Tolerance of Gambian Plasmodium falciparum to Dihydroartemisinin and Lumefantrine Detected by Ex Vivo Parasite Survival Rate Assay

Abstract

Monitoring of Plasmodium falciparum sensitivity to antimalarial drugs in Africa is vital for malaria elimination. However, the commonly used ex vivo/in vitro 50% inhibitory concentration (IC₅₀) test gives inconsistent results for several antimalarials, while the alternative ring-stage survival assay (RSA) for artemisinin derivatives has not been widely adopted. Here, we applied an alternative two-color flow cytometry-based parasite survival rate assay (PSRA) to detect ex vivo antimalarial tolerance in P. falciparum isolates from The Gambia. The PSRA infers parasite viability by quantifying reinvasion of uninfected cells following 3 consecutive days of drug exposure (10-fold the IC₅₀ of drug for field isolates). The drug survival rate is obtained for each isolate from the slope of the growth/death curve. We obtained parasite survival rates of 41 isolates for dihydroartemisinin (DHA) and lumefantrine (LUM) out of 51 infections tested by ring-stage survival assay (RSA) against DHA. We also determined the genotypes for known drug resistance genetic loci in the P. falciparum genes Pfdhfr, Pfdhps, Pfmdr, Pfcrt, and Pfk13 The PSRA results determined for 41 Gambian isolates showed faster killing and lower variance after treatment with DHA than after treatment with LUM, despite a strong correlation between the two drugs. Four and three isolates were tolerant to DHA and LUM, respectively, with continuous growth during drug exposure. Isolates with the PfMDR1-Y184F mutant variant showed increased LUM survival, though the results were not statistically significant. Sulfadoxine/pyrimethamine (SP) resistance markers were fixed, while all other antimalarial variants were prevalent in more than 50% of the population. The PSRA detected ex vivo antimalarial tolerance in Gambian P. falciparum This calls for its wider application and for increased vigilance against resistance to artemisinin combination therapies (ACTs) in this population.

Keywords: Plasmodium falciparum; antimalarial agents; artemisinin combination therapies; drug resistance; drug tolerance; ex vivo; ex vivo survival rates; flow cytometry; genotypes.

FIG 1

(a) Percent ring survival of 51 isolates determined using conventional microscopy to assess viable parasites and flow cytometry to assess the number of reinvaded parasites following pulse exposure and withdrawal of DHA with RSA. Each point on the plot represents an isolate. The median survival rates of the isolates for each method are shown as the red broken lines. t test statistics gave a P value of <0.0001 using the Wilcoxon rank sum test. (a and b) Correlation analysis of percent ring survival determined using flow cytometry and microscopy with a Pearson correlation coefficient of R = 0.83 and a P value of <0.0001 (b) and correlation analysis of percent ring survival using RSA and parasite survival rates using PSRA (c). The Pearson correlation coefficient gave an R value of 0.53 and a P value of 0.00036. (d) Distribution of the parasite survival rates of 41 isolates treated with DHA and LUM at 3 time points over 72 h with PSRA. Each point shows the rate at which each isolate survived following drug exposure with reference to a DMSO-treated control. The red dotted lines represent the median survival rates for both drug treatments with P = 0.35. A P value of <0.05 represents statistical significance. All ex vivo assays were performed in triplicate.

FIG 2

(a and b) Individual trajectories of 41 isolates following exposure to (a) DHA and (b) LUM relative to DMSO-treated controls at the 24-, 48-, and 72-h time points. A linear mixed-effect model was used, and a linear trend was fitted for each isolate across time points. The dotted blue and red lines show the isolates with decreasing and increasing responses over time, respectively. The thick blue and red lines represent the mean log responses of isolates with decreasing and increasing responses, respectively. (c) Mean predicted parasite responses of all isolates following exposure to DHA (red broken line), LUM (blue broken line), and DMSO control (gray broken line), with the standard errors of the means (SEM) shown as bars. (d) Correlation between parasite survival rates of isolates treated with DHA and LUM with R = 0.77 and a P value of <0.001. All ex vivo assays were performed in triplicate.

FIG 3

Grouped profiles of 41 isolates following exposure to DHA and LUM at 24, 48, and 72 h with PSRA. Each point in the individual plots represent the difference between the predicted response of the (a) DHA-treated and control isolates and (b) LUM-treated and control isolates. The connecting lines give an indication of the response pattern of each isolate. The isolates are grouped based on their response profiles. (i) Linear decrease (−−−). (ii) Nonlinear decrease/increase (−+−). (iii) Linear increase (+++). (iv) Nonlinear increase/decrease (+−+).

FIG 4

(a) Allele frequencies of 41 field isolates for the following drug resistance genes: pfcrt C72/M74/N75/K76, pfmdr1 N86, pfmdr1 Y184, pfdhps S436/A437, and pfk13 C580. (b and c) Rates of parasite survival of (b) DHA and (c) LUM treatment for isolates with wild-type, mutant, and mixed alleles corresponding to pfcrt C72/M74/N75/K76, pfmdr1 Y184, and pfdhps S436/A437. Each point in the graphs represents the parasite survival rate of an isolate. The broken red lines indicate the median survival rates of the isolates with the same alleles.

FIG 5

Schematic representation of ex vivo parasite survival rate assay. (Step 1) iRBCs at 0.5% parasitemia and 2% hematocrit are incubated with 10-fold median IC₅₀s of dihydroartemisinin and lumefantrine for 24, 48, and 72 h. (Step 2) uRBCs are labeled with the intracellular dye DDAO-SE (uRBC^DDAO-SE). (Step 3) Drugs are washed off from the reaction mixtures from step 1 every 24 h, aliquots taken, and drugs replenished. (Step 4) Postexposure drug-free aliquots are incubated with 2× uRBC^DDAO-SE for a further 48 h. (Steps 5 and 6) The aliquots are then counterstained with SYBR green I for flow cytometric analysis (step 5), and 100,000 cells are acquired (step 6). (Step 7) Doubly positive stained cells are analyzed.