Ex vivo susceptibility to antimalarial drugs and polymorphisms in drug resistance genes of African Plasmodium falciparum, 2016-2023: a genotype-phenotype association study

Abstract

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.

Funding: 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.

Keywords: Antimalarial drug resistance; IC50 assay; ex-vivo susceptibility; genotype.

Figure 1.. Geographical origin and collection year of isolates.

A) Map of Africa showing the origin of malaria cases imported into France. Colors indicate sample size, and countries with fewer than three isolates are not shown. B) Bar plot showing the temporal distribution of imported malaria cases included in this study.

Figure 2.. Half-maximal inhibitory concentration (IC₅₀) for six antimalarial drugs.

A) Distribution of IC₅₀ for the six antimalarial drugs in 2016–18 and 2019–23. Box plots show the median IC₅₀ (in nM) and interquartile range. Change in susceptibility over time was tested by Mann-Whitney test. P-values were adjusted using Benjamini-Hochberg correction.

Figure 3.. Prevalence of key mutations associated with resistance to different drugs.

A) Prevalence of key mutations in pfcrt, pfdhfr, pfdhps and pfmdr1 genes detected in isolates from this study. B) Prevalence of mutations in the pfkelch13 gene. Green bar plots indicate the prevalence of mutations in the N-terminal-coding domain, while orange bar plots indicate the prevalence of mutations in the propeller domain. SNPs marked with an asterisk (*) are validated or candidate SNPs by WHO. C) Prevalence of the main key mutations in pfcrt and pfmdr1 over time. D) Prevalence of pfcrt 76-pfmdr1 86 haplotypes over time. Color code for the 4 haplotypes: purple, wild-type K76-N86; green, single mutant K76–86Y; blue, single mutant 76T-N86; red, double mutant 76T-86Y. P-values were calculated by Fisher’s exact test.

Figure 4.. Manhattan plot showing the significance of SNPs associated with six antimalarial drugs.

Each dot represents 1 of 362 SNPs with MAF>0·01 colored by chromosome. The x-axis represents the chromosomal location of the SNPs, and the y-axis represents the −log10 of the P-value obtained from the linear regression model analysis. The blue line represents the nominal P-value (P ≤ 0·05), and the red line represents the P-value after Bonferroni correction (P ≤ 1×10⁻⁴). The full list of SNPs associated with IC50 is shown in Table S13.

Figure 5.. Effect of pfcrt 76-pfmdr1 86 haplotypes on IC₅₀ for six drugs.

A) IC₅₀ for mefloquine (MFQ), lumefantrine (LMF) and dihydroartemisinin (DHA) disaggregated by pfcrt-pfmdr1 haplotypes. B) IC₅₀ for chloroquine (CQ), monodesethylamodiaquine (MDAQ) and piperaquine (PPQ) disaggregated by pfcrt-pfmdr1 haplotypes. Haplotypes were built with pfcrt K76T and pfmdr1 N86Y using the major allele per position. n: number of isolates carrying the haplotype. Differences in the IC₅₀ of wild-type (WT|WT, crt-K76|mdr1-N86) versus single mutants (Mut|WT, crt-76T|mdr1-N86), (WT|Mut, crt-K76|mdr1–86Y), and double mutant (Mut|Mut, crt-76T|mdr1–86Y) were calculated with a Pairwise Wilcoxon tests with Benjamini-Hochberg correction. *: p<0·05; **: p<0·001.