Blood samples were collected by the IPD-4S in November 2018 (Surveillance des fièvres, 2015). The IPD-4S network is an important surveillance system initially built to strengthen influenza sentinel surveillance in partnership with the Senegalese Ministry of Health since 2012. This network of researchers, medical doctors, and nurses is implemented in all 14 regions of Senegal. Among its top priorities, the IPD-4S network also helps to routinely identify unusual health events to provide a rapid and appropriate medical response to the communities. Between 2017 and 2018 a surveillance program in response to a Dengue outbreak was carried out (Dieng et al., 2021). For this investigation, all declared PfRDT (SD Bioline malaria AG P.F) positive samples from the surrounding index case (ID 316443) area were collected from Ndoffane and other health care centers from the same region Kaolack as well as in the neighborhood region Diourbel. Malaria-positive samples were collected following the IPD-4S infection transmission control plan. Fifteen malaria-positive samples were collected, and 2 ml of venous blood was then shipped at 4°C to IPD the following day. Clinical information of the 18-year-old girl at day 9 post ACT treatment (ID 316443) is presented in Table 1b . DNA from the erythrocyte pellet was extracted using Quick-gDNA Blood MiniPrep kit from ZYMO research following the manufacturer’s instructions. Nested primers were designed to amplify the propeller domain of Kelch13. Full-length PfKelch13 was also amplified from biological replicates. Chromatograms of all PCR products were analyzed and the multiplicity of infection rate was determined using msp1 and msp2 typing protocol ( Figure 1 and Supplementary Figure 1 ). Human malaria genius typing was done using a light cycler and LightMix modular Plasmodium genus (Malaria) Cat # 53-0694-96 and 40-0694-24 respectively (TIB BioMol). All primers are listed in Supplementary files. ( Supplementary Tables 2A, B ).

The two-plasmid approach was employed to express Cas9, sgRNA, and a donor template (Ghorbal et al., 2014). SpCas9 was delivered on the pUF1 plasmid, which also contains a yeast dihydroorotate dehydrogenase (ydhodh) expression cassette which confers resistance to PfDHODH inhibitors such as DSM1. The sgRNA and the donor DNA template for homologous recombination repair were placed in the same plasmid pL7 (here pL7-238, have already cloned with the seed so we only need to clone the donor DNA). pL7 also expresses human dihydrofolate reductase (hdhfr) allowing positive selection with WR99210. The donor DNA to be used as a template to repair the double-strand breaks (DSBs) generated by Cas9 was designed from the kelch13 gene of Plasmodium falciparum 3D7 (PlasmoDB ID PF3D7_1343700). Donor’s DNA includes the homologous regions (HR1 and HR2) flanking the region of interest (ROI). The ROI carries the desired mutation and an additional modification (defined here as shield mutations) at the Cas9-target site. The shield’s mutations are silent but abolish recognition by Cas9, thereby protecting the modified locus from repeated cleavage. Additional silent mutations spanning the gap between the shield mutations and the desired modification can be introduced to help drive the repair event beyond the mutation-of-interest. Homology regions with plasmid and restriction sites were added for cloning. Donor DNA (synthetic DNA by Integrated DNA technologies) was cloned in pL7 plasmid at SpeI-AflII sites using Infusion cloning technology (Takara-Bio) ( Supplementary Figure 2A ).

P. falciparum asexual blood-stage parasites 3D7 wild-type were cultured in A⁺ human red blood cells (RBCs) in RPMI-1640 culture medium containing 25 mM Hepes + l-glutamine, supplemented with 10% Albumax II (Gibco Life Technologies), Human Sera (HS), hypoxanthine (C.C.Pro GmbH) and gentamicin (Sigma). Parasites were maintained at 37°C in 5% O₂, 5% CO₂, and 90% N₂. Cultures were monitored by blood smears fixed in methanol, stained with Giemsa, and viewed by light microscopy. Synchronous cultures were obtained by sorbitol treatment. Prior to transfection, 50 μg of each plasmid circular DNA (pUF1-Cas9 and recombinant plasmid pL7) were ethanol-precipitated and resuspended in 30 μl of Tris-EDTA. The DNA precipitated plasmids were co-transfected into 100 μl rings stages parasites at 4.87% parasitemia and 270 μl cytomix, by electroporation using the Bio-Rad GenePulse Xcell™ electroporator, at 310 V, with a resistance of 950 μF and a transfection time of less than 10s.

Drug pressure was applied 15–20 h after transfection: WR99210 for pL7-238_Insert was used at 2.5 nM and DMS1 for pUF1-Cas9 was used at 1.5 μM. Media and drugs were renewed every 24 h for the first 5 days, then every other day for a week, and twice a week until parasites are visually detected by microscopy. Parasites came back during the third week post-transfection.

To test PfKelch13 single nucleotide integration, genomic DNA (gDNA) of bulk culture for each transfection (Pf3D7Kelch13^R515K) was extracted from infected RBCs using the Mini NucleoSpin Blood QuickPure kit (MACHEREY-NAGEL). The high-fidelity polymerase PfuUltra II Fusion HS DNA polymerase was used for PCR amplification to detect the integration of locus. PCR conditions are as follows: 95°C for 2 min, followed by 30 cycles of 95°C for 30 s, 48°C for 20 s, 62°C for 15 s, and a final extension cycle of 72°C for 3 min. PCR products were migrated on 1% agarose gel for 20 min at 100V. NucleoSpin Gel and PCR Clean-up kit (MACHEREY-NAGEL) were used to extract amplified DNA at the expected size. Sequencing of the purified PCR products was done by Eurofins Genomics (TubeSeq service). All primers used in this study are listed in an additional file ( Supplementary Table 1 ). Bulk-edited cultures were cloned via limiting dilution. For in vitro malaria culture synchronous culture of trophozoites stages was used to start the cloning dilution. A 1/1000 dilution was parasitemia checked by microscopy in a 2% hematocrit flask. Parasites were gassed and incubated at 37°C, in a static condition. Genomic DNA from selected clones after serial dilutions of transgenic lines were then amplified and sequence aligned with Pf3D7Kelch13^WT parental sequence ( Supplementary Figure 2B ).

In vitro RSA^0-3hpi were conducted on very early ring-stage parasites (0-3 hours post-invasion; hpi) as previously described (Witkowski et al., 2013). Pf3D7Kelch13^R515K clones 1 and 4 were randomly picked and came back from sequencing with correct SNP integration in the Art-r gene marker ( Supplementary Figure 2B ). RSA^0-3hpi level of transgenic parasite clones were studied. Parental laboratory strains Pf3D7Kelch13^WT and PfNF54Kelch13^C580Y were used as negative and positive controls respectively. In vitro RSA^0-3hpi ring parasites were subjected to sorbitol treatment to eliminate remaining schizonts. Synchronized 0–3hpi rings were next adjusted to 0.5% parasitemia and 2% hematocrit in 1 mL volumes (in 48-well plates), and exposed to DHA (700nM) or 0.1% of its solvent dimethyl sulfoxide (DMSO) as previously described. Duplicate wells were established for each parasite line ± drug. Following wash out, parasites were maintained in complemented drug-free medium in an incubator condition for an additional 66h.

Parasite viability was assessed by microscopic examination of Giemsa-stained thin blood smears by counting viable parasites ( Supplementary File 2C ). A measure of 2 μL of the pellet was then used for each smear. Parasitemia was calculated from a total of at least 10,000 erythrocytes per assay. Slides were read from the two duplicate wells per assay. Percent survival was calculated as the parasitemia in the drug-treated sample divided by the parasitemia in the untreated sample ×100. The assay was done three times to confirm the results. The Mean (SD) of RSA^0-3hpi based on three replicates was done.

PfKelch13 polymorphism was studied using the DNA of malaria-infected blood received to investigate a malaria index case reported with a fever persistence upon 3 days ACT treatment (ASAQ) in Kaolack, a low malaria endemic region in Senegal. A total of 15 malaria PfRDT-positive samples were collected including the index case. Sanger sequencing for PfKelch13 was carried out with both a nested PCR and full-length amplification methods. Table 1a shows the parasite ART-r gene marker distribution. The index case was infected by parasites carrying a major arginine (AAA coding for K) peak at position 515 of PfKelch13. Both the chromatogram and the msp1 typing confirm a mixed infection with a second clone carrying a low peak of wild-type PfKelch13 (AGA coding for R) ( Figures 1A, B and Supplementary Figure 1 ). Plasmo typing assay shows 14 out of 15 patients had P. falciparum infections and 3 of the 15 had P. malariae co-infections ( Supplementary Table 1B ). None of the three remaining human malaria parasites (P. ovale, P. knowlesi, and P. vivax) were detected.

The lack of viable parasites (shipped to IPD a day after sample collection) to be culture-adapted and phenotypically tested was a limiting factor to further investigating ART-r phenotype. We next assessed an in vitro phenotypic assay to study the functional relevance of PfKelch13^R515K.

pL7-2386R515K plasmid construct was used to generate a transgenic line ( Supplementary Figure 2A ). The resulting bulk cultures were cloned by serial limiting dilution ( Supplementary Figure 2B ). Individual isolated clones were sequenced to verify the creation of the R515K mutation. Pf3D7Kelch13^R515K clone sequence is displayed and aligned with its parental Pf3D7 sequence ( Figure 2A ). The most frequent SEA ART-r mutation (Kelch^C580Y) was used as a positive control line also transfected in an African background P. falciparum counterpart PfNF54 (Dondorp et al., 2012; Mbengue et al., 2015; Miotto et al., 2015; Mok et al., 2015; Tun et al., 2015; Bhattacharjee et al., 2018).

To validate PfKelch13 R515K mutation as a cause of the fever persistence in the malaria index case reported in Kaolack, we carried out in vitro drug sensitivity assay using the standard RSA^0-3hpi protocol (Witkowski et al., 2013). Pf3D7Kelch13^R515K showed an increased RSA^0-3hpi value ( Figure 2B ). Pf3D7Kelch13^R515K clones 1 and 4 showed a median RSA of 3.9% and 3.7% respectively while the parental Pf3D7 was sensitive to DHA (median RSA^0-3hpi = 0.2%). The parasitemia assessed by microscopy showed a 5.12% RSA^0-3hpi value for the positive control NF54Kelch13^C580Y ( Supplementary Figure 2C ). In conclusion, our findings demonstrate that 3D7PfKelch13^R515K results in an increased in vitro RSA^0-3hpi value.