity and mortality. As drug resistance is widespread, there is a need to identify the mechanisms of action and resistance of current and novel compounds to leverage this knowledge for monitoring the emergence of resistance and to accelerate development of new drugs. Using a genomics approach, we show how whole-genome microarrays and deep sequencing can be used to advance our understanding of parasite biology. First, a custom high-density tiling array for Plasmodium falciparum was developed and validated, detecting nearly all mutations in the parasite genome. This technology was used to show that in vitro-derived P. falciparum parasites resistant to fosmidomycin have acquired of extra copies of 1-deoxy-D-xylulose 5-phosphate reductoisomerase, the presumed target. Second, we describe how whole-genome microarray analysis of 14 P. falciparum patient isolates from the Peruvian Amazon led to the discovery of clindamycin resistance, which is used for malaria treatment in Peru. We discovered a highly related parasite population in this region and identified the progeny of a natural cross. Additionally, we identified the genomic deletion of an important antigen for rapid diagnostic tests for malaria. Third, we describe using a custom microarray-based approach as well as deep sequencing for the study of a patient-derived P. vivax isolate. We show that microarray-based methods can detect thousands of SNPs in a patient isolate of P. vivax and the utility of whole-genome methods for genetic diversity studies. Last, we discuss ongoing work to characterize the mechanisms of action or resistance to several antimalarial compounds: piperaquine, decoquinate, mupirocin, and thiaisoleucine. The work described here provides a framework for future studies of in vitro drug resistance in and populations of malaria parasites.