Search

Objectives: Despite a continued search for novel antileishmanial drugs, treatment options remain restricted to a few standard drugs, e.g. antimonials, miltefosine, amphotericin B and paromomycin. Although these drugs have now been used for several decades, their mechanism of action still remains partly hypothetical and their dynamics of cidal action and time-to-kill are still poorly documented., Methods: An in vitro time-to-kill assay on intracellular amastigotes of the laboratory reference strains Leishmania donovani (MHOM/ET/67/L82) and Leishmania infantum [MHOM/MA(BE)/67/ITMAP263] evaluated the cidal action dynamics of the listed reference drugs at three different concentrations: at IC 50, 2 × IC 50 and the near cytotoxic dose level (CC 90 : determined on MRC-5 cells). This assay focused on identifying the minimal exposure time needed to completely eliminate viable intracellular amastigotes, using the standard microscopic Giemsa assay and the promastigote back-transformation assay., Results: While 100% reduction was microscopically apparent for most drugs, the promastigote back-transformation assay clearly demonstrated a concentration- and time-dependent cidal mechanism. The time-to-kill at 2 × IC 50 was ≥240 h for pentavalent antimony (77 μg eq./mL), 96 h for trivalent antimony (44 μg eq./mL), 168 to >240 h for miltefosine (10 μM), 168 h for paromomycin (100 μM) and >240 h for amphotericin B (2 μM). No differences were noted between both Leishmania species., Conclusions: Evaluation of the concentration- and time-dependent cidal activity using the promastigote back-transformation assay revealed striking differences in efficacy of the different antileishmanial reference drugs. This assay should allow in-depth pharmacodynamic evaluation of novel drug leads in comparison with the existing antileishmanial drug repertoire., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objectives: Although miltefosine and paromomycin were only recently introduced to treat visceral leishmaniasis, increasing numbers of miltefosine treatment failures and occasional primary resistance to both drugs have been reported. Understanding alterations in parasite behaviour linked to drug resistance is essential to assess the propensity for emergence and spread of resistant strains, particularly since a positive effect on fitness has been reported for antimony-resistant parasites. This laboratory study compared the fitness of a drug-susceptible parent WT clinical Leishmania infantum isolate (MHOM/FR/96/LEM3323) and derived miltefosine and paromomycin drug-resistant lines that were experimentally selected at the intracellular amastigote level., Methods: Parasite fitness of WT, paromomycin-resistant and miltefosine-resistant strains, in vitro and in vivo parasite growth, metacyclogenesis, infectivity and macrophage stress responses were comparatively evaluated., Results: No significant differences in promastigote fitness were noted between the WT and paromomycin-resistant strain, while clear benefits could be demonstrated for paromomycin-resistant amastigotes in terms of enhanced in vitro and in vivo growth potential and intracellular stress response. The miltefosine-resistant phenotype showed incomplete promastigote metacyclogenesis, decreased intracellular growth and weakened stress response, revealing a reduced fitness compared with WT parent parasites., Conclusions: The rapid selection and fitness advantages of paromomycin-resistant amastigotes endorse the current use of paromomycin in combination therapy. Although a reduced fitness of miltefosine-resistant strains may explain the difficulty of miltefosine resistance selection in vitro, the growing number of miltefosine treatment failures in the field still requires further exploratory research., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)