Miltefosine and Benznidazole Combination Improve Anti-Trypanosoma cruzi In Vitro and In Vivo Efficacy.

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.

Evaluation of the Chagas Western Blot IgG Assay for the Diagnosis of Chagas Disease.

Chagas disease is a debilitating and often fatal pathology resulting from infection by the protozoan parasite Trypanosoma cruzi. In its recommendations, the World Health Organization states that the diagnosis of T. cruzi infection is usually based on the detection of antibodies against T. cruzi antigens and performed with two methodologically different assays. An inconclusive result can be resolved with a third "confirmatory" assay. The objective of this article is to evaluate the effectiveness of the Chagas Western Blot IgG assay (LDBio Diagnostics, Lyon, France) as a confirmatory serologic test. The Chagas Western Blot IgG assay was performed with native antigens derived from a T. cruzi strain of the TcVI genotype. Retrospective sera were provided by two parasitology laboratories (France and Argentina). The sensitivity, specificity, positive predictive value and negative predictive value of the Chagas blot were all 100% in our sera collection. The Chagas blot is an easy and qualitative method for the diagnosis of Chagas disease, with results in less than 2 h. This immunoblot has potential as a supplemental test for the confirmation of the presence of antibodies against T. cruzi in serum specimens. Nonetheless, the very good initial results presented here will need to be confirmed in larger studies.

Optimization and biological validation of an in vitro assay using the transfected Dm28c/pLacZ Trypanosoma cruzi strain.

There is an urgent need to develop safer and more effective drugs for Chagas disease, as the current treatment relies on benznidazole (BZ) and nifurtimox (NFX). Using the Trypanosoma cruzi Dm28c strain genetically engineered to express the Escherichia coli ß-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 (lytic concentration 50%) 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 ß-galactosidase substrate. The drug concentrations resulting in 50% amastigote development inhibition 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 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 programmes aiming to identify compounds with anti-T. cruzi in vitro activity.

Refining drug administration in a murine model of acute infection with Trypanosoma cruzi.

BACKGROUND: In animal research, "refinement" refers to modifications of husbandry or experimental procedures to enhance animal well-being and minimize or eliminate pain and distress. Evaluation of drug efficacy in mice models, such as those used to study Trypanosoma cruzi infection, require prolonged drug administration by the oral route (e.g. for 20 consecutive days). However, the orogastric gavage method can lead to significant discomfort, upper digestive or respiratory tract lesions, aspiration pneumonia and even accidental death. The aim of this work was to evaluate the effect of two administration methods (conventional oral gavage vs. a refined method using a disposable tip and automatic pipette) on the efficacy of benznidazole in a murine model of T. cruzi infection. RESULTS: Both administration methods led to a rapid and persistent reduction in parasitaemia. Absence of T. cruzi DNA (evaluated by real-time PCR) in blood, cardiac and skeletal muscle confirmed that treatment efficacy was not influenced by the administration method used. CONCLUSIONS: The proposed refined method for long-term oral drug administration may be a suitable strategy for assessing drug efficacy in mice models of Chagas disease and can be applied to similar murine infection models to reduce animal discomfort.

In vitro and in vivo activity of voriconazole and benznidazole combination on trypanosoma cruzi infection models.

Combination therapy has been proposed as an ideal strategy to reduce drug toxicity and improve treatment efficacy in Chagas disease. Previously, we demonstrated potent in vivo anti-Trypanosoma cruzi activity of voriconazole. In this work, we aimed to study the synergistic effect of voriconazole (VCZ) and benznidazole (BZ) both in vitro and in vivo models of T. cruzi infection using the Tulahuen strain. Combining VCZ and BZ at fixed concentrations, the inhibitory concentration 50% (IC50) on amastigotes was lower than the obtained IC50 for BZ alone and the Fractional Inhibitory Concentration Index (∑FIC) suggested an in vitro additive effect on T. cruzi amastigotes inhibition at concentrations devoid of cytotoxic effects. Treatment response in the in vivo model was evaluated by comparing behavior and physical aspects, parasitemia and mortality of mice infected with Tulahuen strain. VCZ and BZ treatments alone or in combination were well tolerated. All treated animals displayed significantly lower mean peak parasitemia and mortality compared to infected non-treated controls (p< 0.05). However, VCZ + BZ combination elicited no additional benefits over BZ monotherapy. VCZ efficacy was not enhanced by combination therapy with BZ at the doses studied, requiring further and astringent non-clinical studies to establish the VCZ efficacy and eventually moving forward to clinical trials.

Molecular and biological characterization of a highly pathogenic Trypanosoma cruzi strain isolated from a patient with congenital infection.

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.

Secretome analysis of Trypanosoma cruzi by proteomics studies.

BACKGROUND: Chagas disease is a debilitating often fatal disease resulting from infection by the protozoan parasite Trypanosoma cruzi. Chagas disease is endemic in 21 countries of the Americas, and it is an emerging disease in other countries as a result of migration. Given the chronic nature of the infection where intracellular parasites persist for years, the diagnosis of T. cruzi by direct detection is difficult, whereas serologic tests though sensitive may yield false-positive results. The development of new rapid test based on the identification of soluble parasitic antigens in serum would be a real innovation in the diagnosis of Chagas disease. METHODS: To identify new soluble biomarkers that may improve diagnostic tests, we investigated the proteins secreted by T. cruzi using mass spectrometric analyses of conditioned culture media devoid of serum collected during the emergence of trypomastigotes from infected Vero cells. In addition, we compared the secretomes of two T. cruzi strains from DTU Tc VI (VD and CL Brener). RESULTS: Analysis of the secretome collected during the emergence of trypomastigotes from Vero cells led to the identification of 591 T. cruzi proteins. Three hundred sixty three proteins are common to both strains and most belong to different multigenic super families (i.e. TcS, GP63, MASP, and DGF1). Ultimately we have established a list of 94 secreted proteins, common to both DTU Tc VI strains that do not belong to members of multigene families. CONCLUSIONS: This study provides the first comparative analysis of the secretomes from two distinct T. cruzi strains of DTU TcVI. This led us to identify a subset of common secreted proteins that could potentially serve as serum markers for T. cruzi infection. Their potential could now be evaluated, with specific antibodies using sera collected from patients and residents from endemic regions.

Quality of Reporting and Adherence to ARRIVE Guidelines in Animal Studies for Chagas Disease Preclinical Drug Research: A Systematic Review.

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.