Histopathology Diagnosing Fungal Infections Using Histopathology Histopathology remains one of the major tools of diagnosis in mycology.

The major advantages of histopathology are speed, low-cost, and the ability to provide a presumptive identification of the infecting fungus, as well as demonstrating the tissue reaction. However, unless special techniques such as immunofluorescence are used, or the infecting fungus possesses unique structures such as spherules, definitive identification of the etiologic agent by histopathology is difficult. Nevertheless, it usually provides essential information before the fungus can be isolated in a mycology laboratory. Furthermore, histopathology is the only way to diagnose infections caused by Loboa loboi or Rhinosporidium seeberi, as cultivation techniques have to date been unsuccessful. In this section, we provide information about the commonly used histopathologic staining methods and the usual appearances of the frequently encountered fungi in infected tissues [415, 602, 654, 1173, 2041, 2064]. For histopathologic appearances of fungi other than those mentioned below or for more detailed information about fungi noted below, please refer to the related page for that specific fungus. The microscopic appearance in tissue of each of the fungi is also shown on the related page. Stains Used in Histopathology A number of histologic stains are available that are routinely used to visualize fungi in tissue sections. While some of these are special fungal stains, others are of more general use but still help to observe the tissue reactions and/or the infecting fungus. The reactions involved and comments of particular interest for some of these stains are summarized in the TABLE. Gomori methenamine silver (GMS), Gridley's fungus (GF), and periodic acid-Schiff (PAS) are special for and very efficient to visualize the fungi. Among these three, GMS is more advantageous since it stains old and nonviable fungal elements more efficiently than the other two. Hematoxylin and eosin (H&E) stain, on the other hand, is very useful to visualize the host's response but is not a special fungal stain. It does not stain most of the fungi, except the Aspergillus spp. and the zygomycetes. Thus, a combination of GMS and H&E is usually employed to visualize both the tissue reaction and the infecting fungus. Mucin stains, like Mayer's mucicarmine, Southgate's mucicarmine and Alcian blue, stain the mucopolysaccharide capsule of Cryptococcus neoformans. However, they are not specific for this particular fungus. Blastomyces dermatitidis Rhinosporidium and Rhinosporidium seeberi may also be stained with the mucin stains. seeberi Histopathologic Features of the Most Commonly Encountered Fungi Achieving a successful histopathologic diagnosis begins with selection of the tissue sample to be examined. While some fungi are more readily located at the periphery of the infected lesion, others are more prominent at the center. Then comes the use of appropriate stains and examination by

experienced eyes. The major growth forms of the fungi that help in histopathologic diagnosis are the yeast cells, hyphae, pseudohyphae, arthroconidia, chlamydoconidia, and spherules. The characteristics of each of these (shape, size, location, color...) help in identification of the fungus. Tissue components such as Russell bodies, karyorrhectic debris, calcified bodies, elastic fibers, and small blood vessels may resemble fungi. Careful examination by experienced specialists significantly reduces such an overdiagnosis. Candida: Budding yeast cells (blastoconidia), pseudohyphae and septate hyphae are observed. Cryptococcus neoformans: Pleomorphic yeast-like cells and formation of narrowbased buds are typical. The encapsulated strains have capsular material detected with mucin stain. Cryptococcus neoformans

Aspergillus: Hyaline, septate, dichotomously branched hyphae of uniform diameter are observed. In cavitary lesions, conidial heads are occasionally observed. Purulent, necrotizing inflammation is present. Calcium oxalate crystals, probably originating from fungal oxalic acid, are present near hyphae in necrotic tissue, if the Aspergillus niger infecting species is Aspergillus niger.

Fusarium and Pseudallescheria: Hyaline, septate, randomly branched hyphae are observed. The hyphal contours are regular.

Fusarium

Mucor, Rhizopus and other fungi of the Order Zygomycetes: Broad, thin-walled, hyaline, often aseptate or sparsely septate hyphae are observed. The contours of the hyphae are typically non-parallel and branches are irregular. Invasion of blood vessels is noteworthy. Pyogenic inflammation, abscess formation and suppurative necrosis are observed.

Rhizopus

Microsporum, Epidermophyton, and Trichophyton: These fungi, the dermatophytes, produce septate, branched hyphae that break into chains of arthroconidia. Coccidioides immitis: Spherule containing endospores is the typical structure. The transition form of C. immitis producing septate hyphae that develop into arthroconidia, may be observed in necrotic nodules and misdiagnosed as one of the fungi in hyphomycetes group, when spherules are not yet evident.

Coccidioides immitis

Histoplasma capsulatum: Intracellular budding yeast cells are typical. It should be differentiated from the other intracellular dimorphic fungus, Penicillium marneffei. The yeast cells of P. marneffei divide by fission, but not by budding. Histoplasma capsulatum predominates at the central portion of the lesions. Thus, the selection of the tissue section to be excised for histopathologic and mycologic examination is of particular significance.

Histoplasma capsulatum

Blastomyces dermatitidis: Thick-walled yeast-like cells and single broad-based buds are typical. In contrast to Histoplasma capsulatum, Blastomyces dermatitidis is located more at the periphery of the infected lesion, rather than the center.

Blastomyces dermatitidis

Paracoccidioides brasiliensis: Yeast-like cells with multiple buds are typical. The buds are attached to the mother cell with narrow necks. The appearance resembles Paracoccidioides a "steering-wheel". brasiliensis

Fungi causing eumycotic mycetoma (Madurella, Acremonium, Pseudallescheria, Exophiala, Curvularia, Aspergillus, and others): Granule formation is typical. The granules are composed of hyaline or phaeoid (depending on the fungus involved), septate, branching hyphae. Chlamydoconidia may be formed.

Madurella

Fungi causing chromoblastomycosis (Fonsecaea, Phialophora, Cladophialophora, and others): Spherical or polyhedral, dark brown, thick-walled sclerotic bodies (muriform cells) are typical. Phaeoid hyphae may also be observed.

Fonsecaea

TABLE

Stain Fontana-Masson

Reaction Chromaffin reaction probably oxidizes melanin or melanin precursor as it reduces silver. Cell walls black. Background pale pink.

Comments Useful for staining cell walls of dematiaceous fungi when the nature of their cell walls is not evident, and useful for Cryptococcus neoformans. Natural color of cell walls masked. Enhances visualization of fungi and their morphology. Natural color of cell walls masked. Erythrocytes and naked nuclei can mimic yeasts. Tissue response cannot be studied. Best contrast of the special fungal stains. Enhances visualization of fungi and their morphology. Natural color of cell walls masked. Tissue response cannot be studied.

Grocott's modification of Gomori's Chromic acid oxidizes cell-wall methenamine silver method polysaccharides to aldehydes. Aldehyde products reduce methenamine silver nitrate to metallic silver. Cell walls brown to black. Background pale green.

Gridley's stain

Chromic acid oxidizes adjacent hydroxyl groups of cell-wall polysaccharides to aldehydes. Aldehyde groups combine with Schiff reagent. Aldehyde fuchsin occupies uninvolved linkages of the Schiff reagent. Cell walls purple to magenta, yeasts rose to purple, and capsules deep purple. Background yellow. Hematein-mordant stains DNA in the nucleus and nuclear proteins. Nuclei blue, cartilage and calcium deposits dark blue, cytoplasm and other components shades of red, and erythrocytes bright red.

Hematoxylin and eosin

Visualizes host tissue response to the fungus. Aspergillus spp. and Zygomycetes stain well. Difficult to distinguish some types of fungi, as well as locate fungi in tissue if they are present in small numbers.

Immunofluorescence

Direct technique: Some use the fluoresceinlabeled antibody reacts immunoperoxidase with fungal antigens in cell wall. technique. Specific

Indirect technique: unlabeled antibody complexes with fungal antigens. Fluorescein-labeled conjugate reacts with globulins attached to fungal antigens. Cell walls yellow-green.

and highly sensitive. Can be used to detect and measure antibodies.

References 415. Chandler, F. W. 1985. Pathology of the mycoses in patients with the acquired immunodeficiency syndrome (AIDS). Curr Top Med Mycol. 1:1-23. 602. Dixon, D. M., and A. Polak-Wyss. 1991. The medically important dematiaceous fungi and their identification. Mycoses. 34:1-18. 654. El Nageeb, S., and R. J. Hay. 1981. Immunoperoxidase staining in the recognition of Aspergillus infections. Histopathology. 5:437-444. 1173. Kaufman, L. 1992. Immunohistologic diagnosis of systemic mycoses: an update. Eur J Epidemiol. 8:377-82. 2041. Schell, W. A. 2000. Histopathology of fungal rhinosinusitis. Otolaryngol Clin N Amer. 33:251276,VII,VIII,NIL_5. 2064. Schwarz, J. 1982. The diagnosis of deep mycoses by morphologic methods. Human Pathol. 13:519-533.

If you cannot necropsy the animal immediately:

At the very least, put it at 4C (DO NOT FREEZE) Preferably place entire animal in fixative (flat bottomed container) lOpen abdominal & thoracic viscera and snip skull lfix ( gently inflating lungs and GI with fix) lSingle small incision in kidneys and liver if possible

Fixation methods
Perfusion fixation followed by immersion fixation Immersion fixation (do not re-use fixative) Ideal fixative-tissue ratio 10:1thus the mouse in a 50 mL conical is not ideal (use a flat bottomed container) Tissues should be no thicker than 5 mm for optimal fixation Bones should be cracked to allow for fixation of marrow
lInfusion of nasal cavities for optimum fixation of nasal structures lInflate lungs and intestinal tract with fix lIf eyes are primary interest, need to identify appropriate fixative for your interests lIf testes are primary interest, consider Bouins

Fixative options
10% Neutral Buffered Formalin Zinc Formalin 4% Paraformaldehyde Bouin's Solution Frozen tissues (Frozens)

10% Neutral Buffered Formalin

lRoutine fixative (Supplied free in our facility!) Dont use 10% formalinthis will destroy your data Done at room temperature lAdequate for many antibodies (know before you fix!) Not adequate for most lymphocyte markers lFix no longer than 16-24 hours (for most samplesexcluding bone), transfer to 70% EtOH for samples for IHC Best to embed as soon as possible, but can stay in EtOH 4% Paraformaldehyde (a buffered solution!) Commonly used in perfusion protocols Good for in situ hybridization (formalin works well, too) More gentle and considered slightly better than formalin for IHC, FISH Must be made just prior to use Keep it a 4C

Zinc Formalin (a buffered solution!) Commonly used for IHC when frozen not a good option More gentle and considered slightly better than 10% neutral buffered formalin for IHC Fix at room temperature Bouins Solution (yellow) Contains picric acid l Maintains good morphology (good for brain) Decalcifies bone while fixing tissue May result in slight hardening of tissue if over-exposed (get into 70% EtOH within 48 hours) Colors tissue and affects some histochemical stains

Frozen tissues Freeze fresh or fixed tissues Good for immunohistochemistry for problem antibodies Decreased quality of morphology, better if pre-fixed Not good for bone (cant cut the tissue) Store at -80

Paraffin Wax Histology

 Rapid and correct fixation of small pieces of tissue are the key to well preserved morphology. Tissue processing through graded alcohols and xylene are automated in our Leica Tissue Processor, while paraffin embedding in the orientation of the principle investigators choice is done by hand at the embedding center. Sections of the resulting wax blocks can then be cut at 2-10m to provide single cell layer sections that are ready for chemical stains, immunohistochemistry, ISH, as well as PCR and laser capture. Store unused wax blocks and sections at 4C.

Frozen Sections
Frozen section histology requires the use of fixed or un-fixed tissue samples
If tissue is not being preserved by a fixative, it is imperative that the tissue is frozen as quickly as possible after removal from the animal Freeze in isopentane chilled in a slurry of dry ice with acetone Tissue samples should be small (3mm x 3mm) to avoid slow freezing of the sample, which can result in artifacts Dab tissue on paper towel to dry to reduce crystal formation during freezing Place tissue into mold and surround with OCT Remove bubbles Place mold in isopentane with flat face down, when frozen, drop mold into chilled isopentane If not pre-fixed, fix sections in cold 100% methanol or acetone for 2-3 minutes and store at 80 or 20C Frozen tissues with special needs Frozen brainshow to get rid of bubble artifact lBest to fix brains by immersion or perfusion followed by immersion lPlace brains in a 30% sucrose solution in PBS O/N (no longer) lEmbed in OCT and freeze as described Frozen muscle lDont pull or break muscle during dissection lPlace elongated muscle on cork and cover with OCT lPlace into isopentane perpendicularly