03/01a Diagnostic Mycology

Question 1

What are the three categories of mycotic afflictions?

Answer 1

Allergic - can be major or minor
Toxicosis - ingestion of mycotoxins
Mycosis - infection by fungi, can range from colonization to disseminated

Question 2

What are the four portals of entry for mycoses?

Answer 2

Skin - simple contact or implantation by trauma
Inhalation
Mucous membranes (usually normal flora)
Iatrogenic - prolonged broad-spectrum antibiotic treatment, immune suppression, indwelling catheters

Question 3

What are the four major categories of clinical classifications of mycoses?

Answer 3

Superficial and cutaneous - infect by casual direct contact
Subcutaneous - infect by traumatic implantation
Endemic/systemic - geographically restricted primary pathogens
Opportunistic - often normal flora

Question 4

What are the characteristics of a superficial mycosis?

Answer 4

Colonizes non-living material
Causes no immune response
Very common, usually non-fatal

Question 5

What are the characteristics of a cutaneous mycosis?

Answer 5

Colonizes non-living material

Immune responses can be present

Question 6

What are the characteristics of an endemic mycosis?

Answer 6

Usually have a limited geographic distribution
Infect by inhalation of spores
Low mortality
Some infections can be chronic and serious, especially in immuncompromised patients

Question 7

What is fungal dimorphism?

Answer 7

Fungi (usually endemic) that can exist in two different forms - a mold in nature, and a non-mold in the host (e.g. yeast)

Question 8

What genera of fungi exhibit fungal dimorphism? Name six

Answer 8

Blastomyces
Coccidioides
Histoplasma
Paracoccidioides
Sporothrix
Penicillium marneffei

Question 9

What are the characteristics of opportunistic mycoses?

Answer 9

Much higher incidence than endemic mycoses (global distribution)
Often normal flora
High mortality

Question 10

What are six common opportunistic mycoses?

Answer 10

Candidiasis
Cryptococcosis
Mucormycosis
Aspergillosis
Hyalohyphomycosis

Question 11

What are the four laboratory methods used to diagnosis fungal infections?

Answer 11

Microscopy of specimens
Culture
Nucleic acid-based methods
Serologic testing

Question 12

What are the advantages and disadvantages of microscopy as a diagnostic tool for fungi?

Answer 12

Advantages - fast and immediate results
Disadvantages - less sensitive than culture, consumes part of specimen, definite ID is not possible without a positive culture

Question 13

What specimens are not usually suitable for microscopy?

Answer 13

Specimens of insufficient quantity

Blood

Question 14

How does a KOH exam work?

Answer 14

KOH dissolves human material, but has no effect on fungal material - makes fungi easier to see
Disadvantage - preparations are not permanent

Question 15

How can you improve a KOH exam?

Answer 15

By the addition of calcofluor white, an optical brightener that fluoresces under UV light
Calcofluor white binds to cellulose and chitin and makes them appear much brighter
Highly sensitive, but not entirely specific for fungi

Question 16

How useful is a Gram stain for detecting fungi? Why?

Answer 16

Not very (not suitable for molds)
Gram stain detects cytoplasm, but fungi cells have minimal cytoplasm

Question 17

For what types of infections is India ink stain useful in diagnosis? Why?

Answer 17

Encapsulated yeasts such as Cryptococcus
Carbon particles of India ink cannot penetrate the extracellular capsule, and thus serve as a negative stain for Crytococcus
Sensitivity and specificity is high

Question 18

What are the six major histological stains for fungi?

Answer 18

1) Silver stain (GMS) - most sensitive because it precipitates on fungal cell walls
2) Gridley - no longer widely used
3) PAS - less sensitive because it poorly stains dead or senescent cells
4) H&E - least sensitive because it does not stain the fungal cell wall
5) Mucicarmine and Alcian Blue - for Cryptococcus
6) Fontana-Masson - for Cryptococcus, highly sensitive

Question 19

What does Cryptococcosis look like in a clinical specimen?

Answer 19

Encapsulated

Fairly large budding yeast

Question 20

What does Candidiasis look like in a clinical specimen?

Answer 20

Budding yeast + pseudohyphae

Question 21

What does Blastomycosis look like in a clinical specimen?

Answer 21

Budding yeast with NO filaments
Very large
Thick-walled
Broad points of attachment between mother and daughter cells

Question 22

What does Histoplasmosis look like in a clinical specimen?

Answer 22

Small oval yeast
Often found within macrophages
Looks like it has a capsule on H&E, but it is actually a staining artifact

Question 23

What does Paracoccidioidomycosis look like in a clinical specimen?

Answer 23

Large yeast
Produces multiple buds in a wheel-like appearance
Grows as a mould in the lab but as a yeast in humans

Question 24

What does Penicilliosis look like in a clinical specimen

Answer 24

Yeast cells that divide by fission (can be identified by a heavy septum between dividing cells)

Question 25

What fungal genera cause Hyalohyphomycosis?

Answer 25

Aspergillus

Fusarium

Question 26

What fungal genus cause most cases of Mucormycosis?

Answer 26

Rhizopus

Question 27

What structure is characteristic of Coccidioidomycosis?

Answer 27

Spherules - grow in size and release endospores

Question 28

What are the advantages of culture as a diagnostic tool for fungi?

Answer 28

More sensitive than direct exam
Can provide definitive identification
Antifungal susceptibility testing is possible

Question 29

What are the three different media used for culturing fungi?

Answer 29

Non-selective (everything can grow)
Selective against bacteria
Selective against certain other fungi

Question 30

At what temperature should you culture most fungi? Why?

Answer 30

30C - many fungi don’t grow very well at body temperature (37C)

Question 31

How quickly do clinically relevant fungal isolates appear?

Answer 31

Within 1-5 days

Question 32

How are yeast isolates identified?

Answer 32

Colony morphology

Primarily biochemical characteristics

Question 33

What are the quickest ways to screen yeasts?

Answer 33

India ink for capsule detection

Germ tube test - no constriction indicates Candida albicans

Question 34

What tests can you use to identify Candida albicans?

Answer 34

Germ tube test

Enzyme detection

Question 35

What test is used to identify a non-Candida organism?

Answer 35

Carbohydrate assimilation - tests whether the yeast can use specific carbohydrates as a carbon source

Question 36

How are mould infections usually identified?

Answer 36

Mostly through morphology

Also biochemical and nucleic acid sequencing tests

Question 37

What is a DTH test? What infections is it useful for?

Answer 37

Delayed-type hypersensitivity skin test

Used for endemic mycoses such as Coccidioidin, Hystoplasmin, and Paracoccidioidin

Question 38

How is a DTH test interpreted?

Answer 38

Must have a history of recent conversion for a meaningful positive test, because once a patient is positive, they are positive for life
Negative skin test can exclude that mycosis in an immunocompetent patient

Question 39

Describe three serological tests for fungal antigens, and which genera they can detect

Answer 39

Capsular antigen, detected in CSF via latex agglutination; highly sensitive for Cryptococcus
Carbohydrate antigen can detect Histoplasma
Cell wall component beta-glucan can detect Candida, Aspergillus, Fusarium, Pneumocystis, and Coccidioides