Quantification of Plasmodium ex vivo drug susceptibility by flow cytometry

Study site and subjects

The study was conducted at a field laboratory in Timika, Papua Province, Indonesia,
a region where multidrug-resistant P. falciparum and CQ-resistant P. vivax are highly prevalent 10], 30], 31]. Plasmodium species isolates were collected between 2012 and 2015, from patients with malaria
attending an outpatient clinic. Patients with symptomatic malaria were recruited into
the study if they had a microscopically confirmed peripheral parasitaemia with monospecies
of either P. falciparum or P. vivax between 2000 and 80,000 µL
^?1
. Patients were excluded from the study when they were younger than 2 years of age,
had a haemoglobin level below 7 g/dL, or had anti-malarial or antibiotic treatment
during the previous month. After obtaining written informed consent, blood was collected
by venepuncture. Host white blood cells (WBC) were removed by cellulose column filtration
as previously described 32] and packed infected red blood cells (IRBC) were used for the ex vivo drug susceptibility
assay.

Ex vivo drug susceptibility assay

Plasmodium drug susceptibility was measured using a protocol modified from the World Health
Organization (WHO) microtest as described previously 12], 13]. In brief, two hundred microlitres of a 2 % haematocrit blood medium mixture (BMM),
consisting of RPMI 1640 medium plus 10 % matched human serum (P. falciparum) or McCoy’s 5A medium plus 20 % matched human serum (P. vivax), was added to each well of pre-dosed drug plates containing 11 serial concentrations
(two-fold dilutions) of the anti-malarials (maximum concentration shown in parentheses)
CQ (2992 nM), piperaquine (PIP, 1029 nM), mefloquine (MFQ, 338 nM), amodiaquine (AQ,
158 nM), and artesunate (AS, 49 nM). A candle jar was used to mature the parasites
at 37 °C for 35–56 h. Incubation was stopped when 40 % of ring stage parasites had
reached the mature schizont stage (i.e., ?5 distinct nuclei per parasite) in the drug-free
control well as determined by light microscopy (LM).

Anti-malarial compounds

The anti-malarial drugs CQ, PIP, MFQ, AQ, and AS were obtained from the Worldwide
Anti malarial Research Network (WWARN) QA/QC Reference Material Programme 33]. The drugs were prepared as 1 mg/mL stock solutions in distilled water (CQ, PIP,
MFQ, and AQ) or 70 % ethanol (AS). Drug plates were pre-dosed by diluting the compounds
in 50 % methanol, followed by lyophilization, and stored at 4 °C. All drugs tested
were assayed in duplicates. Drug plates were quality controlled by measuring drug
response profiles in the CQ-resistant and CQ-sensitive laboratory strains K1 and FC27,
respectively, using the same method.

Plasmodium falciparum culture strains

Laboratory strains K1 and FC27 were obtained from MR4 (BEI Resources, ATCC Manassas,
Virginia, USA). The parasites were kept in continuous in vitro culture as described
previously 34]. To obtain highly synchronous parasite cultures, sorbitol treatment was applied once
every week as described elsewhere 35].

Quantification of parasites by light microscopy

Thick blood films made from each well were stained with 5 % Giemsa solution for 30 min
and examined microscopically. The number of mature schizonts per 200 asexual stage
parasites was determined for each drug concentration and normalized to that of the
control well.

Quantification of parasites by flow cytometry

Staining of IRBCs for FC

The vital dye hydroethidine (HE, Sigma-Aldrich; excitation
_max
/emission
_max
 = 535/610 nm) was used to stain IRBCs for P. falciparum infections and the nucleic acid dye SYBR Green I (SG, Invitrogen-Molecular Probes;
excitation
_max
/emission
_max
 = 497/520 nm) for P. vivax infections. HE was prepared as 10 mg/mL stock solution in dimethyl sulphoxide (DMSO)
and stored at ?20 °C. SG stock solution (10,000 × concentrate in DMSO) was stored
as per the instruction manual at ?20 °C. The working solution for HE was made by diluting
the HE stock solution 1:250 in phosphate buffered saline (PBS, pH 7.4). The 1× working
solution for SG was made by diluting the SG stock solution 1:10,000 in Tris-saline
buffer (TSB, pH 8.5). Staining was performed by adding 50 µL HE or 50 µL SG working
solution to pelleted IRBCs (i.e., centrifugation at 1500 rpm, for 2 min) from 50 µL
of the 2 % haematocrit BMM (final HE and SG concentration: 0.63 mM and 1:2, respectively)
for 25 min in the dark at 37 °C. After the incubation, IRBCs were washed (i.e., centrifugation
at 1500 rpm, for 2 min) once with 200 ?L PBS, followed by final re-suspension in 200
µL PBS.

To further validate the single stain method, comparison was made with the double staining
method published previously 27] using a subset of isolates obtained within the whole study. For the double staining
method, 20 µL of BMM from each well at harvest were transferred to an empty 96-well
plate and stained with a mixture of 2 ?L HE 10 mg/mL solution, 30 ?L of SG 1× working
solution, and 48 ?L PBS (final HE and SG concentration: 0.63  mM and 1:3.3, respectively).
The mixture was incubated for 20 min in the dark at room temperature. The reaction
was stopped by adding 200 ?L PBS to all wells.

Gating and quantification of parasites by FC

Samples were analysed using a dual-laser (blue: 488 nm, 50 mW solid state; red: 640 nm,
30 mW diode) 3/1 emission detection configuration BD Accuri C6â„¢ cytometer system equipped
with a high-throughput 96-well sampler (BD Accuri CSamplerâ„¢). The blue laser was used
for the detection of both dyes (SG in FL1: 530 ± 15 nm; HE in FL2: 585 ± 20 nm). The
gating strategy is depicted in Fig. 1. First, cells were gated according to their FSC-H/SSC-H profile to exclude debris
from the red blood cell (RBC) population. Gating was then applied on the FSC-A/FSC-H
profile to differentiate cell doublets from single cells. The gated single cell population
was analysed further in either the SSC-H/FL2-H (P. falciparum) or the SSC-H/FL1-H (P. vivax) dot plot profiles. The mature schizont gate was set based on the drug-free control
well and applied to all drug-treated wells. Gating procedure for HE and SG double
staining was applied as described by Russell et al. 27]. For data acquisition, 100,000 events were analysed. In the case of low parasitaemia
(i.e., less than 0.2 %), the number of events analysed was increased to 200,000. Automated
analysis was performed using the BD Accuri CFlow Samplerâ„¢ software.

Fig. 1. Gating strategy for mature schizonts. Red blood cells (RBCs) were identified and gated
based on the forward/side scatter (FSC-H/SSC-H) dot plot in gate ‘RBCs’. The RBCs
were visualized in a FSC-A/FSC-H dot plot to select cell singlets in gate ‘Single
RBCs’. The single RBCs were then analysed in a SSC-H/Hydroethidine-H (FL2-H) dot plot
for P. falciparum and a SSC-H/SYBR Green 1-H (FL1-H) dot plot for P. vivax. Mature schizonts were identified in gate ‘MS’ based on fluorescence intensity and
side scatter values (i.e., increased DNA content and cell complexity) produced by
schizonts compared to other Plasmodium life cycle stages

Statistical analysis

Dose–response data were analysed using nonlinear regression analysis (WinNonLn 4.1;
Pharsight Corporation) and the 50 % inhibitory concentration (IC
₅₀
) derived using an inhibitory sigmoid maximum effect (E
_max
) model. IC
₅₀
ex vivo data were only accepted if the E
_max
and E
₀
of the predicted curve were within 15 % of 100 and 0, respectively. IC
₅₀
data with a high coefficient of variation (100 %) and/or a high sum of squared residuals
(1) were also rejected.

Agreement between quantification methods was assessed by Bland–Altman analysis using
log-transformed IC
₅₀
data obtained with both methods 36]. Wilcoxon signed-rank test or Student’s t-test on log-transformed data was used to assess the difference between paired samples
using the different methods. All statistical analyses were carried out using Stata
(version 13.1, College Station, Texas) and GraphPad Prism software (version 6.0).

Ethics

Ethical approval for this project was obtained from the Human Research Ethics Committee
of the Northern Territory Department of Health and Families and Menzies School of
Health Research (HREC 2010-1396), Darwin, Australia, and the Eijkman Institute Research
Ethics Commission (EIREC-47), Jakarta, Indonesia.