As a biomedical science graduate who has been training at MVRU for just under a month, I’d say that there is hardly a parasite more interesting than P. vivax. To me, what is so attractive about this parasite is not what we know about it, but what we do not. Our knowledge gap is both wide and deep, and one of the factors that hamper our efforts to mitigate this gap, is the fact that we cannot culture this little critter (yet).
To culture a parasite, one must provide for the parasite a specific environmental condition that fools the parasite into thinking that it has infected a host (for example; human large roundworm cultures) or if this “deception” should fail –for any reason- then the only other option would be to provide the preferred host cells to the parasite culture. This sounds straightforward and in a lot of cases it certainly is (for example; providing mature erythrocytes to a Plasmodium falciparum culture) but in some cases one encounters difficulties in the only option available. This is indeed the case with culturing Plasmodium vivax.
The problem with culturing blood-stage P. vivax stems from its’ preferred type of red blood cell, the reticulocyte. Reticulocytes are immature red blood cells that constitute 0.5% to 2.5% of all red blood cells in adults at any given time. The low percentage of reticulocytes in circulation brings along a host of problems such as; drawing impractical amounts of blood and finding an efficient method of isolating/concentrating reticulocytes.
Even if one possesses a sufficiently large reserve of reticulocytes, it doesn’t mean that the parasite will propagate indefinitely. In fact, scientists have found that after culturing P. vivax for a certain amount of time the parasite gradually loses its ability to invade target cells. The reason why this happens is unclear (Bermudez et al. 2018). However, just because we are unable to continuously culture P.vivax long-term doesn’t mean that we should abandon P. vivax culture systems altogether. On the contrary, there are still useful things we can do with time-constrained culture systems such as single-cycle schizogony, and reticulocyte invasion assays.
Another challenge in culturing P. vivax is the issue of its’ in vitro environment. Namely, what are the in vitro environmental requirements in which to optimally culture P. vivax. Fortunately, the problem is much less critical than that of P.vivax’s host specificity. Nonetheless, the environment in which the parasite is cultured can either play a greatly facilitating or debilitating role in parasite viability. The standard media used in P. vivax cultures seem to be either RPMI640 or McCoy’s medium (Noulin et al. 2013). The culture is usually kept at 37⁰c in a 90% N2 – 5% O2 – 5% CO2 although there is a lack of evidence supporting this particular composition of gases. Regardless, it is the most commonly utilized configuration (Noulin et al. 2013). One other crucial component is serum. Noulin et al. performed a survey of the P. vivax culture literature and found that serum concentration ranged from 10 – 50% with no substantial difference in parasite density.
The inability to maintain a continuous P. vivax culture hinders research into its biology and thus research into new therapeutics, treatments, and prevention measures for P. vivax, which remains the most frequent and widespread cause of recurring malaria. In this respect, one sees how public health interventions tie back to basic research and therefore impediments of basic research can negatively impact such interventions. Which goes on to show how crucial it is to maintain support for basic research.
On the bright side, headways are being made into how P. vivax can be continuously cultured. Panichakul et al. showed in 2007 that they were able to sustain 5 out of 14 P. vivax isolates for more than 1 month using blood derived from hematopoietic stem cells (HSC’s) taken from umbilical cord blood, albeit with low parasite density. Another more recent study by Scully et al. showed that P. vivax was able to efficiently invade immortalized erythroid progenitor cells generated from peripheral blood mononuclear cells (PBMC). The authors performed lentiviral transduction of E6 and E7 genes from Human Papillomavirus on PBMC’s. This yielded a cell line that continuously undergoes erythropoiesis in a manner similar –but not identical- to primary HSC’s with respect to both morphology and expression of stage-specific surface proteins. The differences lie mainly in; the rate of erythropoiesis, with the erythroid progenitor cell line developing faster than HSC’s, and the percentage of enucleation, which is significantly lower (5%) in the cell line.
Other strategies do away with trying to culture P. vivax, opting for in vivo models that closely mimics human vivax malaria infections. These models include non-human primates (NHP’s), and more recently humanized mice models. The latter of which seems promising as a new tool to investigate vivax malaria and by some measures is better than NHP models, as parasite isolates do not need to be adapted for use. This means that isolates gathered from fieldwork can be directly investigated without any alterations to its biology. In fact, Schäfer et al. has utilized humanized mice models infused with reticulocytes to test a particular vaccine candidate that inhibits reticulocyte invasion.
As research progresses, we will continue to gain more and more insights into P. vivax’s biology; either by finding better ways to culture P. vivax or smarter ways to circumvent the problem. In any case, we have never gotten closer to eliminating P. vivax and we can be certain that the issues presented in this article will be looked at in the future as an challenge overcome by the collective effort of a global network of scientists working towards the same goal. That of bringing about a malaria-free world.
Feature Image reference: Bermudez et al. 2018