Abstract
In 2019 almost half of the global population was at risk of malaria infection and caused the death of ~409, 000 people, mostly children under 5 years of age in sub-Saharan Africa. While these statistics are seen as an improvement over those of the 1980s, the rise and spread of antimalarial drug resistance over the past decade has resulted in plateauing control efforts. It is also important to acknowledge that malaria elimination has primarily focused on deadly human malaria, caused by Plasmodium falciparum. To this end, many anti-malarial therapeutics, rapid diagnostic tests and elimination strategies have been tailored for P. falciparum, to the neglect of the other most important cause of malaria, Plasmodium vivax. The cause of relapsing malaria, P. vivax is the most widely distributed and difficult to treat form of malaria (due to dormant liver stages). Reasons for the past neglect of P. vivax are multifaceted; in addition to the fundamental differences between the parasite biology (which make P. vivax more difficult to detect, treat and eradicate); P. vivax cannot be continuously cultured, limiting our ability to conduct high throughput drug discovery programs and studies into the molecular mechanisms of P. vivax biology (which require tractable cultures to conduct reverse genetic studies). The recent development of a tractable continuous erythrocytic culture of Plasmodium cynomolgi (P. vivax’s sister species), provides an opportunity to better understand the biology of P. vivax and paves the way for the development of diagnostics and therapeutics targeting vivax malaria.
This study primarily leverages the erythrocytic P. cynomolgi Berok model for P. vivax to investigate a range of novel (i.e. 6-substituded amiloride analogues) and repurposed therapeutics (i.e. clofazimine and TB47). The P. cynomolgi model was also used to investigate, standard delayed action antimalarial compounds, such as doxycycline and the effect of prodrugs such as proguanil (neither of which would have been possible using P. vivax which is limited to short duration ex vivo maturation assays). Using the optimised flow cytometry sensitivity assays developed in this thesis, we confirmed the sensitivity phenotype of the first ever integrative recombinant clone of P. cynomolgi, namely the Pcbermdr1Y976F clone (putative molecular marker for P. vivax chloroquine resistance). Therefore, the central hypothesis of this work is that P. cynomolgi Berok provides a useful model to examine drug sensitivity phenotypes of P. vivax. As most P. vivax infections in low transmission settings are at sub-microscopic parasitaemia (and often below the detection threshold of standard PCR methods) the final part of this thesis involves the development of an ultra-sensitive RNA methodology to detect the presence of Plasmodium spp. infection. While this final part of the thesis seems at first to be unrelated to the in vitro sensitivity studies; it should be noted that the next logical step in the drug development pipeline requires clinical (in vivo) drug efficacy studies; which require the sensitive detection of parasites pre and post treatment.
The results of this study confirm the utility of the P. cynomolgi Berok as useful model to examine the drug sensitivity phenotype of P. vivax. Disparities between the antimalarial sensitivity of P. cynomolgi and P. falciparum highlight the necessity for drug development programs targeting vivax malaria not to solely rely on P. falciparum sensitivity data (as disastrously discovered during Phase II trials of the new antimalarial DSM265, which was found to have less potency against P. vivax). The novel P. cynomolgi Berok culture methodologies developed in this study further increase the utility of this important in vitro model, especially for future studies of the molecular mechanisms in drug resistant P. vivax.