Artemisinin pressure in field isolates can select highly resistant Plasmodium falciparum parasites with unconventional phenotype and no K13 mutation.

Artemisinin resistance, which poses a serious threat to malaria control efforts, is monitored in the field by delayed parasite clearance in patients, elevated parasite survival rate in the ring-stage survival assay, and mutations in the Plasmodium falciparum kelch13 gene. However, sporadic cases of artemisinin-resistant malaria do not meet all of these criteria, highlighting that our understanding of artemisinin resistance is still incomplete. Here, we selected for artemisinin resistance in vitro in nine P. falciparum field isolates from Africa, Asia, and South America. Artemisinin pressure selected highly resistant parasites in all lineages, but only one acquired a validated pfkelch13 mutation. Phenotypic tests evidenced an unconventional response of selected parasites that can be artemisinin-resistant only at an advanced ring age, making them undetectable by the standard "0-3 h" test used to monitor resistance. These results highlight the need for broader approaches to artemisinin resistance monitoring.