Multiple Trypanosoma cruzi antigens containing tandemly repeated amino acid sequence motifs.

Chromosomal DNA from Trypanosoma cruzi, the agent of the American trypanosomiasis (Chagas' disease), was used for construction of a DNA library, employing the expression vector lambda gt11. Nine clones encoding different parasite antigens were isolated from this library by screening with an antiserum from a Chagasic patient. Nucleotide sequence analysis showed that seven out of the nine isolated clones code for antigens which contain tandemly repeated amino acid sequence motifs. Each of the seven antigens contains a unique repeat, ranging in length between 5 and 68 amino acids. The length of the repeats is highly conserved within each clone. Fusion proteins, expressed from two of the clones, reacted with a large proportion of sera collected from Chagasic patients in Argentina, Brazil and Chile. These clones appear thus to encode antigens which are shared between different strains of T. cruzi. Immunofluorescence experiments with live parasites showed that three of the antigens were detectable on the surface of trypanosomes.

Cloning and characterization of a gene encoding a novel immunodominant antigen of Trypanosoma cruzi.

A Trypanosoma cruzi genomic expression library was screened with a pool of sera obtained from chronic chagasic patients. The recombinant antigen (Tc40) isolated from this library reacted with a large number of serum samples of chronic chagasic patients, suggesting that the presence of anti-Tc40 antibodies may be specifically associated to Chagas' disease. The full-length sequence of the Tc40 gene was determined after isolation of genomic and cDNA clones. The Tc40 cDNA includes a large open reading frame (2745 bp-long) that encodes a polypeptide of 100 kDa without any homology with previously described T. cruzi sequences. In contrast with other T. cruzi antigens whose immunodominant B-cell epitopes are composed by amino acid repetitive motifs, Tc40 does not show any amino acid repetition. Antibodies against the Tc40 recombinant protein reacted with three native polypeptides of 100, 41 and 38 kDa which are tightly associated with membranes or cytoskeleton and expressed in all developmental stages of the parasite life cycle. A transcript of 3.9-kb was detected in Northern blot analysis which is large enough to encode a 100 kDa polypeptide. Tc40 genes were mapped on a chromosomal band of 1.1 Mbp and in a few copies per haploid genome in the G strain.

Organization and expression of the gene encoding an immunodominant repetitive antigen associated to the cytoskeleton of Trypanosoma cruzi.

We have studied the genomic organization and expression of the gene encoding a high molecular mass (300 kDa) repetitive antigen associated with the cytoskeleton of Trypanosoma cruzi. Protease digestion of the native protein, restriction analysis of genomic DNA and sequencing of genomic and cDNA clones indicated that most of the protein is built up by tandemly arranged, nearly identical repeats of 68 amino acids. The gene size was estimated to be approx. 9.4 kb based on the sizes of the transcript and the native protein. The nucleotide sequence conservation among the repeats indicates that selective sequence homogenization, presumably through gene conversion, maintained the amino-acid sequence conservation. Two duplicated allelic forms of this gene were mapped in fragments of about 20 kb. In some strains an additional allele was located in a fragment of 9.4 kb. Our results suggest that this repetitive antigen is a structural protein which could be involved in the attachment of the flagellum to the cell body.

Serodiagnosis of hepatitis C virus. Effect of new evaluation of cutoff values for enzyme-linked immunosorbent assay in Brazilian patients.

With the goal of reducing false-positive results in enzyme-linked immunosorbent assay (ELISA) serodiagnosis of hepatitis C virus in clinical practice, a study was undertaken to establish better cutoff values. We examined 277 serum samples from patients with hepatitis (non-A, non-B; B; autoimmune); subjects with antinuclear antibodies or rheumatoid factor, anticytomegalovirus or Epstein-Barr virus IgG or IgM antibodies, or parasitic disease (Chagas disease, leishmaniasis); and healthy volunteers. Concordant positive results in 2 different immunoblot assays in 250 samples were taken as indicative of true-positive, and when negative, of absence of infection. Reactivity in 3 ELISA tests were evaluated for the manufacturer recommended cutoff (CO) and for 2CO, 3CO, and 4CO; and corresponding sensitivity and specificity were calculated for single or combined pairs of ELISA tests. Although CO is adequate for blood bank screening, because it provides maximal sensitivity, the frequently observed false-positive results could be significantly reduced by increasing the cutoff value to 2CO, with no significant loss in sensitivity either in relation to pairs of immunoenzymatic tests or to a single ELISA.

Plasmodium gallinaceum-parasitized chicken erythrocytes in a practical hemagglutination test for IgM antibodies in human malaria.

A new hemagglutination test for human malaria, done with Plasmodium gallinaceum-parasitized, aldehyde-fixed, chicken erythrocytes as a stable lyophilized reagent, is described. The test was positive in every human case of falciparum or vivax malaria in which there was parasitemia. It detected only IgM anti-plasmodial antibodies and usually became negative within a few weeks after treatment. As a practical and sensitive test for active malaria, the P. gallinaceum hemagglutination test should be complementary tool for seroepidemiological studies.

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.

Comparison of IgG and IgM contents in serum and filter paper blood eluates.

By the indirect immunoperoxidase radial immunodiffusion technique the IgG and IgM contents in serum and filter paper eluates were found to remain essentially at equivalent levels for at least 2.5 months after collection.

Class specific antibodies and fluorescent staining patterns in acute and chronic forms of schistosomiasis mansoni.

Sera from 40 patients with acute (10) and chronic (30) forms of schistosomiasis mansoni were studied in order to correlate class specific circulating antibodies with fluorescent patterns developed in sections of both worms and liver granulomata. At the acute stage of the infection, IgA antibodies were present, IgM titers were high (about 10 times those found in the chronic stage), and IgG, IgM, IgA, and IgE antibodies were shown by focal fluorescent staining of worms and granulomata. At the chronic stage, IgA antibodies were absent and IgG antibodies showed mostly a diffuse staining pattern in both kinds of sections. The relevance of these observations to diagnosis in clinical cases or epidemiological surveys is discussed.

An unusually high prevalence of ocular toxoplasmosis in southern Brazil.

Because of the frequency of ocular toxoplasmosis and its occurrence in multiple siblings in southern Brazil, a population-based household survey was performed to better understand the epidemiologic characteristics of the disease in this region. Of 1,042 individuals examined, 184 (17.7%) were deemed to have ocular toxoplasmosis on the basis of conservative assessment of ophthalmic findings. Of those with ocular toxoplasmosis, 183 (99.5%) had specific IgG antibodies, compared with only 140 of 181 age-matched control subjects (77.4%; P less than .001). The prevalence of ocular toxoplasmosis was 0.9% in 1- to 8-year-olds, 4.3% in 9- to 12-year-olds, 14.3% in 13- to 16-year-olds, and 21.3% (95% confidence interval, 18.6% to 24.2%) in all individuals 13 years or older. The prevalence of ocular toxoplasmosis in this population was more than 30 times higher than previous estimates for the same condition elsewhere. The low prevalence in the young children we studied supplements previous data suggesting that, in this population, ocular toxoplasmosis is a sequela of postnatal rather than congenital infection.

Sero-epidemiology of Toxoplasma gondii infection in young children in Nairobi, Kenya.

The exposure of 127 pre-school and young schoolchildren to Toxoplasma gondii was investigated by a serological survey of its antibody by means of the passive haemagglutination technique. The significant rise of prevalence of the antibody from 35% in pre-school to 60% in the early school age group suggests that poor sanitary habits and conditions and water shortage in primary schools may cause parasitic infection through contact between children which has not been previously suspected but should be investigated. The strange difference of prevalence of the antibody in the two sexes in the pre-school age children cannot be explained by any social aspects of life. It is therefore suggested the initial exposure of the two sexes to the protozoan is the same but that it acts selectively as a killer disease in the pre-school males either as a primary infection or, more probably, secondary to other lethal paediatric killer diseases. Further studies of the latter aspects in the tropics are needed.

Quantification of crotamine, a small basic myotoxin, in South American rattlesnake (Crotalus durissus terrificus) venom by enzyme-linked immunosorbent assay with parallel-lines analysis.

Intraspecific variation in Crotalus durissus terrificus venom composition was studied in relation to crotamine activity. Crotamine induces paralysis in extension of hind legs of mice and myonecrosis in skeletal muscle cells. To determine whether the venom of crotamine-negative rattlesnake contains a quantity of myotoxin incapable of inducing paralysis, we have developed a very sensitivity immunological assay method, an enzyme-linked immunoabsorbent assay (ELISA), capable of detecting 0.6 ng of purified crotamine. The parallel-lines analysis of ELISA data showed to be useful because it shows the reliability of the experimental conditions. A variation in the amount of myotoxin in the crotamine-positive venom was observed, but not less than 0.1 mg of crotamine per mg of venom. It was not possible to detect it in crotamine-negative venom even at high venom concentrations.

Nucleotide sequence of crotamine isoform precursors from a single South American rattlesnake (Crotalus durissus terrificus).

A cDNA phage library was constructed from venom glands of a single adult specimen of crotamine-plus Crotalus durissus terrificus (South American rattlesnake) captured in a known region. Fifteen crotamine positive clones were isolated using a PCR-based screening protocol and sequenced. These complete cDNAs clones were grouped for maximal alignment into six distinct nucleotide sequences. The crotamine cDNAs, with 340-360 bases, encompass open reading frame of 198 nucleotides with 5' and 3' untranslated regions of variable size, signal peptide sequence, one crotamine isoform message, and putative poly(A+) signal. Of these six different crotamine cDNA precursors, two predict the identical amino acid sequence previously described by Laure (1975), and the other four a crotamine isoform precursor where the Leucine residue at position 19 is replaced by isoleucine by a single base change. On the other hand, nucleotide variation was observed in the 5' and 3' untranslated regions, with one interesting variant containing an 18 base pair deletion at the 5' untranslated region which results in the usual ATG initiator being replaced by the rarely used GUG start codon. Comparison by Northern blot analysis of poly(A+) RNA from venom glands of a crotamine-plus specimen to total and poly(A+) RNA from a crotamine-minus snake indicated that crotamine transcripts were not expressed in the crotamine-minus specimen.

Lectin used in the purification process of Toxoplasma gondii tachyzoites.

Toxoplasma gondii tachyzoites were purified from mouse peritoneal exudates using lectins to remove the host cells. Best results were obtained with phytohemagglutinin P at a concentration of 0.01% (W/V), which provided a 99.7% pure Toxoplasma suspension. The process was reproducible and easy to perform. Toxoplasmas so obtained were infective and served as sources of high quality antigens for immunofluorescence, hemagglutination, and complement fixation tests.

Experimental toxoplasmosis in turkeys.

Fourteen 2-3-wk-old turkeys were inoculated orally with 10(5) or 10(4) infective oocysts of the ME 49 strain of Toxoplasma gondii. Of the 8 turkeys given 10(5) oocysts in experiment 1, 3 died or were killed 12 or 14 days after inoculation (DAI) because of respiratory distress associated with a concomitant Aspergillus-like fungus infection. The remaining 5 turkeys remained normal and were killed 62 DAI. Toxoplasma gondii was isolated in mice from the heart of all 5, from the breast muscles of 2, leg muscles of 3, and from the brains and livers of none of the turkeys. All 6 turkeys fed 10(4) oocysts in experiment 2 remained clinically normal until necropsy on 41 DAI; T. gondii was isolated from pooled tissues from each turkey. All 14 turkeys developed high antibody titers to T. gondii in the modified agglutination test (MAT) using formalinized tachyzoites. The enzyme-linked immunosorbent assay (ELISA) was as sensitive as MAT for detecting T. gondii antibodies in turkey sera. The latex agglutination and indirect hemagglutination tests were less sensitive than the MAT and ELISA. No dye-test-measurable antibodies were found in sera of any turkey.

Serologic and parasitologic responses of domestic chickens after oral inoculation with Toxoplasma gondii oocysts.

Four-week-old chickens were inoculated orally with 1,000 or 100,000 oocysts of the ME-49 or GT-1 strain of Toxoplasma gondii, and their antibody responses were measured, using the direct modified agglutination test, latex agglutination test, indirect hemagglutination test, ELISA, and the Sabin-Feldman dye test. Antibodies against T gondii were detected by use of the modified agglutination test and ELISA within 2 weeks of oocyst inoculation, and antibodies persisted until termination of the study by postinoculation day 68. The latex agglutination test was insensitive in detecting T gondii antibodies, and antibodies were not detected by use of the dye and indirect hemagglutination tests. Of tissues bioassayed in mice for tissue cysts by pepsin digestion of individual organs of chickens on postinoculation day 68, tissue cysts were found in the brain of all 5, heart of 3, and leg muscles of 2, but not in the liver and breast muscles. None of the birds developed clinical toxoplasmosis.

Toxoplasmosis serology: an efficient hemagglutination procedure to detect IgG and IgM antibodies.

In search of an efficient but simple, low cost procedure for the serodiagnosis of Toxoplasmosis, especially suited for routine laboratories facing technical and budget limitations as in less developed countries, the diagnostic capability of Hematoxo, an hemagglutination test for toxoplasmosis, was evaluated in relation to a battery of tests including IgG- and IgM-immunofluorescence tests, hemagglutination and an IgM-capture enzymatic assay. Detecting a little as 5 I.U. of IgG antitoxoplasma antibodies, Hematoxo showed a straight agreement as to reactivity and non-reactivity for the 443 non-reactive and the 387 reactive serum samples, included in this study. In 23 cases presenting a serological pattern of acute toxoplasmosis and showing IgM antibodies, Hematoxo could detect IgM antibodies in 18, indicated by negativation or a significant decrease in titers as a result of treating samples with 2-mercapto-ethanol. However, a neat increase in sensitivity for IgM specific antibodies could be achieved by previously removing IgG from the sample, as demonstrated in a series of acute toxoplasmosis sera. A simple procedure was developed for this purpose, by reconstituting a lyophilized suspension of Protein A--rich Staphylococcus with the lowest serum dilution to be tested. Of low cost and easy to perform, Hematoxo affords not only a practical qualitative procedure for screening reactors and non-reactors, as in prenatal services, but also quantitative assays that permit to titrate antibodies as well as to identify IgM antibodies.

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.

DOT-ELISA for detection of anti-Cysticercus cellulosae antibodies in human cerebrospinal fluid using a new solid-phase (resin-treated polyester fabrics). Preliminary report.

A dot enzyme-linked immunosorbent assay (DOT-ELISA) was developed to detect specific antibodies in cerebrospinal fluid (CSF) for human neurocysticercosis immunodiagnosis, with Cysticercus cellulosae antigen dotted on a new solid-phase. This was represented by sheets of a synthetic polyester fabric impregnated with a polymerized resin (N-methylol-acrylamide). A very stable preparation was thus obtained, the antigen being covalently bound by cross-linking with free N-methylol groups on the resin. Since robust, no special care was necessary for handling the solid-phase. The test could be performed at room-temperature. From 30 CSF samples assayed, 14 were positive, from a group of 15 cases of neurocysticercosis, with titers from 1 to 128; 15 other samples, from normals or other neurological diseases, were all negative. Test characteristics seem to indicate it as adequate for epidemiological surveys. A more detailed study on sensitivity, specificity, reproducibility and the use in serum samples is being conducted.

[Standardization of serological tests for Chagas disease: an immunoenzymatic test for blood donors triage]. / Aspectos da padronização de testes sorológicos para a doença de Chagas: um teste imunoenzimático para a triagem de doadores de sangue.

In the serological diagnosis of Chagas disease large divergences may be found even between laboratories with experience, as a consequence of different criteria for the standardization of the tests. To standardize a immunoenzymatic test developed primarily for screening blood donors, serum panels were carefully chosen so as to best represent chagasic and non-chagasic populations. Produced for the highest sensibility and stability, the new reagent (bioELISA cruzi, Biolab Diagnóstica S/A, Brasil), was tested in serum from 1648 patients 219 with Chagas disease and 104 with other diseases, plus a comparison with well standardized immunofluorescence and hemagglutination tests in 1325 sera. In the immunoenzymatic assays, the cut off was indicated by the absorbance value of a chagasic serum showing a minimal reactivity. ELISA sensibility was 0.9954 and specificity 0.9969, as co-negativity. False positive results were absent with sera from syphilis, toxoplasmosis, mononucleosis and high titered sera for antistreptolysin 0 antibodies. However they were seen in 5 to 15 cases of tegumentar leishmaniasis, 1 of 12 Kala-azar 1 of 15 rheumatoid arthritis and 1 of 12 systemic lupus erythematosus. The high sensibility in chagasics and high specificity in the general population indicate the confiability of the immunoenzymatic assay for screening blood donors and even to confirm a clinical diagnosis of Chagas' disease.

[Avidity of specific IgG antibodies as markers of recent primary infection caused by Toxoplasma gondii]. / Avidez de anticorpos IgG específicos como marcadores de infecção primária recente pelo Toxoplasma Gondii.

For serologically characterizing a recent primary toxoplasma infection, the low avidity of IgG specific antibodies was studied. Avidity was evaluated as the decrease of IgG antibody titers in ELISA after treating plates with 6 M urea, as a dissociating solution of low avidity antigen-antibody complexes. Sixty nine serum samples were studied, presenting characteristic patterns of recent, transitional or chronic toxoplasmosis. Serological patterns were determined according to results of IgG and IgM immunofluorescence, IgM-capture, and hemagglutination tests. Twenty three serum samples from each of the referred patterns I, II and III were titrated. For chronic toxoplasmosis infections, which presented a serological pattern III, observed decrease of titers was 3% +/- 3%. For pattern I recent toxoplasmosis sera it was 34% +/- 12%, and for transition pattern II, 12% +/- 9%. Thus, a low avidity of IgG specific antibodies can be applicable for the diagnosis of a recent toxoplasmosis infection.