Dot-ELISA para detecção de antígenos polissacarídicos de pneumococos em amostras de líquido pleural: Comparação com cultura bacteriana, contraimunoeletroforese e látex-aglutinação

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 0.1 M EDTA pH 7.5 at 90°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.Dot-ELISA para detecção de antígenos polissacarídicos de pneumococos foi padronizado em vista da necessidade de se ter um diagnóstico rápido e eficaz para pneumonia pneumocócica aguda. Um total de 480 amostras de líquido pleural sendo 442 de crianças com diagnóstico clínico e laboratorial de pneumonia bacteriana e 38 de pacientes com tuberculose, mais 20 amostras de soros sanguíneos de crianças sadias foram avaliadas no Dot-ELISA. As amostras foram tratadas previamente a 90°C por 10 min com EDTA 0,1 M de pH 7,5 e aplicadas sobre membrana de nitrocelulose. Para a detecção de antígeno pneumocócico foi empregado omniserum pneumocócico diluído a 1:200. Os resultados de Dot-ELISA avaliados em comparação com os resultados de cultura bacteriana, contra-imunoeletroforese e látex-aglutinação apresentaram índices de 0,940 para sensibilidade, 0,830 para especificidade e 0,760 para concordância. Omniserum pneumocócico mostrou ser um ótimo soro polivalente para a detecção de antígenos pneumocócicos em Dot-ELISA e, essa técnica provou ser uma alternativa prática e eficaz para o diagnóstico de pneumonias pneumocócicas

Evolution of an International External Quality Assurance Model To Support Laboratory Investigation of Streptococcus pneumoniae, Developed for the SIREVA Project in Latin America, from 1993 to 2005▿

In 1993 the Pan American Health Organization initiated a laboratory-based surveillance system, called the SIREVA project, to learn about Streptococcus pneumoniae invasive disease in Latin American children. In 1994, National Laboratories in six countries were trained to perform serotyping and antibiotic susceptibility testing using broth microdilution to determine the MIC for specified antibiotics. An international External Quality Assurance (EQA) program was developed to monitor and support ongoing laboratory performance. The EQA program was coordinated by the National Centre for Streptococcus (NCS), Edmonton, Canada, and included external proficiency testing (EPT) and a validation process requiring regular submission of a sample of isolates from each laboratory to the NCS for verification of the serotype and MIC. In 1999, the EQA program was decentralized to use three of the original laboratories as regional quality control centers to address operational concerns and to accommodate the growth of the laboratory network to more than 20 countries including the Caribbean region. The overall EPT serotyping accuracies for phase I (1993 to 1998) and phase II (1999 to 2005) were 88.0 and 93.8%, respectively; the MIC correlations within ±1 log2 dilution of the expected result were 83.0 and 91.0% and the interpretive category agreements were 89.1 and 95.3%. Overall, the validation process serotyping accuracies for phases I and II were 81.9 and 88.1%, respectively, 80.4 and 90.5% for MIC agreement, and 85.8 and 94.3% for category agreement. These results indicate a high level of testing accuracy in participating National Laboratories and a sustained increase in EQA participation in Latin America and the Caribbean

Cytomegalovirus in colorectal cancer and idiopathic ulcerative colitis Citomegalovírus em câncer coloretal e colite idiopática ulcerativa

Cytomegalovirus (CMV) is a genus in the family Herpesviridae that has been associated with gastrointestinal syndromes. In this work we looked for a possible association of CMV infection with colorectal cancer and ulcerative colitis (UC). Blood and enteric tissue samples of 14 patients with colorectal cancer and of 21 with UC were subjected to a nested-PCR that amplifies part of the gB gene of CMV and also to immunohistochemistry using a specific monoclonal antibody to IE 76kDa protein of CMV. CMV was detected by nested-PCR in the blood and/or the enteric tissue of nine (64.3%) colorectal cancer and 16 (76.2%) ulcerative colitis patients. In the immunohistochemistry it was observed that 12 (12/21, 57.1%) positive enteric tissue samples of patients with UC and none from patients with colorectal cancer (0/14) were positive to CMV. The positivity of CMV infections in the UC patient group (12/21, 57.1%) showed by both techniques, was significantly higher (p = 0.015) than that observed for colorectal cancer patients (2/14, 14.3%). These results suggest an association of ulcerative colitis with CMV infection of the enteric tissue.<br>Os Cytomegalovírus (CMV) são um gênero da família Herpesviridae, que pode estar associado a síndromes gastrointestinais. No presente trabalho buscamos uma possível associação da infecção por CMV com câncer coloretal e retocolite ulcerativa (RCU). Amostras de sangue e tecido entérico de 14 pacientes com câncer coloretal e 21 com RCU foram submetidas a uma nested-PCR que amplifica parte do gene gB do CMV e a uma imunohistoquímica utilizando um anticorpo monoclonal específico para proteína IE 76Kda de CMV. CMV foi detectado pela nested-PCR em sangue e/ou tecido entérico de 9 (64,3%) dos pacientes com câncer coloretal e 16 (76,2%) dos pacientes com RCU. Na imunohistoquímica foram observadas 12 (57,1%) amostras positivas para CMV nos pacientes com RCU e nos pacientes com câncer coloretal o CMV não foi detectado em nenhuma amostra. A positividade das infecções no grupo de pacientes com RCU (12/21, 57.1%) foi significantemente mais alta (p = 0,015) que aquela observada nos pacientes com câncer coloretal (2/14, 14.3%). Estes resultados sugerem uma associação da presença de CMV no tecido entérico com RCU