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 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

Stepwise in vitro screening of MMV pathogen box compounds against Plasmodium falciparum to identify potent antimalarial candidates.

Development of resistance to deployed antimalarial drugs is inevitable and needs prompt and continuous discovery of novel candidate drugs. Therefore, the antimalarial activity of 125 compounds from the Medicine for Malaria Ventures (MMV) pathogen box was determined. Combining standard IC50 and normalised growth rate inhibition (GR50) analyses, we found 16 and 22 compounds had higher potencies than CQ respectively. Seven compounds with relatively high potencies (low GR50 and IC50) against P. falciparum 3D7 were further analysed. Three of these were tested on 10 natural P. falciparum isolates from The Gambia using our newly developed parasite survival rate assay (PSRA). According to the IC50, GR50 and PSRA analyses, compound MMV667494 was most potent and highly cytotoxic to parasites. MMV010576 was slow acting but more potent than dihydroartemisinin (DHA) 72 h after exposure. MMV634140 was potent against the laboratory-adapted 3D7 isolate, but 4 out of 10 natural Gambian isolates survived and replicated slowly despite 72 h of exposure to the compound, suggesting potential drug tolerance and risk of resistance development. These results emphasise the usefulness of in vitro testing as a starting point for drug discovery. Improved approaches to data analyses and the use of natural isolates will facilitate the prioritisation of compounds for further clinical development

Long-distance transmission patterns modelled from SNP barcodes of Plasmodium falciparum infections in The Gambia.

Malaria has declined significantly in The Gambia and determining transmission dynamics of Plasmodium falciparum can help targeting control interventions towards elimination. This can be inferred from genetic similarity between parasite isolates from different sites and timepoints. Here, we imposed a P. falciparum life cycle time on a genetic distance likelihood model to determine transmission paths from a 54 SNP barcode of 355 isolates. Samples were collected monthly during the 2013 malaria season from six pairs of villages spanning 300 km from western to eastern Gambia. There was spatial and temporal hierarchy in pairwise genetic relatedness, with the most similar barcodes from isolates within the same households and village. Constrained by travel data, the model detected 60 directional transmission events, with 27% paths linking persons from different regions. We identified 13 infected individuals (4.2% of those genotyped) responsible for 2 to 8 subsequent infections within their communities. These super-infectors were mostly from high transmission villages. When considering paths between isolates from the most distant regions (west vs east) and travel history, there were 3 transmission paths from eastern to western Gambia, all at the peak (October) of the malaria transmission season. No paths with known travel originated from the extreme west to east. Although more than half of all paths were within-village, parasite flow from east to west may contribute to maintain transmission in western Gambia, where malaria transmission is already low. Therefore, interrupting malaria transmission in western Gambia would require targeting eastern Gambia, where malaria prevalence is substantially higher, with intensified malaria interventions