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.

Background Artemether/lumefantrine is the most commonly used artemisinin-based combination treatment (ACT) for malaria in sub-Saharan Africa. Drug resistance to ACT components is a major threat to malaria elimination efforts. Therefore, rigorous monitoring of drug efficacy is required for adequate management of malaria and to sustain the effectiveness of ACTs. Objectives This study identified and described genomic loci that correlate with differences in ex vivo responses of natural Plasmodium falciparum isolates from The Gambia to antimalarial drugs. Methods Natural P. falciparum isolates from The Gambia were assayed for IC50 responses to four antimalarial drugs (artemether, dihydroartemisinin, amodiaquine and lumefantrine). Genome-wide SNPs from 56 of these P. falciparum isolates were applied to mixed-model regression and network analyses to determine linked loci correlating with drug responses. Genomic regions of shared haplotypes and positive selection within and between Gambian and Cambodian P. falciparum isolates were mapped by identity-by-descent (IBD) analysis of 209 genomes. Results SNPs in 71 genes, mostly involved in stress and drug resistance mechanisms correlated with drug responses. Additionally, erythrocyte invasion and permeability loci, including merozoite surface proteins (Pfdblmsp, Pfsurfin), and high-molecular-weight rhoptry protein 2 (Pfrhops2) were correlated with responses to multiple drugs. Haplotypes of pfdblmsp2 and known drug resistance loci (pfaat1, pfcrt and pfdhfr) from The Gambia showed high IBD with those from Cambodia, indicating co-ancestry, with significant linkage disequilibrium between their alleles. Conclusions Multiple linked genic loci correlating with drug response phenotypes suggest a genomic backbone may be under selection by antimalarials. This calls for further analysis of molecular pathways to drug resistance in African P. falciparum.

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2Two-color flow cytometry(2cFCM) is the most accessible method for phenotyping parasite 3 invasion. However, current protocols require samples of field isolates at ~1% parasitemia for 4 assay set-up, which are becoming more uncommon in low transmission settings. Current 5 protocols, therefore, have to be adapted for low parasitemia if the method must have 6 continued applicability in this era of elimination. Optimizing the protocol requires 7 addressing; interference from young uninfected RBCs background fluorescence and biased 8 phenotypes due to limited labeled RBCs availability and/or parasite density per assay. Here, 9 we used SYBR Green I and CTFR Proliferation fluorescent dyes to set-up invasion assays 10 with Plasmodium falciparum 3D7, Dd2 and field isolates cultures (diluted at 0.05% to 2.0% 11 parasitemia) against varying unlabeled to labeled RBC ratios (1:1 to 1:4). We showed that a 12 shorter SYBR Green I staining time of 20 minutes, down from 1hour, minimized background 13 fluorescence from uninfected RBCs (mean= 0.03% events) and allowed 2cFCM to accurately 14 quantify reinvasion for an assay at 0.02% parasitemia. An increase in labeled target RBCs to 15 1:3 per assays significantly increased heterologous reinvasion (p<0.001). This resulted in a 16 10% greater invasion inhibition by enzyme treatments (p<0.05). Strain-specific invasion 17 phenotype could be accurately determined for samples with as low as 0.3% parasitemia. 18 Samples above 0.8% parasitemia were less accurate. These findings show that invasion 19 pathway phenotypes can be obtained for field samples with low parasitemia at greater 20 sensitivity and reproducibility by increasing the proportion of labeled RBCs per assay by at 21 least 2-fold what is in current methods. 22 23 24 25 26 27 28 29 Keywords: Malaria, Flow cytometry, Sensitivity, Low parasitemia, RBC Invasion pathway 30 phenotypes. 31 32 BACKGROUND: 33 Invasion of human red blood cells (RBCs; Erythrocytes) by Plasmodium falciparum 34 merozoite is vital for malaria parasite multiplication and transmission between hosts and it is 35 considered a key target for the development of malaria interventions (Burns et al. 2019). 36 Invasion mechanism makes use of multiple and redundant parasite ligand and erythrocyte 37 receptor combinations (Perkins and Holt 1988; Binks and Conway 1999), whose dynamics 38 are affected by host, transmission rates and environmental factors such as interventions. 39 Studies on parasite invasion mechanisms and invasion phenotypes could, therefore, help in 40 monitoring the effects of interventions and in designing alternative intervention strategies 41 against natural parasite populations (Stubbs et al. 2005; Beeson et al. 2019). Such studies 42 have recently been scaled up by the application of sensitive high throughput two-color Flow 43 Cytometry (2cFCM)-based methods (Theron et al. 2010; Bei et al. 2010; Bei and Duraisingh 44 2015). These methods quantify the total number of successfully invaded merozoites 45 (reinvasion), rather than just the total numb...