Very Low Prevalence of Validated kelch13 Mutations and Absence of Histidine-Rich Protein 2/3 Double Gene Deletions in South African Malaria-Eliminating Districts (2022–2024)

South Africa is one of 25 countries identified by the WHO as having the potential to eliminate malaria in the near future. In response to the emerging threat of antimalarial resistance, South Africa has enhanced its surveillance programs to enable the mapping of the prevalence of resistance markers at the facility level. A total of 4,471 samples, collected between January 2022 and August 2024 from healthcare facilities and during active surveillance in malaria-eliminating districts in KwaZulu-Natal and Mpumalanga Provinces, were assessed for drug (mutations in the kelch13, chloroquine resistance transporter [crt], multidrug resistance 1 [mdr1], dihydrofolate reductase [dhfr], and dihydropterate synthase [dhps] genes) and diagnostic (histidine-rich protein 2/3 [hrp2/3] gene deletions) resistance markers using polymerase chain reaction testing and sequencing (Sanger or targeted amplicon) protocols. Validated markers of artemisinin partial resistance were rare, with the P574L mutation detected as a minor allele in two samples and the P553L mutation present in one sample. Of the 60 additional nonsynonymous mutations detected, the A578S (30/999) and the I494V (13/951) mutations were most prevalent. Almost all parasites assessed carried the crtK76 (99.8%) and mdr1N86 (99.0%) alleles, suggesting susceptibility to chloroquine. The dhfr triple (99.9%) and dhps double (98.2%) mutations associated with pyrimethamine and sulfadoxine resistance, respectively, were close to fixation. No single or double hrp2/3 gene deletions were detected. These findings suggest that the recommended treatments and diagnostics in South Africa are effective. However, the strong selection for antimalarial drug resistance markers across southern Africa, combined with high regional interconnectedness, emphasizes the need for sustained malaria molecular surveillance to support South Africa and the southern Africa region in achieving their elimination goals.