Anna Maria S. Stolf

Predominance of lineage I among Trypanosoma cruzi isolates from Venezuelan patients with different clinical profiles of acute Chagas’ disease

Trypanosoma cruzi isolates from 23 acute chagasic patients from localities of Western Venezuela (state of Barinas) where Chagas’ disease is endemic were typed using ribosomal and mini‐exon gene markers. Results showed that isolates of the two major phylogenetic lineages, T. cruzi I and T. cruzi II, were isolated from these patients. Six isolates (26%) were typed as T. cruzi II and 17 (74%) as belonging to T. cruzi lineage I. Analysis of random amplified polymorphic DNA (RAPD) patterns confirmed these two groups of isolates, but did not disclose significant genetic intra‐lineage polymorphism. Patients infected by both T. cruzi I or T. cruzi II showed different clinical profiles presenting highly variable signs and symptoms of acute phase of Chagas’ disease ranging from totally asymptomatic to severe heart failure. The predominance of T. cruzi I human isolates in Venezuela allied to the higher prevalence of severe symptoms of Chagas’ disease (heart failure) in patients infected by this lineage do not corroborate an innocuousness of T. cruzi I infection to humans. To our knowledge, this is the first study describing predominance of T. cruzi lineage I in a large number of acute chagasic patients with distinct and well‐characterized clinical profiles.

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 Galα1–3Gal 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.

Trypanosoma cruzi: Identification of proteinases in shed components of trypomastigote forms

Trypanosoma cruzi trypomastigotes were shown to predominantly release high molecular weight components (above 50 kDa) when allowed to shed for 1 hour in protein-free media. Under these conditions, parasites were not damaged or lysed, as was indicated by: (a) their normal mobility; (b) their retaining of some of the labelled proteins; (c) the unchanged pattern of biotinylated surface proteins after shedding. Shed components were shown to display proteinase activities, detected at 97 and 50/60 kDa in gelatin gels. These proteolytic activities were completely inhibited by E-64, indicating that they were due to cysteine proteinases.

Short communication: Trypanosoma cruzi lineage I in endomyocardial biopsy from a north-eastern Brazilian patient at end-stage chronic Chagasic cardiomyopathy.

Trypanosoma cruzi, the agent of Chagas disease, is genetically classified into two major evolutionary lineages, T. cruzi I and T. cruzi II. In Southern American Cone countries it is T cruzi II which causes most cases of severe chronic Chagas disease. Contrary to this, we isolated T. cruzi I nested in endomyocardial biopsies of a chronic chagasic patient with end‐stage heart failure. Our finding should alert clinicians to the possibility of severe Chagas disease in all regions where T. cruzi circulates, regardless of its lineage.

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.

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.

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: 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.

Reactivity of Trypanosoma cruzi strains with peanut agglutinin (PNA) correlates with number of in vitro infected host cells.

Reactivities of 4 lectins with intact trypomastigote forms derived from 8 different Trypanosoma cruzi strains were compared with their capacity to infect in vitro cultured LLC-MK(2)cells. A sensitive and reproducible titration method for lectin binding sites (ELLA: Enzyme Linked Lectin Assay) was employed, in which reactivities were scored through optical densities in an ELISA reader. Tissue culture trypomastigotes from the strains Y, CL, SC4, SC24, SC25, SC28, SC32 and SC33 were investigated for expression of different cell surface carbohydrate residues using Concanavalin A (ConA), Peanut agglutinin (PNA), Soybean agglutinin (SBA) and Wheat germ agglutinin (WGA) conjugated to peroxidase. The reactivity of the strains to PNA lectin was SC28 > SC32 > SC33 > SC25> SC24 > Y> CL> SC4. The optical density values obtained were highly correlated (r2=0.986, p< 10(-4)) with the number of parasitized LLC-MK(2) cells 24 hours after infection by trypomastigotes from each corresponding strain. We concluded that galactose and N-acetyl-D-galactosamine residues that are present on the surface of trypomastigotes are important in host-cell recognition.