Basic Mycology

Question 1

Basic characteristic of Fungi: cell wall and cell membrane components, classifications

Answer 1

Eukaryotic, ubiquitous

Fungal cell wall glycans: mannan, glucan and chitin
Fungal cell membrane: phospholipid bilayer with ergosterols
(both these are possible targets of antifungals)

Unicellullar –> Yeasts
Multicellular –> Moulds
Dimorphic: change with temperature e.g. candida (yeast-like at 37 degrees, mould-like at 25 degrees)

Question 2

Yeasts: general staining characteristics (4)

Answer 2

Gram +ve staining
Budding (daughter cell)
Oval shaped (vs round bacteria)
4-5 micrometre (vs 1 micrometre bacterial)

Question 3

Yeast: Candida - normal sites, growth conditions, morphology (3), diagnosis (2), disease (2)

Answer 3

Candida albicans (and non-albicans)

General characteristics:

• filamentation (pseudohyphae): virulence factor
• commensals of human intestinal tract and vaginal tract
• growth best at 37 degrees, aerobic
• Sabouraoud dextrose agar (4% w/v dextrose) for 48 hrs –> round white colonies, smooth edge, 3-4 micrometers

Morphology: (on unstained wet mount)

• budding
• pseudohyphae (elongated, constriction at budding site)
• true hyphae (elongate through process of apical synthesis that doesn’t involve budding)

Diagnosis:

• GERM TUBE TEST - suspend yeast cells in horse serum at 37 degrees for 2 hrs
• -> germ tube formation +ve in Candida albicans/ -ve in non-albicans
• -> speciation predictive of anti fungal susceptibility results
• KOH wet mount

Disease:

• oral thrush
• candida vulvovaginitis

Question 4

Yeast: Pneumocystis jirovecii - growth conditions, morphology (2), diagnosis (2), disease (1)

Answer 4

Previously known as pneumocystis carinii and thought to be parasite (trophozoite and cyst forms)

General:

• appear in clusters - CUP SHAPED (collapsed basketball)
• obligate intracellular org –> NON-CULTIVABLE in lab
• no ergosterol

Diagnosis:

• BAL/ induced sputum –> TOLUIDINE BLUE O STAIN
• microscopy: >5/high power field (as org is ubiquitous)

Disease:

• via inhalation
• pneumonia in severely immunocompromised e.g. AIDS

Question 5

Yeast: Cryptococcus neoformans species complex - morphology (2), variants and their serotypes/ source/ at risk groups (2), diagnosis (1), disease (2)

Answer 5

General:

• unicellular, oval, 5 micrometres
• large extracellular CAPSULE (similar size to a cell)
• wide ecological niche
• capsular polysaccharides serotypes A-D

var. neoformans
- serotypes A/D
- worldwide
- PIGEON FAECES, soil
- immunosuppressed patients e.g. AIDS

var. gattii
- serotypes B/C
- tropics, subtropics
- eucalyptus tree in Australia
- immunocompetent patients, poor prognosis

Diagnosis:

• CSF for INDIA INK (negative stain) –> budding with large capsular halo
• Latex agglutination assay for capsular polysaccharide antigen

Disease:

• via inhalation (aerosolisation of contaminated soil)
• neurotropism –> meningoencephalitis (confusion and fever)
• extra-neural disease is rare e.g. pneumonia, prostate infection

Question 6

Yeast: Malassezia furfur - normal site (2), biochemical properties, diagnosis (1), disease (1)

Answer 6

Dimorphic, yeast-like

General:

• normal skin flora
• LIPOPHILIC
• associated with sebum (glands in face and upper trunk)

Diagnosis:
- SKIN SCRAPING for KOH preparation and culture (requires olive oil supplement)

Disease:
- superficial skin infections (hyper or hypopigmentation) – Tinea versicolor

Question 7

KOH wet mount preparation: method, purpose

Answer 7

10% KOH to digest keratin from skin scrapings – blur the cell junctions so filaments easier to visualise

Lactophenol cotton blue counterstain (highlight edge of fungal filaments)

Examine at 10x or 40x objective lenses

–> visualise fungal filaments (differentiate M. furfur, candida and dermatophytes from other skin disorders)

Question 8

Mould: Zygomycetes - morphology, diagnosis (2), disease

Answer 8

(genus: rhizomucor, muco, rhizopus)
Commonly known as bread mould

Morphology:
- irregular, NON-SEPTATED hyphae

Diagnosis:

• fragile and easily crushed in specimen –> low culture yield (difficult to dx)
• molecular or serological dx

Disease:

• via inhalation, skin infection
• immunocompromised patients e.g. DM, HIV, transplants
• high mortality

Question 9

Mould: Aspergillus fumigatus - morphology (3), diagnosis (2), disease (4)

Answer 9

Ubiquitous

Morphology:

• SEPTATED HYPHAE
• ACUTE ANGLE BRANCHING
• CONIDIA (spore-like, thick-walled and resist unfavourable conditions)

Diagnosis:
- biopsy for KOH wet mount
- CULTURE: top smoky grey-green with conidia, bottom yellow
(CXR for aspergilloma; IgE)

Disease:
- allergic reactions, aspergilloma, invasive pulmonary infection, aflatoxin (HCC)

Question 10

Mould: Dermatophytes- pathology, morphology (3), diagnosis (3), disease (1)

Answer 10

Trichophyton, epidermophyton, microsporum

Infects the keratin layer of skin, hair, nail (doesn’t invade into deeper tissue due to inhibition by serum) – not life threatening

Morphology:

• Microsporum: macroconidia (thick-walled, fusiform shape), microconidia
• Trichophyton: macroconida rare, microconidia
• Epidermophyton: macroconidia (smooth, thin-walled), no microconidia

Diagnosis:
- SKIN SCRAPINGS (advancing edge) for KOH and CULTURE
- NAIL CLIPPING (proximal part, subungal) for KOH AND CULTURE
+/- plucked hair at alopecia or scarring areas

Disease:

• superficial skin infections (itchy, red, cosmetic concern, persistent for wks)
• –> Tinea infection

Question 11

Dimorphic: Sporothrix schenckii - mode of infection, disease (2)

Answer 11

Cutaneous infection

Disease:

• direct inoculation of rose thorns
• sporotrichosis/ Rose Gardener disease – lymphocutaneous spread and lymphangitis

Question 12

Dimorphic: Talaromyces (penicillium) marneffei - morphology (2), disease

Answer 12

Dimorphic – Yeast at 37 degrees, Mould at 25 degrees
Endemic in SE Asia

Morphology:

• diffusable RED WINE COLOUR pigment (on culture)
• hyphae, conidia

Disease:

• affect HIV seropositive patients
• soil exposure (rainy/ inhalation of conidia)
• skin infection, potentially lethal systemic infection

Question 13

Geographical mycosis - examples of species (2), morphologies

Answer 13

Histoplasma capsulatum - large round macroconidia
Coccidioides immitis - spherules (daughter cells inside)

–> travel history essential (common in America)

Question 14

Overall Yeasts, Moulds and Dimorphic

Answer 14

Yeasts

• candida
• pneumocystis jirovecii
• cryptococcus neoformans species complex

Moulds

• Aspergillus fumigatus
• Zygomycetes
• Dermatophytes (microsporum, trichophyton, epidermophyton)

Dimorphic

• sporothrix schenckii
• talaromyces (penicillium) marneffei

Geographical mycosis