A modified two-color flow cytometry assay to quantify in-vitro reinvasion and determine invasion phenotypes at low Plasmodium falciparum parasitemia

Two-color flow cytometry(2cFCM) is the most accessible method for phenotyping parasite invasion. However, current protocols require samples of field isolates at [~]1% parasitemia for assay set-up, which are becoming more uncommon in low transmission settings. Current protocols, therefore, have to be adapted for low parasitemia if the method must have continued applicability in this era of elimination. Optimizing the protocol requires addressing; interference from young uninfected RBCs background fluorescence and biased phenotypes due to limited labeled RBCs availability and/or parasite density per assay. Here, we used SYBR Green I and CTFR Proliferation fluorescent dyes to set-up invasion assays with Plasmodium falciparum 3D7, Dd2 and field isolates cultures (diluted at 0.05% to 2.0% parasitemia) against varying unlabeled to labeled RBC ratios (1:1 to 1:4). We showed that a shorter SYBR Green I staining time of 20 minutes, down from 1hour, minimized background fluorescence from uninfected RBCs (mean= 0.03% events) and allowed 2cFCM to accurately quantify reinvasion for an assay at 0.02% parasitemia. An increase in labeled target RBCs to 1:3 per assays significantly increased heterologous reinvasion (p<0.001). This resulted in a 10% greater invasion inhibition by enzyme treatments (p<0.05). Strain-specific invasion phenotype could be accurately determined for samples with as low as 0.3% parasitemia. Samples above 0.8% parasitemia were less accurate. These findings show that invasion pathway phenotypes can be obtained for field samples with low parasitemia at greater sensitivity and reproducibility by increasing the proportion of labeled RBCs per assay by at least 2-fold what is in current methods.