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.

Thymoquinone (TQ), 2-isopropyl-5-methyl-1,4-benzoquinone, a natural product isolated from Nigella sativa L., has previously been demonstrated to exhibit antiproliferative activity in vitro against a range of cancers as well as the human malarial parasite Plasmodium falciparum. We describe here the synthesis of a series of analogues of TQ that explore the potential for nitrogen-substitution to this scaffold, or reduction to a hydroquinone scaffold, in increasing the potency of this antiproliferative activity against ovarian cancer cell lines and P. falciparum. In addition, alkyl or halogen-substituted analogues were commercially sourced and tested in parallel. Several TQ analogues with improved potency against ovarian cancer cells and P. falciparum were found, although this increase is suggested to be moderate. Key aspects of the structure activity relationship that could be further explored are highlighted.

Ethnopharmacological relevance and aim: A decoction of the bark of Cylicodiscus gabunensis Harms is used as a traditional medicine in the treatment of malaria in Nigeria. This study aims to validate the antimalarial potency of this decoction in vitro against Plasmodium falciparum and define potential bioactive constituents within the C. gabunensis bark. Materials and methods:A bioassay-guided separation and fractionation protocol was applied to C. 4

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.