Clonal Transmission of Emerging Novel Plasmodium falciparum Kelch13 Mutations and Increasing Complexity of Infection in Libreville, Gabon, 2021-2023

Artemisinin partial resistance (ART-R) in Plasmodium falciparum, due to mutations in the Kelch13 (K13) propeller domain, is spreading across Africa. However, data from Central Africa remain sparse. This study performed molecular surveillance in a peri-urban sentinel site in Libreville, Gabon, from 2021 to 2023 to assess emerging resistance markers and parasite population dynamics. Febrile patients with confirmed P. falciparum infection were enrolled at the Melen sentinel site. Dried blood spots were collected and isolated DNA sequenced using molecular inversion probes (MIPs) targeting drug resistance genes and genome-wide SNPs. We assessed the prevalence of mutations in K13, DHFR, DHPS, CRT, and MDR1. Complexity of infection (COI) and identity-by-descent (IBD) were used to evaluate transmission intensity and parasite relatedness, respectively. Among 468 genotyped samples, no validated or candidate K13 mutations were detected. However, 21 carried K13 mutations of unknown significance, including E433D (n=14), Q613H (n=5), V520I, and V637I. Interestingly, E433D prevalence rose from 0.7% in 2022 to 5.5% in 2023. Parasites with E433D or Q613H showed significantly higher IBD than wild-type (P<0.001) and chains of clonal transmission. Recent DHFR and DHPS mutations associated with higher-level sulfadoxine-pyrimethamine resistance were at low prevalence. MDR1 Y184F increased from 52.6% to 68.8%, while CRT K76T remained rare. IBD estimates support clonal transmission of parasites carrying emerging K13 mutations, particularly E433D and Q613H. In parallel, COI estimates increased over time, suggesting intensifying malaria transmission, potentially with a seasonal component. These findings highlight the need for expanded genomic surveillance and functional validation of these novel mutations to inform malaria control strategies in Gabon and Central Africa.