By sequencing P. simium DNA from Brazil using both Oxford Nanopore and Illumina platforms, we assembled the first reference genome (Ps_SaoPaulo; 21.8 Mb).. Novel zoonotic cases of Plasmodium simium from Sao Paulo with the first reference genome of the Brazilian strain.

Zoonotic Plasmodium simium infections pose a significant threat to malaria control and elimination efforts in the Americas, which currently focus on the high-burden species P. falciparum and P. vivax. Genomic tools are essential for the accurate identification and tracking of P. simium, yet a high-quality reference genome is urgently needed to enable comparative genomics and highlight key differences with P. vivax. By sequencing P. simium DNA from Brazil using both Oxford Nanopore and Illumina platforms, we assembled the first reference genome (Ps_SaoPaulo; 21.8 Mb). Comparative genomic analysis confirmed known differences with P. vivax, including fixed SNP mutations in the mitochondrion, deletions in RBP2a and DBP, and variants in blood stage antigen genes (e.g., msp3.1, msp3.2). Genome-wide variant analysis across 38 P. simium samples from Brazil revealed marked divergence from P. vivax and unique signatures in genes involved in hepatocyte egress, red blood cell invasion and transcription and translation. This study provides the most complete P. simium reference genome, offering insights into its genomic diversity, complex population structure, with implications for infection control in Brazil.