University Hospital Leipzig, Section of Hepatology, Clinic of Gastroenterology and Rheumatology, University Hospital Leipzig, Leipzig, Germany.
Helmholtz Centre for Environmental Research-UFZ, Department of Environmental Microbiology, Leipzig, Germany.
PLoS One. 2018 Jun 13;13(6):e0197319. doi: 10.1371/journal.pone.0197319. eCollection 2018.
Patients with liver cirrhosis are susceptible to fungal infections. Due to low sensitivity of culture-based methods, we applied a real-time PCR assay targeting the 18S rRNA gene in combination with direct sequencing and terminal-restriction fragment length polymorphism (T-RFLP) in order to establish a novel tool to detect fungal DNA and to quantify and differentiate Candida DNA, also in polyfungal specimens. In total, 281 samples (blood n = 135, ascites n = 92, duodenal fluid n = 54) from 135 patients with liver cirrhosis and 52 samples (blood n = 26, duodenal fluid n = 26) from 26 control patients were collected prospectively. Candida DNA was quantified in all samples. Standard microbiological culture was performed for comparison. Blood and ascites samples, irrespective of the patient cohort, showed a method-independent low fungal detection rate of approximately 1%, and the Candida DNA content level did not exceed 3.0x10(1) copies ml-1 in any sample. In contrast, in duodenal fluid of patients with liver cirrhosis high fungal detection rates were discovered by using both PCR- and culture-based techniques (81.5% vs. 66.7%; p = 0.123) and the median level of Candida DNA was 3.8x10(5) copies ml-1 (2.3x10(2)-6.3x10(9)). In cirrhosis and controls, fungal positive culture results were confirmed by PCR in 96% and an additional amount of 44% of culture negative duodenal samples were PCR positive. Using T-RFLP analysis in duodenal samples, overall 85% of results from microbial culture were confirmed and in 75% of culture-negative but PCR-positive samples additional Candida species could be identified. In conclusion, PCR-based methods and subsequent differentiation of Candida DNA might offer a quick approach to identifying Candida species without prior cultivation.
肝硬化患者易发生真菌感染。由于基于培养的方法敏感性低，我们应用实时 PCR 检测 18S rRNA 基因，并结合直接测序和末端限制性片段长度多态性（T-RFLP），以建立一种新的工具来检测真菌 DNA，并定量和区分念珠菌 DNA，也可用于多真菌标本。总共前瞻性收集了 135 例肝硬化患者的 281 个样本（血液 n=135，腹水 n=92，十二指肠液 n=54）和 26 例对照患者的 52 个样本（血液 n=26，十二指肠液 n=26）。所有样本均定量检测了念珠菌 DNA。同时进行了标准微生物培养以作比较。无论患者队列如何，血液和腹水样本的真菌检测率均较低，独立方法检测率约为 1%，任何样本的念珠菌 DNA 含量均未超过 3.0x10(1) 拷贝/ml-1。相比之下，肝硬化患者的十二指肠液中使用 PCR 和基于培养的技术检测到高真菌检出率（81.5% vs. 66.7%；p=0.123），念珠菌 DNA 的中位数水平为 3.8x10(5) 拷贝/ml-1（2.3x10(2)-6.3x10(9)）。在肝硬化和对照组中，96%的真菌阳性培养结果通过 PCR 得到了确认，另外还发现 44%的培养阴性十二指肠样本 PCR 阳性。在十二指肠样本中使用 T-RFLP 分析，微生物培养的总体 85%结果得到了确认，在 75%的培养阴性但 PCR 阳性样本中，还可以鉴定出其他念珠菌物种。总之，基于 PCR 的方法和随后对念珠菌 DNA 的区分可能提供了一种无需事先培养即可快速识别念珠菌的方法。
