Emergence of artemisinin-based combination therapy resistance markers in <i>Plasmodium falciparum</i> from the Brazilian tri-border region of the Guiana Shield

<i>Plasmodium falciparum</i> 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 <i>P</i>. <i>falciparum</i> isolates collected in Roraima State, northern Brazil (2016-2020), using targeted deep sequencing. Variants were assessed in <i>pfcrt</i>, <i>pfmdr1</i>, and <i>pfk13</i>, as well as copy number variation in <i>pfpm2</i> and <i>pfpm3</i>, including detection of the <i>pfpm2/3</i> hybrid. High coverage enabled the detection of both dominant and minority alleles. Among the 99 isolates, the <i>pfmdr1</i>-NFD haplotype was found in 6% (6/99, all likely originating from Venezuela), <i>pfcrt</i>-C350R in 7% (7/99), and <i>pfpm2</i> and <i>pfpm3</i> amplifications in 5% (5/94) and 6% (6/97), respectively, and the <i>pfpm2/3</i> hybrid in 4% (4/99). No validated or candidate <i>pfk13</i> mutations associated with artemisinin partial resistance were detected. This study provides the first report of the <i>pfpm2/3</i> hybrid and the <i>pfmdr1</i>-NFD haplotype in Brazil and confirms the presence of <i>pfcrt</i>-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 artemisinin-based combination therapy partner drugs.