Opportunistic Fungi In Cytopathology

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Opportunistic Fungi: Use of Histochemical Stains for Accurate Cytopathologic Interpretation Suzanne L. Adams, CT(ASCP), Artist Kalamazoo, Michigan Fungal infections in humans and animals may manifest after exposure has occurred from physical contact with fungal particles found growing on organic or decaying matter in nature. Infections may also be contracted by inhalation of airborne fungal spores dislodged from soil by disruption or wind erosion. Of the more than 100,000 species of fungi, which are considered ubiquitous in the environment, only about 150 have been shown to cause disease. Many are considered “opportunistic” because they only rarely cause serious infections in individuals. When they do, it is because the conditions are perfect for their growth. This often occurs when the host’s immune system is compromised by serious infections such as HIV or drug therapy for cancer or chronic diseases. The duration of a specific infection depends upon many factors such as its virulence, location in the body, condition of the host’s immune system, and efficacy of available treatments. Fungal infections can be identified by laboratory procedures in microbiology, immunology, cytology, and histology. Examination of fungal organisms in cytologic and histologic samples is often the most successful method of revealing a fungus’ specific type and significance. The methods employed can rapidly and clearly differentiate contaminant forms of fungi from true opportunistic invasion, and they can determine the extent and type of involvement in various organs of the body. The identification of pulmonary

infections in the cytopathology laboratory is a frequent request. Often histochemical stains are used in the interpretation of these organisms. Immunocompromised patients, especially those diagnosed with AIDS, constitute the majority of this patient population. Special stains such as Grocott’s Methenamine-Silver (GMS), Mucicarmine, Periodic Acid Schiff (PAS) and Giemsa can be easily employed in cytology to help differentiate between various types of fungi as well. (Table 1) Dimorphic fungi are a cause for opportunistic infections. Blastomyces dermatitidis, Coccidioides immitis, Histoplasma capsulatum, Cryptococcus neoformans, and Pneumocystis carinii can be identified in a variety of pulmonary specimen types. Blastomycosis is the result of inhalation of spores of Blastomyces dermatitidis. In the United States, the highest incidence occurs along the Mississippi and the Ohio River valleys and along the western shore of Lake Michigan. Its natural habitat is unclear, but spores have been found in moist soils around farms, animals, and abandoned buildings. Canine infection is common. Patients may present with pulmonary disease or may be asymptomatic. The cytologic findings include inflammation, clusters of epithelioid histiocytes, macrophages, multinucleated giant cells, and often debris in the background. The organisms are found in the background. The yeast forms are identified in vivo. Yeast forms of Blastomyces dermatitidis are large (8-

Suzanne L. Adams, CT(ASCP)

15µm), spherical and double contoured. They have a rigid cell wall with a centrally retracted cytoplasm. (Figures 1a and 1b) Papanicolaou-stained bronchial specimens may reveal yeast-like fungal organisms, but special stains are needed to confirm their identity. The continued on page 100

in this issue ▼ Opportunistic Fungi: Use of Histochemical Stains

for Accurate Cytopathologic Interpretation yeast forms will stain pale orange 97to 2007 ASC Award Recipients Announced 99 pink or blue with the Pap stain and may Self-Assessment Exercise #3–Ultrasound display fuzzy outer cell borders 104 and Guided Fine Needle Aspiration Gallbladder inner thicker membranes with periphPEC Update 105 eral clear zones around the cell bodies. CLIA ‘’88 Part III: Sometimes be Where does that internal come from???structures can106 seen within these bodies which repreImportant ASC Annual Meeting Dates 106 sent organism’s multiple nuclei. Annualthe Scientific Meeting Social Events 109 Grocott’s Book Review Methenamine-Silver (GMS); 110 stains walls and bodies jet black, Position cell Available–Michigan 110 varying in intensity. The cell bodies 55th Annual Scientific Meeting Exhibitors 111 may stain or clear, Geraldine Colbylighter Zeiler Award Winners with darker 111 cell Newlymembranes. Elected Members The Periodic Acid 112 Schiff (PAS) Forthcoming Eventswill stain the yeast forms 114 bright pinkBusiness to redMeeting with darker cell walls. ASC Annual and ACytotechnologist clear zoneMember is seen between cell Business Meeting the 114 wall membrane. Multiple blue Make aand Star of Your Mentor! 115 nuclei be seen in well-stained specAdvocacymay Auction 116

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imens. The Mucicarmine stain reveals a negative or slight reaction. This stain rules out capsulated forms of cryptococcus which stain very positive. Buds remain persistently attached to parent cells, often until completely mature. Attachments to new buds appear thick Fungus

B. dermatitidis

C. immitis

C. neoformans

Coccidioides immitus is more frequent and often more severe during pregnancy and in certain darker skinned races. It is found as a mold in desert soil, predominantly in south western United States and contact is through inhalation of the endospores released from its spherules by the wind. It is the largest of the round fungi, with large spherules

spherules appear as if they are double The older, rupturing contoured. spherules have much thinner walls. The identification of well-formed spherules is necessary for accuracy. Hyphae may be identified, but are not commonly seen. The endospores are round to oval, thick walled and are non-budding. They vary in size from 1-5 microns. The

Size (microns)

Distinguishing Features

Best Stain

8-15 (occasional up to 20)

Broad-based budding, Single budding, Rounder buds, Thick refrac.walls, Granular cytoplasm, Multinucleated

GMS PAS

Spherules- 20-60+ (occasional >100) Endospores-1-5

GMS PAS

Cell bodies- 4-10 Capsules-1-30+ C. bodies and Capsules-5-20 ave.

True capsules, Narrow-necked buds Tear drop buds, Variation in size, Pseudohyphae possible

Mucicarmine India ink PAS GMS

H. capsulatum

1-5 (2-4 ave.)

P. carinii

Cysts- 4-10 (6-8 ave.) Trophozoites-0.5-1

Non-budding, Cell wall microdots Inner trophozoites Alveolar casts

C. immitis –small spherules C. neoformans

Large spherules, Non-budding, Thick cell walls, No microdots, Variation in size, Hyphae possible

Tiny size, Within macrophages, Uninucleated, Pseudohyphae possible

Differential Diagnosis

H. capsulatumfree endospores B. dermatitidislarge spherules-

B. dermatitidis Contaminants Talc

GMS PAS

C. immitis spores Candida spores Toxoplasma Leishmania Hemosiderin Protein deposits Calcified debris Carbon

GMS Romanovsky Giemsa

RBC ghosts, Cell debris

Table 1

and protruding. New buds often have thinner cell walls. When interpreting the findings one may have to search diligently to find cells with attached buds. No hyphae or truly demonstrable capsules are found (unlike C. neoformans). The yeast forms are often confused with Cryptococcus neoformans. 100

staining well with the Papanicolaou, GMS, and PAS stains. (Figures 2a and 2b) The characteristic spherical cystic cells usually contain numerous endospores that vary from 20 to 60 microns in diameter. The mature cells may reach greater than 100 microns in size. Thick refractile cell walls in young

endospores quickly mature into new spherules. Both intact and ruptures spherules will be seen and can often be mistaken for H. capsulatum. They can mimic hemosiderin, pollen and vegetable cells. The Papanicolaou stain reveals blue to pink endospores and spherules, continued on page 101

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often with thick refractile appearing cell walls which actually represent retracted cytoplasm. A common pitfall is to mistake them for pollen. The GMS stain demonstrates black endospores with the walls picking up the deepest stain. The (Above) Figure 1a: GMS stain, Blasto-myces dermatitidis PAS stain displays bright (Right) Figure 1b: Illustrative magenta endospore bodies and drawing, Blastomyces dermaticell walls. Although the granular tidis content of the spherules will stain magenta, the spherule cell walls will stain non-specifically (mature forms often do not stain at all). The endospores may resemble Pneumocystis carinii cysts (look closely for the microdot structures). Histoplasma capsulatum may be confused with the endospores (look for buds found in H. capsulatum). Histoplasmosis has a strong predilection for the reticuloendothelial system and is frequently identified in immunocompromised patients. Organisms are found in certain moist soils and concentrate in bird or bat fecal material. When disrupted, the spores may become airborne and inhaled by the lungs where they are transformed into tiny yeast-like structures which (Above) Figure 3a: GMS stain, get captured by histiocytes and Histoplasma capsulatum; (Right) then proliferate within them. Figure 3b: Illustrative drawing, The protoplasm within yeast Histoplasma capsulatum cells often retracts from the cells walls during staining techniques, They occur intra or extracellularly. The creating peripheral clear zones that extracellular yeasts occur singly or in mimic capsules (hence the name capsulaclusters. They may mimic calcified tum). They do not display true capsular debris, protein deposits, hemosiderin carproperties and are not considered encapbon, spores of Candida or C. immitis and sulated. H. capsulatum is the smallest of cysts of P. carinii. They do not stain well the fungi. The organisms must be preswith the Papanicolaou stain. Special ent within a histiocyte or neutrophil to be stains are required to distinguish them diagnostic. The yeast cells are tiny, 2-4 from other fungi and parasites. GMS microns in diameter and round to oval.

(Above) Figure 2a: GMS stain, Coccidioides immitis (Left) Figure 2b: Illustrative drawing, Coccidioides immitis

stain is the preferred stain. Well stained black to brown cell walls are seen with tightly packed organisms often identified within histiocytes. The GMS stains the endospores black with walls demonstrating intense black staining (Figures 3a and 3b) whereas other organisms, such as Leishmania donovani and Toxoplasma continued on page 102

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gondii, do not stain with this silver stain. PAS demonstrates this fungus positively with pink cell bodies that exhibit slight peripheral clear zones (not true capsules). Leishmania donovani and Toxoplasma gondii can be ruled out because they are PAS negative. H. capsulatum does not stain with Mucicarmine. Cryptococcosis is a frequent opportunistic mycosis caused by the non-dimorphic yeast-like fungus, Cryptococcus neoformans. It is ubiquitous in soil, and concentrates in avian feces, particularly pigeon feces, where it is considered a main source of transmission via spore inhalation. Initiation often begins in the lungs where mucoid material accumulates from the organisms’ capsules. It is particularly seen in immunocompromised populations. The yeast (spore) characteristics include round to oval cells with relatively thin walls. The cells vary in size from 2-20 microns in diameter. Single tear-drop budding is a hallmark feature. (Figure 4a) They may occur singly or in small clusters. Mature capsules may have fuzzy or spiculated edges demonstrating fibrils. New buds often have immature capsules. They may mimic mineral concretions and other artifacts. The yeasts stain poorly on Papanicolaou stained material. Special stains help clarify the thick yet often “cryptic” (invisible) mucopolysaccharide capsules that are so unique to this pathogenic fungus. Mucicarmine demonstrates the unique mucopolysaccharide component of the fungus. The cell walls and inner portions of the capsule stain deep rose red in color with this stain. The cell bodies may appear clear with deeply stained walls (peripheral red rings). (Figure 4b) The GMS stain is commonly used to demonstrate the cell walls of this fungus in dark brown to black. (Figure 4c) India ink is also used to demonstrate the fungus cell walls and is a preferred stain for this purpose. PAS often stains the yeast forms well, in a bright magenta color. Overall, mucicarmine is the stain of choice to verify this fungus as it reveals the organism’s true capsule which 102

is unique to the pathogenic fungi. Pneumocystis is an opportunistic organism that causes respiratory pneumonia in the immunocompromised

(Left) Figure 4a: Illustrative drawing, Cryptococcus neoformans (Above) Figure 4b: Mucicarmine stain, Cryptococcus neoformans (Below) Figure 4c: GMS stain, Cryptococcus neoformans

patient. Cyst filled alveolar casts, foamy debris and trophozoites are hallmark features of this fungus. GMS stained bronchoalveolar lavage (BAL) samples are often preferred as the cyst walls stain clearly and display central microdot structures that distinguish them from other entities. The alveolar casts (circlets) are seen on Papanicolaou stain. The cysts do not stain or stain faintly

folded balls. GMS stains the alveolar casts a greenish-blue with many cysts visible within. The cysts stain brown to black with well-defined membranes. The cell walls of the cysts often display a central micro dot structure, representing a thickened area. (Figures 5a and 5b) They may mimic other yeasts and red blood cells so look for the microdot structure. The intracystic inclusions or trophozoites are not visible as they do not stain with GMS. The Giemsa stain reveals the characteristic trophozoites.

(Above) Figure 5a: GMS stain, Pneumocystis carinii (Right) Figure 5b: Illustrative drawing, Pneumocystis carinii

pink. They may mimic red blood cell ghosts. The cysts measure 4-10 microns in size and appear round to oval and often cupshaped. They may look like squashed or

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