Real-time PCR Assay For Diagnosis Of Pcp Using Oral Wash

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Pneumocystis jiroveci (Pneumocycstis carinii) is an important cause of pneumonia (PCP) in immunocompromised individuals. The standard approach for diagnosing PCP is a microscopic examination of smears prepared from induced sputum or bronchial alveolar lavage (BAL) samples. Recently, investigators have been designing and testing PCR assays for the detection of P. jiroveci in respiratory samples. Of particular interest is the use of PCR with oral wash samples as a means of detecting P. jiroveci in the respiratory tract. These noninvasive specimens could prove to be of value for use in screening tests to rule out PCP. Microscopic methods are too insensitive to be useful with oral wash samples. The increased sensitivity of the PCR method, however, generates some positive results with samples obtained from patients who are only colonized or infected at a sub-clinical level. A precise quantitative method could help differentiate low level colonization from infection and, consequently, improve the clinical usefulness of PCR performed on oral washes and other respiratory samples. We have developed a rapid quantitative real time PCR assay targeting the MSG genes of P. jiroveci using fluorescence resonance energy transfer (FRET) detection probes for signal detection. A mimic has also been constructed for inclusion in PCR reaction tubes as an indicator of false negative reactions. The assay has a detection limit of one to five MSG gene copies per amplification reaction. Results with the real time assay using replicate samples of cloned target material vary by 10% or less. A blinded retrospective study was conducted using 49 lower respiratory tract samples and 49 oral wash samples collected from 51 patients with 24 episodes of PCP and 34 episodes of other respiratory disease. The average number of P. jiroveci copies detected in lower respiratory tract samples and oral washes from patients with PCP was significantly higher than for PCR positive patients without Pneumocystis pneumonia. The results of this preliminary study have been published. A blinded, prospective study has been conducted with collaborators at UCSF to further evaluate the performance of the real time PCR assay in detecting PCP. The results of this study are being compiled. A second collaborative study with UCSF is underway to attempt to detect P. jiroveci colonization of the upper airways of health care workers after exposure to patients with PCP.