Autoantibodies to neurotrophic receptors TrkA, TrkB and TrkC in patients with acute Chagas' disease.

Neurotrophic receptors TrkA and TrkC double up as receptors that Trypanosoma cruzi uses to invade cells and as autoantigen in T. cruzi-infected individuals (with Chagas' disease). Consequently, autoantibodies against TrkA and TrkC (ATA) potently block T. cruzi invasion in vitro and in ATA-immunized mice. Thus, ATA could keep T. cruzi invasion in check in Chagas' disease. However, ATA has been examined only in patients with chronic Chagas' disease. To determine whether ATA potentially participate in the early stage of infection, we analysed the sera of 15 patients with acute Chagas' disease, 4-66 years of age. We find that all sera contain high antibody titres to TrkA, TrkB and TrkC, but not to other growth factor receptors, indicating that ATA are produced relatively soon after T. cruzi infection by an age-independent process. One individual, who acquired the disease after an accidental laboratory infection, converted to Trk-antibody (Ab)-seronegative when progressing to the chronic phase. ATA from acute patients were of low avidity (K(0) <24.8 x 10(-8) m) and of IgM and IgA isotypes. In contrast, ATA from chronic patients were of high avidity (K(o) = 1.4 to 4.5 x 10(-8) m) and of the IgG2 isotype. Therefore, ATA underwent affinity maturation and class switch when patients progressed from acute to chronic disease. Thus, it may be that Trk autoimmunity, which starts in the acute Chagas' disease, plays a role in attenuating parasitemia and tissue parasitism that characterizes the acute/chronic phase transition of Chagas' disease.

Auto-antibodies to receptor tyrosine kinases TrkA, TrkB and TrkC in patients with chronic Chagas' disease.

The Chagas' disease parasite Trypanosoma cruzi promotes survival and differentiation of neurones by binding and activating nerve growth factor (NGF) receptor TrkA. The functional mimic of NGF in T. cruzi is a surface-bound and shed immunogenic protein [neurotrophic factor/trans-sialidase (TS)], which raised the possibility that immune response to T. cruzi in general and to neurotrophic factor/TS in particular leads to loss of immunological tolerance to host NGF and/or the NGF-binding partner TrkA. In testing this hypothesis, we found that sera of individuals with chronic Chagas' disease bear unique IgG2 autoantibodies that bind TrkA and TrkA family members TrkB and TrkC (ATA). Binding of ATA to Trk receptors is specific because the autoantibodies did not cross-react with five other growth factor receptors, NGF and other neurotrophins, and T. cruzi. Thus, individuals with chronic Chagas' disease produce unique antibodies that react with pan-Trk receptors, one of which (TrkA) T. cruzi exploits to inhibit host cell apoptosis and to promote cellular invasion.

Neuronal plasticity of the enteric nervous system is correlated with chagasic megacolon development.

Chagas' disease is one of the few functional gastrointestinal disorders for which a causative agent has been identified. However, some pathological aspects of the chagasic megasyndromes are still incompletely understood. Chagasic megacolon is characterized by an inflammatory process, organ dilatation and neuronal reduction in both plexuses of the enteric nervous system (ENS). Although some studies on the ENS in Chagas' disease have been performed, the process of neuronal destruction and neuronal regeneration still remains unclear. Our hypothesis is that the regeneration process of the ENS may be involved with the mechanisms that prevent or retard organ dilatation and chagasic megacolon development. For that reason, we evaluated the neuronal regeneration with the marker GAP-43 in the colon's neuronal plexuses from chagasic patients with megacolon, and from non-infected individuals. Visual examination and quantitative analysis revealed an increased neuronal regeneration process in the dilated portion from chagasic patients when compared with the non-dilated portion and with non-infected individuals. We believe that this increased regeneration can be interpreted as an accentuated neuronal plasticity that may be a response of the ENS to avoid megacolon propagation to the entire organ and maintain the colon functional innervation.

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.

Identification of a Trypanosoma cruzi antigen that is shed during the acute phase of Chagas' disease.

A Trypanosoma cruzi antigen which is shed into the culture medium by the trypomastigote stage of the parasite and detected in blood of acutely infected mice was cloned and characterized. We designate this antigen shed acute phase antigen (SAPA). Five protein bands with apparent molecular masses ranging from 160 to 200 kDa were detected by immunoblotting of plasma from infected mice and in supernatants of cultured trypomastigotes upon reaction with antibodies against SAPA. A serum obtained from a patient acutely infected with Chagas' disease revealed a similar set of polypeptides in supernatants of cultured trypomastigotes when tested by immunoblotting. SAPA seems thus to be a major shed protein during the acute period of the disease. Twenty-six of 28 sera from human acute cases of Chagas' disease tested reacted with SAPA. Conversely, only 8-10% of sera from chronic cases of the disease contained detectable levels of antibody against SAPA. Sera from rabbits infected with six different parasite strains all contained antibodies against SAPA. Antibodies against SAPA are detectable 15 days after the manifestation of acute Chagas' disease symptoms in humans and 15 days post-infection in sera from mice and rabbits. The nucleotide sequence of a genomic clone encoding the 3' end of the SAPA gene revealed the presence of 14 tandemly arranged 12-amino acid-long repeats. A 39-amino acid-long region that is very hydrophobic precedes the stop codon. Due to its early appearance it might be possible to design diagnostic assays which are based on SAPA for identification of recently infected cases of Chagas' disease.

Seroprevalence of Trypanosoma cruzi infection among unskilled urban workers in central Brazil.

A cross-sectional survey of the prevalence of Trypanosoma cruzi infection was carried out among urban unskilled workers in an endemic area in central Brazil as part of a study to assess the health impact of Chagas disease and to identify risk factors for the evolution of cardiopathy. Blood samples from 5425 male and female workers, aged 15-61 years, from 5 public institutions, were screened by indirect immunofluorescence, enzyme-linked immunosorbent assay and indirect haemagglutination for antibodies to T. cruzi. Seroprevalence varied from 8.8% to 15.6% in the different institutions and increase with age up to 55 years. More stable jobs were associated with lower seroprevalence. Migrants from São Paulo and Minas Gerais presented higher prevalence and a relative risk associated with seropositivity in relation to workers from Goiás of 2.2 (95% confidence limits, 1.4-3.5) and 1.9 (1.6-2.3), respectively.

Further evaluation of lectin affinity purified glycoprotein (GP90) in the enzyme linked immunosorbent assay (ELISA) for diagnosis of Trypanosoma cruzi infection.

Sera from 143 patients considered to be infected with Trypanosoma cruzi on the basis of epidemiological, clinical and standard serological evidence gave positive results in the enzyme-linked immunosorbent assay (ELISA) using a lectin affinity purified 90,000 molecular weight glycoprotein (GP90) antigen preparation. Levels of antibody did not discriminate between clinically classified groups of patients in the chronic phase of infection. The GP90 preparation was found to be heterogeneous.

Specific chemotherapy of Chagas disease: a comparison between the response in patients and experimental animals inoculated with the same strains.

Eleven strains of Trypanosoma cruzi were isolated from patients with Chagas disease in central Brazil by xenodiagnosis and inoculation into newborn mice. Biological characterization and isoenzyme analysis showed that 6 strains were type II (zymodeme 2) and 5 were type III (zymodeme 1). Patients were treated with benznidazole or benznidazole plus nifurtimox. Mice infected with each isolated strain were treated for comparison with the results obtained in the respective patient. Evaluation of cure of the patients was based on the indirect immunofluorescence test, complement fixation reaction and xenodiagnosis. For the mice, haemoculture, indirect immunofluorescence testing, xenodiagnosis and inoculation of blood into newborn mice were used. Tests were performed 3-6 months after the end of treatment. The cure rate was 66-100% in mice infected with type II strains and 0-9% in those infected with type III strains. The correlation between treatment results in patients and mice was 81.8% (9 of 11 cases). Type II strains were more susceptible to treatment, in contrast to type III strains which yielded the majority of therapeutic failures.

Trypanosoma cruzi stocks isolated from acute Chagas disease patients lead to lethal murine infection.

Ten Trypanosoma cruzi stocks recently isolated from patients in acute and chronic phases of Chagas disease were inoculated to susceptible (A/Sn) mice. The mice were inoculated with 10(4) trypomastigotes intraperitoneally and monitored for parasitaemia and mortality for up to 300 d. The results demonstrated that (i) T. cruzi stocks isolated from patients in the acute phase killed animals, while stocks from patients in the chronic phase did not; (ii) survival curves differed statistically among mice infected with lethal stocks, and (iii) parasite burden did not affect the mortality rate of mice.

Trypanosoma cruzi: zymodemes associated with acute and chronic Chagas' disease in central Brazil.

The clinical characteristics of acute and chronic Chagas' disease in central Brazil are described (29 acute cases and 111 chronic cases). The geographical distribution of Trypanosoma cruzi zymodemes in this region was mapped. Zymodeme (Z) 1 was identified in 12 acute cases, Z2 in 13 and repeated xenodiagnosis gave the same zymodeme identification. The clinical pictures of the Z1 and Z2 acute phases were similar. Resistance to benznidazole treatment occurred after either Z1 or Z2 acute infections. Only 14 positive xenodiagnosis were obtained from the 111 chronic phase patients examined. For 12 of these 14 patients the zymodeme was identified. All 12 carried Z2, 10 of whom had mega involvement. There were several possible explanations for the failure to detect T. cruzi Z1 in chronic Chagas' disease with mega syndromes: suggestions were made for follow-up investigations.

[Importance of the method for obtaining feces from triatominae in the assessment of triatomine susceptibility to Trypanosoma cruzi]. / Importância do método de obtenção das dejeções dos triatomíneos na avaliação da suscetibilidade triatomínica para Trypanosoma cruzi.

Two patients on the acute phase of Chagas' disease were submitted to xenodiagnosis examination with different species of triatomine bugs, in order to study bug susceptibility to Trypanosoma cruzi. Species used were Dipetalogaster maximus, Rhodnius neglectus, R. prolixus, R. robustus, Triatoma infestans and T. rubrovaria. Both the number of infected bugs, and the number of excreted trypanosomes by bug, were used as parameters for evaluation of species susceptibility. Xenodiagnosis reading was performed by two methods, the classic abdominal compression and the spontaneous dejection method; this was more efficient than the former in relation to the number of parasites per wet smear (by Wilcoxon test). When susceptibility was evaluated by the number of infected bugs, in one of the patients all D. maximus and R. neglectus became infected (100%), 95% of R. robustus, 90% of R. prolixus and T. rubrovaria, and 85% of T. infestans. When evaluation was performed through the number of excreted parasites in this patient with reading by abdominal compression, the higher susceptibility was with R. neglectus, followed by R. prolixus, D. maximus and R. robustus, T. infestans and T. rubrovaria; reading by spontaneous dejection yielded better results for D. maximus and R. prolixus followed by T. rubrovaria, R. robustus, T. infestans and R. neglectus. For the other patient susceptibility evaluated by the number of excreted parasites was similar by both reading methods and did show the same susceptibility pattern (R. neglectus and then T. rubrovaria and T. infestans), but the number of excreted trypanosomes was much higher by the spontaneous dejection method.

Trypanosoma cruzi and experimental Chagas' disease: characterization of a stock isolated from a patient with associated digestive and cardiac form.

A new Trypanosoma cruzi stock isolated from a patient in the chronic phase of Chagas' disease with the digestive and cardiac form of the disease was characterized by experimental infection in isogenic, susceptible, A/Sn strain mice. Parasitemia curves showed up to 1.7 x 10(6) parasites/ml and no mortality was observed up to 300 days post infection. Specific IgM was found in mice in the acute phase up to 40 days and also in the chronic phase. IgG antibodies were detected in the acute and chronic phase. Histopathology examination demonstrated myotropism to the digestive tract muscle layers and to the heart.

Neurochemical coding of the enteric nervous system in chagasic patients with megacolon.

Neuronal destruction has been considered the hallmark of pathogenic mechanisms in chagasic megacolon. Characterization of neuropeptides in the enteric nervous system from chagasic patients with megacolon could elucidate some aspects of the development of this syndrome. In the present work we demonstrate the changes in expression of neuropeptides and neurochemical markers present in neuronal plexuses from the colons of chagasic patients with megacolon. Sections of frozen tissue samples were immunohistochemically labeled for anticalretinin, cChaT, substance P, VIP, NOS, and NPY. Immunoreactivity was observed using a confocal microscope. Our results demonstrate that in chagasic patients with megacolon, inhibitory motor neurons (VIP and NOS immunoreactive) are preferentially destroyed by Trypanosoma cruzi and/or the inflammatory process. These results suggest a selective destruction of enteric neurons in the colon of chagasic patients with megacolon, pointing to an important discovery in the mechanism of pathogenesis of Chagas' disease.

Mass screening for Trypanosoma cruzi infections using the immunofluorescence, ELISA and haemagglutination tests on serum samples and on blood eluates from filter-paper.

Methods used to diagnose Trypanosoma cruzi infection differ in their ability to discriminate between sera from infected and uninfected individuals. We compared the results of an immunofluorescence (IF) test, a haemagglutination (HA) test, and an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of T. cruzi infections in a large population-based survey in central Brazil using blood eluates from filter-paper and venous blood samples. The sensitivities of the tests on eluates, compared with results on serum samples, were low: ELISA (78.1%), IF (69.2%) and HA (64.6%). The level of agreement between the tests on eluates was very poor, with the best co-positivity for IF and ELISA. Both the positive and negative predictive values of the three tests on eluates were similar (around 96%) to those for sera. Higher co-positivity values were obtained for the three tests on sera. The implications of these results are discussed in relation to blood screening, routine medical practice, sero-epidemiological surveys, and the follow-up of patients admitted to therapeutic trials.

Shift of excretory-secretory immunogens of Trypanosoma cruzi during human Chagas' disease.

We studied secreted-excreted immunogens in human patients infected with Trypanosoma cruzi. A pair of 45- to 55-kDa antigens and a family of shed acute-phase antigens characterized the acute phase, while antibodies against a 160- to 170-kDa immunogen appeared at the chronic phase of the disease.

Blood culture and polymerase chain reaction for the diagnosis of the chronic phase of human infection with Trypanosoma cruzi.

In the present study, we evaluated for the first time the profile of blood parasitism in untreated, chronic Chagas' disease. The study was conducted on 60 patients and a control group of nine serologically negative individuals. Analysis of three blood samples showed 70% cumulative positivity for blood culture and 86.7% positivity for PCR. The comparison of the two tests revealed that 41.1% (74/180) of the samples presented positive results for both PCR and blood culture, 22.2% (40/180) were positive for PCR alone, and 4.4% (8/180) were positive for blood culture and negative for PCR. The addition of the second sample raised positivity significantly for both blood culture ( P=0.0000) and PCR ( P=0.0369). Addition of the third sample was also statistically significant for blood culture ( P=0.0001) but not for PCR ( P=0.1186). These data point to the importance of studying the parasitemia of Trypanosoma cruzi-infected individuals before specific treatment. They also suggest that at least two blood samples should be collected and that two tests should be used, if possible--a procedure that considerably improves the parasitologic diagnosis of Chagas' disease and the evaluation of therapeutic efficacy.

Prevalence of antibodies to 72-kilodalton glycoprotein (GP72) in patients with Chagas' disease and further evidence of zymodeme-associated expression of GP72 carbohydrate epitopes.

Three competitive inhibition enzyme-linked immunosorbent assays were developed to examine the expression of the 72-kilodalton glycoprotein (GP72) and of a GP72 carbohydrate epitope in Trypanosoma cruzi strains and clones. A total of 148 strains and clones of known isozyme phenotype (principal zymodeme, Z) were tested. With monoclonal antibody 8G2B9 the enzyme-linked immunosorbent assay confirmed that the majority of Z1 strains and clones derived from them had undetectable levels of the carbohydrate epitope identified by antibody 8G2B9. This epitope was, however, readily detectable in all Z2, Z2(h), and Z3 strains and clones (P less than 0.001; 148 strains and clones tested). Zymodeme-associated differences in GP72 expression were not apparent from the enzyme-linked immunosorbent assay with monoclonal antibody WIC 226.4 (raised against periodate-treated GP72) or from that with rabbit anti-GP72 antiserum (84 or 119 strains and clones tested, respectively). Mice infected with culture-form metacyclic trypomastigotes of Z1, Z29, and Z3 or with blood-form trypomastigotes of Z1 and Z3 developed antibodies to affinity-purified GP72, showing that at least some GP72 epitopes are neither zymodeme specific nor stage specific. A total of 128 serum samples from patients with acute or clinically classified chronic Chagas' disease were assayed for immunoglobulin G (IgG) or IgM anti-GP72 antibodies. During the acute phase anti-GP72 IgM antibodies were elevated, whereas anti-GP72 IgG antibodies were low. There were no significant differences in anti-GP72 antibody levels among chronic-phase patient groups. Anti-GP72 antibodies were detected irrespective of the geographical origin of patients and irrespective of whether acute-phase blood parasitemias were due to Z1 (four patients) or Z2 (two patients).

Serological diagnosis of Chagas' disease: a potential confirmatory assay using preserved protein antigens of Trypanosoma cruzi.

The diagnosis of Chagas' disease relies mostly on data provided by immunologic tests, but inconclusive results often require elucidation, especially in blood banks. When six different types of Trypanosoma cruzi epimastigote antigens were studied by an immunoblotting assay (IBA), a preserved protein antigen (Ag PP) was found to present the most interesting immunochemical features because of its high reactivity with anti-T. cruzi antibodies. Thus, the IBA with Ag PP (PP IBA) was assessed with panels of coded and noncoded serum samples prepared in different laboratories, including the Brazilian Reference Laboratory for Chagas' Disease. It was found that serum samples from patients proved (clinically, eletrocardiographically, serologically, and epidemiologically) to have Chagas' disease consistently recognized 12 bands (140, 100, 85, 78, 59, 57, 46, 35, 27, 23, 20, and 18 kDa) of Ag PP. In contrast, sera from nonchagasic patients, including patients with mucocutaneous leishmaniasis, were negative or reacted weakly, and one serum sample did not have more than five different bands. These bands were 78, 57, 46, 35, 27, 23, 20, or 18 kDa. A criterion was adopted to interpret the results obtained in the PP IBA. The criterion considered positive a serum sample recognizing all 12 bands and considered negative a serum sample that did not recognize any of the bands except the eight nonspecific bands mentioned above. The PP IBA indicated maximum sensitivity and specificity as well as high positive and negative predictive values. The data demonstrate that the PP IBA discriminates chagasic from nonchagasic infections and seems to be applicable as a confirmatory assay for elucidating inconclusive results obtained by standard serology.