Special Stains Use in Fungal Infections - Dako

Special Stains Use in Fungal Infections
Abida Haque, MD
Professor of Pathology
Weill Medical School of Cornell University, New York
Attending Surgical Pathologist –
Department of Pathology Methodist Hospital
Houston, TX, USA
Histologic evaluation of tissues is a quick and easy way to identify
fungal organisms, and a strong adjunct to microbiologic culture for
diagnosis of fungal infections. Histologic evaluation of granulomatous
inflammation and granulomas must include special stains to exclude or
include presence of fungi and acid fast bacteria. Gomori Methenamine
Silver (GMS) and Periodic acid-Schiff (PAS) are the two most common
stains used to look for fungi in tissues and in cytology specimens in the
daily practice of pathology. The presence of fungus in the tissue sections
provides an indisputable evidence of invasive infection. Because of
their size and morphologic diversity, many fungi can be seen in tissue
sections by conventional light microscopic examination of Hematoxylin
and Eosin (H&E) stained sections. In cytology specimens, fungi can be
identified by their size and specific morphology.
In the tissues, fungi usually occur either as hyphae, budding yeast,
endosporulating spherules, or a combination of these forms (1, 2). In some
groups of fungi only one species of fungus is the cause of mycosis, and
therefore when classic forms are observed, an etiologic diagnosis can
be made. These groups of fungal diseases include adiaspiromycosis,
blastomycosis, coccidioidomycosis, cryptococcosis, Histoplasmosis
capsulati, Histoplasmosis duboisii, paracoccidioidomycosis, Penicilliosis
marneffei, protothecosis, rhinosporidiosis, and sporotrichosis.
Other mycoses are caused by any of the several species of a genus,
all of which are morphologically similar in tissue sections. Although
these fungi cannot be identified as to the species by conventional
histology, the disease that they cause can be diagnosed generically;
Application
for example, aspergillosis, candidiasis, and trichosporonosis. Still other
mycoses are caused by any of a number of fungi belonging to different
genera. These fungi appear similar, if not identical to one another in
tissues. With these fungi, it is not possible to identify the etiologic agent,
however, the mycosis can be named; for example, phaeohyphomycosis
and zygomycosis.
Hematoxylin & Eosin is a versatile stain that enables the pathologist
to evaluate the host response, including the Splendore-Hoeppli
phenomenon, and to detect other micro-organisms (3). It is the stain
of choice to confirm the presence of naturally pigmented fungi, and to
demonstrate the nuclei of yeast-like cells. However, there are drawbacks
to using just the H&E stain for fungal diagnosis. It is often difficult to
distinguish poorly stained fungi from tissue components, even at higher
magnifications. When sparse, fungi are easily overlooked in H&E stained
sections. The morphologic features may not be evident and sometimes
may be misleading. For example, Histoplasma, Blastomyces, and
Paracoccidiodes may have cytoplasmic retraction artifact in the
sections, making morphologic evaluation difficult. Some of the fungal
variants may have different sizes, such as the large form variant (African)
histoplasma, and microform blastomycosis. Some of the dimorphic fungi
can form pseudohyphae in tissues. Sometimes the fungal morphology
may be altered by therapy. Special stains for fungi are therefore essential
for histopathologic evaluation of unexplained inflammatory processes
(4, 5). Most fungi can be readily demonstrated with the common special
stains, Gomori’s methenamine silver (GMS), Gridley’s fungus (GF), and
periodic acid-Schiff (PAS), also referred to as “broad spectrum” fungal
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Figure 1. Hematoxylin and Eosin staining of
Aspergillus.
Figure 2. GMS staining of Aspergillus.

Figure 3. Hematoxylin and Eosin staining of
Pneumocystis jirovici.
Figure 4. GMS staining of
Pneumocystis jirovici.
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Figure 5. Alcian Blue staining
of Cryptococcus.
Figure 6. PAS staining of Cryptococcus.

Figure 7. Hematoxylin and Eosin staining of
Malassezia.
Figure 8. PAS staining of Malassezia.
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Figure 9. Hematoxylin and Eosin staining of
Histoplasma.
Figure 10. AFB staining of Histoplasma.

“ Gomori Methenamine Silver (GMS) and
Periodic Acid-Schiff (PAS) are the two most
common stains used to look for fungi in tissues
and in cytology specimens in the daily practice
of pathology. ”
stains. GMS is preferred for screening, because it gives better contrast,
and stains even degenerated and nonviable fungi that are sometimes
refractory to the other two stains (Fig. 1, 2).
GMS also stains algae (Prototheca and Chlorella spp.), cyst walls of
Pneumocystis jiroveci (Fig. 3, 4), pathogenic free living soil amebas,
the spore coat of most microsporidian parasites, intracytoplasmic
granular inclusions of Cytomyeolovirus, Actinomyces Israeli and related
species, Nocardia spp., most Mycobacterium spp., and nonfilamentous
bacteria with polysaccharide capsules such as Klebsiella pneumoniae
and Streptococcus pneumoniae. Prolonged staining in the silver nitrate
solution may be required to adequately demonstrate degenerated
fungal elements such as the yeast-like cells of Histoplasma capsulatum
var. capsulatum in granulomas.
The disadvantage of GMS and GF fungal stains is that they mask the
natural color of pigmented fungi, making it impossible to determine
whether a fungus is colorless hyaline or dematiaceous (pigmented).
Such a determination is crucial in the histologic diagnosis of mycosis
caused by dematiaceous fungi such as phaeohyphomycosis (6). Except
for the PAS reaction, fungal stains GMS and GF do not adequately
demonstrate the inflammatory response to fungal invasion. To counteract
this, a GMS-stained section can be counterstained with H&E for a
simultaneous study of the fungus and the host response.
The PAS stain performs almost as well as GMS, in screening for fungi.
It actually demonstrates fungal morphology better than the silver stains.
PAS can stain degenerated fungi that may not be visible on H&E stain.
Calcific bodies that are sometimes found in caseating granulomas are
also stained with PAS, and can be mistaken for yeast-like fungi. This
is especially true when calcific bodies are apposed to give the false
impression of budding yeasts, or when the bodies are laminated to give
the appearance of a capsule or thick cell wall. Best stains to avoid this
misinterpretation are GMS and GF stains, because the chromic acid
used as an oxidizer in these stains dissolves the calcium, leaving the
calcific bodies unstained. Conversely, there are artifacts that mimic fungi
on GMS and GF stains that are not seen on PAS stain, therefore the use
of both silver and PAS can reduce the incidence of false positive results.
Narrow-Spectrum Fungal Stains
The differential diagnosis of fungi may require the use of additional
special stains that stain some fungal organism and not others. These
are sometimes referred to as “narrow-spectrum” fungal stains (7, 8).
Some of the stains in this category are mucin stains such as alcian blue
and Mayer’s, or Southgate’s mucicarmine, that readily demonstrate the
mucoid capsule of Cryptococcus neoformans (Fig. 5, 6).
This staining reaction differentiates Cryptococcus from other fungi of
similar morphology, such as Coccidiodes, Candida, and Histoplasma.
These mucin stains are not specific for C. neoformans; the cell walls
of B. dermatitidis and Rhinosporidium seeberi are often stained
to varying degrees with mucin stains. However, these two fungi
are nonencapsulated and morphologically distinct, and not ordinarily
mistaken for Cryptococcus. In some cases, poorly encapsulated
cryptococci in tissue sections may not stain positive with mucicarmine
stain. In these cases, since the cell wall of C. neoformans contains silver
reducing substances, possibly melanin precursors, it can be stained
with Fontana-Masson’s silver procedure for melanin (9, 10). This stain is
especially useful in those cases of Cryptococcosis with invasive yeast
forms that do not have readily detectable capsules, the so-called dry
variants. Such forms could possibly be confused with non-encapsulated
yeasts of similar morphology. Fontana-Masson and Lillie’s ferrous iron
stains for melanin can also be used to confirm and accentuate the
presence of melanin or melanin-like pigments in the cell walls of poorly
pigmented agents of phaeohyphomycosis in tissue sections (11). PAS
may be used as a narrow-spectrum fungus stain. For example, in the
differential diagnosis of small budding yeast forms, a weak PAS and a
strong GMS staining favors a diagnosis of Histoplasma, since Candida,
microforms of Blastomyces, and yeast forms of Malassezia show a
strong cell wall staining with PAS (Fig. 7, 8).
Another narrow-spectrum fungus stain is Ziehl-Neelson (ZN). In one
study, 60% of blastomyces and 47% of histoplasma organism showed
positive cytoplasmic staining of the yeast-like cells with ZN stain
(Fig. 9, 10).
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No staining was seen in Cryptococcus or Candida, and very rare acid
fast staining was seen in coccidiodomyces endospores (12). However,
these staining properties are inconsistent and should not be used for
primary diagnosis. The cell walls of fungi are in general, not acid fast.
Autoflourescent Fungi
Some fungi or fungal components in the H&E stained tissue sections
are autofluorescent when examined under ultraviolet light source
(13). Candida species, Coccidioides immitis and Aspergillus species
can exhibit bright green to yellow-green autofluorescence (14). When
sections of these fungi are stained with the PAS, bright yellow fungal
autofluorescence against a deep red-orange background is seen (15).
Autofluorescence may help delineate sparse or poorly stained fungi in
H&E stained sections, however this property is inconsistent and should
not be used for definitive diagnosis. Most fungi in frozen or paraffin
embedded tissue sections also stain nonspecifically with Calcofluor
white, a cotton whitener that fluoresces under ultraviolet light (16). This
rapid and a simple fluorescence procedure can be routinely used in the
intraoperative examination of fresh-frozen tissues for fungi.
Immunoperoxidase stains can be used to identify certain fungi in
smears and in formalin fixed, paraffin embedded tissue section. This
technique, however, has only limited diagnostic use (17).
References
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4. Schwartz J. The diagnosis of deep mycoses by
morphologic methods. Hum Pathol. 1982; 13:519-33.
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York: Raven; 1985.
6. Chandler FW, Kaplan W, Ajello L. Color Atlas and Text of
the Histopathology of Mycotic Disease. Chicago: Year
Book Medical; 1980.
7. Youngberg GA, Wallen EDB, Giorgadze TA. Narrow-
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8. Hussain Z, Martin A, Youngberg GA. Blastmyces
dermatitides with large yeast forms. Arch Pathol Lab
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for histopathological diagnosis of cryptococcosis. J Clin
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10. Lazcano O, Speights VO Jr, Stickler JG, Bilbao JE,
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11. Wood C, Russel-Bell B. Characterization of pigmented
fungi by melanin staining. Am J Dermatopathol. 1983;
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12. Wages DS, Wear DJ. Acid-fastness of fungi in
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in tissue sections. Am J Clin Pathol. 1983; 79:587-90.
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Direct Immunofluoresence (IF)
Direct immunofluoresence (IF) can improve the diagnostic capability
of conventional histopathology in the diagnosis of fungal diseases
(18). The IF procedure, which can be performed on smears and on
formalin fixed paraffin embedded tissue sections is helpful in confirming
a presumptive histologic diagnosis, especially when fresh tissues
are not available for culture or when atypical fungus forms are seen.
The Division of Mycotic Diseases, Center for Disease Control, Atlanta
(United States) and others have a broad battery of sensitive and specific
reagents available for identifying the more common pathogenic fungi.
The immunofluoresence procedure has several advantages. Final
identification of an unknown fungus is possible within hours after
H&E and GMS stained sections are initially examined. The need
for time consuming and costly cultures is often obviated by IF, and
the hazards of handling potentially infectious materials are reduced
when microorganisms are inactivated by formalin prior to IF staining.
Prolonged storage of formalin fixed tissues, either wet tissue or paraffin
embedded, does not appear to affect the antigenecity of fungi. This
antigenic stability makes possible retrospective studies of paraffin
embedded tissue and the shipment of specimens to distant reference
laboratories for confirmatory identification. Most service laboratories,
however, do not routinely use IF, since the special stains have been very
reliable for diagnosis in the day to day pathology practice.
15. Jackson JA, Kaplan W, Kaufman L. Development of
fluorescent-antibody reagents for demonstration of
Pseudallescheria boydii in tissue. J Clin Microbiol. 1983;
18:668-73.
16. Monheit JE, Cowan DF, Moore DG. Rapid detection of
fungi in tissue using calcofluor white and fluorescence
microscopy. Arch Pathol Lab Med. 1984; 108:616-18.
17. Kobayashi M, Kotani S, Fujishita M, et al.
Immunohistochemical identification of Trichosporon
beigelii in histologic sections by Immunoperoxidase
method. Am J Clin Pathol. 1988; 89:100-05.
18. Reiss E, de-Repentgny L, Kuykendall J, et al.
Monoclonal antibodies against Candida tropicalis
mannans antigen detection by enzyme immunoassay
and immunofluorescence. J Clin Microbiol. 1986;
24:796-802.