多重荧光PCR-熔解曲线法同时检测常见病原真菌方法学的建立及诊断价值

目的建立多重荧光PCR-熔解曲线法,并评价其同时检测假丝酵母菌、曲霉菌及新型隐球菌的诊断价值。 方法建立多重荧光PCR-熔解曲线法并对其检测体系进行优化。收集临床高度怀疑为侵袭性真菌感染（IFI）的各类临床样本179例,其中血液65例、深部痰液35例、尿液30例、脑脊液18例、胸腹水12例、肺泡灌洗液10例、新鲜肺组织9例。通过敏感性、特异性、重复性实验验证多重荧光PCR-熔解曲线法的检测性能,并应用受试者工作特征（ROC）曲线评价该方法的诊断效能。 结果建立的多重荧光PCR-熔解曲线法可同时检测8种常见病原真菌,包括4种假丝酵母菌（白假丝酵母菌、热带假丝酵母菌、光滑假丝酵母菌、克柔假丝酵母菌）、3种曲霉菌（烟曲霉、黄曲霉、黑曲霉）和新型隐球菌。其最低检测限为1 × 10⁴ cfu/mL,且常见细菌无扩增反应,重复性实验解链温度波动小于0.5 ℃。179例临床样本中,以培养法、镜检法、病理诊断等"金标准"方法诊断IFI阳性为112例,多重荧光PCR-熔解曲线法检测阳性为96例。多重荧光PCR-熔解曲线法同时检测假丝酵母菌、曲霉菌及新型隐球菌的敏感度为0.857,特异度为0.970,阳性预测值为0.980,阴性预测值为0.802,ROC曲线下面积为0.914,95%置信区间为0.868 ~ 0.959,P < 0.001。 结论本研究建立的多重荧光PCR-熔解曲线法可快速、准确、高通量同时检测假丝酵母菌、曲霉菌及新型隐球菌,对于IFI的早期诊断具有重要意义。

Establishment and diagnostic value of multiplex fluorescent PCR-melting curve method for detection of common pathogenic fungi

ObjectiveTo establish a multiplex fluorescent PCR-melting curve method for simultaneous detection of Candida, Aspergillus and Cryptococcus, and to evaluate its diagnostic value. MethodsA multiplex fluorescent PCR-melting curve method was established and its detection system was optimized. Totally 179 clinical specimens including 65 blood specimens, 35 deep sputum specimens, 30 urine specimens, 18 cerebrospinal fluid specimens, 12 hydrothorax and ascites specimens, 10 alveolar lavage fluid specimens and 9 fresh lung tissue specimens for highly suspected invasive fungal infection (IFI) were enrolled in this study. The sensitivity, specificity and repeatability experiments were used to verify the detection performance of this method, and the receiver operating characteristic (ROC) curve was used to evaluate its diagnostic efficiency. ResultsThe multiplex fluorescent PCR-melting curve method could simultaneously detect 8 common pathogenic fungi, including 4 kinds of Candida (Candida albicans, Candida tropicalis, Candida glabrata, Candida krusei), 3 kinds of Aspergillus (Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger) and Cryptococcus neoformans. The minimum detection limit was 1 × 10⁴ cfu/mL. Common bacteria had no amplification reaction, and the melting temperature fluctuation of repetitive experiment was less than 0.5 ℃. Among 179 clinical specimens, 112 were positive by "gold standard" methods such as culture, microscopy and pathological diagnosis, and 96 were positive by the multiplex fluorescent PCR-melting curve method. The sensitivity, specificity, positive predictive value and negative predictive value for simultaneous detection of Candida, Aspergillus and Cryptococcus neoformans by this method were 0.857, 0.970, 0.980 and 0.802, respectively. The area under ROC curve was 0.914, and the 95% confidence interval was 0.868 ~ 0.959 (P < 0.001). ConclusionThe multiplex fluorescent PCR-melting curve method can detect Candida, Aspergillus and Cryptococcus neoformans rapidly and accurately with high throughput, which is of great significance for the early diagnosis of IFI.