This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Larsen, H. H.
Right arrow Articles by Fischer, S. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Larsen, H. H.
Right arrow Articles by Fischer, S. H.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, February 2002, p. 490-494, Vol. 40, No. 2
0095-1137/01/$04.00+0     DOI: 10.1128/JCM.40.2.490-494.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Development and Evaluation of a Quantitative, Touch-Down, Real-Time PCR Assay for Diagnosing Pneumocystis carinii Pneumonia

Hans Henrik Larsen,1 Henry Masur,2 Joseph A. Kovacs,2 Vee J. Gill,1 Victoria A. Silcott,1 Palaniandy Kogulan,3 Janine Maenza,4,{dagger} Margo Smith,3 Daniel R. Lucey,3 and Steven H. Fischer1*

Departments of Laboratory Medicine,1 Critical Care, Clinical Center, National Institutes of Health, Bethesda ,2 Department of Medicine, Johns Hopkins University, Baltimore, Maryland,4 Division of Infectious Diseases, Washington Hospital Center, Washington, D.C.3

Received 29 May 2001/ Returned for modification 29 August 2001/ Accepted 9 November 2001

A rapid (time to completion, <4 h, including DNA extraction) and quantitative touch-down (QTD) real-time diagnostic Pneumocystis carinii PCR assay with an associated internal control was developed, using fluorescence resonance energy transfer (FRET) probes for detection. The touch-down procedure significantly increased the sensitivity of the assay compared to a non-touch-down procedure. Tenfold serial dilutions of a cloned target were used as standards for quantification. P. carinii DNA has been detected in respiratory specimens from patients with P. carinii pneumonia (PCP) and from patients without clinical evidence of PCP. The latter probably represents colonization or subclinical infection. It is logical to hypothesize that quantification might prove helpful in distinguishing between infected and colonized patients: the latter group would have lower copy numbers than PCP patients. A blinded retrospective study of 98 respiratory samples (49 lower respiratory tract specimens and 49 oral washes), from 51 patients with 24 episodes of PCP and 34 episodes of other respiratory disease, was conducted. PCR-positive samples from colonized patients contained a lower concentration of P. carinii DNA than samples from PCP patients: lower respiratory tract samples from PCP and non-PCP patients contained a median of 938 (range, 2.4 to 1,040,000) and 2.6 (range, 0.3 to 248) (P < 0.0004) copies per tube, respectively. Oral washes from PCP and non-PCP patients contained a median of 49 (range, 2.1 to 2,595) and 6.5 (range, 2.2 to 10) (P < 0.03) copies per tube, respectively. These data suggest that this QTD PCR assay can be used to determine if P. carinii is present in respiratory samples and to distinguish between colonization and infection.

* Corresponding author. Mailing address: Department of Laboratory Medicine, Microbiology Service, National Institutes of Health, Bldg. 10, Rm. 2C-385, 10 Center Drive, Bethesda, MD 20892. Phone: (301) 496-4433. Fax: (301) 402-1886. E-mail: sfischer{at}nih.gov.

{dagger} Present address: Primary Infection Clinic, University of Washington, Seattle, Wash.

Journal of Clinical Microbiology, February 2002, p. 490-494, Vol. 40, No. 2
0095-1137/01/$04.00+0     DOI: 10.1128/JCM.40.2.490-494.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Takahashi, T., Tamura, M., Asami, Y., Kitamura, E., Saito, K., Suzuki, T., Takahashi, S. N., Matsumoto, K., Sawada, S., Yokoyama, E., Takasu, T. (2008). Novel Wide-Range Quantitative Nested Real-Time PCR Assay for Mycobacterium tuberculosis DNA: Development and Methodology. J. Clin. Microbiol. 46: 1708-1715 [Abstract] [Full Text]  
  • 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 [Abstract] [Full Text]  
  • Uemura, N., Makimura, K., Onozaki, M., Otsuka, Y., Shibuya, Y., Yazaki, H., Kikuchi, Y., Abe, S., Kudoh, S. (2008). Development of a loop-mediated isothermal amplification method for diagnosing Pneumocystis pneumonia. J Med Microbiol 57: 50-57 [Abstract] [Full Text]  
  • Davis, A. H. T., Jianhua Wang, , Tsang, T. C., Harris, D. T. (2007). Direct Sequencing Is More Accurate and Feasible in Detecting Single Nucleotide Polymorphisms than RFLP: Using Human Vascular Endothelial Growth Factor Gene as a Model. Biol Res Nurs 9: 170-178 [Abstract]  
  • Linssen, C. F. M., Jacobs, J. A., Beckers, P., Templeton, K. E., Bakkers, J., Kuijper, E. J., Melchers, W. J. G., 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 [Abstract] [Full Text]  
  • Takahashi, T., Nakayama, T. (2006). Novel Technique of Quantitative Nested Real-Time PCR Assay for Mycobacterium tuberculosis DNA.. J. Clin. Microbiol. 44: 1029-1039 [Abstract] [Full Text]  
  • Espy, M. J., Uhl, J. R., Sloan, L. M., Buckwalter, S. P., Jones, M. F., Vetter, E. A., Yao, J. D. C., Wengenack, N. L., Rosenblatt, J. E., Cockerill, F. R. III, Smith, T. F. (2006). Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing. Clin. Microbiol. Rev. 19: 165-256 [Abstract] [Full Text]  
  • Fedorko, D. P., Preuss, J. C., Fahle, G. A., Li, L., Fischer, S. H., Hohman, P., Cohen, J. I. (2005). Comparison of Methods for Detection of Vaccinia Virus in Patient Specimens. J. Clin. Microbiol. 43: 4602-4606 [Abstract] [Full Text]  
  • Nyamande, K., Lalloo, U. G., York, D., Naidoo, M., Irusen, E. M., Chetty, R. (2005). Low Sensitivity of a Nested Polymerase Chain Reaction in Oropharyngeal Washings for the Diagnosis of Pneumocystis Pneumonia in HIV-Infected Patients. Chest 128: 167-171 [Abstract] [Full Text]  
  • Flori, P., Bellete, B., Durand, F., Raberin, H., Cazorla, C., Hafid, J., Lucht, F., Sung, R. T. M. (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 [Abstract] [Full Text]  
  • Wright, T. W., Pryhuber, G. S., Chess, P. R., Wang, Z., Notter, R. H., Gigliotti, F. (2004). TNF Receptor Signaling Contributes to Chemokine Secretion, Inflammation, and Respiratory Deficits during Pneumocystis Pneumonia. J. Immunol. 172: 2511-2521 [Abstract] [Full Text]  
  • Shrestha, N. K., Tuohy, M. J., Hall, G. S., Reischl, U., Gordon, S. M., Procop, G. W. (2003). Detection and Differentiation of Mycobacterium tuberculosis and Nontuberculous Mycobacterial Isolates by Real-Time PCR. J. Clin. Microbiol. 41: 5121-5126 [Abstract] [Full Text]  
  • O'Neil, K. M. (2002). The Changing Landscape of HIV-Related Lung Disease in the Era of Highly Active Antiretroviral Therapy. Chest 122: 768-771 [Full Text]  
  • Larsen, H. H., Kovacs, J. A., Stock, F., Vestereng, V. H., Lundgren, B., Fischer, S. H., Gill, V. J. (2002). Development of a Rapid Real-Time PCR Assay for Quantitation of Pneumocystis carinii f. sp. carinii. J. Clin. Microbiol. 40: 2989-2993 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»