Tehran University of Medical Sciences Iranian Journal of Parasitology 1735-7020 20 3 2025 09 01 444 453 Elham Rezaee 1. Student Research Committee, Shiraz University of Medical Sciences, Shiraz, Iran 2. Department of Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran Hamed Taghavi Department of Chemical Engineering, Shiraz University, Shiraz, Iran Gholamreza Hatam 1. Department of Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran 2. Basic Sciences in Infectious Diseases Research Center, Shiraz University of Medical Sciences, Shiraz, Iran Kamiar Zomorodian Department of Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran Bahador Sarkari 1. Department of Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran 2. Basic Sciences in Infectious Diseases Research Center, Shiraz University of Medical Sciences, Shiraz, Iran 2024 11 10 2025 04 13 Background: This study aimed to assess the In vitro effects of Cold Atmospheric Multiple Plasma Jet (CAMPJ) on Leishmania major. Methods: A plasma jet device was designed in the Department of Chemical Engineering, Shiraz University, Shiraz, Iran, incorporating a high-purity air supply, an air flow controller, a DC power supply, a 9-10 Farad capacitor, an oscilloscope, and a cold plasma reactor.  The CAMPJ was applied to L. major promastigotes and amastigotes under various plasma conditions, including different flow rates, voltages, and exposure times. The effectiveness of CAMPJ was compared to amphotericin B and a combination of both therapies. Viability and cytotoxicity were assessed on L. major and macrophage cell lines, using MAT assay, while apoptosis was quantified through flow cytometry. Results: The optimal experimental conditions were identified as 2 million L. major promastigotes in 500 μL of culture, a flow rate of 500 mL/min, a voltage of 8.6 kV, a distance of 1 cm, and an exposure time of 15 minutes. The CAMPJ showed limited cytotoxicity to macrophage cells.  The CAMPJ treatment significantly reduced the viability of L. major and induced apoptosis. CAMPJ-amphotericin B combination treatment significantly increased the treatment efficacy, when compared with the CAMPJ alone or negative controls. Conclusion: CAMPJ, alone or in combination with amphotericin B, effectively induces apoptosis in L. major. CAMPJ might be a promising alternative or adjunct therapy for cutaneous leishmaniasis, warranting further investigation in animal models and clinical settings. https://ijpa.tums.ac.ir/index.php/ijpa/article/view/4428 https://ijpa.tums.ac.ir/index.php/ijpa/article/download/4428/1451