Optimization of the Parasite Rescue and Transformation Assay for Leishmania tropica: Analysis of Preconditioning and Back Transformation Dynamics.

OBJECTIVE: This study aimed to compare the effects of three culture media [RPMI-1640, Schneider's insect medium (SIM), and M199] on Leishmania tropica promastigote propagation, preconditioning, and back-transformation of intracellular amastigotes, with the aim of establishing a standardized parasite rescue and transformation assay (PRTA) capable of consistently generating infective metacyclic forms. METHODS: Nine experimental groups were designed by combining the three media during the preconditioning and back-transformation phases. Promastigotes were expanded in the test media and then used to infect differentiated THP-1 macrophages to obtain intracellular amastigotes. Back-transformation was monitored morphologically under an inverted phase-contrast microscope, and metabolic activity was quantified using the ATP-based CellTiter-Glo assay. RESULTS: The highest back-transformation efficiency was observed in test 5 (SIM→SIM) [relative luminescence units (RLU): 49.567±9.099, p<0.05], with microscopic analysis revealing abundant viable transformed promastigotes. In contrast, other medium combinations produced either limited or no transformation. Test 9 (M199→M199; RLU: 0.089±0.071) and test 1 (RPMI→RPMI; RLU: 0.118±0.080) showed minimal back-transformation without statistical significance (p>0.05). CONCLUSION: These findings demonstrate that culture medium composition is a key determinant of L. tropica promastigote differentiation. SIM medium supported the most efficient generation of metacyclic promastigotes, likely because it approximates vector-like conditions. The PRTA model described here offers a reproducible in vitro system for studying parasite differentiation and provides a practical framework to support infection biology and antileishmanial drug development.