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

Artemisinin partial resistance (ART-R) has emerged in eastern Africa, necessitating regular surveillance of susceptibility of <i>Plasmodium falciparum</i> 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 <i>P. falciparum</i> 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 <i>P. falciparum</i> 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 [<i>r</i>_<i>s</i>] = -0.7411, <i>P</i> < 0.0001), with modest associations between the presence of validated <i>P. falciparum</i> Kelch13 ART-R mutations (C469Y or A675V) and RSA (median survival 2.6% for wild type [WT] vs 4.1% for mutant, <i>P</i> = 0.01), or eRRSA (median fold change 63.4 for WT vs 30.9 for mutant, <i>P</i> = 0.003) results. Significant correlations were also observed between DHA IC₅₀ values and both RSA percent survival (<i>r_s</i> = 0.4235, <i>P</i> < 0.0001) and eRRSA fold changes (<i>r_s</i> = -0.4116, <i>P</i> < 0.0001). The eRRSA is a scalable alternative for phenotyping fresh <i>P. falciparum</i> isolates, providing similar results with improved throughput.