Cross-border molecular epidemiology of Plasmodium falciparum drug susceptibility: insights from the Uganda–Democratic Republic of the Congo border

BACKGROUND: Cross-border parasite movement drives resistance emergence in endemic regions. In Uganda, Plasmodium falciparum with artemisinin partial resistance emerged early and spread rapidly, making it a focal point for surveillance. In northeastern Democratic Republic of Congo (DRC), a key trade corridor bordering Uganda, we investigated molecular markers of artemisinin partial resistance and resistance to partner drugs used in artemisinin-based combination therapies (ACTs). Monitoring these resistance markers in DRC is essential for guiding regional containment efforts. METHODS: From May to June 2023, we conducted active and passive malaria case detection in 20 villages and a central hospital, respectively. Molecular analyses included copy number variation (CNV) in pfmdr1 and pfpm2/3, and genotyping of pfkelch13, pfmdr1, and pfcrt. For pfmdr1 CNV determination, we adopted a quantitative real-time PCR threshold of 1.4, recently proposed for parasite populations with frequent multiclonal infections. Whole-genome sequencing (WGS) was performed on hospital-derived samples to assess parasite relatedness and trace resistance spread. RESULTS: Among the 536 individuals sampled from the villages, 49.8% tested positive for P. falciparum by PCR. The pfmdr1 N86 allele was fixed, and 50.3% of samples carried the NFD haplotype (amino acid positions 86, 184, and 1246), a marker associated with reduced lumefantrine susceptibility in Africa. Pfmdr1 CNV was detected in 4.4% of samples using the conventional threshold (≥ 1.5), increasing to 11.7% with the 1.4 threshold. Pfpm2/3 CNVs, which may influence piperaquine susceptibility, were identified in 7.0% of samples. The validated pfkelch13 mutations A675V and C469Y, associated with artemisinin partial resistance, were detected in 4.3% and 1.3% of village samples, respectively. Principal component analysis of WGS data showed that DRC parasites carrying A675V or C469Y clustered closely with Ugandan strains, supporting cross-border spread of resistant parasites between DRC and Uganda. CONCLUSION: Our findings identified the presence of molecular markers associated with artemisinin partial resistance and reduced susceptibility to lumefantrine in northeastern DRC, along with evidence of cross-border parasite migration from Uganda. Given opposing selection pressures of lumefantrine and amodiaquine on pfmdr1 and pfcrt, alternative ACTs such as artesunate-amodiaquine may merit further evaluation. To sustain ACT efficacy, integrated genomic surveillance, clinical monitoring, and coordinated regional policy responses are essential.