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Benznidazole and nifurtimox-treatments regimens currently used in human are supported by very limited experimental data. This study was designed to evaluate the time and dose dependence for efficacy of the most important nitroheterocyclic drugs in use for Chagas disease. In order to evaluate time dependence, Y strain-infected mice received benznidazole for a total of 1, 3, 7, 10, 20, and 40 days. Treatment courses of 3-10-day were effective in clearing parasitaemia and suppressing mortality, but parasitological cure was not achieved. Extending the treatments to 20 or 40 days clearly improved benznidazole efficacy. The 20-day treatment induced cure in 57.1% of Y strain infections (partially drug resistant) but failed to cure Colombian strain infections (full drug resistant), while the 40-day treatment resulted in cure of 100% of Y and 50% of Colombian strain infected mice. The increased cure rates in T. cruzi infected animals that received nifurtimox for 40 days confirm the relationship between the length of treatment and efficacy. An improvement in efficacy was observed with increasing benznidazole doses; cure was verified in 28.6% (75 mg/kg), 57.1% (100 mg/kg) and 80% (300 mg/kg). Overall, these nonclinical study data provide evidence that the efficacy of benznidazole is dose and time dependent. These findings may be relevant for optimizing treatment of human Chagas disease., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The factors involved in the reactivation of chronic Chagas disease infection are not clear enough and may be related to host immune unbalance and/or parasite genetic diversity. To evaluate the role of the Trypanosoma cruzi genetic background in the Chagas disease reactivation, we inoculated Cyclophosphamide-immunosupressed (CyI) Swiss mice with clonal stocks from T. cruzi I (Cuica cl1, P209 cl1, Gamba cl1, SP104 cl1), T. cruzi II (IVV cl4, MVB cl8) and T. cruzi (Bug2148 cl1, MN cl2) lineages. We used the parasitemia as the parameter for Chagas disease reactivation and observed that CyI animals infected with T. cruzi stocks showed no reactivation and those infected with T. cruzi II stocks showed only 5% of reactivation. In contrast, immunosuppressed mice infected with stocks from T. cruzi I lineage showed 77.5 and 51.25% reactivation of the infection when Cyclophosphamide treatment was performed 60 and 180 days after inoculation, respectively. Next, we evaluated the efficacy of the Benznidazole (Bz) pre-treatment in reducing or preventing the recurrence of the infection in these CyI animals. In general, the percentage of the parasite recurrence was not altered among the CyI mice that received the Bz pre-treatment during the acute phase of the infection. Interestingly, when pre-Bz treatment was performed during the chronic phase, we observed two different patterns of response: (i) an increased protection among the animals inoculated with the SP104 cl1 (genotype 19) and Cuica cl1 (genotype 20) stocks; (ii) an increased percentage of parasitemia reactivation among mice inoculated with Gamba cl1 (genotype 19) and P209 cl1 (genotype 20) T. cruzi stocks. Our results corroborate our hypothesis by showing that the T. cruzi genetic background in combination with specific Bz treatment has an important role in the Chagas disease reactivation in immunosuppressed animals., (Copyright 2009 Elsevier B.V. All rights reserved.)

Through a continuous in vivo drug pressure protocol, using mice as experimental model, we induced benznidazole resistance in Trypanosoma cruzi stocks. Full resistance was obtained for four out of five T. cruzi stocks analyzed. However, the number of benznidazole doses (40-180), as well as the time (4-18 months) necessary to induce resistance varied among the different T. cruzi stocks. The resistance phenotype remained stable after T. cruzi stocks has been maintained by 12 passages in mice (six months) and in acellular culture for the same time. However, the maintenance of resistant parasite for 12 months in acellular culture induces a reduction in its level of benznidazole resistance, while no alteration was detected in parasite maintained for the same time in mice. The data showed the stability of the resistance acquired by drug pressure, but suggest the possibility of reversible changes in the resistance levels after maintenance for long time in acellular culture.

The goals of the present study were to evaluate the kinetics of blood parasitism by examination of fresh blood, blood culture (BC) and PCR assays and their correlation with heart parasitism during two years of infection in Beagle dogs inoculated with the Be-78, Y and ABC Trypanosoma cruzi strains. Our results showed that the parasite or its kDNA is easily detected during the acute phase in all infected animals. On the other hand, a reduced number of positive tests were verified during the chronic phase of the infection. The frequency of positive tests was correlated with T. cruzi strain. The percentage of positive BC and blood PCR performed in samples from animals inoculated with Be-78 and ABC strains were similar and significantly larger in relation to animals infected with the Y strain.Comparison of the positivity of PCR tests performed using blood and heart tissue samples obtained two years after infection showed two different patterns associated with the inoculated T. cruzi strain: (1) high PCR positivity for both blood and tissue was observed in animals infected with Be-78 or ABC strains; (2) lower and higher PCR positivity for the blood and tissue, respectively, was detected in animals infected with Y strains. These data suggest that the sensitivity of BC and blood PCR was T. cruzi strain dependent and, in contrast, the heart tissue PCR revealed higher sensitivity regardless of the parasite stock.

We analyzed the influence of Trypanosoma cruzi maintenance in different hosts (dog and mouse) on its susceptibility to benznidazole treatment. Five T. cruzi stocks were isolated from dogs inoculated with Be-62 or Be-78 strain (both sensitive to benznidazole) 2-10 years ago, and the benznidazole sensitivity was then determined using the mouse as experimental model. The different T. cruzi stocks obtained from long-term infected dogs showed 50-90% drug resistance right after isolation. However, maintenance of these T. cruzi stocks in mice, by successive blood passages (2.5 years), led to either a decrease or stability of the drug resistance pattern and an increase in parasite virulence. We also demonstrated the effectiveness of the induction of parasitemia reactivation by cyclophosphamide immunosuppression in the evaluation of the response to the specific drug treatment.

Albaconazole is an experimental triazole derivative with potent and broad-spectrum antifungal activity and a remarkably long half-life in dogs, monkeys, and humans. In the present work, we investigated the in vivo activity of this compound against two strains of the protozoan parasite Trypanosoma (Schizotrypanum) cruzi, the causative agent of Chagas' disease, using dogs as hosts. The T. cruzi strains used in the study were previously characterized (murine model) as susceptible (strain Berenice-78) and partially resistant (strain Y) to the drugs currently in clinical use, nifurtimox and benznidazole. Our results demonstrated that albaconazole is very effective in suppressing the proliferation of the parasite and preventing the death of infected animals. Furthermore, the parasitological, PCR, serological, and proliferative assay results indicated parasitological cure indices of 25 and 100% among animals inoculated with T. cruzi strain Y when they were treated with albaconazole at 1.5 mg/kg of body weight/day for 60 and 90 days, respectively. On the other hand, although albaconazole given at 1.5 mg/kg/day was very effective in suppressing the proliferation of the parasite in animals infected with the Berenice-78 T. cruzi strain, no parasitological cure was observed among them, even when a longer treatment period (150 doses) was used. In conclusion, our results demonstrate that albaconazole has trypanocidal activity in vivo and is capable of inducing radical parasitological cure, although natural resistance to this compound was also indicated. Furthermore, the compound can be used in long-term treatment schemes (60 to 150 days) with minimal toxicity and thus represents a potentially useful candidate for the treatment of human Chagas' disease.

One of the greatest concerns in Chagas' disease is the absence of reliable methods for the evaluation of chemotherapy efficacy in treated patients. The tests available to evaluate cure after the specific treatment are the complement-mediated lysis (CoML) and flow cytometry tests, but they are not feasible for routine clinical use. In this study, we evaluated an enzyme-linked immunosorbent assay (ELISA) based on the recombinant Trypanosoma cruzi complement regulatory protein (rCRP) as a method to determine parasite clearance in comparison to the CoML and other methods such as conventional serology, hemoculture, and PCR in serum samples of 31 patients collected before and after the treatment, monitored for an average of 27.7 months after chemotherapy. The results showed that the percentage of patient samples that were positive by rCRP ELISA was reduced from 100 to 70.3, 62.5, 71.4, and 33.4% in the first, second, third, and fourth years after treatment, respectively, while the samples positive by CoML were reduced to 85.2, 81.2, 71.4, and 33.4% during the same period, demonstrating the same significant tendency in the reduction of positive samples. On the other hand, the conventional serology (CS) tests did not present this reduction. The percentage of samples positive by PCR was initially 77.4% and decreased to 55.5, 68.7, 47.7, and 50.0% at the fourth year after treatment, confirming the drastic clearance of circulating parasites after treatment. Our results strongly suggest that the rCRP ELISA was capable of detecting the early therapeutic efficacy in treated patients and confirmed its superiority over the CS tests and parasitologic methods.

Currently, diagnosis of Chagas' disease is based on serological methods, but due to the high occurrence of inconclusive results, more reliable methods are needed. The use of recombinant antigens for serodiagnosis of Chagas' disease is recommended in order to increase the sensitivity and specificity of the serological tests. The Trypanosoma cruzi complement regulatory protein (CRP) is a surface glycoprotein present on the trypomastigote forms of the parasite, and the recombinant CRP (rCRP) was cloned in a mammalian expression system and purified by affinity chromatography. The purified recombinant protein was used as an antigen in an enzyme-linked immunosorbent assay (rCRP ELISA) in order to verify its sensitivity and specificity compared with other established methods. In this evaluation, a panel of 184 serum samples distributed among chronic chagasic patients (n = 65), blood bank donors (n = 100), and patients infected with Leishmania spp. (n = 19) was used. The sensitivity and specificity of the rCRP ELISA were 100% when compared to conventional serology and complement-mediated lysis tests from these groups. When hemoculture and PCR tests were evaluated for diagnosis of chronic chagasic patients, using the rCRP ELISA as a reference test, the positivities were found to be 64.62 and 81.54%, respectively, showing a higher degree of sensitivity of the test. The data demonstrate that rCRP ELISA was able to discriminate between chronic chagasic patients and nonchagasic individuals, such as blood donors and patients with leishmaniasis. Thus, the rCRP is an excellent antigen for use in Chagas' disease diagnosis, due to the absence of false-negative or false-positive results.