Development and validation of flow cytometric measurement for parasitemia in cultures of P. falciparum vitally stained with YOYO‐1

Abstract

Background: The need for improved malaria diagnostics has long been recognized. Methods: Human parasitized erythrocytes based on the principles of flow cytometry (FCM) method is described for the determination of parasitemia in Plasmodium falciparum cultures using the fluorescence activated cell sorter and DNA-binding fluorescent dye, YOYO-1. The same assay samples can be also evaluated both microscopically and by scintillation counting after use of 3 H-hypoxanthine-labeled parasites. Results: The counts of uninfected, infected, and nucleated cells occurred with high precision. The cells were categorized into different populations according to their physical or chemical properties such as RNase treatment and compensation required optimization. The detection and quantitation limits in the assay were 0.003% and 0.008% parasitemia, respectively. Overall, the parasite counts by FCM measurement correlated highly (r 2 5

Pharmacokinetic Profiles of Artesunate After Single Intravenous Doses at 0.5, 1, 2, 4, and 8 mg/kg in Healthy Volunteers: A Phase I Study

“…However, because of its poor solubility in water or oils, DHA has only been formulated as an oral preparation and has been used primarily as a semisynthetic compound for derivatization to the oil-soluble drugs, artemether and artemether, and the water-soluble drugs, artesunate (AS). 1 DHA is similar to AS 1 and is 3-to 5-fold more active and more toxic than other artemisinin derivatives. 2,3 It can completely inhibit parasite growth within 2-4 hours and is the only artemisinin derivative with activity against all asexual blood-stage parasites.…”

Flow cytometry as a tool for analyzing changes in Plasmodium falciparum cell cycle following treatment with indol compounds

“…A comparison between microscopic analysis following Giemsa staining and FCM shows a very similar distribution (Fig. 1D) (14,23,24), YOYO-1 presented an excellent discrimination between uninfected and infected RBCs and was able to discriminate between mononucleated and multinucleated stages with high resolution. The fluorescence intensities of multinucleated-stage parasites (Fig.…”

“…Infected erythrocytes with parasitemia between 3 and 7% and hematocrit of 2% were incubated with 0.1 lM or 1 lM of melatonin, 0.1 lM or 1 lM N-acetyl-serotonin (NAS), 0.25 lM or 1 lM serotonin and 0.5 lM or 1 lM tryptamine for 24 h. Parasites then were fixed in 2% formaldehyde in phosphatebuffered saline (PBS) for 24 h at room temperature and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) and 20 lg mL 21 RNAse (Invitrogen Life Technologies), incubated for 15 minutes at 378C and stained with 5 nM YOYO-1 (Molecular Probes) in agreement with previous published methods (14). Parasitemia and proportions of parasites at each stage were determined from dot plots [side scatter (SSC) versus fluorescence] of 10 5 cells acquired on a FACS Calibur flow cytometer using CELLQUEST software (Becton & Dickinson).…”

“…Flow cytometry (FCM) is a powerful method for evaluation of human erythrocyte infection rates as well as for discrimination of Plasmodium falciparum developmental stages (12)(13)(14)(15)(16). Several methods of detection of malaria parasite by flow cytometry have been developed taking advantage of the absence of DNA in erythrocytes.…”

“…Different dyes such as acridine orange (17,18), hydroethidine (19,20), SYTO 16 (21) and thiazole orange (22) were already employed for the determination of parasitemia in cultures of P. falciparum by FCM. Some previous studies using YOYO-1 showed that this dye possesses high affinity for DNA and discriminates infected RBC from the uninfected cells in a very effective way (14,23,24). Melatonin and derivatives mediating effects on the cell cycle of P. falciparum-infected human erythrocytes were studied using YOYO-1 staining followed by flow cytometry analysis.…”