Real-time PCR assay-based strategy for differentiation between active Pneumocystis jirovecii pneumonia and colonization in immunocompromised patients
Real-time PCR assay-based strategy for differentiation between active Pneumocystis jirovecii pneumonia and colonization in immunocompromised patients
复制标题
DOI：
10.1111/j.1469-0691.2010.03400.x
复制
发表时间：
2011-10-01
影响因子：
14.2
通讯作者：
Menotti, J.
中科院分区：
文献类型：
--
作者：
Alanio, A.;Desoubeaux, G.;Menotti, J.
肺孢子虫病的诊断通常依赖于呼吸道样本中肺孢子虫的显微镜检查。常规PCR可以检测到低水平的耶氏疟原虫DNA，但不能区分活动性肺炎和定植。在这项研究中，我们使用了一种新的实时定量PCR（qPCR）检测来识别和区分这些实体。从238例连续出现肺炎体征的免疫功能低下患者中前瞻性获得了163例支气管肺泡灌洗液和115例诱导性支气管肺泡灌洗液。根据临床和放射学表现，将每例患者分为具有高或低概率的耶氏肺孢子虫肺炎。通过显微镜和qPCR测定法处理样品，扩增耶氏毕赤酵母线粒体大亚基rRNA基因; qPCR结果表示为营养型当量（TFEq）/mL，参考从编号的营养型悬浮液获得的标准曲线。从16例极有可能患耶氏肺孢子虫肺炎的患者获得的21份样本中，21份通过qPCR检测为阳性，而仅16份通过显微镜检测为阳性。真菌载量范围为134 - 1.73 x 10（6）TFEq/mL。在从222例极有可能患耶氏疟原虫肺炎的患者中采集的257份标本中，222份经两种技术检测均为阴性，但35份经qPCR检测为阳性（0.1-1840 TFEq/mL），表明耶氏疟原虫定殖。提出了120和1900 TFEq/mL的两个临界值来区分活动性肺炎和定植，两者之间有一个灰色区域。总之，该qPCR测定区分活动性肺炎与定植。这对于患者管理特别相关，特别是在非人类免疫缺陷病毒（HIV）感染的免疫功能低下患者中，这些患者通常表现出显微镜下无法诊断的低负荷Jirovecii感染。
Diagnosis of pneumocystosis usually relies on microscopic demonstration of Pneumocystis jirovecii in respiratory samples. Conventional PCR can detect low levels of P. jirovecii DNA but cannot differentiate active pneumonia from colonization. In this study, we used a new real-time quantitative PCR (qPCR) assay to identify and discriminate these entities. One hundred and sixty-three bronchoalveolar lavage fluids and 115 induced sputa were prospectively obtained from 238 consecutive immunocompromised patients presenting signs of pneumonia. Each patient was classified as having a high or a low probability of P. jirovecii pneumonia according to clinical and radiological presentation. Samples were processed by microscopy and by a qPCR assay amplifying the P. jirovecii mitochondrial large-subunit rRNA gene; qPCR results were expressed as trophic form equivalents (TFEq)/mL by reference to a standard curve obtained from numbered suspensions of trophic forms. From 21 samples obtained from 16 patients with a high probability of P. jirovecii pneumonia, 21 were positive by qPCR whereas only 16 were positive by microscopy. Fungal load ranged from 134 to 1.73 x 10(6) TFEq/mL. Among 257 specimens sampled from 222 patients with a low probability of P. jirovecii pneumonia, 222 were negative by both techniques but 35 were positive by qPCR (0.1-1840 TFEq/mL), suggesting P. jirovecii colonization. Two cut-off values of 120 and 1900 TFEq/mL were proposed to discriminate active pneumonia from colonization, with a grey zone between them. In conclusion, this qPCR assay discriminates active pneumonia from colonization. This is particularly relevant for patient management, especially in non-human immunodeficiency virus (HIV)-infected immunocompromised patients, who often present low-burden P. jirovecii infections that are not diagnosed microscopically.