Alternative methods for the Plasmodium falciparum artemisinin ring-stage survival assay with increased simplicity and parasite stage-specificity

Parasites

To evaluate RSA methods P. falciparum lines carrying the K13 propeller polymorphism C580Y, which is associated with prolonged
PCT and approximately 10 % RSA survival 15], 16], and K13 propeller C580 lines, known to have less than 1 % viable parasites after
RSA 8], were chosen. Parasite line 803 contains the K13 C580Y polymorphism and was isolated
from a Cambodian patient presenting an extended PCT while treated with three daily
oral doses of 4 mg/kg artesunate (AS) 7]. Clone GB4, which does not contain any polymorphisms in the Kelch propeller domain
associated with increased PCTs or RSA survival (K13 C580), was isolated from Ghana
prior to widespread use of ART in Africa 20]. Progeny from a genetic cross between 803 and GB4 containing either K13 C580 (clones
36F11, 46G9, 24G11, 39E5, and 34F5) or K13 C580Y (clones 61E8 and 76H10) were included
in the analysis, along with the parental lines.

Culture conditions

Parasite cultures were maintained at 5 % haematocrit between 0.5–3.0 % parasitaemia,
in complete media (cRPMI): RPMI-1640 (KD Medical, MD, USA) containing 25 mM HEPES
and 50 µg/mL hypoxanthine, supplemented with 0.21 % sodium bicarbonate, 20 mg/L gentamicin,
and 1 % Albumax II (Life Technologies, CA, USA). Cultures were incubated at 37 °C
under a 90 % N
₂
, 5 % CO
₂
, and 5 % O
₂
gas mixture. Erythrocytes were purchased from the Interstate Blood Bank (Memphis,
TN, USA), passed through a Sepacell R-500 filter (Baxter, Deerfield, IL, USA) to remove
leukocytes and platelets, washed, re-suspended in RPMI 1640 medium, and stored at
4 °C until use.

Percoll gradient ring-stage survival assay

To perform the 0–3 h RSA protocol developed by Witkowski, Amaratunga, and colleagues
8] (henceforth designated as the Percoll gradient RSA), 300 µL of packed parasitized
RBCs containing 2–3 % mature schizonts (10 well-segmented nuclei) were collected
from a P. falciparum culture previously synchronized twice with 5 % sorbitol (Sigma-Aldrich, MO, USA)
dissolved in distilled tissue culture-grade water (Gibco, MD, USA), 10 min, 37 °C,
over two parasite intra-erythrocytic cycles. The parasites were then incubated in
RPMI-1640 containing 15 U/mL heparin (Sigma-Aldrich), but no other supplements, for
15 min at 37 °C. After incubation, parasites were placed onto a 35/65 % Percoll (GE
Healthcare Life Sciences, Pittsburgh, PA, USA) gradient, centrifuged at 3000 rpm for
10 min (966×g, Eppendorf Centrifuge 5810, rotor A-4-81, no brake). The inter-phase band of late-stage
schizonts was collected and incubated in media with fresh RBCs for 3 h, at 37 °C under
90 % N
₂
, 5 % CO
₂
, and 5 % O
₂
gas mixture (see Additional file 1). This gradient is slightly modified from the original Percoll gradient RSA, as per
recommendation of Chanaki Amaratunga to improve separation (personal communication).
After incubation, some schizonts had yet to invade; thus, parasites were treated with
5 % sorbitol again to remove remaining schizonts and isolate 0–3 h ring-stage parasites.
The parasite mixture was adjusted to 1 % parasitaemia in 20 µl with fresh packed RBCs,
and suspended in 1 mL of cRPMI in a 24-well plate containing either 0.1 % dimethyl
sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) or 700 nM DHA (Sigma-Aldrich,
MO, USA) dissolved in 0.1 % DMSO for 6 h. After drug pulse, cells were washed twice
with 10 mL cRPMI with 3 min centrifugation at 2500 rpm (671×g, 9 acceleration, 5 de-acceleration, Eppendorf Centrifuge 5810, rotor A-4-81). Parasites
were further incubated with drug-free cRPMI for 66 h, when thin blood smears were
prepared, methanol fixed, and stained with 20 % Giemsa (Sigma-Aldrich, MO, USA) for
15 min for independent survival assessment by at least two experienced microscopists.
Evaluation consisted of counting the number of parasitized cells in an estimated 10,000
RBCs and comparing survival to DMSO drug-free incubation.

Filtration ring-stage survival assay

Parasite cultures were twice synchronized with 5 % sorbitol over two parasite cycles
and grown to 40 mL of 5 % haematocrit cultures with at least 2 % parasitaemia. When
the majority of parasites were late-stage schizonts, judged as overtly segmented and
within minutes of bursting (Fig. 1), cultures were collected, centrifuged at 2500 rpm for 3 min (671×g, 9 acceleration, 5 de-acceleration, Eppendorf Centrifuge 5810, rotor A-4-81), combined,
and suspended in 10 mL cRPMI. After transferring to a 12 mL syringe, parasites were
pushed through a 1.2 ?m filter (Pall Corporation, Port Washington, NY, USA) into 200–250 µL
fresh RBCs (packed cells) in 10 mL fresh cRPMI 19]. After filtration, the mixture was incubated for 45 min on a plate shaker (speed
3, Boekel Scientific Orbritron V, model 281111), with a 90 % N
₂
, 5 % CO
₂
, and 5 % O
₂
gas mixture at 37 °C. Cells were then washed with 10 mL cRPMI with 3 min centrifugation
at 2500 rpm (671×g, 9 acceleration, 5 de-acceleration, Eppendorf Centrifuge 5810, rotor A-4-81) to remove
remnants of lysed parasites and RBCs, then further incubated until 3 h post-filtration.
Resulting 3 h ring cultures of 0.07–0.15 % initial parasitaemia were adjusted to 2 %
haematocrit in 1 mL cRPMI in a 24-well plate containing either 0.1 % DMSO or 700 nM
DHA in 0.1 % DMSO and further incubated at 37 °C with gas conditions above (Fig. 2a). After 6 h incubation, cells were washed twice in 10 mL drug-free cRPMI, and then
placed in new wells with 1 mL fresh cRPMI for 66 h cultivation. After incubation,
thin blood smears of each well were methanol fixed and stained with 20 % Giemsa for
15 min, coded for “blind” parasitaemia assessment, and counted independently by two
microscopists, with percent survival adjusted to the DMSO parasitaemia (by counting
the number of parasitized cells in estimated 10,000 RBCs). Given the precise age necessary
for survival of merozoite filtration, the majority of parasites perish in the filtering
process. When too few parasites survive filtration, only low parasitaemias are achieved
by 66 h incubation, even in DMSO control wells. In cases of particularly low DMSO
parasitaemia (0.40 %), the cultures were incubated with fresh cRPMI for an additional
24 h and final smears were made at the 96 h time point.

Fig. 1. Target morphology of segmented schizonts for filtration. a Cultures (1–2 mL packed RBCs) need to be at least 2–3 % mature schizonts for filtration
method to work efficiently. b–d Schizont nuclei are distinct and separated prior to filtration, allowing parasites
to burst upon passing through the filter and enter the assay as viable merozoites

Fig. 2. Preparations for drug pulse in filtration and sorbitol-only assays. a Steps for filtration RSA are depicted. Late-stage, segmented schizonts are collected,
pushed through a 1.2 µm filter, incubated, washed, incubated again, and finally incubated
with either 700 nM DHA in 0.1 % DMSO or 0.1 % DMSO only. b Sorbitol-only RSA involves a single step. Early post-invasion rings (approximately
3 h old) are collected, synchronized with sorbitol to remove remaining schizonts,
and plated at 1 % parasitaemia with either 700 nM DHA in 0.1 % DMSO or 0.1 % DMSO

Sorbitol-only ring-stage survival assay

Parasite cultures were synchronized with 5 % sorbitol twice at exactly 46 h intervals
to tighten synchronicity. When parasites were between very late schizonts and very
early rings, cultures were synchronized with 5 % sorbitol once more to remove remaining
mature forms. Immediately after sorbitol treatment, parasite cultures were adjusted
to 1 % parasitaemia in 20 µL RBCs in 1 mL of cRPMI in a 24-well plate, with either
0.1 % DMSO or 700 nM DHA dissolved in 0.1 % DMSO (Fig. 2b). After 6 h incubation, cells were washed and incubated with drug-free cRPMI for
66 h, after which thin blood smears were prepared, methanol fixed, stained with 20 %
Giemsa for 15 min and read by at least two microscopists. Percent survival was calculated
by counting the number of parasitized cells in estimated 10,000 RBCs and comparing
survival to that of the drug-free DMSO incubation.

Results and discussion

As shown in Fig. 3, all three methods produce comparable results, distinguishing survival of parasites
with K13 C580 and C580Y. With the Percoll gradient and sorbitol-only methods, 1–2 %
of K13 C580 parasites are viable after the DHA pulse, compared to 6–15 % of K13 C580Y
parasites. With the filtration method, the ranges of survival increase, with 1–3 %
of K13 C580 parasites and as much as 20 % of K13 C580Y parasites persisting after
the DHA pulse. Across all three RSA methods, the distinction between high and low
survival remains clear.

Fig. 3. Comparison of results obtained with three RSA methods. The Percoll method is shown
in black, the filtration method in gray, and the sorbitol-only method in pattern. Across the three methods, results are consistent
for each line and the distinction between sensitive and surviving is clear. GB4 and
progeny 39E3, 34F5, 36F11, and 24G11 all contain K13 C580, while 803 and progeny 76H10
and 61E8 contain K13 C580Y. Survival of all K13 C580 lines is significantly less than
those containing the K13 C580Y polymorphism. Values are presented in detail in Additional
file 2

With all three methods producing consistent outcomes, the methodologies can be directly
compared for their respective benefits. Table 1 delineates the distinguishing features of each of the three methods. The two alternative
methods presented here eliminate the need for heparin and Percoll. While the required
number of parasitized cells is increased fivefold in the filtration protocol, it is
reduced tenfold with the sorbitol-only RSA. The filtration RSA may include an additional
24 h incubation in the case of a low initial parasitaemia; however, in response to
a significant burden of conducting RSAs, the time required to set up each assay is
decreased in both alternative methods.

Table 1. Comparison of RSA protocols

The variation observed between experiments involves several possible confounding factors.
In the Percoll and sorbitol-only protocols, variation can be attributed to older rings
and even some mature stages surviving 5 % sorbitol synchronization; older rings of
sensitive lines have been shown to have decreased sensitivity, and mature stages of
both K13 C580 and C580Y lines are highly sensitive to DHA in vitro, thus potentially
skewing results 4], 8]. Despite the heightened stage-specificity of the filtration RSA, variation between
experiments remains similar to the other methods. This variation could occur because
of invasion variability in the 66–90 h post-drug pulse incubation. Measuring survival
24 h after drug pulse would eliminate this variable; however, as the initial parasitaemia
of the filtration method can be as low as 0.07 %, measuring survival prior to one
or even two rounds of invasion can be difficult.

The large number of pyknotic forms present in the 72 h smears after using the Percoll
or sorbitol-only methods may further confound results. Judgment of the viability of
the forms by microscopy introduces an additional level of uncertainty and room for
error. However, the use of flow cytometry for determination of percent survival may
help address this issue 4]. For laboratories without access to flow cytometry, the filtration method eases microscopy
assessment as no pyknotic forms are present in the final smears. A possible explanation
based on the homogeneity of age distribution suggests highly sensitive and 3 h tightly
synchronized rings may simply lyse after drug exposure, whereas slightly older rings
present in Percoll or sorbitol-based methods may become pyknotic. These features offer
confidence in the results produced by the filtration method, as there is less room
for error both in the stage of the exposed parasites and in microscopy assessment.

Others have addressed the need for optimization through highly time-sensitive sorbitol
synchronizations 21]. However, while the involved synchronization improves start times for the Percoll
assay, it still requires large time investments with over a week of long, irregular
hours. In contrast, the proposed alternatives decrease time and resource investment.
With the sorbitol-only method, time investment per assay can be decreased to as little
as 30 min from collecting parasites to starting the drug pulse. The simpler setup
of the assay allows multiple lines to be tested simultaneously with ease, thereby
increasing the efficiency of the protocol even further. The 46 h spacing of sorbitol
synchronizations is based on the specific in vitro cell cycle of the lines presented
here and may not suit the cell cycles of other isolates. Individual laboratories should
address the timing of synchronization to align with the cell cycles of their isolates
and prevent selection of older rings from isolates with shorter cycles.

Apart from improving RSA stage-specificity, the potential precision of the filtration
method opens the door to wider applications of the assay. ART’s mechanism of action
may be related to cell stress responses and delayed cell cycles, investigated through
time-specific drug pulses and carefully tracking parasite growth after ART exposure
14], 22]. For these assays, accurately staging parasites and maintaining a high level of synchronicity
is essential. While estimates of delayed growth are possible through sorbitol synchronized
parasites, the stage-specificity of the filtration RSA provides an accurate platform
upon which to examine growth-related phenomenon.