Emerging threat of Partial Artemisinin Resistance Markers in P. falciparum Parasite Populations in multiple geographical locations in high Transmission Regions of Uganda

Background : Artemisinin-based combination therapies (ACTs) is the only recommended treatment option for uncomplicated malaria, however the emergency and spread of partial artemisinin resistance threatens their effectiveness for malaria treatment in sub-Saharan Africa where the burden of malaria is highest. Early detection and reporting of validated P. falciparum Kelch 13 (PfK13) mutation markers is useful for tracking the emergence and spread of artemisinin resistance to inform containment. Methods : Genomic surveillance was conducted at 50 sites across four regions of Uganda in Karamoja, Lango, Acholi and West Nile from June 2021 to August 2023 for assessing prevalence of pfhrp2/3 deletions and pfk13 mutations. Symptomatic malaria suspected patients were recruited and screened for presence of parasites. Out of 563 available dried blood spots (DBS), a random subset of 240 P. falciparum mono-infections, confirmed by a conventional multiplex PCR, were selected and used for detecting mutations in the propeller region of the pfk13 gene by Sanger sequencing using Big Dye Terminator method. Regional variations in the proportions of PfK13 mutations were assessed using the chi square or Fisher’s exact tests while Kruskal-Wallis test was used to compare absolute parasite DNA levels between wild type and mutants parasites. Results : Overall, 238/240 samples (99.2%) contained sufficient DNA and were successfully sequenced. Three mutations were identified within the sequenced samples; PfK13 C469Y in 32/238 (13.5%) samples, PfK13 A675V in 14/238 (5.9%) and PfK13 S522C in (1/238 (0.42%) samples across the four surveyed regions. The prevalence of PfK13 C469Y mutation was significantly higher in Karamoja region (23.3%) compared to other regions, P=0.007. Majority of parasite isolates circulating in West Nile are of wild type (100%), P=0.002. Relative parasite DNA quantity did not differ in samples carrying the wild type, C469Y and A675V alleles (Kruskal-Wallis test, p=0.6373). Conclusion : The prevalence of validated markers of artemisinin resistance PfK13 A675V and PfK13 C469Y in multiple geographical locations in this high transmission setting provides additional evidence for the emerging threat of artemisinin resistance in Uganda. In view of these findings, country-specific artemisinin resistance management strategy should be developed including periodic genomic surveillance to detect and monitor levels of PfK13 mutations in other regions in parallel with in-vivo therapeutic efficacy studies (TES) to assess potential implication on delayed parasite clearance and associated treatment failure in this setting.