Artemisinin-based combination therapies (ACT) are essential malaria treatment worldwide. However, resistant Plasmodium falciparum strains have emerged and resistance is spreading, posing a great threat to malaria control efforts. Surveillance of genetic resistance markers is highly important, but traditional clinical sampling often comes with logistical constraints. Therefore, this study evaluated two alternative approaches: wastewater-based surveillance (WBS) and molecular xenomonitoring (MX) using mosquitoes. In addition, these methods were combined with Hyperplex PCR (hpPCR) for detection of six selected markers consisting of single nucleotide polymorphisms (SNPs) known to be associated with Artemisinin-resistance: C469F, C469Y, R539T, P574L, Q661E, and A675V.

Fresh and frozen wastewater samples from several Ugandan locations from 2023-2024 and 2026 were analyzed for the presence of Plasmodium spp. after DNA extraction using qPCR, with Cryptosporidium spp. used as a control.

Pools of mosquitoes collected in central Uganda in 2017 were analyzed in a similar way to screen for Plasmodium spp. DNA by qPCR. Positive samples were subsequently analyzed by hpPCR.

No Plasmodium spp. was detected in wastewater samples, regardless of concentration or extraction method. In contrast, Cryptosporidium spp. was successfully detected in concentrated wastewater samples, demonstrating that the wastewater processing and molecular detection workflows were functional.

Among 27 mosquito pools analyzed, 21 were positive for Plasmodium spp. and were selected for hpPCR analysis. No confirmed artemisinin resistance markers were detected. The results indicate that molecular xenomonitoring combined with hpPCR has potential for surveillance of malaria resistance markers, although further optimization and validation are required.