Paper Information

The elimination of Plasmodium falciparum malaria in Eswatini remains elusive because of 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 who tested positive for malaria on a rapid diagnostic test (RDT) and from symptomatic RDT-negative individuals across Eswatini. Multiplexed amplicon deep sequencing was used to genotype 12 genes associated with antimalarial resistance, detect histidine-rich protein (hrp) 2/3 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 four infections (0.9% of all samples), likely linked to importation. The dihydropteroate synthase/dihydrofolate reductase sextuple mutant haplotype, associated with high-level sulfadoxine-pyrimethamine resistance, was found in 7.8% of the samples, also within clusters. The multidrug resistance 1 N86 genotype associated with reduced susceptibility to lumefantrine was nearly fixed. More than 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 coinfections were 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 for detecting, tracking, and responding to resistance threats in countries nearing elimination.