The development of chloroquine resistance in Plasmodium vivax parasites

Plasmodium vivax, one of 5 malaria species occurring in humans, is the most predominant malaria species outside Africa, causing 7.5 million cases annually as of 2018. Chloroquine resistance (CQR) in Plasmodium vivax, which was first reported in 1989, continues to emerge globally while the mechanism of drug resistance and molecular markers remain unknown; hampering molecular surveillance and accurate diagnosis of CQR. Now is the time to address PvCQR as technological applications are finally available to overcome the challenges that traditionally plague P. vivax research including: (1) the inability to culture this parasite in vitro to study its molecular biology, (2) the low density of P. vivax infections, and (3) the complexity of clinical infections (i.e. mixed life-stage and clone). For many years research has focused on the orthologs of Plasmodium falciparum CQR; however, a lack of association of these orthologs’ SNPs and CNVs to resistance and recently published data obtained from P. vivax parasites adapted to a non-human primate model have implicated transcriptional regulation as a potential mechanism of resistance. My hypothesis, based also on our results from a clinical efficacy study in Vietnam, isthat altered gene expression of transporter genes plays a major role in PvCQR. This PhD project will capitalize on a large collection of P. vivax clinical samples (including CQR) at the Malariology Unit and utilize cutting-edge RNA sequencing technologies, including single-cell transcriptomics, to unravel the transcriptional network of genes underlying PvCQR and the impact of infection complexity (parasite life-stages and mixed clones present in natural infections) in treatment outcome. Single-cell RNA-seq will then be combined to deconvolute bulk RNAseq data generated from our collected samples to identify gene expression markers associated with resistance. Whether altered gene expression of identified markers is directly associated with a resistant phenotype will then be tested in a transgenic P. knowlesi model using CRISPR-Cas genome editing. Outcomes of this doctoral research will directly benefit P. vivax patients and drug resistance surveillance, while advancing research with new protocols, tools, datasets, and transgenic lines to address additional key biological questions in P. vivax research.