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

Leishmaniasis is a tropical disease threatening 350 million people from endemic regions. The available drugs for treatment are inadequate, with limitations such as serious side effects, parasite resistance or high cost. Driven by this need for new drugs, we developed a high-content, high-throughput image-based screening assay targeting the intracellular amastigote stage of different species of Leishmania in infected human macrophages. The in vitro infection protocol was adapted to a 384-well-plate format, enabling acquisition of a large amount of readouts by automated confocal microscopy. The reading method was based on DNA staining and required the development of a customized algorithm to analyze the images, which enabled the use of non-modified parasites. The automated analysis generated parameters used to quantify compound activity, including infection ratio as well as the number of intracellular amastigote parasites and yielded cytotoxicity information based on the number of host cells. Comparison of this assay with one that used the promastigote form to screen 26,500 compounds showed that 50% of the hits selected against the intracellular amastigote were not selected in the promastigote screening. These data corroborate the idea that the intracellular amastigote form of the parasite is the most appropriate to be used in primary screening assay for Leishmania

Etude preliminaire de l'association isoniazide-epiroprim dans un modele murin de tuberculose

International audienceThe purpose of this study regarding isoniazid-epiroprim's association applied to antituberculosis chemotherapy, carried through murine model, initiated into Institut Pasteur of Côte d'Ivoire and worked out at Institut Pasteur of Paris was to evaluate the epiroprim's effect alone and associated with isoniazid on Mycobacterium tuberculosis. Sixteen mouses (lineage C57Bl/6) were inoculated by venous way with 10(5) viable bacillus (strain H37Rv) suspended in 500 microliters sterile physiological aqueous solution and were shared out into 4 sets. Fifteen days later the sets have been submitted or not to a daily treatment by gavage during three weeks (epiroprim, isoniazid, isoniazid plus epiroprim). The mouses were euthanasied, spleen and lung were removed from each animal. The titres of determined bacillus into those organs prove that isoniazid and epiroprim associated seem more efficacious than the isoniazid monotherapy for mouses pulmonary tuberculosis. Bacillus obtained are sensitive to isoniazid.Cette étude de chimiothérapie antituberculeuse expérimentale de l'association isoniazide-épiroprim dans un modèle murin initiée à l'Institut Pasteur de Côte d'Ivoire et réalisée à l'Institut Pasteur à Paris avait pour objectif d'évaluer l'effet de l'épiroprim, un inhibiteur de la dihydrofolate reductase seul et en association avec l'isoniazide sur Mycobacterium tuberculosis. 16 souris (lignée C57Bl/6) recevant chacune par voie veineuse un inoculum contenant 10 5 bacilles viables (souche H37RV) en suspension dans 500 µl de soluté physiologique stérile ont été réparties en quatre lots. 15 jours après l'inoculation, les lots ont été pendant trois semaines soumis ou non à un traitement journalier (épiroprim, isoniazide, isoniazide épiroprim) par gavage. Au terme du traitement, les souris ont été euthanasiées, la rate et le poumon prélevés chez chaque animal. Les titres de bacilles déterminés dans ces organes montrent que l'association isoniazide-épiroprim semble plus efficace que la monothérapie à l'isoniazide pour le traitement de la tuberculose pulmonaire de la souris. Les bacilles obtenus à partir de ces organes sont sensibles à l'isoniazide