Yeast Identification

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Yeast Reproduction

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• Fission: splitting of a cell into 2 daughter cells
  • Budding: a small out growth formed on the parental cell. The bud increases in size and eventually breaks off

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Arthoconidia (Arthrospore)

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Thallic conidium is released by either the splitting of a double septum or by the fragmentation of a disjunctor cell

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Blastoconidia (Blastospore)

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Asexual reproductive spore that originates by budding (single asexual spore)

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Pseudohyphae

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A string of elongated blastoconidia formed in some yeast

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Chlamydoconidia (Chlamydospore)

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Thick walled thallic conidium formed within vegetative hyphae

For penetration not dissemination

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Germ tube test

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• Presence of true hyphae when incubated in serum at 37C
• + C. albicans and C. dubliniensis: short hyphal strands when incubated for up to two hours in serum
• Polymorphic yeast: hyphae and pseudohyphae produced
  • 0.5mL of horse or rabbit sera

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Carbohydrate fermentation patterns

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yeast utilize carbon sources anaerobically; produce ethanol and CO2

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Carbon assimilator patterns

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use of carbon sources aerobically, growth/no growth (API)

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Cornmeal

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• Inoculated using the Dalmau plate technique
• Incubate overnight at 25C
  • Examine at 10X for presence/absence of hyphae, size and shape of blastoconidia

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Birdseed agar morphology

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production of melanin

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Caffeic acid disk

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production of melanin

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PNA FISH

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peptide nucleic acid fluorescence in-situ hybridization= tagged PNA probe to the rRNA

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Yeast Morphology

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• Unicellular
  • Transient polarized growth to produce a bud (blastoconidia) or daughter cell. The junction between the bud and mother is called the neck
  • Pseudohyphae
    • True hyphae

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Pseudohyphae

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Formed by the extension of the polarized growth period

Elongated buds are extended opposites the septation site; clear constrictions at septation sites

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True filamentous hyphal cells have

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Continuous width with no constriction around the septae

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Yeast the can produce pseudohyphae

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• Candida albicans
• Candida parapsilosis
• Candida tropicalis
  • Candida krusei

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No pseudohyphae

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• Cryptococcus neoformans
• Candida glabrata
  • Malasezzia furfur

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Chromagar Candida media

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• Selective and differential
• Chromogenic substrates
• Enzymes secreted by microorganisms
• C. albicans: Green
• C. krusei: Dark purple
• C. tropicalis: Steel blue
  • C. glabrata: Light pink

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Dalmau technique

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• Cornmeal with Tween 80
• Tween 80 stimulates conidiation in fungi
• Incubate at 25C
• Examined at low power objective 10X and 40X
• 25C for 3 days in dark
  • 35C for 24-48 hours

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API20C

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• Plastic strip with 20 cupules which contained dehydrated substrate for assimilation testing with cornmeal morphology
• Strips are inoculated and incubated according to manufacturer's instructions
  • Profile number is based upon the reactions observed is generated at 48-72 hr of incubation

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Characteristics of Candidiasis

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• Normal flora, person-to-person, fomite
• endogenous
• exogenous
• any organ
• superifical to disseminated
• hemotogenous route
  • Candida albicans most recognized pathogen

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Candidiasis Clincial Manifestations

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• Spreading moist, erythematous rash of the cheeks and nose
• multi-focal, spreading macular rash
• onychomycosis- fingernails typical cracking, crumbling, and poor growth
• Intertriginous areas
  • thrush, cheilitis

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Candida Spp/Invasive Candidiasis

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• Grows on outer portion of skin and mucosal surfaces: inflammatory response, cell mediated immunity
• At risk populations: age, pregnant, antibiotics, oral contraceptive use, DM, HIV, altered iron metabolism, steroid use
  • C. albicans 50%, C. tropicalis 15-30%

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Candida spp

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• lack of arthroconidia production
• colonies cream colored to yellowish
• Grow rapidly and mature in 3 days
• Colony varies: smooth, glistening, pasty, dry, wrinkled
• Balstoconidia singly or small clusters; round or elongated
• Pseudohyphae: long branched or curved
  • True hyphae and chlamydospores are produced by strains of some Candida spp

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Diagnosis of Candidiasis

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• Stained smear taken from suspicious lesions, from exudates in cases of thrush
  • Key to identify is the presence of pseudohyphae and blastoconidia

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Candida albicans

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• Most common yeast
• Germ tube positive (5-10% negative)
• Growth on cycloheximide (Mycosel Agar)
• Grows at 42C
• Methyl-D-glucoside positive
  • Ferments: Glucose, maltose, galactose

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Candida albicans morphology on SDA

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• Smooth, white, pasty colonies
  • Feet-like extensions

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Candida albicans on cornmeal

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• Blastoconidia at base of sparse hyphae with 90 angle branching, terminal chlamydospores

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Chlamydospores and Blastoconidia: Aniline blue stain

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• C. albicans produce thick walled chlamydospores on cornmeal agar
  • Large spherical, blue-staining spores shown in the photograph, compared to the smaller clear blastoconidia scattered in the background

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Candida dubliniensis

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• Germ tube positive yeast
• Growth on cycloheximide
• No growth at 42C
• Methyl-D-glucoside negative
• Dark green on Chromagar
• Ferments: Glucose, Maltose, sucrose, galactose
  • Distinguish from C. albicans by API

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Pseudohyphae, Chlamydospores and Blastoconidia in clusters

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• cornmeal agar
• balstoconidia in clusters regularly along the pseudohyphae; production of large, spherical, smooth-walled chlamydospores, at the tip of hyphal strand
• Number of chlamydospores at end of the strand
  • C. dubliniensis (abundant) C. albicans (single)

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Candida glabrata

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• commonly isolated yeast
• quite small 2.5-4.0x3.0-6.0um
• SBA<EMB
• Grows at 37C
• Germ tube negative
• No growth on cycloheximide
• Resistant to azole antifungals
  • Ferment: Glucose, trehalose
    • Cornmeal agar: small ovoid budding

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Candida tropicalis

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• Causes candidemia in oncology patients
• Germ tube negative
• Growth on cycloheximide
• Grow at 37C
• Ferments: Glucose, maltose, trehalose
  • Sparse single or short chained blastoconidia at or between septa of pseudohyphae

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Candida parapsilosis

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• Fake fingernails and outbreaks in pediatric centers
• most common cause of candidemia in pediatric patients
• High MICs for Echinocandins
• Germ tube negative
• No growth on cyclcoheximide
• Grows at 37C
  • Ferments: glucose, variable sucrose, galactose

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Candida parapsilosis colonies

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• Many short, much-branched pseudohyphae in spider-like or tree-like pattern
  • 2-3 blastoconidia in small clusters at intervals along the pseudohyphae

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Candida krusei

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• Intrinsically resistant to fluconazole
• germ tube negative
• no growth on cycloheximide
• Grow at 42C
• Ferments: Glucose
• Fallen tree or matchstick "hyphae"
  • Blastoconidia are large and oval

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Candida kefyr

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• Emerging pathogen in oncology centers
• Germ tube negative yeast
• growth on cycloheximide
• Grows at 37C
• Very resistant to azole antifungals
• Ferments: glucose, sucrose, galactose, lactose
  • Cigar shaped and highly branched pseudohyphae, "logs in the stream"

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Candida guilliermondii complex

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• Complex: C. guilliermondii, fermentati, carpophila
• Cutaneous infections
• rarely associated with: endocarditits, osteomyelitis, cutaneous and UTI, fatal immunocompromised
• Germ tube negative
• Growth on cycloheximide
• Grows at 37C
  • Clusters of yeast cells with short pseudohyphae, sm grps of blastoconidia at the septa

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Candida lusitaniae

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• opportunistic pathogen immunocompromised
• Germ tube negative yeast
• no growth on cycloheximide
• grows at 37C
• can develop resistance following a treatment with amphotericin B
• Ferment: G, M, S, galactose, trehalose
  • Pseudohyphae slender, branched and curved short chains of elongate blastoconidia

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Cryptococcus neoformans

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• Worldwide distribution
• Pulmonary entry can spread CNS
• Fecal material of birds: pigeons, chickens or turkey
• High pH and concentration
• airborne due to sweeping, cleaning, construct
• dissemination HIV +pts CD4<100
• rarely occur in immunocompromised
  • laboratory aquired risk

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Cryptococcus spp

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• Basidiomycete- resistant to Echinocandins
• Large round to oval blastoconidia- lateral budding, no pseudohyphae, appear mucoid or slimy on solid media
• Grows at 37C
  • Inositol postive: C. neoformans, C. gattii, C. albidus, C. laurentii

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Cryptococcus neoformans

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• Germ tube negative
• Slow growing (48-72hr)
• polysaccharide capsule
• urea (rapidly positive, 30min)
• no growth on cycloheximide
• grows at 37C
• non-fermenter
• produces melanin
• inositol positive
• caffeic acid disk positive (brown, 4hr)
  • india ink positive

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Cryptococcus neoformans: Niger seed

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• Cryptococcus neoformans; growing on the surface of niger seed agar
• pigmentation indicates phenoloxidase activity; caffeic acid substrate contained in the medium
  • other cryptococcus species are phenoloxidase negative and do not produce the pigmented colonies seen in the photograph

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Cryptococcus gattii

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• Outbreaks in NW US, BC
• Commonly associated with Eucalyptus in India
• Chronic cryptococcus meningitis
• May effect non-immunocompromised host: large mass lesions in lung or brain
  • Canavanine-glycine-bromthymol agar: blue color when creatine is degraded to ammonia

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Serotypes

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• 80-85% of isolates being either A or B
• C. neoformans var. neoformans corresponds to serotypes A and D
  • B and C are associated with C. gattii

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Cryptococcus latex agglutination system (CALAS)

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• Simple, sensitive, rapid latex test for detecting the capsular polysaccharide of C. neoformans in serum and CSF
• Qualitative and Quantitative: prognostic value
• Sensitivity: 90-100%
  • Std to minimize false positive

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CALAS results

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• = a homogenous suspension of particle with no visible clumping
• 1+ fine granulation against a milky background
• 2+ small but definite clumps against a slightly cloudy background
• 3+ large and small clumps against a clear background
  • 4+ large clumps against a very clear background