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

SIMPLE SUMMARY: Pythium insidiosum is a waterborne fungus-like organism commonly present in tropical and subtropical areas that causes a disease named pythiosis in dogs and other animals. This disease can cause inflammatory lesions on the skin and gastrointestinal tract and, if left untreated, may be fatal. Because this mold lacks the distinctive features of true fungi, it requires specific treatment. Diagnosis of pythiosis is commonly achieved by microscope visualization after staining the tissue sections (histopathology). Although some stains highlight hyphae, these techniques are challenging to distinguish P. insidiosum from other fungi. Our study aimed to develop a molecular technique (nested PCR) to specifically detect P. insidiosum DNA in biopsy specimens to aid in diagnosing this organism. Archived biopsies from 26 dogs suspected of pythiosis were examined by histopathology with special stains and tested by the novel nested PCR. Agreement between histopathology and nested PCR occurred in 18/26 cases. The microscopic examination identified hyphae consistent with Pythium sp. in 57.7% of the samples, whereas the nested PCR detected P. insidiosum DNA in 76.9% of samples, aiding in the sensitivity of the diagnosis of pythiosis in dogs. Using this combination of techniques, we report 20 canine cases of pythiosis over 18 years in Indiana and Kentucky, an unexpectedly high incidence for temperate climatic regions. Thus, we recommend using our nested PCR test in addition to the microscopic examination to increase the sensitivity of the diagnosis. ABSTRACT: Pythium insidiosum is an infectious oomycete affecting dogs that develop the cutaneous or gastrointestinal form of pythiosis with a poor prognosis. If left untreated, pythiosis may be fatal. This organism is not a true fungus because its cell wall and cell membrane lack chitin and ergosterol, respectively, requiring specific treatment. Identifying the organism is challenging, as a hematoxylin and eosin (H&E) stain poorly stain the P. insidiosum hyphae and cannot be differentiated conclusively from other fungal or fungal-like organisms (such as Lagenidium sp.) morphologically. Our study aimed to develop a nested PCR to detect P. insidiosum and compare it with the traditional histopathologic detection of hyphae. Formalin-fixed, paraffin-embedded (FFPE) tissue scrolls from 26 dogs with lesions suggesting the P. insidiosum infection were assessed histologically, and DNA was extracted from the FFPE tissue sections for nested PCR. Agreement between the histologic stains, (H&E), periodic acid–Schiff (PAS), and/or Grocott methenamine silver (GMS) and the nested PCR occurred in 18/26 cases. Hyphae consistent with Pythium sp. were identified via histopathology in 57.7% of the samples, whereas the nested PCR detected P. insidiosum in 76.9% of samples, aiding in the sensitivity of the diagnosis of pythiosis in dogs. Using this combination of techniques, we report 20 canine cases of pythiosis over 18 years in Indiana and Kentucky, an unexpectedly high incidence for temperate climatic regions. Using a combination of histopathology evaluation and nested PCR is recommended to aid in the accurate diagnosis of pythiosis.