Week 9

Question 1

Sporotrichosis

Answer 1

Granuloma ulcer at a skin puncture e.g. from a thorn prick, may produce secondary lesions along draining lymphatics is normally self-limiting but may exist in chronic form
Treated with oral itraconazole

Question 2

What causes sporotrichosis?

Answer 2

Sporothrix schenckii
* Fungus found in soil, on plant material
* Dimorphic fungus: Mould in the environment (ambient temp) and yeast during human infection (37°C)

Question 3

How do we diagnose sporotrichosis?

Answer 3

• Biopsy of infected area
• Culture on Sabourauds agar
• Microscopy

Question 4

Histoplasmosis

Answer 4

Infection results after inhalation of the conidia
Majority of cases are subclinical, or completely benign
Minority of cases develop a chronic progressive lung disease, a chronic systemic disease or an acute fulminating fatal systemic disease (more common in children)
Diagnosis by X-ray (areas of pulmonary calcification),
histoplasmin skin test, or culture

Question 5

Ocular histoplasmosis syndrome

Answer 5

Spores may spread from lungs to the eye lodging in a layer of blood vessels called the choroid
Abnormal blood vessels form lesions, which can turn into scar tissue in the
central part of the retina (yellow
discolouration seen on the eye)
Scar tissue formation results in vision loss

Question 6

Histoplasma capsulatum

Answer 6

Dimorphic pathogen i.e.
At room temperature it takes on a mycelial (filamentous) form forming macroconidia and microconidia
At human body temperature, it converts into a yeast form

Question 7

Treatment for histoplasmosis

Answer 7

Amphotericin B binds to ergosterol in the cell membrane
Intraconazole inhibits enzymes involved in the biosynthesis of ergosterol

Question 8

Aspergillus

Answer 8

 filamentous, thermally monomorphic, saprophytic moulds
 wide spread in environment (colonise grain, leaves, soil, living plants)
 conidia easily dispersed into environment, humans became
usually infected by inhaling them
 among the most frequently encountered organisms in clinical laboratory

Question 9

Aspergillosis

Answer 9

Aspergillosis
* Pneumonia
* Septicaemia
* Multi-organ disease

Treated with voriconazole OR
amphotericin B

Question 10

What causes aspergillosis?

Answer 10

– Aspergillus fumigatus and other
Aspergillus species
– A filamentous mould

Question 11

How does one get aspergillosis?

Answer 11

Aspergilli are found in organic debris
* Dust, soil

Immunosuppressed people
* Inhale or ingest fungal spores

Question 12

How does one diagnose aspergillosis?

Answer 12

– Chest X-ray
– Sputum culture
– Blood culture
– Serology based on galactomannan which is a component of the Aspergillus cell wall
– Macroscopic (colony)
morphology after culture on
Sabourauds agar at 37°C
– Microscopic morphology

Question 13

Clinical significance Aspergillosis

Answer 13

 incidence of infection relatively rare (high degree of natural
resistance in healthy host)
 particular predilection to invade blood vessels, causing
thrombosis and haemorrhagic infarction

 Aspergillosis
− invasive lung infection, pulmonary fungus ball (fungus
colony grows within congenital or inflammatory lung cyst)
− allergic bronchopulmonary aspergillosis
− external otomycosis, mycotic keratitis, sinusitis
− endocarditis, central nervous system infections
− onychomycosis, disseminated infections

Aflatoxins
− powerful secondary metabolites produced by growth of
Aspergillus flavus complex
− toxic, cause aflatoxicosis after consumption of affected
foodstuff
− carcinogenic, strong evidence for causal role in primary hepatocarcinoma

Question 14

Specimens Aspergillus

Answer 14

 biopsy material
 transtracheal aspirates, sputum
 skin scrapings, infected nails

 significance of isolation
− detection in specimen of hyphal elements compatible with morphology of isolated mould
− isolation of several colonies of fungus or repeat isolation of same fungus from multiple specimens from patients having compatible clinical picture

Question 15

Direct examination Aspergillus

Answer 15

in acute invasive infection:
− hyaline, closely septate hyphae, 3µm to 6µm in diameter
− branch dichotomously at acute (45o) angles
− have smooth parallel walls with no or slight constriction at septa

in chronic infection:
− short, distorted hyphae may be wide 12µm

 when hyphal elements typical of aspergilli present,
presumptive diagnosis can be made
 must be confirmed by culture or immunologic techniques

Question 16

Aspergillus Isolation and identification

Answer 16

 most species of Aspergillus are susceptible to cycloheximide (media containing this compound should not be used)

 Growth in culture
− septate, hyaline, branched hyphae give rise to upright conidiophores terminating in swollen cell - vesicle
− conidiophore varies in colour, length, wall ornamentation in different species; usually aseptate
− conidiophore arises either directly from vegetative hyphae or from specialised hyphal cell - foot cell
− vesicle: globose, subglobose, hemispherical, or pyriform (pear
shaped)
− vesicle or a portion of it covered with phialides, which form conidia

 Growth temperature studies
− Aspergillus fumigatus complex can grow up to 50oC
− other species are less thermotolerant

 Exoantigen tests
− useful for rapid identification to genus level

 MALDI-TOF MS-based systems

Question 17

Characteristics of Aspergillus species

Answer 17

 Growth rate
− usually rapid, mature within 3 days
− some species slower growing
 Colony morphology
− surface at first white, later turns yellow, green, brown, or black depending on species
− texture velvety or cottony
− reverse white, yellow, or brown

 Microscopic morphology
− morphology of conidia / spores, spore bearing structures, type of hyphae

Question 18

Immunodiagnosis Aspergillus

Answer 18

serologic tests which detect circulating antibodies:
− Enzyme-linked immunosorbent assay (ELISA) - highly
sensitive
− Indirect immunofluorescence - highly sensitive
− Agar gel immunodiffusion (ID)
 detects presence of precipitins to various antigen
preparations of Aspergillus spp.
 lacks sensitivity
 gives no quantitative information on antibody
concentrations

tests which detect soluble antigens of Aspergillus spp. (e.g., galactomannan) in serum, urine, or other body fluids:
− ELISA
− Immunoblotting

Question 19

Therapy aspergillosis

Answer 19

 Allergic forms of aspergillosis
− corticosteroids and antifungal agents
 Aspergilloma
− treatment varies considerably with its severity
 asymptomatic patients may not need treatment
 other patients may require surgical resection
− both amphotericin B and flucytosine recommended
 Local, superficial aspergillosis
− nystatin
 Invasive aspergillosis
− aggressive treatment with amphotericin B initiated as soon as possible

Question 20

Yeasts of medical importance

Answer 20

 species of unicellular fungi capable of producing diseases of humans and animals
 ubiquitous in environment
 found on fruits, vegetables, other plant materials - exogenous
 normal flora in and on human and animal bodies - endogenous
 most common fungi isolated from human patients
 considered opportunistic pathogens
 may be cultured from specimens of patients immunocompromised

Question 21

Candida Natural habitat

Answer 21

− part of normal flora of skin, mucous membranes,
gastrointestinal tract
− potential pathogens
− present in clinical specimens as result of: environmental contamination, colonisation, actual disease processes

Question 22

Candida albicans

Answer 22

 Virulence factors
− adherence to epithelial cell membranes
 germ-tubes more adhesive than yeast cells
− protease(s)
 enzyme(s) capable of digesting host immunoglobulins and other substrates
 Dimorphism
− budding yeast cells (produced when part of normal flora)
− pseudohyphae
− true hyphae (produced only during tissue invasion)

Question 23

candidiasis Clinical significance (Candida albicans)

Answer 23

 candidiasis occurs world-wide
 most common cause of systemic mycosis
− physiologic (pregnancy, old age, infancy)
− traumatic (maceration, burns, other infection)
− haematological (leukaemia, lymphoma)
− endocrine (diabetes mellitus, hypoparathyroidism, iron
metabolic disorders)
− iatrogenic (antibacterial antibiotics, steroid treatment)
− other (AIDS)

candidiasis grouped into 3 categories:
− Cutaneous and mucosal
 e.g., oral thrush, stomatitis, oesophagitis, intertriginous candidiasis, onychomycosis, vaginal thrush, balanitis can be caused by conditions that result in chronic
maceration of these areas
− Systemic
 follow introduction of Candida species into bloodstream
 e.g., meningitis, endophtalmitis, myocarditis,
endocarditis, bronchopulmonary candidiasis, intestinitis, infant diarrhoea, pyelonephritis, cystitis, osteomyelitis, arthritis
− Chronic mucocutaneous
 infection of any or all of epithelial surfaces of body (skin, oral mucosa, upper respiratory tract, gastrointestinal, urinary, genital epithelium)
 invasion of bloodstream or deeper tissues unusual

 allergic reactions to Candida antigens may occur

Question 24

Candida albicans Isolation and identification

Answer 24

Germ-Tube Test
− simplest, most valuable test for rapid presumptive
identification
− not all isolates of Candida albicans germ-tube “+” (5%
isolates “-”)
− some isolates of Candida tropicalis may rarely produce
germ-tubes
− Candida albicans blastoconidia smaller, no constricturewhere hyphal initial (germ-tube) joins blastoconidia
− Candida tropicalis blastoconidia larger, constricture where hyphal initial joins blastoconidia

Cornmeal agar morphology (Dalmau plate technique)
− polysorbate (Tween) 80 added to reduce surface tension to promote germination and sporulation of yeasts
− certain species of yeasts develop characteristic
morphological features
− Candida albicans produces:
 chlamydospores
 clusters of blastoconidia arranged at regular intervals along pseudohyphae

Question 25

Characteristics of Candida albicans

Answer 25

 Growth rate
− rapid, mature in 3 days

 Colony morphology
− Yeast-like, pasty, smooth, opaque, cream coloured,
accompanied by distinctive yeast odour
− on chromogenic agar medium (e.g., CandiSelect,
CHROMagar™ Candida, Candida ID) produces blue green, green, blue colonies respectively

− yeast identification systems (API 20C, Vitek Biochemical Card, etc.)
− RAPIDEC albicans Test

 Carbohydrate assimilation tests
− yeasts contain enzyme systems that determine their ability to utilise carbohydrate as sole source of carbon in presence of O2

 Carbohydrate fermentation tests
− yeasts contain enzyme systems that allow for anaerobic
degradation of specific carbohydrates with production of CO2 and alcohol as end by-products

Question 26

Yeast identification systems

Answer 26

 API 20C Yeast System (bioMérieux - Vitek)
 MALDI-tof
 RAPIDEC albicans Test
 Vitek Biochemical Card (bioMérieux - Vitek)

Question 27

Therapy Candida albicans

Answer 27

 Cutaneous candidiasis
− treatment with topical antibiotics - ketoconazole, nystatin, or miconazole
− treatment with chemical solutions - gentian violet
 Systemic candidiasis
− treatment with amphotericin B, flucytosine, or both
 Chronic mucocutaneous candidiasis
− treatment with flucytosine, amphotericin B, miconazole,
topical chemical solutions

Question 28

Culture media Fungi

Answer 28

 cycloheximide prevents overgrowth of rapidly growing
environmental moulds that may contaminate culture plates
 all media should contain antibacterial agents to inhibit growth of bacteria
 combinations of antibiotics used, including:
− penicillin (20U/mL), streptomycin (40U/mL)
− gentamicin (5µg/mL), chloramphenicol (16µg/mL)

 Sabouraud dextrose agar (SDA) - Emmons modification
− has neutral pH (6.9 instead of 5.6 in original formula)
− contains only 2% dextrose (4% in original formula)
 Dextrose 20g - 2%
 Peptone 10g - 1%
 Agar 17g - 1.7%
 Distilled water 1000mL

Question 29

Incubation requirements Fungi

Answer 29

 at room temperature (25oC or preferably at 30oC)
 humidity of 40% to 50% (achieved by placing open pan of water in incubator)
 for 4 weeks before being considered negative
 cultures for yeasts need to be held 5 days
 culture suspected of containing thermally dimorphic fungi
incubated for 8 weeks
 cultures examined at least 3 times weekly during incubation