Miltefosine was the first oral compound approved for
visceral leishmaniasis chemotherapy, and its efficacy against Leishmania donovani has been well documented. Leishmania amazonensis is the second most prevalent species causing
cutaneous leishmaniasis and the main etiological agent of
diffuse cutaneous leishmaniasis in Brazil. Driven by the necessity of finding alternative therapeutic strategies for a chronic
diffuse cutaneous leishmaniasis patient, we evaluated the susceptibility to
miltefosine of the Leishmania amazonensis line isolated from this patient, who had not been previously treated with
miltefosine. In vitro tests against promastigotes and intracellular amastigotes showed that this parasite isolate was less susceptible to
miltefosine than L. amazonensis type strains. Due to this difference in susceptibility, we evaluated whether genes previously associated with
miltefosine resistance were involved. No mutations were found in the
miltefosine transporter gene or in the Ros3 or
pyridoxal kinase genes. These analyses were conducted in parallel with the characterization of L. amazonensis mutant lines selected for
miltefosine resistance using a conventional protocol to select resistance in vitro, i.e., exposure of promastigotes to increasing
drug concentrations. In these mutant lines, a single
nucleotide mutation G852E was found in the
miltefosine transporter gene. In vivo studies were also performed to evaluate the correlation between in vitro susceptibility and in vivo efficacy.
Miltefosine was effective in the treatment of BALB/c mice infected with the L. amazonensis type strain and with the
diffuse cutaneous leishmaniasis isolate. On the other hand, animals infected with the resistant line bearing the mutated
miltefosine transporter gene were completely refractory to
miltefosine chemotherapy. These data highlight the difficulties in establishing correlations between in vitro susceptibility determinations and response to
chemotherapy in vivo. This study contributed to establish that the
miltefosine transporter is essential for
drug activity in L. amazonensis and a potential molecular marker of
miltefosine unresponsiveness in
leishmaniasis patients.