Bolehovska Radka, Pliskova Lenka, Buchta Vladimir, Cerman Jaroslav, Hamal Petr
Department of Clinical Biochemistry and Diagnostics, Faculty of Medicine and University Hospital, Charles University, Hradec Kralove, 500 05, Czech Republic.
Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2006 Nov;150(2):245-8. doi: 10.5507/bp.2006.035.
Recently, the proportion of invasive infections caused by the filamentous fungi of the Aspergillus genus are growing in immunocompromised persons particularly in transplant recipients and neutropenic patients. Unfortunately, laboratory diagnostics of invasive aspergillosis remains extremely difficult, mainly with regard to the sensitivity of the methods and to the correct interpretation of the results in particular.
The aim of this work was to design a standard and reproducible Aspergillus DNA detection method and its validation. The second aim was to practically use this method for diagnosis of Aspergillus DNA in various samples in patients.
Real-time PCR with two hybridization probes. Amplification and on-line quantification was carried out on a LightCycler 1.5 Instrument.
Specificity of the reaction was tested for A. fumigatus, A. flavus, A. niger and A. terreus, and its sensitivity was determined at 5 copies per ml. The reproducibility of the results was comparable to other methods, reported in the literature. Applicability of the real-time PCR was assessed for detection of Aspergillus DNA in 354 various clinical samples taken from 179 patients at risk of invasive aspergillosis over the period of 33 months. Of 354 samples 103 (29.10 %) taken from 65 patients (36.31 %) were evaluated as positive. Over one year, the percentage of positive samples was mostly about 30 % or less per month.
Our results demonstrate the high sensitivity, specificity and reproducibility of this technique, and its usefulness for rapid laboratory diagnosis of invasive aspergillosis.
最近，曲霉属丝状真菌引起的侵袭性感染在免疫功能低下者中所占比例不断上升，尤其是在移植受者和中性粒细胞减少患者中。不幸的是，侵袭性曲霉病的实验室诊断仍然极其困难，主要体现在方法的敏感性以及结果的正确解读方面。
本研究旨在设计一种标准化且可重复的曲霉DNA检测方法并进行验证。第二个目的是将该方法实际应用于患者各种样本中曲霉DNA的诊断。
采用带有两种杂交探针的实时荧光定量PCR。在LightCycler 1.5仪器上进行扩增和在线定量。
对烟曲霉、黄曲霉、黑曲霉和土曲霉的反应特异性进行了检测，其灵敏度确定为每毫升5个拷贝。结果的可重复性与文献报道的其他方法相当。在33个月期间，对179例有侵袭性曲霉病风险的患者采集的354份各种临床样本进行实时荧光定量PCR检测曲霉DNA的适用性评估。在354份样本中，有103份（29.10%）来自65例患者（36.31%）的样本被评估为阳性。在一年多的时间里，每月阳性样本的百分比大多约为30%或更低。
我们的结果证明了该技术具有高灵敏度、特异性和可重复性，以及其在侵袭性曲霉病快速实验室诊断中的实用性。
