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Nested PCR Detection of Pythium sp. from Formalin-Fixed, Paraffin-Embedded Canine Tissue Sections.

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  • 1Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907, USA.

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|August 25, 2022
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Summary

A new nested PCR test improves the diagnosis of pythiosis in dogs by detecting Pythium insidiosum more effectively than traditional methods. This molecular technique enhances diagnostic sensitivity for this serious oomycete infection.

Keywords:
FFPEPCRPythium insidiosumbiopsydiagnosisdogfungi

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Area of Science:

  • Veterinary Mycology
  • Molecular Diagnostics

Background:

  • Pythiosis, caused by the oomycete Pythium insidiosum, is a severe infection in dogs with a poor prognosis.
  • Traditional diagnostic methods like H&E staining have limitations in accurately identifying P. insidiosum due to poor staining and morphological similarity to other organisms.

Purpose of the Study:

  • To develop and evaluate a nested PCR assay for detecting P. insidiosum in formalin-fixed, paraffin-embedded (FFPE) tissues.
  • To compare the diagnostic sensitivity of the nested PCR with traditional histopathologic techniques.

Main Methods:

  • DNA was extracted from FFPE tissue scrolls of 26 dogs with suspected pythiosis.
  • Nested PCR was performed to detect P. insidiosum DNA.
  • Histopathological evaluation using H&E, PAS, and GMS stains was conducted for comparison.

Main Results:

  • Nested PCR detected P. insidiosum in 76.9% of samples, while histopathology identified Pythium sp. hyphae in 57.7%.
  • Agreement between histopathology and nested PCR was observed in 18 out of 26 cases.
  • The study identified an unexpectedly high incidence of 20 canine pythiosis cases in Indiana and Kentucky over 18 years.

Conclusions:

  • Nested PCR significantly enhances the diagnostic sensitivity for canine pythiosis compared to histopathology alone.
  • Combining histopathology with nested PCR is recommended for accurate diagnosis of P. insidiosum infections.
  • The findings highlight the importance of considering pythiosis in dogs, even in temperate regions.