Tolerance of Gambian Plasmodium falciparum to Dihydroartemisinin and Lumefantrine detected by Ex vivo Parasite Survival Rate Assay

Monitoring of Plasmodium falciparum sensitivity to antimalarial drugs in Africa is vital for malaria elimination. However, the commonly used ex-vivo/in-vitro IC50 test is inconsistent 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-colour flow-cytometry based parasite survival rate assay (PSRA) to detect ex-vivo antimalarial tolerance in P. falciparum isolates from The Gambia.PSRA infers parasite viability from quantifying re-invasion of uninfected cells following 3 consecutive days of drug exposure (10-fold the IC50 drug concentration of field isolates). The drug survival rate for each isolate is obtained from the slope of the growth/death curve. We obtained PSRA of 41 isolates for DHA and LUM, out of 51 infections tested by RSA against DHA. We also determined the genotypes for known drug resistance genetic loci in Pfdhfr, Pfdhps, Pfmdr, Pfcrt and Pfk13 genes.The PSRA for 41 Gambian isolates showed faster killing and lower variance by DHA compared to LUM, despite a strong correlation between both drugs. Four and three isolates were respectively tolerant to DHA and LUM, with continuous growth during drug exposure. Isolates with the PfMDR1-Y184F mutant variant had increased LUM survival though this was not statistically significant. Sulphodoxine/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 increased vigilance against resistance to ACTs in this population.