Immunosensor for the diagnosis of Chagas' disease

Trypanosoma cruzi proteins from epimastigote membranes, herein referred as antigens, have been used for the construction of an amperometric immunosensor for serological diagnosis of Chagas' disease. The proteins used had a molecular mass ranging from 30 to 100 kDa. The gold electrode was treated with cysteamine and glutaraldehyde prior to antigen immobilization. Antibodies present in the serum of patients with Chagas' disease were captured by the immobilized antigens and the affinity interaction was monitored by chronoamperometry at a potential of -400 mV (versus Ag pseudo-reference electrode) using peroxidase-labeled IgG conjugate and hydrogen peroxide, iodide substrate. The incubation time to allow maximum antigen-antibody and antibody-peroxidase-labeled IgG interactions was 20 min with a reactivity threshold at -0.104 microA.     

Trypanosoma cruzi proteins which are antigenic during human infections are located in defined regions of the parasite

Polyclonal antibodies obtained against antigenic proteins encoded by six recombinant DNA clones of Trypanosoma cruzi were used for the ultrastructural localization of the respective antigens in thin sections of parasites (epimastigote, amastigote and trypomastigote forms of T. cruzi) embedded at low temperature in Lowicryl K4M resin. Antigens of high molecular weight containing tandemly repeated amino acid sequence motifs and recognized by sera from patients with Chagas' disease, were located only in the flagellum, where it contacts the parasite cell body. Other antigens were located on the surface of the parasite while still others were found within the flagellar pocket, as is the case with an antigen released during the acute phase of Chagas' disease. Thus, we conclude that some of the T. cruzi proteins which are antigenic in human infections are located in defined regions of the parasite. Some of the antigens were not expressed to the same extent in the three different developmental stages of the parasite.     

Screening for Chagas disease by immunoenzymology: comparison of ELISA with immunofluorescence and indirect hemagglutination in 976 blood donors

The efficiency of an immunoenzymatic technique (ELISA) for the systematic research of Chagas' disease in blood donors was compared with one of 2 well-known methods, indirect haemagglutination (IHA) and indirect fluoro immuno assay (IFA). For the ELISA technique two different antigenic extracts from epimastigote culture forms of T. cruzi, were used for sensitizing the polystyrene plates: a crude extract (Ag R) and a delipidized one (Ag B). Firstly the authors tested these 3 techniques in 5 control groups: sera from Chagas' disease, negative control sera, sera from visceral leishmaniasis, african trypanosomiasis and finally monoclonal gammapathies, the high levels of blood proteins being a possible cause of false positives. Secondly the screening of Chagas' disease was performed in the same way in 976 blood donors from Recife, Brazil. In the case of the Ag-R extract used in the ELISA technique a high cross-reactivity was found with visceral leishmaniasis sera, along a risk of false positives with gammapathic ones. The sensitivity of this technique was found to be high (3,3 +/- 1 p. cent of positive blood donors) and a very good correlation was found with the reference techniques, IFA and IHA, the sensitivity of which is lower (2,3 +/- 1 and 1,7 +/- p. cent). The use of a delipidized antigenic extract (Ag B) for the ELISA technique is not suitable, in spite of an apparent higher specificity: indeed, the positives rate is high (11,5 +/- 0,2 p. cent), but the correlation is very weak or non existent in the case of IHA or IFA. In conclusion, the ELISA technique using a crude extract of T. cruzi appears to be a very convenient method for screening blood donors with Chagas' disease, the lack of specificity due to leishmaniasis or monoclonal blood proteins not posing any real problem to blood banking.     

Evaluation of antibody responses in American visceral leishmaniasis by ELISA and immunoblot

American visceral leishmaniasis (AVL) is an important disease among children of northeast Brazil. In order to characterize antibody responses during AVL, sera of hospitalized patients were analyzed by ELISA and Western blot using a Leishmania chagasi antigen preparation. The ELISA was positive (absorbance greater than or equal to 0.196) at a serum dilution of 1:1024 in all patients at presentation, and fell to ward control levels over the following year. Only one of 72 control subjects tested positive, and that donor had a sibling with AVL. Immunoblots of the patients' sera recognized multiple bands, the most frequent of which were at approximately 116 kDa, 70 kDa, and 26 kDa. Less frequently observed were bands at approximately 93 kDa, 74 kDa, 62 kDa, 46 kDa and 32 kDa. The ELISA responses and patterns of banding were distinctive for AVL, and could be used to differentiate patients with AVL from those with Chagas' disease or cutaneous leishmaniasis. Sera from six AVL patients followed for up to six weeks after treatment identified no new bands. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of surface iodinated parasite proteins showed one major band and four minor bands, whereas SDS-PAGE of biotinylated parasite proteins revealed a banding pattern similar to those of patient sera. AVL appears to produce characteristic immunoblot patterns which can be used along with a sensitive screening ELISA to diagnose AVL.     

Antigenic significance of a Trypanosoma rangeli sialidase

The Trypanosoma rangeli-secreted sialidase was purified by bovine submaxillary gland mucin-sepharose affinity chromatography. In immunoblotting analysis, antibodies raised against this molecule recognized polypeptides of 73 kDa in T. rangeli medium supernatant (TrSialr) and of 70 kDa in the cell lysates of T. rangeli (TrSials) and T. cruzi (TcSialL) epimastigotes. TrSialr, TrSials, and TcSialL were subjected to proteolytic cleavage with papain; the resultant peptide pattern displayed differences in the immunoblotting profiles. TrSials was purified by immunoprecipitation, and this protein band was recognized by sera from T. cruzi-infected chronic mice and Chagas' disease patients. In contrast, TrSialr was not recognized by these sera. The antibodies from the infected mice also recognized a band of 70 kDa present in the medium. These preliminary observations imply that the released and somatic sialidases are partially different molecules, with probably different biological roles. The related proteins recognized in T. rangeli and T. cruzi epimastigotes share many antigenic characteristics but have some structural differences, probably related to their function in the parasitic cell. On the basis of the strong antigenicity of TrSials, this molecule is proposed as the antigen for the detection of antibodies arising during T. cruzi infection.     

Rapid determination of Trypanosoma cruzi urinary antigens in human chronic Chagas disease by agglutination test

Detection of Trypanosoma cruzi in man becomes particularly difficult during the chronic stage of Chagas disease because of the low parasitemia. We were able to develop a simple and straightforward method for determining the concentration of T. cruzi antigens in urine using nitrocellulose micellar suspension (Nitrocell-Mr, Polychaco Argentina) and for their subsequent detection through a "latex" type agglutination test. The latex used was an esferocell nitrocellulose suspension (Esferocell-Mr, Polychaco). Specific antigens for T. cruzi were detected in 54 of 58 urine samples from chronic chagasic patients. The antigens characterized by affinity chromatography and SDS-PAGE were glycoproteins with apparent molecular weights (and pIs) of 100 kDa (pI 5 to 5.5), 80 kDa (pI 6.0), and 50 kDa (pI 6.5 to 7.0). This method is practical and fulfills the requirement of large-scale epidemiological studies. It is also helpful in cases of conflictive serology.     

Trypanosoma cruzi: cloning and expression of an antigen recognized by acute and chronic human chagasic sera.

Here we describe the characterization of a Trypanosoma cruzi DNA sequence (clone A13) that codes for a polypeptide recognized by IgM and IgG antibodies from sera of acute and congenital chagasic patients. Antibodies to A13 antigen are also detected in the sera of chronic patients with different clinical forms of Chagas' disease, but not in sera of patients with leishmaniasis or other parasitic diseases. The antigenic determinants encoded by clone A13 are found in amastigotes and trypomastigotes of several T. cruzi strains, but not in the noninfective epimastigotes. The DNA sequence of the recombinant clone reveals one open reading frame encoding 251 amino acids without tandemly repeated sequences. Our data suggest that the A13 antigen may be useful for the development of serodiagnostic procedures.     

Immunoenzyme assay using micro Dot on nitrocellulose (Dot-ELISA) in the diagnosis of Chagas' disease. I. Comparative study of 2 antigenic preparations of Trypanosoma cruzi

Using the Dot-ELISA technique, two antigenic preparations of Trypanosoma cruzi epimastigote forms have been compared for the diagnosis of Chagas' disease: (1) The cytoplasmic fraction (cytoplasmic antigen) and (2) whole formalin fixed epimastigotes (integral antigen). There was been used sera from 95 chagasic patients with chronic cardiomyopathy, positive conventional serology and either positive or negative xenodiagnosis; 74 subjects with negative conventional serology, and either clinically normal or presenting cardiomyopathy; 74 patients with different diseases including syphilis, toxoplasmosis, leishmaniasis or autoantibodies such as rheumatoid factor and antinuclear antibodies. By defining the diagnostic titers (cut off): 1:512 for cytoplasmic antigen and 1:128 for the integral antigen, a sensitivity of 100% has been obtained with both antigenic preparations, being the specificity of 96% for the former and 100% for the latter when leishmaniasis sera were not included. A comparative study with conventional serology was carried out using 147 sera from a Laboratory of Chagas' diagnosis; Dot-ELISA with cytoplasmic antigen showed co-positivity index of 1.0, co-negativity 0.989 and efficiency of 0.993, and Dot-ELISA with integral antigen 1.0, 0.979 and 0.986 respectively. According to this evaluation, Dot-ELISA using whole formalin fixed epimastigotes might be a practical alternative for the serological diagnosis of Chagas' disease.     

Synthesis and antikinetoplastid activity of a series of N,N'-substituted diamines

A series of 25 N,N'-substituted diamines were prepared by controlled reductive amination of free aliphatic diamines with different substituted benzaldehydes. The library was screened in vitro for antiparasitic activity on the causative agents of human African trypanosomiasis, Chagas' disease and visceral leishmaniasis. The most potent compounds were derived from a subset of diamines that contained a 4-OBn substitution, having a 50% parasite growth inhibition in the submicromolar (against Trypanosoma cruzi) or nanomolar (against Trypanosoma brucei and Leishmania donovani) range. We conclude that members of this series of N,N'-substituted diamines provide new lead structures that have potential to treat trypanosomal and leishmanial infections.     

Immunological consequences of infection and vaccination in South American trypanosomiasis

Trypanosoma cruzi infection provokes a vigorous immune response that terminates the parasitaemia associated with the acute stage within two to three months of initial infection. Even so, a variable proportion of patients may develop severe Chagas' disease, often decades after initial infection. Recent experimental findings suggest that trypomastigotes of T. cruzi possess a surface bound neuraminidase and sugar binding protein by means of which they invade host cells--a mechanism very reminiscent of influenza virus. Studies of the antibody response to trypomastigotes in patients or murine models have identified a series of antibodies able to mediate lysis of live parasites in a complement mediated lysis (c.m.l.) assay. These antibodies have also been linked to resistance to infection in vivo and disappear following successful parasitological 'cure' in drug-treated animals and human patients. Immunochemical studies have shown that sera from infected patients or mice lacking this c.m.l. activity also lack those antibodies able to bind trypomastigote surface components of 85 and 160 kDa relative molecular mass. The availability of rabbit and mouse models of Chagas' disease have produced data that suggest that chronic stage pathology may have an immunological basis dependent on the known cross reactivity between host and parasite cells. Delivery of the lethal hit leading to host cell destruction is probably facilitated by the ability of parasite antigens to bind to host cells thus exposing them to the host's own anti-parasite immune response. If Chagas' disease does indeed have an immunological basis, then this might be controlled in turn by immunoregulation, in a manner analogous to that achieved in experimental allergic encephalomyelitis.     

T cell epitope characterization in tandemly repetitive Trypanosoma cruzi B13 protein

Proteins containing tandemly repetitive sequences are present in several immunodominant protein antigens in pathogenic protozoan parasites. The tandemly repetitive Trypanosoma cruzi B13 protein is recognized by IgG antibodies from 98% of Chagas' disease patients. Little is known about the molecular mechanisms that lead to the immunodominance of the repeated sequences, and there is limited information on T cell epitopes in such repetitive antigens. We finely characterized the T cell recognition of the tandemly repetitive, degenerate B13 protein by T cell lines, clones and PBMC from Chagas' disease cardiomyopathy (CCC), asymptomatic T. cruzi infected (ASY) and non-infected individuals (N). PBMC proliferative responses to recombinant B13 protein were restricted to individuals bearing HLA-DQA1*0501(DQ7), -DR1, and -DR2; B13 peptides bound to the same HLA molecules in binding assays. The HLA-DQ7-restricted minimal T cell epitope [FGQAAAG(D/E)KP] was identified with an overlapping combinatorial peptide library including all B13 sequence variants in T. cruzi Y strain B13 protein; the underlined small residues GQA were the major HLA contact residues. Among natural B13 15-mer variant peptides, molecular modeling showed that several variant positions were solvent (TCR)-exposed, and substitutions at exposed positions abolished recognition. While natural B13 variant peptide S15.9 seems to be the immunodominant epitope for Chagas' disease patients, S15.4 was preferentially recognized by CCC rather than ASY patients, which may be pathogenically relevant. This is the first thorough characterization of T cell epitopes of a tandemly repetitive protozoan antigen and may suggest a role for T cell help in the immunodominance of protozoan repetitive antigens.     

Production and characterization of species-specific monoclonal antibodies against Leishmania donovani for immunodiagnosis

Sixteen species-specific monoclonal antibodies were produced against membranes of Leishmania donovani. These antibodies only reacted with determinants present on L. donovani. No cross-reactions were found with any other species of Leishmania or with membranes of Trypanosoma cruzi. An extensive analysis of the binding specificities of selected antibodies was carried out by using whole promastigote homogenates as antigen. Monoclonal antibodies D-1, D-2, D-3, and D-4 correctly identified all 44 L. donovani stocks from a cross-panel of 84 New and Old World Leishmania stocks. Antibodies D-1 and D-2 were also useful for species classification by immunofluorescence. No cross-reactions were observed with any other Leishmania species examined. Based on either Western blot and/or radioimmunoprecipitation analyses, five distinct groups of molecules associated with L. donovani-specific antigenic determinants were identified. These molecules range in m.w. from 18 to 84 kilodaltons. The antigenic molecules recognized by antibodies D-2, D-10, and D-13 are also recognized by antibodies present in sera from patients with visceral leishmaniasis (kala-azar). Kala-azar sera obtained from cases in both the Old and New World specifically compete with these monoclonal antibodies for the appropriate antigenic determinants in Western blot analysis. These monoclonal antibodies and/or the purified protein antigens may be useful in the development of a serologic assay for the clinical diagnosis of visceral leishmaniasis caused by L. donovani and in epidemiologic studies of leishmaniasis.     

Idiotype stimulation of T lymphocytes from Trypanosoma cruzi-infected patients

Anti-Trypanosoma cruzi epimastigote antibodies (anti-epi) from pooled and individual sera from patients with chronic Chagas' disease were purified on immunoaffinity columns of epimastigotes antigens (epi) coupled to activated Sepharose 4B. SDS-PAGE analysis of purified anti-epi preparations showed only the presence of human IgG H and L chains. These antibodies preparations showed similar Western blotting profiles as the sera pools from which they originated. The main polypeptides recognized by anti-epi had apparent molecular masses 31, 46, 51, 75 and 85 kDa. No difference in these patterns were detected between anti-epi from pooled sera of cardiac (anti-epiC) and indeterminate (anti-epiI) clinical forms. Anti-epi preparations (20 to 60 micrograms/ml) of pooled and individual sera stimulated proliferation of homologous and autologous PBMN or T-lymphocyte-enriched population. The stimulatory ability was dependent upon the PBMN-anti-epi combinations. There is no direct correlation between the level of PBMN response to epi and anti-epi stimuli. Comparison of the stimulatory activities of anti-epiC vs anti-epiI on PBMN of either cardiac or indeterminate group of patients indicate that anti-epiC is significantly more active than anti-epiI (p less than 0.025). These data demonstrate the presence of auto-anti-idiotypic-T cells in chagasic patients and lead to the possibility that idiotype/anti-idiotype interactions may play a role in determining the pathogenesis of chagasic cardiopathy.     

Trypanosoma cruzi ubiquitin as an antigen in the differential diagnosis of Chagas disease and leishmaniasis

In the present report we describe Trypanosoma cruzi ubiquitin as an antigen to be utilized in the differential diagnosis of Chagas disease and leishmaniasis. Initially, recombinant T. cruzi ubiquitin was evaluated against a panel of sera by phage dot immunoassay, showing a good performance against chagasic sera. However, the presence of a carboxy-terminal tail region encoding a ribosomal protein homologous to a related protein present in the genome of Leishmania sp. gave significant cross-reactivity with leishmanial sera. Therefore, ubiquitin was purified by a simple biochemical protocol and its immunoreactivity was studied by enzyme-linked immunosorbent assay. Analysis of 104 sera indicates that the response to ubiquitin is very sensitive towards chronic chagasic sera (98%) and, more important, highly species-specific, presenting better performance compared to the use of the recombinant protein or the total epimastigote extracts when tested against a panel of leishmanial sera, where out of a total of 70 sera tested, only five sera from the mucocutaneous form of the disease reacted with T. cruzi ubiquitin. On the other hand, Leishmania ubiquitin was not recognized by chagasic sera, but was recognized by sera from different forms of leishmaniasis. These results make ubiquitin an excellent candidate to be used in the differential diagnosis of these two parasitic diseases. The molecular basis for this highly species-specific response is discussed.     

Impedimetric evaluation for diagnosis of Chagas' disease: antigen-antibody interactions on metallic electrodes

A polypeptide chain formed by recombinant antigens, cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA) (CF-Chimera) of Trypanosoma cruzi, was adsorbed on gold and platinum electrodes and investigated by electrochemical impedance spectroscopy on phosphate buffer saline solutions (PBS) containing a redox couple. It was found that the adsorption is strongly sensitive to the oxide layer on the electrode surface. In the majority of the experiments the antigens retained their activity as observed through their interaction with sera from chronic chagasic patients. The results expressed in terms of the charge transfer resistance across the interface, indicate the viability of using the impedance methodology for the development of a biosensor for serological diagnosis of Chagas' disease.     

Leishmania, Trypanosoma and monoxenous trypanosomatids as emerging opportunistic agents

Immunosuppression is associated with the occurrence of a large variety of infections, several of them due to opportunistic protozoa. The parasitic protozoa of the family Trypanosomatidae vary greatly in their importance as potential opportunistic pathogens. African trypanosomiasis is no more common nor severe during AIDS. The situation with Chagas' disease, however, is much different. Although the process is not clearly understood, there appears to be a reactivation of Trypanosoma cruzi infection, which can lead to severe meningoencephalitis. In persons with AIDS, leishmaniasis is often exacerbated, particularly Leishmania infantum, which causes visceral leishmaniasis in southern Europe. Since 1990, 1,616 cases of visceral leishmaniasis/HIV co-infection have been reported, mainly from southern Europe, and particularly from Spain, southern France, and Italy. The co-infected patients are primarily young adults and belong to the risk group of intravenous drug users. Isoenzymatic identification of 272 isolates showed 18 different L. infantum zymodemes, of which 10 represent new zymodemes hitherto found only during HIV co-infection. New foci of co-infection are emerging in various parts of the world, including Brazil and East Africa. Moreover, since 1995, non-human monoxenous trypanosomatids have been found in AIDS patients, causing both diffuse cutaneous lesions and visceral infections. In countries where visceral leishmaniasis is endemic, particularly in southern Europe, immunosuppressive treatments for organ transplants or malignant diseases often result either in reactivation of asymptomatic visceral leishmaniasis or in facilitation of new infections.     

Usefulness of the ELISA-IgG test in sera and filter paper blood eluates in the Chagas disease immunodiagnosis]

An ELISA-IgG test using a soluble extract of Trypanosoma cruzi antigen has been developed. One hundred and seventy eight sera (74 healthy people, 44 chagasic patients with positive xenodiagnosis and 60 with clinical symptoms), and 64 paper filter blood eluates (35 healthy people, 10 chagasic patients and 19 with clinical symptoms) were tested by ELISA-IgG and an indirect hemagglutination test. Both types of methods presented an overall average concordance of 98.3% and 100.0% with sera and filter paper blood eluates respectively. A cut off (average of optical density plus 3 standard deviations) of 0.365 and 0.379 for sera and eluates was found. The use of ELISA-IgG in Chagas' disease clinical diagnosis and seroepidemiological studies is discussed.     

The antibody response in Lyme disease.

We determined the antibody response against the Ixodes dammini spirochete in Lyme disease patients by indirect immunofluorescence and an enzyme-linked immunosorbent assay (ELISA). The specific IgM response became maximal three to six weeks after disease onset, and then declined, although titers sometimes remained elevated during later disease. Specific IgM levels correlated directly with total serum IgM. The specific IgG response, often delayed initially, was nearly always present during neuritis and arthritis, and frequently remained elevated after months of remission. Although results obtained by indirect immunofluorescence and the ELISA were similar, the ELISA was more sensitive and specific. Cross-reactive antibodies from patients with other spirochetal infections were blocked by absorption of sera with Borrelia hermsii, but titers of Lyme disease sera were also decreased. To further characterize the specificity of the humoral immune response against the I. dammini spirochete, 35S-methionine-labeled spirochetal antigens were identified by immunoprecipitation with sera from Lyme arthritis patients. These polypeptides had molecular weights of 62, 60, 47, 37, 22, 18, and 15 kDa, and were not recognized by control sera. We conclude that the ELISA, without absorption, is the best method to assay the humoral immune response in Lyme disease, and we have identified methionine-containing spirochetal polypeptides that may be important in Lyme arthritis.     

The genome of Trypanosoma cruzi contains a constitutively expressed, tandemly arranged multicopy gene homologous to a major heat shock protein.

cDNA libraries have been constructed in the plasmid vector pUC18 with mRNA isolated from both epimastigotes and trypomastigotes of the Peru strain of Trypanosoma cruzi. Pools of randomly selected clones were analyzed by hybridization-selection-translation. Translation products were immunoprecipitated either with normal human sera or with sera from patients with Chagas' disease (chagasic sera), and the immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With this approach, a cDNA clone (pEC5) was identified which encodes a portion of an 85,000-Mr polypeptide. A genomic clone was subsequently isolated (FG1) by using oligonucleotide probes derived from the DNA sequence of this cDNA clone. A portion of this clone was isolated and sequenced, and the coding region for the protein was identified. Computer analysis of the predicted protein sequence indicates that this protein is closely related to the 83,000-Mr heat shock protein (hsp83) of Drosophila melanogaster, the hsp90 of Saccharomyces cerevisiae, and the hsp90 of chicken. This gene is tandemly organized in the T. cruzi genome as a cluster of 6 to 10 copies.     

Immunological and structural characterization of an epitope from the Trypanosoma cruzi KMP-11 protein

The K1 peptide is an HLA-A*0201-restricted cytotoxic epitope derived from the Trypanosoma cruzi KMP-11 protein, this being the etiological agent of Chagas' disease. This work describes the K1 peptide's secondary structure and its recognition by sera from chagasic patients. Circular dichroism and NMR spectroscopy analysis revealed that the K1 peptide adopts an alpha-helical conformation. Fifty-six percent of individuals had anti-K1 and 86% anti-KMP-11 antibodies by ELISA in the chronic Chagas' group and 28 and 68% in the indeterminate Chagas' group, respectively. By contrast, no reactivity was observed in sera from healthy individuals and tuberculosis patients. Antibody response subclass specificity to the K1 peptide was IgG1 and IgG3. Taken together these results support the idea that the K1 peptide acts as a B-cell-inducer epitope during Chagas' disease.