Improvement of the indirect hemagglutination test for the detection of antibodies to Streptococcus pyogenes

An indirect hemagglutination test for a seroepidemiological survery of Streptococcus pyogenes infection was standardized. This is an improved modification of the indirect hemagglutination test which utilizes an unstable reagent prepared with fresh blood cells. Two types of bacterial antigens represented by extracellular products and purified streptolysin O were assayed but only the former antigen gave good results. Pretreatment of the bacterial antigen with 0.15 M NaOH and neutralization to pH 5.5, as well as postfixation of sensitized red cells with 0.1 per cent glutaraldehyde at 56 degrees Celsius for 30 min were found to be essential to give long stability to the reagent in liquid suspension, at least 9 months at 4 degrees Celsius. A total of 564 serum samples with high, moderate and low anti-streptolysin 0 antibodies as determined by the neutralization assay were studied by the indirect hemagglutination test using the new reagent. The sensitivity, specificity, efficiency, positive predictive value and negative predictive value of the test in relation to the neutralization assay were 0.950, 0.975, 0.963, 0.973, and 0.955, respectively. The kappa agreement index between the two techniques was high (0.926) and ranked as "almost perfect". Antibody levels detected by both techniques also presented a high positive correlation (r(s)=0.726). Five reagent batches sucessively produced proved to be reproducible. Thus, the improved indirect hemagglutination test seems to be useful for public health laboratories.

Identification of Toxoplasma gondii antigens involved in the IgM and IgG indirect hemagglutination tests for the diagnosis of toxoplamosis

Extratos brutos de Toxoplasma gondii constituem materia prima antigenica para o preparo de reagentes empregados em diferentes testes sorologicos para o diagnóstico da toxoplasmose, incluindo entre estes as reaçöes de hemaglutinaçäo indireta para a detecçäo de anticorpos IgM (HA IgM) e IgG (HA IgG). Até o presente momento, moleculas antigenicas do parasita que realmente estäo envolvidas na interaçäo com anticorpos aglutinantes, anti-T. gondii, näo säo ainda bem conhecidas. O processo de absorçäo de soros de pacientes com toxoplasmose, utilizando o reagente de HA IgG (HA-toxo-G), possibilitou a demonstraçäo de que hemacias deste reagente estavam sensibilizadas com antigeno do parasita associado as bandas de massa molecular relativa de 39, 35, 30, 27, 22 e 14 kDa...

Detection of antibody isotypes to streptolysin O by dot ELISA

The presence of antibody isotypes (IgG, IgA and IgM) to streptolysin O was determined by dot ELISA in 222 serum samples from patients with different levels of anti-streptolysin O (SLO) antibodies as measured by the neutralizing assay (NA), from patients with diseases not related to nonsuppurative complications of Streptococcus pyogenes infection, and from clinically healthy individuals. Immunoglobulin G antibodies were found in 72 per cent of sera from patients with SLO antibodies higher than 333 Todd units (TU), and IgA antibodies were also detected in 53 per cent, but no IgM antibodies were demonstrable. High copositivity (0.94), conegativity (0.97), and positive (0.96) and negative (0.96) predictive values were observed when IgG and IgA findings were combined. The dot ELISA gave highly reproducible results. The present data suggest that the assay may be of practical value for routine detection of SLO antibodies when employed with an anti-human immunoglobulin light chain peroxidase conjugate.

Improvement of the slide hemagglutination test for rapid Chagas' disease screening in epidemiological surveys

A slide hemagglutination test, here called SHAT, which is practical and economical for seroepidemiological surveys was standardized. This is an improved modification of the rapid hemagglutination test (RHA) which utilizes a short-lived reagent prepared with fresh blood cells. The reagent and conditions of the test were considerably modified and, most importantly, an alkaline-solubilized Trypanosoma cruzi epimastigote antigen reagent is proposed. The stability of the SHAT reagent was at least one year at 4 degrees Celsius, in an appropriate liquid suspension. The SHAT was applied to 71 serum samples from patients with Chagas' disease and from 235 clinically healthy blood donors. Sensitivity, specificity and positive and negative predictive values for the selected cutoff titer corresponding to 1:4 dilution were 0.972 (0.903-0.992)9 O.983 (O.957-0.993) 0.945 (O.867-0.979) and O.991 (O.969-0.998), respectively. These values were comparable to those found for the RHA, immunofluorescence (IFT), indirect hemagglutination (IHAT) and complement fixation (CFT) tests. These data suggest that the SHAT should be useful for seroepidemiological surveys conducted at public health laboratories in developing countries.

Production and immunochemical characterization of Neisseria meningitidis group B antiserum for the diagnosis of purulent meningitis

Unlike Neisseria meningitidis groups A, C, Y and W135, the group B capsular polysaccharide has been shown to be chemically and immunologically identical to the capsular polysaccharide of Escherichia coli K1. Both components are sialic acid homopolymers and are poorly immunogenic. Nevertheless, due to the high incidence of Neisseria meningitidis group B meningitis in the population of the State of São Paulo, preparing antiserum to this serogroup for diagnostic purposes has become a matter of high priority. Of the many immunization schemes proposed, intravenous inoculation of whole bacteria previously inactivated with formaldehyde and simultaneous intradermal inoculation with a mixture of the bacterial polysaccharide fraction and whole bacteria in complete Freund;s adjuvant have produced the best results. The antiserum was treated with immunoadsorbents prepared with aluminum chloride and protein and/or polysaccharide antigens from each of the following heterologous bacteria: Haemophilus influenzae type b, Streptococcus pneumoniae, Escherichia coli other than K1, and Staphylococcus aureus, in order to eliminate cross-reactivity. For quality control analysis, the antiserum was assessed by the immunodiffusion, counterimmunoelectrophoresis, dot-ELISA, and immuno-blot techniques against homologous antigens. Specificity was obtained after treating the antiserum with Haemophilus influenzae type b polysaccharide immunosorbent.

Studies on the efficiency of measles virus antigen production using VERO cell culture in a microcarrier system

1. A large amount of antigen is required to conduct seroepidemiologic surveys of measles. Thus, a process to obtain measles virus antigen using a bioreactor was standardized. 2. The virus was grown in a 3.7-1 culture of VERO cells using a Celligen cell culture system containing 2 mg/ml of microcarriers (cytodex I) at 37 degrees C and 60 rpm. The cultures infected with 0.5 m.o.i. of measles virus were harvested after the appearance of the cytopathic effect. The virus suspension was clarified and concentrated by ultracentrifugation. Intracellular and extracellular virus titers were determined by hemagglutination (HA) and by induction of a cytopathic effect in cell culture (TCID50). 3. Intracellular virus presented 5-7 x 10(6) TCID50/0.1 ml, HA activity per 50 microliters equal to 32, with a total HA activity of 4,480 HA units (HAU) and specific activity of 116 HAU/mg. In the concentrated supernatants, the HA titer of extracellular virus was 64, with a total HA activity of 1,024 HAU and a specific activity of 1,600 HAU/mg. 4. The antigen obtained was suitable for the detection of antibodies against measles virus in assays such as ELISA and DOT-ELISA (using 1 micrograms/well to ELISA and 2 micrograms/DOT). 5. The microcarrier system produced antigen sufficient for 26 ELISAs/ml compared to 5.7 ELISAs/ml obtained for the static culture system.

Seroepidemioplogy of schistosomiasis mansoni

In population surveys in wich the Schistosoma mansoni intensity of infection is low, or in localities where the schistosomiasis control program had success the parasitologic methods lack in sensitivity. Despite of some limitations the immunological methods are useful to provide valuable information in such field conditions. Thus, the prevalaence of schistosomiasis in untreated population can be determined by the detection of IgG or IgM antibodies, as well as the incidence by the IgA antibodies , employing mainly immunofluorescence (IF) and immunoenzymatic (ELISA), and in some extent hemagglutination (HA) or even skin test. The true prevalence and incidence of schistosomiasis can be estimated using a probabilistic model equation, since knowing before-hand the sensitivity and specificity of emploved test. The sensitivity and the specificity of serologic test become higher in low aged group, under 14. The geometric mean IF titers also gives a positive correlation with the intensity of infection. Presently there are need of serologic tests wich are economic and pratical in soroepidemiologic inquires, requiring no specialized personnel to collect population blood or serum and also easily interpret the test results. The reagents for such tests are desired to be stable and reproducible. Moreover, it is expected that the tests can distinguish an ative infection

Diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA) for Chagas'disease serodiagnosis

Diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA) was standardized and evaluated for the diagnosis of Chagas'disease in comparison with the conventional serological tests indirect immunofluorescence (IFI), passive hemagglutination (PHA) and complement fixation (CF). A total of 236 serum samples positive and negative for the serodiagnosis of Chagas'disease were studied. The group included 50 serum samples serologically positive for leishmaniasis and 36 positive for malaria. The best diagnostic performance of DIG-ELISA was observed when serum samples were diluted to 1:8 and a diameter of zero mm (no color) was taken as the cut-off. Under these conditions, the relative indices of sensitivity, specificity and agreement were 100%. High positive correlation coeficients were obtained between DIG-ELISA and IFI (r1=0.9010), PHA (r2=0.8943) and CF (r3=0.8269). We conclude that DIG-ELISA provides an alternative technique for screening chagasic infections, as well as for seroepidemiological surveys mainly because it is simple, easy to carry out and does not require expensive equipment

Schistosomiasis mansoni in the municipality of Pedro de Toledo (Säo Paulo, Brazil) where the Biomphalaria tenagophila is the snail host. I-Prevalence in human population

Devido à escassez de dados epidemiológicos sobre esquistossomose mansônica onde Biomphalaria tenagophila é vetor foi desenvolvido em 1980 o presente trabalho, no município paulista de Pedro de Toledo. Foram examinadas fezes de 4741 pesssoas (Método de Kato-Katz) com prevalência de 22,8%; entre essas, 583 foram tratadas para a endemia anteriormente e 4158 näo medicadas; as prevalências nos dois grupos foram, respectivamente, 31,7% e 21,6%. Por investigaçäo epidemiológica constatou-se que 83,6% dos casos foram autóctones da área estudada. Prevalência dos autóctones näo tratados foram maiores no homem do que na mulher, a intensidade no último grupo foi baixa: 58,5 ovos g de fezes (média geométrica). De acordo com grupos etários, se correlacionaram bem (rs=0,745) as intensidades de infecçäo e as prevaleência. A infecçäo ocorreu, na zona rural, principalmente, durante lazer e trabalho doméstico. Somente 0,4% de 1137 moluscos foram positivos para Schistosoma mansoni. Esse índice foi, aparentemente, o mesmo em estudo de 1978 quando a prevalência humana era de 12,0%. A área estudada apresentou difernças e semelhanças epidemiológicas em relaçäo às áreas onde B. glabrata é o principal hospedeiro intermediário

A study on the reproducibility of a stable, lyophilized reagent for the Chagas' disease hemagglutination test: proposals for quality control analysis.

Para se efetuar rigoroso controle de partidas sucessivas de reagentes liofilizados, usados na reacao de hemaglutinacao passiva, sao aqui propostos processos estatisticos simples. Para se avaliar qualitativamente os niveis de sensibilidade e de especificidade do reagente foi empregada a analise sequencial truncada.Os criterios adotados na avaliacao foram definidos com base em estudos comparativos efetuados com o teste de hemaglutinacao passiva em relacao as reacoes de imunofluorescencia, fixacao do complemento e floculacao de 3.264 soros de individuos, infectados ou nao pelo T. cruzi. Onze partidas de reagente de hemaglutinacao, produzidas durante um periodo de 3 anos, foram submetidas a esta analise de qualidade. O processo aqui proposto mostrou-se satisfatorio para selecionar reagentes que forneceram resultados reprodutiveis. Esta observacao foi confirmada quando se procedeu a analise de variancia dos resultados obtidos com 7 partidas de reagentes, aprovados pela analise de qualidade, contra 20 soros de pacientes chagasicos titulados em duplicata

An immunoepidemiological study of schistosomiasis mansoni in Paraiba's Valley, Sao Paulo, Brazil.

Inquerito imunoepidemiologico foi realizado na fazenda Santa Helena, localizada no Vale do Rio Paraiba do Sul, Sao Paulo. A prevalencia de esquistossomose determinada pela reacao de imunofluorescencia foi de 25,5% e de 29,8% pela reacao intradermica. O exame coprologico revelou 10,6% de infectados, cerca de tres vezes mais do que observado no inquerito preliminar de 1977. A influencia de poliparasitismo na sensibilidade e na especificidade das reacoes imunologicas nao foi significativo. Resultados satisfatorios foram obtidos na reacao de imunofluorescencia com cortes de figado de hamster infectado com S.mansoni, tendo granuloma e ovos do parasita como antigeno

Solubilization of antigens of S. mansoni adult worms for the passive hemagglutination test. Preliminary reports.

No preparo do antigeno de S. mansoni para a reacao de hemaglutinacao passiva, a solubilizacao dos componentes do verme adulto melhorou consideravelmente pela adicao de NaOH 0,2 M e neutralizacao com HCl. Este procedimento permitiu a obtencao de cerca de 10 vezes mais antigeno do que na extracao usual feita com solucao salina (NaCl 0,15 M). Os resultados preliminares obtidos em 55 soros de pacientes com formas cronicas de esquistossomose e em 8 com formas agudas mostraram sensibilidade semelhante a do antigeno de extrato salino. A especialidade foi tambem comparavel quando 30 soros de individuos normais foram testados. O tratamento dos vermes adultos com solucao alcalina parece nao ter influido na antigenicidade, podendo com vantagem substituir o antigeno de extracao salina