Seroprevalence of Toxoplasma gondii IgG antibody in HIV/AIDS-infected individuals in Maputo, Mozambique

OBJECTIVE To analyze the prevalence of IgG antibodies to Toxoplasma gondii in patients infected with HIV/AIDS and the association of demographic and social variables. METHODS Descriptive cross-sectional study that included the analysis of sociodemographic data and laboratory findings of 200 patients infected with HIV/AIDS treated in a laboratory unit in Maputo, Mozambique, in 2010. Individual data for all participants were collected with a self-administered questionnaire. Plasma samples were tested for IgG testing of anti- T. gondii using hemagglutination for the analysis of antibodies. RESULTS The seroprevalence of IgG anti- T. gondii was 46.0% (95%CI 39.2;52.9), 39.3% (95%CI 29.5;50.0) in men and 50.9% (95%CI 41.9;59.8) in women, with no difference between sex (OR 1.30; 95%CI 0.95;1.77; p = 0.12). Ages ranged from 10 to 60 years, with a higher prevalence of infection in older age groups, but with no significant difference between them. Regularly consuming cattle meat (OR 1.74; 95%CI 1.04;2.89, p = 0.05), breeding cats/dogs (OR 6.18; 95%CI 3.60;10.62, p < 0.000) and having regular contact with soil (OR 3.38; 95%CI 2.19;5.21; p < 0.000) were significantly associated with risk of latent infection. CONCLUSIONS Toxoplasmosis is an infection with high prevalence in Mozambique. Cultural and behavioral aspects increase the risk. Toxoplasmosis can be responsible in our environment by the great burden of morbidity and mortality associated with meningoencephalic injuries in patients with HIV/AIDS.

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.

Avidez de anticorpos IgG específicos como marcadores de infecção primária recente pelo Toxoplasma gondii

A caracterização de infecção primária recente pelo Toxoplasma gondii se apoia principalmente na presença, no soro, de anticorpos específicos IgM. Para fins diagnósticos de toxoplasmose aguda, ou de contágio recente, a possibilidade de outros marcadores é altamente desejável. Um marcador de infecção recente atualmente referido é a baixa afinidade ou avidez de anticorpos específicos IgG. Para avaliação do novo marcador, titularam-se os soros contra poliantígenos do T. gondii pelo teste imunoenzimático (ELISA), antes e após tratamento dos complexos antígeno-anticorpo formados, com solução de ureia 6 M como agente dissociante. O deslocamento de anticorpos de baixa avidez foi indicado por uma queda de títulos, calculada em porcentagem em relação aos títulos iniciais. Foram estudados 69 soros, 23 de cada um dos 3 perfis sorológicos sucessivos, observados na infecção, e que a caracterizam respectivamente como recente, em fase de transição e crônica. Os perfis foram determinados segundo os resultados de uma bateria de testes, incluindo os de imunofluorescência IgG e IgM, de captura de anticorpos IgM e de hemaglutinação. Para os soros de infecção crônica a queda observada foi de 3% ± 3%, de 34% ± 12% para toxoplasmose recente e de 12% ± 9% para a fase de transição. Conclue-se que a determinação da avidez de anticorpos IgG pode ser utilizada como marcador de infecção primária recente pelo T. gondii.

Performance indexes of a dot-enzime-linked immunosorbent assay (dot-ELISA) and an ezyme-linked immunosorbent assay (IgG-ELISA) for field surveys of new world leishmaniasis

Diagnostic performance indexes of sensitivity, specificity, positive predictive value and efficiency were determined for dot-ELISA and IgG-ELISA tests in 340 leishmaniasis sera. Sensitivity of the dot-ELISA was significantly lower than IgG-ELISA's; the two tests had indexes of specificity and positive predictive value of the same magnitude. Seventy-eight sera gave a negative dot-ELISA test result and a positive IgG-ELISA test result. When sera were classified according to different criteria as how to interpret this diversity, the kappa statistic did not corroborate the classification indicating that the two tests display a substantial strength of agreement. The results presented indicate that performance indexes accrued in a survey where variables arc well known may be extrapolated to other population studies if the disease presents itself as highly prevalent (due to a selection bias or not) and may be expected to discriminate a disease status among test positives.

Comparison on the performance of Leishmania major-like and Leishmania braziliensis braziliensis as antigen for new world leishmaniasis IgG-immunofluorescence test

The performance of an antigen of L. major-like promastigotes for the serological diagnosis of mucocutaneous leishmaniasis in the IgG-immunofluorescent test was compared to that of an antigen of L. braziliensis braziliensis. Each antigen was used to test two hundred and twenty-four sera of etiologies such as mucocutaneous leishmaniasis, deep mycoses, toxoplasmosis, malaria, Chagas' disease, visceral leishmaniasis, anti-nuclear factor, schistosomaiasis, rheumatoid factor and normal controls. Agreement between responses to each antigen was high: 77.2% of leishmaniases sera agreed on a positive or a negative result to both antigens and 91.1 % of control sera. Cross reactivity was restricted to Chagas' disease sera, visceral leishmaniasis, anti-nuclear factor and paracoccidiodomycosis. The quantitative response of leishmaniasis and Chagas' disease sera to both antigens was evaluated by a linear regression; although the y-intercept and the slope were different for each antigen, neither was better than the other in the disclosure of anti-Leishmania antibodies. In the case of Chagas' disease sera the L. major-like antigen was better than L. b. braziliensis' to disclose cross-reacting antibodies.

Total serum IgE and parasite: specific IgG and IgA antibodies in human strongyloidiasis

Total serum IgE, and Strongyloides - specific IgG and IgA antibodies were studied in 27 patients with parasitologically proven strongyloidiasis. Clinical manifestations in this case series were investigated by a restrospective study of the patient's records. Total serum IgE levels were elevated (greater than 250 IU/ml) in 59% of the patients (mean concentration = 1364 IU/ml). Parasite - specific IgG and IgA antibodies were detected by ELISA in the serum of 23 (85.2%) and 21 (77.8%) patients, respectively. Elevated serum IgE and clinical manifestations were not useful indexes of the presence of strongyloidiasis. On the other hand, our results support the view that serologic tests, particularly ELISA for detecting Strongyloides - specific IgG antibodies, can be usefully exploited for diagnostic purposes in strongyloidiasis.

Malaria serology: performance of six Plasmodium falciparum antigen extracts and of three ways of determining serum titers in IgG and IgM-ELISA

The study evaluated six Plasmodium falciparum antigen extracts to be used in the IgG and IgM enzyme-linked immunosorbent assays (ELISA), for malaria diagnosis and epidemiological studies. Results obtained with eighteen positive and nine negative control sera indicated that there were statistically significant differences among these antigen extracts (Multifactor ANOVA, p< 0.0001). Urea, sodium deoxycholate and Zwittergent antigen extracts performed better than did the three others, their features being very similar for the detection of IgG antibodies. Urea, alkaline and sodium deoxycholate antigen extracts proved to be better than the others for the detection of IgM antibodies. A straight line relationship was found between the optical densities (or their respective log 10) and the log 10 of antibody dilutions, with a very constant slope. Thus serum titers could be determined by direct titration and by two different equations, needing only one serum dilution. For IgM antibody detections, log 10 expression gave results that better correlated with direct titration (95% Bonferroni). For IgG antibody detections, the titer differences were not significant. The reproducibility of antibody titers and antigen batches was also evaluated, giving satisfactory results.

Generation of high-affinity monoclonal antibodies of IgG class against native β-d-glucans from basidiomycete mushrooms

β-d-glucans from basidiomycete strains are powerful immunomodulatory agents in several clinical conditions. Therefore, their assay, purification and characterization are of great interest to understand their structure-function relationship. Hybridoma cell fusion was used to raise monoclonal antibodies (Mabs) against extracellular β-d-glucans (EBGs) from Pleurotus ostreatus. Two of the hybridoma clones (1E6-1E8-B5 and 3E8-3B4) secreting Mabs against EBGs were selected. This hybridoma cell line secreted Mabs of the IgG class which were then purified by hydroxyapatite chromatography to apparent homogeneity on native and SDS-PAGE. Mabs secreted by 1E6-1E8-B5 clone were found to recognize a common epitope on several β-d-glucans from different basidiomycete strains. This Mab exhibited high affinity constant (KA) for β-d-glucans from several mushroom strains in the range of 3.20 × 109 ± 3.32 × 103-1.51 × 1013 ± 3.58 × 107 L/mol. Moreover, they reacted to some heat-treated β-d-glucans in a different mode when compared with the native forms; these data suggest that this Mab binds to a conformational epitope on the β-d-glucan molecule. The epitope-binding studies of Mabs obtained from 1E6-1E8-B5 and 3E8-3B4 revealed that the Mabs bind to the same epitope on some β-d-glucans and to different epitopes in other antigen molecules. Therefore, these Mabs can be used to assay for β-d-glucan from basidiomycete mushrooms. © 2015 Elsevier Ltd. All rights reserved.

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.

Identification of Toxoplasma gondii antigens involved in the IgM AND IgG indirect hemagglutination tests for the diagnosis of toxoplasmosis

Crude Toxoplasma gondii antigens represent raw material used to prepare reagents to be employed in different serologic tests for the diagnosis of toxoplasmosis, including the IgM and IgG indirect hemagglutination (IgG-HA and IgM-HA) tests. So far, the actual antigenic molecules of the parasite involved in the interaction with agglutinating anti-T. gondii antibodies in these tests are unknown. The absorption process of serum samples from toxoplasmosis patients with the IgG-HA reagent (G-toxo-HA) demonstrated that red cells from this reagent were coated with T. gondii antigens with Mr of 39, 35, 30, 27, 22 and 14 kDa. The immune-absorption process with the IgM-HA reagent (M-toxo-HA), in turn, provided antibody eluates which recognized antigenic bands of the parasite corresponding to Mr of 54, 35 and 30 kDa, implying that these antigens are coating red cells from this reagent. The identification of most relevant antigens for each type of HA reagent seems to be useful for the inspection of the raw antigenic material, as well as of reagent batches routinely produced. Moreover the present findings can be used to modify these reagents in order to improve the performance of HA tests for the diagnosis of toxoplasmosis

Evolution of IgG antibody response against Toxoplasma gondii tissue cyst in acute and chronic human infections

The recognition profile of the tissue cysts antigens by IgG antibodies was studied during acute and chronic human toxoplasmic infection. Thus the IgG response against Toxoplasma gondii was investigated by immunoblotting in two patients accidentally infected with the RH strain as well as in group of naturally infected patients at acute and chronic phase. There was an overall coincidence of molecular mass among antigens of tachyzoites and tissue cysts recognized by these sera, however, they appear not to be the same molecules. The response against tissue cysts starts early during acute infection, and the reactivity of antibodies is strong against a wide range of antigens. Six bands (between 82 and 151 kDa) were exclusively recognized by chronic phase sera but only the 132 kDa band was positive in more than 50% of the sera analysed. A mixture of these antigens could be used to discriminate between the two infection phases. The most important antigens recognized by the acute and the chronic phase sera were 4 clusters in the ranges 20-24 kDa, 34-39 kDa, 58-80 kDa and 105-130 kDa as well as two additional antigens of 18 and 29 kDa. Both accidentally infected patients and some of the naturally infected patients showed a weak specific response against tissue cyst antigens.

REPRODUCIBILITY OF ALKALINE ANTIGENS OF Leishmania major-LIKE AND Leishmania (V.) braziliensis EVALUATED BY IgG-ELISA. COMPARISON OF ANTIGENS ADDED OF A PROTEIN INHIBITOR (PMSF) OR NOT

This paper deals with the analysis of 10 batches of L.major-like and L.(V.) braziliensis antigens added or not of a proteases inhibitor evaluated by means of an IgG-ELISA on three consecutive days using positive standard sera from patients with diagnosis of American Leishmaniasis previously tested for the presence of IgG antibodies by means of ELISA. The statistical analysis showed that for L. (V.) braziliensis the PMSF-containing antigen did not show any difference among batches or days of testing; the L.(V.) braziliensis antigen without PMSF showed statistical significance for differences among batches and a two-way ANOVA showed significant differences between antigens. L.major-like antigen prepared with or without PMSF showed differences among batches; all 3 days of testing displayed differences for the PMSF antigen but only for days 1 and 2 for the antigen without inhibitor. A two-way ANOVA showed differences among batches of the antigens but not for antigens with and without the protein inhibitor. According to the statistical analysis the L.major-like antigen added or not of PMSF has shown that it is the choice antigen for mucocutaneous leishmaniasis serology.