CONICET | Buscador de Institutos y Recursos Humanos

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi (T. cruzi), constitutes an important public health problem in Latin America. Current treatment options are limited to benznidazole (BZ) and nifurtimox which have significant side effects and limited access. In this context, there is a considerable need for the search of new compounds to improve the chemotherapy of Chagas disease. The aim of this work is to evaluate the in vivo trypanocidal activity of voriconazole (VCZ) in a murine model of acute T. cruzi infection, a drug previously shown to possess in vitro activity against T. cruzi. Two-month-old female BALB/c mice were infected by intraperitoneal injection of 500 trypomastigotes of Tulahuén strain T. cruzi. At 10 days post infection (dpi), mice with detectable parasitemia were randomly divided into the following groups: non treated (NT, n=6); BZ, 100 mg/kg, once daily for 20 days (n=8); and VCZ, 40 mg/kg, once daily for 30 days (n=8). Procedures used for housing and handling animals were in accord with the National Research Council´s Guide for Laboratory Animal Use and Care and the protocol was approved by the internal Ethics Committee at Ricardo Gutiérrez Children Hospital. During the acute phase of infection, parasitemia was evaluated three days/week in 5μl of tail vein blood examined microscopically at X400 magnification. Mortality was registered daily. Surviving mice were sacrificed at completion of study (60 dpi). Segments of heart and skeletal muscle were collected from all animals in order to evaluate the inflammatory process in the myocardium and the presence of amastigote nests in both tissues. Samples were fixed in buffered 10% formaldehyde, dehydrated, and embedded in paraffin. Then, 5µm-thick sections where stained with hematoxylin and eosin.Survival curves of the three experimental groups were compared to determine if treatment led to significantly longer life spans and decreased mortality using the Mantel-Cox log rank test and Fisher?s exact test, respectively. Peak parasitemia for each mouse was established in order to compare treatment response in BZ and VCZ groups to NT mice using the nonparametric Kruskal-Wallis rank sum test, followed by multiple pair-wise comparisons with the Wilcoxon rank sum test. The significance threshold of all results was adjusted post hoc using the Bonferroni correction. NT group exhibited the classical pattern of parasitemia, peaking at 26 dpi, and had significantly higher parasitemia levels than BZ (p=0.0023) or VCZ groups (p=0.0126). Although BZ proved to be significantly more effective than VCZ (p=0.00091), treatment with VCZ lowered peak parasitemia by 81%. Survival rates at the conclusion of the trial were 16.77%, 75% and 100% for NT, VCZ and BZ treated groups, respectively. Mice treated with BZ lived significantly longer than NT mice (p=0.0010) and had a lower risk of mortality (p=0.0030). On the other hand, survival in the VCZ group was not higher or significantly longer than NT mice. This result are most likely due to the small number of animals used, as qualitatively the survival curves show that VCZ prolongs survival and decreases risk of mortality compared to NT mice. The degree of inflammatory process in myocardium was scored as (0) absent, (1) focal, (2) multifocal, (3) diffuse with partial wall involvement and (4) diffuse with total wall involvement. BZ and VCZ treated animals displayed scores that ranged from 0 to 2, whereas the NT group had scores ranging from 0 to 3. In VCZ group, parasites were detected in myocardium and skeletal muscle in 2 out of 8 animals (25%). Amastigote nests were not observed in skeletal muscle from BZ group, however 1 of the 8 mice (12.5%) displayed parasites in myocardium. In NT animals, 5 out of 6 (83%) and 4 out of 6 (66.7%) were positive in myocardium and skeletal muscle, respectively.In our murine model of acute T. cruzi infection, VCZ was effective at lowering parasitemia and mortality. The histological studies showed that VCZ treatment also reduced inflammation and the parasite load in heart and skeletal muscles. Although the efficacy of VCZ was not as pronounced as BZ, additional studies need to be performed with a greater number of subjects, varying doses, and different treatment methods. Furthermore, this experiment presents a reliable model to search for new trypanocidal agents, as the results obtained with BZ are consistent with the human response. In conclusion, our findings emphasize the importance of identifying, among commercially available compounds, drugs that improve the treatment of Chagas disease.Acknowledgements: we thank Dr. Mónica Esteva (National Institute of Parasitology Dr. Mario Fatala Chabén, Buenos Aires, Argentina) for kindly providing the Tulahuén strain of T. cruzi.Financial support: this independent research was supported by grants from Pfizer Laboratory.

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