BackgroundThe two available drugs for treatment of T. cruzi infection, nifurtimox and benznidazole (BZ), have potential toxic side effects and variable efficacy, contributing to their low rate of use. With scant economic resources available for antiparasitic drug discovery and development, inexpensive, high-throughput and in vivo assays to screen potential new drugs and existing compound libraries are essential.MethodsIn this work, we describe the development and validation of improved methods to test anti-T. cruzi compounds in vitro and in vivo using parasite lines expressing the firefly luciferase (luc) or the tandem tomato fluorescent protein (tdTomato). For in vitro assays, the change in fluorescence intensity of tdTomato-expressing lines was measured as an indicator of parasite replication daily for 4 days and this method was used to identify compounds with IC50 lower than that of BZ.FindingsThis method was highly reproducible and had the added advantage of requiring relatively low numbers of parasites and no additional indicator reagents, enzymatic post-processes or laborious visual counting. In vivo, mice were infected in the footpads with fluorescent or bioluminescent parasites and the signal intensity was measured as a surrogate of parasite load at the site of infection before and after initiation of drug treatment. Importantly, the efficacy of various drugs as determined in this short-term (<2 weeks) assay mirrored that of a 40 day treatment course.ConclusionThese methods should make feasible broader and higher-throughput screening programs needed to identify potential new drugs for the treatment of T. cruzi infection and for their rapid validation in vivo.

BackgroundIn contrast to the essentially fully assembled genome sequences of the kinetoplastid pathogens Leishmania major and Trypanosoma brucei the assembly of the Trypanosoma cruzi genome has been hindered by its repetitive nature and the fact that the reference strain (CL Brener) is a hybrid of two distinct lineages. In this work, the majority of the contigs and scaffolds were assembled into pairs of homologous chromosomes based on predicted parental haplotype, inference from TriTryp synteny maps and the use of end sequences from T. cruzi BAC libraries.ResultsUltimately, 41 pairs of chromosomes were assembled using this approach, a number in agreement with the predicted number of T. cruzi chromosomes based upon pulse field gel analysis, with over 90% (21133 of 23216) of the genes annotated in the genome represented. The approach was substantiated through the use of Southern blot analysis to confirm the mapping of BAC clones using as probes the genes they are predicted to contain, and each chromosome construction was visually validated to ensure sufficient evidence was present to support the organization. While many members of large gene families are incorporated into the chromosome assemblies, the majority of genes excluded from the chromosomes belong to gene families, as these genes are frequently impossible to accurately position.ConclusionNow assembled, these chromosomes bring T. cruzi to the same level of organization as its kinetoplastid relatives and have been used as the basis for the T. cruzi genome in TriTrypDB, a trypanosome database of EuPathDB. In addition, they will provide the foundation for analyses such as reverse genetics, where the location of genes and their alleles and/or paralogues is necessary and comparative genome hybridization analyses (CGH), where a chromosome-level view of the genome is ideal.

BackgroundDiagnosis of Trypanosoma cruzi infection by direct pathogen detection is complicated by the low parasite burden in subjects persistently infected with this agent of human Chagas disease. Determination of infection status by serological analysis has also been faulty, largely due to the lack of well-characterized parasite reagents for the detection of anti-parasite antibodies.MethodsIn this study, we screened more than 400 recombinant proteins of T. cruzi, including randomly selected and those known to be highly expressed in the parasite stages present in mammalian hosts, for the ability to detect anti-parasite antibodies in the sera of subjects with confirmed or suspected T. cruzi infection.FindingsA set of 16 protein groups were identified and incorporated into a multiplex bead array format which detected 100% of >100 confirmed positive sera and also documented consistent, strong and broad responses in samples undetected or discordant using conventional serologic tests. Each serum had a distinct but highly stable reaction pattern. This diagnostic panel was also useful for monitoring drug treatment efficacy in chronic Chagas disease.ConclusionsThese results substantially extend the variety and quality of diagnostic targets for Chagas disease and offer a useful tool for determining treatment success or failure.