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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