Evaluation Of Real-time Pcr Assay For Diagnosis Of Pcp

卡氏肺孢子虫是免疫功能低下者肺炎(PCP)的重要病因。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检测呼吸道样本中支原体的聚合酶链式反应方法。特别令人感兴趣的是，使用口腔洗液样本的聚合酶链式反应作为检测呼吸道中支原体的一种手段。这些非侵入性标本在排除PCP的筛查试验中可能被证明是有价值的。显微镜方法太不灵敏，不适用于口腔洗液样本。然而，聚合酶链式反应方法的灵敏度提高，对仅在亚临床水平上被殖民或感染的患者的样本产生了一些阳性结果。一种精确的定量方法可以帮助区分低水平的定植和感染，因此，提高对口腔洗涤剂和其他呼吸道样本进行PCR的临床实用性。利用荧光共振能量转移(FRET)检测探针进行信号检测，建立了针对日本血吸虫msg基因的实时定量聚合酶链式反应快速检测方法。加州大学旧金山分校的合作者进行了一项盲法前瞻性研究，以进一步评估实时聚合酶链式反应检测五氯酚的性能。对于PCP治疗开始后1天内采集的样本，QTD-PCR检测PCP的敏感性大于90%。这项研究的结果已经出版了。与加州大学旧金山分校的第二项合作研究正在进行中，试图检测接触PCP患者后医护人员上呼吸道中的吉罗维氏肺孢子虫定植情况。 最近对一种改进的样品处理方法的研究表明，DTT的使用显著改善了处理后的口腔洗涤材料的均质性。如果如预期的那样，这导致了更具重复性的定量结果，那么与某些信号水平相关的PCP的阳性和阴性预测值应该会有相当大的改善。我们正在启动一系列样本的测试，并进行处理修改，以测试这一假设。

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 QTD-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 on an improved method for sample processing has demonstrated that use of DTT markedly improves the homogeneity of the processed oral wash material. If, as expected, this results in more reproducable quantitative results the positive and negative predictive values for PCP associated with certain levels of signal should improve considerably. We are initiating testing of a series of samples with the processing modifications to test this hypothesis.