Trypomastigote Excreted-secreted Antigens Research Articles

Characterization and Diagnostic Application of Trypanosoma cruzi Trypomastigote Excreted-Secreted Antigens Shed in Extracellular Vesicles Released from Infected Mammalian Cells.

Chagas disease, caused by Trypanosoma cruzi, although endemic in many parts of Central and South America, is emerging as a global health threat through the potential contamination of blood supplies. Consequently, in the absence of a gold standard assay for the diagnosis of Chagas disease, additional antigens or strategies are needed. A proteomic analysis of the trypomastigote excreted-secreted antigens (TESA) associated with exosomal vesicles shed by T. cruzi identified ∼80 parasite proteins, with the majority being trans-sialidases. Mass spectrometry analysis of immunoprecipitation products performed using Chagas immune sera showed a marked enrichment in a subset of TESA proteins. Of particular relevance for diagnostic applications were the retrotransposon hot spot (RHS) proteins, which are absent in Leishmania spp., parasites that often confound diagnosis of Chagas disease. Interestingly, serological screens using recombinant RHS showed a robust immunoreactivity with sera from patients with clinical stages of Chagas ranging from asymptomatic to advance cardiomyopathy and this immunoreactivity was comparable to that of crude TESA. More importantly, no cross-reactivity with RHS was detected with sera from patients with malaria, leishmaniasis, toxoplasmosis, or African sleeping sickness, making this protein an attractive reagent for diagnosis of Chagas disease.

Trypanosoma cruzi Infection in an Indigenous Kariña Community in Eastern Venezuela

We investigated the seroprevalence of Trypanosoma cruzi infection in an indigenous Kariña population in eastern Venezuela. A total of 175 serum samples were collected in the community of Piñantal during February 2009. Interviews targeting socioeconomic and environmental factors associated with the T. cruzi transmission were also conducted. Samples were evaluated using trypomastigote excreted/secreted antigens (TESAs) in an ELISA format. TESA-ELISA positive samples were confirmed by indirect haemagglutination (HAI) (Wiener). A nonsystematic collection of vectors was also undertaken. T. cruzi seroprevalence was 7.43% according to both assays, and the mean age of infected patients was 48.61±10.40 years (range 34 to 73 years). The vector infection rate was 20.00% (2/10). T. cruzi seropositivity was associated with a history of triatomine bites, the ability to recognize the vector and poor knowledge about Chagas disease, but no associations were found with gender, house type, knowledge of how the disease is transmitted, or the presence of vectors or animals inside dwellings. To our knowledge, this is the first study of the seroprevalence of T. cruzi in an indigenous population in eastern Venezuela. All of the epidemiological variables required for the establishment of active vectorial transmission of T. cruzi were present in this community.

Inconclusive results in conventional serological screening for Chagas’ disease in blood banks: evaluation of cellular and humoral response

To find the most reliable screening method for Trypanosoma cruzi infection in blood banks. Epidemiological data, lymphoproliferation assay, parasitological, conventional serological tests: immunofluorescence, haemagglutination, ELISA with epimastigote and trypomastigote antigens and reference serological tests: trypomastigote excreted-secreted antigens (TESA) blot and chemiluminescent ELISA assay with mucine from trypomastigote forms were applied to individuals with inconclusive serology, non-chagasic individuals and chronic chagasic patients. TESA blot had the best performance when used as a single test in all the groups. In the inconclusive group 20.5% of individuals were positive for TESA blot, 23.3% for either lymphoproliferation or TESA blot, and 17.8% for lymphoproliferation only. Positive lymphoproliferation without detectable antibodies was observed in 5.47% of all inconclusive serology cases. Analysis of six parameters (three serological assays, at least one parasitological test, one lymphoproliferation assay and epidemiological data) in the inconclusive group showed that diagnosis of Chagas' disease was probable in 15 patients who were positive by two or more serological tests or for whom three of those six parameters were positive. TESA blot is a good confirmatory test for Chagas' disease in the inconclusive group. Although lymphoproliferation suggests the diagnosis of Chagas' disease in the absence of antibodies when associated with a high epidemiological risk of acquiring Chagas' disease, the data from this study and the characteristics of the lymphoproliferation assay (which is both laborious and time-consuming) do not support its use as a confirmatory test in blood-bank screening. However, our findings underscore the need to develop alternative methods that are not based on antibody detection to improve the diagnosis when serological tests are inconclusive.

Immunoblotting analyses using two-dimensional gel electrophoresis of Trypanosoma cruzi excreted-secreted antigens

Trypanosoma cruzi trypomastigotes excrete-secrete a complex mixture of antigenic molecules. This antigenic mixture denominated trypomastigote excreted-secreted antigens contains a 150-160 kDa band that shows excellent performance in Chagas' disease diagnosis by immunoblotting. The present study partially characterized by two-dimensional gel electrophoresis the immunoreactivity against the 150-160 kDa protein using sera samples from chagasic patients in different phases of the disease. Trypomastigote excreted-secreted antigen preparations were subjected to high-resolution two-dimensional (2D) gel electrophoresis followed by immunoblotting with sera from chagasic and non-chagasic patients. The 150-160 kDa protein presented four isoforms with isoelectric focusing ranging from 6.2 to 6.7. The four isoforms were recognized by IgM from acute phase and IgG from chronic phase sera of chagasic patients. The 150-160 kDa isoform with IF of approximately 6.4 became the immunodominant spot with the progression of the disease. No cross-reactivity was observed with non-chagasic or patients infected with Leishmania sp. In this study we provide basic knowledge that supports the validation of trypomastigote excreted-secreted antigens for serological diagnosis of Chagas' disease.

An improved serodiagnostic test for Chagas' disease employing a mixture of Trypanosoma cruzi recombinant antigens.

Blood transfusion is one of the most important transmission routes of Chagas' disease, a major parasitic infection in Latin America. Therefore, screening for antibodies to Trypanosoma cruzi is mandatory in blood banks in South America. Most of the commercial serologic tests employ epimastigote antigens and show a high number of inconclusive and false-positive results, with high economic and social costs. An ELISA using a mixture of three T. cruzi recombinant antigens, B13, 1F8, and H49 (mix-ELISA), was evaluated, first with a panel of well-characterized sera from 617 patients with Chagas' disease and 277 nonchagasic individuals, living in nine countries of South and Central America. Subsequently, the mix-ELISA was evaluated with 451 samples, from an endemic area of Brazil (Goiás), that were rejected from several blood banks because they presented discrepant results by two commercially available kits (indirect immunofluorescence assay, indirect hemagglutination assay, and/or ELISA). The mix-ELISA exhibited 99.7 percent sensitivity and 98.6 percent specificity in the first evaluation with the 894 samples. In the second evaluation, 451 sera that had discrepant results in the first screening for Chagas' disease were further analyzed with the mix-ELISA. Upon consideration of the consensus results obtained with the trypomastigote excreted-secreted antigens blot test, a confirmatory test for Chagas' disease, the mix-ELISA led to a reduction in 99.6 percent in the number of discordant sera. The combination of three T. cruzi recombinant antigens in a multiantigen immunoassay was highly sensitive and specific for Chagas' disease diagnosis. It is proposed that it can be applicable in blood bank screening in conjunction with the conventional serologic tests.

Trypanosoma cruzi: Isolation of an immunodominant peptide of TESA (trypomastigote excreted-secreted antigens) by gene cloning

Trypomastigote forms of Trypanosoma cruzi excrete-secrete several molecules, which are immunodominant during the human infection. This complex antigenic mixture termed TESA (Trypomastigote Excreted-Secreted Antigens) presents a 150–160 kDa band that shows excellent specificity and sensitivity in Chagas’ disease diagnosis by immunoblotting. Here we describe the isolation and the antigenic characterization of a recombinant peptide ( TESA-1) containing a 10 kDa T. cruzi peptide that belongs to the 150–160 kDa TESA fraction. The clone was isolated by screening a T. cruzi genomic expression library with chagasic antibodies reactive to the 150–160 kDa band of TESA immunoblots. After expression, the recombinant peptide TESA-1 was purified and used to immunize rabbits. Anti- TESA-1 immunesera specifically recognized the 150–160 kDa fraction of TESA-blots from eight different T. cruzi strains. The TESA-1 peptide reacted with 82.2% of chagasic patient sera by immunoblotting, showing that it harbors most of the antigenic epitopes that account for the high reactivity of the 150–160 kDa band of TESA.

Excretory-secretory antigens of Trypanosoma cruzi are potentially useful for serodiagnosis of chronic Chagas' disease.

The reactivities of sera from chronic chagasic patients against the trypomastigote excreted-secreted antigens (TESA) of Trypanosoma cruzi strains with different biodemes were analyzed by TESA-blot and TESA-enzyme-linked immunosorbent assay (ELISA). Although both tests presented high sensitivity and specificity, TESA-ELISA is more appropriate for screening a larger number of samples.

Enzyme-linked immunosorbent assay with Trypanosoma cruzi excreted-secreted antigens (TESA-ELISA) for serodiagnosis of acute and chronic Chagas’ disease

In the present report we describe the use of Trypomastigote Excreted-Secreted Antigens (TESA) as antigen in ELISA for Chagas’ disease serodiagnosis. The study was carried out on 284 patients, 164 of whom were nonchagasic subjects including individuals with leishmaniasis or other pathologies, and 120 chagasic patients, being 53 in the acute (with positive IgA and IgM antibodies to T. cruzi) and 67 in the chronic phase. TESA-ELISA showed 100% positivity in the survey of IgG antibodies in chagasic patients (acute and chronic) and 100% positivity for IgM antibodies in acute phase sera. TESA preparation does not require biochemical purification procedures and does not present the cross-reactivity of leishmaniasis sera observed when ELISA with epimastigote alkaline extract (EAE) is performed. ELISA competition assays showed that anti- T. cruzi antibodies of sera from chagasic patients that react with TESA are different from those that react with EAE. Besides, partial characterization of TESA showed that several epitopes present in this fraction are absent in EAE.

Immunoblot Analysis of Trypomastigote Excreted-Secreted Antigens as a Tool for the Characterization of Trypanosoma cruzi Strains and Isolates

Immunoblot Analysis of Trypomastigote Excreted-Secreted Antigens as a Tool for the Characterization of Trypanosoma cruzi Strains and Isolates

Immunoblot analysis of trypomastigote excreted-secreted antigens as a tool for the characterization of Trypanosoma cruzi strains and isolates.

An analysis of antibody recognition of Trypanosoma cruzi exoantigens by immunoblotting revealed a unique banding pattern that seems to be characteristic of each strain or isolate. Trypomastigote excreted-secreted antigens (TESA) present in supernatants of LLC-MK2 cells infected with 5 strains and 10 isolates of T. cruzi produced 13 different immunoblotting patterns. The same bands were observed when probed with acute-phase Chagas' disease serum or with serum from a rabbit immunized with the repetitive domain of T. cruzi transialidase recombinant protein (anti-shed acute-phase antigens). Three similar patterns were observed with TESA from 3 human isolates that probably belong to the same T. cruzi strain. When clone CL Brener, clone CL-14, and CL parental strain were analyzed, the same bands were observed, although they presented different biological behavior. These results suggest that immunoblotting analysis of TESA may be a useful tool for characterization of T. cruzi strains and isolates.

A survey of congenital Chagas' disease, carried out at three health institutions in São Paulo City, Brazil.

The congenital transmission of Chagas' disease was evaluated in 57 pregnant women with Chagas' disease and their 58 offspring. The patients were selected from three Health Institutions in São Paulo City. The maternal clinical forms of Chagas' disease were: indeterminate (47.4%), cardiac (43.8%) and digestive (8.8%); 55 were born in endemic areas and two in São Paulo City. The transmission of Chagas' disease at fetal level was confirmed in three (5.17%) of the 58 cases studied and one probably case of congenital Chagas' disease. Two infected infants were born to chagasic women with HIV infection and were diagnosed by parasitological assays (microhematocrit, quantitative buffy coat-QBC or artificial xenodiagnosis). In both cases the placenta revealed T. cruzi and HIV p24 antigens detected by immunohistochemistry. In one case, a 14-week old abortus, the diagnosis of congenital T. cruzi infection was confirmed by immunohistochemistry. The other probable infection, a 30-week old stillborn, the parasites were found in the placenta and umbilical cord. The Western blot method using trypomastigote excreted/secreted antigens of T. cruzi (TESA) was positive for IgG antibodies in 54/55 newborns and for IgM in 1/55 newborns. One of the two newborns with circulating parasites had no detectable IgG or IgM antibodies. The assessment of IgG antibodies in the sera of pregnant women and their newborns was performed by ELISA using two different T. cruzi antigens: an alkaline extract of epimastigotes (EAE) and trypomastigote excreted/secreted antigens (TESA). The analysis showed a linear correlation between maternal and newborn IgG antibody titers at birth.

Immunoblot assay using excreted-secreted antigens of Trypanosoma cruzi in serodiagnosis of congenital, acute, and chronic Chagas' disease.

Immunoblotting with trypomastigote excreted-secreted antigens (TESA blot) of Trypanosoma cruzi was evaluated as a method for diagnosis of chronic and acute phases as well as congenital (in newborn children) Chagas' disease. Serum samples from acute-phase and congenital infections were considered to be positive when they reacted with ladder-like bands of 130- to 200-kDa antigens, recognized by immunoglobulin M (IgM) and IgG antibodies, while IgG from chronic-phase sera recognized a broad band antigen of 150 to 160 kDa. Nonchagasic sera were not reactive to these antigens. The study was carried out on 512 patients, 111 of whom were nonchagasic but included cases of leishmaniasis or other pathologies, and 401 chagasic patients. The latter group comprised 361 chronic cases, 36 acute cases, and 4 congenital cases in newborn children. Among the chronic cases, 256 were from areas in which T. cruzi is endemic but which differed widely in the pathogenic expression of T. cruzi infection and in parasitemia levels. These patients at the same time showed a broad range of low, medium, and high reactivity to conventional enzyme-linked immunosorbent assays and indirect immunofluorescence serotests for Chagas' disease. For these reasons they may better represent the universe of chagasic patients than would a sample of highly reactive sera obtained from chagasic patients in a single area endemic for T. cruzi. All acute and congenital cases showed positivity in the IgM and IgG TESA blots, while chronic cases were 100% positive for IgG antibodies. In nonchagasic sera, including 30 cases of visceral and muco-cutaneous leishmaniasis, the specificity index was 1.000, and no cross-reactions were observed. The TESA blot thus seems to be useful as a sensitive and specific diagnostic assay in cases of suspected acute or congenital T. cruzi infection and as a general confirmatory test for conventional Chagas' disease serology.