Flow cytometry-based analysis of artemisinin-resistant Plasmodium falciparum in the ring-stage survival assay

Abstract

The ring-stage survival assay (RSA) is a powerful tool for phenotyping artemisinin-resistant Plasmodium falciparum but requires experienced microscopists to count viable parasites among 10,000 erythrocytes in Giemsa-stained thin blood smears. Here we describe a rapid flow cytometric assay that accurately counts viable parasites among 250,000 erythrocytes in suspension. This method performs as well as light microscopy and can be used to standardize the collection of RSA data between research groups in laboratory and field settings.

FIG 1

Flow cytometry scatterplot and corresponding histograms for DHA-exposed Plasmodium falciparum parasites stained with SYBR green I (SYBR) and MitoTracker Deep Red FM (MTDR). (A) SYBR clearly differentiates infected and uninfected erythrocytes. In histogram overlays of the upper quadrants in panel A, MTDR clearly differentiates viable and pyknotic parasites (B), while SYBR does not (C).

FIG 2

Flow cytometry scatterplots of DHA (A)- and DMSO (B)-exposed parasites. In each plot, the percentage of viable parasites in 250,000 events is shown in the upper right quadrant. (C) In a mode-normalized histogram overlay of these upper quadrants, populations of viable and pyknotic parasites are clearly separated in both samples. Percent survival was calculated by multiplying the ratio of viable parasites in DHA- and DMSO-exposed samples (e.g., 0.11%/2.17% multiplied by 100). (D) Percent survival values for four Cambodian parasite isolates that differ in KH subpopulation (KH1, KH2, KH3, and KH4) and K13-propeller allele (wild type, C580Y, R539T, and Y493H) are shown. For each isolate, percent survival values calculated from microscopy or flow cytometry data were not significantly different. Data from three independent experiments per isolate are shown.