Chronic Trypanosoma cruzi infections lead to cardiomyopathy in 20-30% of cases. A causal link between cardiac infection and pathology has been difficult to establish because of a lack of robust methods to detect scarce, focally distributed parasites within tissues. We developed a highly sensitive bioluminescence imaging system based on T. cruzi expressing a novel luciferase that emits tissue-penetrating orange-red light. This enabled long-term serial evaluation of parasite burdens in individual mice with an in vivo limit of detection of significantly less than 1000 parasites. Parasite distributions during chronic infections were highly focal and spatiotemporally dynamic, but did not localize to the heart. End-point ex vivo bioluminescence imaging allowed tissue-specific quantification of parasite loads with minimal sampling bias. During chronic infections, the gastro-intestinal tract, specifically the colon and stomach, was the only site where T. cruzi infection was consistently observed. Quantitative PCR-inferred parasite loads correlated with ex vivo bioluminescence and confirmed the gut as the parasite reservoir. Chronically infected mice developed myocarditis and cardiac fibrosis, despite the absence of locally persistent parasites. These data identify the gut as a permissive niche for long-term T. cruzi infection and show that canonical features of Chagas disease can occur without continual myocardium-specific infection.

SUMMARY: Trypanosoma cruzi is the protozoan agent of Chagas disease, and the most important parasitic disease in Latin America. Protozoa of the genus Leishmania are global agents of visceral and cutaneous leishmaniasis, fatal and disfiguring diseases. In the 1970s multilocus enzyme electrophoresis demonstrated that T. cruzi is a heterogeneous complex. Six zymodemes were described, corresponding with currently recognized lineages, TcI and TcIIa-e – now defined by multiple genetic markers. Molecular epidemiology has substantially resolved the phylogeography and ecological niches of the T. cruzi lineages. Genetic hybridization has fundamentally influenced T. cruzi evolution and epidemiology of Chagas disease. Genetic exchange of T. cruzi in vitro involves fusion of diploids and genome erosion, producing aneuploid hybrids. Transgenic fluorescent clones are new tools to elucidate molecular genetics and phenotypic variation. We speculate that pericardial sequestration plays a role in pathogenesis. Multilocus sequence typing, microsatellites and, ultimately, comparative genomics are improving understanding of T. cruzi population genetics. Similarly, in Leishmania, genetic groups have been defined, including epidemiologically important hybrids; genetic exchange can occur in the sand fly vector. We describe the profound impact of this parallel research on genetic diversity of T. cruzi and Leishmania, in the context of epidemiology, taxonomy and disease control.

Trypanosoma cruzi is the most important parasitic infection in Latin America and is also genetically highly diverse, with at least six discrete typing units (DTUs) reported: Tc I, IIa, IIb, IIc, IId, and IIe. However, the current six-genotype classification is likely to be a poor reflection of the total genetic diversity present in this undeniably ancient parasite. To determine whether epidemiologically important information is “hidden” at the sub-DTU level, we developed a 48-marker panel of polymorphic microsatellite loci to investigate population structure among 135 samples from across the geographic distribution of TcI. This DTU is the major cause of resurgent human disease in northern South America but also occurs in silvatic triatomine vectors and mammalian reservoir hosts throughout the continent. Based on a total dataset of 12,329 alleles, we demonstrate that silvatic TcI populations are extraordinarily genetically diverse, show spatial structuring on a continental scale, and have undergone recent biogeographic expansion into the southern United States of America. Conversely, the majority of human strains sampled are restricted to two distinct groups characterised by a considerable reduction in genetic diversity with respect to isolates from silvatic sources. In Venezuela, most human isolates showed little identity with known local silvatic strains, despite frequent invasion of the domestic setting by infected adult vectors. Multilocus linkage indices indicate predominantly clonal parasite propagation among all populations. However, excess homozygosity among silvatic strains and raised heterozygosity among domestic populations suggest that some level of genetic recombination cannot be ruled out. The epidemiological significance of these findings is discussed. The arrival of the Trypanosoma cruzi online genome now provides vital information for the study of Chagas disease. Using this resource, we identified and developed a genome-scale panel of rapidly evolving microsatellite markers that can be used to unravel the micro-epidemiology of this parasite. We then tested these against a panel of isolates belonging to the most widely occurring and ancient major lineage, T. cruzi I (TcI). Our study includes samples from across the geographical distribution of this lineage, including isolates from wild vectors, domestic vectors, as well as wild mammalian reservoirs and human hosts. This is the first time T. cruzi has been subjected to such high-resolution population genetic analysis. Our study shows that important epidemiological information lies at the intra-lineage level, especially when wild and domestic populations of parasite are compared. Crucially, in Venezuela, where Chagas disease may be resurgent despite decades of control effort, genotypes of parasites found in the wild are rarely represented in humans, despite evidence that infected wild vectors do invade houses. In this manuscript, we examine the epidemiological implications of this finding and others, and suggest how the approach we have developed can now be used to investigate the true nature of parasite transmission at Chagas disease foci throughout the Americas.

The antifungal drug posaconazole has shown significant activity against Trypanosoma cruzi in vitro and in experimental murine models. Despite this, in a recent clinical trial it displayed limited curative potential. Drug testing is problematic in experimental Chagas disease because of difficulties in demonstrating sterile cure, particularly during the chronic stage of infection when parasite burden is extremely low and tissue distribution is ill defined. To better assess posaconazole efficacy against acute and chronic Chagas disease, we have exploited a highly sensitive bioluminescence imaging system which generates data with greater accuracy than other methods, including PCR-based approaches. Mice inoculated with bioluminescent T. cruzi were assessed by in vivo and ex vivo imaging, with cyclophosphamide-induced immunosuppression used to enhance the detection of relapse. Posaconazole was found to be significantly inferior to benznidazole as a treatment for both acute and chronic T. cruzi infections. Whereas 20 days treatment with benznidazole was 100% successful in achieving sterile cure, posaconazole failed in almost all cases. Treatment of chronic infections with posaconazole did however significantly reduce infection-induced splenomegaly, even in the absence of parasitological cure. The imaging-based screening system also revealed that adipose tissue is a major site of recrudescence in mice treated with posaconazole in the acute, but not the chronic stage of infection. This in vivo screening model for Chagas disease is predictive, reproducible and adaptable to diverse treatment schedules. It should provide greater assurance that drugs are not advanced prematurely into clinical trial.

Trypanosoma cruzi is the causative agent of Chagas disease, which affects more than 9 million people in Latin America. We have generated a draft genome sequence of the TcI strain Sylvio X10/1 and compared it to the TcVI reference strain CL Brener to identify lineage-specific features. We found virtually no differences in the core gene content of CL Brener and Sylvio X10/1 by presence/absence analysis, but 6 open reading frames from CL Brener were missing in Sylvio X10/1. Several multicopy gene families, including DGF, mucin, MASP and GP63 were found to contain substantially fewer genes in Sylvio X10/1, based on sequence read estimations. 1,861 small insertion-deletion events and 77,349 nucleotide differences, 23% of which were non-synonymous and associated with radical amino acid changes, further distinguish these two genomes. There were 336 genes indicated as under positive selection, 145 unique to T. cruzi in comparison to T. brucei and Leishmania. This study provides a framework for further comparative analyses of two major T. cruzi lineages and also highlights the need for sequencing more strains to understand fully the genomic composition of this parasite. Chagas disease is a major health problem in Latin America and it is caused by the protozoan parasite Trypanosoma cruzi. The genome sequence of the T. cruzi strain CL Brener (TcVI) has revealed a genome with large repertoires of genes for surface antigens, among other features. In the present study, we sequenced the genome of a representative member of TcI, the predominant agent of Chagas disease North of the Amazon and performed comparative analyses with CL Brener. Genetic variation between strains can potentially explain differences in disease pathogenesis, host preferences and aid the identification of drug targets. Our analysis showed that the two genomes have very similar sets of genes, but contain large differences in the relative size of several important gene families. Moreover, an abundance of allelic sequence variation was found in a large fraction of genes, and an evolutionary analysis indicated that many genes have evolved at different rates.

Polymorphisms in several DNA repair genes have been described. These polymorphisms may affect DNA repair capacity and modulate cancer susceptibility by means of gene-environment interactions. We investigated DNA repair capacity and its association with acute myeloblastic leukemia (AML). We studied polymorphisms in 3 DNA repair genes: XRCC1, XRCC3, and XPD. We also assessed the incidence of a functional polymorphism in theNQO1 gene, which is involved in protection of cells from oxidative damage. We genotyped the polymorphisms by using polymerase chain reaction–restriction fragment-length polymorphism analysis in 134 patients with de novo AML, 34 with therapy-related AML (t-AML), and 178 controls. The distributions of theXRCC3 Thr241Met and NQO1 Pro187Ser genotypes were not significantly different in patients and controls. However, the distribution of the XRCC1 Arg399Gln genotypes was significantly different when comparing the t-AML and control groups (χ², P = .03). The presence of at least oneXRCC1 399Gln allele indicated a protective effect for the allele in controls compared with patients with t-AML (odds ratio 0.44; 95% confidence interval, 0.20-0.93). We found no interactions between the XRCC1 or XRCC3 and NQO1genotypes. We also found no differences in the distribution of the XPD Lys751Gln or XRCC1 Arg194Trp genotypes. Our data provide evidence of a protective effect against AML in individuals with at least one copy of the variant XRCC1 399Gln allele compared with those homozygous for the common allele.