Plasmodium falciparum isolates: ex vivo drug response

Abstract

Objectives: While artemisinin-based combination therapies (ACTs) are effective in sub-Saharan Africa, clinical isolates that are refractory to artemisinin derivatives are emerging in East Africa and ACT partner drugs are becoming less effective in West Africa. We investigated the ex vivo responses of Plasmodium falciparum clinical isolates to frontline antimalarials and the contribution of validated molecular markers of antimalarial drug resistance.

Methods: Ex vivo susceptibility was measured for 66 clinical isolates collected from uncomplicated malaria patients. IC50 was measured for dihydroartemisinin, artesunate, lumefantrine, amodiaquine and chloroquine using a SYBR Green I growth inhibition assay. We also assessed known drug resistance-mediating polymorphisms in pfcrt, pfmdr1 and pfkelch13 using Oxford Nanopore amplicon sequencing.

Results: P. falciparum clinical isolates were susceptible to dihydroartemisinin and artesunate. Clinical isolates showed a wide distribution of susceptibility to lumefantrine and amodiaquine, with some parasites having IC50 values above reference cut-offs for resistance to lumefantrine (150 nM) and amodiaquine (60 nM), suggesting decreased drug susceptibility. Ninety-seven percent of the isolates carried WT pfcrt K76 and pfmdr1 N86 alleles, reported to mediate reduced response to lumefantrine and artemether/lumefantrine. pfmdr1 N86 and 184F haplotype was carried by 62.1% of parasites. None of the clinical isolates carried validated pfkelch13 mutations known to mediate artemisinin partial resistance.

Conclusions: Clinical isolates from coastal Ghana remain susceptible to artemisinin derivatives in commonly used ACTs in Ghana. However, we observed lower susceptibility to the ACT partner drugs lumefantrine and amodiaquine, suggesting the emergence of drug-tolerance phenotypes. Consistent surveillance of drug phenotype-genotype is needed to support ACT efficacy in Ghana.

Figure 1.

Distribution of ex vivo responses of P. falciparum clinical isolates to (a) dihydroartemisinin (DHA), (b) artesunate (AS), (c) lumefantrine (LUM), (d) amodiaquine (AQ) and (e) chloroquine (CQ). P. falciparum clinical isolates showed variable ex vivo responses to DHA, AS, LUM, AQ and CQ. Almost all the clinical isolates tested had IC₅₀ values below the resistance cut-offs (indicated by blue line) for DHA, AS, LUM, AQ and CQ. Thick black line indicate median with IQR.

Figure 2.

Temporal trends in ex vivo responses of P. falciparum clinical isolates from 2007, 2013 and 2023.^, There was a minimal increase in the geometric mean IC₅₀ values for dihydroartemisinin and artesunate from 2013 to 2023, and 2007 to 2023, respectively (blue and green lines). There was an increase in the geometric mean IC₅₀ value for lumefantrine and amodiaquine from 2013 to 2023, and 2007 to 2023, respectively (purple and orange lines). There was a decrease in the geometric mean IC₅₀ value for chloroquine from 2007 to 2023 (black line). Artesunate amodiaquine was the only ACT evaluated in the 2007 study, thus no data were present for dihydroartemisinin and lumefantrine.

Figure 3.

Phylogenetic relationship between the phenotyped P. falciparum clinical isolates and distribution of IC₅₀ values of phylogenetic groups. Phylogenetic analysis of the clinical isolates revealed three distinct phylogenetic groups (a). A comparison of IC₅₀s of the three phylogenetic groups revealed no statistically significant difference among the groups for (b) dihydroartemisinin (DHA), (d) lumefantrine (LUM), (e) amodiaquine (AQ) and (f) chloroquine (CQ). There was a statistically significant difference among the phylogenetic groups for (c) artesunate (AS) (group 1 versus group 2, and group 2 versus group 3).

Figure 3.

Phylogenetic relationship between the phenotyped P. falciparum clinical isolates and distribution of IC₅₀ values of phylogenetic groups. Phylogenetic analysis of the clinical isolates revealed three distinct phylogenetic groups (a). A comparison of IC₅₀s of the three phylogenetic groups revealed no statistically significant difference among the groups for (b) dihydroartemisinin (DHA), (d) lumefantrine (LUM), (e) amodiaquine (AQ) and (f) chloroquine (CQ). There was a statistically significant difference among the phylogenetic groups for (c) artesunate (AS) (group 1 versus group 2, and group 2 versus group 3).