1887

Abstract

The development and clinical evaluation of a LightCycler PCR assay, including an internal process control (IPC), to detect the autolysin gene in clinical samples is reported. The assay was developed to provide a second target for use in conjunction with existing pneumolysin PCR assays to increase the reliability of non-culture PCR diagnosis of pneumococcal infection. Primers amplify a 173 bp fragment of the autolysin gene (), which is detected by fluorescence-labelled hybridization probes. An IPC was designed to check for the presence of PCR inhibitors and loss of assay sensitivity. The IPC product was amplified by the primers and detected by a second set of hybridization probes. The analytical specificity of the autolysin PCR assay was 100 % against 39 other bacterial species tested; these included related streptococci and other organisms. The assay, which could reliably detect 50 fg purified pneumococcal DNA per reaction, was capable of distinguishing between and atypical and strains known to contain the gene. Using DNA extracts from a panel of EDTA bloods from patients with blood-culture-confirmed pneumococcal infection, the autolysin PCR had a sensitivity of 42.9 %, which was similar to a previously reported TaqMan pneumolysin PCR (43.8 %) run in parallel. Total agreement was shown between the autolysin assay and the pneumolysin TaqMan assay when used to test 23 culture-negative clinical samples, of which eight were positive by PCR, adding valuable clinical information. A specific autolysin-based LightCycler assay has been developed to complement pneumolysin PCR for the detection of in clinical samples. This should be a particularly useful tool for the rapid and sensitive diagnosis of pneumococcal meningitis, even after an antibiotic has been administered. However, poor sensitivity on blood samples limits its usefulness in other bacteraemic infections.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.05460-0
2004-03-01
2019-11-15
Loading full text...

Full text loading...

/deliver/fulltext/jmm/53/3/JM530303.html?itemId=/content/journal/jmm/10.1099/jmm.0.05460-0&mimeType=html&fmt=ahah

References

  1. Al-Soud, W. A. & Rådström, P. ( 2001;). Purification and characterization of PCR-inhibitory components in blood cells. J Clin Microbiol 39, 485–493.[CrossRef]
    [Google Scholar]
  2. Al-Soud, W. A., Jönsson, L. J. & Rådström, P. ( 2000;). Identification and characterization of immunoglobulin G in blood as a major inhibitor of diagnostic PCR. J Clin Microbiol 38, 345–350.
    [Google Scholar]
  3. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. ( 1990;). Basic local alignment search tool. J Mol Biol 215, 403–410.[CrossRef]
    [Google Scholar]
  4. 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]
  5. Dominguez, J., Gali, N., Blanco, S., Pedroso, P., Prat, C., Matas, L. & Ausina, V. ( 2001;). Detection of Streptococcus pneumoniae antigen by a rapid immunochromatographic assay in urine samples. Chest 119, 243–249.[CrossRef]
    [Google Scholar]
  6. Dowell, S. F., Garman, R. L., Liu, G., Levine, O. S. & Yang, Y. H. ( 2001;). Evaluation of Binax NOW, an assay for the detection of pneumococcal antigen in urine samples, performed among pediatric patients. Clin Infect Dis 32, 824–825.[CrossRef]
    [Google Scholar]
  7. Lorente, M. L. L., Falguera, M., Nogués, A., González, A. R., Merino, M. T. & Caballero, M. R. ( 2000;). Diagnosis of pneumococcal pneumonia by polymerase chain reaction (PCR) in whole blood: a prospective clinical study. Thorax 55, 133–137.[CrossRef]
    [Google Scholar]
  8. Marcos, M. A., Martinez, E., Almela, M., Mensa, J. & Jimenez de Anta, M. ( 2001;). New rapid antigen test for diagnosis of pneumococcal meningitis. Lancet 357, 1499–1500.[CrossRef]
    [Google Scholar]
  9. McAvin, J. C., Reilly, P. A., Roudabush, R. M. & 9 other authors ( 2001;). Sensitive and specific method for rapid identification of Streptococcus pneumoniae using real-time fluorescence PCR. J Clin Microbiol 39, 3446–3451.[CrossRef]
    [Google Scholar]
  10. Michelow, I. C., Lozano, J., Olsen, K., Goto, C., Rollins, N. K., Ghaffar, F., Rodriguez-Cerrato, V., Leinonen, M. & McCracken, G. H., Jr ( 2002;). Diagnosis of Streptococcus pneumoniae lower respiratory infection in hospitalized children by culture, polymerase chain reaction, serological testing, and urinary antigen detection. Clin Infect Dis 34, E1–E11.[CrossRef]
    [Google Scholar]
  11. Musher, D. M. ( 1992;). Infections caused by Streptococcus pneumoniae: clinical spectrum, pathogenesis, immunity, and treatment. Clin Infect Dis 14, 801–807.[CrossRef]
    [Google Scholar]
  12. Povlsen, K., Jensen, J. S. & Lind, I. ( 1998;). Detection of Ureaplasma urealyticum by PCR and biovar determination by liquid hybridization. J Clin Microbiol 36, 3211–3216.
    [Google Scholar]
  13. Rudolph, K. A., Parkinson, A. J., Black, C. M. & Mayer, L. W. ( 1993;). Evaluation of polymerase chain reaction for diagnosis of pneumococcal pneumonia. J Clin Microbiol 31, 2661–2666.
    [Google Scholar]
  14. Rys, P. N. & Persing, D. H. ( 1993;). Preventing false positives: quantitative evaluation of three protocols for inactivation of polymerase chain reaction amplification products. J Clin Microbiol 31, 2356–2360.
    [Google Scholar]
  15. Sachadyn, P. & Kur, J. ( 1998;). The construction and use of a PCR internal control. Mol Cell Probes 12, 259–262.[CrossRef]
    [Google Scholar]
  16. Samra, Z., Shmuely, H., Nahum, E., Paghis, D. & Ben-Ari, J. ( 2003;). Use of the NOW Streptococcus pneumoniae urinary antigen test in cerebrospinal fluid for rapid diagnosis of pneumococcal meningitis. Diagn Microbiol Infect Dis 45, 237–240.[CrossRef]
    [Google Scholar]
  17. Sheppard, C. L., Harrison, T. G., Kearns, A. M. & 7 other authors ( 2003;). Diagnosis of invasive pneumococcal infection by PCR amplification of Streptococcus pneumoniae genomic fragments in blood: a multi-centre comparative study. Commun Dis Public Health 6, 221–227.
    [Google Scholar]
  18. Smieja, M., Mahony, J. B., Goldsmith, C. H., Chong, S., Petrich, A. & Chernesky, M. ( 2001;). Replicate PCR testing and probit analysis for detection and quantitation of Chlamydia pneumoniae in clinical specimens. J Clin Microbiol 39, 1796–1801.[CrossRef]
    [Google Scholar]
  19. Smith, M. D., Derrington, P., Evans, R., Creek, M., Morris, R., Dance, D. A. & Cartwright, K. ( 2003;). Rapid diagnosis of bacteremic pneumococcal infections in adults by using the Binax NOW Streptococcus pneumoniae urinary antigen test: a prospective, controlled clinical evaluation. J Clin Microbiol 41, 2810–2813.[CrossRef]
    [Google Scholar]
  20. Toikka, P., Nikkari, S., Ruuskanen, O., Leinonen, M. & Mertsola, J. ( 1999;). Pneumolysin PCR-based diagnosis of invasive pneumococcal infection in children. J Clin Microbiol 37, 633–637.
    [Google Scholar]
  21. Udaykumar, Epstein, J. S. & Hewlett, I. K. ( 1993;). A novel method employing UNG to avoid carry-over contamination in RNA-PCR. Nucleic Acids Res 21, 3917–3918.[CrossRef]
    [Google Scholar]
  22. Whatmore, A. M., Efstratiou, A., Pickerill, A. P., Broughton, K., Woodard, G., Sturgeon, D., George, R. & Dowson, C. G. ( 2000;). Genetic relationships between clinical isolates of Streptococcus pneumoniae, Streptococcus oralis, and Streptococcus mitis: characterization of ‘‘atypical’’ pneumococci and organisms allied to S.mitis harboring S. pneumoniae virulence factor-encoding genes. Infect Immun 68, 1374–1382.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.05460-0
Loading
/content/journal/jmm/10.1099/jmm.0.05460-0
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