Tehran University of Medical Sciences Iranian Journal of Parasitology 1735-7020 21 1 2026 05 19 94 104 Maryam Saeed 1. Wildlife Epidemiology and Molecular Microbiology Laboratory (One Health Research Group), Department of Wildlife and Ecology, University of Veterinary and Animal Sciences, Lahore, Ravi Campus, Pattoki, Pakistan 2. Department of Wildlife & Ecology, University of Veterinary and Animal Sciences, Lahore, Ravi Campus, Pattoki, Pakistan Asia Iqbal Department of Wildlife & Ecology, University of Veterinary and Animal Sciences, Lahore, Ravi Campus, Pattoki, Pakistan Shahzad Ali 1. Wildlife Epidemiology and Molecular Microbiology Laboratory (One Health Research Group), Department of Wildlife and Ecology, University of Veterinary and Animal Sciences, Lahore, Ravi Campus, Pattoki, Pakistan 2. Department of Wildlife & Ecology, University of Veterinary and Animal Sciences, Lahore, Ravi Campus, Pattoki, Pakistan Haroon Akbar Department of Parasitology, University of Veterinary and Animal Sciences, Lahore, Pakistan Saba Sana Institute of Microbiology, University of Veterinary and Animal Sciences, Lahore, Pakistan Usama Saeed Department of Biology, University of Copenhagen, Denmark 2025 08 11 2026 06 07 Background: We investigated the prevalence of Trypanosoma lewisi in Black rats (Rattus rattus) and Brown rats (R. norvegicus) using both microscopic and molecular detection methods, along with the analysis of associated risk factors. Methods: A total of 178 rodents were trapped in Kasur district between November 2023 and November 2024, with epidemiological data and geographical coordinates recorded. Rodents were identified, euthanized and blood samples were collected. T. lewisi was confirmed through microscopy, PCR, sequencing and phylogenetic analysis. A GIS map was generated using ArcGIS 10.5.1 to illustrate geographical distribution. Results: A total of 178 blood samples were examined by microscopically and by PCR. PCR confirmed to be either microscopically positive or negative samples. Overall prevalence of T. lewisi was 10(5.62%) and the findings were consistent across both diagnostic methods.  At the species level, T. lewisi prevalence was higher in R. rattus (7.59%) compared to R. norvegicus (4.04%). Gender and the presence of ectoparasites were identified as potential risk factors. Conclusion: These findings confirm the presence of T. lewisi in black and brown rats and serve as a baseline for further surveillance and control strategies. https://ijpa.tums.ac.ir/index.php/ijpa/article/view/4728 https://ijpa.tums.ac.ir/index.php/ijpa/article/download/4728/1483