Stage-Regulated GFP Expression in Trypanosoma cruzi: Applications from Host-Parasite Interactions to Drug Screening

Trypanosoma cruzi is the etiological agent of Chagas disease, an illness that affects about 10 million people, mostly in South America, for which there is no effective treatment or vaccine. In this context, transgenic parasites expressing reporter genes are interesting tools for investigating parasite biology and host-parasite interactions, with a view to developing new strategies for disease prevention and treatment. We describe here the construction of a stably transfected fluorescent T. cruzi clone in which the GFP gene is integrated into the chromosome carrying the ribosomal cistron in T. cruzi Dm28c. This fluorescent T. cruzi produces detectable amounts of GFP only at replicative stages (epimastigote and amastigote), consistent with the larger amounts of GFP mRNA detected in these forms than in the non replicative trypomastigote stages. The fluorescence signal was also strongly correlated with the total number of parasites in T. cruzi cultures, providing a simple and rapid means of determining the growth inhibitory dose of anti-T.cruzi drugs in epimastigotes, by fluorometric microplate screening, and in amastigotes, by the flow cytometric quantification of T. cruzi-infected Vero cells. This fluorescent T. cruzi clone is, thus, an interesting tool for unbiased detection of the proliferating stages of the parasite, with multiple applications in the genetic analysis of T. cruzi, including analyses of host-parasite interactions, gene expression regulation and drug development.     

The Trypanosoma cruzi Satellite DNA OligoC-TesT and Trypanosoma cruzi Kinetoplast DNA OligoC-TesT for Diagnosis of Chagas Disease: A Multi-cohort Comparative Evaluation Study

Background

The Trypanosoma cruzi satellite DNA (satDNA) OligoC-TesT is a standardised PCR format for diagnosis of Chagas disease. The sensitivity of the test is lower for discrete typing unit (DTU) TcI than for TcII-VI and the test has not been evaluated in chronic Chagas disease patients.

Methodology/Principal Findings

We developed a new prototype of the OligoC-TesT based on kinetoplast DNA (kDNA) detection. We evaluated the satDNA and kDNA OligoC-TesTs in a multi-cohort study with 187 chronic Chagas patients and 88 healthy endemic controls recruited in Argentina, Chile and Spain and 26 diseased non-endemic controls from D.R. Congo and Sudan. All specimens were tested in duplicate. The overall specificity in the controls was 99.1% (95% CI 95.2%–99.8%) for the satDNA OligoC-TesT and 97.4% (95% CI 92.6%–99.1%) for the kDNA OligoC-TesT. The overall sensitivity in the patients was 67.9% (95% CI 60.9%–74.2%) for the satDNA OligoC-TesT and 79.1% (95% CI 72.8%–84.4%) for the kDNA OligoC-Test.

Conclusions/Significance

Specificities of the two T. cruzi OligoC-TesT prototypes are high on non-endemic and endemic controls. Sensitivities are moderate but significantly (p = 0.0004) higher for the kDNA OligoC-TesT compared to the satDNA OligoC-TesT.     

The Attraction of Trypanosoma cruzi to Vertebrate Cells In Vitro

SYNOPSIS. A video technic is described that permits a quantification of the degree of attraction of Trypanosoma cruzi trypomastigotes to vertebrate cells in vitro. Bovine embryo skeletal muscle cells (BESM), HeLa cells and Vero cells all attract a myotropic strain of T. cruzi trypomastigotes. BESM cells, however, are 2-fold more attractive to trypomastigotes than HeLa cells and 10-fold more attractive than Vero cells. Heat-inactivation of BESM cells abolishes their ability to respire and also to attract T. cruzi trypomastigotes. As there is no difference in the endogenous oxygen consumption between BESM, HeLa, and Vero cells, it is unlikely that differences in the attraction of trypomastigotes to the 3 cell types are due to variations in the magnitude of pO₂ or pCO₂ gradients in the milieu around the cells.     

Aptamer Based,Non-PCR,Non-Serological Detection of Chagas Disease Biomarkers in Trypanosoma cruzi Infected Mice

Chagas disease affects about 5 million people across the world. The etiological agent, the intracellular parasite Trypanosoma cruzi (T. cruzi), can be diagnosed using microscopy, serology or PCR based assays. However, each of these methods has their limitations regarding sensitivity and specificity, and thus to complement these existing diagnostic methods, alternate assays need to be developed. It is well documented that several parasite proteins called T. cruzi Excreted Secreted Antigens (TESA), are released into the blood of an infected host. These circulating parasite antigens could thus be used as highly specific biomarkers of T. cruzi infection. In this study, we have demonstrated that, using a SELEx based approach, parasite specific ligands called aptamers, can be used to detect TESA in the plasma of T. cruzi infected mice. An Enzyme Linked Aptamer (ELA) assay, similar to ELISA, was developed using biotinylated aptamers to demonstrate that these RNA ligands could interact with parasite targets. Aptamer L44 (Apt-L44) showed significant and specific binding to TESA as well as T. cruzi trypomastigote extract and not to host proteins or proteins of Leishmania donovani, a related trypanosomatid parasite. Our result also demonstrated that the target of Apt-L44 is conserved in three different strains of T. cruzi. In mice infected with T. cruzi, Apt-L44 demonstrated a significantly higher level of binding compared to non-infected mice suggesting that it could detect a biomarker of T. cruzi infection. Additionally, Apt-L44 could detect these circulating biomarkers in both the acute phase, from 7 to 28 days post infection, and in the chronic phase, from 55 to 230 days post infection. Our results show that Apt-L44 could thus be used in a qualitative ELA assay to detect biomarkers of Chagas disease.     

Distantiae Transmission of Trypanosoma cruzi: A New Epidemiological Feature of Acute Chagas Disease in Brazil

Background

The new epidemiological scenario of orally transmitted Chagas disease that has emerged in Brazil, and mainly in the Amazon region, needs to be addressed with a new and systematic focus. Belém, the capital of Pará state, reports the highest number of acute Chagas disease (ACD) cases associated with the consumption of açaí juice.

Methodology/Principal Findings

The wild and domestic enzootic transmission cycles of Trypanosoma cruzi were evaluated in the two locations (Jurunas and Val-de Cães) that report the majority of the autochthonous cases of ACD in Belém city. Moreover, we evaluated the enzootic cycle on the three islands that provide most of the açaí fruit that is consumed in these localities. We employed parasitological and serological tests throughout to evaluate infectivity competence and exposure to T. cruzi. In Val-de-Cães, no wild mammal presented positive parasitological tests, and 56% seroprevalence was observed, with low serological titers. Three of 14 triatomines were found to be infected (TcI). This unexpected epidemiological picture does not explain the high number of autochthonous ACD cases. In Jurunas, the cases of ACD could not be autochthonous because of the absence of any enzootic cycle of T. cruzi. In contrast, in the 3 island areas from which the açaí fruit originates, 66.7% of wild mammals and two dogs displayed positive hemocultures, and 15.6% of triatomines were found to be infected by T. cruzi. Genotyping by mini-exon gene and PCR-RFLP (1f8/Akw21I) targeting revealed that the mammals and triatomines from the islands harbored TcI and Trypanosoma rangeli in single and mixed infections.

Conclusion/Significance

These findings show that cases of Chagas disease in the urban area of Belém may be derived from infected triatomines coming together with the açaí fruits from distant islands. We term this new epidemiological feature of Chagas disease as “Distantiae transmission”.     

A Simple Strain Typing Assay for Trypanosoma cruzi: Discrimination of Major Evolutionary Lineages from a Single Amplification Product

Background

Trypanosoma cruzi is the causative agent of Chagas'' Disease. The parasite has a complex population structure, with six major evolutionary lineages, some of which have apparently resulted from ancestral hybridization events. Because there are important biological differences between these lineages, strain typing methods are essential to study the T. cruzi species. Currently, there are a number of typing methods available for T. cruzi, each with its own advantages and disadvantages. However, most of these methods are based on the amplification of a variable number of loci.

Methodology/Principal Findings

We present a simple typing assay for T. cruzi, based on the amplification of a single polymorphic locus: the TcSC5D gene. When analyzing sequences from this gene (a putative lathosterol/episterol oxidase) we observed a number of interesting polymorphic sites, including 1 tetra-allelic, and a number of informative tri- and bi-allelic SNPs. Furthermore, some of these SNPs were located within the recognition sequences of two commercially available restriction enzymes. A double digestion with these enzymes generates a unique restriction pattern that allows a simple classification of strains in six major groups, corresponding to DTUs TcI–TcIV, the recently proposed Tcbat lineage, and TcV/TcVI (as a group). Direct sequencing of the amplicon allows the classification of strains into seven groups, including the six currently recognized evolutionary lineages, by analyzing only a few discriminant polymorphic sites.

Conclusions/Significance

Based on these findings we propose a simple typing assay for T. cruzi that requires a single PCR amplification followed either by restriction fragment length polymorphism analysis, or direct sequencing. In the panel of strains tested, the sequencing-based method displays equivalent inter-lineage resolution to recent multi- locus sequence typing assays. Due to their simplicity and low cost, the proposed assays represent a good alternative to rapidly screen strain collections, providing the cornerstone for the development of robust typing strategies.     

The Improbable Transmission of Trypanosoma cruzi to Human: The Missing Link in the Dynamics and Control of Chagas Disease

Chagas disease has a major impact on human health in Latin America and is becoming of global concern due to international migrations. Trypanosoma cruzi, the etiological agent of the disease, is one of the rare human parasites transmitted by the feces of its vector, as it is unable to reach the salivary gland of the insect. This stercorarian transmission is notoriously poorly understood, despite its crucial role in the ecology and evolution of the pathogen and the disease. The objective of this study was to quantify the probability of T. cruzi vectorial transmission to humans, and to use such an estimate to predict human prevalence from entomological data. We developed several models of T. cruzi transmission to estimate the probability of transmission from vector to host. Using datasets from the literature, we estimated the probability of transmission per contact with an infected triatomine to be 5.8×10⁻⁴ (95%CI: [2.6 ; 11.0]×10⁻⁴). This estimate was consistent across triatomine species, robust to variations in other parameters, and corresponded to 900–4,000 contacts per case. Our models subsequently allowed predicting human prevalence from vector abundance and infection rate in 7/10 independent datasets covering various triatomine species and epidemiological situations. This low probability of T. cruzi transmission reflected well the complex and unlikely mechanism of transmission via insect feces, and allowed predicting human prevalence from basic entomological data. Although a proof of principle study would now be valuable to validate our models'' predictive ability in an even broader range of entomological and ecological settings, our quantitative estimate could allow switching the evaluation of disease risk and vector control program from purely entomological indexes to parasitological measures, as commonly done for other major vector borne diseases. This might lead to different quantitative perspectives as these indexes are well known not to be proportional one to another.     

Trypanosoma cruzi: allopurinol in the treatment of mice with experimental acute Chagas disease

The therapeutic effect of allopurinol was studied in an experimental Trypanosoma cruzi infection (Chagas disease) in outbred IVIC-NMRI and inbred C57B1/6J mice intraperitoneally inoculated with the parasites 2–6 days before drug treatment. Allopurinol protected against T. cruzi infection. This effect was evidenced by highly significant reductions in both parasitemias and mortality rates and increased survival time in allopurinol-treated animals compared with untreated infected mice. Allopurinol protected effectively when administered in 10 daily doses of 32–64 mg/kg body wt/day injected intraperitoneally. Using direct methods, parasitemia remained undetectable for at least 310 days. An indirect method, subinoculation to susceptible mice, showed a few circulating trypanosomes which decreased greatly in number after a second schedule of allopurinol treatment; finally no trypanosomes were detectable 275 days after treatment initiation. Allopurinol also induced a strong trypanostatic effect when tested in vitro on five different Trypanosoma cruzi strains (optimal inhibitory concentration: 3 μg/ml). These results suggest that allopurinol protects mice with acute Chagas infection by a direct trypanostatic effect. The low toxicity of this drug suggests its use in more chronic experimental Chagas infections.     

Ecological Connectivity of Trypanosoma cruzi Reservoirs and Triatoma pallidipennis Hosts in an Anthropogenic Landscape with Endemic Chagas Disease

Traditional methods for Chagas disease prevention are targeted at domestic vector reduction, as well as control of transfusion and maternal-fetal transmission. Population connectivity of Trypanosoma cruzi-infected vectors and hosts, among sylvatic, ecotone and domestic habitats could jeopardize targeted efforts to reduce human exposure. This connectivity was evaluated in a Mexican community with reports of high vector infestation, human infection, and Chagas disease, surrounded by agricultural and natural areas. We surveyed bats, rodents, and triatomines in dry and rainy seasons in three adjacent habitats (domestic, ecotone, sylvatic), and measured T. cruzi prevalence, and host feeding sources of triatomines. Of 12 bat and 7 rodent species, no bat tested positive for T. cruzi, but all rodent species tested positive in at least one season or habitat. Highest T. cruzi infection prevalence was found in the rodents, Baiomys musculus and Neotoma mexicana. In general, parasite prevalence was not related to habitat or season, although the sylvatic habitat had higher infection prevalence than by chance, during the dry season. Wild and domestic mammals were identified as bloodmeals of T. pallidipennis, with 9% of individuals having mixed human (4.8% single human) and other mammal species in bloodmeals, especially in the dry season; these vectors tested >50% positive for T. cruzi. Overall, ecological connectivity is broad across this matrix, based on high rodent community similarity, vector and T. cruzi presence. Cost-effective T. cruzi, vector control strategies and Chagas disease transmission prevention will need to consider continuous potential for parasite movement over the entire landscape. This study provides clear evidence that these strategies will need to include reservoir/host species in at least ecotones, in addition to domestic habitats.     

Therapeutic Efficacy of a Subunit Vaccine in Controlling Chronic Trypanosoma cruzi Infection and Chagas Disease Is Enhanced by Glutathione Peroxidase Over-Expression

Trypanosoma cruzi-induced oxidative and inflammatory responses are implicated in chagasic cardiomyopathy. In this study, we examined the therapeutic utility of a subunit vaccine against T. cruzi and determined if glutathione peroxidase (GPx1, antioxidant) protects the heart from chagasic pathogenesis. C57BL/6 mice (wild-type (WT) and GPx1 transgenic (GPx^tg) were infected with T. cruzi and at 45 days post-infection (dpi), immunized with TcG2/TcG4 vaccine delivered by a DNA-prime/Protein-boost (D/P) approach. The plasma and tissue-sections were analyzed on 150 dpi for parasite burden, inflammatory and oxidative stress markers, inflammatory infiltrate and fibrosis. WT mice infected with T. cruzi had significantly more blood and tissue parasite burden compared with infected/GPx^tg mice (n = 5-8, p<0.01). Therapeutic vaccination provided >15-fold reduction in blood and tissue parasites in both WT and GPx^tg mice. The increase in plasma levels of myeloperoxidase (MPO, 24.7%) and nitrite (iNOS activity, 45%) was associated with myocardial increase in oxidant levels (3-4-fold) and non-responsive antioxidant status in chagasic/WT mice; and these responses were not controlled after vaccination (n = 5-7). The GPx^tg mice were better equipped than the WT mice in controlling T. cruzi-induced inflammatory and oxidative stress markers. Extensive myocardial and skeletal tissue inflammation noted in chagasic/WT mice, was significantly more compared with chagasic/GPx^tg mice (n = 4-6, p<0.05). Vaccination was equally effective in reducing the chronic inflammatory infiltrate in the heart and skeletal tissue of infected WT and GPx^tg mice (n = 6, p<0.05). Hypertrophy (increased BNP and ANP mRNA) and fibrosis (increased collagen) of the heart were extensively present in chronically-infected WT and GPx^tg mice and notably decreased after therapeutic vaccination. We conclude the therapeutic delivery of D/P vaccine was effective in arresting the chronic parasite persistence and chagasic pathology; and GPx1 over-expression provided additive benefits in reducing the parasite burden, inflammatory/oxidative stress and cardiac remodeling in Chagas disease.     

Trypanosoma cruzi IV Causing Outbreaks of Acute Chagas Disease and Infections by Different Haplotypes in the Western Brazilian Amazonia

Background

Chagas disease is an emergent tropical disease in the Brazilian Amazon Region, with an increasing number of cases in recent decades. In this region, the sylvatic cycle of Trypanosoma cruzi transmission, which constitutes a reservoir of parasites that might be associated with specific molecular, epidemiological and clinical traits, has been little explored. The objective of this work is to genetically characterize stocks of T. cruzi from human cases, triatomines and reservoir mammals in the State of Amazonas, in the Western Brazilian Amazon.

Methodology/Principal Findings

We analyzed 96 T. cruzi samples from four municipalities in distant locations of the State of Amazonas. Molecular characterization of isolated parasites from cultures in LIT medium or directly from vectors or whole human blood was performed by PCR of the non-transcribed spacer of the mini-exon and of the 24 S alfa ribosomal RNA gene, RFLP and sequencing of the mitochondrial cytochrome c oxidase subunit II (COII) gene, and by sequencing of the glucose-phosphate isomerase gene. The T. cruzi parasites from two outbreaks of acute disease were all typed as TcIV. One of the outbreaks was triggered by several haplotypes of the same DTU. TcIV also occurred in isolated cases and in Rhodnius robustus. Incongruence between mitochondrial and nuclear phylogenies is likely to be indicative of historical genetic exchange events resulting in mitochondrial introgression between TcIII and TcIV DTUs from Western Brazilian Amazon. TcI predominated among triatomines and was the unique DTU infecting marsupials.

Conclusion/Significance

DTU TcIV, rarely associated with human Chagas disease in other areas of the Amazon basin, is the major strain responsible for the human infections in the Western Brazilian Amazon, occurring in outbreaks as single or mixed infections by different haplotypes.     

Lysine Uptake in Cultured Trypanosoma cruzi: Interactions of Competitive Inhibitors*

SYNOPSIS Three sites are involved in lysine transport in Trypanosoma cruzi, as inferred from interactions of inhibitory neutral amino acids. Phenylalanine and tyrosine inhibit one site; proline and the straight-chain amino acids, alanine, methionine and cysteine inhibit another; and glycine and the branched-chain valine, leucine, and isoleucine inhibit a 3rd. The curved rather than straight line obtained with a Lineweaver-Burk plot of uptake rates presumably results from the functioning of qualitatively different transport sites. Blocking every site but one results in the linear double-reciprocal plot characteristic of adsorption kinetics. The partial competitiveness of most of the inhibitions appears to denote qualitatively different sites for lysine transport and the reaction of the inhibitor with more than one site. Since most of the inhibitions were partially competitive, the specificities of the 3 lysine transport sites must overlap considerably.     

Trypanosoma cruzi IIc: Phylogenetic and Phylogeographic Insights from Sequence and Microsatellite Analysis and Potential Impact on Emergent Chagas Disease

Trypanosoma cruzi, the etiological agent of Chagas disease, is highly genetically diverse. Numerous lines of evidence point to the existence of six stable genetic lineages or DTUs: TcI, TcIIa, TcIIb, TcIIc, TcIId, and TcIIe. Molecular dating suggests that T. cruzi is likely to have been an endemic infection of neotropical mammalian fauna for many millions of years. Here we have applied a panel of 49 polymorphic microsatellite markers developed from the online T. cruzi genome to document genetic diversity among 53 isolates belonging to TcIIc, a lineage so far recorded almost exclusively in silvatic transmission cycles but increasingly a potential source of human infection. These data are complemented by parallel analysis of sequence variation in a fragment of the glucose-6-phosphate isomerase gene. New isolates confirm that TcIIc is associated with terrestrial transmission cycles and armadillo reservoir hosts, and demonstrate that TcIIc is far more widespread than previously thought, with a distribution at least from Western Venezuela to the Argentine Chaco. We show that TcIIc is truly a discrete T. cruzi lineage, that it could have an ancient origin and that diversity occurs within the terrestrial niche independently of the host species. We also show that spatial structure among TcIIc isolates from its principal host, the armadillo Dasypus novemcinctus, is greater than that among TcI from Didelphis spp. opossums and link this observation to differences in ecology of their respective niches. Homozygosity in TcIIc populations and some linkage indices indicate the possibility of recombination but cannot yet be effectively discriminated from a high genome-wide frequency of gene conversion. Finally, we suggest that the derived TcIIc population genetic data have a vital role in determining the origin of the epidemiologically important hybrid lineages TcIId and TcIIe.     

Unusual Structures in the Flagellar Apparatus of a Strain of Trypanosoma cruzi*

Certain structures, associated with the flagellum, and which had hitherto been described as appearing occasionally in some species of trypanosomes, were found very frequently in epimastigote forms of strain F of Trypanosoma cruzi: (a) a group of tubular elements in an electron-dense mass enclosed within a swelling of the flagellar membrane as the flagellum emerges from its reservoir; (b) an expansion of the flagellar membrane at the point of the above swelling, which in cross-sections appears as a ring; and (c) an electron dense band in the body of the organism alongside the border of the flagellar pocket. The possible significance of these structures and the fact that so far they have been found only in one strain of T. cruzi are discussed.     

The potential economic value of a Trypanosoma cruzi (Chagas disease) vaccine in Latin America

Background

Chagas disease, caused by the parasite Trypanosoma cruzi (T. cruzi), is the leading etiology of non-ischemic heart disease worldwide, with Latin America bearing the majority of the burden. This substantial burden and the limitations of current interventions have motivated efforts to develop a vaccine against T. cruzi.

Methodology/Principal Findings

We constructed a decision analytic Markov computer simulation model to assess the potential economic value of a T. cruzi vaccine in Latin America from the societal perspective. Each simulation run calculated the incremental cost-effectiveness ratio (ICER), or the cost per disability-adjusted life year (DALY) avoided, of vaccination. Sensitivity analyses evaluated the impact of varying key model parameters such as vaccine cost (range: $0.50–$200), vaccine efficacy (range: 25%–75%), the cost of acute-phase drug treatment (range: $10–$150 to account for variations in acute-phase treatment regimens), and risk of infection (range: 1%–20%). Additional analyses determined the incremental cost of vaccinating an individual and the cost per averted congestive heart failure case. Vaccination was considered highly cost-effective when the ICER was ≤1 times the GDP/capita, still cost-effective when the ICER was between 1 and 3 times the GDP/capita, and not cost-effective when the ICER was >3 times the GDP/capita. Our results showed vaccination to be very cost-effective and often economically dominant (i.e., saving costs as well providing health benefits) for a wide range of scenarios, e.g., even when risk of infection was as low as 1% and vaccine efficacy was as low as 25%. Vaccinating an individual could likely provide net cost savings that rise substantially as risk of infection or vaccine efficacy increase.

Conclusions/Significance

Results indicate that a T. cruzi vaccine could provide substantial economic benefit, depending on the cost of the vaccine, and support continued efforts to develop a human vaccine.