Abstract
BACKGROUND: Artemisinin partial resistance (ART-R) has spread throughout Southeast Asia and mutations in Pfkelch13, the molecular marker of resistance, are widely reported in East Africa. Effective in vitro assays and robust phenotypes are crucial for monitoring populations for the emergence and spread of resistance. The recently developed extended Recovery Ring-stage Survival Assay used a qPCR-based readout to reduce the labour intensiveness for in vitro phenotyping of ART-R and improved correlation with the clinical phenotype of ART-R. Here, the assay is extended and refined to include measurements of parasite growth and recovery after drug exposure. Clinical isolates and progeny from two genetic crosses were used to optimize and validate the reliability of a straight-from-blood, SYBR Green-based qPCR protocol in a 96-well plate format to accurately measure phenotypes with this new Growth, Resistance, and Recovery assay (GRRA).
RESULTS: The assay determined growth between 6 and 96 h, resistance at 120 h, and recovery from 120 to 192 h. Growth can be accurately captured by qPCR and is shown by reproduction of previous growth phenotypes from HB3 × Dd2. Resistance measured at 120 h continually shows the most consistent phenotype for ring stage susceptibility. Recovery identifies an additional response to drug in parasites that are determined sensitive by replicative viability at 120 h. Comparison of progeny phenotypes for Growth versus Resistance showed a minor but significant correlation, whereas Growth versus Recovery and Resistance versus Recovery showed no significant correlation. Additionally, dried blood spot (DBS) samples matched replicative viability measured from liquid samples demonstrating Resistance can be easily quantified using either storage method.
CONCLUSIONS: The direct-from-blood qPCR-based methodology provides the throughput needed to quickly measure large numbers of parasites for multiple relevant phenotypes. Growth can reveal fitness defects and illuminate relationships between proliferation rates and drug response. Recovery serves as a complementary phenotype to resistance that quantifies the ability of sensitive parasites to tolerate drug exposure. All three phenotypes offer a comprehensive assessment of parasite-drug interaction each with potential independent genetic determinants of main effect and overlapping secondary effects. By adapting the method to include DBS, readouts can be easily extended to ex vivo surveillance applications.