Target Amplicon Deep Sequencing of Pfk13 gene in patients with Plasmodium falciparum malaria during the therapeutic efficacy study trials from 2020 to 2022 in Senegal

Abstract Background: Artemisinin-based combination therapies (ACTs) remain the cornerstone of malaria treatment in Africa. However, the emergence of Plasmodium falciparum artemisinin partial resistance associated with mutations in the pfk13 gene in Southeast Asia and East Africa raises concern about its potential emergence or spread to West Africa. This study aimed to investigate whether therapeutic efficacy study (TES)-defined treatment failures in Senegal are associated with pfk13 mutations using targeted amplicon deep sequencing (TADS). Methods: A total of 182 P. falciparum isolates collected during TES conducted between 2020 and 2022 in Senegal were analyzed. These included paired Day 0 (D0)/Day of Failure (DF) samples (n=112) and additional D0 samples (n=70) analyzed in pools. The pfk13 gene was sequenced using TADS, and single nucleotide polymorphisms (SNPs) were identified and compared with WHO-validated artemisinin resistance markers. Results: Among the 182 samples, 23 SNPs were identified in the pfk13 gene, including both synonymous and non-synonymous variants. Six non-synonymous mutations (D547Y, V566L, A578S, V589I, E596D, and V637I) were detected at very low within-sample allele frequencies (<1%). None corresponded to validated or candidate artemisinin resistance-associated mutations. Analysis of paired D0/DF samples showed no enrichment or selection of pfk13 mutations in post-treatment samples, indicating that observed treatment failures were not associated with pfk13 -mediated artemisinin resistance. Conclusion: This study provides no evidence of pfk13 -mediated artemisinin resistance in Senegal between 2020 and 2022. The absence of validated resistance mutations and the lack of association between pfk13 variants and treatment failure support the continued efficacy of ACTs in this setting. These findings highlight the importance of integrating TES outcomes with genomic surveillance to detect early signals of resistance and inform malaria control strategies in West Africa.