Mycology Flashcards
Introduction – The Fungi
Among the most abundant and widely
distributed of all organisms.
~ 1.5 million named and unnamed species,
2nd only to insects.
All people continuously exposed to large
numbers of fungi.
Only 12-15 fungi commonly cause serious
human diseases
– illustrates the effectiveness of our host
defenses.
Serious fungal infections have increased
markedly in frequency in recent decades
The Rise of the Fungi
7th cause of deaths - rising
Basic characteristics
Eukaryotic (membrane-bound nucleus, complex organelles) Energy and nutrition - parasitic or saprophytic. Reproduce by budding, fission or spore formation Cell walls (polysaccharide and glycoproteins). Principal cell wall sterol is ergosterol Can grow as yeast-like cells or as molds.
Traditional Classification
Zygomycetes (Rhizopus, Mucor, Absidia, Basidiobolus)
Ascomycetes (Histoplasma, Blastomyces, some Candida)
Basidiomycetes (Cryptococcus)
Deuteromycetes (most pathogenic Candida species, pathogenic
Aspergillus species, Coccidioides)
Morphologic Classification
yeast (Candida, Cryptococcus)
molds (Aspergillus, Zygomycetes, many others).
dimorphic fungi (Histoplasma, Blastomyces, Coccidioides, Paracoccidioides, Sporothrix)
Diagnosis of Fungal Diseases
Culture causative fungus.
Microscopic morphology
Demonstrate specific host immune response
Demonstrate fungal antigen(s)
Demonstrate fungal nucleic acid sequence(s)
Demonstrate distinctive fungal metabolite(s)
Culture
Culture – Yeast-like fungi (Candida spp. and related) grow on routine bacterial media – Filamentous fungi (molds) may grow on routine media should be cultured on mycologic media for optimum recovery special requirements (M. furfur)
Mycologic Media
Culture media for primary isolation): a
selective & non-selective agars, that can
include:
– Sabouraud’s glucose agar (SAB), non-selective
– Selective SAB, with chloramphenicol
– Selective SAB, with chloramphenicol plus
cycloheximide (Actidione)
– Blood Brain Heart Infusion (BBHI), non-selective
– BBHI with gentamicin (G) & chloramphenicol (C)
– Selective BBHI with G, C & Actidione
Usually incubated at 30oC
– For possible dimorphs sub at 37 to convert to yeast
CanWe Do Fungal Blood Cultures?
Yes!
90% of the time looking for yeast fungemia;
usually from a urinary source
– Conventional blood culture is perfectly adequate
– Some conventional systems are insensitive for Cryptococcus
Occasionally looking for fungemia with a mold or dimorph; Aspergillus, Fusarium,
Histoplasma.
– Special culture procedures
– Biphasic bottle
– Lysis-centrifugation (Isolator) system
Microscopy
Microscopy
– budding yeast or mycelia are often
evident on Gram stains
Histopatholological stains
GMS (Gomori/Grocott Methenamine Silver)
– Cell walls stain black, but so can collagen fibers, RBCs, etc.
Staining pattern plus morphology are indispensable.
PAS (Periodic Acid Schiff)
– Cell walls stain pink/red. Other carbohydrate material & small lipid droplets may also take up the stain.
Mucicarmine (Mayer’s or Southgate’s)
– Used to demonstrate the capsular material of C. neoformans, which stains pink/red. There are hypocapsular, or (rarely) acapsular isolates of C. neoformans
H&E or tissue Gram stains most fungi, although some stain faintly. Not always dependable!
Host antibody responses
More useful in endemic than opportunistic mycoses Cross-reactions between fungi very common Recombinant fungal protein antigens may be useful
Diagnostically-useful fungal antigens
Commonly Used – Cryptococcus neoformans galactoxylomannan –Histoplasma capsulatum surface antigens Used in specific/complex situations – Candida albicans enolase – Aspergillus fumigatus galactomannan.
Outline
Molds; Aspergillus, the zygomycetes,
and others
Yeasts; Candida and Cryptococcus
Dimorphic and endemic fungal infections
Aspergillosis: Microbiology
Pathogenic species
– A. fumigatus
– A. flavus
– A. niger
Common features
– narrow septate hyphae that branch at 30-
45 degrees in tissue (acute-angle branching)
– produce asexual spores in environment and in cultures, but not in mammalian tissues
Aspergillosis: Epidemiology/ecology
Ubiquitous environmental saprophytes (dust, soil, on plants, etc).
– Everyone is exposed to spores every day.
Disease when natural defenses break down.
– Neutropenia or phagocyte dysfunction (eg, CGD) is the key risk factor for invasive aspergillosis.
– T-cell immunity is less important.
– Environment influences incidence - with filtered air, increase with construction and demolition.
Aspergillosis Allergic Bronchopulmonary
Disease
hypersensitivity leads to allergic pneumonitis or allergic bronchopulmonary aspergillosis (ABPA). allergic aspergillosis – bronchospasm – fleeting pulmonary infiltrates – tends to be chronic or recurrent refractory asthma
Aspergillosis Pulmonary colonization
saprophytic colonization - usually no symptoms, but hemoptysis can result from local invasion saprophytic colonization of preexisting lung lesions > superficial invasion > hemoptysis
Invasive Aspergillosis
Invasive aspergillosis:
– infection via inhalation of airborne spores.
– spores survive and germinate (produce hyphae) if local phagocytes are absent or dysfunctional.
– hyphae invade locally and spread via blood vessels.
– dissemination to distant organs via bloodstream.
– vascular invasion and occlusion produces extensive tissue necrosis and infarction.
Pulmonary
Sinusitis
Other and disseminated disease
Aspergillosis Diagnosis
Diagnosis is difficult
Cultures
– blood negative, even with disseminated
disease
– sputum/respiratory - colonization vs invasion
Biopsy & microscopy - KOH of fresh tissue or histopathology
Antibody testing seldom useful
Immunologic detection of cell surface antigens
– Galactomannan test becoming more
available, often used for screening at-risk
patients
PCR of rDNA
sequences in blood and/or bronchial fluids
All limited in availability and data to support use
MORPHOLOGY
Aspergillus fumigatus
The most common pathogen A. fumigatus has a dark-green colony Flask-shaped vesicle with conidia swept away from stalk Single row of phialides (uniseriate)
Aspergillus flavus & niger
A. flavus – Biseriate – Yellow colony – Phialides surround vesicle A. niger – White base with densely black conidia – Dark, rough conidia Also potential pathogens
Mucormycosis (Zygomycosis) Microbiology
Pathogenic species -- Zygomycetes – Rhizopus sp. –Mucor sp. – Absidia sp. Common properties – broad, aseptate hyphae, branching at 90 degrees in tissue – produce sexual and asexual spores in the environment and in cultures, but not in mammalian tissues.
Mucormycosis Epidemiology/ecology
Ubiquitous environmental molds (decaying
organic matter, fruits, etc).
People are regularly exposed to spores of
these fungi.
Disease develops when natural defenses are deficient. Major risk factors include:
– diabetic ketoacidosis, other metabolic acidoses (eg, uremia)
– organ transplantation and abnormal CMI
– neutropenia
– burns
– iron chelation therapy
Mucormycosis Rhinocerebral
Rhinocerebral
– rapidly progressive necrotizing pansinusitis
– spreads into cranium via blood vessels if
untreated.
– can disseminate to distant sites via bloodstream
Mucormycosis Pulmonary and other
Pulmonary
– necrotizing pneumonitis with secondary blood vessel invasion, thrombosis & infarction
– Dissemination to distant organs is common
Cutaneous
– complication of extensive burns or other wounds
Gastrointestinal (rare)
– invasion of gastrointestinal mucosa in malnourished children in developing countries.
Mucormycosis - Diagnosis
Cultures often negative, even in
specimens containing visible fungal forms
Direct microscopy
(KOH preps or histology) is most important diagnostic modality.
No reliable serologic tests for antibodies or
antigens, no DNA tests at this time.
Zygomycetes in Culture
Wooly, rapidlygrowing mold
Large, distinctive sporangium
Mucormycosis - Treatment
Multiple treatment modalities are essential – correct underlying host defense abnormality – surgical debridement/removal of necrotic tissues is essential – amphotericin B, ? Newer azoles
Other Important Molds
Pseudallescheria boydii, Penicillium marneffei, Fusarium spp. can all
cause invasive disease in compromised hosts
A variety of fungi (the dermatophytes)
cause skin infections
Many rare/unusual syndromes caused by a
variety of organisms
Fusarium
Opportunistic pathogen, esp in
neutropenic patients
Common in fungal keratitis
Most commonly F. solani complex
Fusiform macroconidia; microconidia produced as well
Fluffy, white or colored colonies in culture
Dermatophyte infections
Taenia whatever… – Capitis; head and hair – Corporis, cruris, pedis; skin of body, groin, or feet Onychomycosis – nail infections Three major genera – Trichophyton – Epidermophyton – Microsporum
Trichophyton
Colonies
– Slow to moderately rapid growth
– Waxy, glabrous to cottony.
– Front, white to bright yellowish beige or red violet.
– Reverse pale, yellowish, brown, or reddish-brown
Microscopic
– Microconidia, macroconidia, and arthroconidia
Miroconidia numerous; onecelled and round or pyriform in shape.
Macroconidia are multicellular smooth-walled and cylindrical, clavate or cigar-shaped.
Produced in very few numbers or not at all.
Epidermophyton
Colonies
– Grow moderately rapidly (10d or so)
– Front: brownish yellow to olive gray or khaki
– Reverse: orange to brown with an occasional yellow border.
– Flat and grainy initially; then radially grooved and velvety.
Microconidia are typically absent.
Macroconidia (10-40 x 6-12 μm), thin walled, 3- to 5- celled, smooth, and clavate-shaped with rounded ends
Microsporum
Colonies
– Glabrous, downy, wooly or powdery.
– Growth variable
– Color varies depending on the species
Front: white to beige or yellow to cinnamon.
Reverse: yellow to red-brown.
Microconidia
– Unicellular, solitary, oval to clavate in shape, smooth, hyaline and thin-walled.
Macroconidia
– hyaline, echinulate to roughened, thin- to thickwalled, typically fusiform and multicellular, often with an annular frill.
The Dermatophytes Compared
Trichophyton differs from Microsporum and
Epidermophyton by having cylindrical, clavate to cigar-shaped, thin-walled
or thick-walled, smooth macroconidia.
Epidermophyton is differentiated from
Microsporum and Trichophyton by the
absence of microconidia.
Microsporum differs from Trichophyton and
Epidermophyton by having spindle-shaped
macroconidia with echinulate to rough walls
Candidiasis Microbiology
– Causative agents Candida albicans Candida tropicalis Candida parapsilosis Candida glabrata Candida krusei Others – Properties All yeast-like fungi that reproduce by budding All except C. glabrata also produce pseudohyphae and/or true hyphae.
Candidiasis Epidemiology/ecology
Most Candida species colonize normal humans – C. albicans, C. tropicalis and C. glabrata - GI and female GU tract – C. parapsilosis and others - skin Some also present in soil, plant matter. High carriage on skin of HCW Disease occurs when normal host defenses break down Skin and mucosal barriers Cell-mediated immunity Granulocytes Person-to-person transmission can occur, Frequency of serious Candida infections increased >10-fold from 1980-89
Candidiasis
Clinical and pathologic features –
Thrush and esophageal disease
Skin and mucosal surfaces involved when CMI is abnormal – ulcerations with surrounding inflammation – thrush – esophagitis – gastrointestinal ulcers Usually C. albicans HIV infection, inhaled steroids
Candidiasis
Clinical and pathologic features, other mucosal disease
Vaginitis – a common disease
– Edema, pruritis, thick discharge (sometimes thin or scanty)
– Extension to vulva/perineum common
– Diabetes, antibiotic therapy, pregnancy
– ?Oral contraceptives
– 75% lifetime incidence, some with no identifiable risk
– Local or systemic azole therapies
– KOH prep to diagnose – office procedure
Diaper rash
Skin & nail infections
Candidiasis
Clinical and pathologic features –
Organ system and nosocomial
Urinary tract infections – Most common with Foley catheters Intravenous catheterrelated infections Pneumonia quite rare, but a frequent colonizer
Candidiasis
Clinical and pathologic features – disseminated disease
Hematogenous infections – no characteristic syndrome, depends on organ or organs involved and host’s ability to respond Some species tend to attack particular hosts (eg, C. glabrata in diabetics, C. tropicalis in neutropenic patients) Blood cultures may be negative; repeated cultures and empiric therapy in at-risk patients
Candidiasis Diagnosis
Culture (blood cultures may be neg in patients with disseminated infection)
Direct microscopy (yeast and/or
pseudohyphae demonstrable by KOH
preparation, Gram stain, histology)
Antibody responses seldom useful.
Immunologic detection of distinctive antigens, PCR rarely used.
Candidiasis
The germ tube test
– Grow yeast in serum 2h
– Separates C. albicans and other Candida
species
C. albicans is generally fluconazole susceptible
Other Candida include species with a higher frequency of resistance.
Candida Species – ID
Biochemical tests
– API, others
Correlate to morphology on cornmeal
agar
Candida albicans
Budding spherical to ovoid blastoconidia
Candida glabrata
No pseudohyphae – also seen with
Cryptococcus, but capsule usually evident as
space surrounding cell for Crypto.
Candida parapsilosis
Short, curving pseudohyphae with round to
oval blastoconidia
Candida tropicalis
Multibranched pseudohyphae, blastoconidia borne singly or in chains from along pseudohyphae
Candida lusitaniae
Short, curved pseudohyphae with
blastoconidia at or between septae
Candida krusei
Branching pseudohyphae with elongated
blastoconidia
Candidiasis Treatment
azoles (fluconazole, ketoconazole,
itraconazole),
– Resistance in C. glabrata and C. krusei
Caspofungin & other echinocandins
amphotericin B
Some Candida species resistant to specific
antifungals (e.g. C. lusitaniae & Ampho B)
– Susceptibility testing increasingly important
Germ tube test used for C. albicans
– C. albicans most common pathogen, usually susceptible to all agents
Cryptococcosis Microbiology
Cryptococcus neoformans
Properties
– budding yeast-like cells in culture and in tissues
– polysaccharide capsule
– ‘perfect’ stage - Filobasidiella neoformans
– 2 varieties: neoformans and gattii
Cryptococcosis Epidemiology/ecology
worldwide distribution
var. neoformans associated with pigeon
droppings and soil.
var. gattii associated with Eucalyptus trees
in Australia and Southern California;
current epidemic evolving in British Columbia and Pacific Northwest
>80% of infections in people with serious
abnormalities of cell-mediated immunity
(eg, AIDS, transplantation, others).
Cryptococcosis Clinical manifestations
pulmonary (self-limited or progressive) CNS (subacute - chronic) meningitis or meningoencephalitis, mass lesions rare extraneural disseminated disease (skin, bone, etc)
Cryptococcosis Diagnosis
Culture
Direct microscopy (India ink, Gram stain)
Test for polysaccharide antigen.
Cryptococcal Histopathology
Mucicarmine +
• Purulent, granulomatous or inert inflam. reaction
• If granulomatous, could be either caseating or “sarcoid” type granulomas
Malasessia furfur
A lipophilic yeast associated with catherter
infections, typically in neonates on extended
courses of parenteral lipid.
– Also seen in adults with severe immunocompromise
Fever, signs of sepsis, catheter blockage.
Diagnosis requires lipid in culture; alert lab.
Treated by catheter removal and D/C lipids
if possible.
Sporotrichosis Microbiology & Epidemiology
Sporothrix schenckii – dimorphic (hyphae at ambient temp, round to cigar-shaped yeast at 37C and in tissues – natural habitat: soil and plant matter –Worldwide distribution – Most frequent in gardeners or others exposed to plant material.
Sporotrichosis Pathogenesis and clinical
features
Pathogenesis
– inoculation via trauma (thorns or splinters
common) > local inflammation > spread
via lymphatics
– Clinical and pathologic features
lymphocutaneous disease in normal hosts
extracutaneous (bone, lung, meninges)
disseminated disease in compromised hosts (rare).
Sporotrichosis Diagnosis and treatment
Diagnosis – culture – microscopy of aspirates or biopsy Treatment – iodides – amphotericin B – ?azoles.
Sporothrix in Culture
Black mold with ‘florettes’ of conidia
Histoplasmosis
Histoplasma capsulatum – dimorphic (hyphae at ambient to 30 degrees C; small budding yeast at 37 degrees C and in tissues) – present in soil (especially if heavy bird or bat droppings) throughout the world – prevalence highest in Midwestern USA.
Histoplasmosis Pulmonary Disease
Most infections are asymptomatic
M:F 4:1
Primary pulmonary infection
– usually self-limited
– severe diffuse pneumonia with large inoculum
Chronic pulmonary infection
– Histoplasmoma
– Cavitary (resembles TB)
– Mediastinal fibrosis
Disseminated (primary or recrudescent infection)
– severe and rapidly progressive in AIDS patients, other immunocompromised
hosts and infants
Fever, malaise, anemia, hepatosplenomegaly
– subacute but progressive in normal adults (mucosal ulcerations common); more common in older patients
GI, CV, CNS involvement
Histoplasmomas
Histoplasmosis Diagnosis
culture (may take 2-4 weeks)
direct microscopy (tissue sections, periph blood)
– epithelioid granulomas, tuberculoid, caseating
– can also produce noncaseating granulomas
– old histoplasmomas (“fibrocaseous or
fibrocalcific lesions”): yeasts dead or dying
skin testing (epidemiology only)
serum antibodies (CF or ID to H or M antigens)
immunoassay for antigens in urine, serum, CSF.
Histoplasma capsulatum at 30C:
fluffy mold with TUBERCULATED MACROCONIDIA (spiny large spores)
Histoplasma capsulatum at 37C
dry, coral-like colony with small budding yeasts, 2-4 uM, narrow neck
Leishmania vs. Histoplasma
Leishmania Discrete organisms with nucleus and kinetoplast PAS(-)
Histoplama Yeast, often budding Parent and bud not within a discrete membrane PAS(+)
Blastomycosis (North American)
Blastomyces dermatitidis – dimorphic fungus: hyphae at ambient to 30 degrees C highly refractile yeast with broad-based budding at 37 degrees C or in tissues – soil fungus in Eastern US and Africa – M:F ratio approx 10:1.
Blastomycosis Pathogenesis
Infection is by inhalation
– Pulmonary disease most common
– Dissemination via bloodstream
Control via specific T-cell mediated
immunity, but little increase in frequency or severity in patients with CMI deficiency
Histopathology - acute inflammation and granuloma formation.
Blastomycosis - Clinical manifestations
Pulmonary - progressive pneumonia, can be severe Extrapulmonary - necrotic skin ulcers most common – Subcutaneous nodules – also bones, joints and other organs. – GU disease in men
Blastomycosis Diagnosis
Diagnosis
– culture sputum, skin lesion, etc (may take 2- 4 weeks)
– look for broad-based budding yeast by
microscopy (stained sections or KOH preps)
– antibodies to crude B. dermatitidis antigens cross-react with other fungi, but antibodies to recombinant protein
antigen (WI-1) are specific – not widely
available
Blastomyces morphology
Microbiology Culture at 30°C:
– Fluffy, white-buff mold, “prickly stage”
– Pear-shaped (“pyriform”) conidia, resembles Chrysosporium, P. boydii. “Lollipops”.
Convert to yeast – broad-based budding is more diagnostic
Coccidioidomycosis (Valley Fever)
Coccidioides immitis
– dimorphic fungus hyphae that produce
arthrospores at ambient to 30 degrees C
endosporulating spherules in tissues
– Soil fungus in lower Sonoran life zone (California, Texas, Arizona, Baja Calif, etc)
– Variable acquisition rates depending on weather, earthquakes, etc.
– most dangerous fungus in laboratory.
Coccidioidomycosis
Pulmonary
– Most cases are asymptomatic
– Symptomatic pneumonia occurs
usually self-limited
can progress to severe necrotizing/cavitary pneumonia
Extrapulmonary
– more likely if CMI is abnormal (AIDS, imunosuppressive drugs, pregnancy) and in African-Americans and Filipino racial groups
– many tissues can be involved, including bones, joints, kidneys, meninges
– hypersensitivity reactions (Erythema nodosum in 5%)
Coccidioidomycosis Diagnosis
culture sputum, material from extrapulmonary lesions, etc BE CAREFUL!
demonstrate spherules by histology
skin test (negative in disseminated disease)
antibody tests (high titers of serum antibodies in progressive or disseminated
disease, antibodies disappear with effective therapy)
Coccidiomycosis in Culture
Fluffy white mold
Thick-walled barrel-shaped hyphae
Coccidioides Histopathology
- Thick-walled spherules (10-80 μM, ave. 50 μM), with endospores; look for all stages of development in acute lesions, & fragmented spherules in old lesions
- Granulomatous inflamm., with caseation. Pyogenic Rx at areas where endospores are discharged
- Fibrocaseous/fibrocalcific coccidioidomas with calcification
Penicillium marneffii
Endemic to SE Asia
Causes disease in HIV-infected patients.
– Pulmonary, disseminated, and cutaneous disease
– Found in blood and bone marrow in
disseminated disease
Yeast phase in tissue with cross-wall in a
single cell.
Colony with diffusible orange pigment
Paracoccidioidomycosis (S. American blastomycosis)
Paracoccidioides brasiliensis – dimorphic fungus hyphae at ambient to 30 degrees C large yeast with multiple buds at 37 degrees C – Central and S. America only ? soil or plants more cases in rural than urban areas.
Paracoccidiodomycosis Pathogenesis & Clinical Manifestations
Pathogenesis - little is known except
that T-cell immunity is important for
control and that lesions are granulomatous
Clinical manifestations - pulmonary, lymph
nodes, skin, oral mucosal lesions most common
Paracoccidiodomycosis Diagnosis
Culture – Slow, white-buff fluffy mold – Pyriform conidia, like B. dermatidis or P. boydii (thus, like Blasto at 30C: not fully pathognomonic) Direct microscopy – yeast form with multiple buds – Also seen in culture at 37 degrees C
