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

Requejo, Henry I.Z.; Alkmin, Maria das Graças A.; Almeida, Regina G.; Casagrande, Silvana T.; Cocozza, Ana Maria; Lotufo, João Paulo B.; Waetge, Aurora R.P.; Rodrigues, Joaquim C.

doi:10.1590/S0036-46651994000600010

Abstracts

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; Pneumococcal antigen detection; Streptococcus pneumoniae; Pleural fluid effusion; Bacterial 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.

IMMUNOLOGY

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

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

Henry I.Z. Requejo^I; Maria das Graças A. Alkmin^I; Regina G. Almeida^II; Silvana T. Casagrande^II; Ana Maria Cocozza^III; João Paulo B. Lotufo^IV; Aurora R.P. Waetge^V; Joaquim C. Rodrigues^V

^ISeção de Imunologia, Instituto Adolfo Lutz, São Paulo, Brasil

^IISeção de Bacteriologia, Instituto Adolfo Lutz, São Paulo, Brasil

^IIIHospital Infantil Menino Jesus, São Paulo, Brasil

^IVHospital Universitário da Universidade de São Paulo, São Paulo, Brasil

^VInstituto da Criança do Hospital das Clínicas de São Paulo, São Paulo, Brasil

^{Correspondence to} Correspondence to: Dr. Henry I. Z. Requejo Instituto Adolfo Lutz, Seção de Imunologia Av. Dr. Arnaldo 355 01246-902 São Paulo, SP, Brasil

SUMMARY

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.

Keywords: Dot-ELISA; Pneumococcal antigen detection; Streptococcus pneumoniae, Pleural fluid effusion; Bacterial pneumonia.

RESUMO

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.

Full text available only in PDF format.

Texto completo disponível apenas em PDF.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the contribution of Dr. Sumie H. Shimizu, in the evaluation of the present work.

Recebido para publicação em 01/02/1994.

Aceito para publicação em 16/06/1994.

1. BURMAN, L.A.; TROLLFORS, B.; ANDERSON, B. et al. - Diagnosis of pneumonia by cultures, bacterial and viral antigen detection tests, and serology with special reference to antibodies against pneumococcal antigens. J. infect. Dis., 163: 1087-1093, 1993.
2. CAPUTO, G.M.; APPELBAUM, P.C. & LIU, H.H. - Infections due to penicilin-resistant pneumococci. Clinical, epidemiologic, and microbiologic features. Arch. intern. Med., 153: 1301-1310, 1993.
3. CENTERS FOR DISEASE CONTROL. - Recommendation of the public health service advisory commitee on immunization practice. Increase in pneumonia mortality among young adults and the HIV epidemic. New York City, United States. M.M.W.R., 37: 593-596, 1988.
4. CEROSALETTI, K.M.; ROGHMANN, M.C. & BENTLEY, D.W. - Comparison of latex agglutination and counterimmunoelectrophoresis for the detection of pneumococcal antigen in elderly pneumonia patients. J. clin. Microbiol., 22: 553-556, 1985.
5. COCOZZA, A.M.; REQUEJO, H.I.Z.; SERSON, S. & GUIMARĂES, B.R. - Avaliaçăo da efetividade da utilizaçăo clínica de métodos imunodiagnósticos para etiología de pneumonias agudas na infância. In: CONGRESSO BRASILEIRO DE PNEUMOLOGIA PEDIÁTRICA, 5, CONGRESSO LATINO-AMERICANO DE FIBROSE CÍSTICA, 5., JORNADA BRASILEIRA DE FIBROSE CÍSTICA, 5, Olinda, 1993. Anais. p. 28.
6. CONTROL of acute respiratory infections in the Americas. Bull. Pan Amer. Hlth. Org., 26: 274-278, 1992.
7. COONROD, J.D. & RYTEL, M.W. - Detection of type-specific pneumococcal antigens by counterimmunoelectrophoresis. I. Methodology and immunologic properties of pneumococcal antigens. J. Lab. clin. Med., 81: 770-777, 1973.
8. DOSKELAND, S.O. & BERDAL, B.P. - Bacterial antigen detection in body fluids: methods for rapid antigen concentration and reduction of nonspecific reactions. J. clin. Microbiol., 11: 380-384, 1980.
9. FELDMAN, W.E. - Effect of prior antibiotic therapy on concentrations of bacteria in CSF. Amer. J. Dis. Child., 132: 672-674, 1978.
10. FINLAND, M. - Diagnostic lung puncture. Pediatrics, 44: 471-473, 1969.
11. FLEISS, J.L. - The measurement of interrater agreement. In: STATISTICAL METHODS FOR RATES AND PROPORTIONS. 2 ed. New York, John Wiley & Sons, 1981. p. 212-236.
12. FLETCHER, R.M.; FLETCHER, S.W. & WAGNER, E.H. - Clinical epidemiology, the essentials. Baltimore, Waverly, 1983.
13. FRANSEN, H. & TUNEVALL, G. - Bacteria and serological reactions against bacteria in patients hospitalized with acute respiratory illness. Scand. J. infect. Dis., 1: 191-202, 1969.
14. GADOMSKI, A.M. - Potential interventions for preventing pneumonia among young children: lack of effect of antibiotic treatment for upper respiratory infections. Pediat. infect. Dis. J., 12: 115-120, 1993.
15. HEIDELBERGER, M. - Cross-reactions of polysaccharides of Staphylococci and Streptococci in anti-pneumococcal and other antisera. Molec. Immunol., 21: 1011-1013, 1984.
16. HEIDELBERGER, M. & NIMMICH, W. - Immunochemical relationships between bacteria belonging to two separate families: Pneumococci and Klebsiellae. Immunochemistry, 13: 67-80, 1976.
17. HELDRICH, F.J. - Diplococcus pneumoniae bacteremia. Amer. J. Dis. Child., 119: 12-17, 1970.
18. HOLLOWAY, Y.; BOERSNA, W.G.; KUTTSCHRUTTER, H. & SNIJDER, J.A.M. - Detection of pneumococcal capsular antigen in the presence of penicilin in vitro. Scand. J. infect. Dis., 25: 317-322, 1993.
19. INGRAM, D.L.; PEARSON, A.W. & OCCHIUTI, A.R. - Detection of baterial antigens in body fluids with the Wellcogen Haemophilus influenzae type b, Streptococcus pneumoniae and Neisseria meningitidis (ACYW 135) latex agglutination tests. J. clin. Microbiol., 18: 1119-1121, 1983.
20. KALIN, M. & LINDBERG, A.A. - Diagnosis of pneumococcal pneumonia: a comparison between microscopic examination of expectorate, antigen detection and cultural procedures. Scand. J. infect. Dis., 15: 247-255, 1983.
21. KORPPI, M.; KOSKELA, M.; JALONEN, E. & LEINONEN, M. -Serologically indicated pneumococcal respiratory infection in children. Scand. J. infect. Dis., 24: 437-443, 1992.
22. LUND, E. & RASMUSSEN, P. - Omniserum, a diagnostic pneumococcus serum, reacting with the 82 known types of Pneumococcus. Acta path. microbiol. scand., 68: 458-460, 1966.
23. MACONE, A.B.; ARAKERE, G.; LETOURNEAU, J.M. & GOLDMANN, D.A. - Comparison of a new, rapid enzyme-linked immunosorbent assay with latex particle agglutination for the detection of Hemophilus influenzae type b infections. J. clin. Microbiol., 21: 711-714, 1985.
24. MELLES, C.E.A.; LEE, I.M.L. & TAUNAY, A.E. - Pesquisa de antibacterianos no líquido cefalorraquidiano. Rev. Inst. Adolfo Lutz, 44: 155-159, 1984.
25. MUFSON, M. A. - Streptococcus pneumoniae. In: MANDELL, G.L.; DOUGLAS Jr., R.G. & BENNETT, J.E., ed. Principles and practice of infectious diseases. New York, Churchill Livingstone, 1990. p. 1538-1550.
26. MUFSON, M.A.; KRUSS, D.M.; WASOL, R.E. & METZER, W.I. - Capsular types and outcome of bacteremic pneumococcal disease in the antibiotic era. Arch. intern. Med., 134: 505-510, 1974.
27. REQUEJO, H.I.Z. - Polyvalent pneumococcal polysaccharide vaccines. A review of literature. Rev. Hosp. Clin. Fac. Med. S. Paulo, 48: 130-138, 1993.
28. REQUEJO, H.I.Z.; MATSUMOTO, T.K.; LOTUFO, J.P.B. et al. - Detecçăo de antígenos bacterianos em pneumonia aguda: métodos de preparaçăo das amostras de urina, soro e líquido pleural para os testes de imunodiagnóstico. Rev. Hosp. Clin. Fac. Med. S. Paulo, 46: 19-25, 1991.
29. REQUEJO, H.I.Z.; NASCIMENTO, C.M.P.C. & FAHRAT, C.K. - Comparison of counterimmunoelectrophoresis, latex agglutination and bacterial culture for the diagnosis of bacterial meningitis using urine, serum and cerebrospinal fluid samples. Braz. J. med. biol. Res., 25: 357-367, 1992.
30. REQUEJO, H.I.Z. & FERREIRA, A.W. - Diagnóstico rápido de meningites bacterianas pelo teste de latex aglutinaçăo. LAES-HAES, 82: 18-26, 1993.
31. TAUNAY, A.E.; AUSTRIAN, R.; LANDGRAF, L.M. et al. - Sorotipos de Streptococcus pneumoniae isolados de líquido cefalorraquidiano no período de 1977-1988 na cidade de Săo Paulo, Brasil. Rev. Inst. Med. trop. S. Paulo, 32: 11-15, 1990.
32. YOLKEN, R.N.; DAVIS, D.; WINKELSTEIN, J.; RUSSELL, H. & SIPPEL, J.E. - Enzyme immunoassay for detection of pneumococcal antigen in cerebrospinal fluid. J. clin. Microbiol., 20: 802-805, 1984.

Correspondence to:

Dr. Henry I. Z. Requejo

Instituto Adolfo Lutz, Seção de Imunologia

Av. Dr. Arnaldo 355

01246-902 São Paulo, SP, Brasil

Publication Dates

Publication in this collection
05 July 2006
Date of issue
Dec 1994

History

Accepted
16 June 1994
Received
01 Feb 1994

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

[1] 1. BURMAN, L.A.; TROLLFORS, B.; ANDERSON, B. et al. - Diagnosis of pneumonia by cultures, bacterial and viral antigen detection tests, and serology with special reference to antibodies against pneumococcal antigens. J. infect. Dis., 163: 1087-1093, 1993.

[2] 2. CAPUTO, G.M.; APPELBAUM, P.C. & LIU, H.H. - Infections due to penicilin-resistant pneumococci. Clinical, epidemiologic, and microbiologic features. Arch. intern. Med., 153: 1301-1310, 1993.

[3] 3. CENTERS FOR DISEASE CONTROL. - Recommendation of the public health service advisory commitee on immunization practice. Increase in pneumonia mortality among young adults and the HIV epidemic. New York City, United States. M.M.W.R., 37: 593-596, 1988.

[4] 4. CEROSALETTI, K.M.; ROGHMANN, M.C. & BENTLEY, D.W. - Comparison of latex agglutination and counterimmunoelectrophoresis for the detection of pneumococcal antigen in elderly pneumonia patients. J. clin. Microbiol., 22: 553-556, 1985.

[5] 5. COCOZZA, A.M.; REQUEJO, H.I.Z.; SERSON, S. & GUIMARĂES, B.R. - Avaliaçăo da efetividade da utilizaçăo clínica de métodos imunodiagnósticos para etiología de pneumonias agudas na infância. In: CONGRESSO BRASILEIRO DE PNEUMOLOGIA PEDIÁTRICA, 5, CONGRESSO LATINO-AMERICANO DE FIBROSE CÍSTICA, 5., JORNADA BRASILEIRA DE FIBROSE CÍSTICA, 5, Olinda, 1993. Anais. p. 28.

[6] 6. CONTROL of acute respiratory infections in the Americas. Bull. Pan Amer. Hlth. Org., 26: 274-278, 1992.

[7] 7. COONROD, J.D. & RYTEL, M.W. - Detection of type-specific pneumococcal antigens by counterimmunoelectrophoresis. I. Methodology and immunologic properties of pneumococcal antigens. J. Lab. clin. Med., 81: 770-777, 1973.

[8] 8. DOSKELAND, S.O. & BERDAL, B.P. - Bacterial antigen detection in body fluids: methods for rapid antigen concentration and reduction of nonspecific reactions. J. clin. Microbiol., 11: 380-384, 1980.

[9] 9. FELDMAN, W.E. - Effect of prior antibiotic therapy on concentrations of bacteria in CSF. Amer. J. Dis. Child., 132: 672-674, 1978.

[10] 10. FINLAND, M. - Diagnostic lung puncture. Pediatrics, 44: 471-473, 1969.

[11] 11. FLEISS, J.L. - The measurement of interrater agreement. In: STATISTICAL METHODS FOR RATES AND PROPORTIONS. 2 ed. New York, John Wiley & Sons, 1981. p. 212-236.

[12] 12. FLETCHER, R.M.; FLETCHER, S.W. & WAGNER, E.H. - Clinical epidemiology, the essentials. Baltimore, Waverly, 1983.

[13] 13. FRANSEN, H. & TUNEVALL, G. - Bacteria and serological reactions against bacteria in patients hospitalized with acute respiratory illness. Scand. J. infect. Dis., 1: 191-202, 1969.

[14] 14. GADOMSKI, A.M. - Potential interventions for preventing pneumonia among young children: lack of effect of antibiotic treatment for upper respiratory infections. Pediat. infect. Dis. J., 12: 115-120, 1993.

[15] 15. HEIDELBERGER, M. - Cross-reactions of polysaccharides of Staphylococci and Streptococci in anti-pneumococcal and other antisera. Molec. Immunol., 21: 1011-1013, 1984.

[16] 16. HEIDELBERGER, M. & NIMMICH, W. - Immunochemical relationships between bacteria belonging to two separate families: Pneumococci and Klebsiellae. Immunochemistry, 13: 67-80, 1976.

[17] 17. HELDRICH, F.J. - Diplococcus pneumoniae bacteremia. Amer. J. Dis. Child., 119: 12-17, 1970.

[18] 18. HOLLOWAY, Y.; BOERSNA, W.G.; KUTTSCHRUTTER, H. & SNIJDER, J.A.M. - Detection of pneumococcal capsular antigen in the presence of penicilin in vitro. Scand. J. infect. Dis., 25: 317-322, 1993.

[19] 19. INGRAM, D.L.; PEARSON, A.W. & OCCHIUTI, A.R. - Detection of baterial antigens in body fluids with the Wellcogen Haemophilus influenzae type b, Streptococcus pneumoniae and Neisseria meningitidis (ACYW 135) latex agglutination tests. J. clin. Microbiol., 18: 1119-1121, 1983.

[20] 20. KALIN, M. & LINDBERG, A.A. - Diagnosis of pneumococcal pneumonia: a comparison between microscopic examination of expectorate, antigen detection and cultural procedures. Scand. J. infect. Dis., 15: 247-255, 1983.

[21] 21. KORPPI, M.; KOSKELA, M.; JALONEN, E. & LEINONEN, M. -Serologically indicated pneumococcal respiratory infection in children. Scand. J. infect. Dis., 24: 437-443, 1992.

[22] 22. LUND, E. & RASMUSSEN, P. - Omniserum, a diagnostic pneumococcus serum, reacting with the 82 known types of Pneumococcus. Acta path. microbiol. scand., 68: 458-460, 1966.

[23] 23. MACONE, A.B.; ARAKERE, G.; LETOURNEAU, J.M. & GOLDMANN, D.A. - Comparison of a new, rapid enzyme-linked immunosorbent assay with latex particle agglutination for the detection of Hemophilus influenzae type b infections. J. clin. Microbiol., 21: 711-714, 1985.

[24] 24. MELLES, C.E.A.; LEE, I.M.L. & TAUNAY, A.E. - Pesquisa de antibacterianos no líquido cefalorraquidiano. Rev. Inst. Adolfo Lutz, 44: 155-159, 1984.

[25] 25. MUFSON, M. A. - Streptococcus pneumoniae. In: MANDELL, G.L.; DOUGLAS Jr., R.G. & BENNETT, J.E., ed. Principles and practice of infectious diseases. New York, Churchill Livingstone, 1990. p. 1538-1550.

[26] 26. MUFSON, M.A.; KRUSS, D.M.; WASOL, R.E. & METZER, W.I. - Capsular types and outcome of bacteremic pneumococcal disease in the antibiotic era. Arch. intern. Med., 134: 505-510, 1974.

[27] 27. REQUEJO, H.I.Z. - Polyvalent pneumococcal polysaccharide vaccines. A review of literature. Rev. Hosp. Clin. Fac. Med. S. Paulo, 48: 130-138, 1993.

[28] 28. REQUEJO, H.I.Z.; MATSUMOTO, T.K.; LOTUFO, J.P.B. et al. - Detecçăo de antígenos bacterianos em pneumonia aguda: métodos de preparaçăo das amostras de urina, soro e líquido pleural para os testes de imunodiagnóstico. Rev. Hosp. Clin. Fac. Med. S. Paulo, 46: 19-25, 1991.

[29] 29. REQUEJO, H.I.Z.; NASCIMENTO, C.M.P.C. & FAHRAT, C.K. - Comparison of counterimmunoelectrophoresis, latex agglutination and bacterial culture for the diagnosis of bacterial meningitis using urine, serum and cerebrospinal fluid samples. Braz. J. med. biol. Res., 25: 357-367, 1992.

[30] 30. REQUEJO, H.I.Z. & FERREIRA, A.W. - Diagnóstico rápido de meningites bacterianas pelo teste de latex aglutinaçăo. LAES-HAES, 82: 18-26, 1993.

[31] 31. TAUNAY, A.E.; AUSTRIAN, R.; LANDGRAF, L.M. et al. - Sorotipos de Streptococcus pneumoniae isolados de líquido cefalorraquidiano no período de 1977-1988 na cidade de Săo Paulo, Brasil. Rev. Inst. Med. trop. S. Paulo, 32: 11-15, 1990.

[32] 32. YOLKEN, R.N.; DAVIS, D.; WINKELSTEIN, J.; RUSSELL, H. & SIPPEL, J.E. - Enzyme immunoassay for detection of pneumococcal antigen in cerebrospinal fluid. J. clin. Microbiol., 20: 802-805, 1984.

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