Michèle Deniau

Real-Time PCR as a New Tool for QuantifyingLeishmania infantum in Liver in Infected Mice

ABSTRACT The parasitic loads of mouse livers experimentally infected withLeishmania infantum were determined using a double real-time quantitative PCR test targeted to the parasite DNA polymerase gene and to the mouse brain-derived neutrophic factor gene. TheLeishmania DNA copy number was normalized to the number of mouse gene copies in order to quantify the former independently of liver weight. The correlation coefficient with the microtitration method was 0.66. This PCR assay can be considered for experimental pharmaceutical studies.

Detection and Identification of Leishmania Species from Clinical Specimens by Using a Real-Time PCR Assay and Sequencing of the Cytochrome b Gene

ABSTRACT Visceral and cutaneous leishmaniases are heterogenous entities. The Leishmania species that a given patient harbors usually cannot be determined clinically, and this identification is essential to prescribe the best species-specific therapeutic regimen. Our diagnosis procedure includes a real-time PCR assay targeted at the 18S rRNA gene, which detects all Leishmania species but which is not specific for a given Leishmania species. We developed a species identification based on sequencing of the cytochrome b (cyt b) gene directly from the DNA extracted from the clinical specimen. The sequences were analyzed using the Sequence Analysis/Seqscape v2.1 software (Applied Biosystems). This software is designed to automatically identify the closest sequences from a reference library after analysis of all known or unknown polymorphic positions. The library was built with the Leishmania cyt b gene sequences available in GenBank. Fifty-three consecutive real-time PCR-positive specimens were studied for species identification. The cyt b gene was amplified in the 53 specimens. Sequencing resulted in the identification of six different species with ≥99% identity with the reference sequences over 872 nucleotides. The identification was obtained in two working days and was in accordance with the multilocus enzyme electrophoresis identification when available. Real-time PCR followed by sequencing of the cyt b gene confirmed the diagnosis of leishmaniasis and rapidly determined the infecting species directly from the clinical specimen without the need for the isolation of parasites. This technique has the potential to significantly accelerate species-adapted therapeutic decisions regarding treatment of leishmaniasis.

Activity of pentamidine-loaded methacrylate nanoparticles against Leishmania infantum in a mouse model

The use of drug delivery systems may reduce the toxicity and improve the activity of antileishmanial compounds. In view of such a strategy, we loaded the antileishmanial agent pentamidine on polymethacrylate nanoparticles. The activity of pentamidine-loaded nanoparticles was compared with that of free pentamidine in a BALB/c mice model of visceral leishmaniasis induced by Leishmania infantum. On day 0, mice were infected intravenously with 10(7) promastigotes and then treated via the tail vein on days 14, 16 and 18 with bound pentamidine, free drug or isotonic saline (control group). On day 21, liver parasite burdens were evaluated using the Stauber method. Livers and spleens were removed and weighed. Effective doses (ED) were determined using the Michaelis-Menten representation relating the percentage of parasite suppression to the dose. The ED50 of bound pentamidine was six times lower than that of free pentamidine (0.17 mg kg-1 vs 1.06 mg kg-1). The ED90 value calculated for bound pentamidine was 1 mg kg-1. It was not possible to obtain the ED90 for free pentamidine because the dose-response curve reached a plateau near 60% of parasite suppression. A significant decrease in liver and spleen weights, probably reflecting the leishmanicidal activity, was observed for treated mice with bound pentamidine. These results showed that bound pentamidine was more potent than the free drug against L. infantum in our BALB/c mice model.

Leishmania major and HIV co-infection in Burkina Faso.

The incidence of cutaneous leishmaniasis (CL) has increased in Ouagadougou, Burkina Faso since 1996. A study was carried out from September to November 2000 to determine the impact of HIV on this outbreak. Of 74 CL patients, 10 were co-infected with HIV. The percentage of CL in patients positive for HIV was slightly higher than the percentage of HIV patients in Ouagadougou. However, the study showed that HIV infection did not increase the risk of CL infection.

Sodium Stibogluconate (Pentostam) Potentiates Oxidant Production in Murine Visceral Leishmaniasis and in Human Blood

ABSTRACT Sodium stibogluconate (Sbb), a leishmanicidal drug, was studied for its in vivo effect on the formation of reactive oxygen species (ROS), assessed by chemiluminescence (CL) in the whole blood of mice infected with Leishmania infantum. Stimulation of ROS formation induced ex vivo by zymosan particles or the protein kinase C activator phorbol myristate acetate (PMA) was reduced by approximately 25% (P < 0.05) after infection of mice. Treatment of infected mice with Sbb (50 to 400 mg/kg of body weight) enhanced the blood CL induced by zymosan and PMA (47 to 96%, P < 0.01). The drug potentiation effect also occurred in uninfected mice. In vitro treatment of normal human blood with Sbb (1, 10, or 100 μg/ml) for 1 h primed the CL response to PMA (29 to 54%). The priming effect of Sbb was also observed on the production of superoxide by isolated polymorphonuclear leukocytes stimulated either by PMA and zymosan or by the chemoattractants N-formyl-Met-Leu-Phe and platelet-activating factor. These data provide the first evidence of priming of the phagocyte respiratory burst by Sbb. This novel property of Sbb may contribute to the drugs leishmanicidal effect.

Therapeutic evaluation of free and liposome-encapsulated atovaquone in the treatment of murine leishmaniasis

The use of drug delivery systems may reduce the toxicity and improve the activity of anti-leishmanial compounds. The activity of atovaquone (ATV)-loaded liposomes was compared by determination of median effective doses (ED(25) and ED(50)), with that of free ATV in a murine model of visceral leishmaniasis induced by Leishmania infantum. On day 0, mice were infected intravenously with 4.10(7) promastigotes and treated via the tail vein on days 15, 17 and 19 by free drug in a DMSO/cremophor/water solution (0.2 to 1.6 mg/kg body weight) or by liposomal drug (0.04 to 0.32 mg/kg body weight). Mice were killed and livers and spleens were removed and weighed on day 21 p.i. and liver parasite burdens evaluated using the Stauber method. Effective doses were determined using the Hill representation relating the percentage of parasite suppression to the dose. Liposomal ATV was significantly more effective than the free drug in reducing liver parasites (61.6% of parasite suppression at a dose of 0.32 mg/kg vs 34.9% at a dose of 1.6mg/kg). Liposomal ATV was 23 times more active than the free drug (ED(25) value=0. 02+/-0.01 mg/kg vs 0.46+/-0.15 mg/kg for free drug). It was not possible to obtain the ED(50) for free ATV because the dose-response curve reached a plateau around 33% of parasite suppression. Conversely, the ED(50) for liposomal ATV was 0.17+/-0.05 mg/kg. 100% efficacy of bound ATV could be obtained with a concentration of 1. 77+/-0.35 mg/kg. A significant decrease in spleen weights was also observed reflecting a leishmanicidal activity of ATV. These results suggest that liposome loaded ATV is more efficacious than the free drug against Leishmania infantum in this murine model.

Performance of serum biomarkers for the early detection of invasive aspergillosis in febrile, neutropenic patients: a multi-state model.

Background The performance of serum biomarkers for the early detection of invasive aspergillosis expectedly depends on the timing of test results relative to the empirical administration of antifungal therapy during neutropenia, although a dynamic evaluation framework is lacking. Methods We developed a multi-state model describing simultaneously the likelihood of empirical antifungal therapy and the risk of invasive aspergillosis during neutropenia. We evaluated whether the first positive test result with a biomarker is an independent predictor of invasive aspergillosis when both diagnostic information used to treat and risk factors of developing invasive aspergillosis are taken into account over time. We applied the multi-state model to a homogeneous cohort of 185 high-risk patients with acute myeloid leukemia. Patients were prospectively screened for galactomannan antigenemia twice a week for immediate treatment decision; 2,214 serum samples were collected on the same days and blindly assessed for (1->3)- β-D-glucan antigenemia and a quantitative PCR assay targeting a mitochondrial locus. Results The usual evaluation framework of biomarker performance was unable to distinguish clinical benefits of β-glucan or PCR assays. The multi-state model evidenced that the risk of invasive aspergillosis is a complex time function of neutropenia duration and risk management. The quantitative PCR assay accelerated the early detection of invasive aspergillosis (P = .010), independently of other diagnostic information used to treat, while β-glucan assay did not (P = .53). Conclusions The performance of serum biomarkers for the early detection of invasive aspergillosis is better apprehended by the evaluation of time-varying predictors in a multi-state model. Our results provide strong rationale for prospective studies testing a preemptive antifungal therapy, guided by clinical, radiological, and bi-weekly blood screening with galactomannan antigenemia and a standardized quantitative PCR assay.

Characterisation of atovaquone resistance in Leishmania infantum promastigotes

Atovaquone, an antiparasitic agent, could possibly represent an alternative therapy after relapse following classical treatment for visceral leishmaniasis. Atovaquone-resistant strains were selected in vitro by stepwise drug pressure to study the mechanism of resistance in Leishmania. Characteristics of a promastigote strain resistant to 250 microg/ml of atovaquone were compared with those of the wild type (WT) strain. Resistant strains were shown to have a high level of resistance (45 times). They were stable in drug-free medium for 6 months, and showed no cross-resistance with other antileishmanial drugs. Rhodamine uptake and efflux were studied. They were not modified in the resistant strain, indicating the absence of P-glycoprotein overexpession. The effect of atovaquone on membrane lipidic composition was determined in both WT and atovaquone-resistant promastigotes. Analysis of lipid composition of the atovaquone-resistant strain showed that sterol biosynthesis was decreased in atovaquone-resistant parasites. Cholesterol was found to be the major membrane sterol as opposed to the WT strain. Cholesterol, due to its ordering effect, could decrease membrane fluidity and subsequently block the passage of atovaquone through the membrane. Increased membrane cholesterol content and altered drug membrane fluidity resulted from possible decrease of ergosterol biosynthesis by atovaquone, incorporation of cholesterol by promastigotes in the culture medium, solubilisation of atovaquone by cholesterol and co-passage of the two compounds or influence of dimethylsulfoxide. These results indicate that different cellular alterations may participate in the resistant phenotype, by altering drug membrane permeability.

Pentamidine-loaded poly(D, L-lactide) nanoparticles : Adsorption and drug release

This work describes the loading capacity of poly(D,L‐lactide) nanoparticles, the factors influencing pentamidine release, and the cytotoxicity of nanoparticles. The nanoprecipitation method was used to prepare pentamidine‐loaded poly(D,L‐lactide) nanoparticles. Various concentrations of pentamidine base and polymer were tested. The influence of the dilution, temperature, and ionic strength was evaluated. The cytotoxicity on J 774 cells of unloaded nanoparticles, pentamidine‐loaded nanoparticles, and pentamidine isethionate were tested. The percentage of binding decreased significantly with drug load. A nonlinear increase in drug uptake per unit mass of polymer with the equilibrium pentamidine concentration was found. A Langmuir‐type sorption was suggested (r = 0.998). 390 μg/ml was found to be the highest level of drug incorporation. The increase of polymer concentration did not improve the pentamidine fixation yield. The increase in temperature or buffer molarity induced a significant release of pentamidine. The increase in dilution also induced an increase in release of pentamidine. The cytotoxicity of pentamidine‐loaded nanoparticles and unloaded nanoparticles was superimposable. After 24 h of incubation, pentamidine‐loaded nanoparticles presented an IC50 value significantly lower than that of free drug (0.39 vs. 6.5 g/ml). Drug Dev. Res. 43:98–104, 1998.

Preparation and physicochemical characterization of atovaquone-containing liposomes

This work describes the preparation and the physicochemical properties of atovaquone‐loaded liposomes. It also describes drug release from the liposomes. As many factors can influence liposome stability, we studied several formulations, including different concentrations of atovaquone, phospholipids, and cholesterol. The effect of atovaquone (ATV) concentration was also evaluated. The highest binding percentage (100±2.5%) was obtained under alkaline conditions for a 2 mg/ml concentration of ATV. The percentage of encapsulation decreased significantly when drug concentrations increased. Drug uptake (expressed per unit mass of phospholipids) was nonlinearly related to equilibrium ATV concentration. A Langmuir‐type sorption was suggested (r = 0.978). In acidic or neutral buffer, the binding percentage reached 42.1 ± 0.02%. The increase of phospholipids and cholesterol concentrations did not significantly improve the ATV binding yield for the lowest ATV concentration. Conversely, ATV binding was significantly increased for the highest ATV concentration. All the formulations tested gave monodispersed liposomes with a mean diameter around 260 nm. The dilution (1/5–1/20) of liposomes in alkaline conditions induced a significant release of ATV (74% release). In acidic or neutral buffer no release was observed, suggesting an encapsulation process. Drug Dev. Res. 47:155–161, 1999.