MYCOLOGY

Margie Morgan, PhD D(ABMM)

Just the basics – Updated 2017

Mycology

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 (Cryptococcus)

Starting point

Molds are:• Produce filaments or hyphae• Produce conidia [spores]• Colonies on solid agar are downy, fluffy, cottony• Most mold colonies produce pigment which aid in

identification

hyphaespores

Specimen collection and Transport• Fungi are very hardy organisms • No requirement for special transport media • Sterile containers required to prevent bacterial

contamination• Numerous anatomic 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

Fungal Culture Media

• Sabouraud’s glucose agar (SABS)All purpose Fungal media – No antibiotics Best used for subculture of fungi for identification workupContains 2% glucose, pH @7.0

• Inhibitory mold agar (IMA) Selective and enriched agar with chloramphenicol and gentamicin to

inhibit bacteria Good for the primary recovery of pathogenic fungi

Saprophytic fungi and dermatophytes somewhat inhibited

Fungal Culture Media• Mycosel/Mycobiotic agar

• Selective SABS agar with chloramphenicol and cycloheximide • Used for selective culture of dermatophytes – fungi that cause skin,

hair and nail infections

• Brain heart infusion agar • Primary recovery of all fungi• Can inhibit bacterial growth by adding chloramphenicol and

cycloheximide• Can add blood to agar to nurture systemic fungi

• Fungal cultures are incubated for 4 weeks at 30˚C • Lower temperature than bacterial culture incubation [35˚C]• If agar plates are used for fungal cultures the plates must be sealed

with air permeable tape to prevent spore dissemination from plates

What effect does Cycloheximide have when added to media?• Prevents rapidly growing environmental molds from overgrowing pathogenic fungal species• 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

• Inoculate fungal media with specimen

• 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 Manual and automated biochemical reactions capable of identifying most but not all yeast species.

Newer methods (1) Mass spectrometry – MALDI-TOF and (2) 16 sRNA sequencing are more precise methods with the expanding taxonomy of yeast species.

Processing of Fungal Cultures

Lactophenol cotton blue [LCB] adhesive tape preparation is 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 LCBon a microscope slide.

Newer (better methods) for unusual molds:MALDI-TOF16sRNA sequencing

Mold Identification methods

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

Direct Exams used to identify fungi directly from patient specimens

• Gram stain – Most specimen types can be Gram stained. Reliable detection of yeast

• KOH preparation – Skin, Hair or Nails examined for both yeast and/or hyphae

• Calcofluor white stain – Most specimen types can be stained and examined for yeast and/or hyphae

• India ink – Primarily used on CSF for the detection of Cryptococcus neoformans and C. gattii

• Yeast cells stain blue [Gram positive]. • Examine stain for budding cells to confirm that it is a

yeast cell and not an artifact. • You can also detect pseudo-hyphae on Gram stain. • Mold can be difficult to detect on a Gram stain.

pseudohyphae

moldpseudohyphae

Gram Stain

X100 oil immersion

• Detect yeast and/or pseudo-hyphae in skin, hair and nail specimens using 40X light microscopy.

• KOH dissolves keratin found in cellular material and freeing fungal hyphae and yeast cells.• KOH exams can be difficult to interpret!

KOH – potassium hydroxide prep

• Yeast, pseudo-hyphae, and mycelial fungi will bind with the Calcofluor white stain and fluoresce.

• Read using a fluorescence microscope (40X)• More Sensitive and Specific than KOH preparation.

Calcofluor white stain

One drop of black ink is placed into one drop of CSFOn microscope slide - examined using light microscope (40X)

It is a “negative” stain – which stains the background not the yeast cell.The clearing around the yeast is due to the polysaccharide capsule of Cryptococcus neoformans and C. gattii.

India Ink

Grocott’s Methenamine Silver Stain [GMS] – yeast and hyphae stain grey to black. Will stain living and dead fungi

• Observe the width of the hyphae, presence of septations and angle of branching.

• Observe the size and budding pattern of yeast.• Observe on later slides how these oservations can assist

in identification.

Examination of fungi in fixed tissue

Positive staining hyphae will appear magenta – will also stain structures containing a high proportion of carbohydrate macromolecules (glycogen, glycoprotein, proteoglycans) Stains living fungi.

Periodic Acid Schiff [PAS]

Mucicarmine stains the polysaccharide capsule pink for Cryptococcus neoformans and C. gatti. Also stains mucin in fixed tissue.

Mucicarmine [Mucin] stain

Great for cellularity, not a specific stain for fungi.

Hematoxylin and Eosin Stain

DIMORPHIC FUNGIImportant systemic pathogens with some unique characteristics

What does Dimorphic mean?

• Depending on temperature and conditions of environment one fungi demonstrates two forms• Mycelial form - Hyphae and conidia (spores)

• Free living form in nature and laboratory temperature <=30˚C• Yeast or yeast like form

• Parasitic phase found in human tissue or in the lab >= 35˚C

Histoplasma capsulatum – moldIncubated at 30˚C

Histoplasma capsulatum – yeastfrom tissue and 35˚C culture

Dimorphic Fungi capable of causing systemic infection – most common

• Histoplasma capsulatum• Blastomyces dermatitidis• Coccidioides immitis• Paracoccidioides brasiliensis• Sporothrix schenckii• Penicillium marneffei (Talaromyces marneffei)

Histoplasma capsulatum• World wide distribution / In USA considered endemic in Ohio, Missouri, and Mississippi River valleys

• Environmental source: Bat guano (Spelunker = cave explorers) and bird droppings

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 a unique & common site of dissemination

Histoplasma capsulatum - YeastIntracellular within macrophagesSmall 2 – 4 um, regular in size, and oval to round.

Do not have a capsule, it is a staining artifact.

H & E stain GMS

H & E PAS

Gram Wright’s

Histoplasma will stain with a variety of stains

Histoplasma rapid diagnosis

• Antigen detection in urine• Quantitative Enzyme immunoassay• Random urine specimen• Most sensitive for disseminated infection and

chronic pulmonary disease• Antigen is detectable in >=85% of these infections• Good for diagnosis of immune suppressed patients

that do not produce a detectable antibody response• Antibody tests are available but have been replaced

mostly by Antigen detection

Histoplasma capsulatum

• Culture incubated at 30˚C• Requires 2 – 8 weeks to grow• Colony - white to brown and cottony • Microscopic appearance – tuberculated macroconidia that

are large and round (8 – 16 µM) and small microconidia (2 - 4µM) • Microconidia are the infectious particle growing in

nature and capable of penetrating deep into the lung• DNA probe necessary to confirm identification • Sepedonium species looks somewhat like Histoplasma usually a contaminate. It is not dimorphic.

Histoplasm capsulatum

Histoplasma capsulatum in tissue

• Granulomas are usually produced – caseating or non-caseating• Infection usually begins by breathing in the

microconidia and infecting 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

Leishmania speciesNote small round kinetoplast next to nucleus

Toxoplasma

Histoplasma capsulatum

Beware of look alike organisms in tissue specimens!!

The mold phase of H capsulatum var. duboisii is identical to H. capsulatum. The difference is the size of the yeast cells: Note yeast cell is 8 – 10 uM which is 2X the size of H. capsulatum yeast cell.

H. capsulatum var duboisii disease is found in Central Africa – differs from H. capsulatum with primaryinfection in skin and bone

Unusual variant of H. capsulatum

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 primarily to the skin and bone

• Well demarcated skin lesions are often seen in disseminated Blastomycosis.

• Skin lesions are full of yeast cells.

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

BlastomycesChrysosporium

• Slow growing yeast colony (4 weeks to form a colony)

• Yeast cell is 8 – 20 um in size• Unique Broad Based Budding pattern and

the double contoured wall.

Blastomyces culture at 35° C

Broad based bud

Blastomyces dermatitidis histopathology• Mixed pyogenic and granulomatous inflammation is

observed in tissue • Broad based budding yeast cells

Coccidioides immitis

• Endemic in SW USA, Mexico, South America, in areas known as the Sonoran life zone/ warm and desert sands• Infection is from inhalation of fungal particles found in the sand

• New: Coccidioides posadasii is genetically related to C. immitis. These two species are located in different endemic regions, but produce the same disease process Coccidioidomycosis

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 carries a high

fatality rate.• Higher incidence of dissemination occurs in patients with:

• defects in cell mediated immunity (HIV), • darker skinned ethnic groups, • pregnancy

Coccidioides immitis [posadasii]

• Culture at 30˚C• Requires 2 – 3 days to grow, colony starts waxy and

becomes wooly in around 7 – 10 days• Under the microscope (40X) one observes foci with

septate hyphae with thick walled barrel shaped arthroconidia - clear spaces in-between. The clear spaces are dead arthroconidia.

• Arthroconidia are the infectious particle in nature• Culture is very infectious to laboratory personnel

Coccidioides 30°C Culture • Malbranchea species mimics C. immitis/posadasii under

the microscope• Because of look-a-like fungi, one needs to confirm

identification of Coccidioides immitis/posadasii with DNA probe (molecular) or similar method to be accurate

Coccidioides Malbranchea

Culture at 30*C and 35*C produce hyphae with barrel shaped alternating arthroconidia.There is no yeast phase for C. immitis [posadasii]

No yeast phase with Coccidioides!

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.

• Spherules are in all stages of development- fragmented spherules to well formed with endospores

• Granulomatous inflammation with caseation is usually observed

Development of Cocci spherules from the inhalation of Arthroconidia from nature

Rhinosporidium seeberi (aquatic parasite) forms spherules but much larger than those of Cocci. Rhinosporidium spherules are usually > 80 uM in size. Also R. seeberi usually cause only oral or nasal mass lesions

Oral or nasal mass lesionsof Rhinosporidium seeberi

Coccidioidesspherules

Coccidioides is not the only spherule forming organism!

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. Pneumonia2. Disseminated infection to 2 or more organs3. Extrapulmonary lesions on the face and oral mucosa

Paracoccidioides

• Cultures at 30°C for mycelial phase are usually not performed - 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 budding yeast cell known as the Mariner’s wheel or

Pilot’s wheel yeast

Tissue Exam ofParacoccidioides brasiliensis

• If more than 2 buds off mother yeast cell – High likelihood it is Paracoccidioides

• Granulomatous inflammation with Giant Cells formed in tissue

Sporothrix schenckii

• Sporotrichosis• Cutaneous inoculation of fungus from penetrating injury with a

spore or thorn (rose bush)• Begins as single skin lesion w/wo ulceration• Lesion can progress to many lesions and lymphocutaneous

spread, possibly to bone and other organs• Pulmonary and CNS infections are rare but reported

Sporothrix schenckii

• 30*˚C culture grows in 3 -5 days as mold• Beige colony/ turns brown to black over time• Septate hyphae with conidia in daisy

wheel pattern

• 37˚C culture small oval yeast cells, elongated 2 – 5 µM, described as cigar bodies

Sporothrix schenckii Histology

• Pyogenic to granulomatous inflammation• Hard to find yeast cells in human tissue• More commonly an Asteroid body known as Splendore-

Hoeppli phenomenon can be seen, but this is not specific to Sporothrix, also seen in:• Zygomycetes (Mucorales)• Aspergillus• Blastomycosis• Candida

Daisy like spore arrangement

Sporothrix schenckiiCigar shaped yeast cell

Splendore-hoeppli

Green colony with red diffusable pigment grows in 2 – 3 days at 30 C°

Uncommon dimorphic fungus – yeast like cellsproduced in tissueOnly dimorphic species of Penicillium Diseases: skin lesions in tropics and Pneumonia or systemic disease in advancedHIV

Penicillium (Talaromyces) marneffei

Penicillium (Talaromyces) marneffei

SUBCUTANEOUS FUNGAL INFECTIONSVery unique structures in tissue!

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

Mycetoma• First observed in India and known as Madura Foot or Maduromycosis • Found in the hot temperate parts of

the world

• Three criteria define Mycetoma:-Traumatic lesion leads to swollen extremity

-Draining sinuses evolve -Sulfur granules observed in tissue and found in the

weeping drainage from sinus tract

• Fungus grows on organic debris in soil• Implanted into subcutaneous tissue from trauma

Swollen extremity with draining sinuses that drain sulfur granules

Sulfur Granule

Mycetoma

Actinomycotic Mycetoma There are two types of Mycetoma:

1. Actinomycotic – caused by higher bacteria species2. Eumycotic – caused by black molds

• Actinomycotic Mycetoma• 98% of cases• Nocardia species most common agent• Sulfur granules formed in tissue. The granules vary in color

based on Nocardia species and contain a matrix of the filamentous bacteria

Edge of granule has thin filamentous bacteria: How can you tell if it is Nocardia?Nocardia is modified acid fast [PAF] positive and is an aerobic bacteria. Actinomyces is PAF negative and grows anaerobically.

Beware! Sulfur granule caused by Actinomyces israelii looks identical to one formed by Nocardia.

Actinomycotic sulfur granule

Gram stain = filamentous Gram positive bacilli can be poorly staining and appear speckled.

Positive [red] on the Modified Kinyoun acid fast stain.

Modified acid-fast stain[modified Kinyoun stain]

Gram stain of sputum

Nocardia

Grows in 3 – 5 days on agar media [Sabs, 5% Sheep’s blood agar]

Colony is dry/crumbly - Musty smell

Total of 85 species:Nocardia asteroides most common

Therapy: Trimethoprim/Sulfa

Identification by Maldi-TOF or 16s rRNA gene sequencing

Nocardia species – Mycetoma, Pulmonary and Brain infections

ThermoactinomycetesGram positive filamentous rods – related to Nocardia

• Thermoactinomyces and Saccharopolyspora • Flourish in areas of high humidity and high temperatures (40-60*C)• Grow in agricultural environments – like stacks of hay

• Farmer’s lung – hypersensitivity pneumonitis or extrinsic allergic alveolitis, occupational disease• Immunologically medicated inflammatory disease of the lung• Inhalation exposure to the thermophilic actinomycetes

• Diagnosis• Detailed environmental history• Serology testing

Eumycotic mycetoma

Caused by pigmented/black fungi due to melanin -Numerous species found in soil -2% of cases of mycetoma -Traumatic implantation implants the mold into the subcutaneous tissue-Most common black molds include: Cladophialophora (Cladosporium) carrionii

Cladophialophora bantianaPhialophora verrucosaFonsecaea pedrosoiExophiala speciesWangiella species

Eumycotic sulfur granule – the granule is composed of a matrix of thick fungal hyphae

Chromomycosis/Chromoblastomycosis• Three characteristics:

• 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

Chromomycosis/Chromoblastomycosis

Wart like/Verrucous lesionsIn subcutaneous tissue

Sclerotic Body/Medlar Body/Copper Penny is the uniquestructure found in tissue

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

Phaeohyphomycosis Traumatic implantation of dark fungi into subcutaneous

tissue• Variety of infections but nodules/lesions most common

with/without dissemination• Dark hyphae observed in tissue

Black mold also known as Dematiaceous mold

-Black colored colonies and the reverse [underside of colony] is also black-Natural-colored brown hyphae and spores (melanin)-One of the common molds that grow due to water damage!

Black Molds – Dematiaceous fungi• Black colonies• Brown hyphae and spores (melanin)• Numerous species • Difficult to identify

• All have one of four types of sporulation• Rhinocladiella-like• Cladosporium-like• Phialophora-like• Acrotheca-like

Rhinocladiella Cladosporium

Phialophora Acrotheca

Exophiala species

Black Molds cause Mycetoma/Chromomycosis/Phaeohyphomycosis plus sinusitis, pulmonary, brain, and disseminated infections.These fungi are difficult to identify using only morphology – require sequencing/MALDI for speciation

Cladophialophora bantiana -Brain infections

Wangiella dermatitidis

Phialophora verrucosa

Alternaria species– Opportunistic fungus - sinusitis in normal host, dissemination in immune suppressed.

Other black molds of importance:

Bipolaris australiensis

Disseminated fungal infection in both normal and immune suppressed hosts:Skin, nasalsinuses, bonebrain

Curvularia lunata

Center cell is the largest

Opportunistic fungal pathogenReported in Bone Marrow Transplantation and Aplastic anemiaCan cause unusual erythematous skin lesions

Exserohilum rostrum• Outbreak associated with compounded pharmaceutical

[steroid] products contaminated with dust/dirt• Used for infections into lumbar spine and knee joints for

pain management and led to infections:• Meningitis• Spinal abscess• Synovial infections

Scedosporium apiospermum/Pseudallescheria boydii Cat fur-like colony/growth in 3 – 5 days, lollipop like sporesOpportunistic pulmonary pathogen. Can be difficult to distinguish from Aspergillus in fixed tissue stains.

IMPORTANT YEAST CAUSING HUMAN INFECTION

Candida speciesCryptococcus neoformans & Cryptococcus gattii

Candida species

• Candida species (@ 10 infect humans)• Yeast are normal flora in GI, GU and skin• Opportunistic pathogen involving skin or mucous

membranes from excessive exposure to moisture, antibiotics, or immune suppression• Thrush, vaginitis, skin lesions, nail, diaper rash

• Also can cause more serious infections such as fungemia, endocarditis, and systemic tissue infection.

Candida species

• Candida albicans – most common species causing @ 60% of human yeast infections

• Candida glabrata, C. krusei, and C. tropicalis are causing higher % of infections than in the past • Fluconazole is a very useful oral and IV anti-fungal • These 3 species are more likely to be resistant to Fluconazole

• New species Candida auris – outbreaks in hospitals, intrinsically resistant to most anti-fungals

• Candida parapsilosis has emerged as a pathogen of children and common in IV line infections (forms biofilms on plastics) • C.parapsilosis is susceptible to fluconazole.

Candida species• Grow in 24 – 48 hours 30-35*C

• Many agars: SABS, IMA, Blood agar• Bacteria-like colony – pasty white• Budding yeast – oval @ 7-8 um in size**• Form pseudohyphae not true hyphae (Yeast do not

detach and form elongated hyhae) • Identify using biochemical rxn, MALDI, or Sequencing

• Exception: **Candida glabrata is only Candida species @ 4 µM in size and does NOT form pseudohyphae – this species breaks all the Candida rules

pseudohyphae

Candida albicansIdentification

• Germ tube formation• Incubate small amount of yeast in serum for 3-4hrs at 35 ˚C• If incubate >4 hrs – C. tropicalis will also form germ tubes• C. dubliniensis is also positive • Specific ID from biochemical reactions, MALDI-TOF or

Sequencing• Chlamydospore formation

• Growth on cornmeal agar at 48 hrs• Rudimentary structures unique to C. albicans

chlamydospore

ChromAgar for the identification of Candida using chromogenic substrates.The unique colors can identify 4 yeast species.

Yeast with pseudohyphae

Candida Histopathology• Pyogenic to granulomatous• Usually observe yeast cells (8 um), pseudohyphae and/or hyphae-

like structures• Candida glabrata = smaller yeast cells (4 um) and absence of

pseudohyphae

GMS stain of Candida glabrataCandida species not glabrata

Cryptococcus neoformans

• In nature forms a 2um non-encapsulated yeast cell. • Associated with bird droppings (esp. pigeon). C neoformans is enriched by the nitrogen in the

heaped droppings.• Non-encapsulated yeast cells are inhaled – travel

through the pulmonary system with hematogenous spread to brain and meninges

• Tropism to the meninges• Infects mostly compromised hosts – one of the major

AIDS defining infections

Cryptococcus gattii – a close relative of C. neoformans• Isolated from forested area of the Pacific Northwest

(British Columbia, Washington, Oregon and California) found in soil debris and tree species

• Infection of normal and immune suppressed hosts• Primarily a pulmonary disease [Cryptococcoma] but can

develop meningitis like C. neoformans• Culture, biochemical & staining identical to C. neoformans • Defining reactions –

• L Canavanine glycine bromthymol blue medium – C. gatti = blue C. neoformans = colorlessMALDI-TOF will also ID both species

Cryptococcus neoformansand C. gattii• Irregularly sized yeast cells (2 – 20 um)• Polysaccharide capsule is the primary virulence factor• Polysaccharide capsule is detected in diagnostic testing:

• India ink exam- CSF placed in one drop of black ink. It is a negative staining method – background is stained not the capsule• Sensitive test for AIDS patients (90% sensitive)

• Cryptococcal antigen test – Titer of capsular polysaccharide detected in either CSF or serum• More sensitive than India ink for all patient groups• Test for diagnosis or can follow recovery with falling titer

• Grows on mycologic agars / sensitive to cycloheximide –• Mucoid colonies due to capsule polysaccharide formation• Urease enzyme + Inositol assimilation +• Brown colonies produced on bird seed agar

Both C. Neoformans & C. gattii producebrown colonies on Birdseed agar

Positive India Ink

Urea medium demonstrating urease enzyme activity of Cryptococcus

Observe Budding cells

Variability in size

Positive

Mucoid colonies ofC. neoformans andC. gattii

Mucicarmin stains the capsular polysaccharide of Cryptococcus Pneumocystis jiroveci (yeast like

fungus) could be confused with C. neoformans – Careful! Central nuclear staining in pneumocystis

C. neoformans/ C. gattii – no nuclear staining

Cryptococcus vs. Pneumocystis

Pneumocystis froth on H & E

Trichosporon• T. asahii most common – 9 species• Commonly inhabit the soil. Colonize the skin/

gastrointestinal tract of humans.• Rectangular shaped yeast cells • Cause of superficial infection - white piedra,

a distal infection of the hair shaft, • Second most commonly reported cause of

disseminated yeast infections in humans• Rare, even among patients with impaired host defenses:

• Corticosteroid use, solid tumors, HIV/AIDS, and intravascular devices, including catheters and prosthetic heart valves

• Mortality 50 – 80%

CUTANEOUS AND SUPERFICIAL MYCOSESMalassezia furfurDermatophytes

Malassezia furfur

• Most superficial of the dermatomycoses • 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 – known as pityriasis versicolor

• Fungemia: caused by skin flora tunneling in with IV lipid feeding lines (parenteral nutrition) – in neonates or rarely adults

Malassezia furfur• Lipophilic yeast – oil required for growth

• Media for isolation must contain oil or us an oil overlay• Small budding yeast 2 – 4 µM with collarette (appears like

necklace at junction of mother and daughter yeast cell)• In tissue described as “Spaghetti and Meatballs”

Spaghetti and meatballs

Size range for Yeast• Candida glabrata/Histoplasma capsulatum/ M. furfur• 2 – 4 um

• Candida species• 8 – 10 um plus pseudohyphae

• Cryptococcus neoformans/gattii• 2 – 20 um

• Blastomyces dermatitidis• 8-15 um

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: For example: tinea capitis (head), t. pedis (foot)

• Usually a clinical diagnosis not requiring culture• KOH prep or Calcofluor white prep can be used to visualize fungal hyphae from skin scrapings

Positive KOH prepShowing thin septate fungal hyphae

Calcofluor white stain with fluorescence – thin fungal hyphae

Microsporum canisRingworm acquired from dog and catWhite colony/ yellow on backside of colonyTuberculate thick walled macroconidia [spiny projections] Few if any microconidia

Microsporum gypseum – skin infection from exposure to contaminated soil, sandy colored colony, large macroconidia are produced.

Trichophytonrubrum

White colony with red diffusable pigment

Pencil shapedmacroconidiamany micro-conidia

Skin infection from fomites

Red diffusible pigment

TrichophytonTonsurans

White colony grows in @ 5 daysNo macroconidiaBallooning microconidia

Primary cause ofepidemic scalpringworm inchildren

Epidermophyton floccosum

Beaver tail large macroconidia – no microconidiaKhaki green colored colony – most often cause infectionsin skin and nails.

Opportunistic Fungal Pathogens

Infections in the immune suppressed host or special circumstances

Hyaline molds

Opportunistic Fungi - hyaline

• Hyaline – no color to the hyphae• Regular septations (walls) in the hyphae • Branching of hyphae in stains of fixed tissue – angle of branching can be helpful in identification

• Usually grow in 3 – 5 days at 35 or 30˚C • ??? of species in the thousands– taxonomy changing daily

Aspergillus species• Hyphae - Hyaline with septations• Numerous round conidia produced• In tissue - Branching at 45 degree angle• Usually pulmonary infection but can disseminate• Invade vessels, cause thrombosis & infarctions

Septation

Aspergillus Structure

Aspergillus species

Four species most common in human infections:1. Aspergillus fumigatus 2. Aspergillus flavus3. Aspergillus niger4. Aspergillus terreus – unique and important – intrinsic

resistance to Amphotericin B

Aspergillus Galactomannan Enzyme immunoassay – detects circulating Aspergillus antigen in the blood and/or bronchial lavage fluid

Low sensitivity and specificity False positive reaction from therapy with Piperacillin/Tazobactam, infection with Histoplasma capsulatum

Aspergillus fumigatus

Blue/Green colony grows in 3 – 5 days

Phialids with colorless spores are directed upward

Aspergillus flavus

Green/yellow colony

Orange colored sporeson phialids that surround the vessicle

Aspergillus niger

Black colony – visible black heads

Black spores supported by phialids surround the vesicle

Aspergillus terreus Sandy colored colonyColorless conidia supported by phialids headed upward

Aleurioconidia produced

Intrinsic resistance to Amphotericin B

Aspergillus – fruiting head seldom seen in fixed tissue usually only dichotomous (continuous) branching septate hyphae which branch at a 45° angle

Can appear much like that of Pseudallescheria boydii(Scedosporium) Growth in culture can differentiate the two fungi.

Fusarium species – Common in nature/plantsDisease related to immune status of hostInfections reported: Disseminated in bone marrow transplantsCorneal infections in contact lens wearers

Scopulariopsis species –found in soil and plantsInfections: Nail, skin, sinusitis, pulmonary and disseminated

Penicillium species – one of the most common mold in the environment , cause of bread mold, uncommon cause of human disease

MUCORMYCOSIS/ ZYGOMYCOSISFungi in the Order Mucorales

HyalineBroad hyphae without septations

Mucormycosis/Zygomycosis• Rhinocerebral mucormycosis

• Infections in diabetics, the elevated glucose enriches fungal growth

• Sinus – orbit of eye - brain• Sinus and pulmonary infection in the immune

suppressed host• Broad, hyaline, aseptate hyphae produced• Cultures grow in 24 hrs, producing coarse aerial

hyphae• Mince infected tissue and place on agar

• Do not grind tissue – grinding will kill the aseptate hyphae

Order Mucorales – coarse, aerial hyphae after 24 hours on SABS agar at 30˚C

Rhizopus Absidia

Distant rhizoids

Mucor

No rhizoids

Rhizoids

Order Mucorales

90˚ angle branching, aseptate, ribbon like hyphae

Invades vessels and can cause infarcts and thrombi

Zygomycetes (Mucorales)