Background: Given the altered responses to both artemisinins and lumefantrine in Eastern Africa, monitoring antimalarial drug resistance in all African countries is paramount. Methods: We measured the susceptibility to six antimalarials using ex vivo growth inhibition assays (IC50) for a total of 805 Plasmodium falciparum isolates obtained from travelers returning to France (2016-2023), mainly from West and Central Africa. Isolates were sequenced using molecular inversion probes (MIPs) targeting fourteen drug resistance genes across the parasite genome. Findings: Ex vivo susceptibility to several drugs has significantly decreased in 2019-2023 versus 2016-2018 parasite samples: lumefantrine (median IC50: 23.0 nM [IQR: 14.4-35.1] in 2019-2023 versus 13.9 nM [8.42-21.7] in 2016-2018, p<0.0001), monodesethylamodiaquine (35.4 [21.2-51.1] versus 20.3 nM [15.4-33.1], p<0.0001), and marginally piperaquine (20.5 [16.5-26.2] versus 18.0 [14.2-22.4] nM, p<0.0001). Only four isolates carried a validated pfkelch13 mutation. Multiple mutations in pfcrt and one in pfmdr1 (N86Y) were significantly associated with altered susceptibility to multiple drugs. The susceptibility to lumefantrine was altered by pfcrt and pfmdr1 mutations in an additive manner, with the wild-type haplotype (pfcrt K76-pfmdr1 N86) exhibiting the least susceptibility. Interpretation: Our study on P. falciparum isolates from West and Central Africa indicates a low prevalence of molecular markers of artemisinin resistance but a significant decrease in susceptibility to the partner drugs that have been the most widely used since a decade -lumefantrine and amodiaquine. These phenotypic changes likely mark parasite adaptation to sustained drug pressure and call for intensifying the monitoring of antimalarial drug resistance in Africa.

The authors have declared no competing interest.

This work was supported by the French Ministry of Health (grant to the French National Malaria Reference Center) and by the Agence Nationale de la Recherche (ANR-17-CE15-0013-03 to JC). JAB was supported by NIH R01AI139520. JR postdoctoral fellowship was funded by Institut de Recherche pour le Développement.

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

Informed consent was not required because the clinical and biological data were collected from the FNMRC database by the common public health mission of all National Reference Centers in France, in coordination with the Santé Publique France organization for malaria surveillance and care. According to article L1221-1.1 of the Public Health Code in France, the study was considered non-interventional research and only required the patient's non-opposition (according to article L1211-2 of the Public Health Code). All data were anonymized before use. The FNMRC got the authorization to use their databases according to the National Data Protection Commission (CNIL) (number 1223103). Human DNA was not analyzed.

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All sequencing data are available under accession no. SAMN42141774 to SAMN42142576 at the Sequence Read Archive (SRA), and the associated BioProject ID is PRJNA1129163. De-identified datasets generated during the current study and used to make all figures are available as supplementary files or tables. Software packages and tools useful when working with MIP data are available at https://github.com/bailey-lab/MIPTools and https://github.com/Mrc-ide/mipanalyzer.