The extended recovery ring-stage survival assay is a scalable alternative for artemisinin susceptibility phenotyping of fresh Plasmodium falciparum isolates.

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Artemisinin partial resistance (ART-R) has emerged in eastern Africa, necessitating regular surveillance of susceptibility of Plasmodium falciparum to artemisinins. The microscopy-based ring-stage survival assay (RSA) provides a laboratory correlate of ART-R but is limited by low throughput and subjectivity of microscopic counts of viable parasites. The extended recovery ring-stage survival assay (eRRSA) replaces microscopy with efficient quantitative PCR (qPCR) readouts but has been studied only with culture-adapted P. falciparum clones. We measured susceptibility to dihydroartemisinin (DHA) after a 6-h incubation with 700-nM DHA, followed by culture without drug, by comparing survival with that of untreated controls by microscopy (the RSA) or qPCR (the eRRSA) and also performed standard growth inhibition (half-maximal inhibitory concentration [IC50]) assays for 122 P. falciparum isolates freshly collected in eastern and northern Uganda from March to July 2022. The median values for RSA survival, eRRSA fold change, and DHA IC50 were 3.0%, 46.2, and 3.2 nM, respectively. RSA percent survival and eRRSA fold changes correlated strongly (Spearman correlation coefficient [rs] = -0.7411, P < 0.0001), with modest associations between the presence of validated P. falciparum Kelch13 ART-R mutations (C469Y or A675V) and RSA (median survival 2.6% for wild type [WT] vs 4.1% for mutant, P = 0.01), or eRRSA (median fold change 63.4 for WT vs 30.9 for mutant, P = 0.003) results. Significant correlations were also observed between DHA IC50 values and both RSA percent survival (rs = 0.4235, P < 0.0001) and eRRSA fold changes (rs = -0.4116, P < 0.0001). The eRRSA is a scalable alternative for phenotyping fresh P. falciparum isolates, providing similar results with improved throughput.