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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, Margo Smith,³ Daniel R. Lucey,³ and Steven H. Fischer^1*

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.

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

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