Isolation and characterization of Plasmodium falciparum blood-stage persisters by improved selection protocols using dihydroartemisinin alone.

Artemisinin-based combination therapies (ACTs) are vital for malaria treatment, but these are threatened by blood-stage persisters-dormant forms of Plasmodium parasites that can survive drug exposure and cause recrudescent infections. Here, we present improved protocols for efficient preparation of pure Plasmodium falciparum persister populations without the need for magnetically activated columns, sorbitol exposure, or prolonged manipulations. Our protocols transformed actively replicating parasites into persister populations by exposing mixed blood-stage parasites to three or four consecutive daily 6 h pulses of 700 nM or 200 nM dihydroartemisinin (DHA). In micrographs of Giemsa-stained cells, we observed different persister morphologies: Type I persisters containing a rounded magenta-stained nucleus accompanied by a local region of blue-stained cytoplasm; and the more-prevalent Type II persisters characterized by a dark round or irregular-appearing nucleus and faded or no detectable cytoplasm. We also observed cells with disorganized nuclear and cytoplasmic structure, suggesting possible autophagic processes of destruction and remodeling. Recrudescence of actively replicating parasites to starting parasitemia or higher occurred around 17-22 days after initial DHA exposure. Differential expression patterns of the acetyl CoA carboxylase (acc) and skeleton binding protein 1 (sbp1) genes during DHA treatment, dormancy, and recrudescence highlighted the evolution of physiologic states and metabolic changes underlying persister formation and recovery. Our findings suggest hypotheses and questions for further research to understand the cellular pathways of dormancy and uncover strategies to thwart parasite survival after drug exposure.