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Mycology

Molecular Detection of Filamentous Fungi in Formalin-Fixed Paraffin-Embedded Specimens in Invasive Fungal Wound Infections Is Feasible with High Specificity

Anuradha Ganesan, Justin Wells, Faraz Shaikh, Philip Peterson, William Bradley, M. Leigh Carson, Joseph L. Petfield, Mary Klassen-Fischer, Kevin S. Akers, Kevin Downing, Ralf Bialek, David R. Tribble, Brian L. Wickes
Kimberly E. Hanson, Editor
Anuradha Ganesan
aInfectious Disease Clinical Research Program, Preventive Medicine and Biostatistics Department, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA
bHenry M. Jackson Foundation for the Advancement of Military Medicine, Inc., Bethesda, Maryland, USA
cWalter Reed National Military Medical Center, Bethesda, Maryland, USA
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Justin Wells
cWalter Reed National Military Medical Center, Bethesda, Maryland, USA
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Faraz Shaikh
aInfectious Disease Clinical Research Program, Preventive Medicine and Biostatistics Department, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA
bHenry M. Jackson Foundation for the Advancement of Military Medicine, Inc., Bethesda, Maryland, USA
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Philip Peterson
cWalter Reed National Military Medical Center, Bethesda, Maryland, USA
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William Bradley
aInfectious Disease Clinical Research Program, Preventive Medicine and Biostatistics Department, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA
bHenry M. Jackson Foundation for the Advancement of Military Medicine, Inc., Bethesda, Maryland, USA
dBrooke Army Medical Center, JBSA Fort Sam Houston, Texas, USA
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M. Leigh Carson
aInfectious Disease Clinical Research Program, Preventive Medicine and Biostatistics Department, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA
bHenry M. Jackson Foundation for the Advancement of Military Medicine, Inc., Bethesda, Maryland, USA
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Joseph L. Petfield
eLandstuhl Regional Medical Center, Landstuhl, Germany
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Mary Klassen-Fischer
fJoint Pathology Center, Silver Spring, Maryland, USA
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Kevin S. Akers
gU.S. Army Institute of Surgical Research, JBSA Fort Sam Houston, Texas, USA
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Kevin Downing
eLandstuhl Regional Medical Center, Landstuhl, Germany
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Ralf Bialek
hLADR GmbH Medizinisches Versorgungszentrum Dr. Kramer und Kollegen, Geesthacht, Germany
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David R. Tribble
aInfectious Disease Clinical Research Program, Preventive Medicine and Biostatistics Department, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA
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Brian L. Wickes
iUniversity of Texas Health Sciences Center at San Antonio, San Antonio, Texas, USA
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Kimberly E. Hanson
University of Utah
Roles: Editor
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DOI: 10.1128/JCM.01259-19
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ABSTRACT

Trauma-related invasive fungal wound infections (IFIs) are associated with significant morbidity and mortality. Early identification and treatment are critical. Traditional identification methods (e.g., fungal cultures and histopathology) can be delayed and insensitive. We assessed a PCR-based sequencing assay for rapid identification of filamentous fungi in formalin-fixed paraffin-embedded (FFPE) specimens obtained from combat casualties injured in Afghanistan. Blinded FFPE specimens from cases (specimens positive on histopathology) and controls (specimens negative on histopathology) were submitted for evaluation with a panfungal PCR. The internal transcribed spacer 2 (ITS2) region of the fungal ribosomal repeat was amplified and sequenced. The PCR results were compared with findings from histopathology and/or culture. If injury sites contributed multiple specimens, findings for the site were collapsed to the site level. We included 64 case subjects (contributing 95 sites) and 102 controls (contributing 118 sites). Compared to histopathology, panfungal PCR was specific (99%), but not as sensitive (63%); however, sensitivity improved to 83% in specimens from sites with angioinvasion. Panfungal PCR identified fungi of the order Mucorales in 33 of 44 sites with angioinvasion (75%), whereas fungal culture was positive in 20 of 44 sites (45%). Saksenaea spp. were the dominant fungi identified by PCR in specimens from angioinvasion sites (57%). Panfungal PCR is specific, albeit with lower sensitivity, and performs better at identifying fungi of the order Mucorales than culture. DNA sequencing offers significant promise for the rapid identification of fungal infection in trauma-related injuries, leading to more timely and accurate diagnoses.

FOOTNOTES

    • Received 31 July 2019.
    • Returned for modification 19 August 2019.
    • Accepted 10 October 2019.
    • Accepted manuscript posted online 16 October 2019.
  • Supplemental material is available online only.

  • Copyright © 2019 American Society for Microbiology.

All Rights Reserved.

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Molecular Detection of Filamentous Fungi in Formalin-Fixed Paraffin-Embedded Specimens in Invasive Fungal Wound Infections Is Feasible with High Specificity
Anuradha Ganesan, Justin Wells, Faraz Shaikh, Philip Peterson, William Bradley, M. Leigh Carson, Joseph L. Petfield, Mary Klassen-Fischer, Kevin S. Akers, Kevin Downing, Ralf Bialek, David R. Tribble, Brian L. Wickes
Journal of Clinical Microbiology Dec 2019, 58 (1) e01259-19; DOI: 10.1128/JCM.01259-19

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Molecular Detection of Filamentous Fungi in Formalin-Fixed Paraffin-Embedded Specimens in Invasive Fungal Wound Infections Is Feasible with High Specificity
Anuradha Ganesan, Justin Wells, Faraz Shaikh, Philip Peterson, William Bradley, M. Leigh Carson, Joseph L. Petfield, Mary Klassen-Fischer, Kevin S. Akers, Kevin Downing, Ralf Bialek, David R. Tribble, Brian L. Wickes
Journal of Clinical Microbiology Dec 2019, 58 (1) e01259-19; DOI: 10.1128/JCM.01259-19
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KEYWORDS

invasive fungal wound infection
trauma
combat
PCR-based assay
mucormycosis
PCR
invasive fungal infection

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