Publication of accurate and detailed descriptions of methods in research articles involving animals is essential for health scientists to accurately interpret published data, evaluate results and replicate findings. Inadequate reporting of key aspects of experimental design may reduce the impact of studies and could act as a barrier to translation of research findings. Reporting of animal use must be as comprehensive as possible in order to take advantage of every study and every animal used. Animal models are essential to understanding and assessing new chemotherapy candidates for Chagas disease pathology, a widespread parasitic disease with few treatment options currently available. A systematic review was carried out to compare ARRIVE guidelines recommendations with information provided in publications of preclinical studies for new anti-Trypanosoma cruzi compounds. A total of 83 publications were reviewed. Before ARRIVE guidelines, 69% of publications failed to report any macroenvironment information, compared to 57% after ARRIVE publication. Similar proportions were observed when evaluating reporting of microenvironmental information (56% vs. 61%). Also, before ARRIVE guidelines publication, only 13% of papers described animal gender, only 18% specified microbiological status and 13% reported randomized treatment assignment, among other essential information missing or incomplete. Unfortunately, publication of ARRIVE guidelines did not seem to enhance reporting quality, compared to papers appeared before ARRIVE publication. Our results suggest that there is a strong need for the scientific community to improve animal use description, animal models employed, transparent reporting and experiment design to facilitate its transfer and application to the affected human population. Full compliance with ARRIVE guidelines, or similar animal research reporting guidelines, would be an excellent start in this direction.

Although many Trypanosoma cruzi (T. cruzi) strains isolated from a wide range of hosts have been characterized, there is a lack of information about biological features from vertically transmitted strains. We describe the molecular and biological characteristics of the T. cruzi VD strain isolated from a congenital Chagas disease patient. The VD strain was typified as DTU TcVI; in vitro sensitivity to nifurtimox (NFX) and beznidazole (BZ) were 2.88 μM and 6.19 μM respectively, while inhibitory concentrations for intracellular amastigotes were 0.24 μM for BZ, and 0.66 μM for NFX. Biological behavior of VD strain was studied in a mouse model of acute infection, resulting in high levels of parasitemia and mortality with a rapid clearence of bloodstream trypomastigotes when treated with BZ or NFX, preventing mortality and reducing parasitic load and intensity of inflammatory infiltrate in skeletal and cardiac muscle. Treatment-induced parasitological cure, evaluated after immunossupression were 41% and 35% for BZ and NFX treatment respectively, suggesting a partial response to these drugs in elimination of parasite burden. This exhaustive characterization of this T. cruzi strain provides the basis for inclusion of this strain in a panel of reference strains for drug screening and adds a new valuable tool for the study of experimental T. cruzi infection.

Drug repurposing and combination therapy have been proposed as cost-effective strategies to improve Chagas disease treatment. Miltefosine (MLT), a synthetic alkylphospholipid initially developed for breast cancer and repositioned for leishmaniasis, is a promising candidate against Trypanosoma cruzi infection. This study evaluates the efficacy of MLT as a monodrug and combined with benznidazole (BZ) in both in vitro and in vivo models of infection with T. cruzi (VD strain, DTU TcVI). MLT exhibited in vitro activity on amastigotes and trypomastigotes with values of IC50 = 0.51 µM (0.48 µM; 0,55 µM) and LC50 = 31.17 µM (29.56 µM; 32.87 µM), respectively. Drug interaction was studied with the fixed-ration method. The sum of the fractional inhibitory concentrations (ΣFICs) resulted in ∑FIC= 0.45 for trypomastigotes and ∑FIC= 0.71 for amastigotes, suggesting in vitro synergistic and additive effects, respectively. No cytotoxic effects on host cells were observed. MLT efficacy was also evaluated in a murine model of acute infection alone or combined with BZ. Treatment was well tolerated with few adverse effects, and all treated animals displayed significantly lower mean peak parasitemia and mortality than infected non-treated controls (p<0.05). The in vivo studies showed that MLT led to a dose-dependent parasitostatic effect as monotherapy which could be improved by combining with BZ, preventing parasitemia rebound after a stringent immunosuppression protocol. These results support MLT activity in clinically relevant stages from T. cruzi, and it is the first report of positive interaction with BZ, providing further support for evaluating combined schemes using MLT and exploring synthetic alkylphospholipids as drug candidates.

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There is an urgent need to develop safer and more effective drugs for Chagas disease, as current treatment relies on benznidazole (BZ) and nifurtimox (NFX). Using the Trypanosoma cruzi Dm28c strain genetically engineered to express the Escherichia coli β-galactosidase gene, lacZ, we have adapted and validated an easy, quick, and reliable in vitro assay suitable for high throughput screening for candidate compounds with anti-T. cruzi activity. In vitro studies were conducted to determine trypomastigotes sensitivity to BZ and NFX from Dm28c/pLacZ strain by comparing the conventional labour-intensive microscopy counting method with the colourimetric assay. Drug concentrations producing the lysis of 50% of trypomastigotes (LC50) were 41.36 and 17.99 µM for BZ and NFX, respectively, when measured by microscopy and 44.74 and 38.94 µM, for the colourimetric method, respectively. The optimal conditions for the amastigote development inhibitory assay were established considering the parasite-host relationship (i.e., multiplicity of infection) and interaction time, the time for colourimetric readout and the incubation time with the β-galactosidase substrate. The drug concentrations resulting in 50% amastigote development inhibition (IC50) obtained with the colourimetric assay were 2.31 µM for BZ and 0.97 µM for NFX, similar to the reported values for the Dm28c wild strain (2.80 µM and 1.5 µM respectively). In summary, a colourimetric assay using the Dm28c/pLacZ strain of T. cruzi has been set up, obtaining biologically meaningful sensibility values with the reference compounds on both trypomastigotes and amastigotes forms. This development could be applied to high throughput screening programs aiming to identify compounds with anti-T. cruzi in vitro activity.

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