Comparative evaluation of isolation and purification strategies for intracellular Leishmania tropica amastigotes in THP-1 macrophages.

Intracellular amastigotes represent the clinically relevant stage of Leishmania infection, yet their recovery with preserved functional integrity and minimal host contamination remains challenging. This study comparatively evaluated five lysis/separation strategies for isolating intracellular Leishmania tropica amastigotes from PMA-differentiated THP-1 macrophages, focusing on recovery efficiency, purity, and metabolic output. Infected cultures were processed by (A) mechanical disruption (Dounce homogenization), (B) filtration (5 µm), (C) mechanical disruption followed by filtration, (D) detergent-based lysis (0.05% SDS), or (E) osmotic lysis (0.1 × PBS), with isotonic PBS samples serving as controls. Method performance was assessed by amastigote yield, host-cell contamination, the Amastigote Purity Index (API), and ATP-dependent luminescence measured after day 7 promastigote transformation, including RLU values normalized to day 0 amastigote counts. Amastigote recovery differed significantly (ANOVA, p<0.0001), with the highest counts in controls (288.7 ± 3.2 amastigotes/500 fields) and Group A (166.3 ± 5.1 amastigotes/500 fields). Filtration-based approaches (Groups B/C) achieved maximal purity (API = 1.00) but a low yield (15.3 ± 0.6 and 17.3 ± 1.5 amastigotes/500 fields). Total metabolic activity (RLU) was highest in controls (110.67 ± 3.14) and Group A (82.25 ± 1.41), whereas no luminescence signal was detected following osmotic lysis (Group E). Normalized analyses (promastigote RLU per initial amastigote counted at day 0) revealed marked method-dependent variation in metabolic output per amastigote, with the highest value observed in Group B (0.79). Overall, filtration maximized sample purity, whereas mechanical disruption better preserved functionally viable amastigote biomass, indicating that isolation strategy selection should be guided by downstream experimental priorities.