Antimalarial Drug Resistance: Mechanisms of Emergence in Low-Transmission Settings

Abstract

Antimalarial drug resistance is a major factor affecting global malaria control, causing treatment failure and significant morbidity and mortality. Resistance has historically emerged in low-transmission areas before spreading to high-transmission areas, and there is consequently widespread interest in genomic surveillance and antimalarial sensitivity profiling of parasites from low-transmission areas. The Greater Mekong Subregion is one such region and has been extensively researched, however other regions such as South America in general, and Colombia in particular, which carries a significant proportion of the malaria burden in South America, are relatively neglected in the literature. Antimalarial drug resistance to multiple drugs, including chloroquine and sulfadoxine-pyrimethamine, has emerged independently in South America and Colombia. In 2006, Colombia adopted Artemisinin Combination Therapies (ACTs) as front-line antimalarial treatments, and withdrew the previous frontline therapy, sulfadoxine-pyrimethamine. While sulfadoxine-pyrimethamine resistance is widespread, no resistance to ACTs has been detected to date, although genomic surveillance is very limited and in vitro phenotypic characterisation of isolates is almost non-existent. I collected contemporary samples of P. falciparum isolates from Colombia and compared these to isolates collected before the withdrawal of sulfadoxine-pyrimethamine to explore if there had been any changes in the proportion of resistance-associated genotypes and/or in vitro drug sensitivity of the population of parasites. I also optimised the process of adapting isolates to culture in Colombia, and trained local staff so that isolate collection can continue, and the laboratory can form a focus of surveillance of parasite sensitivity in Colombia. I contextualised the phenotypic and genetic profiles of these isolates in the body of literature characterising parasite populations along the Pacific Coast of Colombia. Genetic characterisation of these field isolates revealed the presence and subsequent maintenance of a novel allele in a transporter that has been recently associated with chloroquine resistance in Sub-Saharan Africa and Southeast Asia called PfAAT1. I generated a panel of transgenic lines and patient isolates using CRISPR/Cas9 genome editing and demonstrated for the first time the fitness and sensitivity phenotypes of different PfAAT1 haplotypes found uniquely in South America. Through this work, I identified an urgent need for improved in vitro assays of artemisinin sensitivity, and attempted to address this through adapting novel molecular tools into P. falciparum expression systems. These tools would have allowed me to synchronise protein trafficking and therefore explore the molecular mechanism of artemisinin resistance, with the end goal of using the assay to test and validate the role of novel SNPs associated with resistance. These attempts were ultimately unsuccessful, but through optimisation of super-resolution microscopy techniques I validated theories about the localisation of haemoglobinases and contribute to understanding of food vacuolar ultrastructure throughout the parasite life cycle. In sum, I have successfully characterised the genotypic and phenotypic profiles of P. falciparum isolates collected from Colombia across the withdrawal of sulfadoxine-pyrimethamine and introduction of ACTs, and experimentally validated the role of novel mediators of the chloroquine resistance phenotype in South America.