Evaluation Of A Real-time PCR Assay For Diagnosis Of PCP

卡氏肺孢子虫（Pneumocystis carinii）是免疫功能低下个体肺炎（PCP）的重要原因。诊断PCP的标准方法是对诱导痰或支气管肺泡灌洗（BAL）样本制备的涂片进行显微镜检查。最近，研究人员一直在设计和测试用于检测呼吸道样本中的P.jiroveci的PCR检测。特别令人感兴趣的是使用口腔洗涤样品作为检测呼吸道中的耶氏疟原虫的手段。这些非侵入性样本可能被证明在排除PCP的筛查测试中具有价值。显微镜方法太不敏感，不能用于口腔清洗样品。然而，PCR方法的灵敏度增加，对于从仅在亚临床水平定植或感染的患者获得的样品产生一些阳性结果。一种精确的定量方法可以帮助区分低水平的定植感染，从而提高PCR的临床实用性进行口腔清洗和其他呼吸道样本。我们建立了一种快速定量的真实的时间PCR检测方法，以希氏毕赤酵母的MSG基因为靶点，使用荧光共振能量转移（FRET）检测探针进行信号检测。与UCSF的合作者进行了一项盲法前瞻性研究，以进一步评估真实的PCR检测PCP的性能。对于在开始PCP治疗后一天内获得的样本，用PCR检测PCP的口腔洗液样本的灵敏度大于90%。这项研究的结果已付印。 与加州大学旧金山分校的第二项合作研究正在进行中，试图检测卫生保健工作者暴露于PCP患者后上呼吸道的Jiroveci定植。最近的工作，以提高定量测量的再现性呼吸道样本中的P. jiroveci表明，预处理的样品材料与DTT大大降低了连续定量P. jiroveci测定的变异性。对非HIV阳性且无呼吸道症状的健康医护人员进行了盲法前瞻性研究，使用DTT样本预处理，对来自137名受试者的329份样本进行了研究。这些健康个体中的一小部分通过PCR测定重复呈阳性，并且似乎被耶氏疟原虫定殖。该数据于2005年5月在美国胸科学会上发表。

Pneumocystis jiroveci (Pneumocystis 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 blinded, prospective study has been conducted with collaborators at UCSF to further evaluate the performance of the real time PCR assay in detecting PCP. For samples obtained within one day of the initiation of PCP therapy the sensitivity of oral wash samples with PCR for detecting PCP was greater than 90%. The results of this study are in print. 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. Recent work to improve the reproducibility of quantitative measurements of P. jiroveci in respiratory samples indicates that pre-treatment of sample materials with DTT greatly reduces the variability of serial quantitative P. jiroveci determinations. The blinded, prospective study of healthy Health Care Workers, who are not HIV-positive and did not have respiratory symptoms, was undertaken with 329 samples from 137 subjects using the DTT sample pre-treatment. A small percentage of these healthy individuals were repeatedly positive by the PCR assay and appear to be colonized with P. jiroveci. This data was presented at the Amercian Thoracic Society in May, 2005.