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Antimicrob Agents Chemother. 2016 November; 60(11): 6859–6866.
Published online 2016 October 21. Prepublished online 2016 September 6. doi:  10.1128/AAC.01292-16
PMCID: PMC5075096

Ex Vivo Maturation Assay for Testing Antimalarial Sensitivity of Rodent Malaria Parasites

Abstract

Ex vivo assay systems provide a powerful approach to studying human malaria parasite biology and to testing antimalarials. For rodent malaria parasites, short-term in vitro culture and ex vivo antimalarial susceptibility assays are relatively cumbersome, relying on in vivo passage for synchronization, since ring-stage parasites are an essential starting material. Here, we describe a new approach based on the enrichment of ring-stage Plasmodium berghei, P. yoelii, and P. vinckei vinckei using a single-step Percoll gradient. Importantly, we demonstrate that the enriched ring-stage parasites develop synchronously regardless of the parasite strain or species used. Using a flow cytometry assay with Hoechst and ethidium or MitoTracker dye, we show that parasite development is easily and rapidly monitored. Finally, we demonstrate that this approach can be used to screen antimalarial drugs.

INTRODUCTION

The spread of artemisinin-resistant malaria parasites in Southeast Asia requires the urgent development of new antimalarial therapeutics (1). Currently, the discovery of drugs with activity against Plasmodium falciparum follows a defined pathway (2, 3). Central to this pathway is quantifying the intrinsic susceptibility of a standard laboratory strain of P. falciparum (usually strain 3D7) to the candidate antimalarial drugs, specifically by defining their half-maximal (50%) inhibitory concentration (IC50) in vitro. Lead antimalarial candidates with the lowest IC50s (generally ≤1 μM) are then tested against cultures of a range of adapted isolates (often with well-defined resistance phenotypes) or against ex vivo fresh isolates (2, 4,6). Next, to demonstrate efficacy in vivo, rodent malaria parasite models, in particular, Plasmodium berghei ANKA (7) and P. chabaudi (8) in mice, are used. Unfortunately, it is rare for the IC50s of the candidate drugs for rodent malaria parasites to be determined, thus preventing meaningful comparisons of the effects of the candidate drugs on human parasites and rodent malaria parasites.

In vitro drug screening methods have been developed for P. berghei ANKA. However, P. berghei ANKA parasite infections are generally asynchronous, and therefore, an in vivo synchronization step is required prior to in vitro culture of the parasite (9). Additionally, rodent malaria parasites have different infection profiles, i.e., different lethalities and synchronicities, which are also influenced by the mouse genetic background and immunity (2). As the in vivo result is confounded by the choice of rodent malaria species and strain, as well as the mouse genetic background, we need a simplified ex vivo assay to accurately determine the intrinsic profile of the susceptibility of murine Plasmodium spp. to antimalarials.

In the study described here, we developed a single-step protocol to enrich the ring stages of rodent malaria parasites for short-term in vitro culture that can be used for in vitro screening and measurement of parasite sensitivities to different antimalarial compounds using flow cytometry.

MATERIALS AND METHODS

Mice.

Both female and male C57BL/6J mice were used in this study. All mice (age, 6 to 8 weeks) were housed in a specific-pathogen-free (SPF) environment in the A*STAR Biomedical Resource Centre (Biopolis). The experiments and procedures were approved by the A*STAR Biomedical Resource Centre Institutional Animal Care and Use Committee (IACUC), in accordance with Agri-Food and Veterinary Authority (AVA) rules and the guidelines of the National Advisory Committee for Laboratory Animal Research (NACLAR) of Singapore.

Parasite strains and infection.

A Plasmodium berghei ANKA parasite line (231c1l) expressing luciferase and green fluorescent proteins under the control of the ef1-α gene (kindly provided by Christian Engwerda, QIMR, Brisbane, Australia) (10), P. yoelii yoelii 17X clone YM (kindly provided by Anthony Holder, National Medical Research Institute, London, UK) (11), and P. vinckei vinckei S67 (kindly provided by Irene Landau, Paris, France) (12) were used in this study. P. yoelii yoelii 17X clone YM and P. vinckei vinckei S67 were chosen because they differ in terms of synchronicity and red blood cell (RBC) tropism. P. yoelii yoelii 17X clone YM is asynchronous in vivo, and schizonts of P. yoelii yoelii 17X clone YM allow in vitro merozoite egress and RBC infection (13), in contrast to P. berghei ANKA (14). P. berghei ANKA merozoites have a strong preference for invading and growing inside reticulocytes during the early stages of infection; however, after a number of in vivo cycles, they can invade both reticulocytes and normocytes (15). P. vinckei vinckei S67 displays a synchronous infection in vivo (16) and has a strict tropism for normocytes (17). Stabilates containing infected red blood cells (iRBCs) were free from other pathogens, were generated through in vivo passage in C57BL/6J mice, and were stored in liquid nitrogen (107 iRBCs/ml in Alsever's solution). Mice were injected intraperitoneally with 106 iRBCs for P. berghei ANKA and P. vinckei vinckei S67 or 2 × 105 iRBCs for P. yoelii yoelii 17X clone YM. Parasitemia was quantified by flow cytometry, using 1 μl of whole blood diluted in 100 μl of phosphate-buffered saline (PBS). Staining of the blood samples was done with dihydroethidium (Sigma, Singapore), Hoechst 33342 (Sigma), and anti-CD45 coupled to allophycocyanin (APC) as previously described (18).

Parasite sample processing.

Mice with 2 to 5% parasitemia were anesthetized in an oxygen-rich induction chamber with 2% isoflurane, and blood was collected through retro-orbital puncture and placed into a tube containing 5 ml of PBS solution with heparin. White blood cells (WBCs) were removed from infected heparinized blood samples using an NFW filter (19). Briefly, the blood sample solution was transferred to a 10-ml syringe attached to the NFW filter. The eluate was recovered, and the RBCs were washed twice in PBS solution by centrifugation at 1,500 × g for 5 min. Using the protocol adapted from previous work (20, 21), infected heparinized blood was laid over a 65% Percoll gradient to enrich for ring-stage parasites. The pellet with rings and uninfected red blood cells was washed twice with complete RPMI medium supplemented with 20% fetal bovine serum and 1% penicillin-streptomycin (100 U/ml stock solution; Gibco, Life Technologies). The iRBCs were first diluted in a solution containing uninfected RBCs to reach a final parasitemia of 1 to 3% and were then diluted in complete RPMI medium to obtain a 2% hematocrit.

Drug plate preparation.

The antimalarial drugs chloroquine diphosphate salt (CQ; Mw, 515.9 g/mol), artesunate (ART; Mw, 384.4 g/mol), and methylene blue (Mw, 319.85 g/mol) (all from Sigma-Aldrich) were used for the in vitro assays. ART and methylene blue were dissolved in 70% ethanol, and chloroquine was dissolved in ultrapure water. The stock solutions of each drug were used to prepare dilution series in 70% (vol/vol) ethanol or ultrapure water. The dilutions, ranging from 1 to 8,129 nM for artesunate, 0.7 to 6,057 nM for chloroquine, and 4.5 to 9,770 nM for methylene blue, were distributed in 96-well plates (F8 PolySorp Nunc-Immuno Module). The predosed antimalarial drug plates were covered with TopSeal-A Plus sealing tape (PerkinElmer), dried in a biological safety cabinet overnight, and stored at 4°C until they were ready to use.

Antimalarial drug assay.

The diluted infected blood samples were dispensed (100 μl per well) into plates that had been predosed (coated) with drug (CQ, ART, or methylene blue). In each experiment, wells with uninfected and infected red blood cells without drug were included as negative and positive controls of growth, respectively. The plates were incubated in a gas chamber with a gas mixture of 5% CO2, 5% O2, and 90% N2 at 36.9°C for 16 to 24 h, depending on the Plasmodium species. CQ and ART were tested with all 3 species, while methylene blue was tested only with P. berghei ANKA. At the end of the incubation period, thin blood smears and flow cytometry analysis of the different samples were carried out to assess the maturation of schizonts.

Schizont maturation assessment and determination of IC50s.

Twenty microliters of the in vitro parasite culture was collected to carry out flow cytometry, while the remaining 80 μl was used to do a Giemsa-stained thick smear. Twenty microliters of culture was stained for 30 min at 37°C in the dark with a mixed solution of 80 μl of PBS containing 8 μm Hoechst 33342 (Sigma-Aldrich Pte. Ltd., Singapore) and 5 μg/ml dihydroethidium (Sigma-Aldrich Pte. Ltd., Singapore). After the incubation, 400 μl of cold PBS was added. One hundred thousand events were acquired using an LSR II flow cytometer (Becton Dickinson). The data were analyzed using FlowJo (TreeStar) software and were normalized against those for the control well, which was dried overnight and which did not contain any antimalarial drug.

Although Hoechst is a common dye used to quantify parasites by flow cytometry because it reveals DNA synthesis (22), it does not allow the differentiation of rings from trophozoites. Thus, if a drug blocks the transition of a ring into a trophozoite, a Hoechst dye-based assay may not uncover this effect. Merozoites are known to contain a single mitochondrion-like organelle which is reproduced during schizogony (23). The mitochondrial membrane potential is a key indicator of parasite viability that can be revealed using MitoTracker Red FM (MitoT; Invitrogen, Life Technologies), a dye known to stain the intact mitochondrial membrane (24,26). In some experiments, MitoT was used at a concentration of 200 nM. Since the excitation wavelength of MitoT does not interfere with the Hoechst and ethidium dyes, the combination of the 3 dyes allowed us to successfully monitor schizont maturation (indicated by high levels of Hoechst staining [Hoechsthigh] and MitoT staining [MitoThigh]) (see Fig. S1A and B in the supplemental material). The method for the measurement of the IC50 using microscopy was adapted from a procedure described previously (27). The IC50s and curves were plotted using GraphPad Prism (version 6) software (four-parameter dose-response curve variable slope).

Statistics.

Statistical analyses were performed using Prism (version 6) software (GraphPad Software, Inc.). Samples were subjected to a normality test (the D'Agostino and Pearson omnibus normality test). If the results followed a normal distribution for more than two groups, one-way analysis of variance and Bonferroni's multiple-comparison posttest were used. Otherwise, for data from two groups that failed the normality test, the Mann-Whitney test was used. The geometric IC50s were compared using a Wilcoxon matched-pairs test. The agreement between the P. berghei ANKA IC50s obtained by flow cytometry and those obtained by microscopy were assessed using the method of Bland and Altman (1986).

RESULTS

Single-step enrichment of ring-stage of Plasmodium berghei ANKA.

The isolation and enrichment of late-stage parasites from infected red blood cells were achieved after the depletion of WBCs using NFW filters, followed by enrichment on a 65% Percoll gradient (Fig. 1A). Microscopic examination of Giemsa-stained thin smears showed that rings and uninfected RBC (Fig. 1C) were separated from the late-stage parasites (Fig. 1D) better than they were by initial culture prior to enrichment on a Percoll gradient (Fig. 1B). Flow cytometry analysis confirmed the efficacy of the enrichment procedure. The enriched ring-stage parasites were found in the population with low levels of Hoechst staining (Hoechstlow) and ethidium staining (ethidiumlow), while the late-stage parasites were found in the populations with Hoechstlow and high levels of ethidium staining (ethidiumhigh) and in those with Hoechsthigh and ethidiumhigh, corroborating the findings obtained with the Giemsa-stained thin smear (Fig. 1B to toD)D) and those of a previous study where parasites in the different flow cytometry gates were sorted and smeared on glass slides (18). The proportion of ring forms found by flow cytometry was similar to that found by microscopy using Giemsa-stained slides (see Fig. S2 in the supplemental material).

FIG 1
Single-step ring-stage P. berghei ANKA enrichment. (A) Schematic work flow of single-step ring-stage P. berghei ANKA enrichment using a 65% Percoll gradient. P. berghei ANKA-infected C57BL/6 mice were bled retro-orbitally, and the blood was passed through ...

Flow cytometry-based assay for ex vivo characterization of Plasmodium berghei ANKA schizont maturation and antimalarial sensitivity.

Having demonstrated the ability to enrich the ring stage of P. berghei ANKA parasites, we sought to determine the time required for the different parasite species to mature in vitro. We observed the appearance of schizonts in the culture after 22 h postincubation (Fig. 2A to toC).C). Cultures of the parasite demonstrated that the schizont population (Hoechsthigh ethidiumhigh) remained detectable in the culture for up to 24 h postincubation (Fig. 2D).

FIG 2
Short-term in vitro culture of P. berghei ANKA. Dot plots show schizont maturation after 22 h of culture of naive blood (A), in the parasite culture at 0 h (B), and in the parasite culture at 22 h postincubation (C). (B and C, bottom) The micrographs ...

Since we were able to establish full ex vivo maturation of P. berghei ANKA parasites, we investigated whether the synchronous mature schizonts could be used as a reliable endpoint for an ex vivo susceptibility assay. To test this idea, P. berghei ANKA-infected RBCs were incubated for 22 h with increased concentrations of either artesunate (ART) or chloroquine diphosphate salt (CQ), and the extent of schizont maturation was determined on the next day. After treatment with a high drug concentration, the proportion of schizonts was shown to be reduced compared to that in the control well (Fig. 3A and andB).B). Another antimalarial drug, methylene blue (28, 29), was shown to be as effective as artesunate in its ability to inhibit the maturation of P. berghei ANKA schizonts (Fig. 3C; see also Fig. S3C in the supplemental material). Moreover, the IC50s obtained by flow cytometry and microscopy were similar for the antimalarial drugs ART and CQ (for ART, 15.4 nM [95% confidence interval {CI}, 14.44 to 16.55 nM] by flow cytometry versus 14.61 nM [95% CI, 14.11 to 15.13 nM] by microscopy [see Fig. S3A]; for CQ, 53.78 nM [95% CI, 43.38 to 66.68 nM] by flow cytometry versus 20.11 nM [95% CI, 15.70 to 25.76 nM] by microscopy [see Fig. S3B]). Wilcoxon matched-pairs test analysis showed that there was no significant difference between the IC50s of both ART and CQ obtained by flow cytometry and microscopy (see Fig. S4). Bland-Altman analysis indicated good agreement between the two methods, even though there was a slight bias toward the determination of higher IC50s by the flow cytometry method for P. berghei ANKA (1.13 log10 units) (see Fig. S4). Additionally, when we assessed mitochondrial activity using a marker of viability, MitoT, the IC50s determined using Hoechsthigh MitoThigh parasites (see Fig. S5A) correlated well with those determined using Hoechsthigh ethidiumhigh parasites and those determined by microscopy (see Fig. S5B). Hence, MitoT staining confirmed that the association of Hoechst and ethidium staining is suitable for the accurate quantification of live parasites. Therefore, we decided to use this dye combination in all subsequent experiments.

FIG 3
Assay of inhibition of P. berghei ANKA schizont maturation after drug treatment. (Top) Dot plots with ethidium-Hoechst staining of synchronized P. berghei ANKA parasites before and after treatment with different doses of ART (A), CQ (B), and methylene ...

Flow cytometry-based assay for ex vivo characterization of P. yoelii yoelii 17X clone YM and P. vinckei vinckei S67 schizont maturation and antimalarial sensitivity.

The same approach used to enrich rings of P. berghei ANKA parasites was successfully extended to two other rodent parasites, P. yoelii yoelii 17X clone YM and P. vinckei vinckei S67 (Fig. 4A and and5A,5A, respectively). We determined the optimal time for quantifying schizont maturation (the endpoint for this susceptibility assay). Flow cytometry analysis of in vitro parasite cultures showed that schizonts (Hoechsthigh ethidiumhigh) of P. yoelii yoelii 17X clone YM were detectable in the culture at 14 h postincubation. However, after 18 h of culture, a large proportion of the schizonts had already burst (Fig. 4B). In contrast, P. berghei ANKA and P. vinckei vinckei S67 schizonts were detected after 24 h of culture (Fig. 5B).

FIG 4
Single-step ring-stage P. yoelii YM enrichment. (A) P. yoelii YM-infected C57BL/6 mice were bled retro-orbitally, and the blood was layered on a 65% Percoll gradient. (Top) A thin smear of the blood taken before and after the 65% Percoll gradient enrichment ...
FIG 5
Single-step ring-stage P. vinckei enrichment. (A) P. vinckei vinckei-infected C57BL/6 mice were bled retro-orbitally, and the blood was layered on a 65% Percoll gradient. (Top) A thin smear of the blood taken before and after 65% Percoll enrichment ( ...

Using our optimized flow cytometry assay, we determined the ex vivo susceptibility of the murine malaria parasites to ART and CQ. The mean IC50s of ART and CQ against P. yoelii yoelii 17X clone YM were 63.32 nM (95% CI, 56.80 to 70.59 nM) and 56.35 nM (95% CI, 40.33 to 78.73 nM), respectively (Fig. 4C). For P. vinckei vinckei S67, the mean IC50s were 18.45 nM (95% CI, 13.23 to 25.71 nM) for ART and 49.46 nM (95% CI, 27.84 to 87.87 nM) for CQ. We also checked the validity of our flow cytometry results using microscopy (see Fig. S6 in the supplemental material).

DISCUSSION

Here we demonstrate an optimized ex vivo antimalarial susceptibility assay for murine malaria parasites which uses a simplified single-step ring-stage parasite enrichment technique that can be applied to different rodent Plasmodium spp. The single-step 65% Percoll gradient efficiently separated ring-stage parasites from late trophozoites and schizonts, as confirmed by microscopy and a flow cytometry technique. This single-step method provided the fast and efficient synchronization of P. berghei ANKA infections, without the need for an additional in vivo synchronization step (30). Using enriched ring-stage preparations of P. berghei ANKA, P. yoelii yoelii 17X clone YM, and P. vinckei vinckei S67, we set out to define the culture conditions. This is important, as each species has different durations of cycles, schizogony, and synchronicity, all of which are factors to be taken into account when determining the potency of an antimalarial drug against the different parasite stages (31). The maturation of Percoll-enriched ring parasites was synchronous, regardless of the parasite species used (Fig. 2, ,4,4, and and5),5), although the best timing for harvesting of the culture for optimal schizont maturation was parasite species specific (Fig. 2D, ,4B,4B, and and5B).5B). The ability to monitor blood-stage parasite maturation will greatly facilitate further studies on the pathobiology of murine parasite species, especially for P. berghei and P. yoelii yoelii, for which a range of complementary molecular tools exists.

The principal use of the ex vivo maturation assay coupled with double-color flow cytometry is to determine the drug sensitivity of the different parasite species. We confirmed that the IC50s of ART and CQ for P. berghei ANKA were comparable to those determined in previous studies (see Table S1 in the supplemental material). Furthermore, the IC50s measured by flow cytometry were comparable to those measured by microscopy (see Fig. S2). Data acquisition and analysis were faster by flow cytometry than by microscopy, indicating that flow cytometry may be able to be used for the rapid acquisition of the IC50s of different drugs. The differences between the IC50s of ART and CQ noted were expected, as they reflect the fast parasite killing capability of ART, which has the fastest parasite killing capability of all antimalarial drugs, since ART has a parasiticidal effect on rings that chloroquine and other antimalarials do not have (32). To refine the assay, we added the MitoTracker dye, which measures mitochondrial membrane potential and is thus considered a marker of cell viability. The IC50s determined by three-color flow cytometry using Hoechsthigh, ethidiumhigh, and MitoThigh parasites or Hoechst-ethidium were similar (see Fig. S4A). This demonstrates that Hoechst-ethidium staining can accurately quantify live parasites of the 3 parasite species tested (Fig. 4 and and5;5; see also Fig. S4B).

Finally, we made an interesting observation that rodent malaria species were intrinsically less susceptible to ART than human malaria parasites isolated from Thai patients by a factor of 10 to 60 (see Table S1 in the supplemental material). P. yoelii yoelii 17X clone YM was also more resistant to ART than P. vinckei vinckei S67 and P. berghei ANKA. This was also true for CQ, since all rodent parasites had IC50s higher than those of the human parasites. Similarly, P. berghei ANKA parasites were also less susceptible to methylene blue than human parasites (see Table S1). One possible explanation for the reduced susceptibility of murine versus human malaria parasites is that the cycles of the rodent parasites are shorter (18 to 24 h) than those of the human parasites (~48 h), and thus, the drugs may have less time to act during the maturation time. This difference might be an advantage for drug discovery. Indeed, screening first on rodent parasites rather than on human parasites may identify drugs with a higher potency against human parasites. In addition, the collection of mouse blood is less limiting than the collection of human blood, and our flow cytometry approach can be scaled up for high-throughput assays.

In conclusion, we have developed an efficient method to enrich ring-stage murine parasites that, when coupled with a double-color flow cytometry technique, allows monitoring of blood-stage parasite development and determination of antimalarial drug sensitivity profiles. The method developed in this study will help in the testing of new antimalarial drugs and provide data that may be used for meaningful comparisons with in vivo data obtained for rodent malaria parasites and ex vivo/in vitro data obtained for human malaria parasites.

Supplementary Material

Supplemental material:

ACKNOWLEDGMENTS

This study was funded by Singapore's Agency for Science, Technology and Research.

We thank the Singapore Immunology Network's Flow Cytometry Facility and Mutant Mouse Core for all the assistance provided.

Z.W.C., C.C., B.M., B.R., and L.R. conceived and designed the experiments; Z.W.C. performed the experiments; Z.W.C., C.C., B.M., and L.R. analyzed the data; Z.W.C., C.C., B.R., and L.R. wrote the paper; and C.C., B.M., B.R., and L.R. read and corrected the manuscript.

Funding Statement

This study was funded by Singapore's Agency for Science, Technology and Research Core Grant. Zi Wei Chang is supported by a postgraduate award from the Agency for Science, Technology and Research.

Footnotes

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.01292-16.

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