Flow cytometric assessment of amphotericin B susceptibility in Leishmania infantumisolates from patients with visceral leishmaniasis

C. Di Giorgioa,*, F. Faraut-Gambarellib, A. Imbertb, P. Minodierc, M. Gasqueta and H. Dumonb

,aLaboratoire de Parasitologie, Hygiène et Zoologie, Faculté de Pharmacie, 27 Bd. Jean Moulin, 13385 Marseille Cedex 05; ,bLaboratoire de Parasitologie, Faculté de Medecine; ,cService de Pédiatrie Pr. Garnier, Hôpital Nord, Marseille, France


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Amphotericin B susceptibility was measured by a flow cytometric membrane potential assay in Leishmania infantum promastigotes isolated from 11 immunocompetent children treated with liposomal amphotericin B and 19 HIV-infected young adults treated with intralipid amphotericin B. Susceptibility levels were measured by the 90% inhibitory concentrations (IC 90) representing the concentrations of drug that induced a 90% decrease in membrane potential compared with the control culture. In immunocompetent children, treatment was fully effective whatever the susceptibility of isolates to amphotericin B. In immunocompromised adults, on the contrary, unresponsiveness and relapses could be observed in all cases and IC 90 increased in the course of successive treatments: a decrease of amphotericin B susceptibility in both promastigote and amastigote forms could be observed in a patient who had six relapses. These results suggest that the success of amphotericin B treatment depends greatly on patient immunity status, and indicate that successive relapses could enhance emergence of amphotericin B resistant isolates. The results demonstrate that the flow cytometric membrane potential assay can be used as an easy and reliable tool for studying the evolution of interactions between amphotericin B and the parasite membrane during long-term treatments.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Visceral leishmaniasis is a parasitic disease resulting from infection of macrophages with protozoans of the genus Leishmania. In France, the main causative agent of the disease has been identified as Leishmania infantum, endemic in the Mediterranean area. The first antileishmanial chemotherapeutic compounds, the pentavalent antimonial agents, introduced in the 1940s, are still generally accepted as the first-line therapy for all forms of leishmaniasis. 1,2 However, clinical resistances and therapeutic failures in immunocompromised and immunocompetent patients have been increasingly recognized in recent years, leading to the need for second-line drugs. 3

The pore-forming antibiotic amphotericin B, extensively used to treat serious systemic fungal infections, has been shown to have efficacy against leishmaniasis in spite of its toxic side effects. Since 1984, it has been applied successfully to the treatment of both cutaneous and visceral leishmaniasis. 4,5 In spite of therapeutic failures in immunocompromised patients, 6 no primary amphotericin B resistance has been reported in Leishmania sp.; moreover, recent studies have demonstrated that effective doses of amphotericin B were stable after long-term treatments in immunocompromised patients. 7 Nevertheless, the possibility of emergence of amphotericin B resistant isolates has been proven by the experiments of Mbongo et al., 8 who obtained amphotericin B resistant promastigotes after in-vitro drug pressure. We demonstrated in a previous study 9 that flow cytometric determination of membrane potential changes in L. infantum promastigotes constituted an easy and reliable method for studying the interactions between amphotericin B and the parasite membrane and evaluating the susceptibility of isolates to the antibiotic.

In the present study, we planned to explore the effect of amphotericin B on the parasite membrane in the course of amphotericin B treatment in wild strains of L. infantum. For this purpose, amphotericin B induced membrane potential changes were measured in 45 isolates from immunocompetent children and immunocompromised patients with visceral leishmaniasis during the first episode and eventually after relapses.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
Patients and samples

Forty-five isolates of L. infantum were recovered from 30 patients with visceral leishmaniasis living in the area of Marseilles: 11 immunocompetent children (0.5-3 years old) treated with liposomal amphotericin B (liposomal AmB (AmBisome, Nexstar Pharmaceuticals, Paris, France) 3 mg/kg/day for 10 days), and 19 young adults (30-35 years old) infected with the human immunodeficiency virus (HIV), treated with intralipid amphotericin B (intralipid AmB (intralipid 20% emulsion, Pharmacia SA, Paris, France) 1-2 mg/kg/day for 21 days). Leishmania sp. parasites were recovered during the first episode, and eventually after relapses, from bone marrow aspirates and blood samples. They were isolated in NNN medium (Gibco, Paisley, UK) and cultivated in RPMI medium (Eurobio) supplemented with 15% fetal calf serum, France). They were analysed by the isoenzymatic method: all were identified as L. infantum and three zymodems were represented (28 MON 1, one MON 11 and one MON 108). Two referenced isolates that had never been treated with amphotericin B were added to the study: L. infantum MON 1 (MHOM/ FR/78/LEM75) and L. infantum MON 11 (MHOM/FR/80/ LEM189).

Flow cytometric assessment of amphotericin B-membrane interactions

Susceptibility of isolates to amphotericin B (Fungizone, Bristol Myers Squibb, Paris, France) was assessed immediately after isolation and growth of promastigotes (no more than 2 weeks between isolation and test). The method used for evaluating the susceptibility of parasites to amphotericin B was based on a flow cytometric technique extensively used in clinical microbiology 10 and adapted for use with leishmania promastigotes. This technique was established on the basis that the main mechanism of action of polyene antibiotics such as amphotericin B was an interaction with membrane sterols, inducing membrane potential depletion. The method consisted of measuring antibiotic-induced membrane potential changes by using a carbocyanine dye, 3,3'-dipentyloxacarbocyanine iodide (DIOC 5(3)), 11 which is positively charged and accumulates inside the cell according to the Nernst equation: DIOC 5(3)-related fluorescence variations in parasites were proportional to membrane potential changes. Parasites maintained in RPMI medium supplemented with 15% heat-inactivated fetal calf serum were treated with a range of amphotericin B (Sigma, St Louis, MO, USA) concentrations (from 0 to 10 mg/L). After a 3 h incubation period at 25°C, DIOC 5(3) (Molecular Probes, Eugene, OR, USA) was incorporated into each cell culture, at the final concentration of 0.5 µM at room temperature. Cells were run immediately on a FacScan analytical flow cytometer (Becton Dickinson, Paris, France). Ten thousand cells were used for each analysis and their relative fluorescence was estimated by the arithmetic mean of their distribution. Amphotericin B induced fluorescence inhibition was expressed as the percentage of fluorescence observed in amphotericin B treated promastigotes compared with the control culture. Dose-response curves representing the percentage of fluorescence compared with the control culture according to the concentration of amphotericin B incorporated in duplicate parasite cultures were calculated by a non-linear regression. Amphotericin B susceptibility was expressed by the 90% inhibitory concentration (IC 90), representing the concentration of antibiotic that induced 90% of fluorescence decrease compared with the control culture.

Assessment of amphotericin B susceptibility on Leishmania intracellular amastigotes

The activity of amphotericin B on the amastigote form of the parasite was assessed in adherent human monocytes (THP1) infected by the two referenced Leishmania sp. isolates and the isolate from patient 10 during the first episode and the following relapses. The test was performed according to the methods previously described by Ogunkolade et al. 12 Briefly, adherent cells were obtained by treating THP1-monocytes with 1 µM phorbol myristate acetate in RPMI medium. After a 48 h incubation period at 37°C, cells were rinsed and infected by a suspension of promastigotes using an infection ratio of 10/1 parasites to macrophages. Adapted dilutions of amphotericin B were added in duplicate cultures and incubated for 96 h at 37°C. The plates were fixed with methanol and stained with 10% Giemsa stain. The percentage of infected macrophages in each assay was determined microscopically at 1000x magnification. The activity of amphotericin B on amastigotes was expressed by the 50% inhibitory concentrations (IC 50), illustrating the concentrations of drug that produced a 50% reduction of infected macrophage compared with the control culture.

Statistical analysis

Comparison between independent variables such as amphotericin B susceptibilities observed in isolates from immunocompromised and immunocompetent patients was performed by the Mann- Whitney U-test. The Wilcoxon test was used for comparing double variables such as amphotericin B sensitivities observed before and after treatment in immunocompromised patients.


    Results
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 Materials and methods
 Results
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 References
 
Complete data for immunocompetent children are reported in Table I: IC 90 ranged from 0.075 to 0.435 mg/L, and averaged 0.218 ± 0.12 mg/L. Treatment involving liposomal amphotericin B was highly effective whatever the susceptibility of isolates to amphotericin B determined by flow cytometric assessment. In four cases, previous inefficient meglumine treatment was administered to children before liposomal amphotericin B: the corresponding isolates, which demonstrated primary clinical and biological resistance to antimonial agents, 3 did not exhibit a reduced susceptibility to amphotericin B (IC 90 = 0.118 mg/L).


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Table I. Amphotericin B susceptibility in immunocompetent children
 
Results observed in immunocompromised patients are reported in Table II: IC 90 measured during the first episode ranged from 0.065 to 0.654 mg/L and averaged 0.200 ± 0.14 mg/L. These values were not significantly different from those observed in immunocompetent patients (P= 0.865, Mann-Whitney test); nevertheless, treatment with intralipid amphotericin B was not fully effective among this population: 10 cases of treatment failure and 23 cases of relapse were observed out of 33 clinical outcomes.


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Table II. Amphotericin B susceptibility in immunocompromised adult patients
 
Results observed during the first relapse revealed that the susceptibility of promastigotes to amphotericin B was reduced after only one course of treatment: IC 90 values ranged from 0.146 to 1 mg/L and averaged 0.354 ± 0.27 mg/L. These values were significantly different from the initial levels (P= 0.015, Wilcoxon test) and suggested that adaptation to amphotericin B could occur in Leishmania isolates after long-term treatment in immunocompromised patients. Figure 1 displays the evolution of amphotericin B susceptibility in a Leishmaniaisolate from patient 10. A significant increase of IC 90 (from 0.165 to 1.29 mg/L) could be observed in promastigotes during the course of amphotericin B cures, suggesting a rapid adaptation of parasites to therapeutic pressure. Amphotericin B susceptibility levels measured in the amastigote form are shown in Figures 1 and 2. IC 50 values observed during the first episode and during successive relapses were respectively 0.043, 0.0654, 0.101 and 0.152 mg/L: a progressive decrease in amphotericin B susceptibility could also be detected in the course of treatment with amphotericin B.



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Figure 1. Changes in amphotericin B susceptibility in the course of successive relapses in patient 10. •, IC 90 observed in promastigotes; {circ}, IC 50 observed in amastigotes.

 


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Figure 2. Changes in amphotericin B susceptibility in isolates from patient 10 in amastigotes. •, first episode; {blacktriangleup}, third episode; hexagon, fifth episode; {blacksquare}, sixth episode.

 
Amphotericin B susceptibility observed in L. infantum MON 1 (MHOM/FR/78/LEM75) and L. infantum MON 11 (MHOM/FR/80/LEM189) promastigotes were 0.098 and 0.101 mg/L respectively, and they reached 0.0236 and 0.0354 mg/L in the amastigote form. Results were consistent with IC 90 and IC 50 values observed in the wild strains and no difference could be noted between the two zymodems.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
As a consequence of its powerful activity on ergosterol-containing membranes, amphotericin B has been shown to be effective against all forms of leishmaniasis. 13 Recently, the development of new formulations such as liposomes, lipid complexes and colloidal dispersions, which reduce amphotericin B acute and chronic side effects, has multiplied the use of this polyene antibiotic for antileishmanial chemotherapy. 1 In spite of relapses described in immunocompetent patients receiving short courses, 6 the efficacy of all amphotericin B formulations against leishmaniasis has been proven extensively. 14 Nevertheless, treatment failures and relapses have been observed commonly in immunocompromised patients, 13,15 illustrating the problem of the treatment of HIV-Leishmaniasp. coinfections.

Results observed in the present study confirmed the efficacy of liposomal amphotericin B 6,15,16 in the treatment of glucantime-resistant visceral leishmaniasis among immunocompetent patients: no case of treatment failure was seen in children; moreover, an early reduction of inflammatory signs illustrated by fever disappearance after 44 h was observed and no secondary effect was found. In HIV-Leishmania sp. coinfected patients, amphotericin B courses of treatment involving intralipid emulsions were not fully effective: the first relapses occurred 1-15 months following treatment and, after the first episode, additional courses of treatment were similarly ineffective. Nevertheless, during relapses, no pulmonary or cutaneous localizations 17 could be detected. Liposomal formulation of amphotericin B has been shown to be more effective than lipid emulsion in the treatment of murine visceral leishmaniasis; 18 moreover, it has been demonstrated to reduce the acute and chronic side effects of the parent drug, permitting higher dosages to be tolerated. However, its cost was high 19,20 compared with that of intralipid amphotericin B; that was the main reason why, in the present study, liposomal amphotericin B was preferentially used only for immunocompetent children. A comparison between the clinical efficacy of each amphotericin B formulation was therefore not possible in these two populations.

The susceptibility to amphotericin B of Leishmania sp. isolates from patients was assessed by a new flow cytometric method that consisted of measuring drug induced membrane potential changes. This technique has been well validated with fungi; however, its application to Leishmania sp. promastigotes is recent. Nevertheless, we demonstrated in a previous study 9 that the concentrations of amphotericin B inducing a 90% decrease of membrane potential (IC 90) produced a 90% drop in parasite growth. Moreover, a strong correlation could be established between results obtained by flow cytometry and susceptibility levels determined by the conventional assessment of parasite growth, suggesting that this new method could be a reliable tool for estimating the sensitivity of parasites to polyene antibiotics.

Amphotericin B susceptibility measured in promastigotes during the first episode exhibited a weak range of variations in both immunocompromised and immunocompetent patients (0.065 <= IC 90 <= 0.654 mg/L), and no significant difference could be observed between IC 90 determined in each population. No primary resistance to amphotericin B could be detected, as no treatment failure could be observed in immunocompetent children. On the contrary, in HIV-Leishmania sp. coinfected patients, relapses could be observed whatever the susceptibility of isolates. In fact, all HIV-Leishmania sp. coinfected patients exhibited weak levels of T4 cells (<100/mm 3) during relapses, suggesting that the success of the antileishmanial treatment depended greatly on patient immunity status.

After an initial cure, amphotericin B susceptibility was shown to decline in Leishmania sp. promastigotes: in patient 10, more particularly, the IC 90 increased 10-fold after six relapses. This result indicated that after long-term treatment in vivo, interactions between amphotericin B and the parasite membrane could be modulated, leading to a significant decrease of amphotericin B susceptibility in the promastigote form of the parasite. Results observed in infected THP1-cells indicated also that sensitivity to amphotericin B was reduced in the intracellular amastigote form of the parasite: the IC 50 was multiplied by three after six relapses. The mechanisms involved in this adaptation could possibly be similar to those observed in fungi, such as stable qualitative and quantitative alterations in the lipid composition of the membrane 21 or resistance to oxidation-dependent damage. 22 On this basis, the assessment of membrane potential changes by flow cytometry could be useful for the study of resistance to amphotericin B, as it has been demonstrated that the antibiotic killed unicellular parasites by forming aqueous pores permeable to small anions and cations, 23 leading to membrane depolarization. 24 Thus, in the clinical trial, flow cytometric measurement of amphotericin B susceptibility has been shown to be a reliable way of evaluating the susceptibility of yeasts and bacteria to pore-forming antibiotics. 10 Concerning Leishmania sp. parasites, however, although results for patient 10 showed that a reduction of amphotericin B susceptibility in promastigotes could be followed by a similar decrease of amastigote susceptibility, the application of this flow cytometric technique could not have any predictive value concerning leishmaniasis clinical outcome: drug susceptibility in the intracellular amastigote form of the parasite was shown to differ from that in the promastigote form. 25 Amphotericin B activity appeared also to depend on interactions with macrophages 26 and the efficacy of treatment was demonstrated to vary greatly according to patient immunity status. 18 Nevertheless, this technique could be used for examining rapidly the evolution of interactions between amphotericin B and the parasite membrane in the course of amphotericin B treatment and detecting the emergence of amphotericin B resistant isolates.


    Acknowledgments
 
We wish to thank Dr N. Petit (Sce du Pr. Gastaut, IPC Marseille), Dr H.Tissot-Dupont(Sce du Pr. Gallais, Hôpital La Conception, Marseille), Dr P. Brouqui and R. Moreau (Sce du Pr. Bourgeade Hôpital Houphouet-Boigny, Marseille), Dr P. Marty (Hôpital Larchet, Nice), Pr. M. Deniau (Laboratoire de Parasitologie-Mycologie, CHU Henri Mondor, Créteil)and Pr. Perrimond, Unal and Giraud (Hôpital la Timone, Marseille) for providing Leishmania isolates.We are grateful to Dr Pratlong (Laboratoire de Parasitologie et Ecologie médicale, Montpellier, France)for the isoenzymatic analysis of Leishmania isolates.


    Notes
 
* Corresponding author. Tel: +33-04-91-83-55-44; Fax: +33-04-91-80-26-12. Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 28 July 1998; returned 6 October 1998; revised 26 November 1998; accepted 5 March 1999