1887

Abstract

A direct PCR test (DT-PCR) was established to detect DNA in clinical samples from patients with suspected bacterial meningitis. Specific primers for the 16S rDNA of were designed to amplify a 600 bp DNA fragment. One hundred and ninety-three clinical samples were analysed, corresponding to 114 samples from patients diagnosed as positive and 79 as negative for infection by using conventional methods (culture, latex agglutination and counterimmunoelectrophoresis). These samples were submitted to PCR by two different clinical sample preparation approaches (with and without DNA extraction and purification) and submitted to different PCR protocols to improve the results. In agarose gel detection, the sensitivity value for DT-PCR was 88.5 % and, using dot-blot DNA detection, the sensitivity increased to 96.4 %. The detection limit for meningococcus in cerebrospinal fluid was 2×10 c.f.u. ml. Serogroup prediction was done using a multiplex PCR protocol and the sensitivity was 83 % for agarose gel DNA detection and 96.4 % using dot-blot DNA detection.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.05192-0
2003-09-01
2019-11-12
Loading full text...

Full text loading...

/deliver/fulltext/jmm/52/9/JM520911.html?itemId=/content/journal/jmm/10.1099/jmm.0.05192-0&mimeType=html&fmt=ahah

References

  1. Atobe, J. H., Hirata, M. H., Hoshino-Shimizu, S., Schmal, M. R. & Mamizuka, E. M. ( 2000;). One-step heminested PCR for amplification of Neisseria meningitidis DNA in cerebrospinal fluid. J Clin Lab Anal 14, 193–199.[CrossRef]
    [Google Scholar]
  2. Bäckman, A., Lantz, P.-G., Rådström, P. & Olcén, P. ( 1999;). Evaluation of an extended diagnostic PCR assay for detection and verification of the common causes of bacterial meningitis in CSF and other biological samples. Mol Cell Probes 13, 49–60.[CrossRef]
    [Google Scholar]
  3. Ballard, T. L., Roe, M. H., Wheeler, R. C., Todd, J. K. & Glode, M. P. ( 1987;). Comparison of three latex agglutination kits and counterimmunoelectrophoresis for the detection of bacterial antigens in a pediatric population. Pediatr Infect Dis J 6, 630–634.[CrossRef]
    [Google Scholar]
  4. Borrow, R., Claus, H., Guiver, M., Smart, L., Jones, D. M., Kaczmarski, E. B., Frosch, M. & Fox, A. J. ( 1997;). Non-culture diagnosis and serogroup determination of meningococcal B and C infection by a sialyltransferase (siaD) PCR ELISA. Epidemiol Infect 118, 111–117.[CrossRef]
    [Google Scholar]
  5. Coovadia, Y. M., van den Ende, J. & Solwa, Z. ( 1989;). Comparison of gram stain and latex agglutination for diagnosis of meningococcal meningitis. Lancet ii, 677. 677.
    [Google Scholar]
  6. Corless, C. E., Guiver, M., Borrow, R., Edwards-Jones, V., Fox, A. J. & Kaczmarski, E. B. ( 2001;). Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in suspected cases of meningitis and septicemia using real-time PCR. J Clin Microbiol 39, 1553–1558.[CrossRef]
    [Google Scholar]
  7. Diggle, M. A., Edwards, G. F. S. & Clarke, S. C. ( 2001;). Automation of fluorescence-based PCR for confirmation of meningococcal disease. J Clin Microbiol 39, 4518–4519.[CrossRef]
    [Google Scholar]
  8. Gray, L. D. & Fedorko, D. P. ( 1992;). Laboratory diagnosis of bacterial meningitis. Clin Microbiol Rev 5, 130–145.
    [Google Scholar]
  9. Greisen, K., Loeffelholz, M., Purohit, A. & Leong, D. ( 1994;). PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid. J Clin Microbiol 32, 335–351.
    [Google Scholar]
  10. Guiver, M., Borrow, R., Marsh, J., Gray, S. J., Kaczmarski, E. B., Howells, D., Boseley, P. & Fox, A. J. ( 2000;). Evaluation of the Applied Biosystems automated Taqman polymerase chain reaction system for the detection of meningococcal DNA. FEMS Immunol Med Microbiol 28, 173–179.[CrossRef]
    [Google Scholar]
  11. Hall, L. M. C., Duke, B. & Urwin, G. ( 1995;). An approach to the identification of the pathogens of bacterial meningitis by the polymerase chain reaction. Eur J Clin Microbiol Infect Dis 14, 1090–1094.[CrossRef]
    [Google Scholar]
  12. Jenkins, P., Barnes, R. A. & Coakley, W. T. ( 1997;). Detection of meningitis antigens in buffer and body fluids by ultrasound-enhanced particle agglutination. J Immunol Methods 205, 191–200.[CrossRef]
    [Google Scholar]
  13. Kotilainen, P., Jalava, J., Meurman, O., Lehtonen, O.-P., Rintala, E., Seppälä, O.-P., Eerola, E. & Nikkari, S. ( 1998;). Diagnosis of meningococcal meningitis by broad-range bacterial PCR with cerebrospinal fluid. J Clin Microbiol 36, 2205–2209.
    [Google Scholar]
  14. Kristiansen, B.-E., Ask, E., Jenkins, A., Fermer, C., Rådström, P. & Skold, O. ( 1991;). Rapid diagnosis of meningococcal meningitis by polymerase chain reaction. Lancet 337, 1568–1569.[CrossRef]
    [Google Scholar]
  15. La Scolea, L. J., Jr & Dryja, D. ( 1984;). Quantitation of bacteria in cerebrospinal fluid and blood of children with meningitis and its diagnostic significance. J Clin Microbiol 19, 187–190.
    [Google Scholar]
  16. Ley, B. E., Linton, C. J., Bennett, D. M. C., Jalal, H., Foot, A. B. M. & Millar, M. R. ( 1998;). Detection of bacteraemia in patients with fever and neutropenia using 16S rRNA gene amplification by polymerase chain reaction. Eur J Clin Microbiol Infect Dis 17, 247–253.[CrossRef]
    [Google Scholar]
  17. Margall, N., Majó, M., Sánchez, F. & 7 other authors ( 1999;). Evaluation of the polymerase chain reaction technique for the diagnosis of meningitis caused by Neisseria meningitidis and Haemophilus influenzae. Enferm Infecc Microbiol Clin 17, 3–8 (in Spanish).
    [Google Scholar]
  18. McGowan, K. L. ( 1992;). Diagnostic value of latex agglutination tests for bacterial infection. Rep Pediatr Infect Dis 2, 31–32.
    [Google Scholar]
  19. Nato, F., Mazie, J. C., Fournier, J. M., Slizewicz, B., Sagot, N., Guibourdenche, M., Postic, D. & Riou, J. Y. ( 1991;). Production of polyclonal and monoclonal antibodies against group A, B, and C capsular polysaccharides of Neisseria meningitidis and preparation of latex reagents. J Clin Microbiol 29, 1447–1452.
    [Google Scholar]
  20. Ni, H., Knight, A. I., Cartwright, K., Palmer, W. H. & McFadden, J. ( 1992;). Polymerase chain reaction for diagnosis of meningococcal meningitis. Lancet 340, 1432–1434.[CrossRef]
    [Google Scholar]
  21. Orvelid, P., Bäckman, A. & Olcén, P. ( 1999;). PCR identification of the serogroup A Neisseria meningitidis gene in cerebrospinal fluid. Scand J Infect Dis 31, 481–483.[CrossRef]
    [Google Scholar]
  22. Perkins, M. D., Mirrett, S. & Reller, L. B. ( 1995;). Rapid bacterial antigen detection is not clinically useful. J Clin Microbiol 33, 1486–1491.
    [Google Scholar]
  23. Pollard, A. J., Probe, G., Trombley, C. & 10 other authors ( 2002;). Evaluation of a diagnostic polymerase chain reaction assay for Neisseria meningitidis in North America and field experience during an outbreak. Arch Pathol Lab Med 126, 1209–1215.
    [Google Scholar]
  24. Probert, W. S., Bystrom, S. L., Khashe, S., Schrader, K. N. & Wong, J. D. ( 2002;). 5′ exonuclease assay for detection of serogroup Y Neisseria meningitidis. J Clin Microbiol 40, 4325–4328.[CrossRef]
    [Google Scholar]
  25. Rådström, P., Bäckman, A., Qian, N., Kragsbjerg, P., Påhlson, C. & Olcén, P. ( 1994;). Detection of bacterial DNA in cerebrospinal fluid by an assay for simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and streptococci using a seminested PCR strategy. J Clin Microbiol 32, 2738–2744.
    [Google Scholar]
  26. Raskin, S., Phillips, J. A., III, Kaplan, G., McClure, M. & Vnencak-Jones, C. ( 1992;). Cystic fibrosis genotyping by direct PCR analysis of Guthrie blood spots. PCR Methods Appl 2, 154–156.[CrossRef]
    [Google Scholar]
  27. Rossetti, M. L. R., Jardim, S. B., Rodrigues, V. F. S., Moura, A. R., Oliveira, H. & Zaha, A. ( 1997;). Improvement of Mycobacterium tuberculosis detection in clinical samples using DNA purified by glass matrix. J Microbiol Methods 28, 139–146.[CrossRef]
    [Google Scholar]
  28. Saruta, K., Matsunaga, T., Kono, M., Hoshina, S., Kanemoto, S., Sakai, O. & Machida, K. ( 1997;). Simultaneous detection of Streptococcus pneumoniae and Haemophilus influenzae by nested PCR amplification from cerebrospinal fluid samples. FEMS Immunol Med Microbiol 19, 151–157.[CrossRef]
    [Google Scholar]
  29. Seward, R. J. & Towner, K. J. ( 2000;). Evaluation of a PCR-immunoassay technique for detection of Neisseria meningitidis in cerebrospinal fluid and peripheral blood. J Med Microbiol 49, 451–456.
    [Google Scholar]
  30. Sobanski, M. A., Vince, R., Biagini, G. A., Cousins, C., Guiver, M., Gray, S. J., Kaczmarski, E. B. & Coakley, W. T. ( 2002;). Ultrasound enhanced detection of individual meningococcal serogroups by latex immunoassay. J Clin Pathol 55, 37–40.[CrossRef]
    [Google Scholar]
  31. Taha, M.-K. ( 2000;). Simultaneous approach for nonculture PCR-based identification and serogroup prediction of Neisseria meningitidis. J Clin Microbiol 38, 855–857.
    [Google Scholar]
  32. Takeda, A. K., Umekita, L. F., Boscardin, N. B., Melles, C. A. E. & Taunay, A. E. ( 1979;). Imunoeletroforese cruzada no diagnóstico da meningite por Haemophilus influenzae tipo b. Rev Inst Adolfo Lutz 39, 165–169 (in Portuguese).
    [Google Scholar]
  33. Tzanakaki, G., Urwin, R., Musilek, M., Kriz, P., Kremastinou, J., Pangalis, A., Blackwell, C. C. & Maiden, M. C. J. ( 2001;). Phenotypic and genotypic approaches to characterization of isolates of Neisseria meningitidis from patients and their close family contacts. J Clin Microbiol 39, 1235–1240.[CrossRef]
    [Google Scholar]
  34. van Soolingen, D., de Hass, P. E. W., Hermans, P. W. M. & van Embden. J. D. A. ( 1994;). DNA fingerprinting of Mycobacterium tuberculosis. Methods Enzymol 235, 196–205.
    [Google Scholar]
  35. Yakubu, D. E., Abadi, F. J. & Pennington, T. H. ( 1999;). Molecular typing methods for Neisseria meningitidis. J Med Microbiol 48, 1055–1064.[CrossRef]
    [Google Scholar]
  36. Yamamoto, Y. ( 2002;). PCR in diagnosis of infection: detection of bacteria in cerebrospinal fluids. Clin Diagn Lab Immunol 9, 508–514.
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.05192-0
Loading
/content/journal/jmm/10.1099/jmm.0.05192-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error