A simple monochromatic flow cytometric assay for assessment of intraerythrocytic development of Plasmodium falciparum

Plasmodium falciparum strain K1 was used in this study. Parasites were maintained as described previously [15]. Briefly, malaria culture medium (MCM) was prepared that consisted of RPMI 1640 (Sigma-Aldrich Corporation, St. Louis, MO, USA) supplemented with 5.96 g/L HEPES, 2 g/L sodium bicarbonate, and 10% heat-inactivated human AB serum. The parasites were cultured in a T-25 flask containing 5% human O+ erythrocytes in MCM in a 5% CO₂ environment at 37 °C. To assess the developmental stages of the studied parasites, a thin blood smear was prepared on a glass slide. Cells were visualized by staining with Giemsa dye prior to observation under light microscope [16].

Parasites were maintained in a synchronicity manner as described previously [17]. Briefly, parasites were allowed to grow to the ring stage, and they could not be older than 10 to 12 h after merozoite invasion. The parasite culture was spun down at 2000 revolutions per minute (rpm) for 5 min. After removal of the supernatant, an equal volume of sterile 5% d-sorbitol in distilled water was mixed with the packed erythrocytes and the mixture was incubated at 37 °C for 10 min. After incubation, the cell suspension was spun down at 2000 rpm for 5 min and then washed three times with RPMI 1640. The parasitaemia and synchronicity were evaluated by counting the infected cells per 1000 erythrocytes on a Giemsa-stained thin blood smear under a microscope. Ninety percent synchronicity was accepted for this experiment. The synchronized parasites were adjusted to 1% parasitaemia with fresh human O+ erythrocytes and cultured in MCM as described above.

Gametocytes were prepared as described previously [18]. Briefly, the parasites were allowed to grow to the ring stage at 3–5% parasitaemia in MCM and then adjusted to 1% ring-stage parasitaemia with fresh human O+ erythrocytes. To induce gametocyte formation, MCM was replaced with a gametocyte-inducing medium, which is MCM consisting of 0.37 mM hypoxanthine (Sigma-Aldrich) and 10% human AB serum without heat inactivation. A 75% volume of the gametocyte-inducing medium was replaced daily. To assess sexual development of P. falciparum, a thin blood smear was prepared on a glass slide and stained with Giemsa dye prior to observation under a light microscope. Gametocytes were identified as described in a published method [19].

Given that no previous study has used VSG (20 mg/mL; Vivantis Technologies, Salangor, Malaysia) for nucleic staining in viable cells, the fluorescent dye concentration was initially optimized. Briefly, cells were suspended in diluted concentrations of VSG (0.5, 1, 2, 5, 10, and 20 μg/mL) in phosphate-buffered saline (PBS) and kept in the dark at room temperature (RT) for 20 min. Cells were then subjected to flow cytometric analysis and cell sorting using a FACS Aria II Instrument (BD Biosciences, San Jose, CA, USA) without cell washing. A threshold of FSC was set at 10,000 to reduce a contamination of cell debris (Additional file 1: Fig. S1). A type of VSG-activating laser and a suitable fluorescence detector of an emitted fluorescent signal were determined. Given that the FACS Aria II is equipped with 488-, 633-, and 375-nm lasers, all three lasers were used for VSG activation. Fluorescence detectors of FITC (500–560 nm), PE (543–627 nm), PE-Texas Red (593–639 nm), PerCP-Cy5-5 (655–735 nm), PE-Cy7 (720–840 nm), APC (640–680 nm), A700 (685–775 nm), APC-Cy7 (720–840), BV421 (400–500 nm), BV510 (500–560 nm), and BV605 (590–630 nm) were used for detection of the emitted fluorescent signals. The flow cytometric data were analysed using FlowJo version 10 software (Tree Star, Inc., Ashland, OR, USA). To increase the accuracy of flow cytometric analysis, non-single cells were excluded by gating according to forward scatter (FSC) and side scatter (SSC) characteristics of cells. Briefly, cells were first gated using forward scatter area (FSC-A) parameter at the X-axis, and then using forward scatter height (FSC-H) parameter at the Y-axis. Cells having the characteristic of FSC-A equal to FSC-H were gated. Then, side scatter width (SSC-W) and side scatter height (SSC-H) were set at the X-axis and Y-axis, respectively, in order to exclude cells having SSC-W^high, which are not single cells. Cells were then further gated according to forward scatter width (FSC-W) and forward scatter height (FSC-H). Cells were sorted into PBS containing 1% fetal bovine serum (FBS) for morphologic analysis.

Cells were affixed to glass slides using a CytoSpinTM4 Cytocentrifuge (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 450 rpm for 7 min, and then rapidly air-dried. Cells were fixed with absolute methanol and stained using a 1:18 diluted Giemsa solution at RT for 30 min. After one wash with running tap water, slides were air-dried and covered with glass coverslips with one drop of mounting solution. Cell morphology was assessed using an Olympus BX53 using an objective lens at 100× magnification. For Giemsa-stained thin film of the culture, a minimum of 100 fields was examined at 100× magnification with oil immersion [20].

To ensure that VSG is able to pass through the cell membrane and bind to the parasite’s nucleic acid, 50–100 μL of VSG-stained cells was dropped onto a glass slide and covered with a thin glass. The VSG-stained cells were observed under a laser-scanning confocal microscope (ECLIPSE Ti-Clsi4 Laser Unit; Nikon Corporation, Tokyo, Japan). Differential interference contrast and a 488-nm argon-ion laser was used for microscopic imaging.

To test the reliability of the VSG-based flow cytometric assay, the parasitaemia estimated from microscopic examination of the Giemsa-stained blood smear (the standard method) and the percentage of VSG+ cells obtained from flow cytometry were compared. Various concentrations of parasitaemia were prepared by diluting the parasitized erythrocytes in a 5% uninfected erythrocyte suspension. Spearman’s rank correlation coefficient was used to assess the strength of association between the standard microscopic assay and VSG-based flow cytometry. For sensitivity testing, culture of P. falciparum was diluted to 0.001% parasitaemia, which is the limit of detection in routine microscopic diagnosis [20], and then stained with VSG as describe above and analysed by flow cytometry.

Dihydroartemisinin (DHA) (Sigma-Aldrich), which is a primary drug for falciparum malaria treatment, was used in this study to induce lethal form of the parasites.

DHA was prepared at a concentration of 700 nM in dimethyl sulfoxide (DMSO) (Sigma-Aldrich) as described in previous study [21]. In short, 2 mg of DHA was resuspended in 2 mL of DMSO and used as stock solution. The stock solution was then diluted fivefold in DMSO to achieve a drug concentration of 200 µg/mL (700 μM). Synchronized ring stages of P. falciparum K1 strain were diluted with 5% haematocrit O cell and MCM to obtain 1% parasitaemia and 2% haematocrit. Twenty µL of DHA solution (700 μM) or DMSO was mixed with 2 mL of MCM to obtain a concentration of 7 μM. Then, 100 µL of 7-μM DHA was mixed with 900 µL of the infected erythrocytes. Therefore, the final concentration of DHA in the culture was 700 nM. To examine dose-dependent effect of DHA, three different concentrations of DHA (350, 700 and 1400 nM) were prepared. The parasites were then exposed to DHA or DMSO (as control) in a 5% CO₂ atmosphere at 37 °C for 24 h. To access time-dependent effect of DHA, the parasites were exposed to 700-nM DHA for 6 h and then cultured in MCM without DHA [21]. The parasites were collected at different time points (12, 24, 36, 48 and 60 h) and subjected to Giemsa dye stain and VSG-based flow cytometry as described above.

Data analysis and graph generation were performed using GraphPad Prism software version 5.0 (GraphPad Software, Inc., San Diego, CA, USA). Results are expressed as mean ± standard deviation (SD) and coefficient of variation (CV). Spearman’s rank correlation coefficient was used to measure the strength of association between standard microscopy and VSG-based flow cytometry. Statistically significant differences were identified using non-parametric Student’s t-test. A p-value less than 0.05 was regarded as being statistically significant.

To ensure that VSG is cell-permeable and that it binds to nucleic acid, a non-synchronized culture of P. falciparum (Fig. 1a) was incubated with VSG dye without fixation and subjected to laser-scanning confocal microscope imaging in which the emitted fluorescent signal of VSG was displayed as green. To deny the possibility of autofluorescence, a sample of unstained P. falciparum-infected erythrocytes was used as a control. There was no green colour observed in the control (Fig. 1b, lower panels). At lower magnification, cells having green colour were observed, and they accounted for 1.9% of total observed cells (Fig. 1b, upper panels). Higher magnification images revealed green colour inside the erythrocytes (Fig. 1b, yellow and blue arrows in middle panels), which suggests cell membrane permeability of VSG. Moreover, the intensity of green colour was shown to vary, with intensity roughly grouped into low or high intensity (Fig. 1b, yellow and blue arrows, respectively). Two green dots were also observed in a single erythrocyte similar to those found in the Giemsa-stained thin blood smear, which suggests multiple infection of P. falciparum. These findings indicate that VSG was able to permeate the P. falciparum-infected erythrocytes.

Given that VSG has never been used for flow cytometry, a type of VSG-activating laser and a suitable fluorescence detector had first to be identified. The concentration of VSG was then optimized. In flow cytometry analysis, non-single cells were excluded by gating according to forward scatter (FSC) and side scatter (SSC) characteristics of cells. Briefly, cells were first gated using FSC-A parameter at the X-axis, and using FSC-H parameter at the Y-axis (Fig. 2a, upper panel). Cells having the characteristic of FSC-A equal to FSC-H were gated. Then, SSC-W and SSC-H were set at the X-axis and Y-axis, respectively (Fig. 2a, middle panel), in order to exclude cells having SSC-W^high, which are not single cells. Cells were then further gated according to FSC-W and FSC-H (Fig. 2a, lower panel). Based on FSC-A and SSC-A, there were two populations: cells having FSC-A lower or higher than 50 K (Additional file 1: Fig. S1A). Both contained P. falciparum-infected and non-infected erythrocytes (Additional file 1: Fig. S1B). Thus, both populations were included for analysis. These initial gating steps aimed to obtain single cells, which increases the accuracy of flow cytometric analysis. Using a FACS Aria II, the 488-nm laser could activate VSG and effectuate emission of a fluorescent signal, whereas the 633-nm and 375-nm laser could not (Fig. 2b). When a detector of FITC fluorochrome (500–560 nm) was used, VSG+ cells (green-coloured lines) could be separated from the unstained cells (magenta-coloured lines). In contrast, when a detector of PE (543–627 nm) and PE-Texas Red (593–639 nm) was used, VSG+ cells (green-coloured lines) overlapped with the unstained cells (magenta-coloured lines), which limited our ability to analyse the parasitized cells. Therefore, it was decided to use the 488-nm laser for VSG activation, and the FITC detector to read the emitted fluorescent signal.

To determine the optimal concentration of VSG, P. falciparum-infected erythrocytes were incubated with 0.5, 1, 2, 5, 10, and 20 μg/mL of VSG. The optimal VSG concentration was determined based on its ability to fractionate P. falciparum-infected erythrocytes from non-infected cells. As shown in Fig. 2c, 20 and 10 μg/mL of VSG were the concentrations that yielded the highest fluorescence intensity in VSG+ cells. Moreover, different intensity of fluorescence was observed in the 20 and 10 μg/mL VSG-stained samples (Fig. 2c, histogram), which is a finding that is consistent with confocal microscopic data. The 200, 100, and 50 μg/mL VSG concentrations were excluded due to an upward shift in the dots on the flow cytometric profile, which suggested an increase in non-specific staining (high background). Microscopic observation of sorted VSG+ cells showed that 10 μg/mL of VSG yielded all stages of intraerythrocytic development of P. falciparum (Fig. 2d). In agreement with Fig. 2d, Giemsa staining of presorted sample showed 10.4% parasitaemia that consisted of 9.8% ring form, 0.1% trophozoites, and 0.5% schizonts, which strongly suggests the accuracy of VSG at a concentration of 10 μg/mL. Therefore, 10 μg/mL of VSG was used for other experiments in this study.

To test that each stage of intraerythrocytic development of P. falciparum could be fractionated based on the intensity of VSG, a non-synchronized culture of malaria parasites was prepared. As a standard method, Giemsa staining of thin blood film showed 14% parasitaemia that consisted of 13% ring form, 0% trophozoites, and 1.1% schizonts (Fig. 3a). The VSG+ cells were separated according to intensity into low, intermediate, or high (hereafter referred to as VSG^low, VSG^intermediate, and VSG^high, respectively) (Fig. 3b), and their morphologies were examined. Schizonts were observed only in VSG^high fraction, and ring forms and growing trophozoites were observed only in VSG^intermediate and VSG^low fraction (Fig. 3c). Moreover, different morphology of the P. falciparum parasites could be observed in VSG^intermediate and VSG^low fraction. The cytoplasm of P. falciparum in the VSG^intermediate fraction was thicker than that in the VSG^low fraction, and it contained malarial pigment (Fig. 3d). These findings were in agreement with microscopically examined Giemsa-stained thin blood film that revealed ring form, trophozoites, and schizonts in the culture, which suggested that this protocol was optimal. Thus, fluorescence intensity of VSG depends on the stage of in vitro malaria development.

To test whether VSG-based flow cytometric analysis could distinguish gametocytes from schizonts, P. falciparum strain K1 was grown in gametocyte-inducing culture medium and performed VSG-based flow cytometric analysis. Cells in VSG^low, VSG^intermediate, and VSG^high fraction were sorted and stained with Giemsa. In the VSG^high fraction, parasitized erythrocytes could be observed having granular distribution of haemozoin that resembled stage-IB gametocytes. Moreover, some were elongated and D-shaped within erythrocytes, which are key characteristics of stage-II gametocytes. Early schizonts having 2 and 6 divided nucleus, and mature schizonts consisting of 14 merozoites were also observed in the VSG^high fraction, whereas ring forms and trophozoites were observed in the VSG^low and VSG^intermediate fractions, respectively (Additional file 2: Fig. S2). Thus, VSG-based flow cytometric assay is not able to distinguish gametocytes from schizonts.

Given the ability of VSG to differentiate intraerythrocytic stages, the study explored whether change in cell granularity is related to the developmental stages of P. falciparum. VSG^low, VSG^intermediate, and VSG^high cells were gated and analysed for SSC-A, which is an indicator of cell granularity. As shown in Fig. 3e, the median of SSC-A increased about 2 times when VSG^low and VSG^intermediate cells developed into VSG^high cells. These results suggest that change in cell granularity is related to intraerythrocytic development of P. falciparum, and that this change can be assessed using VSG-based flow cytometry.

To evaluate the optimized protocol relative to its ability to enumerate parasitized erythrocytes, detection of malaria-infected erythrocytes was performed in a dose-dependent manner. Various concentrations of malaria-infected erythrocytes were prepared. Two-fold dilutions of infected cells were prepared using non-infected erythrocytes as diluent. That analysis revealed that VSG-based flow cytometry could detect malaria-infected erythrocytes in a dose-dependent manner (Fig. 4a). The relative values correlated well between the two assays (r² = 0.75–0.97; p < 0.05). The same results were observed from three independent experiments (CV = 11.2%), indicating the reproducibility of linearity measurement.

To assess the sensitivity of VSG-based flow cytometry, parasitized erythrocytes were diluted to 0.001%, which is the limit of detection in routine microscopic diagnosis [20]. As shown in Fig. 4b, two independent cultures were analysed for each cytometry run. There were 11% and 9% parasitaemia enumerated using Giemsa-based microscopy. The parasites were diluted to 0.001% using non-infected erythrocytes as diluent. The diluted samples having 0.001% parasitaemia were then subjected to flow cytometry analysis. VSG-based flow cytometry was capable of detecting 0.3% and 1.1% of VSG+ cells, which is 300–1000 times higher than the detection rate (0.001% parasitaemia) by Giemsa-based microscopy. Next, the reproducibility of the developed assay for enumeration of low parasitaemia was examined. Three independent settings of malaria culture was prepared and diluted them to 0.01% parasitaemia, which is a minimum value that correlated well with standard microscopic examination (Fig. 4a). All three independent runs of VSG-based flow cytometry were able to detect 0.9 ± 0.2% of VSG+ cells (CV = 22%, Fig. 4c), implying reproducibility comparable to Giemsa-based microscopy (CV = 21.8%) for detection of low parasitaemia.

To examine the variability of VSG-based flow cytometric assay for enumeration and identification of P. falciparum-infected erythrocytes among different sets of parasite culture, parasite culture was prepared on different dates and compared the enumerated values of parasitized cells (mean ± SD) obtained from Giemsa-based microscopic analysis with those obtained from VSG-based flow cytometric analysis (Table 1). There were two types of culture: ring-form and trophozoite predominant. In both types of culture, the CV of the VSG-based flow cytometric assay for enumeration of parasitaemia was relatively lower than that of the microscopic method, which implies lower variability of the VSG-based flow cytometric assay. When analysing the variability of assays according to developmental stage, high CV values were obtained from both Giemsa-based microscopy and VSG-based flow cytometry, which is likely due to low parasitaemia in each developmental stage. Collectively, VSG-based flow cytometry is a reliable, sensitive, and reproducible assay for enumeration of parasitaemia.

Synchronization of P. falciparum development is a common method used in routine culture, and its aim is to obtain a predominant intraerythrocytic stage of parasites. To explore whether VSG-based flow cytometry is capable of assessing synchronicity of P. falciparum development in a routine culture, synchronized and non-synchronized cultures of P. falciparum were prepared (Fig. 5a), stained with VSG, and subjected to flow cytometry analysis. Given the ability of flow cytometry to detect cell size and granularity using respective FSC and SSC, it was hypothesized that synchronized parasites have the same size and granularity, which suggests homogeneity. Thus, a quantile contour plot, which is an effective way to visualize distinct populations regardless of the numbers of cells displayed [22], was selected to assess cell homogeneity. In Fig. 5b, only VSG+ cells were displayed based on their size (as indicated by FSC-A on the X-axis) and granularity (as indicated by SSC-A on the Y-axis). To enhance the visualization of a distinct cell population having various cell size and granularity, histograms of FSC-A and SSC-A are also shown at the top and left side of the contour plots, respectively. Given the ability of contour plot to visualize cells based on the relative frequencies of sub-populations, distinct populations of VSG+ cells were able to be located using vertical and horizontal lines drawn on the contour plots. There were at least three distinct populations observed in the non-synchronized culture (Fig. 5b, left panel), as follows: (1) cells having small size with various granularity (approximately 0–45 K of FSC-A, and 30–170 K of SSC-A); (2) cells having a relatively large size with high granularity (approximately 45–185 K of FSC-A, and 75–170 K of SSC-A); and, (3) cells having a relatively larger size with low granularity (approximately 45–185 K of FSC-A, and 20–75 K of SSC-A). In contrast, only one minor (indicated as 1) and one major (indicated as 2) population were observed in the synchronized culture. They had a similar size (50–150 K of FSC-A), but different levels of granularity (35–240 K of SSC-A) (Fig. 5b, right panel). In the left panel of Fig. 5b, the population of VSG+ cells having less than 45 K of FSC-A was observed only in the non-synchronized culture (indicated as 1), but not in the synchronized culture (Fig. 5b, lower panel). Based on the intensity of VSG and microscopic images (Additional file 3: Fig. S3A), the population number 1, 2, and 3 in the left panel of Fig. 5b are schizonts, trophozoites, and ring forms, respectively. In contrast to observation in the non-synchronized culture, the minor and major populations of the synchronized culture could be separated based on SSC-A, as follows: (1) minor population with SSC-A higher than 160 K, and (2) major population with SSC-A lower than 160 K (Fig. 5b, right panel). Compared to the non-synchronized culture, a population of VSG+ cells having SSC-A higher than 160 K was observed only in the synchronized culture (indicated as 1 in Fig. 5b, lower panel). Based on microscopic images (Additional file 3: Fig. S3B), the VSG+ cells with more than 160 K SSC-A are infected erythrocytes containing multiple (60%) and single (40%) ring forms, and they had VSG intensity of 11,578; whereas, the VSG+ cells with lower than 160 K SSC-A are infected erythrocytes with multiple (35%) and single (65%) ring forms. Disappearance of the population having more than 160 K of SSC-A on the contour plot of the non-synchronized culture (Fig. 5b, left panel) and less than 45 K of FSC-A on the contour plot of the synchronized culture (Fig. 5b, right panel) resulted from different developmental stage of Plasmodium between the two separate cultures. To confirm heterogeneity in the non-synchronized culture, the CV, which is a measure of relative variability, of FSC-A and SSC-A was statistically analysed. Despite statistical non-significance (p > 0.05), the non-synchronized culture tended to have a higher CV for both FSC-A and SSC-A (Fig. 5c), which confirms the relatively high heterogeneity of VSG+ cells. Thus, VSG-based flow cytometry is an effective alternative method for assessing synchronicity of P. falciparum development in erythrocytes.

To demonstrate the use of VSG for assessment of growth inhibitory effect of anti-malarial drug, malaria-infected erythrocytes were incubated with 700 nM DHA for 24 h following a standard assay [21]. The DHA- and DMSO-treated cells were stained with VSG and analysed by flow cytometry. In both the presence and absence of the drug, there were VSG+ cells exhibiting VSG^intermediate and VSG^low (Fig. 5d), which were likely to resemble trophozoite and ring form, respectively. These results showed that the number of VSG+ cells decreased following DHA treatment (Fig. 5d, right panel) compared to that of the DMSO-treated control cells (Fig. 5d, left panel and p = 0.02). The majority of DHA-treated VSG+ cells appear as VSG^low, implying that ring form was predominant. In contrast, both VSG^intermediate and VSG^low cells were observed in the DMSO-treated control (Fig. 5d, left panel), implying that both ring from and trophozoite were present in the culture. Similar to 700-nM DHA treatment, number of VSG+ cells also decreased after treatment with 350 and 1400 nM DHA for 24 h (p = 0.007 and 0.016, respectively); however, there was no difference in number of VSG+ cells among doses (Fig. 5e). Moreover, VSG-based flow cytometry was able to access effect of 700-nM DHA in time-dependent manner (Fig. 5f). According to the VSG-based flow cytometric data, DHA likely inhibited parasite growth. Therefore, the VSG-based flow cytometric assay can be used as an alternative assay for assessment of P. falciparum growth in the presence of anti-malarial drug in vitro.