In Vitro Susceptibilities of Leishmania donovani Promastigote and Amastigote Stages to Antileishmanial Reference Drugs: Practical Relevance of Stage-Specific Differences

Culture media, products, reagents, and animals.Adenosine, folic acid, d-biotin, hemin, NaHCO₃, potato starch, dimethyl sulfoxide, Giemsa stain, and resazurin were purchased from Sigma, whereas minimal essential medium (MEM), RPMI-1640 medium, l-glutamine, fetal calf serum (FCS), and trypsin-EDTA (0.25%) were supplied by Invitrogen. The powder form of sodium stibogluconate and the marketed injectable formulation Pentostam (GSK) were included along with miltefosine (pure crystalline compound obtained from WHO-TDR) and amphotericin B-deoxycholate (Fungizone; Squibb). The experimental drug PX-6518 was available from previous investigations (19).

Golden hamsters and Swiss mice were supplied by Janvier (France). Animal experiments were approved by the ethical committee of the University of Antwerp.

Parasite and cell cultures.Leishmania donovani MHOM/ET/67/L82 (also known as LV9 or HU3) was obtained as a cryostabilate from the Institute of Tropical Medicine in Antwerp, Belgium, and was used to infect golden hamsters serving as donor animals. The adapted axenic amastigote culture of the same strain was obtained from the Swiss Tropical Institute and further maintained in Schneider's medium. For the specific purpose of this experiment, the axenic amastigotes were converted back to promastigotes and were maintained in MEM supplemented with 200 mM l-glutamine, 16.5 mM NaHCO₃, 10% heat-inactivated FCS, 40 mg/liter adenine, 3 mg/liter folic acid, 2 mg/liter d-biotin, and 2.5 mg/liter hemin. Fresh ex vivo amastigotes were purified from the spleen of a 6-week-infected donor hamster and were immediately kept at 37°C on acidic amastigote medium. Primary mouse peritoneal macrophages (PMM) were collected from Swiss mice 2 days after peritoneal stimulation with a 2% potato starch suspension. Cells were collected and grown in RPMI-1640 medium supplemented with 200 mM l-glutamine, 16.5 mM NaHCO₃ and 5% inactivated FCS. Human simian virus 40-immortalized lung fibroblasts (MRC-5 SV2) were obtained from the European Collection of Cell Cultures (United Kingdom) and maintained in MEM supplemented with 5% FCS and 1% l-glutamine. All amastigote cultures and assays and the cytotoxicity assay were conducted at 37°C under 5% CO₂. The promastigote cultures and assay mixtures were kept at 25°C under normal atmospheric conditions.

Compound solutions/dilutions.Stock solutions of crystalline sodium stibogluconate (NaSb^V [31.3% Sb^V]) were prepared in preheated phosphate-buffered saline at 37°C and were immediately used in the various tests. Pentostam (100 mg Sb^V/ml), miltefosine, amphotericin B, and semipurified PX-6518 were dissolved/diluted in MilliQ-water. All results with crystalline NaSb^V and Pentostam are presented as Sb^V μg/ml equivalents. The stock solutions were kept at −20°C prior to use, except for miltefosin and Pentostam, which were stored at 4°C. For axenic tests, twofold serial dilutions of the reference drugs were spotted in 96-well flat-bottom microplates (Greiner) prior to addition of the biological test system. For intracellular amastigote tests, drugs were added after adhesion of macrophages and proper internalization of Leishmania stages. Therefore, dilutions were spotted in separate V-bottom plates, allowing easy transfer to the test plates after solubilization in 100 μl culture medium.

Axenic drug sensitivity assays.Assays were performed in sterile 96-well microtiter plates, with each well containing 10 μl of the compound dilution and 200 μl of parasite inoculum (1 × 10⁵ promastigotes/well or 2 × 10⁵ amastigotes/well). Parasite multiplication was compared to that for untreated controls (100% growth) and uninfected controls (0% growth). After 72 h of incubation, resazurin was added and cell viability was measured fluorometrically (excitation λ, 550 nm; emission λ, 590 nm) (21, 34). The results were expressed as the percentage of reduction in the parasite burden compared to that in untreated control wells, and the 50% inhibitory concentration (IC₅₀) was calculated by Probit analysis. At least six independent replicates were performed for each observation.

Intracellular drug sensitivity assays.PMM were seeded in 96-well microtiter plates at 3 × 10⁵ cells/well and were left for adhesion and differentiation for 48 h before infection with either promastigotes, axenic amastigotes, or ex vivo amastigotes. To assure optimal metacyclogenesis prior to infection, promastigotes were preconditioned for 24 h at 37°C in acidified promastigote medium (pH 5.4).

For promastigotes, an infection ratio of 15 stages per macrophage was used. Since axenic and ex vivo amastigotes tend to multiply slightly more extensively in PMM, the infection ratio was kept at 5. Compound dilutions were added 2 h after infection, except for promastigotes, which were left undisturbed for another 24 h to allow proper internalization and transformation into amastigotes. At this stage, the medium was replaced to remove the noninternalized stages. After 5 days of incubation, total parasite burdens were microscopically assessed on Giemsa-stained wells and were compared to those for untreated infected controls. The results are expressed as the percentages of reduction in the parasite burden from that in wells with untreated infected controls, and the IC₅₀s were calculated. At least six independent tests were performed for each observation.

Electron microscopic studies.A small-scale ultrastructural study was performed with the knowledge that long-term cultivation of axenic amastigotes may promote the reappearance of promastigote-like features (16, 24). Samples for transmission electron microscopy were obtained after centrifugation of extracellular stages or trypsinized PMM with intracellular stages, fixation in a 0.1 M sodium cacodylate-buffered (pH 7.4) 2.5% glutaraldehyde solution for 2 h, rinsing (three times, for 10 min each time) in 0.1 M sodium cacodylate-buffered (pH 7.4) 7.5% saccharose, and postfixation in 1% OsO₄ solution for 1 h. After dehydration in an ethanol gradient (70% ethanol [20 min], 96% ethanol [20 min], and 100% ethanol [twice, for 20 min each time]), samples were embedded in Durcupan ACM. Ultrathin sections were stained with uranyl acetate and lead citrate and were examined in a Philips CM 10 microscope at 80 kV.

In vitro susceptibility testing.The activity of amphotericin B did not differ much across the different models and stages (Table 1). Mean IC₅₀s against axenic log-phase promastigotes, established axenic, and ex vivo amastigotes ranged from 0.6 to 0.7 μM. Slightly higher potency was observed against the intracellular amastigote stage (IC₅₀s, 0.1 to 0.4 μM), irrespective of the stages that were used for infection.

A similar trend was observed for miltefosine, with almost comparable efficacies against the extracellular and intracellular stages; established axenic and ex vivo amastigotes were slightly more sensitive (IC₅₀s, 0.4 to 0.9 μM).

Pentostam and NaSb^V were not active against promastigotes (IC₅₀, >64 μg Sb^V/ml), but surprisingly, no activity was observed against established axenic amastigotes or freshly collected spleen-derived ex vivo amastigotes (IC₅₀, >64 μg Sb^V/ml). Against intracellular amastigotes, Pentostam yielded IC₅₀s of 22 to 28 μg Sb^V/ml, although with a higher degree of variability among the different replicates. It is noteworthy that crystalline NaSb^V showed about a two- to threefold increase in potency, with IC₅₀s ranging from 8.8 to 10.5 μg Sb^V/ml.

Like Pentostam and NaSb^V, PX-6518 showed no efficacy against promastigotes, established axenic amastigotes, or ex vivo amastigotes. A consistent, high activity was obtained against intracellular stages after infection with either axenic or ex vivo amastigotes (IC₅₀, 0.1 μg/ml). Infection with metacyclic promastigotes resulted in marginally lower efficacy (IC₅₀, 0.7 μg/ml), which may reflect incomplete intracellular transformation of promastigotes into amastigotes.

The applicability of the different models for either drug screening or determination of the resistance of field strains is summarized in Table 2.

Morphological evaluation.Specific attention was given to the established axenic amastigotes in an attempt to ascertain whether the parasite stages were either amastigote-like or promastigote-like. Light microscopy showed amastigote-like organisms, i.e., clear, immobile, rounded stages with dense cell masses, about 2 μm in diameter. However, ultrastructural characteristics indicated the presence of organisms that were more promastigote-like, i.e., more-oval cell bodies, the presence of a strong protruding flagellum, and the absence of megasomes (Fig. 1a and b). In contrast, fresh ex vivo and intracellular amastigote stages (Fig. 1c) did not possess flagella beyond the margins of the cell body, were clearly rounded, and exhibited structures resembling megasomes.

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FIG. 1.

Ultrastructural details of in vitro-established axenic and intracellular amastigotes. Shown are Leishmania donovani L82 axenic amastigotes in cell-free medium (a and b) and 72 h after infection of PMM (c). Established axenic amastigotes show oval cell bodies with a clear nucleus (n) and kinetoplast (k) and with flagella (f) protruding from the flagellar pocket. In contrast, intracellular axenic amastigotes are more rounded and possess short flagella and megasome-like (m) structures.