Emergence of Artemisinin-based Combination Therapy Resistance Markers in Plasmodium falciparum from the Brazilian Tri-Border Region of the Guiana Shield

Plasmodium falciparum malaria remains a public health concern in the Brazilian Amazon, particularly among mobile and remote populations. Antimalarial drug resistance threatens elimination efforts, particularly in the Guiana Shield, which has historically been linked to the spread of resistant parasites. We analyzed 99 P. falciparum isolates collected in Roraima State, northern Brazil (2016-2020), using targeted deep sequencing. Variants were assessed in pfcrt, pfmdr1, and pfk13, and copy number variation in pfpm2 and pfpm3, including detection of the pfpm2/3 hybrid. High coverage enabled the detection of both dominant and minority alleles. Among the 99 isolates, the pfmdr1-NFD haplotype was found in 6% (6/99, all likely originating from Venezuela), pfcrt-C350R in 7% (7/99), and pfpm2 and pfpm3 amplifications in 5% (5/94) and 6% (6/97), respectively, and the pfpm2/3 hybrid in 4% (4/99). No validated or candidate pfk13 mutations associated with artemisinin partial resistance were detected. This study provides the first report of the pfpm2/3 hybrid and the pfmdr1-NFD haplotype in Brazil, and confirms the presence of pfcrt-C350R in isolates from the tri-border area. These variants, associated with reduced susceptibility to lumefantrine and piperaquine, suggest that parasites in northern Brazil already carry resistance markers that are prevalent in neighboring countries. These results highlight the need for continued genomic surveillance to detect early warning signals, guide treatment policies, and contain the spread of parasites with reduced susceptibility to ACT partner drugs.