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

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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 CVIET haplotype declined significantly from 88.1% (59/67) in 2017 to 35.4% (101/285) in 2024 (p < 0.001). The Pfmdr1 N86Y decreased from 11.6% (8/69) to 6.0% (17/285), while the NFD haplotype increased slightly from 56.9% (37/65) to 61.9% (169/273). No mutations were observed at Pfdhfr I164L or Pfdhps K540E. The Pfdhfr triple-mutant IRNI 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 A437G at 98% in 2017 and 2024. The Pfdhps I431V rose from 8.3% (5/60) in 2017 to 10.5% (29/275) in 2024, whereas Pfdhps A581G remained stable at 8%. The combined Pfdhfr/PfdhpsIRN_G haplotype remained highly prevalent (96.5% vs 94.6%). Aside from the significant decline in CVIET, 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.

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