Molecular and Genetic Laboratory, Centre for Cardiovascular Surgery and Transplantation, Brno, Czech Republic.
Medical Genomics Research Group, CEITEC, Masaryk University, Brno, Czech Republic.
J Clin Microbiol. 2019 Mar 28;57(4). doi: 10.1128/JCM.01861-18. Print 2019 Apr.
Molecular techniques in fungal detection and identification represent an efficient complementary diagnostic tool which is increasingly used to overcome limitations of routinely used culture techniques. The aim of this study was to characterize sp. representation in samples from urine, urinary catheter, and ureteral stent biofilm using ribosomal DNA (rDNA) amplification followed by fluorescent capillary electrophoresis (f--PCR-CE) and to compare the results with those obtained by culture. A total of 419 samples were analyzed, and 106 (25.2%) were found positive, out of which 17 (16%) were polyfungal. The positivity rate did not differ between samples from catheters and stents (23.6% versus 20.9%) or between catheter and stent corresponding urine samples (40.2% versus 30.2%). Ten different species were detected, with (31.4%), (26.5%), and (12.4%) predominating. f--PCR-CE was evaluated as substantially less time-consuming and 8.3 times more sensitive than the routinely applied culture technique with 1 µl of urine/sonicated fluid inoculated, detecting 67 (19.9%) versus 8 (2.4%) positive samples out of 337 initially analyzed samples. The culture sensitivity considerably improved to 1.7 times lower than that of f--PCR-CE after the inoculation volume was increased to 100 µl in the additional 82 samples. Moreover, the molecular technique, unlike routine cultivation, enabled precise pathogen composition determination in polymicrobial samples. In conclusion, the f--PCR-CE method was shown to be a quick and efficient tool for culture-independent detection and identification of fungi in urinary tract-related samples, demonstrating a higher sensitivity than culture.
分子技术在真菌检测和鉴定中代表了一种有效的补充诊断工具,它越来越多地被用于克服常规培养技术的局限性。本研究的目的是使用核糖体 DNA(rDNA)扩增结合荧光毛细管电泳(f--PCR-CE)来描述尿液、导尿管和输尿管支架生物膜样本中 sp. 的代表情况,并将结果与培养结果进行比较。共分析了 419 个样本,其中 106 个(25.2%)为阳性,其中 17 个(16%)为多真菌。导管和支架样本的阳性率(23.6%比 20.9%)或导管和支架相应尿液样本的阳性率(40.2%比 30.2%)无差异。检测到 10 种不同的 种,其中 (31.4%)、 (26.5%)和 (12.4%)占主导地位。f--PCR-CE 比常规应用的培养技术耗时少,灵敏度高 8.3 倍,用 1μl 尿液/超声液接种,可检测出 337 个初始分析样本中 67 个(19.9%)阳性样本,而不是 8 个(2.4%)阳性样本。在另外 82 个样本中,将接种体积增加到 100μl 后,培养的灵敏度提高到 f--PCR-CE 的 1.7 倍以下。此外,与常规培养不同,该分子技术可在多微生物样本中精确确定病原体组成。总之,f--PCR-CE 方法被证明是一种快速有效的工具,用于非培养依赖的尿路上皮相关样本中真菌的检测和鉴定,其灵敏度高于培养。