Abstract
Miltefosine (MTF) is currently the only available oral treatment for leishmaniasis. However, increasing reports of therapeutic failure have raised concerns about emerging resistance. This study aimed to investigate the effects of reduced MTF susceptibility loss in the protozoan parasite Leishmania (Leishmania) amazonensis, with a particular focus on its impact on key biological and molecular parameters. Two distinct Leishmania lines (LaR-40 Line 1 and Line 2) were generated through stepwise in vitro selection with increasing concentrations of MTF, reaching up to 40 μM MTF. They were compared to their wild-type counterpart (LaWT). After 12 weeks of selection, LaR-40 promastigotes exhibited IC(50) values that were 4- to 8-fold higher than those of LaWT, with resistance remaining stable even after three months without drug pressure and following passage through BALB/c mice. No cross-resistance was detected against pentamidine, ketoconazole, or amphotericin B. MTF-resistant parasites exhibited reduced reactive oxygen species production, reduced lipid droplets (LD) abundance (in LaR-40 Line 1), delayed lesion onset, and smaller cutaneous lesions in mice, while maintaining normal infectivity in THP-1 macrophages. Quantitative RT-PCR analysis revealed consistent downregulation of the miltefosine transporter (mt) gene in both MTF-resistant lines, indicating that reduced drug uptake is the main mechanism underlying resistance. Line-specific changes, such as the upregulation of the serine palmitoyltransferase (spt) gene or the downregulation of the trypanothione reductase (tryr) gene, suggest that distinct metabolic pathways may act in a compensatory manner to reinforce resistance once transporter function is impaired. These findings indicate that MTF resistance in L. amazonensis is polygenic, stable, and adaptable. Routine monitoring of mt gene expression, combined with therapeutic strategies that target lipid or redox metabolism alongside drug uptake pathways, may help preserve the efficacy of current treatment regimens against leishmaniasis.