Evaluation of Tissue Specimens

The diagnosis of zygomycosis is easily made on tissue section from the margin of a necrotic lesion. Involved tissue demonstrates focal areas of infection and may appear nodular or may produce extensive areas of necrosis with accompanying hemorrhage into the tissue. Specifically, histology demonstrates invasion along the elastic lamina of blood vessels, with hyphae of the fungus extending into and occluding the lumens of the blood vessels they have invaded, followed by thrombosis and tissue necrosis (7). Abscess formation with central tissue necrosis, acute inflammatory exudate, and peripheral tissue invasion by hyphal elements is quite common. An acute inflammatory exudate often accompanies these infections in non-neutropenic patients.

Demonstration of the fungal elements with fungal specific stains such as Calcofluor white stain or Grocott-Gomori methenamine silver stain is recommended. The specimen should not be crushed or ground, because the nonseptate hyphae are prone to damage. The key feature associated with the Zygomycetes on direct examination of cytologic specimens is the presence of wide, ribbon-like, aseptate, hyaline hyphal elements, often in the setting of extensive necrotic debris. The width of the hyphal element varies substantially. Branching of the hyphae is seen, with wide-angle (generally around 90°) bifurcations noted.

Invasion of the blood vessels (angioinvasion) by hyphal elements is generally seen in infections with the Mucorales but usually not with the Entomophthorales. The hallmark of a zygomycosis includes the demonstration of wide, ribbon-like, hyaline, predominantly aseptate hyphae with wide-angle (45-90°) branching. The hyphae often are not preserved well and may become crinkled or gnarled in the tissue sections. This is often referred to as a "crinkled cellophane" appearance of the hyphal elements. To the inexperienced observer, these artifactual folds in the hyphae may be confused with septations. Cross sections of hyphal elements often give tissues a vacuolated appearance. These cross sections vary in diameter and may be confused with yeast cells. In H&E-stained tissue section, the Entomophthorales demonstrate hyphal encasement by eosinophilic material.

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