Real-time PCR in HIV/Trypanosoma cruzi coinfection with and without Chagas disease reactivation: association with HIV viral load and CD4 level

Background

Reactivation of chronic Chagas disease, which occurs in approximately 20% of patients coinfected with HIV/Trypanosoma cruzi (T. cruzi), is commonly characterized by severe meningoencephalitis and myocarditis. The use of quantitative molecular tests to monitor Chagas disease reactivation was analyzed.

Methodology

Polymerase chain reaction (PCR) of kDNA sequences, competitive (C-) PCR and real-time quantitative (q) PCR were compared with blood cultures and xenodiagnosis in samples from 91 patients (57 patients with chronic Chagas disease and 34 with HIV/T. cruzi coinfection), of whom 5 had reactivation of Chagas disease and 29 did not.

Principal Findings

qRT-PCR showed significant differences between groups; the highest parasitemia was observed in patients infected with HIV/T. cruzi with Chagas disease reactivation (median 1428.90 T. cruzi/mL), followed by patients with HIV/T. cruzi infection without reactivation (median 1.57 T. cruzi/mL) and patients with Chagas disease without HIV (median 0.00 T. cruzi/mL). Spearman''s correlation coefficient showed that xenodiagnosis was correlated with blood culture, C-PCR and qRT-PCR. A stronger Spearman correlation index was found between C-PCR and qRT-PCR, the number of parasites and the HIV viral load, expressed as the number of CD4⁺ cells or the CD4⁺/CD8⁺ ratio.

Conclusions

qRT-PCR distinguished the groups of HIV/T. cruzi coinfected patients with and without reactivation. Therefore, this new method of qRT-PCR is proposed as a tool for prospective studies to analyze the importance of parasitemia (persistent and/or increased) as a criterion for recommending pre-emptive therapy in patients with chronic Chagas disease with HIV infection or immunosuppression. As seen in this study, an increase in HIV viral load and decreases in the number of CD4⁺ cells/mm³ and the CD4⁺/CD8⁺ ratio were identified as cofactors for increased parasitemia that can be used to target the introduction of early, pre-emptive therapy.     

Use of a Novel Chagas Urine Nanoparticle Test (Chunap) for Diagnosis of Congenital Chagas Disease

Background

Detection of congenital T. cruzi transmission is considered one of the pillars of control programs of Chagas disease. Congenital transmission accounts for 25% of new infections with an estimated 15,000 infected infants per year. Current programs to detect congenital Chagas disease in Latin America utilize microscopy early in life and serology after 6 months. These programs suffer from low sensitivity by microscopy and high loss to follow-up later in infancy. We developed a Chagas urine nanoparticle test (Chunap) to concentrate, preserve and detect T. cruzi antigens in urine for early, non-invasive diagnosis of congenital Chagas disease.

Methodology/Principal Findings

This is a proof-of-concept study of Chunap for the early diagnosis of congenital Chagas disease. Poly N-isopropylacrylamide nano-particles functionalized with trypan blue were synthesized by precipitation polymerization and characterized with photon correlation spectroscopy. We evaluated the ability of the nanoparticles to capture, concentrate and preserve T. cruzi antigens. Urine samples from congenitally infected and uninfected infants were then concentrated using these nanoparticles. The antigens were eluted and detected by Western Blot using a monoclonal antibody against T. cruzi lipophosphoglycan. The nanoparticles concentrate T. cruzi antigens by 100 fold (western blot detection limit decreased from 50 ng/ml to 0.5 ng/ml). The sensitivity of Chunap in a single specimen at one month of age was 91.3% (21/23, 95% CI: 71.92%–98.68%), comparable to PCR in two specimens at 0 and 1 month (91.3%) and significantly higher than microscopy in two specimens (34.8%, 95% CI: 16.42%–57.26%). Chunap specificity was 96.5% (71/74 endemic, 12/12 non-endemic specimens). Particle-sequestered T. cruzi antigens were protected from trypsin digestion.

Conclusion/Significance

Chunap has the potential to be developed into a simple and sensitive test for the early diagnosis of congenital Chagas disease.     

Parasite load evaluation by qPCR and blood culture in Chagas disease and HIV co-infected patients under antiretroviral therapy

Chagas disease also known as American trypanosomiasis, is caused by Trypanosoma cruzi and transmitted by triatominae-contaminated feces. It is considered a neglected tropical disease that affects 6 to 7 million people worldwide. The reactivation of Chagas disease occurs when the chronically infected hosts are not able to control T. cruzi infection, generating recurrence of the acute phase. HIV is the main immunosuppressive infection that can lead to the reactivation of chronic Chagas disease in AIDS conditions. In co-infected patients, the reactivation of Chagas disease is related to their high parasite load, high HIV viral load, and CD4 T-cell counting less than 200/mm³, which may evolve to meningoencephalitis and myocarditis. Eight T. cruzi/HIV co-infected patients under antiretroviral therapy (ART) and ten Chagas disease patients without HIV infection that attended at Study Group of Chagas Disease, Hospital de Clínicas, University of Campinas (GEdoCh/HC/UNICAMP-SP) and Pontifical Catholic University of Campinas SP (PUCC/SP) were evaluated. Tests for Chagas disease were performed, such as qPCR and T. cruzi blood culture. The patient’s medical records were analyzed to verify clinical and epidemiological data, viral load, and CD4 T-cell counting since the outset of ART. For both groups, we found no statically significant differences between parasite load via blood culture and qPCR. In T. cruzi/HIV co-infected subjects, we observed a significant increase of CD4 T-cells counting and viral load decrease, which became undetectable over the years after ART. Parasites isolated from the patient’s blood culture were genotyped, being the majority of them infected with TcII and one case of mixed infection (TcII and TcV/TcVI). These results were expected according to the region of origin of the patients. We suggest that the parasite load be monitored through qPCR in T.cruzi/HIV co-infected patients. We conclude that ART in people living with HIV improves infection and immunosuppression control, enabling the natural evolution of the American trypanosomiasis.     

Adipose Tissue-Derived Mesenchymal Stromal Cells Protect Mice Infected with Trypanosoma cruzi from Cardiac Damage through Modulation of Anti-parasite Immunity

Background

Chagas disease, caused by the protozoan Trypanosoma cruzi (T.cruzi), is a complex disease endemic in Central and South America. It has been gathering interest due to increases in non-vectorial forms of transmission, especially in developed countries. The objective of this work was to investigate if adipose tissue-derived mesenchymal stromal cells (ASC) can alter the course of the disease and attenuate pathology in a mouse model of chagasic cardiomyopathy.

Methodology/Principal Findings

ASC were injected intraperitoneally at 3 days post-infection (dpi). Tracking by bioluminescence showed that cells remained in the abdominal cavity for up to 9 days after injection and most of them migrated to the abdominal or subcutaneous fat, an early parasite reservoir. ASC injection resulted in a significant reduction in blood parasitemia, which was followed by a decrease in cardiac tissue inflammation, parasitism and fibrosis at 30 dpi. At the same time point, analyses of cytokine release in cells isolated from the heart and exposed to T. cruzi antigens indicated an anti-inflammatory response in ASC-treated animals. In parallel, splenocytes exposed to the same antigens produced a pro-inflammatory response, which is important for the control of parasite replication, in placebo and ASC-treated groups. However, splenocytes from the ASC group released higher levels of IL-10. At 60 dpi, magnetic resonance imaging revealed that right ventricular (RV) dilation was prevented in ASC-treated mice.

Conclusions/Significance

In conclusion, the injection of ASC early after T. cruzi infection prevents RV remodeling through the modulation of immune responses. Lymphoid organ response to the parasite promoted the control of parasite burden, while the heart, a target organ of Chagas disease, was protected from damage due to an improved control of inflammation in ASC-treated mice.     

Development of a Novel Multiplex Immunoassay Multi-cruzi for the Serological Confirmation of Chagas Disease

Background

Chagas disease is due to the parasite Trypanosoma cruzi, a protist disseminated by a Triatome vector. This disease is endemic to Latin America and considered by WHO as one of the 17 world’s neglected diseases. In Europe and in North America, imported cases are also detected, due to migration of population outside of the endemic region. Diagnosis of T. cruzi infection is usually made indirectly by the detection of specific antibodies to T. cruzi antigens. Following initial diagnostic evaluation or screening test (qualifying or discarding blood donation), a confirmation test is performed for samples initially reactive. The test presented in this study aims at the confirmation/refutation of the infectious status of human blood samples and will permit taking appropriate clinical measures.

Methodology/Principal Findings

We designed a novel array of twelve antigens and printed these antigens onto 96-well plates. We tested 248 positive samples T. cruzi, 94 unscreened blood donors’ samples from non-endemic area, 49 seronegative blood donors, 7 false-positive and 3 doubtful samples. The observed reactivities were analyzed to propose a decision-tree algorithm that correctly classifies all the samples, with the potential to discriminate false-positive results and sticky samples. We observed that antibodies levels (Sum of all antigens) was significantly higher for PCR positive than for PCR negative samples in all studied groups with Multi-cruzi.

Conclusion/Significance

The results described in this study indicate that the Multi-cruzi improves the serological confirmation of Chagas disease. Moreover the “sum of all antigens” detected by Multi-cruzi could reflect parasitemia level in patients–like PCR signals does—and could serve as an indicator of parasite clearance in longitudinal follow-ups. Validation of this assay is still required on an independent large collection of well characterized samples including typical false-reactive samples such as Leishmaniasis.     

Combining Public Health Education and Disease Ecology Research: Using Citizen Science to Assess Chagas Disease Entomological Risk in Texas

Background

Chagas disease is a zoonotic parasitic disease well-documented throughout the Americas and transmitted primarily by triatomine ‘kissing bug’ vectors. In acknowledgment of the successful history of vector control programs based on community participation across Latin America, we used a citizen science approach to gain novel insight into the geographic distribution, seasonal activity, and Trypanosoma cruzi infection prevalence of kissing bugs in Texas while empowering the public with information about Chagas disease.

Methodology/Principal Findings

We accepted submissions of kissing bugs encountered by the public in Texas and other states from 2013–2014 while providing educational literature about Chagas disease. In the laboratory, kissing bugs were identified to species, dissected, and tested for T. cruzi infection. A total of 1,980 triatomines were submitted to the program comprised of at least seven species, of which T. gerstaeckeri and T. sanguisuga were the most abundant (85.7% of submissions). Triatomines were most commonly collected from dog kennels and outdoor patios; Overall, 10.5% of triatomines were collected from inside the home. Triatomines were submitted from across Texas, including many counties which were not previously known to harbor kissing bugs. Kissing bugs were captured primarily throughout April-October, and peak activity occurred in June-July. Emails to our dedicated account regarding kissing bugs were more frequent in the summer months (June-August) than the rest of the year. We detected T. cruzi in 63.3% of tested bugs.

Conclusions/Significance

Citizen science is an efficient approach for generating data on the distribution, phenology, and infection prevalence of kissing bugs—vectors of the Chagas disease parasite—while educating the public and medical community.     

The Trypanosoma cruzi Vitamin C Dependent Peroxidase Confers Protection against Oxidative Stress but Is Not a Determinant of Virulence

Background

The neglected parasitic infection Chagas disease is rapidly becoming a globalised public health issue due to migration. There are only two anti-parasitic drugs available to treat this disease, benznidazole and nifurtimox. Thus it is important to identify and validate new drug targets in Trypanosoma cruzi, the causative agent. T. cruzi expresses an ER-localised ascorbate-dependent peroxidase (TcAPx). This parasite-specific enzyme has attracted interest from the perspective of targeted chemotherapy.

Methodology/Principal Findings

To assess the importance of TcAPx in protecting T. cruzi from oxidative stress and to determine if it is essential for virulence, we generated null mutants by targeted gene disruption. Loss of activity was associated with increased sensitivity to exogenous hydrogen peroxide, but had no effect on susceptibility to the front-line Chagas disease drug benznidazole. This suggests that increased oxidative stress in the ER does not play a significant role in its mechanism of action. Homozygous knockouts could proceed through the entire life-cycle in vitro, although they exhibited a significant decrease in their ability to infect mammalian cells. To investigate virulence, we exploited a highly sensitive bioluminescence imaging system which allows parasites to be monitored in real-time in the chronic stage of murine infections. This showed that depletion of enzyme activity had no effect on T. cruzi replication, dissemination or tissue tropism in vivo.

Conclusions/Significance

TcAPx is not essential for parasite viability within the mammalian host, does not have a significant role in establishment or maintenance of chronic infections, and should therefore not be considered a priority for drug design.     

Galectin-1 Prevents Infection and Damage Induced by Trypanosoma cruzi on Cardiac Cells

Background

Chronic Chagas cardiomyopathy caused by Trypanosoma cruzi is the result of a pathologic process starting during the acute phase of parasite infection. Among different factors, the specific recognition of glycan structures by glycan-binding proteins from the parasite or from the mammalian host cells may play a critical role in the evolution of the infection.

Methodology and Principal Findings

Here we investigated the contribution of galectin–1 (Gal–1), an endogenous glycan-binding protein abundantly expressed in human and mouse heart, to the pathophysiology of T. cruzi infection, particularly in the context of cardiac pathology. We found that exposure of HL–1 cardiac cells to Gal–1 reduced the percentage of infection by two different T. cruzi strains, Tulahuén (TcVI) and Brazil (TcI). In addition, Gal–1 prevented exposure of phosphatidylserine and early events in the apoptotic program by parasite infection on HL–1 cells. These effects were not mediated by direct interaction with the parasite surface, suggesting that Gal–1 may act through binding to host cells. Moreover, we also observed that T. cruzi infection altered the glycophenotype of cardiac cells, reducing binding of exogenous Gal–1 to the cell surface. Consistent with these data, Gal–1 deficient (Lgals1 ^-/-) mice showed increased parasitemia, reduced signs of inflammation in heart and skeletal muscle tissues, and lower survival rates as compared to wild-type (WT) mice in response to intraperitoneal infection with T. cruzi Tulahuén strain.

Conclusion/Significance

Our results indicate that Gal–1 modulates T. cruzi infection of cardiac cells, highlighting the relevance of galectins and their ligands as regulators of host-parasite interactions.     

Risk Factors and Screening for Trypanosoma cruzi Infection of Dutch Blood Donors

Background

Blood donors unaware of Trypanosoma cruzi infection may donate infectious blood. Risk factors and the presence of T. cruzi antibodies in at-risk Dutch blood donors were studied to assess whether specific blood safety measures are warranted in the Netherlands.

Methodology

Birth in a country endemic for Chagas disease (CEC), having a mother born in a CEC, or having resided for at least six continuous months in a CEC were considered risk factors for T. cruzi infection. From March through September 2013, risk factor questions were asked to all donors who volunteered to donate blood or blood components. Serum samples were collected from donors reporting one or more risk factors, and screened for IgG antibodies to T. cruzi by EIA.

Results

Risk factors for T. cruzi infection were reported by 1,426 of 227,278 donors (0.6%). Testing 1,333 at-risk donors, none (0.0%; 95%, CI 0.0–0.4%) was seroreactive for IgG antibodies to T. cruzi. A total of 472 donors were born in a CEC; 553 donors reported their mother being born in a CEC; and 1,121 donors reported a long-term stay in a CEC. The vast majority of reported risk factors were related to Suriname and Brazil. Overall, the participants resided for 7,694 years in CECs, which equals 2.8 million overnight stays. Of those, 1.9 million nights were spent in Suriname.

Conclusions/Significance

Asymptomatic T. cruzi infection appears to be extremely rare among Dutch blood donors. Blood safety interventions to mitigate the risk of T. cruzi transmission by transfusion would be highly cost-ineffective in the Netherlands, and are thus not required.     

Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples

Background

Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI–TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR).

Methods/Principal Findings

The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm.

Conclusions/Significance

Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production.     

Clinical profile and mortality in patients with T. cruzi/HIV co-infection from the multicenter data base of the “Network for healthcare and study of Trypanosoma cruzi/HIV co-infection and other immunosuppression conditions”

ObjectiveChagas disease (CD) globalization facilitated the co-infection with Human Immunodeficiency Virus (HIV) in endemic and non-endemic areas. Considering the underestimation of Trypanosoma cruzi (T. cruzi)-HIV co-infection and the risk of life-threatening Chagas Disease Reactivation (CDR), this study aimed to analyze the major co-infection clinical characteristics and its mortality rates.MethodsThis is a cross-sectional retrospective multicenter study of patients with CD confirmed by two serological or one parasitological tests, and HIV infection confirmed by immunoblot. CDR was diagnosed by direct microscopy with detection of trypomastigote forms in the blood or other biological fluids and/or amastigote forms in inflammatory lesions.ResultsOut of 241 patients with co-infection, 86.7% were from Brazil, 47.5% had <200 CD4⁺ T cells/μL and median viral load was 17,000 copies/μL. Sixty CDR cases were observed. Death was more frequent in patients with reactivation and was mainly caused by CDR. Other causes of death unrelated to CDR were the manifestation of opportunistic infections in those with Acquired Immunodeficiency Syndrome. The time between the co-infection diagnosis to death was shorter in patients with CDR. Lower CD4⁺ cells count at co-infection diagnosis was independently associated with reactivation. Similarly, lower CD4⁺ cells numbers at co-infection diagnosis and male sex were associated with higher lethality in CDR. Additionally, CD4⁺ cells were lower in meningoencephalitis than in myocarditis and milder forms.ConclusionThis study showed major features on T. cruzi-HIV co-infection and highlighted the prognostic role of CD4⁺ cells for reactivation and mortality. Since lethality was high in meningoencephalitis and all untreated patients died shortly after the diagnosis, early diagnosis, immediate antiparasitic treatment, patient follow-up and epidemiological surveillance are essentials in T. cruzi/HIV co-infection and CDR managements.     

Phenotypic Features of Circulating Leukocytes from Non-human Primates Naturally Infected with Trypanosoma cruzi Resemble the Major Immunological Findings Observed in Human Chagas Disease

Background

Cynomolgus macaques (Macaca fascicularis) represent a feasible model for research on Chagas disease since natural T. cruzi infection in these primates leads to clinical outcomes similar to those observed in humans. However, it is still unknown whether these clinical similarities are accompanied by equivalent immunological characteristics in the two species. We have performed a detailed immunophenotypic analysis of circulating leukocytes together with systems biology approaches from 15 cynomolgus macaques naturally infected with T. cruzi (CH) presenting the chronic phase of Chagas disease to identify biomarkers that might be useful for clinical investigations.

Methods and Findings

Our data established that CH displayed increased expression of CD32⁺ and CD56⁺ in monocytes and enhanced frequency of NK Granzyme A⁺ cells as compared to non-infected controls (NI). Moreover, higher expression of CD54 and HLA-DR by T-cells, especially within the CD8⁺ subset, was the hallmark of CH. A high level of expression of Granzyme A and Perforin underscored the enhanced cytotoxicity-linked pattern of CD8⁺ T-lymphocytes from CH. Increased frequency of B-cells with up-regulated expression of Fc-γRII was also observed in CH. Complex and imbricate biomarker networks demonstrated that CH showed a shift towards cross-talk among cells of the adaptive immune system. Systems biology analysis further established monocytes and NK-cell phenotypes and the T-cell activation status, along with the Granzyme A expression by CD8⁺ T-cells, as the most reliable biomarkers of potential use for clinical applications.

Conclusions

Altogether, these findings demonstrated that the similarities in phenotypic features of circulating leukocytes observed in cynomolgus macaques and humans infected with T. cruzi further supports the use of these monkeys in preclinical toxicology and pharmacology studies applied to development and testing of new drugs for Chagas disease.     

Oral Outbreak of Chagas Disease in Santa Catarina,Brazil: Experimental Evaluation of a Patient’s Strain

Chagas disease is a worldwide public health problem. Although the vectorial transmission of Chagas disease has been controlled in Brazil there are other ways of transmission, such as the ingestion of T. cruzi contaminated food, which ensures the continuation of this zoonosis. Here, we demonstrate the influence of the inoculation route on the establishment and development of the SC2005 T. cruzi strain infection in mice. Groups of Swiss mice were infected intragastrically (IG) or intraperitoneally (IP) with the T. cruzi SC2005 strain derived from an outbreak of oral Chagas disease. The results revealed that 100% of IP infected mice showed parasitemia, while just 36% of IG infected showed the presence of the parasite in blood. The parasitemia peaks were later and less intense in the IG infected mice. Mortality of the IP infected animals was more intense and earlier when compared to the IG infected mice. In the IP infected mice leucopenia occurred in the early infection followed by leucocytosis, correlating positively with the increase of the parasites. However, in the IG infected mice only an increase in monocytes was observed, which was positively correlated with the increase of the parasites. Histopathological analyses revealed a myotropic pattern of the SC2005 strain with the presence of inflammatory infiltrates and parasites in different organs of the animals infected by both routes as well as fibrosis foci and collagen redistribution. The flow cytometric analysis demonstrated a fluctuation of the T lymphocyte population in the blood, spleen and mesenteric lymph nodes of the infected animals. T. cruzi DNA associated with the presence of inflammatory infiltrates was detected by PCR in the esophagus, stomach and intestine of all infected mice. These findings are important for the understanding of the pathogenesis of T. cruzi infection by both inoculation routes.     

Machine Learning Models and Pathway Genome Data Base for Trypanosoma cruzi Drug Discovery

BackgroundChagas disease is a neglected tropical disease (NTD) caused by the eukaryotic parasite Trypanosoma cruzi. The current clinical and preclinical pipeline for T. cruzi is extremely sparse and lacks drug target diversity.

Methodology/Principal Findings

In the present study we developed a computational approach that utilized data from several public whole-cell, phenotypic high throughput screens that have been completed for T. cruzi by the Broad Institute, including a single screen of over 300,000 molecules in the search for chemical probes as part of the NIH Molecular Libraries program. We have also compiled and curated relevant biological and chemical compound screening data including (i) compounds and biological activity data from the literature, (ii) high throughput screening datasets, and (iii) predicted metabolites of T. cruzi metabolic pathways. This information was used to help us identify compounds and their potential targets. We have constructed a Pathway Genome Data Base for T. cruzi. In addition, we have developed Bayesian machine learning models that were used to virtually screen libraries of compounds. Ninety-seven compounds were selected for in vitro testing, and 11 of these were found to have EC₅₀ < 10μM. We progressed five compounds to an in vivo mouse efficacy model of Chagas disease and validated that the machine learning model could identify in vitro active compounds not in the training set, as well as known positive controls. The antimalarial pyronaridine possessed 85.2% efficacy in the acute Chagas mouse model. We have also proposed potential targets (for future verification) for this compound based on structural similarity to known compounds with targets in T. cruzi.

Conclusions/ Significance

We have demonstrated how combining chemoinformatics and bioinformatics for T. cruzi drug discovery can bring interesting in vivo active molecules to light that may have been overlooked. The approach we have taken is broadly applicable to other NTDs.     

Frequent House Invasion of Trypanosoma cruzi-Infected Triatomines in a Suburban Area of Brazil

Background

The demographic transition of populations from rural areas to large urban centers often results in a disordered occupation of forest remnants and increased economic pressure to develop high-income buildings in these areas. Ecological and socioeconomic factors associated with these urban transitions create conditions for the potential transmission of infectious diseases, which was demonstrated for Chagas disease.

Methodology/Principal Findings

We analyzed 930 triatomines, mainly Triatoma tibiamaculata, collected in artificial and sylvatic environments (forests near houses) of a suburban area of the city of Salvador, Bahia State, Brazil between 2007 and 2011. Most triatomines were captured at peridomiciles. Adult bugs predominated in all studied environments, and nymphs were scarce inside houses. Molecular analyses of a randomly selected sub-sample (n=212) of triatomines showed Trypanosoma cruzi infection rates of 65%, 50% and 56% in intradomestic, peridomestic and sylvatic environments, respectively. We detected the T. cruzi lineages I and II and mixed infections. We also showed that T. tibiamaculata fed on blood from birds (50%), marsupials (38%), ruminants (7%) and rodents (5%). The probability of T. cruzi infection was higher in triatomines that fed on marsupial blood (odds ratio (OR) = 1.95, 95% confidence interval (CI) = 1.22-3.11). Moreover, we observed a protective effect against infection in bugs that fed on bird blood (OR = 0.43, 95% CI = 0.30-0.73).

Conclusions/Significance

The frequent invasion of houses by infected triatomines indicates a potential risk of T. cruzi transmission to inhabitants in this area. Our results reinforce that continuous epidemiological surveillance should be performed in areas where domestic transmission is controlled but enzootic transmission persists.     

Overexpression of Cytoplasmic TcSIR2RP1 and Mitochondrial TcSIR2RP3 Impacts on Trypanosoma cruzi Growth and Cell Invasion

Background

Trypanosoma cruzi is a protozoan pathogen responsible for Chagas disease. Current therapies are inadequate because of their severe host toxicity and numerous side effects. The identification of new biotargets is essential for the development of more efficient therapeutic alternatives. Inhibition of sirtuins from Trypanosoma brucei and Leishmania ssp. showed promising results, indicating that these enzymes may be considered as targets for drug discovery in parasite infection. Here, we report the first characterization of the two sirtuins present in T. cruzi.

Methodology

Dm28c epimastigotes that inducibly overexpress TcSIR2RP1 and TcSIR2RP3 were constructed and used to determine their localizations and functions. These transfected lines were tested regarding their acetylation levels, proliferation and metacyclogenesis rate, viability when treated with sirtuin inhibitors and in vitro infectivity.

Conclusion

TcSIR2RP1 and TcSIR2RP3 are cytosolic and mitochondrial proteins respectively. Our data suggest that sirtuin activity is important for the proliferation of T. cruzi replicative forms, for the host cell-parasite interplay, and for differentiation among life-cycle stages; but each one performs different roles in most of these processes. Our results increase the knowledge on the localization and function of these enzymes, and the overexpressing T. cruzi strains we obtained can be useful tools for experimental screening of trypanosomatid sirtuin inhibitors.     

A Novel Method for Inducing Amastigote-To-Trypomastigote Transformation In Vitro in Trypanosoma cruzi Reveals the Importance of Inositol 1,4,5-Trisphosphate Receptor

Background

Trypanosoma cruzi is a parasitic protist that causes Chagas disease, which is prevalent in Latin America. Because of the unavailability of an effective drug or vaccine, and because about 8 million people are infected with the parasite worldwide, the development of novel drugs demands urgent attention. T. cruzi infects a wide variety of mammalian nucleated cells, with a preference for myocardial cells. Non-dividing trypomastigotes in the bloodstream infect host cells where they are transformed into replication-capable amastigotes. The amastigotes revert to trypomastigotes (trypomastigogenesis) before being shed out of the host cells. Although trypomastigote transformation is an essential process for the parasite, the molecular mechanisms underlying this process have not yet been clarified, mainly because of the lack of an assay system to induce trypomastigogenesis in vitro.

Methodology/Principal Findings

Cultivation of amastigotes in a transformation medium composed of 80% RPMI-1640 and 20% Grace’s Insect Medium mediated their transformation into trypomastigotes. Grace’s Insect Medium alone also induced trypomastigogenesis. Furthermore, trypomastigogenesis was induced more efficiently in the presence of fetal bovine serum. Trypomastigotes derived from in vitro trypomastigogenesis were able to infect mammalian host cells as efficiently as tissue-culture-derived trypomastigotes (TCT) and expressed a marker protein for TCT. Using this assay system, we demonstrated that T. cruzi inositol 1,4,5-trisphosphate receptor (TcIP₃R)—an intracellular Ca²⁺ channel and a key molecule involved in Ca²⁺ signaling in the parasite—is important for the transformation process.

Conclusion/Significance

Our findings provide a new tool to identify the molecular mechanisms of the amastigote-to-trypomastigote transformation, leading to a new strategy for drug development against Chagas disease.     

Testing the efficacy of a multi-component DNA-prime/DNA-boost vaccine against Trypanosoma cruzi infection in dogs

Background

Trypanosoma cruzi, the etiologic agent of Chagas Disease, is a major vector borne health problem in Latin America and an emerging infectious disease in the United States.

Methods

We tested the efficacy of a multi-component DNA-prime/DNA-boost vaccine (TcVac1) against experimental T. cruzi infection in a canine model. Dogs were immunized with antigen-encoding plasmids and cytokine adjuvants, and two weeks after the last immunization, challenged with T. cruzi trypomastigotes. We measured antibody responses by ELISA and haemagglutination assay, parasitemia and infectivity to triatomines by xenodiagnosis, and performed electrocardiography and histology to assess myocardial damage and tissue pathology.

Results

Vaccination with TcVac1 elicited parasite-and antigen-specific IgM and IgG (IgG2>IgG1) responses. Upon challenge infection, TcVac1-vaccinated dogs, as compared to non-vaccinated controls dogs, responded to T. cruzi with a rapid expansion of antibody response, moderately enhanced CD8⁺ T cell proliferation and IFN-γ production, and suppression of phagocytes’ activity evidenced by decreased myeloperoxidase and nitrite levels. Subsequently, vaccinated dogs controlled the acute parasitemia by day 37 pi (44 dpi in non-vaccinated dogs), and exhibited a moderate decline in infectivity to triatomines. TcVac1-immunized dogs did not control the myocardial parasite burden and electrocardiographic and histopatholgic cardiac alterations that are the hallmarks of acute Chagas disease. During the chronic stage, TcVac1-vaccinated dogs exhibited a moderate decline in cardiac alterations determined by EKG and anatomo-/histo-pathological analysis while chronically-infected/non-vaccinated dogs continued to exhibit severe EKG alterations.

Conclusions

Overall, these results demonstrated that TcVac1 provided a partial resistance to T. cruzi infection and Chagas disease, and provide an impetus to improve the vaccination strategy against Chagas disease.     

Course of Chronic Trypanosoma cruzi Infection after Treatment Based on Parasitological and Serological Tests: A Systematic Review of Follow-Up Studies

Background

Chagas disease is caused by the flagellate protozoan Trypanosoma cruzi (T. cruzi). It is endemic in Latin American countries outside the Caribbean. The current criterion for cure in the chronic phase of the disease is the negativization of at least two serological tests such as enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IIF) and indirect hemagglutination assay (IHA). The serological evolution of treated subjects with chronic T. cruzi infection is variable. Treatment failure is indicated by a positive parasitological and/or molecular test (persistence of parasitemia).

Objectives

To summarize the pattern of response to treatment of parasitological, molecular and serological tests performed during the follow-up of subjects with chronic T. cruzi infection.

Methods

Electronic searches in relevant databases and screening of citations of potentially eligible articles were accomplished. Organizations focusing on neglected infectious diseases were asked for help in identifying relevant studies.Included studies were randomized controlled trials (RCTs), quasi-RCTs, and cohort studies involving adults and children with chronic infection who received trypanocidal treatment (benznidazole or nifurtimox) and were followed over time. The assessment of risk of bias was performed separately for each study design. The Cochrane Collaboration’s tool and the guidelines developed by Hayden et al. were used. Two reviewers extracted all data independently. A third review author was consulted in case of discordant opinion.Additional analyses were defined in ad-hoc basis. Scatter plots for percentage of positive parasitological and molecular tests and for negative serological tests were developed by using the lowess curve technique. Heterogeneity was measured by I².The protocol was registered in PROSPERO, an international prospective register of systematic review protocols (Registration Number CRD42012002162).

Results

Out of 2,136 citations screened, 54 studies (six RCTs and 48 cohort studies) were included. The smoothed curves for positive xenodiagnosis and positive polymerase chain reaction (PCR) were characterized by a sharp decrease at twelve month posttreatment. Afterwards, they reached 10–20% and 40% for xenodiagnosis and PCR, respectively. The smoothed curves for negative conventional serological tests increased up to 10% after 48 months of treatment. In the long-term, the rate of negativization was between 20% and 45%.The main sources of bias identified across cohort studies were the lack of control for confounding and attrition bias. In general, RCTs were judged as low risk of bias in all domains. The level of heterogeneity across included studies was moderate to high.Additional analysis were incomplete because of the limited availability of data. In this regard, the country of origin of study participants might affect the results of parasitological and molecular tests, while the level of risk of bias might affect serological outcomes. Subgroup analysis suggested that seronegativization occurs earlier in children compared to adults.

Conclusions

We acknowledge that there is a dynamic pattern of response based on parasitological, molecular and serological tests in subjects chronically infected with T. cruzi after treatment. Our findings suggest a trypanocidal effect in the long-term follow-up.Further research is needed to explore potential sources of heterogeneity and to conduct reliable subgroup analysis.     

MicroRNA Transcriptome Profiling in Heart of Trypanosoma cruzi-Infected Mice: Parasitological and Cardiological Outcomes

Chagas disease is caused by the parasite Trypanosoma cruzi, and it begins with a short acute phase characterized by high parasitemia followed by a life-long chronic phase with scarce parasitism. Cardiac involvement is the most prominent manifestation, as 30% of infected subjects will develop abnormal ventricular repolarization with myocarditis, fibrosis and cardiomyocyte hypertrophy by undefined mechanisms. Nevertheless, follow-up studies in chagasic patients, as well as studies with murine models, suggest that the intensity of clinical symptoms and pathophysiological events that occur during the acute phase of disease are associated with the severity of cardiac disease observed during the chronic phase. In the present study we investigated the role of microRNAs (miRNAs) in the disease progression in response to T. cruzi infection, as alterations in miRNA levels are known to be associated with many cardiovascular disorders. We screened 641 rodent miRNAs in heart samples of mice during an acute infection with the Colombiana T.cruzi strain and identified multiple miRNAs significantly altered upon infection. Seventeen miRNAs were found significantly deregulated in all three analyzed time points post infection. Among these, six miRNAs had their expression correlated with clinical parameters relevant to the disease, such as parasitemia and maximal heart rate-corrected QT (QTc) interval. Computational analyses identified that the gene targets for these six miRNAs were involved in networks and signaling pathways related to increased ventricular depolarization and repolarization times, important factors for QTc interval prolongation. The data presented here will guide further studies about the contribution of microRNAs to Chagas heart disease pathogenesis.