Résumé
Background and Aim: Trypanosoma lewisi is a flea-transmitted protozoan parasite commonly infecting rodents and posing zoonotic risks. Conventional diagnostics such as blood smear and serology often fail in low parasitemia conditions. Molecular diagnostics using polymerase chain reaction (PCR) offer improved sensitivity and specificity, but the optimal primer set for field detection remains unclear. This study aimed to compare the diagnostic performance of three published PCR primer sets–TC121/TC122, CATLew F/CATLew R, and LEW1S/LEW1R–for the detection of T. lewisi in wild Rattus spp. in Indonesia and determine the most reliable tool for field application. Materials and Methods: One hundred rat blood samples obtained from the Badan Riset dan Inovasi Nasional (BRIN), Research Center for Veterinary Science, Bogor, West Java Province, Indonesia were analyzed through PCR using the three primer sets under optimized thermal cycling conditions. DNA amplification products were visualized using agarose gel electrophoresis. Diagnostic performance was evaluated based on sensitivity and specificity calculations using microscopy as the reference standard. Results: The LEW1S/LEW1R primer set demonstrated the highest diagnostic accuracy, detecting T. lewisi in 30 samples with 100% sensitivity and 97.22% specificity. CATLew F/CATLew R detected 29 positives with 96.43% sensitivity and 97.22% specificity, whereas TC121/TC122 detected 21 positives, yielding 67.86% sensitivity and 97.22% specificity. Only the LEW1S/LEW1R primer set consistently produced single, distinct amplicons with no non-specific bands. Conclusion: LEW1S/LEW1R is the most sensitive and diagnostically reliable primer set for PCR-based detection of T. lewisi, particularly suitable for low-resource settings where accurate and early detection is crucial. Its implementation in surveillance programs can strengthen zoonotic disease monitoring and guide timely interventions. Future studies should validate these findings in mixed-infection contexts and explore their application in human and non-rodent hosts. Keywords: diagnostic validation, flea-transmitted protozoa, molecular diagnostics, neglected disease, polymerase chain reaction primers, public health, rodent-borne zoonosis, Southeast Asia, Trypanosoma lewisi.