Genomic Surveillance Reveals Clusters of Plasmodium falciparum Antimalarial Resistance Markers in Eswatini, a Low-Transmission Setting

Elimination of Plasmodium falciparum malaria in Eswatini remains elusive due to ongoing importation and sustained local transmission. The current status of antimalarial and diagnostic resistance in the country remains unknown. Genomic surveillance can complement routine surveillance by characterizing the prevalence and distribution of resistance markers and revealing granular patterns of transmission.

Between March and December 2023, dried blood spots, demographic data, and travel history were collected from individuals testing positive for malaria by rapid diagnostic test (RDT), and symptomatic RDT-negative individuals across Eswatini. Multiplexed amplicon deep sequencing was used to genotype 12 genes associated with antimalarial resistance, detect hrp2 / 3 deletions, estimate genetic relatedness, and detect non- falciparum species.

Data from 437 samples revealed significant clustering of parasites carrying resistance markers. A validated marker of artemisinin partial resistance, kelch13 P553L, was identified in a cluster of 4 infections (0.9% of all samples), likely linked to importation. The dhps / dhfr sextuple mutant haplotype, associated with high-level sulfadoxine-pyrimethamine resistance, was found in 7.8% of the samples, also within clusters. The mdr1 N86 genotype associated with reduced lumefantrine susceptibility was nearly fixed. Over 60% of the infections were polyclonal, with higher complexity observed in imported cases. No hrp2 / 3 deletions were detected; most false-negative RDTs were attributed to subpatent parasitemia. Non- falciparum co-infections were very rare (<1%).

These findings shed light on the dynamics of resistance emergence in low-transmission settings. Integrating routine genomic surveillance into national malaria programs is essential to detect, track, and respond to resistance threats in countries nearing elimination.