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BIOL 4849: Medical Mycology Summer 2011
Lecture 5: Histopathology of Fungal Infections 1
Histopathology of Fungal Infections
BIOL 4849 (Summer 2011) Copyright © 2011 Chester R. Cooper, Jr.
Disclaimer: This lecture slide presentation is intended solely for educational purposes. Many of the images contained herein are the property of the original owner, as indicated within the figure itself or within the figure legend. These images are used only for illustrative purposes within the context of this lecture material. Use of these images outside the purpose of this presentation may violate the rights of the original owner. Dr. Cooper and Youngstown State University assume no responsibility for the unauthorized use of the material contained herein.
BIOL 4849 (Summer 2011) Copyright © 2011 Chester R. Cooper, Jr.
Diagnosis of Fungal Infections
• Diagnosis of a mycotic disease ideally includes: – Observation of typical symptoms – Demonstration of fungus in lesion with
accompanying host reaction – Isolation of causative agent
• Not all the above can be accomplished in every type of fungal disease
BIOL 4849 (Summer 2011) Copyright © 2011 Chester R. Cooper, Jr.
Diagnosis of Fungal Infections (cont.)
• Other methods that can be used to aid in the diagnosis of a fungal infection include the detection of fungal: – Antigens – Antiboides – Metabolites – Cell wall markers
• More modern molecular-based methods are now available
BIOL 4849 (Summer 2011) Copyright © 2011 Chester R. Cooper, Jr.
Diagnosis of Fungal Infections (cont.)
• Some of the above methods are not yet available for many pathogenic fungi, particularly those that are somewhat unusual
• Remaining method for diagnosis includes the histopathological examination of biopsy material in order to observe: – Characteristic features of specific etiological agent – Host response to infection
BIOL 4849 (Summer 2011) Copyright © 2011 Chester R. Cooper, Jr.
Histological Stains for Fungi
• Hematoxylin and eosin (H&E) – Color of fungi: pink to pinkish blue – Applications:
• Demonstrates inflammatory response • Stains some fungi • Allows determination of innate pigmentation by
invading fungus • Demonstrates Splendore-Hoeppli material • Stains most nuclei of yeast-like fungi
BIOL 4849 (Summer 2011) Copyright © 2011 Chester R. Cooper, Jr.
Haematoxylin and eosin (H&E) stained sections of lung tissue showing the broad, infrequently septate, thin-walled hyphae of
Absidia corymbifera (left) and darkly-pigmented sclerotic cells of Fonsecaea pedrosoi. Source: www.doctorfungus.com
BIOL 4849: Medical Mycology Summer 2011
Lecture 5: Histopathology of Fungal Infections 2
BIOL 4849 (Summer 2011) Copyright © 2011 Chester R. Cooper, Jr.
Splendore-Hoeppli material from a case of sporotrichosis. Source: www.histopathology-india.net/ Sporotri.htm
BIOL 4849 (Summer 2011) Copyright © 2011 Chester R. Cooper, Jr.
Histological Stains for Fungi (cont.)
– Limitations: • Does not stain many fungi • Does not stain filamentous bacteria • Is not adequate for screening tissue with
sparse number of fungal elements
BIOL 4849 (Summer 2011) Copyright © 2011 Chester R. Cooper, Jr.
Histological Stains for Fungi (cont.)
• Gomori’s methenamine silver (GMS) [often referred to as ‘silver stain’] – Color of fungi: black brown on a light green
background – Applications:
• Stains most fungi, viable or not • Can stain filamentous bacteria
– Limitations: • May overstain fungi and obscure internal
details • Cannot detect host response
BIOL 4849 (Summer 2011) Copyright © 2011 Chester R. Cooper, Jr.
Methenamine silver (GMS) stained tissue section from a lung showing typical zygomycete hyphae and by
chance a sporangium of Absidia corymbifera. Source: www.doctorfungus.com
BIOL 4849 (Summer 2011) Copyright © 2011 Chester R. Cooper, Jr.
Histological Stains for Fungi (cont.)
• Periodic acid-Shiff (PAS) – Color of fungi: red pink on a green background – Application: stains most fungi, viable or not – Limitations:
• Masks innate color and internal details • Many tissue elements take up the stain • Cannot detect host response • Does not stain filamentous bacteria
BIOL 4849 (Summer 2011) Copyright © 2011 Chester R. Cooper, Jr.
PAS stain of Rhinosporidium seeberi depicting sporangia at different stages of sporangiospore development in the
large sporangium as observed in a human nasal polyp. Source: www.doctorfungus.com
BIOL 4849: Medical Mycology Summer 2011
Lecture 5: Histopathology of Fungal Infections 3
BIOL 4849 (Summer 2011) Copyright © 2011 Chester R. Cooper, Jr.
Histological Stains for Fungi (cont.)
• Gridley fungus (GF) – Color of fungi: purplish red on a yellow
background – Application: stains most fungi – Limitations:
• Masks innate color • Non-viable cells do not stain • Cannot demonstrate host response • Does not stain filamentous bacteria
BIOL 4849 (Summer 2011) Copyright © 2011 Chester R. Cooper, Jr.
Gridley fungus stain of a mycetoma. Source: www.doctorfungus.com
BIOL 4849 (Summer 2011) Copyright © 2011 Chester R. Cooper, Jr.
Histological Stains for Fungi (cont.)
• GMS with H&E counterstain – Stain of choice if only one slide available for
histopathological examination – Color of fungi: black brown fungi on a red-pink
background – Applications:
• Permits study of host response • Excellent for detecting fungi and filamentous
bacteria – Limitation: cannot determine innate fungal color
BIOL 4849 (Summer 2011) Copyright © 2011 Chester R. Cooper, Jr.
Yeast and pseudohyphal cells of Exophiala spinifera in a combined GMS/H&E stain. Source: www.doctorfungus.com
BIOL 4849 (Summer 2011) Copyright © 2011 Chester R. Cooper, Jr.
Histological Stains for Fungi (cont.)
• Mucin (mucicarmine) stains – Mayer’s or Southgate’s preparations – Application: stains of mucopolysaccharide
capsular material of fungi, e.g., Cryptococcus – Limitation: Not specific for Cryptococcus
BIOL 4849 (Summer 2011) Copyright © 2011 Chester R. Cooper, Jr.
Mucicarmine stain of capsules of Cryptococcus neoformans in brain tissue. Source: www.doctorfungus.com
BIOL 4849: Medical Mycology Summer 2011
Lecture 5: Histopathology of Fungal Infections 4
BIOL 4849 (Summer 2011) Copyright © 2011 Chester R. Cooper, Jr.
Histological Stains for Fungi (cont.)
• Modified Gram’s stains – Brown-Hopps’ and MacCallum-Goodpasture
preparations – Application: stains Gram-positive filamentous
bacteria – Limitation: does not selectively stain fungi
• Modified acid-fast stains – Ziehl-Neelsen’s and Kinyoun’s preparations – Application: stains Gram-positive filamentous
bacteria – Limitation: does not stain fungi
BIOL 4849 (Summer 2011) Copyright © 2011 Chester R. Cooper, Jr.
Histological Stains for Fungi (cont.)
• Modified Fontana-Masson – Applications:
• Stains cell walls of Cryptococcus and other melanin producing fungi
• Accentuates weakly pigmented agents of phaeohyphomycosis
– Limitation: may stain fungal elements that are immature or innately not pigmented
Fontana-Masson stain of Lacazia loboi in biospy material. Source: Taborda et al., Rev. Inst. Med. trop. S. Paul vol.4, Jan./Feb. 1999
BIOL 4849 (Summer 2011) Copyright © 2011 Chester R. Cooper, Jr.
Histological Stains for Fungi (cont.)
• Whitening agents – Calcofluor White, Uvitex,
and others – Application: stains cell walls
of fungi – Limitation: need a
fluorescent microscope
Calcofluor White stain of Candida albicans. Source: www.reviberoammicol.com/photo_gallery/Candida/
albicans/
BIOL 4849 (Summer 2011) Copyright © 2011 Chester R. Cooper, Jr.
Histopathological Identification
• Tissue sections can be used to observe fungal elements and particular attributes that may be characteristic of certain species
• Fungi can appear as – Hyaline or pigmented (phaeoid) – One of four broad morphological categories
• Yeast-like • Hyphae • Endosporulating spherules • Granules
BIOL 4849 (Summer 2011) Copyright © 2011 Chester R. Cooper, Jr.
Histopathological Identification (cont.)
• Other defining features of fungal forms in vivo include – Size and shape of cells – Cell wall thickness – Number and shape of blastoconidia (buds) – Presence or absence of septations – Capsules – Number of nuclei – Presence of pseudohyphae, hyphae, or
arthroconidia
BIOL 4849 (Summer 2011) Copyright © 2011 Chester R. Cooper, Jr.
Histopathological Identification (cont.)
• Immunohistological staining is also used to detect and identify fungi in tissue – Can be direct or indirect staining, i.e., one step or
multi-step process – Often fluorescent-tagged antibodies are used – Other ‘tags’ include
• Gold-silver complexes • Enzyme complexes (e.g., peroxidases)
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