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.

Canine pyoderma gangrenosum with recurring skin lesions of unknown origin and splenic involvement

“…Periodic acid Schiff stain and acid‐fast stain did not reveal infectious agents. PCR for bacterial, fungal and mycobacterial pathogens performed on formalin‐fixed paraffin‐embedded skin were negative …”

“…PCR for bacterial, fungal and mycobacterial pathogens performed on formalin-fixed paraffin-embedded skin were negative. [1][2][3] After ruling out other causes of cutaneous ulceration and applying the diagnostic criteria for human pyoderma gangrenosum (PG) 4 and criteria used in another case of canine PG, 5 a presumptive diagnosis of PG was made. The dog was treated with prednisolone 1.5 mg/kg twice daily p.o.…”

Long term survival of a dog with disseminated Aspergillus deflectus infection without definitive treatment

“…This might have led to misidentification in earlier studies. Molecular testing methods using polymerase chain reaction and comparative sequence analysis enable definitive identification of most clinical isolates [5] .…”

Is This Zebra Really a Zebra? The Challenge of Diagnosing Rare Fungal Infections in Veterinary Pathology

“…1,15,19 However, caution needs to be observed in the interpretation of FFPE-PCR results because, as More et al 13 pointed out, this methodology can lead to erroneous results, which become apparent when the species identified by PCR does not match the morphology seen in the tissue or the culture results. 11 Erroneous PCR results are most frequent in specimens from nonsterile body sites such as the nasal cavity. As fungal spores are ubiquitous in the environment and animals, with their noses to the ground, are constantly filling their sinuses with these spores, any species detected by PCR from nasal/fungal specimens may simply represent an environmental contaminant and not the cause of an infection.…”