Measuring Plasmodium falciparum Erythrocyte Invasion Phenotypes Using Flow Cytometry

Abstract

Having the ability to rapidly, accurately, and robustly measure Plasmodium falciparum merozoite invasion is a critical component in effective assessment of a blood stage vaccine's mechanism of action. Being able to measure invasion of erythrocytes accurately, objectively and in a high throughput fashion is of critical importance. Here, we describe a simple and robust flow cytometry method that allows for the measurement of the key invasion parameters of parasite multiplication rate and erythrocyte selectivity-both important determinants of disease severity-from the schizont to the ring stage of the parasite's life-cycle, thus separating invasion from growth of the parasite. Importantly, this method is able to accurately detect low levels of parasitemia and heterogeneity within the population that can be missed by enzymatic methods. Lastly, this method has been successfully adapted and employed in field based research settings for parasitemia measurements in vivo, ex vivo, and in vitro and to measure invasion inhibition by antibodies and the use of alternative pathways for invasion.

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

“…Two-color FCM is the most widely used method for phenotyping parasite invasion pathways as it offers minimal handling of donor infected culture. However, current protocols require samples of field isolates at ∼1% parasitemia for assay set-up (Bei and Duraisingh 2015), which are more uncommon in low transmission settings. This study presents a modification of current protocols to enable the determination of invasion pathways for field isolates at low parasitemia with improved sensitivity and reproducibility.…”

“…SYBR Green I has a low binding affinity for RNA and so, will preferentially bind DNA given the shortest time possible. This study protocol reported a reduced parasite DNA staining time with SYBR Green 1 down to 20minutes, from 1 hour used in the conventional protocols (Theron et al 2010; Bei and Duraisingh 2015). The limit of quantification of reinvasion parasitemia was accurate to 0.05% parasitemia from a 1:1 assay of 0.02% well parasitemia (>2-fold increase after a single cycle of growth).…”

“…Studies on parasite invasion mechanisms and invasion phenotypes could, therefore, help in monitoring the effects of interventions and in designing alternative intervention strategies against natural parasite populations (Stubbs et al 2005; Beeson et al 2019). Such studies have recently been scaled up by the application of sensitive high throughput two-color Flow Cytometry (2cFCM)-based methods (Theron et al 2010; Bei et al 2010; Bei and Duraisingh 2015). These methods quantify the total number of successfully invaded merozoites (reinvasion), rather than just the total number of parasitized erythrocytes (growth).…”

“…Conventional 2cFCM combines equal (1:1) proportions of infected unlabeled donor RBCs with uninfected fluorescently labeled target RBCs (pre-treated with neuraminidase, trypsin and chymotrypsin to remove various surface receptors) to specifically quantify parasite reinvasion into target RBCs (heterologous reinvasion) over a single erythrocytic cycle. Parasitized target RBCs are then detected and counted by flow cytometry following parasite nucleic acid staining with dyes such as SYBR Green I (Theron et al 2010; Bei and Duraisingh 2015). However, current protocols are optimized for testing malaria samples with ∼1% parasitemia (pct), a level becoming less common in low transmission settings following massive interventions for malaria elimination.…”

“…This will require; (a) reducing the high background fluorescence from nucleic acid-containing uninfected (unRBCs) by nucleic acid staining dyes like SYBR Green I (Theron et al 2010), (b) minimizing autologous reinvasion, which can potentially create a bias in the measured phenotype. Autologous reinvasion is high with the high proportion of unlabeled donor unRBCs present in 1:1 ratio protocols (Bei and Duraisingh 2015). (c) determining the lower threshold of parasitemia for invasion phenotyping given that parasite invasion is highly strain-specific and parasite density-dependent (Rovira-Graells et al 2016).…”