Laboratory diagnosis of zygomycosis

Zygomycosis:

1. Clinical Material: Skin scrapings from cutaneous lesions; sputum and needle biopsies from pulmonary lesions; nasal discharges, scrapings and aspirates from sinuses in patients with rhinocerebral lesions; and biopsy tissue from patients with gastrointestinal and/or disseminated disease.

Warning: zygomycetous fungi have primitive coenocytic hyphae that will often be damaged and become non-viable during the biopsy procedure (especially scrapings and aspirates), or by the chopping up or tissue grinding process in the laboratory. This is why zygomycetous fungi that are clearly visible in direct microscopic or histopathological mounts are often difficult to grow in culture from clinical specimens. If on clinical and/or radiological evidence zygomycosis is suspected then try to avoid excessive tissue damage when collecting the specimen and in the laboratory gently tease the tissue apart and inoculate it directly onto the isolation media. If you are not sure hold the specimen in saline or BHI broth until the results of the direct microscopy or frozen histology sections are known. If zygomycetous hyphae are present proceed as above, otherwise homogenised the specimen and plate out.

2. Direct Microscopy: (a) Scrapings, sputum and exudates should be examined using 10% KOH & Parker ink or Calcofluor mounts; and (b) Tissue sections should be stained with H&E and GMS. Examine specimens for broad, infrequently septate, thin-walled hyphae, which often show focal bulbous dilations and irregular branching.

Interpretation: As a rule, a positive direct microscopy, especially from a sterile site, should be considered significant, even if the laboratory is unable to culture the fungus.

Tissue morphology in zygomycosis showing distinctive infrequently septate thin walled hyphae with focal bulbous dilations and irregular branching, typical for those species belonging to the Mucorales.

GMS stained tissue section from a lung showing typical zygomycete hyphae and by chance a sporangium of Mycocladus (Absidia) corymbifera.

H&E stained section of infected tissue showing broad, infrequently septate, hyphae surrounded by an eosinophilic sheath [Splendore-Hoeppli phenomena], typical of zygomycosis caused by Basidiobolus ranarum.

3. Culture: Inoculate specimens onto primary isolation media, like Sabouraud's dextrose agar. Most zygomycetes are sensitive to cycloheximide (actidione) and this agent should not be used in culture media. Look for fast growing, white to grey or brownish, downy colonies.

Interpretation: Despite being recognised as common laboratory contaminants, zygomycetes are infrequently isolated in the clinical laboratory. Therefore, in patients with any of the above predisposing conditions, especially diabetes or immunosuppression and/or clinical symptoms, the isolation of any zygomycete fungus should be regarded as potentially significant. Obviously, in patients without predisposing conditions, the isolation of a zygomycete from a non-sterile site, such as skin or sputum, must be interpreted with caution, especially in the absence of direct microscopy.

4. Serology: There are currently no commercially available serological procedures for the diagnosis of zygomycosis. Although some laboratories have developed ELISA tests for the detection of antibodies to Zygomycetes.

5. Identification: Zygomycetes are usually fast growing fungi characterised by primitive coenocytic (mostly aseptate) hyphae. Asexual spores include chlamydoconidia, conidia and sporangiospores contained in sporangia borne on simple or branched sporangiophores. Sexual reproduction is isogamous producing a thick-walled sexual resting spore called a zygospore.