High-content imaging for automated determination of host-cell infection rate by the intracellular parasite Trypanosoma cruzi

Abstract

Chagas disease affects 8-11 million people, mostly in Latin America. Sequelae include cardiac, peripheral nervous and/or gastrointestinal disorders, thus placing a large economic and social burden on endemic countries. The pathogenesis and the evolutive pattern of the disease are not fully clarified. Moreover, available drugs are partially effective and toxic, and there is no vaccine. Therefore, there is an urgent need to speed up basic and translational research in the field. Here, we applied automated high-content imaging to generate multiparametric data on a cell-by-cell basis to precisely and quickly determine several parameters associated with in vitro infection of host-cell by Trypanosoma cruzi, the causative agent of Chagas disease. Automated and manual quantification was used to determine the percentage of T. cruzi-infected cells in a 96-well microplate format and the data generated was statistically evaluated. Most importantly, this automated approach can be widely applied for discovery of potential drugs as well as molecular pathway elucidation not only in T. cruzi but also other human intracellular pathogens.

An Image-Based Algorithm for Precise and Accurate High Throughput Assessment of Drug Activity against the Human Parasite Trypanosoma cruzi

“…In previous research, thresholding and size-based filtering, median filtering, and top-hat filtering methods have been presented to segment nuclei regions [6], [7]. However, those approaches were not suitable for our T. cruzi images for the following reasons:…”

Current and Future Chemotherapy for Chagas Disease

“…The most recent trend in whole-cell assays has employed the use of high-content screening analyzers -automated microscopes that can image many conditions (compounds) in clear bottom culture microplates. When the technology appeared one assay was developed that made use of mammalian cells expressing GFP and parasites stained with DAPI, and both manual and automated data analysis was performed 50 . With critics of genetically modified parasites/host cells and the further development of technology an improved assay was developed that used whole unmodified cells and parasites.…”

Metal–drug synergy: new ruthenium(II) complexes of ketoconazole are highly active against Leishmania major and Trypanosoma cruzi and nontoxic to human or murine normal cells

“…The cells were fixed with 4% paraformaldehyde and stained with Alexa Fluor 488 Phalloidin and DAPI. The number of infected cells and amastigotes was determined by HCIA using a BD Pathway Bioimager microplate confocal microscope [30, 43]. Subsequently, the infection index was calculated from the mean of triplicate values by multiplying the percentage of infected cells by the constraint used in the HCIA assay, which was of 5 parasites per macrophage (adaptation from Lonardoni et al 2000).…”