Chagas disease: recombinant Trypanosoma cruzi antigens for serological diagnosis.

Diagnosis of individuals infected by Trypanosoma cruzi is performed mainly by serological tests using crude antigens, which might crossreact with other infections. In the past ten years, many recombinant T. cruzi proteins and synthetic peptides have been described, and some are already on the market. Managers of laboratories and blood banks need to make decisions on a cost-benefit basis whether to include these new-generation tests. Here, we indicate antigens that are likely to prove most useful.

A rapid slide flocculation test for the diagnosis of American trypanosomiasis using Trypanosoma cruzi fragments preserved by lyophilization. Comparison with hemagglutination, immunofluorescence, and complement fixation tests.

A slide flocculation test for Chagas' disease is described, which uses a lyophilized, stable antigen obtained by formalin and ultrasonic treatment of culture forms of Trypanosoma cruzi. The test was compared with other tests for the serodiagnosis of American trypanosomiasis and showed a high sensitivity, positive results being obtained in every case of acute or chronic Chagas' disease. In sera from blood donors and from normal individuals with negative T. cruzi antigen complement fixation tests a specificity of 96% was found. False positive flocculation tests were seen, especially in cases of South American blastomycosis and in a few cases of acute toxoplasmosis. Since it is easy and quick to perform, the slide flocculation test can be recommended as a screening procedure, especially for blood banks.

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.

Trypanosoma cruzi: different surface antigens of trypomastigotes are targets of lytic antibodies.

Polyclonal antisera were obtained in rabbits following immunization with disrupted epimastigote or trypomastigote forms; 8-methoxypsoralen-inactivated trypomastigotes; and surface trypomastigote antigens shed into the medium. High antibody levels were induced by all preparations as observed by indirect immunofluorescence and ELISA. However, antibodies promoting complement-mediated lysis of bloodstream forms were only detected in animals immunized with inactivated living trypomastigotes and shed surface antigens. Immunoprecipitation of radioiodinated parasites showed that sera with lytic antibodies bound strongly to a wide range of membrane polypeptides from 72 to 160 kDa. Immunoadsorption of antibodies from a serum with high lytic activity on specific classes of trypomastigote polypeptides indicated that independent antigens are targets of lytic antibodies and that common epitopes may exist in different trypomastigote components.

Trypanosoma cruzi amastigotes: development in vitro and infectivity in vivo of the forms isolated from spleen and liver.

Trypanosoma cruzi amastigotes were isolated from liver and spleen of previously infected mice and purified in discontinuous gradients of Metrizamide and Percoll. The amastigotes were well preserved as judged by electron microscopy. The amastigotes were readily interiorized by macrophages and multiplied actively within these cells in vitro. However, their capacity of differentiation was hampered as estimated by the absence of trypomastigotes until day 6 of cultivation. The purified amastigotes were infective for mice but the onset of parasitemia was somewhat delayed and less intense when compared to mice infected with trypomastigotes.

Trypanosoma cruzi: studies on the reactivity of antibodies bound to the surface of blood forms at the early phase of infection.

The specificity and reactivity of antibodies bound to the surface of Trypanosoma cruzi blood forms at the very early acute phase of murine infection was investigated. Surface-bound antibodies of the IgG and IgM isotypes were recovered from blood forms upon incubation at 37 degrees C. The eluted antibodies immunoprecipitated several trypomastigote surface polypeptides from 80 to 100 kDa. In contrast, for epimastigotes a very faint reactivity was detected only for antigens of 50 and 95 kDa. The shed antibodies promoted in vitro complement-mediated lysis of live blood forms and reacted with fixed trypomastigotes by immunofluorescence. Thus, blood forms are already coated with active trypomastigote-specific antibodies with a potential role in the host defense, although the low levels of serum antibodies have prevented the demonstration of humoral protection at the early stages of infection.

In situ immunoassay for the assessment of Trypanosoma cruzi interiorization and growth in cultured cells.

Interiorization and multiplication of Trypanosoma cruzi within its host cells are usually assessed by counting parasites in fixed and stained cover slip preparations, a subjective and time-consuming method. Here we describe an immunoenzymatic assay (ELISA) for assessing the number of internalized parasites in infected LLC-MK2 seed on chamber slides (NUNC). ELISA was performed employing a rabbit polyclonal serum against trypomastigote components (MOP) and anti-rabbit IgG conjugated to peroxidase. The bottom of the chamber slide was then detached and processed for quantification of internalized parasites by the conventional method. Data analysis showed a linear relationship between optical densities and number of internalized parasites (r2 = 93.99, p < 0.001). The assay was also efficient to assess inhibition of parasite interiorization induced by the monosaccharide NAc-D-glucosamine.

Trypanosoma cruzi genome project: biological characteristics and molecular typing of clone CL Brener.

Clone CL Brener is the reference organism used in the Trypanosoma cruzi Genome Project. CL Brener was obtained by cloning procedures from bloodstream trypomastigotes isolated from mice infected with the CL strain. The doubling time of CL Brener epimastigotes cultured at 28 degrees C in liver infusion-tryptose (LIT) medium is 58 +/- 13 h. Differentiation to metacyclic forms is induced by incubation of epimastigotes in LIT-20% Grace's medium. Metacyclics give very low parasitemia in mice, contrary to what is observed for blood forms which promote 100% mortality of the animals with inocula of 5 x 10(3) parasites. CL Brener blood forms are highly susceptible to nifurtimox, benznidazole and ketoconazole. Allopurinol is inefficient in the treatment of mice experimental infection. The clone infects mammalian cultured cells and performs the complete intracellular cycle at 33 and 37 degrees C. The molecular typing of CL Brener has been done by isoenzymatic profiles; sequencing of a 24S alpha ribosomal RNA gene domain and by schizodeme, randomly amplified polymorphic DNA and DNA fingerprinting analyses. For each typing approach the patterns obtained do not change after prolonged parasite subcultivation in LIT medium (up to 100 generations). The stability of the molecular karyotype of the clone was also confirmed.

Elevated anti-galactosyl antibody titers in endemic goiter.

Anti-Gal is a human polyclonal antibody that constitutes approximately 1% of the circulating immunoglobulin G (IgG), interacts specifically with the mammalian carbohydrate alpha-galactosyl epitope. Furthermore, it was found to mimic in vitro thyrotropin (TSH) effects regarding stimulation for cyclic adenosine monophosphate (cAMP) synthesis, 125I uptake, and cellular proliferation on cultured porcine thyrocytes and on Graves' disease thyrocytes, but not on normal human thyrocytes. As immune activation in sporadic and endemic goiters might play a secondary role in regulating thyrocyte proliferation and function, we evaluated anti-Gal titers in endemic goiter. Serum was obtained from 109 Chagas'-negative patients living in an endemic goiter area of Brazil (Grao Mogol, MG) and 160 controls. The patients were divided into 3 groups, according to their goiter size (World Health Organization [WHO] classification): grade 0 (group 1, n = 24), grade I-II (group 2, n = 41), and grade III-IV (group 3, n = 44). Anti-Gal was assessed by a radioimmunological procedure (results expressed as the percentage of bound radioactivity/total activity [%B/T]). The antibody titer was significantly more elevated in group 1 (mean +/- SEM: 9.27%+/-0.80%), in group 2 (mean +/- SEM: 16.17%+/-0.97%), and in group 3 (20.97%+/-1.30%) than in normal controls (6.46%+/-0.33%). Analysis of the male and female data separately for anti-Gal titer did not substantially alter these results. We concluded that the anti-Gal titer is higher in patients with endemic goiter and presented a possible relationship with the size of goiter. Whether these antibodies contribute to the pathogenesis of the disease needs further clarification.

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.

Alterations of the microcirculatory network during the clearance of epimastigote forms of Trypanosoma cruzi: an intravital microscopic study.

The distribution of epimastigote forms of Trypanosoma cruzi in the microcirculatory network and the vessel alterations were observed using an intravital microscopy technique. Immediately after intravenous inoculation of 2 x 10(6) epimastigote suspension into normal mice, parasites were seen as circulating clumps, and their retention at some sites of the endothelium of venules and capillaries was observed. Injection of 2 x 10(7) and 2 x 10(8) parasite suspensions induced, respectively, intermittent or total stasis of venules and capillaries, probably via obstruction by clumping. The mobility of epimastigotes in the clumps indicates that parasites were alive in the lumen of vessels. The retention of clumps in the capillaries, although intense, could only be observed when labeled parasites were inoculated. These results suggest that the rapid clearance of epimastigote forms of T. cruzi from the blood circulation of mice may be due to the retention of parasites to the endothelium of venules and capillaries that, in turn, may facilitate phagocytosis. This may be a mechanism by which mice are able to eliminate epimastigote forms from the circulation. These findings are consistent with our previous observations showing that epimastigotes are not lysed by complement activation but are phagocytosed and destroyed by a distinct population of blood cells.

Simultaneous identification of Trypanosoma cruzi surface and internal antigens reactive to different immunoglobulin classes (radio-immunoblotting).

A radioactive Western-blotting technique was developed by which the reactivity of Immunoglobulins (Igs) from different classes to both membrane radiolabelled and internal parasite antigens is simultaneously identified. The method includes radioiodination of parasites, polypeptide fractionation by SDS-PAGE, Western-blot transfer and autoradiography of the immunoblots developed with anti-Igs conjugates labelled with enzymes. The analysis is then performed by the comparison of common bands on the autoradiograms and the respective substrate stained nitrocellulose blots. This technique was used to analyse T. cruzi trypomastigote surface labelled antigens reactive to IgM, IgA and IgG specific antibodies. A different pattern of reactivity with acute Chagas' disease patients sera was thus obtained.

Alkaline soluble Trypanosoma cruzi epimastigote antigen (ASEA) applied to Dot-ELISA.

The alkaline soluble Trypanosoma cruzi epimastigote antigen (ASEA) was assessed in dot-ELISA for the diagnosis of Chagas' disease. Serum samples (355) from chagasic and non-chagasic patients were studied, and IgG antibodies to ASEA were found in all patients with chronic Chagas' disease. In non-chagasic patients 95.6% were negative, except for those with leishmaniasis (visceral and mucocutaneous), and some patients from control group reacted in low titers. The data indicate that dot-ELISA using ASEA is suitable for seroepidemiologic surveys to be employed in endemic areas for Chagas' disease.

Chagas'disease: IgA, IgM and IgG antibodies to T. cruzi amastigote, trypomastigote and epimastigote antigens in acute and in different chronic forms of the disease.

In an attempt to find a better T. cruzi antigen and possible immunological markers for the diagnosis of different clinical forms of Chagas'disease, amastigote and trypomastigote antigens obtained from immunosuppressed mice infected with T. cruzi (Y strain) were assessed in comparison with conventional epimastigote antigens. A total of 506 serum samples from patients with acute and with chronic (indeterminate, cardiac and digestive) forms, from nonchagasic infections, and from healthy individuals were assayed in immunofluorescence (IF) tests, to search for IgG, IgM and IgA antibodies. Amastigote proved to be the most convenient antigen for our purposes, providing higher relative efficiency indexes of 0.946, 0.871 and 0.914 for IgG, IgM and IgA IF tests, respectively. Anti-amastigote antibodies presented higher geometric mean titers (GMT) than anti-trypomastigote and anti-epimastigote. Anti-amastigote IgG antibodies were found in all forms of Chagas'disease, and predominantly IgA antibodies, in chronic digestive and in acute forms, as well as IgM antibodies, in latter forms. Thus, tests with amastigote antigen could be helpful for screening chagasic infections in blood banks. Practical and economical aspects in obtaining amastigotes as here described speak in favour of its use in developing countries, since those from other sources require more complex system of substruction, specialized personnel or equipment.

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.

Autochthonous acute Chagas' disease in São Paulo State, Brazil: epidemiological aspects.

Since the beginning of the seventies the natural transmission of Chagas' infection has been considered to be under control in the State of São Paulo and not even a case of American Trypanosomiasis, transmitted by triatomine bugs, has been detected by the epidemiological surveillance system. This situation justifies the report of a case of acute Chagas' disease that occurred in a forest area considered free of domiciliary triatomines along the Southern seacoast of São Paulo State. In May, 1995 the presence of trypomastigote forms of Trypanosoma cruzi had been diagnosed in a retired 57 year-old male patient, born and living in Santos (São Paulo State), complaining of fever, fatigue and malaise. The patient reported that 40 days before he had participated with 17 friends in a 7-day excursion in a forest area of the municipalities of Itanhaém and Peruíbe. During this period the group had been lodged in three houses located within the forest. Eight days after the end of the excursion the patient began to have fever, malaise and fatigue. During the next 31 days he had received medical care both as an inpatient and an outpatient, without any significant improvement. After the detection of T. cruzi trypomastigotes in his blood stream the patient began to be treated with benzonidazole in a hospital but died 8 days after the beginning of treatment. The epidemiological investigation carried out showed no signs of the presence of triatomine bugs in the three houses where the group had been lodged, or any indication of Chagas' infection in other excursionists.

TESA-blot for the diagnosis of Chagas disease in dogs from co-endemic regions for Trypanosoma cruzi, Trypanosoma evansi and Leishmania chagasi.

We standardized serodiagnosis of dogs infected with Trypanosoma cruzi using TESA (trypomastigote excreted-secreted antigen)-blot developed for human Chagas disease. TESA-blot showed 100% sensitivity and specificity. In contrast, ELISA using TESA (TESA-ELISA) or epimastigotes (epi-ELISA) as antigen yielded 100% sensitivity but specificity of 94.1% and 49.4%, respectively. When used in field studies in an endemic region for Chagas disease, visceral leishmaniasis and Trypanosoma evansi (Mato Grosso do Sul state, Central Brazil), positivities were 9.3% for TESA-blot, 10.7% for TESA-ELISA and 32% for epi-ELISA. Dogs from a non-endemic region for these infections (Rondonia state, western Amazonia) where T. cruzi is enzootic showed positivity of 4.5% for TESA-blot and epi-ELISA and 6.8% for TESA-ELISA. Sera from urban dogs from Santos, São Paulo, where these diseases are absent, yielded negative results. TESA-blot was the only method that distinguished dogs infected with T. cruzi from those infected with Leishmania chagasi and/or Trypanosoma evansi.

Changes in isotype composition and antigen recognition of anti-Trypanosoma cruzi antibodies from acute to chronic Chagas disease.

This report describes differences in humoral immune response of acute and chronic phases of human Chagas disease. The reactivities of IgG, IgM, and IgA anti-Trypanosoma cruzi antibodies in serum samples from both groups of patients were compared by enzyme-linked immunosorbent assay (ELISA) employing either one of four antigenic fractions: mouse laminin (LAM), which reacts through Gal alpha 1-3Gal epitopes expressed on trypomastigote surface: whole intact trypomastigotes (TCT); trypomastigotes excreted/secreted antigens (TESA); and epimastigote alkaline extract (EAE). The selection of T. cruzi antigen preparations was based on their relative content of surface and internal antigens found in trypomastigote forms. The proportion of IgG reactive to carbohydrate epitopes was assessed through the decay of IgG reactivity from acute and chronic sera after m-periodate oxidation of solid-phase bound antigens. Trypomastigote and TESA antigens recognized by IgG from acute and chronic sera were also compared by immunoblotting. ELISA and immunoblotting data showed that: (1) the proportion of IgG directed to trypomastigote surface antigens was higher in acute than in chronic sera, whereas the opposite was found for internal antigens, (2) acute sera contained a higher percentage of IgG reactive to trypomastigote carbohydrate epitopes than chronic sera, and (3) anti-T. cruzi IgA was found exclusively in acute sera and led to 100% positivity when LAM, TCT, and TESA were employed as antigens. IgA ELISA with these antigens and IgG immunoblotting pattern with TESA could be useful as serological markers for the acute phase of human Chagas disease.