The emergence of mutant K13-mediated artemisinin (ART) resistance in Plasmodium falciparum malaria parasites has led to widespread treatment failure across Southeast Asia. In Africa, K13-propeller genotyping confirms the emergence of the R561H mutation in Rwanda and highlights the continuing dominance of wild-type K13 elsewhere. Using gene editing, we show that R561H, along with C580Y and M579I, confer elevated in vitro ART resistance in some African strains, contrasting with minimal changes in ART susceptibility in others. C580Y and M579I cause substantial fitness costs, which may slow their dissemination in high-transmission settings, in contrast with R561H that in African 3D7 parasites is fitness neutral. In Cambodia, K13 genotyping highlights the increasing spatio-temporal dominance of C580Y. Editing multiple K13 mutations into a panel of Southeast Asian strains reveals that only the R561H variant yields ART resistance comparable to C580Y. In Asian Dd2 parasites C580Y shows no fitness cost, in contrast with most other K13 mutations tested, including R561H. Editing point mutations in ferredoxin or mdr2, earlier associated with resistance, has no impact on ART susceptibility or parasite fitness. These data underline the complex interplay between K13 mutations, parasite survival, growth and genetic background in contributing to the spread of ART resistance.

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BackgroundGiven the risk of artemisinin resistance spreading from the Greater Mekong sub-region, prospective monitoring in sub-Saharan Africa should be expedited. Molecular biology techniques used for monitoring rely on the detection of k13 validated mutants by using PCR and Sanger sequencing approach, usually not available in malaria endemic areas.MethodsA semi-automated workflow based on the easyMAG® platform and the Argene Solution® (bioMérieux, Marcy l’Etoile, France) as a field-based surveillance tool operable at national level was developed in four steps. Clinical and analytical performances of this tool detecting five of the most frequent and validated k13 mutants (Y493H, I543T, R539T, F446I and C580Y) from dried blood spots (DBS) were compared to the gold standard approach (PCR and Sanger sequencing).ResultsBy using the ARMS (amplification-refractory mutation system) strategy, the best multiplexing options were found in 3 separate real-time PCR duplexes (IC as internal control/I543T, C580Y/Y493H and F446I/R539T) with limits of detection ranging from 50 (C580Y) to 6.25 parasites/µL (Y493H). In field conditions, using 642 clinical DBS (from symptomatic patients and asymptomatic individuals) collected from Cambodia, Myanmar and Africa (Chad), the overall sensitivity and specificity of the K13 bMx prototype assay developed by bioMérieux were ≥ 90%. Areas under the ROC curves were estimated to be > 0.90 for all k13 mutants in samples from symptomatic patients.ConclusionThe K13 ready-to-use bMx prototype assay, considered by the end-users as a user-friendly assay to perform (in shorter time than the K13 reference assay) and easy to interpret, was found to require less budget planning and had fewer logistical constraints. Its excellent performance qualifies the prototype as a reliable screening tool usable in malaria endemic countries recognized to be at risk of emergence or spread of validated k13 mutants. Additional multi-site studies are needed to evaluate the performances of the K13 bMx prototype assay in different epidemiological contexts such as Africa, India, or South America.Electronic supplementary materialThe online version of this article (10.1186/s12936-018-2329-y) contains supplementary material, which is available to authorized users.

The emergence of artemisinin (ART) resistance in Plasmodium falciparum parasites, driven by K13 mutations, has led to widespread antimalarial treatment failure in Southeast Asia. In Africa, our genotyping of 3,299 isolates confirms the emergence of the K13 R561H variant in Rwanda and reveals the continuing dominance of wild-type K13 across 11 countries. We show that this mutation, along with M579I and C580Y, confers varying degrees of in vitro ART resistance in African parasites. C580Y and M579I cause substantial fitness costs, which may counter-select against their dissemination in high-transmission settings. We also define the impact of multiple K13 mutations on ART resistance and fitness in multiple Southeast Asian strains. ART susceptibility is unaltered upon editing point mutations in ferrodoxin or mdr2, earlier resistance markers. These data point to the lack of an evident biological barrier to mutant K13 mediating ART resistance in Africa, while identifying their detrimental impact on parasite growth.

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