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Performances of two different panfungal PCRs to detect mould DNA in formalin-fixed paraffin-embedded tissue: what are the limiting factors?
BACKGROUND: 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...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4272807/ https://www.ncbi.nlm.nih.gov/pubmed/25518949 http://dx.doi.org/10.1186/s12879-014-0692-z |
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author | Babouee Flury, B Weisser, M Prince, S Savič Bubendorf, L Battegay, M Frei, R Goldenberger, D |
author_facet | Babouee Flury, B Weisser, M Prince, S Savič Bubendorf, L Battegay, M Frei, R Goldenberger, D |
author_sort | Babouee Flury, B |
collection | PubMed |
description | BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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. |
format | Online Article Text |
id | pubmed-4272807 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-42728072014-12-22 Performances of two different panfungal PCRs to detect mould DNA in formalin-fixed paraffin-embedded tissue: what are the limiting factors? Babouee Flury, B Weisser, M Prince, S Savič Bubendorf, L Battegay, M Frei, R Goldenberger, D BMC Infect Dis Research Article BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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. BioMed Central 2014-12-18 /pmc/articles/PMC4272807/ /pubmed/25518949 http://dx.doi.org/10.1186/s12879-014-0692-z Text en © Babouee Flury et al.; licensee BioMed Central Ltd. 2014 This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Research Article Babouee Flury, B Weisser, M Prince, S Savič Bubendorf, L Battegay, M Frei, R Goldenberger, D Performances of two different panfungal PCRs to detect mould DNA in formalin-fixed paraffin-embedded tissue: what are the limiting factors? |
title | Performances of two different panfungal PCRs to detect mould DNA in formalin-fixed paraffin-embedded tissue: what are the limiting factors? |
title_full | Performances of two different panfungal PCRs to detect mould DNA in formalin-fixed paraffin-embedded tissue: what are the limiting factors? |
title_fullStr | Performances of two different panfungal PCRs to detect mould DNA in formalin-fixed paraffin-embedded tissue: what are the limiting factors? |
title_full_unstemmed | Performances of two different panfungal PCRs to detect mould DNA in formalin-fixed paraffin-embedded tissue: what are the limiting factors? |
title_short | Performances of two different panfungal PCRs to detect mould DNA in formalin-fixed paraffin-embedded tissue: what are the limiting factors? |
title_sort | performances of two different panfungal pcrs to detect mould dna in formalin-fixed paraffin-embedded tissue: what are the limiting factors? |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4272807/ https://www.ncbi.nlm.nih.gov/pubmed/25518949 http://dx.doi.org/10.1186/s12879-014-0692-z |
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