Diagnostic Accuracy of a Direct Panfungal Polymerase Chain Reaction Assay Performed on Stained Cytology Slides.
Authors: Myers Alexandra N, Jeffery Unity, Seyler Zachary G, Lawhon Sara D, Hoffmann Aline Rodrigues
Journal: Veterinary pathology
Summary
# Diagnostic Accuracy of Direct Panfungal PCR on Cytology Slides Molecular identification of fungi from routine cytology slides could streamline diagnosis of fungal infections across multiple species, yet the reliability of direct PCR approaches applied to stained preparations had never been formally validated. Researchers tested a direct panfungal PCR assay targeting the internal transcribed spacer region on 36 cases with cytologically visible fungi or oomycetes (confirmed by culture or immunoassay) and 29 negative controls, performing the assay directly on stained cytology slides without nucleic acid extraction. The technique achieved 67% sensitivity overall, improving to 73% when excluding oomycetes and 86% when analysing slides with abundant fungal material, but specificity was disappointingly low at 62% owing to amplification of fungal DNA on control slides with negative cultures and no visible organisms. Whilst the assay reliably identifies fungi to genus or species level when adequate material is present, its propensity to detect environmental or non-pathogenic fungal contamination means it should be reserved for slides exhibiting morphologically visible fungi and always interpreted alongside cytopathological assessment rather than used as a standalone diagnostic tool. For equine practitioners, this suggests panfungal PCR could add value to suspected cutaneous or respiratory fungal cases showing clear cytological evidence, though morphological correlation remains essential to distinguish clinically significant infection from incidental contamination.
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Practical Takeaways
- •Direct panfungal PCR on cytology slides can identify fungal species without culture delays, but should only be used on slides showing visible fungi to reduce false positives from contamination
- •A negative PCR result does not exclude fungal infection if organisms are visible on cytology; always correlate molecular results with morphologic assessment
- •This technique is most reliable on samples with abundant fungal elements; sparse fungi may be missed
Key Findings
- •Direct panfungal PCR on stained cytology slides achieved 67% sensitivity overall and 86% sensitivity when slides contained abundant fungi
- •Specificity was only 62% due to amplification of contaminant fungal DNA from negative control slides
- •The assay provided genus- or species-level fungal identification when positive results were obtained
- •Panfungal PCR performance was improved when cytologic evidence of fungi was abundant