Performances of two different panfungal PCRs to detect mould DNA in formalin-fixed paraffin-embedded tissue: what are the limiting factors?

Detection of fungal DNA from formalin-fixed, paraffin-embedded (FFPE) tissue is challenging due to degradation of DNA and presence of PCR inhibitors in these samples. We analyzed FFPE samples of 26 patients by panfungal PCR and compared the results to the composite diagnosis according to the Europea...

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Bibliographic Details
Published in:BMC infectious diseases 2014-12, Vol.14 (1), p.692-692, Article 692
Main Authors: Babouee Flury, B, Weisser, M, Prince, S Savič, Bubendorf, L, Battegay, M, Frei, R, Goldenberger, D
Format: Article
Language:English
Subjects:
Analysis
Antifungal agents
Aspergillosis - diagnosis
Aspergillosis - microbiology
Aspergillus - genetics
Biopsy
Care and treatment
Deoxyribonucleic acid
Diagnosis
DNA
DNA, Fungal - genetics
DNA, Fungal - isolation & purification
Fixatives - chemistry
Formaldehyde
Formaldehyde - chemistry
Genes
Health aspects
Histopathology
Humans
Identification systems
Infectious diseases
Molecular Diagnostic Techniques
Mortality
Mycological Typing Techniques
Mycoses
Paraffin Embedding
Polymerase Chain Reaction - methods
Risk factors
Sensitivity and Specificity
Studies
Tissue Fixation
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Summary:Detection of fungal DNA from formalin-fixed, paraffin-embedded (FFPE) tissue is challenging due to degradation of DNA and presence of PCR inhibitors in these samples. We analyzed FFPE samples of 26 patients by panfungal PCR and compared the results to the composite diagnosis according to the European Organization for Research and Treatment of Cancer (EORTC) criteria. Additionally we analyzed the quality of human and fungal DNA and their level of age-dependent degradation, as well as the existence of PCR inhibition in these tissue samples. We evaluated two 45-cycle panfungal PCR tests that target the internal transcribed spacer 2 (ITS2) as well as the ITS1-5.8S-ITS2 (ITS1-2) region. The PCRs were applied to 27 FFPE specimens from 26 patients with proven invasive fungal disease (IFD), and one patient with culture and histologically negative but PCR-positive fungal infection collected at our institution from 2003 to 2010. Quality of DNA in FFPE tissue samples was evaluated using fragments of the beta-globin gene for multiplex PCR, inhibition of PCR amplification was evaluated by spiking of C. krusei DNA to each PCR premix. In 27 FFPE samples the ITS2 PCR targeting the shorter fragment showed a higher detection rate with a sensitivity of 53.8% compared to the ITS1-2 fragment (sensitivity 38%). Significant time-dependent degradation of human DNA in FFPE sample extracts was detected based on partial beta-globin gene amplification which was not in correlation to successful panfungal PCR identification of fungal organisms. The analytical sensitivity of both assays compared with culture was 60 CFU/ml of a Candida krusei reference strain. The performance of the two tests in an Aspergillus proficiency panel of an international external quality assessment programme showed considerable sensitivity. Panfungal diagnostic PCR assays applied on FFPE specimens provide accurate identification of molds in highly degraded tissue samples and correct identification in samples stored up to 7 years despite sensitivity limitations, mainly caused by partial PCR inhibition and DNA degradation by formalin.
ISSN:1471-2334
1471-2334
DOI:10.1186/s12879-014-0692-z