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

氏肺囊虫（卡氏肺囊虫）是免疫功能低下个体肺炎（PCP）的重要病因。诊断PCP的标准方法是显微镜检查从诱导痰或支气管肺泡灌洗液（BAL）样品中制备的涂片。最近，研究人员一直在设计和测试呼吸样本中检测耶氏疟原虫的PCR方法。特别令人感兴趣的是使用口腔洗涤样品的聚合酶链反应作为检测呼吸道中耶氏疟原虫的手段。这些非侵入性标本可能被证明在筛查试验中排除PCP的使用价值。显微方法太不敏感，不能用于口腔洗液样品。然而，PCR方法的灵敏度提高，对于仅在亚临床水平上定植或感染的患者获得的样本产生了一些阳性结果。精确的定量方法可以帮助区分低水平定植与感染，从而提高对口腔洗液和其他呼吸道样本进行PCR的临床有效性。

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.