Sustained Ex Vivo Susceptibility of Plasmodium falciparum to Artemisinin Derivatives but Increasing Tolerance to Artemisinin Combination Therapy Partner Quinolines in The Gambia

Abstract

Antimalarial interventions have yielded a significant decline in malaria prevalence in The Gambia, where artemether-lumefantrine (AL) has been used as a first-line antimalarial for a decade. Clinical Plasmodium falciparum isolates collected from 2012 to 2015 were analyzed ex vivo for antimalarial susceptibility and genotyped for drug resistance markers (pfcrt K76T, pfmdr1 codons 86, 184, and 1246, and pfk13) and microsatellite variation. Additionally, allele frequencies of single nucleotide polymorphisms (SNPs) from other drug resistance-associated genes were compared from genomic sequence data sets from 2008 (n = 79) and 2014 (n = 168). No artemisinin resistance-associated pfk13 mutation was found, and only 4% of the isolates tested in 2015 showed significant growth after exposure to dihydroartemisinin. Conversely, the 50% inhibitory concentrations (IC₅₀s) of amodiaquine and lumefantrine increased within this period. pfcrt 76T and pfmdr1 184F mutants remained at a prevalence above 80%. pfcrt 76T was positively associated with higher IC₅₀s to chloroquine. pfmdr1 NYD increased in frequency between 2012 and 2015 due to lumefantrine selection. The TNYD (pfcrt 76T and pfmdr1 NYD wild-type haplotype) also increased in frequency following AL implementation in 2008. These results suggest selection for pfcrt and pfmdr1 genotypes that enable tolerance to lumefantrine. Increased tolerance to lumefantrine calls for sustained chemotherapeutic monitoring in The Gambia to minimize complete artemisinin combination therapy (ACT) failure in the future.

Keywords: alleles; antimalarial agents; artemisinin combination therapies; drug resistance evolution; ex vivo susceptibility; haplotypes; malaria elimination.

FIG 1

In vitro drug susceptibilities of Plasmodium falciparum isolates collected from Brikama (western Gambia) during the transmission seasons from years 2013 to 2015. Each plot shows the log₁₀ of the 50% inhibitory concentration (logIC₅₀) of isolates for a drug grouped per year of isolate collection as labeled on the x axes. Each point is the logIC₅₀ for an isolate against amodiaquine (AMD) (a), lumefantrine (LUM) (b), dihydroartemisinin (DHA) (c), and artemether (ARM) (d). The median for each drug per year is shown as broken red lines. Lines connect pairs of years for which the distribution of IC₅₀s were significantly different at a P value of <0.05 (*). The most significant differences had P values of <0.0001 (****).

FIG 2

In vitro drug susceptibilities of Plasmodium falciparum isolates collected in the year 2015 from Brikama determined by flow cytometry (ACCURI) and IVART for standard inhibitory drug concentrations (IC₅₀) and RSA against dihydroartemisinin. The logIC₅₀ for each isolate is shown for quinolines (chloroquine [CQ], quinine [QN], AMD, and LUM) (a), SP drugs sulfadoxine (SD) and pyrimethamine (PYR) (b), and artemisinin derivatives DHA and ARM (c). (d) RSA. The percentage of infected cells with parasite growth after DHA treatment of ring stages for each isolate is shown. Broken red lines are the median value of the isolates for each assay.

FIG 3

Correlation between drug IC₅₀s (y axes) for LUM (a), QN (b), SD (c), and ARM (d) and the percentage of rings surviving after dihydroartemisinin exposure. Each point corresponds to the IC₅₀ plotted against the percentage of infected cells with parasite growth for an isolate, with the confidence interval of the IC₅₀ shown as bars.

FIG 4

Temporal trends in the frequencies of Plasmodium falciparum drug resistance alleles at chloroquine resistance transporter (pfcrt), multidrug resistance protein 1 (pfmdr1), and calcium-transporting ATPase (pfatpase6) (a), pfcrt-pfmdr1 haplotypes (b), pfmdr1 codons 86-184-1246 haplotypes (c), and biallelic haplotypes at pfmdr1 codons 86, 184, and 1246 (d). Isolates were collected across four transmission seasons (years 2012 to 2015) from Brikama, The Gambia. Each point represents the frequency (y axes) plotted against the year (x axes) isolates were collected. Each line shows the trend for frequencies of alleles or haplotypes in the figure legends.

FIG 5

In vitro sensitivity (IC₅₀) of Plasmodium falciparum isolates collected in the year 2015 for mutant or wild-type alleles at pfcrt K76T codon (a) and pfmdr1-86-184-1246 haplotypes of isolates assayed against quinolines (CQ, QN, LUM, and AMD) and artemisinin derivatives (DHA and ARM) (b). Each point in a group represents the logIC₅₀ for an isolate with the specific allele or haplotype and drug labeled on the x axes. Broken red lines show the median logIC₅₀ values for each haplotype group per drug.