Wen Xiaozi, Chen Qiong, Yin Huali, Wu Shenghai, Wang Xianjun
Department of The Fourth Clinical Medical College affiliated to Zhejiang Chinese Medical University.
Department of Clinical Laboratory, Hangzhou First People's Hospital affiliated to Zhejiang Chinese Medical University.
Medicine (Baltimore). 2020 Feb;99(7):e19194. doi: 10.1097/MD.0000000000019194.
The incidence of invasive fungal infections (IFIs) has recently increased, and early and accurate diagnosis of IFIs is important for the rational selection of antifungal drugs with high efficacy. We developed a method for rapid and accurate clinical diagnosis of IFIs and provide a reference for personalized drug treatment.We designed and screened fungal internal transcribed spacer regions with universal primers and designed 8 TaqMan detection probes to establish a multi-channel real-time fluorescent polymerase chain reaction (PCR) melting curve analysis (MCA) method. The sensitivity, specificity, and reproducibility of this method were investigated using standard fungal strains and clinical isolates. Candidemia was detected using the MCA method.The limit of detection and assay cut-off (melting temperature [Tm]) for Candida albicans were 0.05 pg/μL and 66.50 °C; Candida glabrata were 0.1 pg/μL and 66.25 °C; Candida tropicalis were 0.1 pg/μL and 60.15 °C; Candida krusei were 0.1 pg/μL and 72.15 °C; Candida parapsilosis were 0.2 pg/μL and 63.10 °C; Candida guilliermondii were 0.1 pg/μL and 61.84 °C; Cryptococcus neoformans were 0.1 pg/μL and 65.50 °C; Aspergillus flavus were 0.05 pg/μL and 71.50 °C; Aspergillus terreus, Aspergillus fumigatus, and Aspergillus niger were 0.05 pg/μL and 76.80 °C. Analytical specificity was evaluated using 13 clinical pathogens including Streptococcus pneumoniae, Staphylococcus aureus, and Haemophilus influenzae, etc. No false-positive results were obtained for any of these samples. The MCA method can detect and identify different candidemia simulations. The limit detection concentration of C albicans was 44 cfu/mL, C glabrata was 73 cfu/mL, C tropicalis was 29 cfu/mL, C parapsilosis was 21 cfu/mL, C krusei was 71 cfu/mL, and C guilliermondii was 53 cfu/mL.The multi-channel real-time fluorescence PCR melting curve analysis displayed high sensitivity and specificity in detecting various clinically invasive fungi. Furthermore, it simultaneously detected the genera Candida, Cryptococcus, and Aspergillus and identified Candida at the species level. Our method can facilitate early and accurate clinical diagnosis and personalized medication regimens.
侵袭性真菌感染（IFI）的发病率近年来有所上升，IFI的早期准确诊断对于合理选择高效抗真菌药物至关重要。我们开发了一种快速准确的IFI临床诊断方法，为个性化药物治疗提供参考。我们用通用引物设计并筛选真菌内部转录间隔区，设计8种TaqMan检测探针，建立了多通道实时荧光聚合酶链反应（PCR）熔解曲线分析（MCA）方法。使用标准真菌菌株和临床分离株研究了该方法的灵敏度、特异性和可重复性。用MCA方法检测念珠菌血症。白色念珠菌的检测限和分析临界值（熔解温度[Tm]）分别为0.05 pg/μL和66.50°C；光滑念珠菌为0.1 pg/μL和66.25°C；热带念珠菌为0.1 pg/μL和60.15°C；克柔念珠菌为0.1 pg/μL和72.15°C；近平滑念珠菌为0.2 pg/μL和63.10°C；季也蒙念珠菌为0.1 pg/μL和61.84°C；新生隐球菌为0.1 pg/μL和65.50°C；黄曲霉为0.05 pg/μL和71.50°C；土曲霉、烟曲霉和黑曲霉为0.05 pg/μL和76.80°C。使用包括肺炎链球菌、金黄色葡萄球菌和流感嗜血杆菌等13种临床病原体评估分析特异性。这些样本均未获得假阳性结果。MCA方法可检测和鉴定不同的念珠菌血症模拟物。白色念珠菌的最低检测浓度为44 cfu/mL，光滑念珠菌为73 cfu/mL，热带念珠菌为29 cfu/mL，近平滑念珠菌为21 cfu/mL，克柔念珠菌为71 cfu/mL，季也蒙念珠菌为53 cfu/mL。多通道实时荧光PCR熔解曲线分析在检测各种临床侵袭性真菌时显示出高灵敏度和特异性。此外，它同时检测念珠菌属、隐球菌属和曲霉属，并在种水平上鉴定念珠菌。我们的方法有助于早期准确的临床诊断和个性化用药方案。
