Figure 1.
Gating for detection of depolarizing parasitized red blood cells in a Plasmodium falciparum culture.
Flow cytometric analysis of an uninfected and a synchronized P. falciparum (3D7) infected culture (1.5% parasitemia) after 24 hours of incubation, stained with SYBR green I. Plots of forward vs. side scatter for the uninfected and infected cultures appear in Figures A and B; corresponding plots of side scatter vs. depolarized side scatter appear in Figures C and D. The gates in Figures C and D identify the depolarizing events. Figures E and F (see text) illustrate gates defining SYBR green-positive parasitized cells. The blue dots on Figure F represent the depolarizing events. Staining with the red blood cell surface marker (CD235) shows that 99.5% of events in a stained sample (red line) exhibit fluorescence above the highest level measured in an unstained control (black line), indicating that the detected events are red blood cells (G). In the SYBR green I histogram (H) of the infected culture, the overall population (black line) shows a distinct peak with a high fluorescent intensity in the third decade. This peak corresponds mainly to the gated population of depolarizing events (pink line). Because SYBR green I intensity correlates with DNA content and thus with parasite level of maturation, the depolarizing population (pink line) consists mainly (79.4%) of mature parasites. The highly red- and green-fluorescent events visible outside the SYBR green gate just to the right of its apex represent contaminating white blood cells among the donor red cells.
Figure 2.
Growth curves of Plasmodium falciparum (3D7) in culture.
Flow cytometric analysis of a synchronized P. falciparum (3D7) culture (1.4% parasitemia). The percentages of depolarizing events (A) and SYBR green I positive events (B) were followed for 48 hours in uninfected (black lines in A and B) and infected red blood cells (red line in A and green line in B). Analysis of depolarizing events (Hz-containing parasitized erythrocytes) shows an increase at 18 hours, peaking at 30 hours (A). SYBR green I positive events (parasitized RBC) remain unchanged at 1.4% until 30 hours, after which a steady increase can be noted (B). Hz detection reflects parasite maturation with increasing amounts of Hz until 30 hours, while the parasitemia remains unchanged (SYBR green I positive events). After 30 hours, increasing SYBR green I positive events indicate replication and presence of immature forms, which explains the decrease observed in the depolarizing population. Each time point represents the mean value of triplicate samples ± one SD. Red blood cell lysis was excluded by absolute cell counts, which remained stable.
Figure 3.
Effect of chloroquine on the growth curve of P. falciparum sensitive and resistant strains.
Synchronous cultures of sensitive (3D7, parasitemia of 1.3%) and resistant (Dd2, parasitemia of 1.4%) P. falciparum strains were incubated for 48 hours with doubling concentrations of chloroquine and analyzed at 6 hourly intervals. The inhibitory effect of chloroquine at higher concentrations (>25 nM) is clearly visible (arrow) after 18 hours of incubation (A). The resistant strain can easily be distinguished from the sensitive strain with growth curves of all drug concentrations being identical to the drug free control (B). Each time point represents the mean value of triplicate samples ± one SD.
Figure 4.
Effect of quinine, mefloquine, artemisinin and a spiroindolone (NITD246) on the growth curve of P. falciparum (3D7).
Synchronous cultures of a P. falciparum 3D7 strain were incubated for 48 hours with increasing concentrations of quinine (A), mefloquine (B), artemisinin (C) and NITD246 (D). In all cases the dose-dependent inhibitory effect of the drugs could already be detected at 18 hours by comparing the treated samples (solid lines) with the drug free control (dotted red line). The curves allowed the determination of IC50 values at 24 hours. Of note, artemisinin at 32 nM showed a 6 hour delayed growth curve, from 18 to 42 hours, with the peak of maturation occurring at 36 hours. Each time point represents the mean value of triplicate samples ± one SD. DF ctrl – drug free control; UI ctrl – uninfected control.
Figure 5.
Effect of artesunate on the growth curve of P. falciparum (3D7) and the effect of 12 hourly renewing of artesunate during incubation.
Synchronous cultures of a P. falciparum 3D7 strain were incubated for 48 hours with doubling concentrations of artesunate (A) or with a single concentration of 8 nM of artesunate for the whole time or renewed at 12 hour intervals (B and C). Figures A and B show detection of Hz (depolarizing events) while Figure C shows detection of SYBR green I fluorescence (DNA in parasites). The inhibitory effect of artesunate was already detectable after 18 hours of incubation (A). Similar to artemisinin (Figure 4C), the growth curve of artesunate at 4 nM showed a 6 hourly delayed growth curve from 18 to 42 hours, including a 6 hour delay in the peak, occurring at 36 hours. Interestingly, the growth curve at 8 nM seemed to show inhibition until 30 hours, when a slight increase was observed (A and B). However, renewing artesunate at 12 hourly intervals eliminates this effect (green line in B). The percentage of SYBR green I positive events remained approximately the same during the 48 hours of incubation (C). This indicates that parasites at the non-renewed 8 nM concentration showed some maturation as indicated by Hz detection but were unable to replicate. Each time point represents the mean value of triplicate samples ± one SD.
Figure 6.
Growth curve of Plasmodium falciparum (3D7) after treatment with pyrimethamine.
Synchronous cultures of a P. falciparum 3D7 strain were incubated for 72 hours with doubling concentrations of pyrimethamine. No inhibitory effect could be observed during the first 48 hours at any concentration (A). However, an inhibition was clearly visible at 72 hours (B). This could be explained by the fact that pyrimethamine is a slow acting drug and its effect can only be detected on the second generation, after 48 hours. Interestingly, the growth curves at all concentrations follow the drug free control after the 30 hour peak, while the curve for 200 nM shows a later peak at 36 hours with a higher number of depolarizing events, compared to the drug free control (A). See discussion for possible explanation. Each time point represents the mean value of triplicate samples ± one SD. DF ctrl – drug free control; UI ctrl – uninfected control.
Table 1.
Inhibitory concentrations (50%) of several antimalarial drugs against P. falciparum 3D7 strain determined by the Hemozoin detection assay at different times of incubation.
Table 2.
Antimalarial activities of several antimalarial drugs determined by the Hemozoin detection assay and the HRP2 assay.