Clinical Significance of Quantifying Pneumocystis jirovecii DNA by Using Real-Time PCR in Bronchoalveolar Lavage Fluid from Immunocompromised Patients
Clinical Significance of Quantifying Pneumocystis jirovecii DNA by Using Real-Time PCR in Bronchoalveolar Lavage Fluid from Immunocompromised Patients
复制标题
DOI：
10.1128/jcm.06036-11
复制
发表时间：
2012-02-01
影响因子：
9.4
通讯作者：
Bretagne, Stephane
中科院分区：
文献类型：
--
作者：
Botterel, Francoise;Cabaret, Odile;Bretagne, Stephane
定量 PCR (qPCR) 在检测支气管肺泡灌洗 (BAL) 液中的耶氏肺孢子虫时比显微镜更灵敏。因此，我们开发了一种 qPCR 检测方法，并将结果与常规免疫荧光检测 (IFA) 和临床数据进行比较。该检测包括自动 DNA 提取、线粒体大亚基 rRNA 基因和内部对照的扩增，以及借助质粒克隆对拷贝数进行定量。我们研究了 353 份连续的 BAL 液体，这些液体是为了调查 287 名免疫功能低下患者的不明原因发烧和/或肺炎而获得的。没有观察到 qPCR 抑制。 17 份 (5%) 样本 IFA 和 qPCR 均为阳性，63 份 (18%) IFA 阴性且 qPCR 阳性，273 份 (77%) IFA 和 qPCR 均为阴性。 IFA 阳性/qPCR 阳性样品的拷贝数显着高于 IFA 阴性/qPCR 阳性样品的拷贝数（4.2 +/- 1.2 对比 1.1 +/- 1.1 log(10) 拷贝/μl；P < 10(-4)）。以 IFA 为标准，qPCR 测定灵敏度对于 >= 2.6 log(10) 拷贝/μl 为 100%，对于 >= 4 log(10) 拷贝/μl 特异性为 100%。由于在做出决策时无法获得 qPCR 结果，因此这些结果并未触发复方新诺明治疗。全身炎症性疾病且IFA阴性/qPCR阳性BAL液患者的1年生存率低于IFA阴性/qPCR阴性患者(P < 10(-3))，而实体器官移植受者(P = 0.88)和血液恶性肿瘤患者(P = 0.26)则相反。独立于 IFA 阳性对 BAL 液中的 P. jirovecii DNA 进行定量应纳入免疫功能低下患者肺炎的调查中。相关阈值仍有待确定，并且可能根据潜在疾病而有所不同。
Quantitative PCR (qPCR) is more sensitive than microscopy for detecting Pneumocystis jirovecii in bronchoalveolar lavage (BAL) fluid. We therefore developed a qPCR assay and compared the results with those of a routine immunofluorescence assay (IFA) and clinical data. The assay included automated DNA extraction, amplification of the mitochondrial large-subunit rRNA gene and an internal control, and quantification of copy numbers with the help of a plasmid clone. We studied 353 consecutive BAL fluids obtained for investigation of unexplained fever and/or pneumonia in 287 immunocompromised patients. No qPCR inhibition was observed. Seventeen (5%) samples were both IFA and qPCR positive, 63 (18%) were IFA negative and qPCR positive, and 273 (77%) were both IFA and qPCR negative. The copy number was significantly higher for IFA-positive/qPCR-positive samples than for IFA-negative/qPCR-positive samples (4.2 +/- 1.2 versus 1.1 +/- 1.1 log(10) copies/mu l; P < 10(-4)). With IFA as the standard, the qPCR assay sensitivity was 100% for >= 2.6 log(10) copies/mu l and the specificity was 100% for >= 4 log(10) copies/mu l. Since qPCR results were not available at the time of decision-making, these findings did not trigger cotrimoxazole therapy. Patients with systemic inflammatory diseases and IFA-negative/qPCR-positive BAL fluid had a worse 1-year survival rate than those with IFA-negative/qPCR-negative results (P < 10(-3)), in contrast with solid-organ transplant recipients (P = 0.88) and patients with hematological malignancy (P = 0.26). Quantifying P. jirovecii DNA in BAL fluids independently of IFA positivity should be incorporated into the investigation of pneumonia in immunocompromised patients. The relevant threshold remains to be determined and may vary according to the underlying disease.