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Mycology
Just the basics – meant for
board review or brief study of
this fascinating area of
microbiology!
Mycology
Starting point
Yeast are:
 unicellular / produce budding daughter cells
 colony on solid media are usually white to
beig...
Starting point
Molds are:
 Filamentous with hyphae
 Produce conidia [spores]
 Colonies on solid agar are downy, fluffy,...
Appropriate Specimens and Transport
conditions for Fungal disease diagnosis
 Fungi are very hardy organisms
 Specimens d...
Fungal Culture Media
 Sabouraud’s glucose agar (SABS)
Common Fungal Media
 Mycosel/Mycobiotic agars
 Selective SABS with chloramphenicol and cycloheximide
 used for culture ...
What effect does Cycloheximide have
when added to media?
 Prevents rapidly growing molds from overgrowing
dimorphics and ...
Inoculate fungal media
Seal plates with tape to prevent culture
contamination and escape of fungal spores
Incubate at 30˚C...
Lactophenol cotton blue [LCB] adhesive tape preparations are
the standard method used for mold identification.
The LCB mou...
Safety in the Mycology Laboratory
 If a culture is growing a mold, it cannot be
opened on the bench top
 All mold work m...
Direct Exams used to identify fungi
directly from patient specimens
 Gram stain – all specimen types can be Gram
stained....
Yeast cells stain blue [Gram positive]. Examine for
budding cells to confirm that it is a yeast cell and not
an artifact. ...
Used to detect yeast and/or hyphae in skin, hair
and nail specimens using 40X light microscope.
KOH dissolves keratin foun...
Yeast, pseudohyphae, and mycelial fungi will bind
with the Calcofluor white stain.
Prep is interpreted using a fluorescenc...
One drop of black ink is placed
into one drop of CSF and it is
examined using a 40X lens on
light microscope
It is a “nega...
Methenamine Silver Stain [GMS] –
yeast and hyphae stain grey to black.
Examine the hyphae for presence of septations in th...
PAS-positive staining red against a
green or blue background
Periodic Acid Schiff [PAS]
Stain-Cryptococcus neoformans polysaccharide capsule
stains pink
Mucicarmine [Mucin] stain
great for cellularity, it is
the beginning but GMS, or
PAS show features of the
fungi better.
Hematoxylin and Eosin Stain
The Dimorphic Fungi
Important pathogens with some
unique characteristics
What does Dimorphic mean?
 Two forms exist for one fungus species depending on
temperature and conditions of environment
...
Dimorphic Fungi capable of causing
systemic infection
 Histoplasma capsulatum
 Blastomyces dermatitidis
 Coccidioides i...
Histoplasma capsulatum
 World wide distribution / In USA in Ohio,
Missouri, and Mississippi River valleys
 Associate wit...
Histoplasmosis Disease
 95% of infections are subclinical
 5% infections:
 Progressive pulmonary
 Chronic systemic inf...
The yeast of Histoplasma capsulatum prefer to
be intracellular and inhabit Macrophages.
Yeast are small 2 – 4 um, regular ...
H & E PAS
Gram Wright’s
Histoplasma will stain with a variety of stains
Histoplasmosis rapid diagnosis
 Antigen detection in urine
 Quantitative Enzyme immunoassay
 Random urine specimen
 Mo...
Histoplasma capsulatum
 Fungal Culture incubated at 30˚ C
 Very SLOW growing taking 2 – 8 weeks to form colonies
 Colon...
Histoplasma capsulatum
culture at 30˚ C is white and
cottony.
Microscopic exam:
Tuberculate [projections]
macroconidia is ...
Appearance in culture at
35 degrees C
 Culture @ 35˚C is yeast
 Grows as small yeast, round to oval, always
consistent i...
Histoplasma capsulatum in tissue
 Granulomas are usually produced and can be
either caseating or non caseating
 Infectio...
Leishmania species
Note small round
kinetoplast next to nucleus
Toxoplasma
Histoplasma capsulatum
Beware of look alike org...
H capsulatum var duboisii yeast cells are
8 – 10 uM in size, which is 2X the size of
regular Histoplasma capsulatum yeast ...
Blastomyces dermatitidis
 Epidemiology
 Ohio and Mississippi River valleys
 No association with specific animal or acti...
Blastomyces dermatitidis
 Culture at 30˚C
 Grows in 2- 3 weeks
 Fluffy white – buff colored mold, prickly
 Pear shaped...
Slow growing yeast colony taking @ 4
weeks to form a colony
Yeast cell is 8 – 20 um in size and is
unique for it’s Broad B...
Blastomyces dermatitidis histopathology
Mixed pyogenic and granulomatous
inflammation is observed in tissue with
Broad bas...
Coccidioides immitis
 Endemic in SW USA, Mexico,
South America, in areas known
as the Sonoran life zone with a warm clima...
Coccidioidomycosis
 95% of infections are asymptomatic or with limited
symptoms
 The remaining 5% are focal pulmonary, p...
Coccidioides immitis [posadasii]
 Culture at 30˚C
 Requires only 2 – 3 days to grow, colony starts waxy and
becomes wool...
Coccidioides
 Malbranchea species can look like C. immitis under the
microscope
 Because of look-a-like fungi one needs ...
Barrel shaped alternating arthroconidia are
produced in cultures grown at both 30 and 35 C.
There is no yeast phase for C....
Coccidioides Histopathology
 Thick walled spherules (10 – 80 uM) with endospores
are seen in tissue. This is the second f...
Development of Cocci spherules from the
inhalation of Arthroconidia from nature
Rhinosporidium seeberi forms spherules but much larger
than the Cocci spherules - they are usually > 80 uM in size.
Also R...
Paracoccidioides brasiliensis
 South American Blastomycosis –
endemic area Brazil, Venezuela, Columbia
 Inhale infectiou...
Paracoccidioides
 Cultures are usually not used in diagnosis
due to slow growth and nonspecific
sporulation
 Culture @ 3...
Mariner’s wheel yeast of
Paracoccidioides brasiliensis
If more than 2 buds off mother cell –
High likelihood it is Paracoc...
Sporotrichosis
 Sporothrix schenckii
 Cutaneous inoculation of fungus from penetrating
injury with a spore or thorn (ros...
Starts as one ulcerative lesion and then chains
Up the lymphatics – can involve lymph nodes
and bone
Sporothrix schenckii
 Dimorphic fungus
 MOLD PHASE
 30*˚C growth in 3 -5 days
 Turns brown to black over time
 Septat...
Sporothrix schenckii
 Histology –
 Pyogenic – to – granulomatous inflammation
 Hard to find yeast in human tissue
 Ast...
Daisy like spore arrangement
Sporothrix
schenckii
Green colony with red diffusable pigment
Uncommon dimorphic fungus
The only species of Penicillium that is dimorphic
Cause...
Penicillium marneffei yeast like cells in tissue
Subcutaneous Fungal
Infections
Very unique structures in tissue!
Subcutaneous Fungal Infections
Most common will be described
 Mycetoma [2 types]
 Actinomycotic – caused by higher bacte...
Mycetoma
 First observed in India and known
as Madura Foot or Maduromycosis
 Found in the hot temperate parts of
the wor...
Swollen extremity and draining sinus with
sulfur granules
Sulfur Granule
Mycetoma
Mycetoma
 There are two types of Mycetoma:
1. Actinomycotic mycetoma – caused by higher bacteria
species
2. Eumycotic myc...
Gram stain as filamentous Gram positive bacilli – can be
poorly staining and appear speckled.
Nocardia are positive [red] ...
Edge of granule has thin filamentous bacteria
for both bacteria – Nocardia is modified acid fast
[PAF] positive and is aer...
Requires 3 – 5 days to
grow on agar media [Sabs,
5% Sheep’s blood agar
Colony is dry and crumbly
Musty smell
Total of 85 s...
Eumycotic mycetoma – subcutaneous
infection caused by the black molds
Numerous species of pigmented/black fungi found
natu...
Eumycotic sulfur granule – the granule is
full of a matrix of thick fungal hyphae
Chromomycosis/Chromoblastomycosis
 Three characteristics describe Chromomycosis
 Wart like lesions in subcutaneous tissu...
Chromomycosis/Chromoblastomycosis
Wart like/Verrucous lesions
In subcutaneous tissue
Sclerotic Body/Medlar Body/
Copper Pe...
Prototheca wickerhamii – the cause of
Protothecosis
 Algae without chlorophyll
 Causes skin lesions & nodules
 Most com...
Phaeohyphomycosis
This infection is caused by traumatic
implantation of dark fungi into subcutaneous
tissue
 Variety of i...
Black molds/Dark molds
also known as Dematiaceous
fungi
Black colored colonies and the
reverse [back of colony] is also
bl...
Black Molds – Dematiaceous fungi
 Black colonies
 Brown hyphae and spores
 Numerous species
 Difficult to identify
 A...
Rhinocladiella type
sporulation
Phialophora type sporulation
Cladophialophora
[Cladosporium type sporulation]
Acrotheca type sporulation
Exophiala species
Black Molds that can cause
Mycetoma/Chromomycosis/Phaeohyphomycosis
These are difficult to identify but ...
Wangiella dermatitidis
Phialophora verrucosa
Alternaria
Other black molds of importance:
Bipolaris
australiensis
Very invasive
fungal infection:
Skin, nasal
sinuses, bone
brain
Curvularia lunata
Center cell is the
largest
Exserohilum rostrum
 Associated with compounded pharmaceutical
[steroid] products contaminated with dust/dirt
 Used for ...
Scedosporium apiospermum/
Pseudallescheria boydii
Cat fur-like colony
Important Yeast causing human
infection
Candida species
Cryptococcus neoformans &
Cryptococcus gattii
Cutaneous and Superficial Mycoses
 Candida species (@ 10 found in humans)
 Opportunistic pathogen involving skin or muco...
Candida species
 Candida albicans – most common species
causing @ 60% of human yeast infections
 Candida glabrata, C. kr...
Candida species
 Grow in 24 – 48 hours
 SABS, IMA, BAP
 Bacteria-like colony – pasty white
 Budding yeast – oval @ 7-8...
Candida albicans
Identification
 Germ tube formation
 Incubate small amount of yeast in serum for 3-4hr at 35 ˚C
 Do no...
ChromAgar for the
identification of Candida
Chromogenic substrates
Turn different colors with
4 different yeast species
Ye...
Candida Histopathology
 Pyogenic to granulomatous
 Usually observe yeast cells, pseudohyphae
and/or hyphae appearing str...
Cryptococcus
neoformans
 In nature forms a 2um non-encapsulated
yeast cell. It is associated with bird
droppings (esp. pi...
Cryptococcus neoformans
 Irregular sized (2 – 20uM) yeast cells
 Polysaccharide capsule is virulence factor and it’s pre...
Cryptococcus gattii – a closely related
relative of C. neoformans
 Isolated from forested area of the Pacific Northwest
(...
Positive India Ink
Urea medium demonstrating urease
enzyme activity of Cryptococcus
Observe
Budding cells
Variability in s...
C. neoformans/C. gattii forms
brown colonies on Birdseed agar
Mucicarmine stain
Stains the capsular polysaccharide of capsule
Pneumocystis jeroveci could
be confused with C.
neoformans...
Cutaneous and Superficial Mycoses
Malassezia furfur
Dermatophytes
Malassezia furfur
 Pityriasis versicolor
 Most superficial of the dermatomycoses
 Found as normal flora on the skin,
 ...
Malassezia furfur
 Lipophilic yeast – oil required for growth
 Media used for culture must contain oil or have oil
overl...
Size range for Yeast
 Candida glabrata/Histoplasma capsulatum
 2 – 4 um
 Candida species
 8 – 10 um plus pseudohyphae
...
Dermatophytes – Ringworm infections
 Hair, skin and nail infections
 3 genera of fungi
 Microsporum species (many)
 Ep...
Positive KOH prep
Showing thin septate
fungal hyphae
Calcofluor white stain
with fluorescence –
thin fungal hyphae
Microsporum canis
Main cause of ringworm from dog and cat
White colony/ yellow on backside of colony
Tuberculate macroconi...
Microsporum gypseum infection from
exposure to contaminated soil
Trichophyton
Rubrum
White colony with
red diffusable
pigment
Pencil shaped
Macroconidia
Many micro-
Conidia
Infection from...
Trichophyton
tonsurans
Ballooning
Microconidia
Primary cause of
epidemic
ringworm in
children
Epidermophyton floccosum
Beaver tail large spores without microconidia
Khaki green colony
Opportunistic Fungal Pathogens
Infections in the immune suppressed
host or special circumstances
Hyaline molds
Black molds
Opportunistic Fungi - hyaline
 Hyaline – no color to the hyphae
 Regular septations in the hyphae
 Branching – angle ca...
Aspergillus species
 Hyaline with septations
 Numerous round conidia
 In tissue - Branching at 45 degree angle
 Primar...
Aspergillus Structure
Aspergillus species
Four species most common in human infections:
1. Aspergillus fumigatus
2. Aspergillus flavus
3. Asperg...
Aspergillus fumigatus
Blue/Green colony
Phialids with spores are
Directed upward
Aspergillus flavus
Green colony
Orange colored spores
that surround the vesecle
Aspergillus niger
Black colony
Black spores surround the vesicle
Aspergillus terreus
Sandy colored colony
Aleurioconidia
Resistance to Amphotericin B
Aspergillus – fruiting head and dichotomous
branching septate hyphae with branching at 45* angle
Can appear much like that...
Fusarium species –
Common in nature/plants
disease related to immune
status of host
Infections reported:
Disseminated in b...
Scopulariopsis species –found in soil and
plants
Infections: Nail, skin, sinusitis, pulmonary and
disseminated
Paecilomyces species
Isolated from soil and food
Opportunistic pathogen in the
immune suppressed
Penicillium species – most common mold
in the environment, bread mold, uncommon
cause of human disease
Mucormycosis/Zygomycosis
Hyaline
Broad non-septate hyphae
Mucormycosis/Zygomycosis
 Infections in diabetics, the elevated
glucose enriches fungal growth – classic
infection is rhi...
Zygomycete – coarse, aerial hyphae after 24
hours on SABS agar at 30˚C
Rhizopus Absidia
Distant rhizoids
Mucor
No rhizoids
Rhizoids
90˚ angle branching, aseptate, ribbon like
Invades vessels and can cause
infarcts and thrombi
Zygomycetes (Mucorales)
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MYCOLOGY REVIEW

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MYCOLOGY REVIEW

  1. 1. Mycology Just the basics – meant for board review or brief study of this fascinating area of microbiology! Mycology
  2. 2. Starting point Yeast are:  unicellular / produce budding daughter cells  colony on solid media are usually white to beige and appear much like bacterial colonies  some genera produce mucoid colonies
  3. 3. Starting point Molds are:  Filamentous with hyphae  Produce conidia [spores]  Colonies on solid agar are downy, fluffy, cottony  Most mold colonies are pigmented which aid in identification hyphae spores
  4. 4. Appropriate Specimens and Transport conditions for Fungal disease diagnosis  Fungi are very hardy organisms  Specimens do not require special transport media for culture submission  Sterile containers should be used to prevent bacterial contamination and numerous sites are appropriate for culture  Respiratory specimens – sputum, bronchial lavage, brushings, nasal sinuses  Tissue biopsies  Cutaneous - Skin scrapings, material from lesions  Ocular  Sterile body fluids including CSF  Blood, bone marrow
  5. 5. Fungal Culture Media  Sabouraud’s glucose agar (SABS)
  6. 6. Common Fungal Media  Mycosel/Mycobiotic agars  Selective SABS with chloramphenicol and cycloheximide  used for culture of dermatophytes – fungi that cause skin, hair and nail infections  Brain heart infusion agar  Primary recovery of all fungal organisms  Can make it more selective by adding chloramphenicol and cycloheximide  All fungal cultures must be incubated for 4 weeks at 30˚C  Lower temperature than bacterial culture incubation [35˚C]  If plates are used for fungal cultures the plates must be sealed with air permeable tape for laboratory safety
  7. 7. What effect does Cycloheximide have when added to media?  Prevents rapidly growing molds from overgrowing dimorphics and dermatophytes  This is the good aspect of cycloheximide in media  Beware: it is not all good, it can suppress important fungi from growing. Inhibited fungi include:  Trichosporon beigelii  Candida tropicalis  Cryptococcus neoformans  Yeast phase of Blastomyces  Yeast phase of Histoplasma
  8. 8. Inoculate fungal media Seal plates with tape to prevent culture contamination and escape of fungal spores Incubate at 30˚C for 4 wk If growth occurs - perform proper identification methods: Yeast identification methods are very similar to methods used to identify bacteria. There are manual and automated biochemical reactions capable of identifying most yeast species. There are newer methods [Mass spectrometry – MALDI-TOF and 16 sRNA sequencing that can also be used in the more sophisticated mycology laboratory. Processing of Fungal Cultures - stepwise
  9. 9. Lactophenol cotton blue [LCB] adhesive tape preparations are the standard method used for mold identification. The LCB mounting medium consists of phenol lactic acid, glycerol and aniline cotton blue dye. Clear adhesive tape touches a mold colony, picking up fungal hyphae and pressed into one drop of LCB If LCB prep is not able to identify a mold 16sRNA sequencing can be used to identify problematic molds in reference laboratories. Mold Identification
  10. 10. Safety in the Mycology Laboratory  If a culture is growing a mold, it cannot be opened on the bench top  All mold work must be performed in a BSL-2 biosafety cabinet with Hepa filtration  Yeast identification can be performed on the bench top
  11. 11. Direct Exams used to identify fungi directly from patient specimens  Gram stain – all specimen types can be Gram stained. Can only reliably detect yeast by Gram stain.  KOH preparation – Skin, Hair or Nails examined for both yeast and/or hyphae  Calcofluor white stain – all specimen types can be stained and examined for yeast and/or hyphae  India ink – Primarily used for CSF for the detection of Cryptococcus neoformans and C. gattii
  12. 12. Yeast cells stain blue [Gram positive]. Examine for budding cells to confirm that it is a yeast cell and not an artifact. Examination on oil immersion lens. You can also detect pseudohyphae on Gram stain. Mold can be difficult to identify on a Gram stain. pseudohyphae mold pseudohyphae Gram Stain
  13. 13. Used to detect yeast and/or hyphae in skin, hair and nail specimens using 40X light microscope. KOH dissolves keratin found in cell material and frees hyphae from the cell KOH exams can be difficult to interpret! KOH – potassium hydroxide prep
  14. 14. Yeast, pseudohyphae, and mycelial fungi will bind with the Calcofluor white stain. Prep is interpreted using a fluorescence microscope. Sensitivity and specificity is improved over the KOH preparation. Calcofluor white stain
  15. 15. One drop of black ink is placed into one drop of CSF and it is examined using a 40X lens on light microscope It is a “negative” stain because it stains the background not the yeast The clearing is the polysaccharide capsule of Cryptococcus neoformans or C. gattii. Specificity is improved if you look for budding yeast cells. India Ink
  16. 16. Methenamine Silver Stain [GMS] – yeast and hyphae stain grey to black. Examine the hyphae for presence of septations in the hyphae, broad or more narrow width and angle of branching. Examine the size and budding pattern of observed yeast. We will observe on later slides these criteria can assist in identification. Examination of fungi in fixed tissue
  17. 17. PAS-positive staining red against a green or blue background Periodic Acid Schiff [PAS]
  18. 18. Stain-Cryptococcus neoformans polysaccharide capsule stains pink Mucicarmine [Mucin] stain
  19. 19. great for cellularity, it is the beginning but GMS, or PAS show features of the fungi better. Hematoxylin and Eosin Stain
  20. 20. The Dimorphic Fungi Important pathogens with some unique characteristics
  21. 21. What does Dimorphic mean?  Two forms exist for one fungus species depending on temperature and conditions of environment  Mycelial form free living form found in nature and at laboratory temperature <=30˚C  Yeast or yeast like form parasitic phase found in human tissue or in the lab >= 35˚ Histoplasma capsulatum – mold from 30˚C culture Histoplasma capsulatum – yeast from tissue and <=30˚C culture
  22. 22. Dimorphic Fungi capable of causing systemic infection  Histoplasma capsulatum  Blastomyces dermatitidis  Coccidioides immitis  Paracoccidioides brasiliensis  Sporothrix schenckii
  23. 23. Histoplasma capsulatum  World wide distribution / In USA in Ohio, Missouri, and Mississippi River valleys  Associate with Bat guano (Spelunker) and bird droppings
  24. 24. Histoplasmosis Disease  95% of infections are subclinical  5% infections:  Progressive pulmonary  Chronic systemic infection with dissemination to the RES system including bone marrow  Acute fulminating systemic disease (fatal)  Reactivation disease can occur in elderly and immunosuppressed (AIDS is a good example)  Bone marrow exam is useful in diagnosing disseminated infections  Mucocutaneous lesions are common and a site of dissemination
  25. 25. The yeast of Histoplasma capsulatum prefer to be intracellular and inhabit Macrophages. Yeast are small 2 – 4 um, regular in size, and oval to round. Yeast do not have a capsule, this is just a staining artifact.
  26. 26. H & E PAS Gram Wright’s Histoplasma will stain with a variety of stains
  27. 27. Histoplasmosis rapid diagnosis  Antigen detection in urine  Quantitative Enzyme immunoassay  Random urine specimen  Most useful for disseminated infection and chronic pulmonary disease  Antigen is detectable in >=85% of these infections  Good for immune suppressed patients that do not produce a detectable antibody response
  28. 28. Histoplasma capsulatum  Fungal Culture incubated at 30˚ C  Very SLOW growing taking 2 – 8 weeks to form colonies  Colony is white to brown and cottony  Microscopic appearance – tuberculated macroconidia that are large and round (8 – 16 µM) plus small microconidia (2 - 4µM) [see picture]  Microconidia are the infectious particle growing in nature and capable of penetrating deep into the lung  DNA probe must be used to confirm identification so there is definitive identification  Sepedonium species looks somewhat like Histoplasma and is considered a look a like fungus
  29. 29. Histoplasma capsulatum culture at 30˚ C is white and cottony. Microscopic exam: Tuberculate [projections] macroconidia is the structure used for ID. Microconidia are the infectious particle. Appearance in culture at 30 degrees C
  30. 30. Appearance in culture at 35 degrees C  Culture @ 35˚C is yeast  Grows as small yeast, round to oval, always consistent in size and shape (2 -4 uM) narrow neck at the budding juncture
  31. 31. Histoplasma capsulatum in tissue  Granulomas are usually produced and can be either caseating or non caseating  Infection usually begins in the Lung  Infection disseminates to organs of the Reticuloendothelial System (RES) – with high % of dissemination to the Bone Marrow  Intracellular budding yeast (2 – 4 µM) are seen in all tissues
  32. 32. Leishmania species Note small round kinetoplast next to nucleus Toxoplasma Histoplasma capsulatum Beware of look alike organisms in tissue specimens!!
  33. 33. H capsulatum var duboisii yeast cells are 8 – 10 uM in size, which is 2X the size of regular Histoplasma capsulatum yeast cells. H. capsulatum var duboisii disease is found in Central Africa Causes infection in skin and bone The 30˚C culture is identical to H capsulatum. Unusual variant of H. capsulatum
  34. 34. Blastomyces dermatitidis  Epidemiology  Ohio and Mississippi River valleys  No association with specific animal or activity  Forrest and river banks?  Primarily a pulmonary infection which may disseminate to the skin and bone Well demarcated skin lesion is typical Scraping of skin lesions are full of yeast cells
  35. 35. Blastomyces dermatitidis  Culture at 30˚C  Grows in 2- 3 weeks  Fluffy white – buff colored mold, prickly  Pear shaped conidia at the end of supporting hyphae – looks like lollipops  Look alike fungus – Chrysosporium species  Do DNA probe test to confirm identification Blastomyces Chrysosporium
  36. 36. Slow growing yeast colony taking @ 4 weeks to form a colony Yeast cell is 8 – 20 um in size and is unique for it’s Broad Based Budding pattern and the double contoured wall. Blastomyces culture at 37 degrees C
  37. 37. Blastomyces dermatitidis histopathology Mixed pyogenic and granulomatous inflammation is observed in tissue with Broad based budding yeast cells
  38. 38. Coccidioides immitis  Endemic in SW USA, Mexico, South America, in areas known as the Sonoran life zone with a warm climate and desert sands Infection is from inhalation of fungal particles found in the sand  Coccidioides posadasii is a genetically related to C. immitis. The two species are located in different endemic regions, but produce the same disease process
  39. 39. Coccidioidomycosis  95% of infections are asymptomatic or with limited symptoms  The remaining 5% are focal pulmonary, progressive pulmonary or disseminated infections. Dissemination to the central nervous system is difficult to cure and has a high fatality rate.  Higher incidence of dissemination occurs in patients with:  defects in cell mediated immunity (HIV),  darker skinned ethnic groups,  pregnancy
  40. 40. Coccidioides immitis [posadasii]  Culture at 30˚C  Requires only 2 – 3 days to grow, colony starts waxy and becomes wooly in around 7 – 10 days  Under the microscope one looks for foci of septated hyphae with thick walled barrel shaped arthroconidia with clear spaces in between. The clear spaces are dead arthroconidia.  Arthroconidia infectious particle in nature  Very infectious to laboratory personnel
  41. 41. Coccidioides  Malbranchea species can look like C. immitis under the microscope  Because of look-a-like fungi one needs to confirm identification of Coccidioides immitis with DNA probe or similar method to be sure! Coccidioides Malbranchea
  42. 42. Barrel shaped alternating arthroconidia are produced in cultures grown at both 30 and 35 C. There is no yeast phase for C. immitis [posadasii] No yeast phase with Coccidioides!
  43. 43. Coccidioides Histopathology  Thick walled spherules (10 – 80 uM) with endospores are seen in tissue. This is the second form of Cocci. No yeast cells are produced in tissue for this fungus.  Spherules are at all stages of development- fragmented spherules to well formed with endospores  Granulomatous inflammation with caseation is usually observed
  44. 44. Development of Cocci spherules from the inhalation of Arthroconidia from nature
  45. 45. Rhinosporidium seeberi forms spherules but much larger than the Cocci spherules - they are usually > 80 uM in size. Also R. seeberi almost always cause oral or nasal mass lesions, unlike Cocci. Oral or nasal mass lesions of Rhinosporidium seeberi Coccidioides spherules Cocci is not the only spherule forming organism!
  46. 46. Paracoccidioides brasiliensis  South American Blastomycosis – endemic area Brazil, Venezuela, Columbia  Inhale infectious particle from soil  >95% of infections in males, possibly due to estrogen inhibition of mycelial to yeast transformation  Disease presentation: 1. Pneumonia 2. Disseminated infection 3. Extrapulmonary lesions on the face and oral mucosa
  47. 47. Paracoccidioides  Cultures are usually not used in diagnosis due to slow growth and nonspecific sporulation  Culture @ 37˚C  Slow growing yeast  Large yeast (10 – 30uM) with multiple daughter buds (2 – 10 uM) in size  Unique yeast cell known as the Mariner’s wheel or Pilot’s wheel yeast
  48. 48. Mariner’s wheel yeast of Paracoccidioides brasiliensis If more than 2 buds off mother cell – High likelihood it is Paracoccidioides
  49. 49. Sporotrichosis  Sporothrix schenckii  Cutaneous inoculation of fungus from penetrating injury with a spore or thorn (rose bush)  Initial skin lesion w/wo ulceration  Lymph-cutaneous spread – bone – systemic  Pulmonary and CNS infections are rare but reported
  50. 50. Starts as one ulcerative lesion and then chains Up the lymphatics – can involve lymph nodes and bone
  51. 51. Sporothrix schenckii  Dimorphic fungus  MOLD PHASE  30*˚C growth in 3 -5 days  Turns brown to black over time  Septate hyphae with conidia in daisy wheel pattern  YEAST PHASE  At 37˚C small oval yeast cells, elongated 2 – 5 µM, described as cigar bodies
  52. 52. Sporothrix schenckii  Histology –  Pyogenic – to – granulomatous inflammation  Hard to find yeast in human tissue  Asteroid body known as Splendore-Hoeppli phenomenon can be seen – also seen in: Zygomycetes (mucorales) Aspergillus Blastomycosis Candida
  53. 53. Daisy like spore arrangement Sporothrix schenckii
  54. 54. Green colony with red diffusable pigment Uncommon dimorphic fungus The only species of Penicillium that is dimorphic Causes skin lesions in tropics and Pneumonia in immune suppressed Penicillium marneffei
  55. 55. Penicillium marneffei yeast like cells in tissue
  56. 56. Subcutaneous Fungal Infections Very unique structures in tissue!
  57. 57. Subcutaneous Fungal Infections Most common will be described  Mycetoma [2 types]  Actinomycotic – caused by higher bacteria  Eumycotic – caused by dark pigmented molds  Chromomycosis [Chromoblastomycosis]  Phaeohyphomycosis  Sporotrichosis
  58. 58. Mycetoma  First observed in India and known as Madura Foot or Maduromycosis  Found in the hot temperate parts of the world  Three criteria describe Mycetoma: 1. Lesions lead to swollen extremities 2. Draining sinuses 3. Sulfur granules observed in tissue and found in the weeping drainage  Fungus grows on organic debris in soil  Implanted into subcutaneous tissue from trauma
  59. 59. Swollen extremity and draining sinus with sulfur granules Sulfur Granule Mycetoma
  60. 60. Mycetoma  There are two types of Mycetoma: 1. Actinomycotic mycetoma – caused by higher bacteria species 2. Eumycotic mycetoma – caused by the black molds  Actinomycotic Mycetoma  98% of cases of Mycetoma  Nocardia species most common cause  Sulfur granules formed in tissue and the granules vary in color and contain a matrix of the filamentous bacteria
  61. 61. Gram stain as filamentous Gram positive bacilli – can be poorly staining and appear speckled. Nocardia are positive [red] on the Modified Kinyoun stain. Modified acid-fast stain [modified Kinyoun stain] Gram stain of sputum containing Nocardia Nocardia
  62. 62. Edge of granule has thin filamentous bacteria for both bacteria – Nocardia is modified acid fast [PAF] positive and is aerobic bacteria. Actinomyces is PAF negative and grows anaerobically. Beware! Sulfur granule caused by Actinomyces israelii looks identical. Actinomycotic sulfur granule - Nocardia
  63. 63. Requires 3 – 5 days to grow on agar media [Sabs, 5% Sheep’s blood agar Colony is dry and crumbly Musty smell Total of 85 species Nocardia asteroides is the most common species isolated from human infection Identification by HPLC or molecular methods Nocardia species cause mycetoma, and can also cause Pulmonary and Brain infections
  64. 64. Eumycotic mycetoma – subcutaneous infection caused by the black molds Numerous species of pigmented/black fungi found naturally in the soil can cause this type of infection Cause @ 2% of cases of mycetoma Traumatic implantation injects the mold into the subcutaneous tissue Most common species of black mold include: Cladophialophora (Cladosporium) carrionii Cladophialophora bantiana Phialophora verrucosa Fonsecaea pedrosoi Exophiala species Wangiella species
  65. 65. Eumycotic sulfur granule – the granule is full of a matrix of thick fungal hyphae
  66. 66. Chromomycosis/Chromoblastomycosis  Three characteristics describe Chromomycosis  Wart like lesions in subcutaneous tissue  Sclerotic bodies observed in tissue  Growth of dark/pigmented fungi  Black mold naturally found in the soil cause infection through abrasion/ implantation  Black molds that can cause Chromomycosis:  Cladophialophora [Cladosporium] carrionii  C. bantiana  Phialophora verrucosa  Fonsecaea pedrosoi  Exophiala species  Wangiella species
  67. 67. Chromomycosis/Chromoblastomycosis Wart like/Verrucous lesions In subcutaneous tissue Sclerotic Body/Medlar Body/ Copper Penny is the unique structure found in tissue
  68. 68. Prototheca wickerhamii – the cause of Protothecosis  Algae without chlorophyll  Causes skin lesions & nodules  Most common in patients with suppressed immune system  Compare morula of Protothecosis to sclerotic body of Chromomycoses
  69. 69. Phaeohyphomycosis This infection is caused by traumatic implantation of dark fungi into subcutaneous tissue  Variety of infections but nodules/lesions most common with/without dissemination  Dark hyphae only observed in tissue
  70. 70. Black molds/Dark molds also known as Dematiaceous fungi Black colored colonies and the reverse [back of colony] is also black Naturally brown hyphae and spores One of the major causes of mold growth due to water damage!
  71. 71. Black Molds – Dematiaceous fungi  Black colonies  Brown hyphae and spores  Numerous species  Difficult to identify  All have one of four types of sporulation  Rhinocladiella-like  Cladosporium-like  Phialophora-like  Acrotheca-like
  72. 72. Rhinocladiella type sporulation
  73. 73. Phialophora type sporulation
  74. 74. Cladophialophora [Cladosporium type sporulation]
  75. 75. Acrotheca type sporulation
  76. 76. Exophiala species Black Molds that can cause Mycetoma/Chromomycosis/Phaeohyphomycosis These are difficult to identify but viewing is necessary! Cladophialophora bantiana
  77. 77. Wangiella dermatitidis Phialophora verrucosa
  78. 78. Alternaria Other black molds of importance:
  79. 79. Bipolaris australiensis Very invasive fungal infection: Skin, nasal sinuses, bone brain
  80. 80. Curvularia lunata Center cell is the largest
  81. 81. Exserohilum rostrum  Associated with compounded pharmaceutical [steroid] products contaminated with dust/dirt  Used for infections into lumbar spine and knee joints for pain management  Meningitis  Spinal abscess  Synovial infections
  82. 82. Scedosporium apiospermum/ Pseudallescheria boydii Cat fur-like colony
  83. 83. Important Yeast causing human infection Candida species Cryptococcus neoformans & Cryptococcus gattii
  84. 84. Cutaneous and Superficial Mycoses  Candida species (@ 10 found in humans)  Opportunistic pathogen involving skin or mucous membranes from excessive exposure to moisture, antibiotics, or immune suppression  Yeast is from endogenous source – found as normal flora in the GI and GU tracts and skin  Variety of infections including: Thrush, vaginitis, skin lesions, nail, diaper rash, to more serious infections like fungemia and endoarditis.
  85. 85. Candida species  Candida albicans – most common species causing @ 60% of human yeast infections  Candida glabrata, C. krusei, and C. tropicalis are becoming more common in infection  These 3 species are more likely to be resistant to Fluconazole  Candida parapsilosis has emerged as a pathogen of children and IV lines
  86. 86. Candida species  Grow in 24 – 48 hours  SABS, IMA, BAP  Bacteria-like colony – pasty white  Budding yeast – oval @ 7-8 um in size form pseudohyphae (look like sausage links)  Exception **Candida glabrata is @ 4 µM in size and does NOT form any pseudohyphae
  87. 87. Candida albicans Identification  Germ tube formation  Incubate small amount of yeast in serum for 3-4hr at 35 ˚C  Do not incubate >4 hr – this can lead to a false positive reaction with C. tropicalis  C. dubliniensis also positive (uncommon yeast isolate)  Chlamydospore formation  Growth on cornmeal agar >48 hrs  Rudimentary structures C. albicans chlamydospore C. glabrata only forms yeast No pseudohyphae
  88. 88. ChromAgar for the identification of Candida Chromogenic substrates Turn different colors with 4 different yeast species Yeast with pseudohyphae
  89. 89. Candida Histopathology  Pyogenic to granulomatous  Usually observe yeast cells, pseudohyphae and/or hyphae appearing structures  Candida glabrata = smaller yeast cells and no pseudohyphae GMS stain of Candida glabrataCandida species not glabrata
  90. 90. Cryptococcus neoformans  In nature forms a 2um non-encapsulated yeast cell. It is associated with bird droppings (esp. pigeon). C neoformans is enriched by the nitrogen in the droppings.  Yeast cells are inhaled – travels through the pulmonary system with hematogenous spread to brain and meninges  Has tropism to the meninges  Infects mostly compromised hosts - AIDS
  91. 91. Cryptococcus neoformans  Irregular sized (2 – 20uM) yeast cells  Polysaccharide capsule is virulence factor and it’s presence is used in diagnostic tests for C. neoformans  India ink exam of CSF is a negative staining method/capsule not stained,  Sensitive test for AIDS patients (90% sensitive)  Cryptococcal antigen test – capsular polysaccharide is detected in both CSF and serum,  Test for diagnosis and can also follow recovery with falling titer /more sensitive than India ink  Grows on mycologic agars but is sensitive to cycloheximide –  Mucoid colonies due to capsule po;ysaccharide formation  Urease enzyme + Inositol assimilation +  Brown colonies produced on bird seed agar
  92. 92. Cryptococcus gattii – a closely related relative of C. neoformans  Isolated from forested area of the Pacific Northwest (British Columbia, Washington, and Oregon)  Infection of normal and immune suppressed hosts  Mostly Pulmonary disease [Cryptococcoma] but can develop meningitis  Culture and staining identical to C. neoformans except for L Canavanine glycine bromthymol blue medium – C
  93. 93. Positive India Ink Urea medium demonstrating urease enzyme activity of Cryptococcus Observe Budding cells Variability in size Positive Mucoid colonies of C. neoformans and C. gattii
  94. 94. C. neoformans/C. gattii forms brown colonies on Birdseed agar
  95. 95. Mucicarmine stain Stains the capsular polysaccharide of capsule Pneumocystis jeroveci could be confused with C. neoformans – Careful! Central nuclear staining C. neoformans/ C. gattii
  96. 96. Cutaneous and Superficial Mycoses Malassezia furfur Dermatophytes
  97. 97. Malassezia furfur  Pityriasis versicolor  Most superficial of the dermatomycoses  Found as normal flora on the skin,  More common on oily skin or high use of skin oils  Diseases:  Skin: macules, papules, patches, plaques on chest back and shoulders with either hypo or hyper pigmentation – does not invade into deeper tissues  Fungemia in neonates caused by skin flora tunneling in the IV lipid feeding lines
  98. 98. Malassezia furfur  Lipophilic yeast – oil required for growth  Media used for culture must contain oil or have oil overlay  Small budding yeast 2 – 4 µM with collarette Spaghetti and meatballs
  99. 99. Size range for Yeast  Candida glabrata/Histoplasma capsulatum  2 – 4 um  Candida species  8 – 10 um plus pseudohyphae  Cryptococcus neoformans/gattii  2 – 20 um  Blastomyces dermatitidis  8-15 um
  100. 100. Dermatophytes – Ringworm infections  Hair, skin and nail infections  3 genera of fungi  Microsporum species (many)  Epidermophyton floccosum  Trichophyton species (many)  Disease described by area of the body infected: tinea capitis (head), t. pedis (foot)  Usually a clinical diagnosis not requiring culture  Can do a KOH prep or Calcofluor white prep to visualize fungal hyphae
  101. 101. Positive KOH prep Showing thin septate fungal hyphae Calcofluor white stain with fluorescence – thin fungal hyphae
  102. 102. Microsporum canis Main cause of ringworm from dog and cat White colony/ yellow on backside of colony Tuberculate macroconidia [spiny projections] Few if any microconidia
  103. 103. Microsporum gypseum infection from exposure to contaminated soil
  104. 104. Trichophyton Rubrum White colony with red diffusable pigment Pencil shaped Macroconidia Many micro- Conidia Infection from fomites Red diffusible pigment
  105. 105. Trichophyton tonsurans Ballooning Microconidia Primary cause of epidemic ringworm in children
  106. 106. Epidermophyton floccosum Beaver tail large spores without microconidia Khaki green colony
  107. 107. Opportunistic Fungal Pathogens Infections in the immune suppressed host or special circumstances Hyaline molds Black molds
  108. 108. Opportunistic Fungi - hyaline  Hyaline – no color to the hyphae  Regular septations in the hyphae  Branching – angle can be helpful in identification  Usually grow in 3 – 5 days at 30˚C  ??? of species thousands– taxonomy changing daily
  109. 109. Aspergillus species  Hyaline with septations  Numerous round conidia  In tissue - Branching at 45 degree angle  Primarily pulmonary infection in immune suppressed  Invade vessels, cause thrombosis & infarctions
  110. 110. Aspergillus Structure
  111. 111. Aspergillus species Four species most common in human infections: 1. Aspergillus fumigatus 2. Aspergillus flavus 3. Aspergillus niger 4. Aspergillus tereus – unique and important – only Aspergillus species resistant to Amphotericin B Aspergillus Galactomannan Enzyme immunoassay – detects circulating Aspergillus antigen in the blood and/or bronchial lavage fluid Problems with low sensitivity and specificity False positive reaction in patients on therapy with Piperacillin/Tazobactam
  112. 112. Aspergillus fumigatus Blue/Green colony Phialids with spores are Directed upward
  113. 113. Aspergillus flavus Green colony Orange colored spores that surround the vesecle
  114. 114. Aspergillus niger Black colony Black spores surround the vesicle
  115. 115. Aspergillus terreus Sandy colored colony Aleurioconidia Resistance to Amphotericin B
  116. 116. Aspergillus – fruiting head and dichotomous branching septate hyphae with branching at 45* angle Can appear much like that of Pseudallescheria boydii! P. boydii hyphae is a bit thinner. The appearance in culture can differentiate the two fungi.
  117. 117. Fusarium species – Common in nature/plants disease related to immune status of host Infections reported: Disseminated in bone marrow transplants Corneal infections in contact lens wearers
  118. 118. Scopulariopsis species –found in soil and plants Infections: Nail, skin, sinusitis, pulmonary and disseminated
  119. 119. Paecilomyces species Isolated from soil and food Opportunistic pathogen in the immune suppressed
  120. 120. Penicillium species – most common mold in the environment, bread mold, uncommon cause of human disease
  121. 121. Mucormycosis/Zygomycosis Hyaline Broad non-septate hyphae
  122. 122. Mucormycosis/Zygomycosis  Infections in diabetics, the elevated glucose enriches fungal growth – classic infection is rhinocerebral mucormycosis  Sinus and pulmonary infection in the immune suppressed host  Broad, hyaline, aseptate hyphae produced  Cultures grow in 24 hrs, coarse aerial hyphae  Can be difficult to culture – tube like hyphae killed during manipulation and plating  Should not grind tissue  Mince tissue and place on agar
  123. 123. Zygomycete – coarse, aerial hyphae after 24 hours on SABS agar at 30˚C
  124. 124. Rhizopus Absidia Distant rhizoids Mucor No rhizoids Rhizoids
  125. 125. 90˚ angle branching, aseptate, ribbon like Invades vessels and can cause infarcts and thrombi Zygomycetes (Mucorales)

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