The global burden of Plasmodium vivax is being increasingly reevaluated as more fatal cases are identified and drug resistant strains are discovered [1, 2]. Despite the fact that 2.85 billion people live in P. vivax endemic areas, there is a substantial lack of knowledge surrounding the mechanisms of biological features unique to P. vivax, constraining the ability to design appropriate control strategies.
The fact that P. vivax exclusively invades reticulocytes impairs the development of a reliable, long-term in vitro culture method, a technique that has been available for the study of P. falciparum for over 30 years . While some progress has been made in establishing P. vivax culture in the laboratory, the lack of a reproducible in vitro culture method prevents basic laboratory manipulations, such as genetic crosses, and has limited the types of questions that can be answered about P. vivax biology.
The advent of low-cost whole genome technologies allows direct analysis of P. vivax field populations, without the need for in vitro culture. With the completion of the P. vivax reference genome as well as the publication of the first P. vivax resequencing project , single nucleotide variants (SNV) are now being identified that can be used to track parasite populations and investigate parasite population structure on both the regional and global levels. In addition, new whole genome sequencing technologies allow for sequencing hundreds of samples from different geographic locations and thus take advantage of the thousands of natural genetic crosses that occur over time and in the context of parasite movements among regions under different epidemiological contexts (reverse genetics). Using signatures in the genome left by these natural crosses, investigators will be able to identify regions of the genome under selection and, potentially, the genes involved in P. vivax virulence, drug resistance, and immune evasion.
A critical barrier to the whole genome analysis of P. vivax is the ability to obtain sufficient quantities of high quality parasite genomic DNA free of human nucleic acid contamination. Current protocols for obtaining parasite DNA for whole genome studies from P. falciparum field isolates consist of culture adapting the isolated parasites and passaging them for 3–4 weeks. This intermediate step achieves two things: one, it expands the parasite population allowing for isolation of a larger quantity of DNA and, two, it removes human leukocytes containing contaminating DNA. Since there is no reliable culture method to propagate P. vivax in vitro, alternative methods have to be designed to work with nucleic acids from P. vivax field samples.
To address the two issues of low quantities of parasite DNA and human DNA contamination, the standard method adopted by the P. vivax research community is leukocyte filtration using ion-exchange columns followed by whole genome amplification (WGA) [4, 5]. This current method of obtaining P. vivax DNA from field samples is only feasible when the patient blood samples are collected in close proximity to a field laboratory because of the need to filter out the leukocytes before they lyse. This logistical issue precludes the collection of field samples from remote areas where P. vivax is endemic and thus limits our understanding of the population genetics of P. vivax. In addition, there are many samples that were collected before leukocyte depletion became a standard technique. As of now these samples cannot be analyzed via whole genome sequencing prohibiting the use of these samples to study ofhow P. vivax has evolved over time.
Here we demonstrate the feasibility of analyzing P. vivax field samples without on-site leukocyte filtration using an in-solution hybridization capture method [6, 7]. By modifying the whole genome capture protocol designed for P. falciparum by Melnikov et al., we show that Sal1 reference genomic DNA can be used to create whole genome baits, which can then be used to extract P. vivax genomic DNA from the contaminating human DNA in both frozen samples and mock blood spots. After the whole genome extraction of P. vivax DNA and subsequent whole genome sequencing, greater than 90% of the P. vivax assembled genome (~22 million bases) can be confidently assigned a genotype, or “called.” Our whole genome sequencing results are equivalent to previous results published using the leukocyte filtration protocol, and we, therefore, propose that because of its much easier application in the field, whole genome capture is a superior method of analyzing large numbers of P. vivax field samples from diverse geographic areas.