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目的应用靶基因的多重定量PCR方法对肺孢子菌肺炎(PCP)患者呼吸道标本中肺孢子菌进行检测,比较3种方法的检测性能。方法提取PCP患者呼吸道标本DNA,分别以Pj线粒体大亚基rRNA(mtLSUrRNA)、主要表面糖蛋白(Msg)、二氢叶酸合成酶(DHPS)为靶基因进行定量PCR检测,以临床诊断为金标准评价Mt-qPCR,Msg-qPCR和DHPS-qPCR 3种方法的敏感性、特异性、阳性预测值和阴性预测值。结果Mt-qPCR敏感性95.9%,特异性82.9%,阳性预测值83.8%,阴性预测值95.6%,Youden指数0.788,受试者工作特征曲线(ROC)下面积0.942(P<0.01),95%置信区间(CI)0.909-0.975;Msg-qPCR敏感性63.9%,特异性92.4%,阳性预测值88.6%,阴性预测值73.5%,Youden指数0.563,ROC曲线下面积0.795(P<0.01),95%CI 0.730-0.860;DHPS-qPCR敏感性49.5%,特异性98.1%,阳性预测值96.0%,阴性预测值67.8%,Youden指数0.476,ROC曲线下面积0.740(P<0.01),95%CI0.669-0.811。PCP诊断组中Mt-qPCR、Msg-qPCR和DHPS-qPCR同时阳性43例(44.3%),任意2种方法阳性20例(占20.6%),任意1种方法阳性34例(占35.1%);非PCP组中任意2种方法阳性5例(占4.8%),任意1种方法阳性18例(占17.1%),3种方法均为阴性82例(78.1%),两组复合阳性率(100%与21.9%)比较差异有统计学意义(χ2=127.5,P<0.05)。结论以临床诊断为金标准,Mt-qPCR方法优于Msg-qPCR和DHPS-qPCR,联合使用含有MtqPCR在内的2种以上qPCR方法能提高PCP诊断的符合率。
Objective To detect the Pneumocystis pneumoniae in respiratory specimens of patients with pneumocystis pneumonia (PCP) by multiplex quantitative polymerase chain reaction (PCR) of target genes. The detection performance of the three methods was compared. Methods DNA from respiratory specimens of patients with PCP was extracted and quantitative PCR was performed to detect the mRNA of mtLSUrRNA, Msg and DHPS in Pj mitochondria respectively, The sensitivity, specificity, positive predictive value and negative predictive value of Mt-qPCR, Msg-qPCR and DHPS-qPCR were evaluated. Results The Mt-qPCR sensitivity was 95.9%, specificity was 82.9%, positive predictive value was 83.8%, negative predictive value was 95.6%, Youden index was 0.788. The area under the receiver operating characteristic curve (ROC) was 0.942 (95% Confidence interval (CI) 0.909-0.975; Msg-qPCR sensitivity 63.9%, specificity 92.4%, positive predictive value 88.6%, negative predictive value 73.5%, Youden index 0.563, area under the ROC curve 0.795 (P <0.01) % CI 0.730-0.860; DHPS-qPCR sensitivity 49.5%, specificity 98.1%, positive predictive value 96.0%, negative predictive value 67.8%, Youden index 0.476, area under ROC curve 0.740 (P <0.01), 95% CI0. 669-0.811. 43 cases (44.3%) were positive for Mt-qPCR, Msg-qPCR and DHPS-qPCR in the PCP diagnosis group, 20 cases (20.6%) were positive in any two methods and 34 cases (35.1%) in any one method. There were 5 positive cases (4.8%) in any of the 2 non-PCP group, 18 positive cases (17.1%) in any of the 1 methods, 82 negative cases (78.1%) in 3 methods, % And 21.9%, respectively) were statistically significant (χ2 = 127.5, P <0.05). Conclusion The clinical diagnosis is the gold standard. Mt-qPCR is superior to Msg-qPCR and DHPS-qPCR, and the combination of two or more qPCR methods including MtqPCR can improve the coincidence rate of PCP diagnosis.