2. Advantages
• Speed, low cost
• Presumptive identification of the infecting
fungus
• Demonstrating tissue reaction.
• Only way to diagnose- L. loboi or
Rhinosporidium seeberi
3. Special Stains
TISSUE STAINING METHODS FUNGI
Skin and Subcutaneous
Tissue
KOH , Giemsa, GMS Dermatophytes,
Sporothrix,Zygomycetes
Nasal Smear Biopsy H and E, GMS Rhinosporidiosis
Muscle H and E, GMS Zygomycetes( very rare)
Bone Marrow Giemsa, GMS Histoplasma
Lung H & E, Giemsa, GMS and
PAS
Aspergillus, C.Neoformans
Lymph Node H & E, Giemsa , GMS and
PAS
Aspergillus, C.Neoformans
Mucin stains (Mayer’s mucicarmine and Alcian blue)-C.
neoformans,Blastomyces dermatitidis and R. seeberi
4. Major forms
• Yeast cells
• Hyphae
• Pseudohyphae,
• Arthroconidia,
• Chlamydoconidia
• Spherules.
14. Case 2
• 52 year old male
• C/o – Cough and fever since 1 week a/w fatigue,
headaches, joint pains at night .
• No history of pulmonary diseases or smoking
• Travel history to Phoenix 3 weeks ago
• O/E- Mild fever, wheezing in upper left chest
• X ray chest- Hilar adenopathy
• CBC- Normal TLC with eosinophilia
• KOH- Numerous spherules
21. Case 3
• 68 year old male
• C/O- Dysphagia , retrosternal pain since
2 days, was on treatment for UTI
• O/E- Normal
• On Endoscopy- Thick , curdy white patches on
esophageal wall
43. CASE 6
• 27 year old male
• Newly diagnosed HIV (CD 4 count- 7
cells/mm3)
• C/O- fever with chills, night sweats, myalgia,
dry cough, loss of weight x 25 days
• O/E- 2 - 3 mm hyperpigmented papules and
dermal basophilic spherules 2 - 4 microns
• Has been treated for Pneumonia
49. Case 7
• 60 year old farmer
• c/o – Nasal obstruction, epistaxsis since one
year .
• No comorbid conditions
• H/O Chronic smoking , occasional ethanolic
• O/E- Mass noticed in left nostril, reddish in
color, strawberry like in appearence
53. CASE 8
• A 70-year-old male
• C/O- Bad breath and pus discharge from
upper left region of jaw since one month.
• K/C/O - Diabetes mellitus
• Three months came to Emergency
Department in an unconscious state- Diabetic
ketoacidosis with renal failure
60. Artefacts
• Tissue components such as Russell bodies,
calcified bodies, elastic fibres and small blood
vessels .
• Silver precipitates- collagen, fibrin, elastin ,
neutrophils, cytolytic debris.
61. • The KOH preparation – unstained fungal
elements in skin, hair and nails
• Specimens- sputum and vaginal
• Calcofluor white
• Combination of the two.
• India ink preparation - C. neoformans in CSF
However, unless special techniques
such as immunofluorescence are used, or the infecting
fungus possesses unique structures such as spherules,
definitive species identification of the aetiologic agent
by histopathology is difficult. Nevertheless, it usually
provides essential information before the fungus can
be isolated in a mycology laboratory.
H and 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
and E is usually employed to visualize both the tissue
reaction and the infecting fungus [Figures 3 and 4]. GMS is more advantageous
because it stains old and non-viable fungal elements
The major growth
forms of the fungi that help in histopathologic diagnosis
yeast cells- any small single cell which reproduce by budding
hyphae- branching filaments that make up the mycelium of a fungus.
Pseudohyphae-marked by constrictions rather than septa at the junctions
arthroconidia-are a type of fungal spore typically produced by segmentation of pre-existing fungal hyphae
chlamydoconidia -A thallic conidium that is thick-walled
Spherules-a thick-walled spherical structure enclosing endospores and occurring in the parasitic form of fungi of the genus Coccidioides
CUTANEOUS: limited to the dermis
SUBCUTANEOUS : when infection penetrates significantly beneath the skin
SYSTEMIC : when the infection is deep within the body or disseminated to internal organs
Infarct & Asprgilloma
Asperg hyphae lung. Hyphae are at acute angle at each other
GMS Progressive acute angle dichotomous branching of aspergillus hyphae (Gomori methenamine-silver stain, original magnification600).
Aspergillosis - Ubiquaitous fungus causing dis- healthy as well as immunocompromised
Infection – By inhalation or innoculation
ABPA- Hypersenstivity response to inhaled aspergillus
Aspergilloma- Fungal ball mc in sinus or within an old tuberculous cavity.
It has very small spore whch can penetrate deeper
Infected form- arthroconidia- Skin and meninges
This fungus is dimorphic- mold at room temperature and yeast at 35 degree
10 x Granulomatous inflammation, large thick-walled spherules contain variable sized daughter cysts
? Pyogenic reaction occurs with release of endospores from rupture of spherules
? Variable fibrocaseous granulomata, military disease, pyogenic inflammation
? Immature nonendosporulating spherules can resemble nonbudding forms of Blastomyces dermatitidis (Can Respir J 2008;15:377)
GMS Ten x
Barrel shaped structure
arthroconidia-are a type of fungal spore typically produced by segmentation of pre-existing fungal hyphae
This well-formed granuloma has a large Langhans giant cell in the center. Two small spherules of Coccidioides immitis are seen within the cytoplasm of the giant cell.
Histoplasma- thin based pear shaped yeast cells
Coccidomycosis- thick walled non- budding spherule 20 60 micron in diAMETER
Blasto- 5- 15 micron yeast cells that divide by broad based budding . It has thick double contoured cell wall and a visible nucleus
This is Candida esophagitis. Tan-yellow plaques are seen in the lower esophagus, along with mucosal hyperemia. The same lesions are also seen at the upper right in the stomach
Candida albicans- Pseudohyphae
PAS 40 x
Densely matted pseudohyphae and budding spores in squamous debris, fibrinopurulent exudate or necrotic debris
Underlying active esophagitis
HIV patients may have invasion into muscularis propria and adventitia if untreated (Mycoses 1997;40 Suppl 1:81)
PAS 40 x
Cryptococci in perivascular space
Mucicarmine
Alcian Blue
PAS
Mucicrmine stain
CNS
40 x of pnemocystis h e
the organisms spread to the interstitium of the lung, widen alveolar walls, and invade blood vessel walls (arrow). Some authors believe that alveolar walls then undergo lysis to form cavities [3]. Other authors have attributed the lysis to ischemic necrosis caused by a necrotizing angiitis [4]. Note that there is little inflammatory reaction.
An adjacent section was stained with the GMS stain to show the presence of spore cases both in the alveolar space (A) and alveolar wall (W).
The foamy pink exudate stains with the PAS stain after diastase digestion. It is composed of trophic forms, surfactant phospholipids, cell debris, and host-derived proteins [2]. The faint dots in the exudate correspond to nuclei of the trophic forms.
Histoplasma infection skinmicro1breast
Isolated intracellular organisms and large aggregates surrounded by chronic inflammatory cells and fibroblasts (but no neutrophils or eosinophils); also epithelioid granulomas with variable caseation
May be narrow based budding of spores
histoplasma in macrophages
Slide culture with small microconidia and tuberculate macroconidia
Culture shows tan-white-brown wooly mold at 25 - 30 C on Sabouraud dextrose agar
Organisms have delicate, septate hyphae, 1 - 2 microns thick, with large rough-walled macroconidia 5 - 15 microns
Reverts to yeast at 37 C on sheep blood agar
Yeast is 2 - 4 microns, budding, single nuclei, round/oval with thin rigid walls
Nasal rhinosporidiosis. In the insert, globular cyst containing
endospores (Haematoxylin & Eosin, 50×; in the insert, 400×
. Each of these cysts
represented a thick-walled sporangium containing
numerous "daughter spores" in different stages of development
(Fig. 1). The stroma contained
Gomori methenamine silver stain and, in the insert, periodic
acid-Schiff stain (200×; in the insert, 400×).On histological
examination, the lesion showed the characteristic features
of the rhinosporidiosis: the polypoid fibroconnective
stroma, covered by flat multi-stratified squamous epithelium,
contained many globular cysts. Each of these cysts
represented a thick-walled sporangium containing
numerous "daughter spores" in different stages of development
(Fig. 1). The stroma contained a vascular fibroconnective
tissue with fibroblasts and myofibroblasts and
an inflammatory infiltrate (neutrophil granulocytes, lymphocytes,
plasma cells and histiocytes). Histochemical
stains such as PAS, GMS (Fig. 2) and mucicarmine were
used to establish the correct diagnosis of rhinosporidiosis.
Morphological criteria were based on the diameter of the
endospores and sporangia, respectively 5–10 μm and 50–
1000 μm. These findings made easier the distinction of
Rhinosporidium seeberi from another common nasal mycosis
Showing fungal hyphae (H&E, ×40)
Histology of the received specimen showed stratiÞ ed squamous
epithelium which was ulcerated with intraepithelial split
formation; underlying connective tissue showed numerous
minor salivary glands, ducts and muscle tissue. Large amounts
of necrotic tissue were evident with cellular degeneration and
debris [Figure 4]. Fungal hyphae were seen with neutrophil
inÞ ltration and generalized chronic inß ammatory cell inÞ ltrate
within connective tissue. Hyphae were aseptate, branched and
resembled mucormycosis. Areas of hemorrhage were present
Hemotoxylin and Eosin stain histopathology showing necrotic and edematous tissue with neutrophilic infiltrate and hyphae.
Any excess silver precipitate makes interpretation difficult
or impossible because collagen, fragmented elastin
fibres, mucin, neutrophil granules and cytolytic debris
stain with silver precipitation techniques.
sputum and vaginal secretions that contain large
amounts of cellular material, because KOH dissolves
keratin and much of the other interfering background
protein-rich debris
Many laboratories have substituted
calcofluor white for KOH or use a combination of the two
reagents. Calcofluor white is a fluorescent brightener or
whitening agent that binds to cellulose and chitin in fungal
cell walls and fluoresces with a blue white colour when
exposed to ultraviolet radiation
