In vitro selection of ketoconazole-pentamidine-resistant Leishmania (Viannia) braziliensis strains.

The use of ketoconazole (KTZ) plus pentamidine (PMD) could be an interesting treatment option for New World cutaneous leishmaniasis. The aim of this work was to generate KTZ- and PMD-resistant strains and to determine some characteristics of the selection process and the resulting parasites. Resistance to one or two drugs was selected on promastigotes by progressively increasing drug concentrations for eleven months. The resistance levels (IC) to one or two drugs (synergism assay) were determined using a colorimetric resazurin methodology. The stability of the resistance phenotype (without drug pressure or after mouse passage), cross resistance with paromomycin and miltefosine, and resistance transference to intracellular amastigotes were determined. In addition, some parasite attributes compared with WT, such as growth kinetics, amastigogenesis, THP-1 cells, and mouse infection, were determined. Promastigotes resistant to KTZ or PMD were obtained three times earlier than the combined KTZ + PMD-resistant strains. Resistant parasites (promastigotes and intracellular amastigotes) were three to twelve times less susceptible to KTZ and PMD than WT parasites. The resistance phenotype on parasites was unstable, and no cross resistance was observed. Similar parasite fitness related to our evaluated characteristics was observed except for in vivo infection, where a delay of the onset of cutaneous lesions was observed after KTZ + PMD-resistant parasite infection. CONCLUSION: Combined treatment with KTZ and PMD delayed the onset of parasite resistance and was more effective in vitro than each drug separately for WT and all resistant strains. Parasites resistant to KTZ and PMD acquired similar in vitro behaviour to WT parasites, were less virulent to mice and maintained their resistance phenotype on intracellular amastigotes but not without drug pressure or after mouse infection.