Mycology

Question 1

Introduction – The Fungi

Answer 1

 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

Question 2

The Rise of the Fungi

Answer 2

7th cause of deaths - rising

Question 3

Basic characteristics

Answer 3

 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.

Question 4

Traditional Classification

Answer 4

 Zygomycetes (Rhizopus, Mucor, Absidia, Basidiobolus)
 Ascomycetes (Histoplasma, Blastomyces, some Candida)
 Basidiomycetes (Cryptococcus)
 Deuteromycetes (most pathogenic Candida species, pathogenic
Aspergillus species, Coccidioides)

Question 5

Morphologic Classification

Answer 5

 yeast (Candida, Cryptococcus)
 molds (Aspergillus, Zygomycetes, many others).
 dimorphic fungi (Histoplasma, Blastomyces, Coccidioides, Paracoccidioides, Sporothrix)

Question 6

Diagnosis of Fungal Diseases

Answer 6

 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)

Question 7

Culture

Answer 7

 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)

Question 8

Mycologic Media

Answer 8

 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

Question 9

CanWe Do Fungal Blood Cultures?

Answer 9

 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

Question 10

Microscopy

Answer 10

 Microscopy
– budding yeast or mycelia are often
evident on Gram stains

Question 11

Histopatholological stains

Answer 11

 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!

Question 12

Host antibody responses

Answer 12

 More useful in endemic than opportunistic mycoses
 Cross-reactions between fungi very
common
 Recombinant fungal protein antigens
may be useful

Question 13

Diagnostically-useful fungal antigens

Answer 13

 Commonly Used
– Cryptococcus neoformans galactoxylomannan
–Histoplasma capsulatum surface antigens
 Used in specific/complex situations
– Candida albicans enolase
– Aspergillus fumigatus galactomannan.

Question 14

Outline

Answer 14

Molds; Aspergillus, the zygomycetes,
and others
 Yeasts; Candida and Cryptococcus
 Dimorphic and endemic fungal infections

Question 15

Aspergillosis: Microbiology

Answer 15

 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

Question 16

Aspergillosis: Epidemiology/ecology

Answer 16

 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.

Question 17

Aspergillosis Allergic Bronchopulmonary

Disease

Answer 17

 hypersensitivity leads to allergic pneumonitis or allergic bronchopulmonary
aspergillosis (ABPA).
 allergic aspergillosis
– bronchospasm
– fleeting pulmonary infiltrates
– tends to be chronic or recurrent
 refractory asthma

Question 18

Aspergillosis Pulmonary colonization

Answer 18

 saprophytic colonization - usually no
symptoms, but hemoptysis can result
from local invasion
 saprophytic colonization of preexisting
lung lesions > superficial invasion >
hemoptysis

Question 19

Invasive Aspergillosis

Answer 19

 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

Question 20

Aspergillosis Diagnosis

Answer 20

 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

Question 21

Aspergillus fumigatus

Answer 21

 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)

Question 22

Aspergillus flavus & niger

Answer 22

 A. flavus
– Biseriate
– Yellow colony
– Phialides surround vesicle
 A. niger
– White base with densely black conidia
– Dark, rough conidia
 Also potential pathogens

Question 23

Mucormycosis (Zygomycosis) Microbiology

Answer 23

 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.

Question 24

Mucormycosis Epidemiology/ecology

Answer 24

 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

Question 25

Mucormycosis Rhinocerebral

Answer 25

 Rhinocerebral – rapidly progressive necrotizing pansinusitis – spreads into cranium via blood vessels if untreated. – can disseminate to distant sites via bloodstream

Question 26

Mucormycosis Pulmonary and other

Answer 26

 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.

Question 27

Mucormycosis - Diagnosis

Answer 27

 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.

Question 28

Zygomycetes in Culture

Answer 28

 Wooly, rapidlygrowing mold |  Large, distinctive sporangium

Question 29

Mucormycosis - Treatment

Answer 29

```  Multiple treatment modalities are essential – correct underlying host defense abnormality – surgical debridement/removal of necrotic tissues is essential – amphotericin B, ? Newer azoles ```

Question 30

Other Important Molds

Answer 30

 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

Question 31

Fusarium

Answer 31

 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

Question 32

Dermatophyte infections

Answer 32

```  Taenia whatever… – Capitis; head and hair – Corporis, cruris, pedis; skin of body, groin, or feet  Onychomycosis – nail infections  Three major genera – Trichophyton – Epidermophyton – Microsporum ```

Question 33

Trichophyton

Answer 33

 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.

Question 34

Epidermophyton

Answer 34

 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

Question 35

Microsporum

Answer 35

 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.

Question 36

The Dermatophytes Compared

Answer 36

 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

Question 37

Candidiasis Microbiology

Answer 37

``` – 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. ```

Question 38

Candidiasis Epidemiology/ecology

Answer 38

```  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 ```

Question 39

Candidiasis Clinical and pathologic features – Thrush and esophageal disease

Answer 39

```  Skin and mucosal surfaces involved when CMI is abnormal – ulcerations with surrounding inflammation – thrush – esophagitis – gastrointestinal ulcers  Usually C. albicans  HIV infection, inhaled steroids ```

Question 40

Candidiasis | Clinical and pathologic features, other mucosal disease

Answer 40

 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

Question 41

Candidiasis Clinical and pathologic features – Organ system and nosocomial

Answer 41

```  Urinary tract infections – Most common with Foley catheters  Intravenous catheterrelated infections  Pneumonia quite rare, but a frequent colonizer ```

Question 42

Candidiasis | Clinical and pathologic features -- disseminated disease

Answer 42

```  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 ```

Question 43

Candidiasis Diagnosis

Answer 43

 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.

Question 44

Candidiasis

Answer 44

 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.

Question 45

Candida Species – ID

Answer 45

 Biochemical tests – API, others  Correlate to morphology on cornmeal agar

Question 46

Candida albicans

Answer 46

 Budding spherical to ovoid blastoconidia

Question 47

Candida glabrata

Answer 47

 No pseudohyphae – also seen with Cryptococcus, but capsule usually evident as space surrounding cell for Crypto.

Question 48

Candida parapsilosis

Answer 48

 Short, curving pseudohyphae with round to | oval blastoconidia

Question 49

Candida tropicalis

Answer 49

 Multibranched pseudohyphae, blastoconidia borne singly or in chains from along pseudohyphae

Question 50

Candida lusitaniae

Answer 50

 Short, curved pseudohyphae with | blastoconidia at or between septae

Question 51

Candida krusei

Answer 51

 Branching pseudohyphae with elongated | blastoconidia

Question 52

Candidiasis Treatment

Answer 52

 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

Question 53

Cryptococcosis Microbiology

Answer 53

 Cryptococcus neoformans  Properties – budding yeast-like cells in culture and in tissues – polysaccharide capsule – ‘perfect’ stage - Filobasidiella neoformans – 2 varieties: neoformans and gattii

Question 54

Cryptococcosis Epidemiology/ecology

Answer 54

 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).

Question 55

Cryptococcosis Clinical manifestations

Answer 55

```  pulmonary (self-limited or progressive)  CNS (subacute - chronic) meningitis or meningoencephalitis, mass lesions rare  extraneural disseminated disease (skin, bone, etc) ```

Question 56

Cryptococcosis Diagnosis

Answer 56

 Culture  Direct microscopy (India ink, Gram stain)  Test for polysaccharide antigen.

Question 57

Cryptococcal Histopathology

Answer 57

 Mucicarmine + • Purulent, granulomatous or inert inflam. reaction • If granulomatous, could be either caseating or “sarcoid” type granulomas

Question 58

Malasessia furfur

Answer 58

 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.

Question 59

Sporotrichosis Microbiology & Epidemiology

Answer 59

```  Sporothrix schenckii – dimorphic (hyphae at ambient temp, round to cigar-shaped yeast at 37C and in tissues – natural habitat: soil and plant matter –Worldwide distribution – Most frequent in gardeners or others exposed to plant material. ```

Question 60

Sporotrichosis Pathogenesis and clinical | features

Answer 60

 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).

Question 61

Sporotrichosis Diagnosis and treatment

Answer 61

```  Diagnosis – culture – microscopy of aspirates or biopsy  Treatment – iodides – amphotericin B – ?azoles. ```

Question 62

Sporothrix in Culture

Answer 62

 Black mold with ‘florettes’ of conidia

Question 63

Histoplasmosis

Answer 63

```  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. ```

Question 64

Histoplasmosis Pulmonary Disease

Answer 64

 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

Question 65

Histoplasmosis Diagnosis

Answer 65

 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.

Question 66

Histoplasma capsulatum at 30C:

Answer 66

fluffy mold with TUBERCULATED MACROCONIDIA (spiny large spores)

Question 67

Histoplasma capsulatum at 37C

Answer 67

dry, coral-like colony with small budding yeasts, 2-4 uM, narrow neck

Question 68

Leishmania vs. Histoplasma

Answer 68

Leishmania Discrete organisms with nucleus and kinetoplast PAS(-) Histoplama Yeast, often budding Parent and bud not within a discrete membrane PAS(+)

Question 69

Blastomycosis (North American)

Answer 69

```  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. ```

Question 70

Blastomycosis Pathogenesis

Answer 70

 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.

Question 71

Blastomycosis - Clinical manifestations

Answer 71

```  Pulmonary - progressive pneumonia, can be severe  Extrapulmonary - necrotic skin ulcers most common – Subcutaneous nodules – also bones, joints and other organs. – GU disease in men ```

Question 72

Blastomycosis Diagnosis

Answer 72

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

Question 73

Blastomyces morphology

Answer 73

 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

Question 74

Coccidioidomycosis (Valley Fever)

Answer 74

 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.

Question 75

Coccidioidomycosis

Answer 75

 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%)

Question 76

Coccidioidomycosis Diagnosis

Answer 76

 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)

Question 77

Coccidiomycosis in Culture

Answer 77

 Fluffy white mold |  Thick-walled barrel-shaped hyphae

Question 78

Coccidioides Histopathology

Answer 78

* 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

Question 79

Penicillium marneffii

Answer 79

 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

Question 80

Paracoccidioidomycosis (S. American blastomycosis)

Answer 80

```  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. ```

Question 81

Paracoccidiodomycosis Pathogenesis & Clinical Manifestations

Answer 81

 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

Question 82

Paracoccidiodomycosis Diagnosis

Answer 82

```  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 ```