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PLoS One. 2018 Jun 13;13(6):e0197319. doi: 10.1371/journal.pone.0197319. eCollection 2018.

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

Author information

1
University Hospital Leipzig, Section of Hepatology, Clinic of Gastroenterology and Rheumatology, University Hospital Leipzig, Leipzig, Germany.
2
Helmholtz Centre for Environmental Research-UFZ, Department of Environmental Microbiology, Leipzig, Germany.
3
Ludwig Maximilians-University, Max von Pettenkofer-Institute for Hygiene and Clinical Microbiology, Munich, Germany.
4
Department of Gastroenterology and Hepatology, Hospital and Outpatient Clinic for Internal Medicine A, Ernst Moritz Arndt University of Greifswald, Greifswald, Germany.
5
Institute for Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany.
6
Interdisciplinary Endoscopy Unit, Clinic of Gastroenterology and Rheumatology, University Hospital Leipzig, Leipzig, Germany.
7
University College London, Institute for Liver and Digestive Health, Royal Free Campus, London, United Kingdom.

Abstract

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.

PMID:
29897895
PMCID:
PMC5999271
DOI:
10.1371/journal.pone.0197319
[Indexed for MEDLINE]
Free PMC Article

Conflict of interest statement

This research received financial support from Pfizer Pharma GmbH (WS1956204; www.pfizer.de). Pfizer Pharma GmbH had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The authors have declared that no other competing interests exist.

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