Performance of indirect immunofluorescence assay, immunochromatography assay and reverse transcription-polymerase chain reaction for detecting human respiratory syncytial virus in nasopharyngeal aspirate samples

Comparison of the use of indirect immunofluorescence assay (IFA), immunochromatography assay (ICA-BD) and reverse transcription-polymerase chain reaction (RT-PCR) for detecting human respiratory syncytial virus (HRSV) in 306 nasopharyngeal aspirates samples (NPA) was performed in order to assess their analytical performance. By comparing the results obtained using ICA-BD with those using IFA, we found relative indices of 85.0 percent for sensitivity and 91.2 percent for specificity, and the positive (PPV) and negative (NPV) predictive values were 85.0 percent and 91.2 percent, respectively. The relative indices for sensitivity and specificity as well as the PPV and NPV for RT-PCR were 98.0 percent, 89.0 percent, 84.0 percent and 99.0 percent, respectively, when compared to the results of IFA. In addition, comparison of the results of ICA-BD and those of RT-PCR yielded relative indices of 79.5 percent for sensitivity and 95.4 percent for specificity, as well as PPV and NPV of 92.9 percent and 86.0 percent, respectively. Although RT-PCR has shown the best performance, the substantial agreement between the ICA-BD and IFA results suggests that ICA-BD, also in addition to being a rapid and facile assay, could be suitable as an alternative diagnostic screening for HRSV infection in children.

Decay of antibody isotypes against early developmental stages of Schistosoma mansoni after treatment of schistosomiasis patients.

Antibodies to a number of parasite antigens are found in schistosomiasis patients, and antibodies to early developmental stages were demonstrated to be efficient immunologic markers for the diagnosis of schistosomiasis. In the present study, decay patterns of IgM and IgG antibodies against cercariae and schistosomula were investigated, in comparison to antibodies against worms and eggs in schistosomiasis patients after chemotherapy, for an investigation of seroepidemiologic aspects. Data obtained in the study of 359 serum samples from patients with Schistosoma mansoni infection, noninfected individuals, and patients followed-up for a period of 12 to 15 months after treatment provided the basis to postulate a general pattern for the kinetics of antibody decay. Before treatment, the antibody pattern was represented by a unimodal curve, which shifted to a bimodal curve after treatment, and ended with a unimodal curve similar to that for the noninfected group. Different types of antibodies were classified into four categories according to their decay features, and anti-schistosomulum IgM was classified into the moderate-decay category, whereas other antibodies to early parasite stages were classified into the slow-decay category. The present methodology permits the identification of the most suitable antibodies to be detected in field control programs for schistosomiasis or other parasitoses.

High resolution of Trypanosoma cruzi amastigote antigen in serodiagnosis of different clinical forms of Chagas' disease.

The serodiagnosis of Chagas' disease, a highly prevalent disorder in South American countries, is usually made by the detection of antibodies to Trypanosoma cruzi epimastigote antigen. In this study, we assess the diagnostic performance of the immunofluorescence test with T. cruzi (Y strain) amastigote antigen from an LLC-MK2-infected cell supernatant in comparison with a test with the conventional epimastigote antigen. A total of 238 serum samples from patients in the acute and chronic phases of the disease, with the chronic indeterminate, cardiac, and digestive forms, and from nonchagasic individuals were tested for the presence of immunoglobulin G (IgG), IgM, and IgA antibodies. The reactivity of the amastigote antigen in terms of geometric mean titers was 2 to 4 times higher than that of the epimastigote antigen. Clear-cut results were obtained with the amastigote antigen, with no overlapping of true and false positives. IgG antibodies to amastigotes were found in all patients with Chagas' disease, whereas all sera from nonchagasic patients were negative, except for those from patients with visceral leishmaniasis, in which 63% cross-reactivity was observed. IgM antibodies to amastigotes were detected in 100% of sera from patients with acute Chagas' disease and in 7.5% of sera from patients with chronic Chagas' disease, whereas IgA antibodies were found in 60% of sera from patients in the acute phase and in 33% of sera from patients in the chronic phase. Despite the cross-reactivity observed with sera from visceral leishmaniasis patients, the IgG immunofluorescence test with the amastigote antigen had the highest sensitivity, specificity, and efficiency. No relationship was observed between the class-specific antibodies or their titers and the clinical forms of patients in the chronic phase. Amastigotes from the cell culture supernatant proved to be useful as an alternative antigen to epimastigotes because of their high resolution in the serodiagnosis of Chagas' disease.

Deteccao de antigenos bacterianos em pneumonia aguda: metodos de preparacao das amostras de urina, soro e liquido pleural para os testes de imunodiagnostico / Detection of bacterial antigens in acute pneumonia: methods of preparing the urine, serum and pleural fluid samples for immunodiagnostic assays

Foi desenvolvido um metodo de precipitacao de antigenos polissacaridicos de S. pneumoniae e H influenzae tipo b na urina, atraves do tratamento com uma solucao de etnol-acetona 1:1 seguido de um tratamento a quente com EDTA 0,1M. Foram empregadas as tecnicas de contra-imunoeletroforese e latex aglutinacao para a deteccao de antigenos polissacarideos em amostras pareadas de urina e soro e ainda de liquido pleural, de criancas com diagnostico clinico e radiologico de pneumonia aguda. Contra-imunoeletroforese e latex aglutinacao apresentaram melhores indices de sensibilidade em urina do que em soro e tiveram otimo desempenho tanto para urina de volume inicial relativamente pequeno como de grande volume, colhidas antes ou durante os primeiros dias de antibioticoterapia. Os resultados obtidos em contra-imunoeletroforese e latex aglutinacao mostraram que a solucao etanol-acetona 1:1 fornece melhor rendimento na precipitacao de antigeno polissacaridico enquanto que o aquecimento com EDTA diminui a probabilidade de ocorrencia de resultados falso-positivos e de reatividade cruzada entre S. pneumoniae e H. influenzae tipo b. A urina mostrou-se como importante meio de deteccao de antigenos bacterianos no diagnostico de pneumonia bacteriana aguda, principalmente se a antibioticoterapia previa obstrui o crescimento bacteriano nos meios de cultura.

[Detection of bacterial antigens in acute pneumonia: methods of preparing the urine, serum, and pleural fluid samples for immunodiagnostic assays]. / Detecção de antígenos bacterianos em pneumonia aguda: métodos de preparação das amostras de urina, soro e líquido pleural para os testes de imunodiagnóstico.

A method of polysaccharide antigen precipitation in urine treated with 1:1 ethanol-acetone solution, followed by heat treatment with 0.1 M EDTA were developed for detection of S. pneumoniae and H. influenzae type b. Counterimmunoelectrophoresis and latex agglutination were employed to detect the antigens, in paired samples of urine and serum, and also in pleural fluid samples from children with clinical diagnosis of acute pneumonia. Counterimmunoelectrophoresis and latex agglutination showed better results in urine than in serum and also in smaller initial volumes of urine from the onset of illness or during the first days of antibiotic therapy. The results obtained in counterimmunoelectrophoresis and latex agglutination showed that ethanol-acetone solution increased the yield of polysaccharide antigen precipitation while heating with EDTA diminished the probability of false-positive results and cross-reactivity between S. pneumoniae and H. influenzae type b. The results, statistically evaluated, suggest that urine is a body fluid in which the bacterial antigens may be detected in the acute pneumonia. This is of importance in patients previously treated with antibiotics which may inhibit bacterial growth in the culture media.

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

1. 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). 2. 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. 3. 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 coefficients were obtained between DIG-ELISA and IFI (r1 = 0.9010), PHA (r2 = 0.8943) and CF (r3 = 0.8269). 4. 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.

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

[Acute toxoplasmosis: evaluation of a thin-layer immunoassay technic for detection of IgM antibodies, anti-Toxoplasma gondii]. / Toxoplasmose aguda: avaliação da técnica de imunoensaio em camada delgada para a detecção de anticorpos IgM, anti-Toxoplasma gondii.

A solid phase method, thin-layer immunoassay (IgM-TIA) was standardized and evaluated for the immunodiagnosis of acute toxoplasmosis, through the detection of IgM antibodies to Toxoplasma gondii. A total of 300 serum samples from serologically defined acute toxoplasmosis and, from non-related infections, was investigated by IgM-TIA. Statistical analysis were carried out in comparison with conventional tests, the immunofluorescence test for the detection of IgM antibodies (IgM-IFI) and hemagglutination test which uses 2-mercaptoethanol serum treatment (2ME-HA). Also the correlation coefficients were calculated for various Toxoplasma gondii antigen concentrations, as well as, the influence of the antigenic concentration on the relative indices of sensitivity and specificity were verified. The intra and inter test reproducibilities were demonstrated statistically, as well as, the reutilization of T. gondii antigen was proven to be possible for at least 10 times. The data indicated that antigenic concentrations, from 70 to 100 Cmg/ml, were able to provide maximum sensitivity and specificity. IgM-TIA displayed similar diagnostic efficiency to those two conventional tests here utilized, and may be employed to make diagnosis of acute toxoplasmosis, mainly if laboratory animals are available.