The extended recovery ring-stage survival assay provides a superior association with patient clearance half-life and increases throughput

To evaluate the eRRSA methods, P. falciparum isolates with varying kelch13 mutations and PC_1/2 were chosen. These isolates were derived from cloning by limiting dilution from patient samples. A total of 15 parasite isolates were chosen, 9 of which have kelch13 mutations (including one C580Y mutant, the most common kelch13 mutation found in Southeast Asia currently), and a PC_1/2 distribution between 1.67 and 9.24. All 15 parasite isolates were isolated from patients on the Thailand-Myanmar border between 2008 and 2012. 3D7 was used as a control for comparison to the 15 Southeast Asian isolates (Table 1) [18, 19].

Plasmodium falciparum isolates were cultured using standard methods in human red blood cells (RBC) (Biochemed Services, Winchester, VA and Interstate Blood Bank, Memphis, TN) suspended in complete medium (CM) containing RPMI 1640 with l-glutamine (Gibco, Life Technologies.), 50 mg/L hypoxanthine (Calbiochem, Sigma-Aldrich), 25 mM HEPES (Corning, VWR, 0.5% Albumax II (Gibco, Life Technologies.), 10 mg/L gentamicin (Gibco, Life Technologies) and 0.225% NaHCO₃ (Corning, VWR) at 5% haematocrit. Cultures were grown separately in sealed flasks at 37 °C under an atmosphere of 5% CO₂/5% O₂/90% N₂.

Parasites were synchronized using a density gradient method as previously described with slight modifications [14, 15, 20]. Briefly, 350 μl of packed, infected erythrocytes at high schizogony (> 50% schizonts) was suspended in 2 ml of RPMI. Cultures were layered over a single 70% Percoll (Sigma-Aldrich) layer in 1 × RPMI and 13.3% sorbitol in phosphate buffer saline (PBS) and centrifuged (1561×g for 10 min, no brake). The top layer of infected late stage schizonts was then removed and washed with 10 ml of RPMI twice. Cultures were then suspended in 2 ml of CM at 2% haematocrit and placed in culture flasks on a shaker in a 37 °C incubator for 4 h to allow for re-invasion.

Four hours after Percoll synchronization (unless noted otherwise), samples were measured by flow cytometry as previously described with slight modifications to determine parasitaemia [14, 16]. Briefly, 80 μl of culture and an RBC control incubated for at least 8 h at 2% haematocrit in CM were stained with SYBR Green I (SYBR) and SYTO 61 (SYTO) and measured on a guava easyCyte HT (Luminex Co.). Analysis was performed with guavaSoft version 3.3 (Luminex Co.). 50,000 events were recorded for both the RBC control and samples to determine relative parasitaemias.

Two hours post-cytometric quantitation (or 6 h after Percoll synchronization) samples whose stage composition was > 70% rings as determined by flow cytometry were diluted to 2% haematocrit and 0.5% parasitaemia (unless otherwise noted), and 200 μl of culture was aliquoted into 6 wells of a flat bottom 96-well plate. Each treated and untreated sample had three technical replicates: RBC controls were aliquoted into 2 wells at 2% haematocrit and 200 μl. Three wells of parasites and 1 well of the RBC control were treated with 700 nM dihydroartemisinin (DHA) (Sigma-Aldrich); an additional 3 wells of parasites and 1 well of RBC control was treated with 0.02% dimethyl sulfoxide (DMSO) (ThermoFisher) as untreated controls. Parasites were incubated for 6 h, and then washed three times with 150 μl of RPMI to remove drug. Samples were then suspended in CM and placed back in the incubator. Sixty-six hours after drug removal, 20 μl of sample from each well was collected and frozen for qPCR amplification (72 h sample) without any media changes. Plates were then placed back in the incubator for another 48 h, after which 20 μl of sample was again collected and frozen for qPCR amplification (120 h sample).

Ring-stage samples were quantified at 72 and 120 h post-drug treatment. qPCR was performed using the Phusion Blood Direct PCR kit (ThermoFisher, cat # F547L), supplemented with 1 × SYBR. Three microlitres of a 1:3 culture dilution was used in a 10 μl reaction and amplified using forward and reverse primers of the pfcrt gene. PCR amplification was measured using the fast mode of the ABI 7900HT, with a 20 s denaturation at 95 °C, followed by 30 cycles of 95 °C for 1 s, 62.3 °C for 30 s, and 65 °C for 15 s (Additional file 1). Cycle threshold (Ct) values were calculated using the ABI SDS 2.4.1. Fold change (2^ΔCt) was calculated by determining the average ΔCt for the three technical replicates for the untreated and treated samples by applying the following equation:All statistics were performed and figures generated using GraphPad Prism version 8.2.1.

Percent proliferation is the standard RSA measurement to determine whether a parasite is artemisinin resistant or sensitive. This is calculated by dividing the percent parasitaemia in the treated (DHA) sample over the percent parasitaemia in the untreated (DMSO) sample. Parasitaemia is determined either by counting the number of viable and nonviable parasites using blood smears and microscopy or by flow cytometry. Determining parasitaemia with microscopy is cost effective and convenient but is also highly variable and time-consuming. Flow cytometry is typically very accurate; however, the process must begin upon reaching a time point, adding a substantial time investment at the point of sampling. To find another measurement of parasitaemia that could be automated and have less variability, qPCR on parasite genomic DNA was tested. A standard curve of percent parasitaemia (as measured by flow cytometry) shows excellent inverse correlation with Ct values as measured by qPCR (Fig. 1). To quantify the difference between treated and untreated final RSA samples, fold change (2^ΔCt) was calculated according to Eq. 1.

The standard RSA determines parasite viability at 72 h after drug treatment. However, a common problem in the final readout (using either microscopy, flow cytometry, or qPCR) is the difficulty in differentiating between pyknotic (nonviable) parasites and viable parasites 72 h after drug treatment. It is also difficult to measure viable parasitaemia when it can be as low as 0.01% (or even 0% in some cases), especially when measuring ART sensitive parasites [21]. To address these issues, the time to readout was extended by an additional intraerythrocytic development cycle (48 h); previous methods have extended the time to readout by 24 h in specific cases [13, 22]. This extension was added to both allow parasites an additional expansion cycle, creating larger differences to distinguish resistant and sensitive isolates, and to allow erythrocytes to clear pyknotic parasites. This additional cycle provides a much greater separation between resistant and sensitive parasite isolates (Fig. 2).

The RSA is a growth assay targeted at a very narrow window of the parasite intraerythrocytic development cycle. In order to maximize the precision of the assay, the growth and the timing of the target window were carefully optimized. First, as it has been established that growth rates can vary based on parasitaemia, the effect of varying starting parasitaemias on RSA outcome was observed [23]. RSA was performed on three parasite isolates (3D7, 1337, and 4673) at varying starting parasitaemias as determined by a microscopist, and samples were collected at 72 h and 120 h (Additional file 2A, B, respectively). For the three parasite isolates tested, 3D7 was the ART sensitive control and 1337 and 4673 were two ART resistant parasite isolates as determined by their PC_1/2 values (1337 PC_1/2 = 7.84 and 4673 PC_1/2 = 5.34). The 0.25% starting parasitaemia showed the most distinguishable phenotype between the ART sensitive 3D7 control and the two ART resistant isolates at 120 h (Additional file 2B). It was noted internally that determinations of parasitaemia by microscopy varied widely and underestimated parasitaemia compared to flow cytometry, likely due to the difficulty of correctly identifying new invasions. A comparison of RSA results from isolates set up at 0.25% parasitaemia determined by microscopy and 0.5% parasitaemia determined by flow cytometry showed no difference between the two (Additional file 3). As a result, in subsequent setups starting parasitaemia was determined by flow cytometry and was normalized to 0.5% parasitaemia.

A key factor in the RSA is applying the drug treatment in the tight 0–3 h window of the parasite life cycle that can differentiate ART resistant parasites from ART sensitive parasites. Therefore, the time from Percoll synchronization to drug treatment was also varied to find the optimal time for drug treatment. The same three parasite isolates, (3D7, 1337, and 4673), were set up and treated with 700 nM DHA 4 h, 6 h, 8 h, or 10 h after Percoll synchronization and samples collected at 72 h and 120 h post-drug treatment (Additional file 2C, D, respectively). All parasites were set up at a starting parasitaemia of 0.5% as measured by flow cytometry. The time that resulted in the most consistent and distinguishable phenotypes between the ART sensitive and resistant isolates was 6 h post-Percoll synchronization. Controlling these factors resulted in a more consistent and reproducible RSA phenotype.

Based on the previously described optimizations, the eRRSA is defined as a single layer Percoll synchronization, flow cytometry measurement of parasite parasitaemia and stage prior to assay setup, 700 nM DHA treatment of 200 μl of parasites at 2% haematocrit and 0.5% parasitaemia in 96-well plates that are set up at 6 h post-Percoll synchronization, drug washed off 6 h after application, and samples for qPCR readout collected at 120 h post-drug treatment (Additional file 4). This extension to 120 h was added to both allow parasites an additional expansion cycle to recover from drug treatment, creating larger differences to distinguish resistant and sensitive isolates, and to allow erythrocytes to clear pyknotic parasites. This protocol provides the most consistent and higher throughput results. These modifications made to the standard RSA are a new in vitro ART resistance phenotyping method: the Extended Recovery Ring-stage Survival Assay (eRRSA).

The RSA was introduced as an in vitro method that better captures the gold standard for in vivo ART resistance, PC_1/2, than the traditional IC₅₀. For a new assay to be relevant, it should perform at least comparably to the existing standard. Therefore, the eRRSA was used to assay 15 isolates from Southeast Asia with varying known PC_1/2 and kelch13 mutations collected between 2008 and 2012 (Table 1). NHP1337 was used as a resistant parasite isolate control and 3D7 was used as a sensitive parasite control. Three biological replicates were collected (each with three technical replicates) for each isolate and collected samples at 72 h and 120 h post-drug treatment and compared the viability of treated and untreated parasites at each stage.

The fold change data was then compared to PC_1/2: at the 72 h timepoint across 15 isolates, the eRRSA has a Spearman correlation coefficient of − 0.6071 (Fig. 3a). This is comparable to other RSA correlations in the field, showing that the improvements made to the RSA protocol do not significantly affect the outcome while increasing efficiency and ease of the assay [8, 17]. The 120 h correlations, however, improved Spearman correlation between fold change and PC_1/2 to − 0.8393 (Fig. 3b).

Traditional RSA uses a value of  ≥ 1% parasite survival to indicate ART resistance [8]. To determine a value for ART resistance as measured by the eRRSA, the best-fit line for the 120 h post-drug treatment (Fig. 3b) was used to calculate the fold change when PC_1/2 is 5 (given that PC_1/2 ≥ 5 h defines clinical ART resistance [7]. Therefore, a fold change of 30 or less defines an ART resistant parasite by eRRSA.