Changes in molecular markers of Plasmodium falciparum drug resistance between 2017 and 2024 in Southern Benin.

Background Benin remains highly burdened by malaria, and data on antimalarial resistance surveillance are limited. This study assesses the evolution of molecular markers associated with antimalarial resistance in southern Benin between 2017 and 2024. Methods We analysed 358 dried blood spot samples collected from children in southern Benin (Kétonou, Kpomè and Klouékanmè) in 2017 and 2024. Molecular markers in the PfKelch13 , Pfmdr1 , Pfcrt , Pfdhfr , and Pfdhps genes associated with antimalarial drug resistance were investigated. Pfcrt haplotypes were characterised using quantitative PCR, while the remaining markers were analysed by nested PCR followed by Sanger sequencing. Results None of the detected non-synonymous PfKelch13 mutations (7/281) was a WHO-validated, candidate or potential marker of artemisinin partial resistance. The Pfcrt CV IET haplotype declined significantly from 88.1% (59/67) in 2017 to 35.4% (101/285) in 2024 (p < 0.001). The Pfmdr1 N86 Y decreased from 11.6% (8/69) to 6.0% (17/285), while the N F D haplotype increased slightly from 56.9% (37/65) to 61.9% (169/273). No mutations were observed at Pfdhfr I164 L or Pfdhps K540 E . The Pfdhfr triple-mutant IRN I haplotype remained nearly fixed over time at 97.0% (64/66) in 2017 vs 96.4% (268/278) in 2024. A similar trend was observed for Pfdhps A437 G at 98% in 2017 and 2024. The Pfdhps I431 V rose from 8.3% (5/60) in 2017 to 10.5% (29/275) in 2024, whereas Pfdhps A581 G remained stable at 8%. The combined Pfdhfr/Pfdhps IRN _ G haplotype remained highly prevalent (96.5% vs 94.6%). Aside from the significant decline in CV IET , none of the other changes was statistically significant. Conclusion Our findings show no mutations compromising ACT efficacy, nor any immediate threat to the continued use of SP. In addition, the observed re-emergence of chloroquine-sensitive genotypes suggests a shift in parasite population dynamics over time. Nevertheless, continuous molecular surveillance remains essential for detecting emerging resistance and informing timely policy decisions.