title: Panfungal Polymerase Chain Reaction for Identification of Fungal Pathogens in Formalin-Fixed Animal Tissues.
abstract: Identification of fungal organisms often poses a problem for pathologists because the histomorphology of some fungal organisms is not specific, fresh tissues may not be available, and isolation and identification in culture may take a long time. The purpose of this study was to validate the use of panfungal polymerase chain reaction (PCR) to identify fungal organisms from formalin-fixed paraffin-embedded (FFPE) tissues. Formalin-fixed paraffin-embedded curls were tested from 128 blocks containing canine, feline, equine, and bovine tissues with cutaneous, nasal, pulmonary, and systemic fungal infections, identified by the presence of fungi in histologic sections. Quantitative scoring of histologic sections identified rare (11.9%), occasional (17.5%), moderate (17.5%), or abundant (53.1%) fungal organisms. DNA was isolated from FFPE tissues and PCR was performed targeting the internal transcribed spacer 2 (ITS-2) region, a segment of noncoding DNA found in all eukaryotes. Polymerase chain reaction products were sequenced and identified at ≥97% identity match using the Basic Local Alignment Search Tool and the NCBI database of ITS sequences. Of the 128 blocks, 117 (91.4%) yielded PCR products and high-quality sequences were derived from 89 (69.5%). Sequence and histologic identifications matched in 79 blocks (61.7%). This assay was capable of providing genus- and species-level identification when histopathology could not and, thus, is a beneficial complementary tool for diagnosis of fungal diseases.
authors.affiliation: 1 Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.
authors.forename: Courtney
authors.initials: C
authors.lastname: Meason-Smith
authors.affiliation: 1 Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.
authors.forename: Erin E
authors.initials: EE
authors.lastname: Edwards
authors.affiliation: 1 Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.
authors.forename: Caitlin E
authors.initials: CE
authors.lastname: Older
authors.affiliation: 1 Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.
authors.forename: Mackenzie
authors.initials: M
authors.lastname: Branco
authors.affiliation: 1 Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.
authors.forename: Laura K
authors.initials: LK
authors.lastname: Bryan
authors.affiliation: 1 Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.
authors.forename: Sara D
authors.initials: SD
authors.lastname: Lawhon
authors.affiliation: 2 Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.
authors.forename: Jan S
authors.initials: JS
authors.lastname: Suchodolski
authors.affiliation: 3 Texas A&M Veterinary Medical Diagnostic Laboratory, College Station, TX, USA.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Gomez
authors.affiliation: 1 Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.
authors.forename: Joanne
authors.initials: J
authors.lastname: Mansell
authors.affiliation: 1 Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.
authors.forename: Aline Rodrigues
authors.initials: AR
authors.lastname: Hoffmann
mesh_terms: D000818:Animals; D002371:Cat Diseases / Q000175:diagnosis / Q000382:microbiology / Q000473:pathology; D002415:Cats; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis / Q000382:microbiology / Q000473:pathology; D004271:DNA, Fungal / Q000235:genetics; D004283:Dog Diseases / Q000175:diagnosis / Q000382:microbiology / Q000473:pathology; D004285:Dogs; D006734:Horse Diseases / Q000175:diagnosis / Q000382:microbiology / Q000473:pathology; D006736:Horses; D009181:Mycoses / Q000175:diagnosis / Q000382:microbiology / Q000473:pathology / Q000662:veterinary*; D016612:Paraffin Embedding / Q000662:veterinary; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D016415:Sequence Alignment / Q000662:veterinary
chemical_list: D004271:DNA, Fungal
keywords: DNA isolation; formal-fixed paraffin-embedded; fungi; internal transcribed spacer; lyticase; mycology; mycoses; panfungal polymerase chain reaction; pathology