Degree and frequency of inhibition in a routine real-time PCR detecting for the diagnosis of pneumonia in Turkey Free

Abstract

Routine laboratory diagnosis of is currently achieved by PCR in almost all laboratories with sufficient equipment due to its high sensitivity and specificity compared to staining methods. A current issue that limits the reliability and sensitivity of PCR is the degree of inhibition caused by inhibitory substances in respiratory samples. The present study aimed to analyse the degree and frequency of inhibition in real-time PCR detecting in respiratory specimens submitted to a pneumonia (PcP) diagnosis laboratory in Ege University Medical School, Turkey. Between July 2009 and December 2010, 76 respiratory specimens [63 bronchoalveolar lavage (BAL) fluid, 10 sputum samples, two tracheal aspiration fluid and one thoracentesis fluid] obtained from 69 PcP-suspected patients were investigated for the presence of using real-time PCR targeting the gene. Of these samples, 42 of the specimens were stained and examined by microscopy according to the request of the clinicians. PCR was positive in 15 specimens in the initial run. Of the remaining 61 samples, 41 of them were negative with positive internal inhibition controls (i.e. true-negative group). The frequency of inhibition in the initial run was 26.31 % (20/76) as determined by spiked negative controls. All of the inhibited samples were resolved after 1 : 2, 1 : 5, 1 : 10 and 1 : 20 dilutions. was detected by PCR in two inhibited specimens after retesting with diluted samples which were also positive by microscopy. The incidence of in respiratory specimens was 22.36 % (17/76) as determined by real-time PCR and 7.14 % (3/42) by microscopy. Overall, the incidence of in respiratory samples was 23.68 % (18/76) as detected by both methods. In conclusion, inclusion of spiked positive controls in each sample and retesting with diluted samples to resolve inhibition increased the reliability of the real-time PCR assay in terms of determining false-negative results and influencing the treatment of the patient. Furthermore, results of the present study determined for the first time the frequency and degree of inhibition in a real-time PCR detecting in respiratory specimens during routine diagnosis of PcP.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.030775-0
2011-07-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/jmm/60/7/937.html?itemId=/content/journal/jmm/10.1099/jmm.0.030775-0&mimeType=html&fmt=ahah

References

  1. Al-Soud W. A., Jönsson L. J., Râdström P. 2000; Identification and characterization of immunoglobulin G in blood as a major inhibitor of diagnostic PCR. J Clin Microbiol 38:345–350[PubMed]
    [Google Scholar]
  2. Alvarez-Martínez M. J., Miró J. M., Valls M. E., Moreno A., Rivas P. V., Solé M., Benito N., Domingo P., Muñoz C. et al. 2006; Sensitivity and specificity of nested and real-time PCR for the detection of Pneumocystis jiroveci in clinical specimens. Diagn Microbiol Infect Dis 56:153–160 [View Article][PubMed]
    [Google Scholar]
  3. Amicosante M., Richeldi L., Trenti G., Paone G., Campa M., Bisetti A., Saltini C. 1995; Inactivation of polymerase inhibitors for Mycobacterium tuberculosis DNA amplification in sputum by using capture resin. J Clin Microbiol 33:629–630[PubMed]
    [Google Scholar]
  4. Apfalter P., Barousch W., Nehr M., Makristathis A., Willinger B., Rotter M., Hirschl A. M. 2003; Comparison of a new quantitative ompA-based real-time PCR TaqMan assay for detection of Chlamydia pneumoniae DNA in respiratory specimens with four conventional PCR assays. J Clin Microbiol 41:592–600 [View Article][PubMed]
    [Google Scholar]
  5. Arcenas R. C., Uhl J. R., Buckwalter S. P., Limper A. H., Crino D., Roberts G. D., Wengenack N. L. 2006; A real-time polymerase chain reaction assay for detection of Pneumocystis from bronchoalveolar lavage fluid. Diagn Microbiol Infect Dis 54:169–175 [View Article][PubMed]
    [Google Scholar]
  6. Bandt D., Monecke S. 2007; Development and evaluation of a real-time PCR assay for detection of Pneumocystis jiroveci . Transpl Infect Dis 9:196–202 [View Article][PubMed]
    [Google Scholar]
  7. Bencini M. A., van den Brule A. J., Claas E. C., Hermans M. H., Melchers W. J., Noordhoek G. T., Salimans M. M., Schirm J., Vink C. et al. 2007; Multicenter comparison of molecular methods for detection of Legionella spp. in sputum samples. J Clin Microbiol 45:3390–3392 [View Article][PubMed]
    [Google Scholar]
  8. Binnicker M. J., Buckwalter S. P., Eisberner J. J., Stewart R. A., McCullough A. E., Wohlfiel S. L., Wengenack N. L. 2007; Detection of Coccidioides species in clinical specimens by real-time PCR. J Clin Microbiol 45:173–178 [View Article][PubMed]
    [Google Scholar]
  9. Brancart F., Rodriguez-Villalobos H., Fonteyne P. A., Peres-Bota D., Liesnard C. 2005; Quantitative TaqMan PCR for detection of Pneumocystis jiroveci . J Microbiol Methods 61:381–387 [View Article][PubMed]
    [Google Scholar]
  10. Carmona E. M., Limper A. H. 2011; Update on the diagnosis and treatment of Pneumocystis pneumonia . Ther Adv Respir Dis 5:41–59[PubMed] [CrossRef]
    [Google Scholar]
  11. Choukri F., Menotti J., Sarfati C., Lucet J. C., Nevez G., Garin Y. J., Derouin F., Totet A. 2010; Quantification and spread of Pneumocystis jirovecii in the surrounding air of patients with Pneumocystis pneumonia . Clin Infect Dis 51:259–265 [View Article][PubMed]
    [Google Scholar]
  12. Demanche C., Berthelemy M., Petit T., Polack B., Wakefield A. E., Dei-Cas E., Guillot J. 2001; Phylogeny of Pneumocystis carinii from 18 primate species confirms host specificity and suggests coevolution. J Clin Microbiol 39:2126–2133 [View Article][PubMed]
    [Google Scholar]
  13. Deneer H. G., Knight I. 1994; Inhibition of the polymerase chain reaction by mucolytic agents. Clin Chem 40:171–172[PubMed]
    [Google Scholar]
  14. Fillaux J., Malvy S., Alvarez M., Fabre R., Cassaing S., Marchou B., Linas M. D., Berry A. 2008; Accuracy of a routine real-time PCR assay for the diagnosis of Pneumocystis jirovecii pneumonia. J Microbiol Methods 75:258–261 [View Article][PubMed]
    [Google Scholar]
  15. Fischer S., Gill V. J., Kovacs J., Miele P., Keary J., Silcott V., Huang S., Borio L., Stock F. et al. 2001; The use of oral washes to diagnose Pneumocystis carinii pneumonia: a blinded prospective study using a polymerase chain reaction-based detection system. J Infect Dis 184:1485–1488 [View Article][PubMed]
    [Google Scholar]
  16. Flori P., Bellete B., Durand F., Raberin H., Cazorla C., Hafid J., Lucht F., Sung R. T. 2004; Comparison between real-time PCR, conventional PCR and different staining techniques for diagnosing Pneumocystis jiroveci pneumonia from bronchoalveolar lavage specimens. J Med Microbiol 53:603–607 [View Article][PubMed]
    [Google Scholar]
  17. Gianella S., Haeberli L., Joos B., Ledergerber B., Wüthrich R. P., Weber R., Kuster H., Hauser P. M., Fehr T., Mueller N. J. 2010; Molecular evidence of interhuman transmission in an outbreak of Pneumocystis jirovecii pneumonia among renal transplant recipients. Transpl Infect Dis 12:1–10 [View Article][PubMed]
    [Google Scholar]
  18. Gill V. J., Nelson N. A., Stock F., Evans G. 1988; Optimal use of the cytocentrifuge for recovery and diagnosis of Pneumocystis carinii in bronchoalveolar lavage and sputum specimens. J Clin Microbiol 26:1641–1644[PubMed]
    [Google Scholar]
  19. Gupta R., Mirdha B. R., Guleria R., Kumar L., Samantaray J. C., Agarwal S. K., Kabra S. K., Luthra K. 2009; Diagnostic significance of nested polymerase chain reaction for sensitive detection of Pneumocystis jirovecii in respiratory clinical specimens. Diagn Microbiol Infect Dis 64:381–388 [View Article][PubMed]
    [Google Scholar]
  20. Halse T. A., Edwards J., Cunningham P. L., Wolfgang W. J., Dumas N. B., Escuyer V. E., Musser K. A. 2010; Combined real-time PCR and rpoB gene pyrosequencing for rapid identification of Mycobacterium tuberculosis and determination of rifampin resistance directly in clinical specimens. J Clin Microbiol 48:1182–1188 [View Article][PubMed]
    [Google Scholar]
  21. Heginbothom M. L., Magee J. T., Flanagan P. G. 2003; Evaluation of the Idaho Technology LightCycler PCR for the direct detection of Mycobacterium tuberculosis in respiratory specimens. Int J Tuberc Lung Dis 7:78–83[PubMed]
    [Google Scholar]
  22. Helweg-Larsen J., Jensen J. S., Dohn B., Benfield T. L., Lundgren B. 2002; Detection of Pneumocystis DNA in samples from patients suspected of bacterial pneumonia – a case-control study. BMC Infect Dis 2:28 [View Article][PubMed]
    [Google Scholar]
  23. Huang S. N., Fischer S. H., O’Shaughnessy E., Gill V. J., Masur H., Kovacs J. A. 1999; Development of a PCR assay for diagnosis of Pneumocystis carinii pneumonia based on amplification of the multicopy major surface glycoprotein gene family. Diagn Microbiol Infect Dis 35:27–32 [View Article][PubMed]
    [Google Scholar]
  24. Huggett J. F., Taylor M. S., Kocjan G., Evans H. E., Morris-Jones S., Gant V., Novak T., Costello A. M., Zumla A., Miller R. F. 2008; Development and evaluation of a real-time PCR assay for detection of Pneumocystis jirovecii DNA in bronchoalveolar lavage fluid of HIV-infected patients. Thorax 63:154–159 [View Article][PubMed]
    [Google Scholar]
  25. Hunter J. A., Wakefield A. E. 1996; Genetic divergence at the mitochondrial small subunit ribosomal RNA gene among isolates of Pneumocystis carinii from five mammalian host species. J Eukaryot Microbiol 43:24S–25S [View Article][PubMed]
    [Google Scholar]
  26. Iinuma Y., Senda K., Fujihara N., Saito T., Takakura S., Shimojima M., Kudo T., Ichiyama S. 2003; Comparison of the BDProbeTec ET system with the Cobas Amplicor PCR for direct detection of Mycobacterium tuberculosis in respiratory samples. Eur J Clin Microbiol Infect Dis 22:368–371 [View Article][PubMed]
    [Google Scholar]
  27. Jiancheng W., Minjun H., Yi-jun A., Lan S., Zengzhu G., Jianrong S., Xixiong K. 2009; Screening Pneumocystis carinii pneumonia in non-HIV-infected immunocompromised patients using polymerase chain reaction. Diagn Microbiol Infect Dis 64:396–401 [View Article][PubMed]
    [Google Scholar]
  28. Kais M., Spindler C., Kalin M., Ortqvist A., Giske C. G. 2006; Quantitative detection of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis in lower respiratory tract samples by real-time PCR. Diagn Microbiol Infect Dis 55:169–178 [View Article][PubMed]
    [Google Scholar]
  29. Kaiser K., Rabodonirina M., Mayençon M., Picot S. 1999; Evidence for cdc2 gene in Pneumocystis carinii hominis and its implication for culture. AIDS 13:419–420 [View Article][PubMed]
    [Google Scholar]
  30. Kaiser K., Rabodonirina M., Picot S. 2001; Real time quantitative PCR and RT–PCR for analysis of Pneumocystis carinii hominis. J Microbiol Methods 45:113–118 [View Article][PubMed]
    [Google Scholar]
  31. Kern M., Böhm S., Deml L., Wolf H., Reischl U., Niller H. H. 2009; Inhibition of Legionella pneumophila PCR in respiratory samples: a quantitative approach. J Microbiol Methods 79:189–193 [View Article][PubMed]
    [Google Scholar]
  32. Khot P. D., Ko D. L., Hackman R. C., Fredricks D. N. 2008; Development and optimization of quantitative PCR for the diagnosis of invasive aspergillosis with bronchoalveolar lavage fluid. BMC Infect Dis 8:73 [View Article][PubMed]
    [Google Scholar]
  33. Kitada K., Oka S., Kimura S., Shimada K., Serikawa T., Yamada J., Tsunoo H., Egawa K., Nakamura Y. 1991; Detection of Pneumocystis carinii sequences by polymerase chain reaction: animal models and clinical application to noninvasive specimens. J Clin Microbiol 29:1985–1990[PubMed]
    [Google Scholar]
  34. Larsen H. H., Kovacs J. A., Stock F., Vestereng V. H., Lundgren B., Fischer S. H., Gill V. J. 2002a). Development of a rapid real-time PCR assay for quantitation of Pneumocystis carinii f. sp. carinii . J Clin Microbiol 40:2989–2993 [View Article][PubMed]
    [Google Scholar]
  35. Larsen H. H., Masur H., Kovacs J. A., Gill V. J., Silcott V. A., Kogulan P., Maenza J., Smith M., Lucey D. R., Fischer S. H. 2002b). Development and evaluation of a quantitative, touch-down, real-time PCR assay for diagnosing Pneumocystis carinii pneumonia. J Clin Microbiol 40:490–494 [View Article][PubMed]
    [Google Scholar]
  36. Larsen H. H., Huang L., Kovacs J. A., Crothers K., Silcott V. A., Morris A., Turner J. R., Beard C. B., Masur H., Fischer S. H. 2004; A prospective, blinded study of quantitative touch-down polymerase chain reaction using oral-wash samples for diagnosis of Pneumocystis pneumonia in HIV-infected patients. J Infect Dis 189:1679–1683 [View Article][PubMed]
    [Google Scholar]
  37. Leibovitz E., Pollack H., Moore T., Papellas J., Gallo L., Krasinski K., Borkowsky W. 1995; Comparison of PCR and standard cytological staining for detection of Pneumocystis carinii from respiratory specimens from patients with or at high risk for infection by human immunodeficiency virus. J Clin Microbiol 33:3004–3007[PubMed]
    [Google Scholar]
  38. Levidiotou S., Vrioni G., Galanakis E., Gesouli E., Pappa C., Stefanou D. 2003; Four-year experience of use of the Cobas Amplicor system for rapid detection of Mycobacterium tuberculosis complex in respiratory and nonrespiratory specimens in Greece. Eur J Clin Microbiol Infect Dis 22:349–356 [View Article][PubMed]
    [Google Scholar]
  39. Linssen C. F., Jacobs J. A., Beckers P., Templeton K. E., Bakkers J., Kuijper E. J., Melchers W. J., Drent M., Vink C. 2006; Inter-laboratory comparison of three different real-time PCR assays for the detection of Pneumocystis jiroveci in bronchoalveolar lavage fluid samples. J Med Microbiol 55:1229–1235 [View Article][PubMed]
    [Google Scholar]
  40. Lipschik G. Y., Andrawis V. A., Ognibene F. P., Kovacs J. A., Gill V. J., Nelson N. A., Lundgren J. D., Nielsen J. O. 1992; Improved diagnosis of Pneumocystis carinii infection by polymerase chain reaction on induced sputum and blood. Lancet 340:203–206 [View Article][PubMed]
    [Google Scholar]
  41. Louie G. H., Wang Z., Ward M. M. 2010; Trends in hospitalizations for Pneumocystis jiroveci pneumonia among patients with rheumatoid arthritis in the US: 1996–2007. Arthritis Rheum 62:3826–3827 [View Article][PubMed]
    [Google Scholar]
  42. Lu J. J., Chen C. H., Bartlett M. S., Smith J. W., Lee C. H. 1995; Comparison of six different PCR methods for detection of Pneumocystis carinii . J Clin Microbiol 33:2785–2788[PubMed]
    [Google Scholar]
  43. Maaroufi Y., de Bruyne J. M., Duchateau V., Scheen R., Crokaert F. 2006; Development of a multiple internal control for clinical diagnostic real-time amplification assays. FEMS Immunol Med Microbiol 48:183–191 [View Article][PubMed]
    [Google Scholar]
  44. Olsson M., Elvin K., Löfdahl S., Linder E. 1993; Detection of Pneumocystis carinii DNA in sputum and bronchoalveolar lavage samples by polymerase chain reaction. J Clin Microbiol 31:221–226[PubMed]
    [Google Scholar]
  45. Olsson M., Strålin K., Holmberg H. 2001; Clinical significance of nested polymerase chain reaction and immunofluorescence for detection of Pneumocystis carinii pneumonia. Clin Microbiol Infect 7:492–497 [View Article][PubMed]
    [Google Scholar]
  46. Opel K. L., Chung D., McCord B. R. 2010; A study of PCR inhibition mechanisms using real time PCR. J Forensic Sci 55:25–33 [View Article][PubMed]
    [Google Scholar]
  47. Pinlaor S., Mootsikapun P., Pinlaor P., Phunmanee A., Pipitgool V., Sithithaworn P., Chumpia W., Sithithaworn J. 2004; PCR diagnosis of Pneumocystis carinii on sputum and bronchoalveolar lavage samples in immuno-compromised patients. Parasitol Res 94:213–218 [View Article][PubMed]
    [Google Scholar]
  48. Raggam R. B., Leitner E., Mühlbauer G., Berg J., Stöcher M., Grisold A. J., Marth E., Kessler H. H. 2002; Qualitative detection of Legionella species in bronchoalveolar lavages and induced sputa by automated DNA extraction and real-time polymerase chain reaction. Med Microbiol Immunol 191:119–125 [View Article][PubMed]
    [Google Scholar]
  49. Ribes J. A., Limper A. H., Espy M. J., Smith T. F. 1997; PCR detection of Pneumocystis carinii in bronchoalveolar lavage specimens: analysis of sensitivity and specificity. J Clin Microbiol 35:830–835[PubMed]
    [Google Scholar]
  50. Rohner P., Jacomo V., Studer R., Schrenzel J., Graf J. D. 2009; Detection of Pneumocystis jirovecii by two staining methods and two quantitative PCR assays. Infection 37:261–265[PubMed] [CrossRef]
    [Google Scholar]
  51. Rosen P. P., Martini N., Armstrong D. 1975; Pneumocystis carinii pneumonia. Diagnosis by lung biopsy. Am J Med 58:794–802 [View Article][PubMed]
    [Google Scholar]
  52. Rosenstraus M., Wang Z., Chang S. Y., DeBonville D., Spadoro J. P. 1998; An internal control for routine diagnostic PCR: design, properties, and effect on clinical performance. J Clin Microbiol 36:191–197[PubMed]
    [Google Scholar]
  53. Sandhu G. S., Kline B. C., Espy M. J., Stockman L., Smith T. F., Limper A. H. 1999; Laboratory diagnosis of Pneumocystis carinii infections by PCR directed to genes encoding for mitochondrial 5S and 28S ribosomal RNA. Diagn Microbiol Infect Dis 33:157–162 [View Article][PubMed]
    [Google Scholar]
  54. Schluger N., Sepkowitz K., Armstrong D., Bernard E., Rifkin M., Cerami A., Bucala R. 1991; Detection of Pneumocystis carinii in serum of AIDS patients with Pneumocystis pneumonia by the polymerase chain reaction. J Protozool 38:240S–242S[PubMed]
    [Google Scholar]
  55. Tong C. Y., Donnelly C., Harvey G., Sillis M. 1999; Multiplex polymerase chain reaction for the simultaneous detection of Mycoplasma pneumoniae, Chlamydia pneumoniae, and Chlamydia psittaci in respiratory samples. J Clin Pathol 52:257–263 [View Article][PubMed]
    [Google Scholar]
  56. Torres J., Goldman M., Wheat L. J., Tang X., Bartlett M. S., Smith J. W., Allen S. D., Lee C. H. 2000; Diagnosis of Pneumocystis carinii pneumonia in human immunodeficiency virus-infected patients with polymerase chain reaction: a blinded comparison to standard methods. Clin Infect Dis 30:141–145 [View Article][PubMed]
    [Google Scholar]
  57. Wakefield A. E. 1996; DNA sequences identical to Pneumocystis carinii f. sp. carinii and Pneumocystis carinii f. sp. hominis in samples of air spora. J Clin Microbiol 34:1754–1759[PubMed]
    [Google Scholar]
  58. Wakefield A. E. 2002; Pneumocystis carinii . Br Med Bull 61:175–188 [View Article][PubMed]
    [Google Scholar]
  59. Wakefield A. E., Pixley F. J., Banerji S., Sinclair K., Moxon E. R., Miller R. F., Hopkin J. M. 1990; Detection of Pneumocystis carinii with DNA amplification. Lancet 336:451–453 [View Article][PubMed]
    [Google Scholar]
  60. Wakefield A. E., Guiver L., Miller R. F., Hopkin J. M. 1991; DNA amplification on induced sputum samples for diagnosis of Pneumocystis carinii pneumonia. Lancet 337:1378–1379 [View Article][PubMed]
    [Google Scholar]
  61. Walker J., Conner G., Ho J., Hunt C., Pickering L. 1989; Giemsa staining for cysts and trophozoites of Pneumocystis carinii . J Clin Pathol 42:432–434 [View Article][PubMed]
    [Google Scholar]
  62. Wilson I. G. 1997; Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol 63:3741–3751[PubMed]
    [Google Scholar]
  63. Wilson D., Yen-Lieberman B., Reischl U., Warshawsky I., Procop G. W. 2004; Comparison of five methods for extraction of Legionella pneumophila from respiratory specimens. J Clin Microbiol 42:5913–5916 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.030775-0
Loading
/content/journal/jmm/10.1099/jmm.0.030775-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed