Vet World   Vol.18   August-2025  Article - 21 

Comparative evaluation of three polymerase chain reaction primer sets for accurate molecular detection of Trypanosoma lewisi in wild rodents in Indonesia

Aditya Yudhana^1,2,3 , Gusti Ayu Illiyin Putri Santosa¹ , April Hari Wardhana⁴ , Frenky Laksana Putra⁵ , Ryanka Edila⁶ , Dyah Haryuningtyas Sawitri⁴ , Ratih Novita Praja^1,2 , Muhammad Aqil Kurnianto¹ , Aldi Gusnizar Rizaldy Tanjung¹ , Marc Desquesnes^7,8,9 , and Makoto Matsubayashi¹⁰

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.

How to cite this article: Yudhana A, Santosa GAIP, Wardhana AH, Putra FL, Edila R, Sawitri DH, Praja RN, Kurnianto MA, Tanjung AGR, Desquesnes M, and Matsubayashi M (2025) Comparative evaluation of three polymerase chain reaction primer sets for accurate molecular detection of Trypanosoma lewisi in wild rodents in Indonesia, Veterinary World, 18(8): 2395-2405.

Received: 04-04-2025   Accepted: 18-07-2025   Published online: 21-08-2025

Corresponding author: April Hari Wardhana    E-mail: wardhana24id@yahoo.com

DOI: 10.14202/vetworld.2025.2395-2405

Copyright: Yudhana, et al. This article is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.