Exploring Pfkelch13-mediated ART resistance in a West African genetic context

Abstract

Extensive use of artemisinin-based combination therapy (ACT) for antimalarial treatment ultimately led to the emergence of Pfkelch13 (Pfkelch13)-mediated artemisinin (ART) resistance and widespread treatment failure in Southeast Asia. The recent independent emergence of ART-resistant Pfkelch13 variants in Africa (primarily in the East) motivates understanding this threat in West African countries where evidence of ART resistance remains limited. As parasite genetic background is crucial for development of ART resistance, this provides an opportunity to identify genetic variants that contribute to resistance in a West African genetic context to deepen our understanding of this threat. To assess the risk of ART resistance emerging in the West African country of Senegal, we utilized a CRISPR/Cas9 strategy to introduce ART-resistant Pfkelch13 mutations found in other African countries (R561H, M579I, and I543T) and a Senegal-emerging Pfkelch13 variant (C473S) into a culture-adapted field isolate that is representative of Senegal’s extant parasite population – SenTh15.14. Successful introduction of each mutation allowed assessment of these engineered lines via the Ring-stage Survival Assay (RSA), resulting in some of the strongest phenotypes reported for these mutations: R561H and M579I confer high levels of RSA survival (~20% and 27 % respectively) in comparison to low levels conferred by C473S and I543Tb (~4 % and 2 %). Assessing the impact of these mutations on parasite fitness, all mutant lines tested (R561H, M579I, and C473S) outcompeted SenTh15.14 wildtype within 30 days. The resistance and fitness phenotypes indicate that Senegal’s extant parasite population is indeed permissive to Pfkelch13 mutations, and there is high risk of ART resistance associated with some of these variants if they were to emerge. However, testing the parental strain and mutant lines against a panel of standard antimalarial drugs either previously or currently used extensively in Senegal points to the country’s antimalarial efficacy remaining high. This data supports continued use of current ACT and malaria chemo-preventive regimens in country. The use of field isolates in gene-editing studies such as this can present major challenges to phenotypic assessment of these lines. We successfully obtained clonal mutant lines for all Pfkelch13 mutations introduced except I543T for which wildtype parasites consistently outgrew mutant parasites prior to clonal expansion. This limitation resulted in development of a novel sequencing-based RSA approach that confirmed this bulk population was mutant-dominant at the start of RSA and allowed robust measurement of the in vitro survival rate reported. Nanopore sequencing of the SenTh15.14_I543T and R561H (control) bulk populations grown in the absence of drug revealed rapid reversion of the I543T mutant bulk population to wildtype whereas the R561H bulk subpopulations remained constant. Our approach not only provided quantitative evidence of I543T’s genetic instability in the SenTh15.14 background, but it allowed this line to still be phenotypically assessed, expanding the range of experimental isolates that can be risk-assessed via RSA. Together, our findings provide valuable phenotypic insight in a West African genetic context where resistance data is limited, thus serving as an early-warning system for Senegal, and it underscores the need to perform gene-editing and phenotypic studies in local genetic contexts for the best understanding of the emerging ART resistance threats in Africa.