In Vitro and In Vivo High-Throughput Assays for the Testing of Anti-Trypanosoma cruzi Compounds

Abstract

BackgroundThe two available drugs for treatment of T. cruzi infection, nifurtimox and benznidazole (BZ), have potential toxic side effects and variable efficacy, contributing to their low rate of use. With scant economic resources available for antiparasitic drug discovery and development, inexpensive, high-throughput and in vivo assays to screen potential new drugs and existing compound libraries are essential.MethodsIn this work, we describe the development and validation of improved methods to test anti-T. cruzi compounds in vitro and in vivo using parasite lines expressing the firefly luciferase (luc) or the tandem tomato fluorescent protein (tdTomato). For in vitro assays, the change in fluorescence intensity of tdTomato-expressing lines was measured as an indicator of parasite replication daily for 4 days and this method was used to identify compounds with IC50 lower than that of BZ.FindingsThis method was highly reproducible and had the added advantage of requiring relatively low numbers of parasites and no additional indicator reagents, enzymatic post-processes or laborious visual counting. In vivo, mice were infected in the footpads with fluorescent or bioluminescent parasites and the signal intensity was measured as a surrogate of parasite load at the site of infection before and after initiation of drug treatment. Importantly, the efficacy of various drugs as determined in this short-term (<2 weeks) assay mirrored that of a 40 day treatment course.ConclusionThese methods should make feasible broader and higher-throughput screening programs needed to identify potential new drugs for the treatment of T. cruzi infection and for their rapid validation in vivo.

New, Combined, and Reduced Dosing Treatment Protocols Cure Trypanosoma cruzi Infection in Mice

“…We have previously used immunosuppression with cyclophosphamide to demonstrate that treatment of T. cruzi-infected mice with the antifungal POS or the nitrotriazole NTLA-1 suppresses parasitemia but failed to provide parasitological cure in all treated mice [24]. Mice cured with BZ, NTLA-1, or POS exhibited undetectable levels of parasite DNA in skeletal muscle tissue and an absence of histological evidence of infection or disease, whereas drug failure was indicated by relatively high levels of parasite DNA in tissue, amastigote nests, and a significant inflammation in the skeletal muscle after immunosuppression ( Figure 1A-C and data not shown).…”

The overexpression of the trypanosomatid-exclusive TcRBP19 RNA-binding protein affects cellular infection by Trypanosoma cruzi

“…These drugs are primarily effective during the early-acute phase; in chronically infected patients, they are considered less effective and their use can even lead to resistance. The severe side effects associated with benznidazole and nifurtimox and the absence of paediatric formulations (a serious issue because the majority of acute Chagas disease patients are children) underscore the drawbacks of these drugs (Canavaci et al 2010). The characterisation of T. cruzi-specific molecules or processes could provide a basis for the development of novel tools for appropriate disease treatments.…”

Oral Exposure to Trypanosoma cruzi Elicits a Systemic CD8+T Cell Response and Protection against Heterotopic Challenge

“…The orally vaccinated mice were challenged in each footpad with 2.5 ϫ 10 5 CL trypomastigotes transfected with a gene encoding TdTomato fluorescent protein, and parasite load at the site of infection was monitored from 2 to 10 days postchallenge (dpc) by in vivo imaging (9). Orally vaccinated mice displayed a significant reduction in the fluorescence signal compared to control unvaccinated mice at all times measured ( Fig.…”

“…For protection studies, T. cruzi parasites of the CL strain expressing the far-red tdTomato protein (9,64) were subcutaneously inoculated into superficial subcutaneous tissue of the footpads. Mouse feet were imaged every other day using the Maestro 2 in vivo imaging system (CRi, MA) with the green set of filters (acquisition settings: 560 to 750 in 10-nm steps; exposure time of 88.18 ms and 2 ϫ 2 binning).…”