UC San Diego

Plasmodium vivax causes 25-40% of malaria cases worldwide, yet research on this human malaria parasite has been neglected. Nevertheless, the recent publication of the P. vivax reference genome now allows genomics and systems biology approaches to be applied to this pathogen. We show here that whole genome analysis of the parasite can be achieved directly from ex vivo-isolated parasites, without the need for in vitro propagation. Additionally, we show that in-solution hybridization capture can be used to extract P. vivax DNA from human contaminating DNA in the laboratory without the need for on-site leukocyte filtration. Utilizing a whole genome capture method, we were able to enrich P. vivax DNA from bulk genomic DNA from less than 0.5% to a median of 55% (range 20%-80%). This level of enrichment allows for efficient analysis of the samples by whole genome sequencing and does not introduce any gross biases into the data. These techniques were subsequently used to investigate the dormant hepatic stages, known as hypnozoites, in P. vivax. This unique parasite stage is an important reservoir of infection and a critical barrier to malaria eradication. At present there are no biomarkers to identify this tissue stage and estimates of the prevalence of infections due to hypnozoites are confounded by the inability to distinguish between new and relapsing infection. Here we performed whole genome sequencing of consecutive P. vivax relapse infections using material from a patient who experienced three episodes of P. vivax malaria over 33 months in a non -endemic country. Based on patient medical history and analysis of single nucleotide variants (SNV), it was determined that two of the infections were caused by reactivation of single hypnozoites. We observe that the three recurring infections were caused by meiotic siblings. This indicates that a single sexual cross in the mosquito is capable of creating multiple distinct parasite populations, thus definitively demonstrating that the result of parasite sexual replication and meiosis in the mosquito, the oocyst, is comprised of parasites descended from a single tetrad