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% for sensitivity and 91.2% for specificity, and the positive (PPV) and negative (NPV) predictive values were 85.0% and 91.2%, respectively. The relative indices for sensitivity and specificity as well as the PPV and NPV for RT-PCR were 98.0%, 89.0%, 84.0% and 99.0%, 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% for sensitivity and 95.4% for specificity, as well as PPV and NPV of 92.9% and 86.0%, 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.

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.

Immunodiagnosis of pneumococcal meningitis using Dot-enzyme-linked immunosorbent assay.

A diagnosis of bacterial meningitis requires isolation of the pathogen from cerebrospinal fluid (CSF). However, cultures of CSF are usually insensitive, thus, in the majority of patients, the etiology is rarely determined. A total of 90 CSF samples from pediatric patients with clinical diagnosis of bacterial meningitis were evaluated by Dot-ELISA. This method was standardized in order to detect pneumococcal polysaccharide antigen in CSF samples previously treated with 0.1 M EDTA and dotted on nitrocellulose membrane strips. Pneumococcal omniserum diluted 1:200 was employed for pneumococcal antigen detection. Dot-ELISA showed relative indices of 100 and 90 per cent for sensitivity and specificity, respectively. This method is cheaper than counter immunoelectrophoresis for pneumococcal antigen detection.

[Immunologic behavior of meningococcal vaccines]. / Comportamento imunológico das vacinas anti-meningocócicas.

Meningococcal disease continues to be a great health problem on all continents and the meningococcal vaccines have been proposed for their prevention and epidemic control. The polysaccharide A and C vaccines are relatively efficacious with distinct immunological behavior with regard to the different age groups, however, up to the present no highly efficacious vaccine for meningococcal B disease exists. The meningococcal B capsular polysaccharide is not immunogenic due to the structural mimicry of mammalian tissues and efforts to produce carrier proteins have been proposed in order to obtain an immunogenic vaccine for all age groups that would if possible, protect against all the meningococci. This review of the literature presents the study of the development of the immunological behavior of all the meningococcal vaccines undergoing development and reports on the efforts to obtain a safe and efficacious product for the control of meningococcal disease.

Immunodiagnoses of community-acquired pneumonia in childhood.

A diagnosis of bacterial pneumonia requires isolation of the pathogen from blood, lung or tracheal aspirate; however, cultures of blood and pleural fluid samples are usually insensitive. Thus, in the majority of patients the etiology is rarely determined. A total of 840 pleural fluid effusion samples from children with clinical and laboratory diagnoses of acute bacterial pneumonia were evaluated by Dot-ELISA. This method was standardized in order to detect polysaccharide capsular bacterial antigen in pleural fluid samples previously treated with 0.1 M EDTA and dotted on nitrocellulose membrane strips. Pneumococcal omniserum and H. influenzae type b antiserum diluted 1:200 were employed for detection of S. pneumoniae and H. influenzae type b antigens, respectively. Dot-ELISA showed relative indices of 0.913 for sensitivity and 0.552 for specificity, and a total positivity of 75.6 per cent, being 53.21 per cent for S. pneumoniae and of 22.38 per cent for H. influenzae.

Primary community-acquired pneumonia by Escherichia coli. Case report and review of the literature.

A two year old girl with chronic neurologic convulsive disease was admitted with a six day history of pneumonia and, despite treatment, died on hospital day 3. The X-ray revealed right upper lobar pneumonia. The results of pleural effusion and blood cultures drawn on admission yielded a non-typable Escherichia coli. No other source of infection was identified. The authors discuss the clinical and pathophysiological aspects of Escherichia coli pneumonia.

Dot-enzyme-linked immunosorbent assay (Dot-ELISA) for detection of pneumococcal polysaccharide antigens in pleural fluid effusion samples. Comparison with bacterial culture, counterimmunoelectrophoresis and latex agglutination.

A dot-enzyme-linked immunosorbent assay (Dot-ELISA) for pneumococcal antigen detection was standardized in view of the need for a rapid and accurate immunodiagnosis of acute pneumococcal pneumonia. A total of 442 pleural fluid effusion samples (PFES) from children with clinical and laboratory diagnoses of acute bacterial pneumonia, plus 38 control PFES from tuberculosis patients and 20 negative control serum samples from healthy children were evaluated by Dot-ELISA. The samples were previously treated with 0.1M EDTA pH 7.5 at 90 degrees C for 10 min and dotted on nitrocellulose membrane. Pneumococcal omniserum diluted at 1:200 was employed in this assay for antigen detection. When compared with standard bacterial culture, counterimmunoelectrophoresis and latex agglutination techniques, the Dot-ELISA results showed relative indices of 0.940 to sensitivity, 0.830 to specificity and 0.760 to agreement. Pneumococcal omniserum proved to be an optimal polyvalent antiserum for the detection of pneumococcal antigen by Dot-ELISA. Dot-ELISA proved to be a practical alternative technique for the diagnosis of pneumococcal pneumonia.

Polyvalent pneumococcal polysaccharide vaccines; a review of the literature.

Streptococcus pneumoniae is a leading cause of morbidity and mortality, mainly in children, elders and individuals with AIDS or AIDS-related complex, being a frequent bacterial cause of pneumonia, otitis media, sinusitis, bacteremia and meningitis. Polyvalent pneumococcal capsular polysaccharide vaccines contain the 23 most common pneumococcal serotypes causative of pneumococcal infection in several countries. Public Health Service Advisory Committee on Immunization Practice of the Centers for Disease Control (CDC) does not recommend the polyvalent pneumococcal vaccine for general public. However, several investigators have recommended its employment for special population at high risk, such as for HIV infection, who can be at enhanced risk for systemic pneumococcal disease. The objective of the present literature review is to relate the importance of studied different types of polyvalent pneumococcal vaccines as well as their immunological properties in the vaccinated people.

Comparison of counterimmunoelectrophoresis, latex agglutination and bacterial culture for the diagnosis of bacterial meningitis using urine, serum and cerebrospinal fluid samples.

1. Urine, serum and cerebrospinal fluid (CSF) samples from 98 children with clinical and laboratory diagnosis of bacterial meningitis were evaluated by counterimmunoelectrophoresis (CIE) and latex agglutination (LA) methods and the results compared to those obtained with bacterial cultures of the CSF samples. Antigens of Neisseria meningitidis groups A, B and C, Haemophilus influenzae type b and Streptococcus pneumoniae were determined by both immunological methods. Serum was diluted (1:4) with 0.1 M sodium EDTA, pH 7.5, and held at 80 degrees C for 10 min before assay. Polysaccharide of the urine samples was precipitated overnight using an equal volume of 1:1 ethanol-acetone followed by a heat-treatment with 0.1 M sodium EDTA, pH 7.5, at 80 degrees C for 10 min.. 2. Sensitivity indices were 0.772 (CSF), 0.595 (urine) and 0.317 (serum) for CIE, and 0.914 (CSF), 0.930 (urine) and 0.683 (serum) for LA in relation to the 42 positive bacterial cultures. 3. The optimal diagnostic efficacy reached 52% for CIE and 72% for LA when urine was concentrated 20- to 30-fold. 4. These data show that immunological tests of urine samples were more effective than bacterial culture for diagnosing bacterial meningitis and may be indicated when negative results are obtained for CSF tested by bacterial culture and immunoassay methods.

Comparison of counterimmunoelectrophoresis, latex agglutination and bacterial culture for the diagnosis of bacterial meningitis using urine, serum and cerebrospinal fluid samples

Urine, serum and cerebrospinal fluid (CSF) samples from 98 children with clinical and laboratory diagnosis of bacterial meningitis were evaluated by counterimmunoelectrophoresis (CIE) and latex agglutination (LA) methods and the results compared to those obtained with bacterial cultures of the CSF samples. Antigens of Neisseria meningitidis groups A, B and C, Haemophilus influenzae type b and Streptococcus pneumoniae were determined by both immunological methods. Serum was diluted (1:4) with 0.1 M sodium EDTA pH 7.5, and held at 80 grade C for 10 min before assay. Polysaccharide of the urine samples was precipitated overnight using an equal volume of 1:1 ethanol-acetone followed by a heat-treatment with 0.1 M sodium EDTA, pH 7.5, at 80 grade C for 10 min. Sensitivity indices were 0.772 (CSF), 0.595 (urine) and 0.317 (serum) for CIE, and 0.914 (CSF), 0.930 (urine) and 0.683 (serum) for LA in relation to the 42 positive bacterial cultures. The optimal diagnostic efficacy reached 52% for CIE and 72% for LA when urine was concentrated 20-to 30-fold. These data show that immunological tests of urine samples were more effective than bacterial culture for diagnosing bacterial meningitis and may be indicated when negative results are obtained for tested by bacterial culture and immunoassay methods

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.