RESUMO
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.
Assuntos
Cromatografia , Técnica Indireta de Fluorescência para Anticorpo , Infecções por Vírus Respiratório Sincicial/diagnóstico , Vírus Sincicial Respiratório Humano , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Doença Aguda , Pré-Escolar , Cromatografia/métodos , Humanos , Líquido da Lavagem Nasal/virologia , Nasofaringe/virologia , Valor Preditivo dos Testes , RNA Viral/genética , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/imunologia , Sensibilidade e EspecificidadeRESUMO
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.
Assuntos
Pré-Escolar , Humanos , Cromatografia , Técnica Indireta de Fluorescência para Anticorpo , Vírus Sincicial Respiratório Humano , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Infecções por Vírus Respiratório Sincicial/diagnóstico , Doença Aguda , Cromatografia/métodos , Líquido da Lavagem Nasal/virologia , Nasofaringe/virologia , Valor Preditivo dos Testes , RNA Viral/genética , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/imunologia , Sensibilidade e EspecificidadeRESUMO
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.
Assuntos
Antígenos Virais/biossíntese , Vírus do Sarampo/crescimento & desenvolvimento , Animais , Antígenos Virais/imunologia , Chlorocebus aethiops , Ensaio de Imunoadsorção Enzimática , Testes de Hemaglutinação , Humanos , Vírus do Sarampo/imunologia , Ultracentrifugação , Células Vero , Cultura de VírusRESUMO
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.
Assuntos
Humanos , Animais , Antígenos Virais/biossíntese , Vírus do Sarampo/crescimento & desenvolvimento , Antígenos Virais/imunologia , Chlorocebus aethiops , Ensaio de Imunoadsorção Enzimática , Testes de Hemaglutinação , Ultracentrifugação , Células Vero , Cultura de Vírus , Vírus do Sarampo/imunologiaRESUMO
A Dot-ELISA using a measles virus (MV) antigen obtained by sodium deoxycholate treatment was standardized and evaluated for IgM and IgG antibody detection in measles patients and measles-vaccinated subjects. A total of 192 serum samples were studied, comprising 47 from patients with acute and convalescent measles, 55 from 9-month old children prior to measles vaccination and 41 from children of the same age after vaccination, and 49 from patients with unrelated diseases. The diagnostic performances of the IgG Dot-ELISA and IgG immunofluorescence test (IFT) were found to be close, varying from 0.97 to 1.00 in sensitivity and the specificities were maximum (1.00). Nevertheless, the sensitivity of the IgM Dot-ELISA (0.85) was higher than that (0.63) of the IgM IFT, although both assays had comparably high (1.00) specificities. The IgM Dot-ELISA in particular proved to be more sensitive in relation to other assays studied by revealing antibodies in 80.0% (12/15) of vaccinated children on the 15th day after immunization. In contrast, the IgM IFT, failed to detect antibodies in the same group of vaccinated children. The stability of the MV antigen was longer than that of the IFT antigen, and the reproducibility of the Dot-Elisa was satisfactory.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Sarampo/diagnóstico , Ensaio de Imunoadsorção Enzimática/normas , Feminino , Imunofluorescência , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Lactente , Masculino , Sarampo/sangue , Sarampo/tratamento farmacológico , Vacina contra Sarampo/uso terapêutico , Vírus do Sarampo/imunologia , Sensibilidade e EspecificidadeRESUMO
The complement system (C) of Calomys callosus, Rengger, 1830 (Rodentia, Cricetidae), a wild reservoir for several infectious agents in Latin America, was characterized. Sera from normal adult animals lysed sheep erythrocytes (Es) previously sensitized with rabbit serum anti-Es (Ar) in the presence of veronal-buffered saline containing 0.15 mM CaCl2 and 0.5 mM MgCl2, pH 7.4, or unsensitized rabbit erythrocytes (Er) in the presence of one-half isotonic strength veronal-buffered-saline containing 2.5% glucose, 2 mM MgCl2 and 10 mM EGTA, pH 7.4. Both hemolytic curves were sigmoidal in shape, with CH50 values of 30-40 for females and 20-30 for males. C5, determined hemolytically using the intermediate cells EsArClm4m2m3m, was approximately 4.5 x 10(8)/ml and 4.0 x 10(8)/ml for females and males, respectively. Immunochemical serum analyses by double immunodiffusion or by immunoblotting using polyclonal antisera against human C1s, C1q, C2, C3, C4, C5, C8 and factors B, I and H indicated that C. callosus C components factor B, C4 and C3 cross-reacted with the corresponding human C components. Thus, C. callosus was found to contain effective classical and alternative pathways (CP, AP) and common pathways, reasonable amounts of C5 and common epitopes in the key C components, factor B, C4 and C3, which were preserved during evolution.