Very low prevalence of validated kelch13 mutations and absence of hrp2/3 double gene deletions in South African malaria-eliminating districts (2022-2024)

South Africa is one of 25 countries identified by the World Health Organization as having the potential to eliminate malaria in the near future. In response to the emerging threat of antimalarial resistance, South Africa enhanced its surveillance programs to enable the mapping of resistance markers prevalence down to 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-geliminating districts in KwaZulu-Natal and Mpumalanga provinces, were assessed for drug (mutations in the kelch13 , crt , mdr1 , dhfr, and dhps genes) and diagnostic ( hrp 2/3 gene deletions) resistance markers using PCR and sequencing (Sanger and/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 non-synonymous 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 mdr1N 86 (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 double hrp 2/3 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 and southern Africa achieve their elimination goals.