In Vitro Sensitivity Testing of Leishmania Clinical Field Isolates: Preconditioning of Promastigotes Enhances Infectivity for Macrophage Host Cells

Abstract

Diagnostic material from patients with leishmaniasis is generally available as promastigotes, and proper testing for susceptibility to first-line drugs by the intracellular amastigote assay is frequently hampered by the poor infectivity of the promastigotes for the macrophage host cell. Several conditions for optimization of the in vitro metacyclogenesis and cell infectivity of Leishmania donovani, L. guyanensis, and L. braziliensis field strains obtained from patients receiving standard antimony medication were investigated. Triggering logphase promastigotes to become amastigote-like by increasing the temperature or acidifying the culture medium was not successful. Adequate metacyclogenesis and the highest levels of macrophage infection were obtained after 5-day-old late-log-phase promastigote cultures were preconditioned at 25°C to pH 5.4 for 24 h in Schneider's medium prior to infection. The susceptibility assay with primary peritoneal mouse macrophages included pentavalent antimony (Sb V ; sodium stibogluconate), trivalent antimony (Sb III ; potassium antimonyl tartrate), miltefosine, and the experimental drug PX-6518. All strains were sensitive to miltefosine (50% inhibitory concentration [IC 50 ] < 10 M) and PX-6518 (IC 50 < 2 g/ml) but showed distinct susceptibility to Sb V and/or Sb III , depending on whether they were derived from cured, relapse, or nonresponder patients. Within the available set of Leishmania species and strains, simultaneous Sb V -Sb III resistance was clearly associated with treatment failure; however, a larger set of isolates is still needed to judge the predictive value of Sb V -Sb III susceptibility profiling on treatment outcome. In conclusion, the proposed conditioning protocol further contributes toward a more standardized laboratory model for evaluation of the drug sensitivities of field isolates.As the failure of first-line treatment for all clinical forms of leishmaniasis is a growing problem, it is pivotal to monitor the efficacy of standard drugs and map the prevalence of drug resistance in areas where the disease is endemic (7,9,24). Diagnostic field isolates are mostly provided to the laboratory as promastigotes, but it remains an experimental challenge to appropriately adapt them to the amastigote-macrophage model, still considered the "gold standard" for susceptibility evaluation (23, 31). Infection of the macrophage is generally achieved with metacyclic promastigotes, but unfortunately, infection is subject to a high degree of variability, among several other factors affecting the outcome of the sensitivity test (8, 10). These factors strongly suggest the need for the further standardization of susceptibility testing of clinical field isolates.In the normal course of events, infective metacyclic promastigotes are inoculated by the sand fly into the mammalian host, where they rapidly penetrate susceptible cells, undergo intracellular transformation to the amastigote form, and start dividing. The procyclic and metacyclic phases observed during in vitro culture appear to...

An Image-Based High-Content Screening Assay for Compounds Targeting Intracellular Leishmania donovani Amastigotes in Human Macrophages

“…3A illustrates the growth curves and highlights the optimal culture durations for the host cells and parasites prior to infection. The use of 5- or 6-day-old late-log-phase promastigote cultures resulted in higher infection ratios because of the enrichment of metacyclic promastigotes in the culture media, as previously reported [23]. A high infection ratio obtained with this protocol is illustrated in Fig.…”

Imipramine Is an Orally Active Drug against Both Antimony Sensitive and Resistant Leishmania donovani Clinical Isolates in Experimental Infection

“…The average of three untreated cultures was taken as 100% control against which the percentage inhibition of infected MΦs in treated cultures was calculated. The 50% effective concentration (EC 50 ) of imipramine for each of the isolates was estimated as described elsewhere [19], [20].…”

Berberine Chloride Mediates Its Anti-Leishmanial Activity via Differential Regulation of the Mitogen Activated Protein Kinase Pathway in Macrophages

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