DEMONSTRATION OF FUNGI IN TISSUE

SECTIONS

SPEAKER : DR SUPRIYA R KOKANAY

WHY?

1.Increased mobility

2.Immunodeficiency states

3.Adaptive mutations in microbes

DIAGNOSIS• Clinical• Mycologic• Immunologic• Pathologic

ROLE OF HISTOPATHOLOGY• Histopathology should not substitute culture but

both should complement each other

• Only fixed tissues available

• Not yet isolated in culture – lobomycosis, Rhinosporidiosis

• Indisputable evidence of tissue invasion

• Coexisting infections

• Less time

• Formal saline- Fixative- safety

• Clinical details – patient details, immune status, recent travel

• Macroscopic appearance – abcess, cavitation

• Microscopic appearance – lymphocytes, neutrophils, granulomas, necrosis

• Based on morphologic distinctiveness

1.Fungi with distinctive morphology – Blastomycosis, Cryptococcosis

2.Fungi identified at genus level – Aspergillosis, Candidiasis

3.Fungi belonging to various genera – Zygomycosis, Dermatophytosis

4.Mycetomas

HAEMATOXYLIN & EOSIN STAIN• corner stone of histochemical methods• Haematoxylin is extracted from tree hematoxylon

campechianum with hot water & precipitated out with urea

• Counterstain – Eosin Y, Eosin B, Phloxine, Erythrosin

RESULTS –• Stains nuclei blue, cytoplasm pinkADV – • Demo tissue responseDISADV – • Contrast is not great

GOMORI’S METHENAMINE SILVER• PRINCIPLE –

aldehydrogenic groups are uncovered by Chromic acid oxidation which reduce methenamine silver nitrate in alkaline solution

• RESULTS –

fungi, glycogen, mucin, BM, Reticulin, Elastin – brownish black to black

HOTCHKISS – MCMANUS PAS TECHNIQUE• Described by McMannus for demonstration of

mucin• Principle: Periodic acid(0.5-1%) brings oxidative

cleavage of c to c bond in 1-2 glycols or their amino or alkylamine derivative to form dialdehyde which react with Schiff reagent to give magenta colour compound to the tissues.

• RESULTS • PAS Positive - magenta • Nuclei - blue• Tissue - yellow

PAS- Methenamine silver: • used to stain basement membrane of kidney,

fungi.

RESULTS • PAS positive - black brown• Background – light yellow or green.

Versitality of PAS• Intensity of staining is proportional to conc of

reactive groups• Can be combined with other stains

DISADV• Nocardia & Actinomyces stain poorly or not

at all

GRIDLEY STAIN• Modified chromic acid schiff method• Demo of fungal wall

RESULTS – • Mycelia – deep blue• Conidia – deep rose to purple• Background – yellow• Elastic tissue & mucin – deep blue

Southgate’s modification of Mayer’sMucicarmine • Popular and gives consistent result, obsolescent • Demo of capsulePRINCIPLE : • Aluminium salt solution form a chelate compound

with carmine, forms a net positive charge on the molecule and subsequently binds to the tissue polyanion

Larger molecular size is able to penetrate and combine with low density acidic compounds .

Results : Mucin & capsule - red Nuclei - blue

ALCIAN BLUE METHODS

• Water soluble amphoteric copper phthalocyanin dye• Introduced by ICI chemist Haddock in 1948 • Demo of capsules

• PRINCIPLE: forms salt linkages with acidic groups of acid mucins.

A tissue is intensely stained, if the dye is used at low pH at which the reacting groups are fully ionized.

Advantages of Alcian blue• Simplicity• Striking blue color produced & its resistance

to counter staining• Specificity• Insolubility of staining• Permanance of results• Can be combined with PAS

BROWN AND BRENN GRAM’S STAINPROCEDURE – 1. Add mixture of 1 ml crystal violet & 5 drops of 5% aqueous

sodium bicarbonate2. Rinse in water3. Add Gram’s iodine(1 min)4. Rinse in water5. Decolorize with mixture of equal parts of ether & acetone6. Add 0.1% basic fuchsin(1 min)7. Rinse in water8. Dip in acetone9. Diff in 0.1% picric acid in acetone until yellowish pink10.Rinse in acetone then with mixture of equal parts of acetone

& xylene11.Clear in xylene & mount

MODIFIED ZIEHL-NEELSON STAINPRINCIPLE:• Nocardia when stained by strong stain with

the aid of heat resist decolorisation by acid owing to structural incorporation of lipids

RESULTS• Nocardia – red• Background - blue

METACHROMATIC STAIN

Principle - • acid mucosubstances containing carboxylated,

phosphated and sulphated compounds are metachromatic(polymerize dye), neutral mucins are orthochromatic(retain dye colour).

• Body of cryptococcus but not capsule stains metachromatically with toluidine blue

MASSON-FONTANA STAIN• PRINCIPLE –

melanin reduces ammoniacal silver soln to metallic silver

• RESULTS – • Phaeohyphomycosis & cell wall of

Cryptococcus - black

IMMUNOHISTOCHEMISTRY• increases specificity when organism load is

less• Co-infections• Candida, aspergillus, cryptococcos

LECTINS• CON A can be used in detection of some fungi

ELECTRON MICROSCOPY• Ultrastructure of fungi

DIRECT FLUORESCENT ANTIBODY STAINING

• Formalin fixed paraffin embeded• Added dimension of serologic specificity• Increase accuracy of HPE when atypical fungal

forms are present

CLASSIFICATION• Superficial mycosis• Cutaneous & subcutaneous mycosis• Systemic mycosis

BLACK PIEDRA • Piedraia hortae• Pigmented, closely septate hyphae,4-6 micron,

organized as nodules surrounding hair shaft; asci containing ascospores

WHITE PIEDRA • Trichosporon beigelii• Hyaline hyphae, 2-4 micron ( arthroconidia and

blastoconidia)organized as nodules surrounding hair shaft; invades and destroys hair

• Both involves the hair exclusively

TINEA NIGRA

• Phaeoannellomyces werneckii, Stenella araguata

• Pigmented, branched, septate hyphae 1-3 micron , elongated budding cells, 1-5 micron

TINEA VERSICOLOR

• Malassezia furfur

• Short, curved, and bent, hyaline hyphae, 2-4 micron, clusters of oval or round, thickwalled cells (phialoconidia),3-8 microns

• Both cause hyperkeratosis

DERMATOPHYTOSIS

• Epidermophyton,Microsporum and Trichophyton

TINEA CORPORIS(HE,PAS)

MAJOCCHI’S GRANULOMA• Hyaline, septate hyphae that break up into

chains of arthroconidia• Hyperkeratosis, acanthosis, mild mononuclear

infiltrate in dermis; rarely, suppurative or granulomatous, splendore hoeppli material

CUTANEOUS AND SUBCUTANEOUS MYCOSES

CHROMOBLASTOMYCOSIS

• Cladosporium carrionii,Fonsecaea compacta,F. pedrosoi, Phialophora verrucosa, Rhinocladiella aquaspersa

• Large, 6-12 micron, spherical to polyhedral, thick-walled, dark brown muriform cells (sclerotic bodies) with septations along one or two planes; pigmented hyphae

• Suppurative/granulomatous

LOBOMYCOSIS• Loboa loboi

• Spherical, budding yeastlike cells, 5-12 microns, that form chains of cells connected by tubelike isthmuses “string of pearls” secondary budding may be present

• Granulomatous

H & E GMS

EUMYCETOMA

• Pseudallescheria boydii,Madurella grisea, M.mycetomatis, Curvularia geniculata, Exophiala jeanselmei,Leptosphaeria senegalensis

• Granules, 0.2 to several mm composed of broad (2-6 micron), hyaline (white to yellow granules) or dematiaceous (black granules) septate hyphae that often branch and form chlamydoconidia, splender hoeppli

HE GRIDLEY STAIN

PHEAOHYPHOMYCOSIS

• Exophiala jeanselmei,Phialophora parasitica,P. richardsiae, Wangiella dermatitidis

• Wood prick

1.Cutaneous - abcess

2.Systemic – lung, brain

• Granulomatous

• Pigmented (brown) hyphae, 2-6 microns, branched or unbranched,and often constricted at their frequent and prominent septations; yeast forms and chlamydoconidia sometimes present

RHINOSPORODIOSIS

• Rhinosporidium seeberi

• Large sporangia, 100-350 micron, with thin walls that enclose numerous sporangiospores,6-8 microns

• Nonspecific chronic inflammatory or granulomatous

SPOROTHRICOSIS

• Sporothrix schenckii

1.Lymphocutaneous

2.Systemic – lungs,bones

joints

• Pleomorphic, spherical to oval and, at times, cigar-shaped yeastlike cells, 2-10 Microns that produce single and rarely, multiple buds

• Mixed suppurative and granulomatous;Splendore-Hoeppli material (asteroid body), pseudoepitheliomatous hyperplasia

ACTINOMYCOSIS

• Actinomyces israelii,A. naeslundii, A. viscosus, A. odontolyticus,A. bovis, Arachnia propionica,Rothia dentocariosa

• Organized aggregates (granules)composed of delicate,branched filaments about 1microns; entire granules 30-3000 microns

• Suppurative with multiple abscesses,extensive fibrosis,and formation of sinus tracts;Splendore-Hoeppli

• ACTINOMYCOSIS(HE,FITE FARACO,GRAM’S,PAS)

ADIASPIROMYCOSIS

• Crysosporium parvum

• Large adiaconidia, 200-400 microns, with thick (20-70 microns walls

• Granulomatous, fibrotic, and noncaseating

• Doesn’t multiply in humans

ASPERGILLOSIS

• Aspergillus fumigatus, A. flavus ,A. niger

• Septate, dichotomously branched hyphae of uniform width (3-6 microns); conidial heads may be formed in cavitary lesions

• Nodular infarcts; rarely granulomatous or suppurative; tendency for angioinvasion

BLASTOMYCOSIS

• Blastomyces dermatitidis

• Spherical, multinucleated yeastlike cells, 8-15 microns thick walls and single,broad-based buds

1. Cutaneous – ulcerated, verrucous

2. Systemic – bone, joint, brain

• Mixed suppurative and granulomatous,

BLASTOMYCOSIS

CANDIDIASIS

• Candida albicans, C.tropicalis, C. parapsilosis,C. krusei,C. pseudotropicalis,C. guilliermondii,C. stellatoidea

• Oval, budding yeastlike cells,pseudohyphae;septate hyphae

• Suppurative, less commonly granulomatous or infarctive; minimal inflammation

COCCIDIOIDOMYCOSIS• Coccidioides immitis• lung• Spherical, thick-walled,

endosporulating spherules, 30-200 microns; mature spherules contain small, 2-5 microns uninucleate endospores; arthroconidia and hyphae may be formed in cavitary lesions

CRYPTOCOCCUS• Cryptococcus neoformans• Pleomorphic yeastlike cells, 2-20 microns, with

gelatinous, carminophilic capsules and single or multiple narrow-based buds; some strains poorly encapsulated and may not be carminophilic

1.Pulmonary

2.Disseminated• Varies from minimal reaction ("cystic" or "mucoid"

lesion) to granulomatous

MUCORMYCOSIS

• Absidia corymbifera,Apophysomyces elegans,Cunninghamella bertholletiae,Mucorramosissimus,Rhizomucor pusillus, Rhizopus oryzae, R. rhizopod/formis,Saksenaea vasiformis

• Broad, thin-walled, infrequently septate hyphae, 6-25 microns wide, with nonparallel sides and random branches

• Suppurative necrosis, granulomatous, angioinvasion and infarction

PARACOCCIDIODOMYCOSIS

• Paracoccidioides brasiliensis

• Large spherical yeastlike cells,5-60 microns, with multiple buds attached by narrow necks("steering wheel" forms)

1.Lung 2. disseminated

• Mixed suppurative and granulomatous;

HISTOPLASMOSIS

• Histoplasma capsulatum

• Spherical to oval budding yeastlike cells, 2-4 microns; often clustered within mononuclear phagocytes

• Lungs – acute, chronic & disseminated

GEOTRICOSIS

• Geothricosis candidum

• Septate, infrequently branched hyphae, 3-6 microns wide; spherical yeastlike cells; and rectangular or oval arthroconidia,4-10 microns wide, with rounded or squared ends

• Varies from minimal reaction to acute suppurative inflammation and necrosis

PENICILLIOSIS• Penicillium marneffei• Disseminated• Spherical to oval

yeastlike cells,2.5-5 microns with a single transverse septum; short hyphal forms and elongated, curved "sausage" forms with one or more septa may be formed in necrotic and cavitary lesions

CONCLUSION

• Use of special stain is only to elucidate or negate a possibility arrived with H & E stain

• Diagnosis of lesion primarily rests on H & E stain & a good deal of common sense

• Histopathology should not substitute culture but both should complement each other

REFERENCES

• Cellular pathology technique - C.F.A Culling, R.T,Allison

• Theory and practice of Histological techniques- Bancroft

• Histopathological technique- Lynch

• Anderson’s pathology

• Lab Techniques in Surgical Pathology – Shameem Shariff

• Sternberg’s Diagnostic Surgical Pathology

• Robbins & Cotran- Pathologic Basis of Disease

CANDIDA