Chagas disease.

Chagas disease persists as a global public health problem due to the high morbidity and mortality burden. Despite the possibility of a cure and advances in transmission control, epidemiological transformations, such as urbanisation and globalisation, and the emerging importance of oral and vertical transmission mean that Chagas disease should be considered an emerging disease, with new cases occurring worldwide. Important barriers to diagnosis, treatment, and care remain, resulting in repressed numbers of reported cases, which in turn leads to inadequate public policies. The validation of new diagnostic tools and treatment options is needed, as existing tools pose serious limitations to access to health care. Integrated models of surveillance, with community and intersectional participation, embedded in the concept of One Health, are essential for control. In addition, mitigation strategies for the main social determinants of health, including difficulties imposed by migration, are important to improve access to comprehensive health care in a globalised scenario.

Congenital Chagas disease: an update.

Congenital infection with Trypanosoma cruzi is a global problem, occurring on average in 5% of children born from chronically infected mothers in endemic areas, with variations depending on the region. This presentation aims to focus on and update epidemiological data, research methods, involved factors, control strategy and possible prevention of congenital infection with T. cruzi. Considering that etiological treatment of the child is always effective if performed before one year of age, the diagnosis of infection in pregnant women and their newborns has to become the standard of care and integrated into the surveillance programs of syphilis and human immunodeficiency virus. In addition to the standard tests, polymerase chain reaction performed on blood of neonates of infected mothers one month after birth might improve the diagnosis of congenital infection. Recent data bring out that its transmission can be prevented through treatment of infected women before they become pregnant. The role of parasite genotypes and host genetic factors in parasite transmission and development of infection in foetuses/neonates has to be more investigated in order to better estimate the risk factors and impact on health of congenital infection with T. cruzi.

Congenital transmission of Trypanosoma cruzi in central Brazil. A study of 1,211 individuals born to infected mothers.

Transmission of Trypanosoma cruzi during pregnancy is estimated to occur in less than 20% of infected mothers; however, the etiopathogenesis is not completely understood. The Centre for Studies on Chagas Disease provides confirmation of T. cruzi infection for individuals living in central Brazil. In this retrospective hospital-based study, all requests for diagnosis of T. cruzi infection in individuals less than 21 years old from 1994-2014 were searched. We end with 1,211 individuals and their respective infected mothers. Congenital transmission of infection was confirmed in 24 individuals (2%) in central Brazil, an area where the main T. cruzi lineage circulating in humans is TcII. This low prevalence of congenital Chagas disease is discussed in relation to recent findings in the south region of Brazil, where TcV is the main lineage and congenital transmission has a higher prevalence (approximately 5%), similar to frequencies reported in Argentina, Paraguay and Bolivia. This is the first report to show geographical differences in the rates of congenital transmission of T. cruzi and the relationship between the prevalence of congenital transmission and the type of Tc prevalent in each region.

Comparative evaluation of 11 commercialized rapid diagnostic tests for detecting Trypanosoma cruzi antibodies in serum banks in areas of endemicity and nonendemicity.

Chagas disease is one of the main public health issues in Latin America. Increasingly during the past few decades, Trypanosoma cruzi infection has been detected in North America, Europe, and the Western Pacific, mainly as a result of population movement. The limited availability of rapid serological diagnostic tests hinders rapid diagnosis and early treatment in areas of endemicity and nonendemicity. In collaboration with 11 national reference laboratories (NRLs) from different geographical areas, we evaluated the performances of commercialized serological rapid diagnostic tests (RDT) for T. cruzi infection. Eleven commercialized T. cruzi infection RDTs were evaluated on a total of 474 samples extensively tested with at least three different techniques for Chagas disease, maintained at controlled low temperatures, and stored in the serum banks of the 11 NRLs. We measured the sensitivity, specificity, and concordance of each RDT and provided an additional questionnaire to evaluate its ease of use. The selected RDTs in this study were performed under controlled laboratory conditions. Out of the 11 RDTs, we found 8 of them to be useful, with the cassette format favored over the strip. We did not observe significant differences in RDT performances in the different regions. Overall, the performance results were lower than those disclosed by the manufacturers. The results of this evaluation validate the possibility of using RDTs to diagnose Chagas disease, thereby decreasing the time to treatment at a primary health care facility for patients who are willing to be treated. Further studies should be conducted in the laboratory and in the field to confirm these data, expressly to evaluate reproducibility in resource-limited settings, or using whole blood in clinical settings in areas of endemicity and nonendemicity.

Evaluation of Four Biomarkers in Patients Chronically Infected with Trypanosoma cruzi and Their Relationship with Disease Progression.

This study evaluated four biomarkers of inflammation or fibrosis as progression indicators for heart disease in patients with Chagas disease. We compared values of these markers at the time of the first sample collection of blood (first time point) and at the time of the last collection of blood (second time point) for 103 individuals positive for Trypanosoma cruzi antibodies. They were split into two clinical groups: 52 individuals with the indeterminate form of the disease at the first time point and 51 controls that already had either cardiac involvement (N = 25) or megaviscera (megaesophagus and/or megacolon; N = 26) at that time. All individuals had an electrocardiogram performed both at the first and second time point (mean time between time points: 11 years). All samples were blind tested for galectin-3, brain natriuretic peptide (NT-proBNP), lysyl oxidase-like protein 2 (LOXL2), and troponin. Differences in concentrations between samples were analyzed using the months between samples as the covariate. This analysis showed that values for all markers, except troponin biomarkers had a significative increase at the second time point for the 91 patients without progression. A similar result was obtained for NT-proBNP and LOXL2 with sera from 12 patients that progressed with cardiac disease. The single marker that showed a significative difference between groups (P = 0.01) was galectin-3. We concluded that galectin-3 was the only marker with a prognostic value in relation to the progression or worsening of heart disease in patients with Chagas disease.

The CUIDA Chagas Project: towards the elimination of congenital transmission of Chagas disease in Bolivia, Brazil, Colombia, and Paraguay.

BACKGROUND: Mother-to-child transmission of Chagas disease (CD) has become a relevant problem in both endemic and non-endemic areas. METHODS: Description of the CUIDA Chagas Project - Communities United for Innovation, Development and Attention for Chagas disease'. RESULTS: Through innovative and strategic research, this project will provide improved diagnostic and treatment options as well as replicable implementation models that are adaptable to different contexts. CONCLUSIONS: By integrating test, treat and care actions for CD into primary health care practices, the burden of CD on people and health systems may be significantly reduced.

Validation of a rapid immunochromatographic assay for diagnosis of Trypanosoma cruzi infection among Latin-American Migrants in Geneva, Switzerland.

Chagas' disease is a global public health problem due to the recent exchange of population between Latin America and other regions, including Europe. The recent development of rapid diagnostic tests (RDTs) for Trypanosoma cruzi infection may improve patient access to diagnosis and care worldwide. We evaluated the diagnostic accuracy of the Chagas Stat-Pak RDT in a cohort of undocumented Latin-American migrants living in Geneva, Switzerland. Study participants were enrolled in a primary health care center. The Chagas Stat-Pak test was performed independently on blood and serum samples. A combination of two commercialized enzyme-linked immunosorbent assay (ELISA)-based serological tests was used for comparison (reference standard). A total of 999 adults (median age, 36 years) were included in the study; the majority were women (83%) and originally from Bolivia (47%) or Brazil (25%). A total of 125 participants (12.5%) were diagnosed with T. cruzi infection; with the exception of three individuals, all individuals diagnosed with T. cruzi were originally from Bolivia. The sensitivity and specificity of the Chagas Stat-Pak test on blood samples were 95.2% (95% confidence interval [95% CI], 89.2% to 97.9%) and 99.9% (95% CI, 99.3% to 100%), respectively. When the test was performed on serum samples, the sensitivity was 96% (95% CI, 91% to 98.3%), and the specificity was 99.8% (95% CI, 99.2% to 99.9%). The concordance of test results for blood and serum samples was 99.7%. Both negative and positive predictive values were above 98%. The Chagas Stat-Pak is an accurate diagnostic test for T. cruzi infection among Latin-American migrants living in Europe. The mild deficit in sensitivity should be interpreted in light of its ease of use and capacity to provide immediate results, which allow more people at risk to have access to diagnosis and care both in countries where Chagas' disease is endemic and in countries where this disease is not endemic.

Development of a New Lateral Flow Assay Based on IBMP-8.1 and IBMP-8.4 Chimeric Antigens to Diagnose Chagas Disease.

Despite several available methodologies for Chagas disease (CD) serological screening, the main limitation of chronic CD diagnosis is the lack of effective tools for large-scale screening and point-of-care diagnosis to be used in different CD epidemiological scenarios. Taking into account that developing such a diagnostic tool will significantly improve the ability to identify CD carriers, we aimed at performing a proof-of-concept study (phase I study) to assess the use of these proteins in a point-of-care platform using serum samples from different geographical settings of Brazil and distinct clinical presentations. The diagnostic accuracy study was conducted on a panel of two WHO International Standards (IS) and 14 sera from T. cruzi-positive and 16 from T. cruzi-negative individuals. The results obtained with the test strips were converted to digital images, allowing quantitative comparison expressed as a relative band intensity ratio (RBI). The diagnostic potential and performance were also determined. Regardless of the geographical origin or clinical presentation, all sera with T. cruzi antibodies returned positive both for IBMP-8.1 and IBMP-8.4 chimeric antigens. The area under the ROC curve (AUC) values was 100% for both antigens, demonstrating an outstanding overall diagnostic accuracy (100%). Based on the data, we believe that the lateral flow assays based on these antigens are promising methodologies for screening CD.

Human autoantibodies specific for neurotrophin receptors TrkA, TrkB, and TrkC protect against lethal Trypanosoma cruzi infection in mice.

Patients with Chagas' disease remain asymptomatic for many years, presumably by keeping the etiological agent Trypanosoma cruzi in check through protective immunity against. Recently, we found that T. cruzi uses TrkA, a receptor tyrosine kinase responsive to neurotrophin nerve growth factor in vertebrate nervous systems, to invade cells. We also found that TrkA, TrkB, and TrkC, but not T. cruzi, are targets of specific autoantibodies present in the sera of patients with chronic Chagas' disease. Here we show that TrkA-, TrkB-, and TrkC-specific autoantibodies isolated from the sera of four individuals with chronic indeterminate (asymptomatic) Chagas' disease potently blocked invasion of Trk-bearing neuronal PC12 cells, neuroglial astrocytes, enteroglial cells, and Schwann cells and Trk-expressing non-neural smooth muscle and dendritic cells. However, these autoantibodies did not inhibit T. cruzi invasion of mutant PC12 cells lacking TrkA or of normal cells lacking Trk receptors, suggesting that autoantibodies interfered with parasite/Trk cross talk to access the intracellular milieu. Passive immunization of susceptible and resistant mouse strains with very small doses of these autoantibodies reduced parasitemia and transferred resistance to an otherwise lethal trypanosome infection. Hence, this exquisitely sensitive and unique regulatory immunity against the host (instead of parasite) could benefit infected individuals by blocking cellular invasion of the obligatory intracellular pathogen, resulting in attenuation of tissue infection and clinical manifestations. Such action is contrary to the horror autotoxicus frequently associated with microbe-related autoimmune responses.

Characterization of the presence and distribution of Foxp3(+) cells in chagasic patients with and without megacolon.

Patients with Chagas's disease in the chronic phase regularly present with the chagasic megacolon. This form is characterized by inflammation, neuronal destruction, and organ dilatation. Chagasic patients with megacolon always present with inflammatory process near the enteric plexuses of the colon, as previously demonstrated. The aim of this study is to characterize the presence and distribution of Foxp3(+) cells in the muscle layers and neuronal plexuses area of the colon from chagasic patients with and without megacolon. Our results demonstrated that chagasic patients without megacolon presented with an increased concentration of Foxp3(+) cells in all colon layers compared with chagasic patients with megacolon and noninfected individuals. These cells were situated mainly near the blood vessels and rarely were associated with the inflammatory foci. We believe that the presence of Foxp3(+) cells may help to control the inflammatory process through the management of lymphocyte migration and, consequently, prevent neuronal destruction and chagasic megacolon development.

Diagnosis of Chagas disease: what has been achieved? What remains to be done with regard to diagnosis and follow up studies?

In the acute phase and in the chronic forms of Chagas disease, the etiological diagnosis may be performed by detection of the parasite using direct or indirect parasitological methods and by the presence of antibodies in the serum by way of serological tests. Several techniques are easily available, ranging from the simplest wet smear preparation to immuno-enzymatic assays with recombinant antigens that will meet most diagnostic needs. Other tests under evaluation include a molecular test using polymerase chain reaction, which has shown promising results and may be used as a confirmatory test both in the acute and chronic phases of the disease. Better rapid tests are needed for diagnosis, some of which are already under evaluation. Additionally, there is a need for tools that can identify patients cured shortly after specific treatment. Other needs include a marker for prognosis and early diagnosis of congenital transmission.

Performance levels of four Latin American laboratories for the serodiagnosis of Chagas disease in Mexican sera samples.

In nearly all of the previous multicentre studies evaluating serological tests for Trypanosoma cruzi infection, sera samples from Central or South American countries have been used preferentially. In this work we compared the reliability of the serological tests using Mexican sera samples that were evaluated in four independent laboratories. This included a reference laboratory in Brazil and three participant laboratories, including one in Central America and two in Mexico. The kappa index between Brazilian and Honduran laboratories reached 1.0 and the index for the Mexican laboratories reached 0.94. Another finding of this study was that the source of antigen did not affect the performance of the serological tests.

The CUIDA Chagas Project: towards the elimination of congenital transmission of Chagas disease in Bolivia, Brazil, Colombia, and Paraguay

ABSTRACT Background: Mother-to-child transmission of Chagas disease (CD) has become a relevant problem in both endemic and non-endemic areas. Methods: Description of the CUIDA Chagas Project - Communities United for Innovation, Development and Attention for Chagas disease'. Results: Through innovative and strategic research, this project will provide improved diagnostic and treatment options as well as replicable implementation models that are adaptable to different contexts. Conclusions: By integrating test, treat and care actions for CD into primary health care practices, the burden of CD on people and health systems may be significantly reduced.

Involvement of Ssp-4-related carbohydrate epitopes in mammalian cell invasion by Trypanosoma cruzi amastigotes.

We examined whether the expression of Ssp-4-related carbohydrate epitopes defined by monoclonal antibodies 1D9 and 2B7 was related to cell invasion by Trypanosoma cruzi amastigotes from different isolates and whether the highest expression of the epitope defined by MAb 1D9 would confer greater infectivity. Confocal microscopy showed that both epitopes localize to the membrane of amastigotes from 569, 588, 573, 587 and SC2005 isolates, similar to the G isolate, whereas the CL isolate showed a punctate and diffuse staining. Flow cytometry revealed inter- and intra-isolate variable expression of these epitopes. Apart from the lower expression of MAb 2B7 epitope by intracellular amastigotes of the SC2005 isolate, amastigotes from chagasic patient isolates expressed both epitopes similar to the G isolate, in contrast to CL isolate, that showed lower expression of both epitopes. MAb 1D9 did not react with CL isolate on immunoblots and reacted poorly with 588 and 587 parasites. MAb 2B7 preferentially reacted with an epitope on an 84 kDa component in G and 573 isolates. Invasion assays revealed that despite the fact that amastigotes from chagasic patient isolates displayed high levels of the epitope defined by MAb 1D9, only isolate 588 invaded host cells in levels comparable to that of isolate G. Both MAbs specifically inhibited cell invasion by G and 588, but not CL. These results suggested that the highest expression of MAb 1D9 epitope was not sufficient to confer higher infectivity on the isolate, and besides the two epitopes, other factors may modulate the invasiveness of extracellular amastigotes from the different isolates.

Trypanosoma cruzi surface molecule gp90 downregulates invasion of gastric mucosal epithelium in orally infected mice.

Experiments were performed to elucidate why Trypanosoma cruzi isolates 573 and 587 differ widely in their efficiency to infect gastric mucosal epithelium when administered orally to mice. These isolates have the same surface profile and a similar capacity to enter host cells in vitro. Metacyclic forms of isolates 573 and 587 and the control CL isolate expressed similar levels of gp82, which is a cell invasion-promoting molecule. Expression of gp90, a molecule that downregulates cell invasion, was lower in the CL isolate. Consistent with this profile, approximately threefold fewer parasites of isolates 573 and 587 entered epithelial HeLa cells, as compared to the CL isolate. No difference in the rate of intracellular parasite replication was observed between isolates. When given orally to mice, metacyclic forms of isolate 573, like the CL isolate, produced high parasitemia (>10(6) parasites per ml at the peak), killing approximately 40% of animals, whereas infection with isolate 587 resulted in low parasitemia (<10(5) parasites per ml), with zero mortality. On the fourth day post-inoculation, tissue sections of the mouse stomach stained with hematoxylin and eosin showed a four to sixfold higher number of epithelial cells infected with isolate 573 or CL than with isolate 587. The rate of intracellular parasite development was similar in all isolates. Mimicking in vivo infection, parasites were treated with pepsin at acidic pH and then assayed for their ability to enter HeLa cells or explanted gastric epithelial cells. Pepsin extensively digested gp90 from isolate 573 and significantly increased invasion of both cells, but had minor effect on gp90 or infectivity of isolates 587 and CL. The profile of g82 digestion was similar in isolates 573 and 587, with partial degradation to a approximately 70 kDa fragment, which preserved the target cell binding domain as well as the region involved in gastric mucin adhesion. Gp82 from CL isolate was resistant to pepsin. Assays with parasites recovered from the mouse stomach 2 h after oral infection showed an extensive digestion of gp90 and increased infectivity of isolate 573, but not of isolate 587 or CL. Our data indicate that T. cruzi infection in vitro does not always correlate with in vivo infection because host factors may act on parasites, modulating their infectivity, as is the case of pepsin digestion of isolate 573 gp90.

Chagas disease. The use of serology to establish drug efficacy. Value and limitations

Presented at the "Consultative Meeting on the Strategic and Operational Aspects for the Clinical Development of Trypanocidal Drugs for Chagas Disease, 23-24 April 2007, Buenos Aires, Argentina.", sponsored by TDR, WHO.

Multi-epitope proteins for improved serological detection of Trypanosoma cruzi infection and Chagas Disease.

We previously reported that tandem repeat (TR) proteins from Trypanosoma cruzi could serve as targets of the antibody response and be useful as diagnostic indicators. To optimize reagents for detecting T. cruzi infection we evaluated individual TR proteins and identified several that were recognized by the majority of Chagas patient's sera collected from individuals form Brazil. We then produced novel, recombinant fusion proteins to combine the reactive TR proteins into a single diagnostic product. Direct comparison of the antibody response of serum samples that were readily detected by the established fusion antigen used in commercial detection of Chagas disease, TcF, revealed strong responses to TcF43 and TcF26 proteins. While the TcF43 and TcF26 antigens enhanced detection and strength of signal, they did not compromise the specificity of detection compared to that obtained with TcF. Finally, it was apparent by testing against a panel of 84 serum samples assembled on the basis of moderate or weak reactivity against TcF (mostly signal:noise <5) that TcF43 and TcF26 could more strongly detected by many of the sera that had low TcF antibody levels. Taken together, these data indicate that TcF43 and TcF26 could be used to enhance the detection of T. cruzi infection as well as supporting a diagnosis of Chagas disease.

[Diagnosis and treatment of congenital infection caused by Trypanosoma cruzi in Brazil]. / Diagnóstico y tratamiento de la infección congénita por Trypanosoma cruzi en Brasil.

Diagnosis of congenital infection should be performed preferentially by the search of the parasite, through microhematocrit. Nevertheless, most of the infected mothers are living now in non-endemic areas, where the expertise for identification of the parasite is low. Furthermore, transmission near delivery or even at delivery do not allow that enough parasites will be detected when the baby is at the maternity. So, if parasites are not found or not searched, it is imperative that, in serologically confirmed mothers (two positive tests), the babies are recalled at six/eight months of age, to look for IgG antibodies. If they are present, the baby is infected and should be treated. Treatment in Brazil is with benznidazol, 10 mg/Kg/day, during 60 days. Certification of cure is obtained once a negative serology is obtained, as a rule after one year. A follow up each six months is suggested.

[Congenital transmission of Trypanosoma cruzi in Brazil: estimation of prevalence based on preliminary data of national serological surveys in children under 5 years old and other sources]. / Transmisión congénita del Trypanosoma cruzi en Brasil: estimativa de prevalencia baseada en resultados preliminares de la encuesta nacional serológica en niños menores de cinco años, así como otras fuentes.

A prevalence estimation of congenital transmission in Brazil is performed, based on several sources of recent data. From a serological survey conducted now in Brazil, with children below 5 years old, preliminary data from the state of Minas Gerais only 19/9,556 children did have antibodies against Trypanosoma cruzi. All 19 mothers were infected, but only one child persisted with antibodies on a second blood collection, hence diagnosed as congenital. The other were just passive transference of maternal antibodies. From a recent publication, 278 children born from 145 infected mothers were studied. Two cases (0.7%) were congenital. In other source, from 1,348 blood donors, 35 were born in non endemic areas. When 10 of them were called, 8 were born from infected mothers and five may be congenital. Finally, no infection was detected in 93 children born from 78 infected mothers. The reasons for this low prevalence are discussed, are lower than in other countries of the South Cone, that harbor also T. cruzi 2, but are unrecognized up to now.