Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2002 Feb;40(2):490-4.
doi: 10.1128/JCM.40.2.490-494.2002.

Development and evaluation of a quantitative, touch-down, real-time PCR assay for diagnosing Pneumocystis carinii pneumonia

Hans Henrik Larsen  1 , Henry Masur, Joseph A Kovacs, Vee J Gill, Victoria A Silcott, Palaniandy Kogulan, Janine Maenza, Margo Smith, Daniel R Lucey, Steven H Fischer
Affiliations

Development and evaluation of a quantitative, touch-down, real-time PCR assay for diagnosing Pneumocystis carinii pneumonia

Hans Henrik Larsen et al. J Clin Microbiol. 2002 Feb.

Abstract

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.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
QTD PCR-positive lower respiratory samples from PCP and non-PCP patients. Symbols: ○, BAL fluid; ×, induced sputum; —, median; ∗, two-sided P < 0.0004.
FIG. 2.
FIG. 2.
QTD PCR-positive oral wash samples from PCP and non-PCP patients. Symbols: ○, oral washes; —, median; ∗, two-sided P < 0.03.
See this image and copyright information in PMC

References

    1. Aztori, C., F. Agostini, E. Angeli, A. Mainini, G. Orlando, and A. Cargnel. 1998. Combined use of blood and oropharyngeal samples for noninvasive diagnosis of Pneumocystis carinii pneumonia using the polymerase chain reaction. Eur. J. Clin. Microbiol. Infect. Dis. 17:241-246. - PubMed
    1. Aztori, C., E. Angeli, F. Agostini, A. Mainini, V. Micheli, and A. Cargnel. 1999. Biomolecular techniques to detect Pneumocystis carinii f. sp. hominis pneumonia in patients with acquired immunodeficiency syndrome. Int. J. Infect. Dis. 3:76-81. - PubMed
    1. Calderon, E. J., C. Regordan, F. J. Medrano, M. Ollero, and J. M. Varela. 1996. Pneumocystis carinii infection in patients with chronic bronchial disease. Lancet 347:977. - PubMed
    1. Cartwright, C. P., N. A. Nelson, and V. J. Gill. 1994. Development and evaluation of a rapid and simple procedure for detection of Pneumocystis carinii by PCR. J. Clin. Microbiol. 32:1634-1638. - PMC - PubMed
    1. Chouaid, C., P. Roux, I. Lavard, J. L. Poirot, and B. Housset. 1995. Use of the polymerase chain reaction technique on induced-sputum samples for the diagnosis of Pneumocystis carinii pneumonia in HIV-infected patients. Am. J. Clin. Pathol. 104:72-75. - PubMed

Publication types

MeSH terms

LinkOut - more resources