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New assay designs are needed to improve the predictive value of the Trypanosoma cruzi in vitro tests used as part of the Chagas' disease drug development pipeline. Here, we employed a green fluorescent protein (eGFP)-expressing parasite line and live high-content imaging to monitor the growth of T. cruzi amastigotes in mouse embryonic fibroblasts. A novel assay design allowed us to follow parasite numbers over 6 days, in four-hour intervals, while occupying the microscope for only 24 hours per biological replicate. Dose-response curves were calculated for each time point after addition of test compounds, revealing how EC50 values first decreased over the time of drug exposure, and then leveled off. However, we observed that parasite numbers could vary, even in the untreated controls, and at different sites in the same well, which caused variability in the EC50 values. To overcome this, we established that fold change in parasite number per hour is a more robust and informative measure of drug activity. This was calculated based on an exponential growth model for every biological sample. The net fold change per hour is the result of parasite replication, differentiation, and death. The calculation of this fold change enabled us to determine the tipping point of drug action, i.e. the time point when the death rate of the parasites exceeded the growth rate and the fold change dropped below 1, depending on the drug concentration and exposure time. This revealed specific pharmacodynamic profiles of the benchmark drugs benznidazole and posaconazole. Author summary: Chagas' disease, caused by Trypanosoma cruzi, is a chronic debilitating infection occurring mostly in Latin America. There is an urgent need for new, well tolerated drugs. However, the latest therapeutic candidates have yielded disappointing outcomes in clinical trials, despite promising preclinical results. This demands new and more predictive in vitro assays. To address this, we have developed an assay design that enables the growth of T. cruzi intracellular forms to be monitored in real time, under drug pressure, for 6 days post-infection. This allowed us to establish the tipping point of drug action, when the death rate of the parasites exceeded the growth rate. The resulting pharmacodynamics profiles can provide robust and informative details on anti-chagasic candidates, as demonstrated for the benchmark drugs benznidazole and posaconazole. [ABSTRACT FROM AUTHOR]

Background: The long term and complex nature of Chagas disease in humans has restricted studies on vaccine feasibility. Animal models also have limitations due to technical difficulties in monitoring the extremely low parasite burden that is characteristic of chronic stage infections. Advances in imaging technology offer alternative approaches that circumvent these problems. Here, we describe the use of highly sensitive whole body in vivo imaging to assess the efficacy of recombinant viral vector vaccines and benznidazole-cured infections to protect mice from challenge with Trypanosoma cruzi. Methodology/Principal findings: Mice were infected with T. cruzi strains modified to express a red-shifted luciferase reporter. Using bioluminescence imaging, we assessed the degree of immunity to re-infection conferred after benznidazole-cure. Those infected for 14 days or more, prior to the onset of benznidazole treatment, were highly protected from challenge with both homologous and heterologous strains. There was a >99% reduction in parasite burden, with parasites frequently undetectable after homologous challenge. This level of protection was considerably greater than that achieved with recombinant vaccines. It was also independent of the route of infection or size of the challenge inoculum, and was long-lasting, with no significant diminution in immunity after almost a year. When the primary infection was benznidazole-treated after 4 days (before completion of the first cycle of intracellular infection), the degree of protection was much reduced, an outcome associated with a minimal T. cruzi-specific IFN-γ+ T cell response. Conclusions/Significance: Our findings suggest that a protective Chagas disease vaccine must have the ability to eliminate parasites before they reach organs/tissues, such as the GI tract, where once established, they become largely refractory to the induced immune response. Author summary: Chagas disease, which is caused by the protozoan parasite Trypanosoma cruzi, is a major public health problem throughout Latin America. Attempts to develop a vaccine have been hampered by technical difficulties in monitoring the extremely low parasite burden during the life-long chronic stage of infection. To circumvent these issues, we used highly sensitive bioluminescence imaging to assess the ability of recombinant viral vector vaccines and drug-cured infections to confer protection against experimental challenge in mice. We observed that drug-cured infections were much more effective than subunit vaccines. Efficacy was independent of the route of infection or size of the challenge inoculum, and was undiminished after almost a year. In addition, drug-cured infections conferred a high level of cross-strain protection. The highly sensitive imaging procedures enabled us to visualise parasite distribution in mice where full protection was not achieved. This suggested that to confer protection, vaccines must prevent the infection of organs/tissues that act as parasite reservoirs during the chronic stage. Once established at these sites, parasites become largely refractory to vaccine-induced elimination. [ABSTRACT FROM AUTHOR]

The protozoan parasite Trypanosoma cruzi causes Chagas disease, an important public health problem throughout Latin America. Current therapeutic options are characterised by limited efficacy, long treatment regimens and frequent toxic side-effects. Advances in this area have been compromised by gaps in our knowledge of disease pathogenesis, parasite biology and drug activity. Nevertheless, several factors have come together to create a more optimistic scenario. Drug-based research has become more systematic, with increased collaborations between the academic and commercial sectors, often within the framework of not-for-profit consortia. High-throughput screening of compound libraries is being widely applied, and new technical advances are helping to streamline the drug development pipeline. In addition, drug repurposing and optimisation of current treatment regimens, informed by laboratory research, are providing a basis for new clinical trials. Here, we will provide an overview of the current status of Chagas disease drug development, highlight those areas where progress can be expected, and describe how fundamental research is helping to underpin the process. [ABSTRACT FROM AUTHOR]

Focuses on the proline racemase in the form of Trypanosoma cruzi (T. cruzi) B-cell mitogen which activates in Chagas disease. Impact of Chagas disease on the public health in Latin America; Features of the T.cruzi parasites; Deductibility of proline racemase on the parasite membrane; Emphasis on the biological functions of the racemase.