Molecular quantification and differentiation of Candida species in biological specimens of patients with liver cirrhosis

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 3.0x101 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 3.8x105 copies ml-1 (2.3x102-6.3x109). 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

Patient characteristics at baseline.

<p>Patient characteristics at baseline.</p

Fungal pathogen and <i>Candida</i> DNA detection rates from microbial culture, real-time PCR analysis, and its statistical significance.

<p>Fungal pathogen and <i>Candida</i> DNA detection rates from microbial culture, real-time PCR analysis, and its statistical significance.</p

Receiver operating characteristic curve (ROC) of 18S rRNA gene based quantitative PCR from biological samples of patients with liver cirrhosis and controls.

<p>Receiver operating characteristic curve (ROC) of 18S rRNA gene based quantitative PCR from biological samples of patients with liver cirrhosis and controls.</p

Fungal species analysis using either the T-RFLP or conventional culture methods on duodenal fluid samples from patients with liver cirrhosis and controls (n = 56).

<p>Each bar represents a single species. A star indicates that a fungal species could not be confirmed by the alternative method. The x-axis presents single duodenal samples so that multiple culture or T-RFLP-identified pathogens per sample are stacked in columns. Samples with culture-positive and PCR/T-RFLP-positive samples are compared in A (n = 40). Culture-negative but PCR/T-RFLP-positive samples are displayed in B (n = 9) and culture-positive and PCR/T-RFLP-negative samples are presented in C (n = 2). The duodenal samples that were culture-positive and PCR-positive but T-RFLP-negative are represented in area D (n = 5). Additional duodenal samples that were culture negative but PCR-positive with DNA not being sufficient for T-RFLP analysis (n = 3) have been left out in analysis.</p

Detection of fungal species in blood and duodenal fluid of patients with liver cirrhosis and in control patients by using microbial culture techniques.

<p>The number of each sample group equals 100%. Stacked bars without percentage value correspond to 1.6%.</p