Immunofluorescent antibody to rotavirus and antigen excretion in infantile acute diarrhoea

A serological study was carried out to evaluate the causal relationship of acute diarrhoea and rotavirus in children under five years of age. The rotaviral infection was demonstrated in paired sera by determining the antibody titers by immunofluorescence test (IF). Bovine rotavirus-infected MA-104 cell culture was used as substratum for IF. Out of 80 paired sera it was shown that 23 (28.75%) presented seroconversion, 19 (23.75%) samples showed a twofold increase in their titers and 38 (47.5%) had no increase in rotavirus antibody. This result is discussed on the light of previously obtained results on viral antigen detection by counterimmunoelectrosmophoresis (CIEOP).

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.

Evaluation of enzyme-linked immunosorbent and alternative assays for detection of HIV antibodies using panels of Brasilian sera

Sera from 472 Brazilian subjects, confirmed to be either positive or negative for HIV antibodies and comprising the total clinical spectrum of HIV infection, were utilized in the evaluation of six commercially available enzyme-linked immunosorbent assays (ELISA), as well as of four alternative assays, namely indirect immunofluorescence (IIF), passive hemagglutination (PHA), dot blot and Karpas AIDS cell test. The sensitivities ranged from 100% (Abbott and Roche ELISA) to 84.2% (PHA) and the specificities ranged from 99.3% (IIF) to 80.2% (PHA). The sensitivity and specificity of the PHA and the sensitivity of the Karpas cell test were significantly lower than those of the other tests. Although the IFF and dot blot had good sensitivities and specificities, the six ELISA were more attractive than those tests when other parameters such as ease of reading and duration of assay were considered.

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.

Epidemiological survey of Trypanosoma cruzi infection in North-Eastern Brazil using different diagnostic methods

A survey of the prevalence of Trypanosoma cruzi infection was carried out in Oitis, a small community in the State of Piaui, Brazil. Two hundred and sixty five individuals were screened by microscopic examination, hémoculture, indirect immunofluorescence (IFA), enzyme-linked immunosorbent assay (ELISA), and competitive enzyme-linked immunosorbent assay (C-ELISA) using the monoclonal antibody TCF87 against to a 25kd T. cruzi antigen. Seropositivity was 14.3% by the IFA test, 14.7% by ELISA, and 13.2% by C-ELISA. The C-ELISA using the TCF87 monoclonal antibody seems to be applicable in serodiagnosis of Chagas' disease.

The sensitivity, specificity and efficiency values of some serological tests used in the diagnosis of paracoccidioidomycosis

This work reports on the results of double immunodiffusion (ID), counterimmunoelectrophoresis (CIE), complement fixation (CF) and indirect immunofluorescence (IIF) techniques in the serodiagnosis of paracoccidioidomycosis. The study was undertaken on four groups of individuals: 46 patients with untreated paracoccidioidomycosis, 22 patients with other deep mycoses, 30 with other infectious diseases (tuberculosis and cutaneous leishmaniasis) and 47 blood donors as negative controls. Data were obtained using Paracoccidioides brasiliensis antigens, i.e.,a yeast culture filtrate for ID, CIE and CF, and a yeast cell suspension for IIF. The sensitivity, specificity and efficiency values were measured according to GALEN & GAMBINO8.The gel precipitation tests (ID and CIE) showed the greatest sensitivity (91.3 and 95.6%, respectively), maximum specificity (100%) and the highest efficiency values when compared to the CF and IIF tests.

Comparative study of adenoviruses with monoclonal antibodies

The obtainment of monoclonal antibodies for adenovirus species 4(Ad4) is described.The specificities of selected monoclonal antibodies were determined by means of viral neutralization test in cell culture, immunofluorescence and Enzyme-Linked Immunosorbent Assay (ELISA), in the presence of the following species of human adenovirus: 1, 2, 5 (subgenus C), 4 (subgenus E), 7 and 16 (subgenus B) and 9 (subgenus D). Two monoclonal antibodies species specific to adenovirus 4 (1CIII and 3DIII) and one monoclonal antibody that cross reacted with adenovirus species 4 and 7 (2HIII) were obtained.

Nephrotic syndrome associated with hepatointestinal schistosomiasis

Schistosomal nephropathy has long been related to the hepatosplenic form of schistosomiasis. In the last few years, 24 patients with hepatointestinal schistosomiasis and the nephrotic syndrome were studied. Aiming at evaluating a possible etiologic participation of schistosomiasis in the development of the nephropathy, this group was comparatively studied with a group of 37 patients with idiopathic nephrotic syndrome. Both groups had a different distribution of the histologic lesions. In the group with schistosomiasis there was a statistically significant prevalence of proliferative mesangial glomerulonephritis (33.3%), whereas in the control group there was prevalence of membranous glomerulonephritis (32.4%). On immunofluorescence, IgM was positive in 94.4% of the patients with schistosomiasis versus 55.0% in the control group (p<0.01). In the group with schistosomiasis, 8 patients evidenced mesangial proliferative glomerulonephritis and 5, membra-noproliferative glomerulonephritis. In both histological types immunofluorescence showed IgM and C3 granular deposits in the glomeruli. The data in this study suggests that mesangial proliferative and membranoproliferative glomerulonephritis, with glomerular granular IgM and C3 deposits, represent the renal lesions of the schistosomiasis associated nephropathy.

Evaluation of different methods for Plasmodia detection, in well defined population groups in an endemic area of Brazil

In Brazil, more than 500,000 new cases of malaria were notified in 1992. Plasmodium falciparum and P.vivax are the responsible species for 99.3% of the cases. For adequate treatment, precoce diagnosis is necessary. In this work, we present the results of the traditional Plasmodia detection method, thick blood film (TBF), and the results of alternative methods: Immunofluorescence assay (IFA) with polyclonal antibody and Quantitative Buffy Coat method (QBC)® in a well defined population groups. The analysis were done in relation to the presence or absence of malaria clinical symptoms. Also different classes of immunoglobulins anti-P.falciparum were quantified for the global analysis of the results, mainly in the discrepant results. We concluded that alternative methods are more sensitive than TBF and that the association of epidemiological, clinical and laboratory findings is necessary to define the presence of malaria.

Schistosoma mansoni: identification of a 46KDa antigen of the schistosomular surface by monoclonal antibody

An IgG2a subclass monoclonal antibody, C6G9, was obtained by immunization of BALB/c mice with Schistosoma mansoni egg antigens. With this monoclonal antibody, it was possible to identify a schistosomular antigen with a molecular weight of 46 kilodaltons (KDa), and its expression being evaluated by means of indirect immunofluorescence. The antigen persisted in the integument of the developing schistosomulum, for at least 96 hours post-transformation. The monoclonal antibody also reacted with the cercaria surface, but not with that of adult worm. The C6G9 was also able to mediate significant levels of cytotoxicity in the presence of complement for newly transformed schistosomula.

Legionnaires' disease in the renal transplant unit of "Hospital das Clinicas, FMUSP": during a five year period (1988-1993)

Several reports have related Legionella pneumophila with pneumonia in renal transplant patients, however this association has not been systematically documented in Brazil. Therefore this paper reports the incidence, by serologycal assays, of Legionella pneumophila serogroup 1 in these patients during a five year period. For this purpose sera from blood samples of 70 hospitalized patients with pneumonia from the Renal Transplant Unit of Hospital das Clinicas, FMUSP collected at the acute and convalescent phase of infection were submitted to indirect immunofluorescence assay (IFA) to demonstrate anti-Legionella pneumophila serogroup 1 antibodies. Of these 70 patients studied during the period of 1988 to 1993,18 (25.71 %) had significant rises in specific antibody titers for Legionella pneumophila serogroup 1. Incidence was interrupted following Hospital water decontamination procedures, with recurrence of infections after treatment interruption. In this study, the high susceptibility (25.71%) of immunodepressed renal transplant patients to Legionella pneumophila serogroup 1 nosocomial infections is documented. The importance of the implementation and maintenance of water decontamination measures for prophylaxis of the infection is also clearly evident.

Cell and humoral immunity in endemic pemphigus foliaceus

A study was conducted on 16 patients with pemphigus foliaceus, ten of them with the localized form (group G1) and six with the disseminated form (group G2). These patients were submitted to full blood counts, quantitation of mononuclear cell subpopulations by monoclonal antibodies, study of blastic lymphocyte transformation, and quantitation of circulating antibodies by the indirect immunofluorescence test, in order to correlate their clinical signs and symptoms and laboratory data with their immunological profile, and to determine the relationship between circulating autoantibody titers and lesion intensity and course of lesions under treatment. Leucocytosis was observed especially in group G2. All patients showed decreased relative CD3+ and CD4+ values and a tendency to decreased relative values of the CD8+ subpopulation. Blastic lymphocyte transformation indices in the presence of phytohemagglutinin were higher in patients (group G1+G2) than in controls. The indirect immunofluorescence test was positive in 100% of G2 patients and in 80% of G1 patients. The median value for the titers was higher in group G2 than in group G1. Analysis of the results as a whole permits us to conclude that cell immunity was preserved and that there was a relationship between antibody titers detected by the direct immunofluorescence test and extent of skin lesions.

Chagas' disease in the Brazilian Amazon: II. A serological survey

A serological survey, involving indirect immunofluorescence testing of blood sera samples, was carried out on the residents of one in every five dwellings in the town of Barcelos (in the northern part of the State of Amazonas, on the right bank of the Rio Negro, 490 Km from Manaus by river) and on the rural populations of the villages of Piloto and Marará (also on the right bank of the Rio Negro, 30 minutes by boat from Barcelos). A total of 710 sera samples were tested, 628 from the resident population in the town of Barcelos, 35 from Piloto and 47 from Marará. The tests were carried out using human anti-gammaglobulin type IgG (Biolab) and antigen from formolized culture of T.cruzi Y strain. The sera were serially diluted from 1:40 to 1:320 in PBS 7.2. Of the 710 samples examined 89(12.5%) were positive for anti-T.cruzi antibodies: 2 of these (2.2%) at a dilution of 1:320; 12(13.4%) at 1:160; 38 (42.6%) at 1:80; and the remainder at 1:40, giving a median serological dilution of 1:80. The following questions are discussed: the high serological prevalence for Chagas'infection found in our survey; the possibility of serological cross-reactions; the need for confirmatory tests for the positives reactions; and the strong correlation between our results and preliminary epidemiological data (such as the level of human contact with wild triatominae, know locally as "Piacava's lice". We draw attention to the isolation by xenodiagnosis of one strain of T.cruzi from a patient with positive serology for Chagas' infection.

Dot-ELISA for the detection of IgM and IgG antibodies to Schistosoma mansoni worm and egg antigens, associated with egg excretion by patients

Human schistosomiasis, caused by Schistosoma mansoni, is highly prevalent in Brazil and usually diagnosed by time consuming stool analysis. Serological tests are of limited use in this disease, mainly for epidemiological studies, showing no discrimination between previous contact with the parasite and active infections. In the present study, we standardized and compared a Dot-ELISA for IgM and IgG antibodies against S. mansoni antigens from eggs and worms with a routine IgG and IgM immunofluorescence assay using similar antigens, in the study of sera from 27 patients who had quantified egg stool excretion. The positivity obtained for IgG Dot-ELISA was 96.3% and 88.9% for IgM Dot-ELISA with worm antigen and 92.6% and 90.9% with egg antigen. The IFI presented similar positivities using worm antigen, 92.6% (IgG) and 96.3% (IgM),and lower results with egg antigen, 77.8% (IgG and IgM). The patients studied were divided into two groups according to their egg excretion, with greater positivity of serological tests in higher egg excreters. When comparing the quantitative egg excretion and the serological titers of the patients, we detected a correlation only with IgM Dot-ELISA, with r=0.552 (p=0.0127). These data show that Dot-ELISA can be used for the detection of specific antibodies against S. mansoni in sera from suspected patients or in epidemiological studies and, with further purification of egg antigen and larger samples, IgM Dot-ELISA could be a possible tool for rough estimates of parasite burden in epidemiological studies.

Diagnosis of human toxoplasmosis by a dot enzyme-linked immunosorbent assay

A Dot enzyme-linked immunosorbent assay (Dot-ELISA) was standardized and evaluated for the serodiagnosis of human toxoplasmosis. Out of 538 serum samples tested by the immunofluorescence test for toxoplasmosis (IFAT-IgG) as reference test, 183 (34%) were positive at cut off 1:16 and 192 (36%) were positive for Dot-ELISA-IgG at cut-off 1:256. For Dot-ELISA, co-positivity was 0.94, co-negativity 0.94 and concordance 0.88 in relation to IFAT-IgG. These results suggest the usefulness of Dot-ELISA (cut-off titer of 1:256) for the serodiagnosis of human toxoplasmosis. The main advantage of this technique is simplicity, positive test can be visually identified (colored precipitate). It does not require a special equipment and it can be used as a qualitative test to screen large numbers of samples or as a quantitative assay to determine end-point titration of individual sera.