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

Abstract

Background: Tracking and understanding artemisinin resistance is key for preventing global setbacks in malaria eradication efforts. The ring-stage survival assay (RSA) is the current gold standard for in vitro artemisinin resistance phenotyping. However, the RSA has several drawbacks: it is relatively low throughput, has high variance due to microscopy readout, and correlates poorly with the current benchmark for in vivo resistance, patient clearance half-life post-artemisinin treatment. Here a modified RSA is presented, the extended Recovery Ring-stage Survival Assay (eRRSA), using 15 cloned patient isolates from Southeast Asia with a range of patient clearance half-lives, including parasite isolates with and without kelch13 mutations.

Methods: Plasmodium falciparum cultures were synchronized with single layer Percoll during the schizont stage of the intraerythrocytic development cycle. Cultures were left to reinvade to early ring-stage and parasitaemia was quantified using flow cytometry. Cultures were diluted to 2% haematocrit and 0.5% parasitaemia in a 96-well plate to start the assay, allowing for increased throughput and decreased variability between biological replicates. Parasites were treated with 700 nM of dihydroartemisinin or 0.02% dimethyl sulfoxide (DMSO) for 6 h, washed three times in drug-free media, and incubated for 66 or 114 h, when samples were collected and frozen for PCR amplification. A SYBR Green-based quantitative PCR method was used to quantify the fold-change between treated and untreated samples.

Results: 15 cloned patient isolates from Southeast Asia with a range of patient clearance half-lives were assayed using the eRRSA. Due to the large number of pyknotic and dying parasites at 66 h post-exposure (72 h sample), parasites were grown for an additional cell cycle (114 h post-exposure, 120 h sample), which drastically improved correlation with patient clearance half-life compared to the 66 h post-exposure sample. A Spearman correlation of - 0.8393 between fold change and patient clearance half-life was identified in these 15 isolates from Southeast Asia, which is the strongest correlation reported to date.

Conclusions: eRRSA drastically increases the efficiency and accuracy of in vitro artemisinin resistance phenotyping compared to the traditional RSA, which paves the way for extensive in vitro phenotyping of hundreds of artemisinin resistant parasites.

Keywords: Artemisinin resistance; Ring-stage survival assay; kelch13.

Fig. 1

qPCR standard curve for detecting parasitaemia of P. falciparum infected RBCs. A standard curve of samples ranging from 0.0001% parasitaemia to 9% parasitaemia were measured using qPCR amplification on the ABI 7900HT. Ct values were calculated based on three technical replicates using the ABI SDS 2.4.1. Ct value is inversely related to percent parasitaemia (R² = 0.9699) and therefore can be used as a measurement of percent parasitaemia in final RSA samples

Fig. 2

Comparison of 72 h and 120 h perturbations. Parasites were set-up using the eRRSA protocol: 0.5% parasitaemia at early ring-stage and 700 nM DHA was applied and washed off after 6 h. Samples were collected at a 72 h post-drug treatment and at b 120 h post-drug treatment. Parasites are ordered from the smallest PC_1/2 (least resistant) to the largest PC_1/2 (most resistant) from left to right (with the sensitive control, 3D7 on the far left). Sensitive and resistant parasites were more distinguishable based on their 120 h post-drug treatment sample fold changes

Fig. 3

120 h eRRSA increases correlation with PC_1/2 compared to 72 h. a 15 isolates from Southeast Asia with varying PC_1/2 were assayed using the eRRSA. 72 h post-drug treatment samples were measured to give a fold change for each isolate and those fold changes were correlated with each isolate’s PC_1/2 (Spearman r = − 0.6071). Isolates with red boxes are kelch13 mutants and the 95% confidence interval around the best-fit line ($y=-6.025x+61.14$) is denoted with dotted lines. b The same 15 isolates were assayed using the eRRSA and 120 h post-drug treatment samples were measured to calculate a fold change for each isolate. The fold changes were correlated with PC_1/2 (isolates marked with red boxes are kelch13 mutants and the 95% confidence interval of the best-fit line is denoted with dotted lines) (Spearman r = − 0.8393); the 120 h eRRSA samples showed an increased correlation with PC_1/2 compared to the 72 h samples