Yeast and yeast-like organisms

Yeast and Yeast-like organisms

Candida species

Identification

• Any yeast that has growth on culture (blood, fluid, tissue) or seen on Gram stain gets subcultured to SAB-CG at 35º C.
  • Growth rate: <7 days is rapid
  • Texture
  • Colour of surface and reverse
• Do wet mount to confirm features of Candida.
• Microscopy for chlamydospores, pseudohyphae, hyphae, arthroconidia, blastoconidia formation, budding, capsules, pigmentation.
  • C. glabrata is smaller, does not produce hyphae or pseudohyphae, produces blastoconidia, and grows as creamy yeast colonies.
  • Non-glabrata spp. usually exhibit single buddings and can have pseudohyphae (rarely true septate hyphae). Cannot identify species based on microscopy alone.
• MALDI-ToF (Vitek MS), which provides a species. If C. haemolunii or C. famata identified on Vitek, need to rule out C. auris.
  • If Vitek not ≥95% match, or identifies one of the two species above, then repeat the MALDI-ToF and set up Dalmau on cornmeal agar.
• Dalmau technique: growth in adverse conditions (bile oxgall and corn meal) to help identify differences between species of yeast.
  • Light inoculum of single colony in a #-sign pattern with lines 1 inch apart. Cover streaks with coverslip and tamp down gently. Incubate at room temperature 18-72 hours.
  • Examine after 18-24 hours. Look for thick-walled chlamydospores (terminal refractory circles), blastoconidia morphology, and presence of pseudohyphae. Then continue incubating, examining daily.
• Old-school enzymatic tests and assimilation assays.
• Temperature tolerance test at 35-37º C, 42º C, and 45º C
• Germ tube test: if positive, either C. albicans or C. dubliniensis.

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Species	Colony	Conidia	Pseudohyphae	Other	Other Tests
C. albicans	White to creamy, raised, pasty, smooth and soft, shiny and moist. May produce mycelial growth called “feet” or “roots”.	Blastoconidia globose to oval.	Well-developed, abundant with blastoconidia in clusters or grape-like arrangement at septa.	Chlamydospores: round, large, thick-walled, usually single and mostly terminal forming on the tip of pseudohyphae, but some may be sessile. True hyphae may be present in older cultures.	Growth at 42-45º C.
C. dubliniensis	Cream-coloured, glistening, waxy, usually smooth.	Blastoconidia subspherical, identical to C. albicans.	Well-developed with blastoconidia in grape-like arrangement.	Chlamydospores: round, large, thick-walled, usually in pairs, triplets, and clusters of 1-3 and mostly terminal. True hyphae may be present, especially in older culture.	Usually no growth at 42-45º C.
C. glabrata	Small, white to cream-coloured, shiny, pasty, and smooth.	Terminal single budding, oval, and small. Typically arranged in dense groups.	Absent, or rudimentary if present
C. krusei	Cream-coloured to tannish-white. Flat, dry, ground-glass appearance. Spreading edge with a delicate feathery periphery.	Elongate, ellipsoidal to cylindrical. Cells are liberated and arranged parallel to the main axis appearing like logs on the stream.	Initially sparse, often present on prolonged incubation. Elondated and slender, with blastoconidia forming a cross-matchstick or treelike branching appearance at the septa.
C. parapsilosis	White to creamy, shiny, moist, slightly flat, mostly smooth or partly or entirely wrinkled.	Ovoid, single or in small clusters.	Usually abundant, but may be slow to grow. Crooked or curved, relatively short, branched chains of pseudohyphae with clusters of blastoconidia at or between septa. Christmas-tree-like arrangement.	Occasional presence of large hyphal elements called giant cells.
C. tropicalis	Cream-coloured to semiwhite, dull, dry, soft, smooth and creamy. May be wrinkled or have a mycelial fringe near the edge. Can have feet (root mycelium) similar to C. albicans.	Oval. Single or in small groups or short chains at or between septa of pseudohyphae.	Very active growth. Abundant, long and branched. Blastoconidia produced in verticils from the pseudohyphae.	True hyphae may be present.
C. famata	White to cream coloured.	Ellipsoidal.	Absent.		Weak growth at 40º C. Does not grow at 42º C.
C. auris	White to cream-coloured. Pink to beige on chromogenic agar (depending on the agar).	Oval or elongated yeast cells, singly or in pairs or groups.	Absent.		Grows well at 42º C. Variable growth at 45º C. Cycloheximide-susceptible. Usually fluconazole-resistant.
C. guilliermondii	White to tan, slightly heaped, shiny, moist, and usually mucoid with smooth edge.	Spherical to ellipsoidal.	May be slow growing (up to 10 days), radiating from centre of masses of budding cells.	Pseudohyphae not produced.	May grow at 42 º C.

Epidemiology

• Refer to Cleveland et al; CID 2012 55:1352.
• The predominant species worldwide (and in Canada) is C. albicans, by a wide margin, followed by C. glabrata, C. tropicalis, and C. parapsilosis.
• Non-candida species are becoming more common, globally, over the past three decades.
  • C. dubliniensis associated with HIV esophagitis.
  • C. lusitaniae can develop resistance to amphotericin.
  • C. guilliermondii is multidrug resistant.
  • C. auris still rare but may be emerging. It can be misidentified as other yeasts (most commonly C. haemulonii, but also C. famata, Saccharomyces cerevisiae, and Rhodotorula glutinis).

Sources: Laverdiere et al. J Crit Care. 2007;22(3):245–250, Guinea. Clin Microbiol Infect. 2014;20(Suppl 6):5-10.

Cryptococcus species

Diagnosis of Cryptococcus meningitis

• Microscopy:
  • India ink can help identify encapsulated yeasts (50% of non-AIDS and 80% of AIDS will have CSF positive by India ink). India ink no longer done because antigen LFA way more sensitive and specific.
  • GMS may help identify narrow-based budding.
• From culture:
  • Christensen’s Urea: urease-positive yeast include Cryptococcus, Trichosporon, and Malassezia furfur.
  • Capsule present on India ink preparation.
  • Characteristics include:
    • Colonies: Cream to tan in colour, slightly heaped, chiny, moist, and usually mucoid with smooth edge.
    • Conidia: Blastoconidia are round, budding attached to parent cells by narrow necks.
    • Pseudohyphae: absent
    • Extra tests: India ink to demonstrate capsule; urease positive.
  • Other methods to presumptively identify the yeast are to perform a rapid urease test or to inoculate the yeast onto Staib’s birdseed, DOPA, or caffeic acid media (in which colonies will produce melanin and turn brown to black).
• Antigen detection (cryptococcal polysaccharide antigen)
  • Latex agglutination (Sn & Sp 90%)
  • ELISA (Sn & Sp 90%)
  • Lateral flow assay (LFA) (Sn & Sp 95-100%); sensitivity higher in AIDS

Performance characteristics of cryptococcal diagnostic assays in persons with suspected meningitis, Uganda and South Africa

The composite reference standard was defined as a CSF culture-positive (n = 459) or a culture-negative sample with ≥2 positive test results (e.g., India ink microscopy, CRAG latex, or CRAG LFA) and without an alternative etiologic explanation (n = 60). Source: Boulware et al. EID. 2014;20(1):45-53.

Cryptococcus gattii and C. neoformans

Epidemiology of C. gattii

• Unlike C.neoformans, C. gattii has never been found from bird/bat guano. It is usually found in eucalyptus (River red and Forest red gum trees) and coniferous trees, specifically in Vancouver Island and the West Coast
• No specific seasonality, one study reports more cases in the fall and winter
• No human-to-human transmission (unless in solid organ transplantation of an infected organ)
• Unlike C. neoformans, C. gattii usually affects immunocompetent individuals
• Incubation period is 1 year
• More likely to cause cryptococcomas

Distinguishing C. gattii and C. neoformans

• A color reaction on concanavalin-glycine-thymol agar
  • Creatinine deiminase is repressed by ammonia in C. neoformans but not in C. gattii. This difference is manifested by a change in the color of the diagnostic media as the pH changes
  • Media containing L-canavanine and glycine with bromothymol blue as the color indicator for growth (CGB medium) can efficiently differentiate between these two species (C. gattii will produce a color change and C.neoformans will not)
• They produce different sexual spores (impractical)
• MALDI-TOF may be able to identify the different species

Other yeasts of clinical importance

Trichosporon

• Clinical Significance:
  • Can cause Tinea blanca/White piedra which is a mycosis of the hair. Soft nodules composed of yeast cells and arthroconidia encompass the hair shaft. (Immunocompetent)
  • Invasive human infection is most commonly due to Trichosporon asahii or, less commonly, T. mucoides. This usually occurs in immunocompromised patients.
    • Trichosporonosis is an acute febrile illness with dissemination to deep organs with a 64% mortality rate.
    • Often hematuria and funguria given common renal involvement
    • May present with multiple red papules on skin
• Identification:
  • Colonies are white to cream colored, moist and soft. Become irregularly wrinkled, dry, powdery or crumb-like with age.
  • Blastoconidia are ovoid to ellipsoidal, 3.5-3.5-14 um, singly or in short chains but few in number
  • RESISTANT to cyclohexidine
  • ++ True hyphae
  • Urease positive
  • Treatment with voriconazole

Malassezia furfur

• Clinical Significance:
  • Common skin colonizer
  • Causes catheter-related sepsis, almost always in patients who are receiving parenteral lipids through a central line
  • Described in neonates in the ICU, immunocompromised adults
  • Can present with fever, bradycardia, thrombocytopenia
  • M.globosa causes tinea versicolor
• Identification:
  • Will NOT grow on routine media
  • With olive oil overlay or use of specialized media (Dixon, Leeming-Notman): colonies are smooth, cream to yellowish brown, dry with age
  • RESISTANT to cyclohexamide
  • Yeast cells are phialides with colarettes, broad based buds

Saccharomyces cerevisiae (“Baker’s Yeast”)

• Clinical Significance:
  • Considered an occasional digestive commensal organism
  • Can cause fungemia especially in patients on probiotics with central lines/catheter-related infections
  • S. cerevisiae invasive infection usually seen in immunocompromised patients, while S. boulardii usually seen in immunocompetent patients
  • Has been very rarely described in esophagitis
• Identification:
  • Colonies are white to cream, smooth and glabrous
  • Evidence of multilateral budding
  • Blastoconidia are globose to ellipsoidal
  • Absence of hyphae

Rhodotorula

• Clinical Significance:
  • Can cause fungemia usually in immunocompromised patients or patients with central lines
  • The most common underlying diseases included solid and haematologic malignancies in patients who were receiving corticosteroids and cytotoxic drugs, the presence of CVC, and the use of broad-spectrum antibiotics.
  • Can also cause localized infections i.e. meningitis, SSTI, prosthetic joint, peritoneal and ocular infections
• Identification:
  • Colonies are pink, reddish or yellow. They are either slimy or dry.
  • May have rudimentary hyphae, but ballistoconidia are absent
  • Urease positive

Candida auris

• Review the paper Clin Micro Rev January 2018 31(1).

Identification Methods

• C. auris can often be misidentified using the usual methods, often mistaken for C. haemulonii
• Use chromogenic agar to differentiate between C. auris and C. haemulonii isolates using growth characteristics
  • C. auris forms pink-to-beige colonies on chromogenic agar
  • Grows well at 42 degrees
  • No growth in cyclohexidine
  • No hyphae or pseudohyphae
• MALDI-TOF is more accurate now that it’s been added to the database
• PCR assays are in development (likely most helpful in an outbreak setting)
• Sequencing

Susceptibility Patterns

• There are no antifungal clinical breakpoints
• Demonstrated failures and resistance to: fluconazole and other azoles i.e. voriconazole, itraconazole and isavuconazole
  • Resistance to azoles likely caused by the Erg11 mutation
• Variability of susceptibility to Amphotericin B
• Therefore empiric therapy is suggested with echinocandins, especially Micafungin (unless it’s a CNS infection as there is no penetration)
• Combination therapy is being studied with Micafungin + Voriconazole
• CNS infections: Amphotericin B + 5-flucytosine

Infection Control Methods

• Chlorhexidine is active against it
  • Decolonize patients with chlorhexidine gluconate body wash, chlorhexidine mouthwash, and chlorhexidine-impregnated pads on CVC exit sites
  • Note: C. auris can survive up to 14 days on plastic
• Sodium hypochlorite and hydrogen peroxide can be used for cleaning surfaces
  • With the exception of a water-based QAC and a 1:50 dilution of sodium hypochlorite, our data demonstrate that most disinfectants used in healthcare facilities are effective (>3-log10 reduction) against C. auris (source).
• Screening (new PIDAC 2019 guidelines exist, essentially CPE risk factor screening):
  • Axilla and groin screening; additional sites as directed clinically or by previously positive sites
  • Periodic reassessment for presence of colonization at 1- to 3-mo intervals
  • For deisolation, 2 or more assessments 1 wk apart with negative results (off antifungals)
• Isolation (per CDC):
  • Single room with standard and contact precautions;
  • gown and gloves
  • hand hygiene precautions
• Cleaning:
  • Thorough daily and terminal cleaning/disinfection using Environmental Protection Agency-registered disinfectant effective against C. difficile spores

Antifungal Susceptibility Testing of Yeast

Patterns of resistance

• Candida spp: most species are susceptible to azoles including fluconazole, except C. glabrata (which may be dose-dependently susceptible) C. krusei, and C. auris. C. lusitaniae is resistant to ampho B. All are generally susceptible to echinocandins.
• Trichosporon: azoles tested appeared to be more potent than amphotericin B, and were likely fungicidal. However, a more recent series reports resistance to fluconazole (though continued susceptibility to other azoles).
• Saccharomyces: in one review of case reports, there was in vitro resistance to ampho B and dose-dependent susceptibility to fluconazole. However, rates of clinical response were similar and other researchers have found in vitro susceptibility to amphotericin. A review of 7 cases describes successful treatment with micafungin, caspofungin, and voriconazole.
• Cryptococcus neoformans: routinely susceptible to fluconazole and amphotericin, but not to echinocandins (does not have beta-D-glucan).

Organism	AmB	Fluc	Itra	Vori	Posa	Anidula	Caspo	Mica	Flucyt
Aspergillus spp.	+	–	+	+	+	+	+	+	–
A. flavus	±	–	+	+	+	+	+	+	–
A. fumigatus	+	–	+	+	+	+	+	+	–
A. terreus	–	–	+	+	+	+	+	+	–
A. niger	+	–	±	+	+	+	+	+	–
Candida spp.	+	+	+	+	+	+	+	+	+
C. albicans	+	+	+	+	+	+	+	+	+
C. glabrata	+	SDD	±	+	+	+	+	+	+
C. krusei	+	–	±	+	+	+	+	+	±
C. lusitaniae	–	+	+	+	+	+	+	+	+
C. parapsilosis	+	+	+	+	+	±	±	±	+
C. tropicalis	+	+	+	+	+	+	+	+	+
Crypto. neoformans	+	+	+	+	+	–	–	–	+
Coccidioides spp.	+	+	+	+	+	±	±	±	–
Blastomyces	+	+	+	+	+	±	±	±	–
Histoplasma spp.	+	+	+	+	+	±	±	±	–
Fusarium spp.	±	–	–	+	+	–	–	–	–
Saccharomyces spp.	±	SDD	SDD
Scedosporium apiospermum	±	–	±	+	+	–	–	–	–
Scedosporium prolificans	–	–	–	±	±	–	–	–	–
Trichosporon spp.	±	+	+	+
Zygomycetes	±	–	–	–	+	–	–	–	–

Source: Dodds Ashley et al. CID 2006;43(Suppl 1):S28-S39 (Candida etc.), Enache-Angoulvant, et al. CID 2005;41(11):1559-1568 (Saccharomyces), Paphitou AAC 2002;46(4):1144-1146 (Trichophyton).

Mechanisms of resistance

• Azole resistance: triazoles inhibit C-14α demethylase (Erg11p), which is required for the synthesis of ergosterol from lanosterol. Ergosterol is required for proper cell membrane function. There are several mechanisms of resistance:
  • Induction of efflux pumps: most common mechanism, with various possible pumps
  • Altered or overexpressed C-14α demethylase
  • Rarely, alterations in sterol synthesis pathway
• Echinocandin resistance: echinocandins inhibit synthesis of 1,3-β-d-glucan, which is the main component of the cell wall. Resistance is conferred by mutations in the FKS gene (Fks1 for all species, as well as Fks2 in C. glabrata) which encodes a subunit of glucan synthase. Ser-641 or Ser-645 account for more than 90% of resistance in C. albicans. Cryptococcus, by comparison, does not use that pathway and therefore has inherent resistance to echinocandins.
• Sources: Lancet ID series, Mandell, Sanglard and Odds. Lancet ID 2002;2(2):73-85.

Susceptibility testing

• Sensitire plates: used for Candida susceptibility testing, does colorimetric testing of broth microdilution.
• Definitions
  • Epidemiological cut-off value (ECV or ECOFF): the minimum MIC or MEC that separates fungi with acquired or mutational resistance and those without. They can be used to detect antimicrobial resistance in the lab, essentially discriminating wild-type from non-wild-type strains.
  • Clinical breakpoint: the MIC or MEC that predicts clinical response to the antimicrobial, which is often based on the MIC distribution, PK/PD data, and clinical outcomes from trials.
  • Wild-type strains: isolates with no mechanisms of acquired resistance or reduced susceptibility for the antifungal agent being evaluated.
• Amphotericin and Candida: there is no clinical breakpoint for amphotericin B with Candida, so it is reported instead as an MIC. You should consult the ECV (CLSI or other) to make an educated guess at susceptibility.

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Source: Lockhart, et al. J Clin Microbiol 2017;55:1262-1268.

• Species-specific breakpoints: (by CLSI and EUCAST) the distribution of wild-type MICs differs between species, with the previous species-independent breakpoints often very far from the ECV. Using the species-independent breakpoints, then, may not detect the development of new resistance until much later.
• Further Reading: Pfaller J Clin Microbiol 2012 50(9): 2846, CLSI M59 and M27, the SOP for yeast susceptibility reporting.