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

Objective: 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: system for studying parasite differentiation and provides a practical framework to support infection biology and antileishmanial drug development.