1887

Abstract

Real-time PCR was employed to detect a region of the P1 cytadhesin gene of in clinical samples. An internal processing control was included that could be co-amplified simultaneously in the same reaction tube. The assay could reproducibly detect 1×10 organisms ml in clinical samples. There was no amplification of DNA or signal production from 15 other species of human mycoplasmas and 19 other bacterial species. Using a panel of 175 respiratory samples taken from patients with pneumonia of proven aetiology, the sensitivity was found to be 60 % and the specificity of the assay 96·7 % when compared with serology. This assay is suitable for same-day diagnosis of infection and batch processing of respiratory samples for clinical screening.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.46281-0
2006-02-01
2020-04-04
Loading full text...

Full text loading...

/deliver/fulltext/jmm/55/2/149.html?itemId=/content/journal/jmm/10.1099/jmm.0.46281-0&mimeType=html&fmt=ahah

References

  1. Abele-Horn M., Busch U., Nitschko H., Jacobs E., Bax R., Pfaff F., Schaffer B., Heesemann J. 1998; Molecular approaches to diagnosis of pulmonary diseases due to Mycoplasma pneumoniae . J Clin Microbiol 36:548–551
    [Google Scholar]
  2. 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]
  3. Cloud J. L., Hymas W. C., Turlak A., Croft A., Reischl U., Daly J. A., Carroll K. C. 2003; Description of a multiplex Bordetella pertussis and Bordetella parapertussis LightCycler® PCR assay with inhibition control. Diagn Microbiol Infect Dis 46:189–195 [CrossRef]
    [Google Scholar]
  4. Clyde W. A. Jr 1993; Clinical overview of typical Mycoplasma pneumoniae infections. Clin Infect Dis 17 (Suppl. 1):S32–S36 [CrossRef]
    [Google Scholar]
  5. Deneer H. G., Knight I. 1994; Inhibition of the polymerase chain reaction by mucolytic agents. Clin Chem 40:171–172
    [Google Scholar]
  6. Dorigo-Zetsma J. W., Zaat S. A. J., Wertheim-van Dillen P. M. E., Spanjaard L., Rijntjes J., van Waveren G., Jensen J. S., Angulo A. F., Dankert J. 1999; Comparison of PCR, culture and serological tests for diagnosis of Mycoplasma pneumoniae respiratory tract infection in children. J Clin Microbiol 37:14–17
    [Google Scholar]
  7. Foy H. M. 1993; Infections caused by Mycoplasma pneumoniae and possible carrier state in different populations of patients. Clin Infect Dis 17 (Suppl. 1):S37–S46 [CrossRef]
    [Google Scholar]
  8. Friedlaender R. P., Grizzard M. B., Helms C. M., Barile M. F., Senterfit L., Chanock R. M. 1976; Experimental production of respiratory tract infection with Mycoplasma pneumoniae in rhesus monkeys. J Infect Dis 133:343–346 [CrossRef]
    [Google Scholar]
  9. Gnarpe J., Lundback A., Sundelof B., Gnarpe H. 1992; Prevalence of Mycoplasma pneumoniae in subjectively healthy individuals. Scand J Infect Dis 24:161–164 [CrossRef]
    [Google Scholar]
  10. Hardegger D., Nadal D., Bossart W., Altwegg M., Dutly F. 2000; Rapid detection of Mycoplasma pneumoniae in clinical samples by real-time PCR. J Microbiol Methods 41:45–51 [CrossRef]
    [Google Scholar]
  11. Ievens M., Ursi D., van Bever H., Quint W., Niesters H. G. M., Goossens H. 1996; Detection of Mycoplasma pneumoniae by two polymerase chain reactions and role of M. pneumoniae in acute respiratory tract infections in pediatric patients. J Infect Dis 173:1445–1452 [CrossRef]
    [Google Scholar]
  12. Khanna M., Fan J., Pehler-Harrington K., Waters C., Douglass P., Stallock J., Kehl S., Henrickson K. J. 2005; The pneumoplex assays, a multiplex PCR-enzyme hybridization assay that allows simultaneous detection of five organisms, Mycoplasma pneumoniae ,Chlamydia ( Chlamydophila ) pneumoniae , Legionella pneumophila , Legionella micdadei , and Bordetella pertussis , and its real-time counterpart. J Clin Microbiol 43:565–571 [CrossRef]
    [Google Scholar]
  13. Loens K., Ursi D., Ieven M., van Aarle P., Sillekens P., Oudshoorn P., Goosens H. 2002; Detection of Mycoplasma pneumoniae in spiked clinical samples by nucleic acid sequence-based amplification. J Clin Microbiol 40:1339–1345 [CrossRef]
    [Google Scholar]
  14. Loens K., Ursi D., Goossens H., Leven M. 2003; Molecular diagnosis of Mycoplasma pneumoniae respiratory tract infections. J Clin Microbiol 41:4915–4923 [CrossRef]
    [Google Scholar]
  15. Longo M. C., Berninger M. S., Hartley J. L. 1990; Use of uracil DNA glycosylate to control carry-over in polymerase chain reaction. Gene 93:125–128 [CrossRef]
    [Google Scholar]
  16. Ludwig W., Schleifer K.-H. 2000; How quantitative is quantitative PCR with respect to cell counts?. Syst Appl Microbiol 23:556–562 [CrossRef]
    [Google Scholar]
  17. McMillan J. A. 1998; Mycoplasma pneumoniae infection in pediatrics. Semin Pediatr Infect Dis 9:112–119 [CrossRef]
    [Google Scholar]
  18. Michelow I. C., Olsen K., Lozano J., Duffy L. B., McCracken G. H., Hardy R. D. 2004; Diagnostic utility and clinical significance of naso- and oropharyngeal samples used in a PCR assay to diagnose Mycoplasma pneumoniae infection in children with community-acquired pneumonia. J Clin Microbiol 42:3339–3341 [CrossRef]
    [Google Scholar]
  19. Miyashita N., Saito A., Kohno S., Yamaguchi K., Watanabe A., Oda H., Kazuyama Y., Matsushima T. the CAP Study Group 2004; Multiplex PCR for the simultaneous detection of Chlamydia pneumoniae , Mycoplasma pneumoniae and Legionella pneumophila in community-acquired pneumonia. CAP Study Group. Respir Med 98:542–550 [CrossRef]
    [Google Scholar]
  20. Mygind T., Birkelund S., Falk E., Christiansen G. 2001; Evaluation of real-time quantitative PCR for identification and quantification of Chlamydia pneumoniae by comparison with immunohistochemistry. J Microbiol Methods 46:241–251 [CrossRef]
    [Google Scholar]
  21. Nour M., Trabelsi A., Maatouk N., Hammami M. 2005; Amplification of P1 and 16S rRNA genes by nested PCR for detection of Mycoplasma pneumoniae in paediatric patients. Pathol Biol (Paris) 53:9–14 [CrossRef]
    [Google Scholar]
  22. Principi N., Esposito S., Blasi F., Allegra L. the Mowgli Study Group 2001; Role of Mycoplasma pneumoniae and Chlamydia pneumoniae in children with community-acquired lower respiratory tract infections. Clin Infect Dis 32:1281–1289 [CrossRef]
    [Google Scholar]
  23. Raggam R. B., Leitner E., Berg J., Mühlbauer G., Marth E., Kessler H. H. 2005; Single-run, parallel detection of DNA from three pneumonia-producing bacteria by real-time polymerase chain reaction. J Mol Diagn 7:133–138 [CrossRef]
    [Google Scholar]
  24. Razin S. 1994; DNA probes and PCR in diagnosis of mycoplasma infections. Mol Cell Probes 8:497–511 [CrossRef]
    [Google Scholar]
  25. Sillis M. 1993; Modern methods for diagnosis of Mycoplasma pneumoniae . Rev Med Microbiol 4:24–31 [CrossRef]
    [Google Scholar]
  26. Stralin K., Backman A., Holmberg H., Fredlund H., Olcen P. 2005; Design of a multiplex PCR for Streptococcus pneumoniae , Haemophilus influenzae , Mycoplasma pneumoniae and Chlamydophila pneumoniae to be used on sputum samples. APMIS 113:99–111 [CrossRef]
    [Google Scholar]
  27. Su C. J., Tryon V. V., Baseman J. B. 1987; Cloning and sequence analysis of cytadhesin P1 gene from Mycoplasma pneumoniae . Infect Immun 55:3023–3029
    [Google Scholar]
  28. Svenstrup H. F., Nielsen P. K., Drasbek M., Birkelund S., Christiansen G. 2002; Adhesion and inhibition assay of Mycoplasma genitalium and M. pneumoniae by immunofluorescence microscopy. J Med Microbiol 51:361–373
    [Google Scholar]
  29. Ursi D., Dirven K., Loens K., Ieven M., Goossens H. 2003; Detection of Mycoplasma pneumoniae in respiratory samples by real-time PCR using an inhibition control. J Microbiol Methods 55:149–153 [CrossRef]
    [Google Scholar]
  30. Whiley D. M., LeCornec G. M., Mackay I. M., Siebert D. J., Sloots T. P. 2002; A real-time PCR assay for the detection of Neisseria gonorrhoeae by LightCycler. Diagn Microbiol Infect Dis 42:85–89 [CrossRef]
    [Google Scholar]
  31. Wreghitt T. G., Sillis M. 1985; A micro-capture ELISA for detecting Mycoplasma pneumoniae IgM: comparison with indirect immunofluorescence and indirect ELISA. J Hyg 94:217–227 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.46281-0
Loading
/content/journal/jmm/10.1099/jmm.0.46281-0
Loading

Data & Media loading...

Most cited this month

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