Changing Prevalence of Potential Mediators of Aminoquinoline, Antifolate, and Artemisinin Resistance Across Uganda.

BACKGROUND: In Uganda, artemether-lumefantrine is recommended for malaria treatment and sulfadoxine-pyrimethamine for chemoprevention during pregnancy, but drug resistance may limit efficacies. METHODS: Genetic polymorphisms associated with sensitivities to key drugs were characterized in samples collected from 16 sites across Uganda in 2018 and 2019 by ligase detection reaction fluorescent microsphere, molecular inversion probe, dideoxy sequencing, and quantitative polymerase chain reaction assays. RESULTS: Considering transporter polymorphisms associated with resistance to aminoquinolines, the prevalence of Plasmodium falciparum chloroquine resistance transporter (PfCRT) 76T decreased, but varied markedly between sites (0-46% in 2018; 0-23% in 2019); additional PfCRT polymorphisms and plasmepsin-2/3 amplifications associated elsewhere with resistance to piperaquine were not seen. For P. falciparum multidrug resistance protein 1, in 2019 the 86Y mutation was absent at all sites, the 1246Y mutation had prevalence ≤20% at 14 of 16 sites, and gene amplification was not seen. Considering mutations associated with high-level sulfadoxine-pyrimethamine resistance, prevalences of P. falciparum dihydrofolate reductase 164L (up to 80%) and dihydropteroate synthase 581G (up to 67%) were high at multiple sites. Considering P. falciparum kelch protein propeller domain mutations associated with artemisinin delayed clearance, prevalence of the 469Y and 675V mutations has increased at multiple sites in northern Uganda (up to 23% and 41%, respectively). CONCLUSIONS: We demonstrate concerning spread of mutations that may limit efficacies of key antimalarial drugs.

Single cell transcriptional changes across the blood stages of artemisinin resistant K13 580Y Plasmodium falciparum upon dihydroartemisinin exposure.

Artemisinins have been a cornerstone of malaria control, but resistance in Plasmodium falciparum , due to mutations in the Kelch13 (K13) protein, threaten these advances. Artemisinin exposure results in a dynamic transcriptional response across multiple pathways, but most work has focused on ring stages and ex vivo transcriptional analysis. We applied single cell RNAseq to two unsynchronized coisogenic parasite lines (K13 C580 and K13 580Y ) over 6 hrs after a pulse exposure to dihydroartemisinin (DHA). Transcription was altered across all stages, with the greatest occurring at the trophozoite and ring stage in both lines. This response involved the arrest of metabolic processes, support for a dormancy phenomenon upon treatment, and the enhancement of protein trafficking and the unfolded protein response. While similar, the response was consistent across stages in K13 580Y , with enhanced parasite survival to drug induced stress. Increased surface protein expression was seen in K13 580Y parasites at baseline and upon drug exposure, highlighted by the increased expression of PfEMP1 and GARP, a potential therapeutic target. Antibody targeting GARP maintained anti-parasitic efficacy in K13 580Y parasites. This work provides single cell insight of gene transcription across all life cycle stages revealing transcriptional changes that could initiate a dormancy state and mediate survival upon treatment. IMPORTANCE: Single cell RNA sequencing allows deconvolution of the cellular stages of malaria and investigation of their response to treatment conditions. Utilizing two different genetic backgrounds of a key resistance marker to artemisinin treatment, we compared the response to dihydroartemisinin between these genetic backgrounds. We found a distinct transcriptional profile post treatment in both genetic backgrounds, with downregulation of metabolic process genes and upregulation of stress response genes. Comparing these two genetic backgrounds post treatment using traditional differential expression, and a novel computational method called MELD, we found consistent increased expression of GARP and pathogenesis related genes, like PfEMP1 . This study identifies possible gene dependencies of parasite survival post artemisinin treatment, providing targets for inhibiting the dormancy state.