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

Artemisinin partial resistance (ART-R) has emerged in eastern Africa, necessitating regular surveillance of susceptibility of 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 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 [IC]) assays for 122 isolates freshly collected in eastern and northern Uganda from March to July 2022. The median values for RSA survival, eRRSA fold change, and DHA IC were 3.0%, 46.2, and 3.2 nM, respectively. RSA percent survival and eRRSA fold changes correlated strongly (Spearman correlation coefficient [] = -0.7411, < 0.0001), with modest associations between the presence of validated Kelch13 ART-R mutations (C469Y or A675V) and RSA (median survival 2.6% for wild type [WT] vs 4.1% for mutant, = 0.01), or eRRSA (median fold change 63.4 for WT vs 30.9 for mutant, = 0.003) results. Significant correlations were also observed between DHA IC values and both RSA percent survival ( = 0.4235, < 0.0001) and eRRSA fold changes ( = -0.4116, < 0.0001). The eRRSA is a scalable alternative for phenotyping fresh isolates, providing similar results with improved throughput.