A modified two-color flow cytometry assay to quantify in-vitro reinvasion and determine invasion phenotypes at lowPlasmodium falciparumparasitemia

Abstract

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...