Yeasts Flashcards
Yeasts
Eukaryotic, unicellular organisms. They are oval, elongate, or irregular shaped. They multiple by producing Blastoconidia – budding, pseudohyphae. Colonies are moist, creamy, and glabrous. They may produce a capsule (mucoid) or may be darkly pigmented (melanin production). Inhibited by cyclohexamide
How are yeasts identified?
Macro and microscopic methods- corn meal, rapid biochemical testing, and molecular methods in development
Yeasts as normal flora
Yeast are normal microbiota and common contaminants. Interpretation of a significant isolate difficult from site where usually considered normal microbiota- sputum and stool vs sterile body sites (blood, CSF, etc.)
Considerations for patients with yeast infections
Need to consider the quantity of yeast vs. bacteria (normally less than bacteria). Also, if the patient is on antimicrobial treatment, this could increase yeast. Not reporting Genus and species may lead to consequences-Candida tropicalis can be aggressive in immunocompromised people.
Isolation from sterile body sites is always considered a significant isolate.
Specimen collection and transport
Special transport is not needed, but it should be less than 2 hours. If there is a delay for sterile body site specimens place at 37°C. If there is a delay for specimen containing normal flora bacteria, the recommendation is 4°C. Yeast can withstand refrigeration
Special plating considerations (2)
There are special considerations if ruling out:
1. Coccidiodes immitis – highly infectious
2. Malassezia furfur – lipid growth requirement
Microscopic identification of direct yeast specimens (5)
- Gram stain of specimen-budding yeast, pseudohyphae OR true hyphae
- Wet prep – patient sample and saline for review
- 0-40% KOH
- Calcofluor white stain
- India Ink – capsule visualization
Growth characteristics of yeast
Most grow well on both yeast/fungus and bacteria specific media, but Malassezia sp. is an exception. 48 to 72 hours is necessary for good colony growth. Temperature-
30 or 37°C. Yeast form smooth, creamy colonies. Morphology-pigment, “Feet”, Mucoid – capsule
Sabouraud dextrose agar
Mycology specific media lacking cyclohexamide- primary plated media
Germ tube
Hyphal-like extension of yeast cells that are not constricted. Yeasts are incubated with serum at 37oC for up to 3 hours and examined microscopically. Candida albicans, tropicalis =(>2hrs), dubliniensis (not in commercial media). Rapid and inexpensive test, incubation of less than 3hr is a false positive
Rapid Systems
Use carbohydrate or nitrate assimilation for full identification. Identification systems include Vitek-Yeast Biochemical Card, Microscan Rapid Yeast ID and API 20C, API Candida, Uni-Yeast Tek. Also includes Cornmeal Tween 80
Assimilation
Utilization of carbon or nitrogen source by a microorganism in the presence of oxygen
Biochemical identification of yeast (9)
- Germ tube
- Rapid systems- MALDI-TOF, PCR
- Chromogenic agars - CHROMagar Candida
- Phenol oxidase test
- Urease test
- PNA-FISH
- Molecular methods
- Rapid nitrate assimilation
- Rapid trehalose
Cornmeal Tween 80
basic media utilized to cultivate the growth of many fungal isolates while also inhibiting the growth of contaminating bacterial flora and some saprophytic fungi. Identification is confirmed with morphology. Chlamydospore development and Blastoconidia, arthroconidia, pseudohyphae and true hyphae. Used for Candida Albicans.
Nitrate assimilation is used for which species? (3)
Best for ID of Candida, Cryptococcus or Rhodotorula spp.
Chromogenic agars
Includes CHROMagar Candida. Differentiation of 10 species, presumptive ID of C. albicans, C. tropicalis, C. krusei. Identifications are only presumptive, variation in colony color is found
Phenol oxidase test
Detects ability of Cryptococcus neoformans to produce phenol oxidase on substrates containing caffeic acid. Uses bird seed agar - niger or thistle seed. Dark brown to black growth - Cryptococcus neoformans
Urease test
Test for urease using a urea slant, takes 24 to 72 hours. Turning a pink color is a positive result. ID for Cryptococcus sp., Rhodotorula sp., Trichosporon sp.
PNA-FISH
Full identification in 2.5 hours- commercial peptide nucleic acid fluorescent in situ hybridization. Identification of C. albicans from blood cultures only using 26S rRNA. Peptide-nucleic acid chains mimic rRNA, hybridize to target rRNA
Molecular methods (3)
- T2 Biosystem
- NAAT
- BioFire for blood culture
Rapid nitrate assimilation
Use of KNO3 as a sole source of nitrogen- reduction of nitrate to nitrite. Differentiate between Cryptococcus spp. isolates and Rhodotorula
Rapid trehalose
Utilization of trehalose in the presence of a protein inhibitor. C. glabrata degrades trehalose quicker, producing increased acid as compared to other yeasts.
Commonly isolated pathogenic yeasts (7)
- Candida species
- Cryptococcus sp.
- Trichosporon sp.
- Malassezia sp.
- Rhodotorula sp
- Saccharomyces sp.
- Geotrichum sp.
Candida species (10)
- Albicans/dubliniensis
- Auris
- Tropicalis
- Glabrata complex
- Parapsilosis complex
- Pichia kudriavzevii
- Kluyveromyces marxianus
- Clavispora lusitaniae
- Meyerozyma guilliermondii
- Yarrowia lipolytica
Cryptococcus species (4)
- Neoformans
- Gattii
- Albidus
- Laurentii
Malassezia species (5)
- Furfur
- Globosa
- Sympodialis
- Obtusa
- Restricta
Which Candida species are most invasive? (5)
95% of all invasive Candida infections are caused by 5 species:
1. Albicans
2. Glabrata complex
3. Tropicalis
4. Parapsilosis
5. Pichia kudriavzevii (formerly Candida krusei)
Infections caused by Candida species (8)
- Oral candidiasis (thrush)
- Vaginal candidiasis
- Cutaneous candidiasis
- Onychomycosis
- Bronchopulmonary
- Urinary tract infection
- Intestinal candidiasis
- Systemic
Oral candidiasis (thrush) risk factors (9)
Risk factors- infants or elderly, immunosuppressed (AIDS), transplant recipients
leukemia, cancer, TB, diabetes
oral contraceptives, antibacterial treatment
Vaginal candidiasis risk factors
Pregnancy or diabetes. Needs to be isolated repeatedly
Cutaneous candidiasis
Red, inflamed patches that can be localized or disseminated. Yeast are isolated in large numbers. Risk factors- obese, alcoholic, profuse sweating
Onychomycosis
Candidiasis of nail
Bronchopulmonary candidiasis
A secondary infection following antimicrobial therapy. Symptoms- irritating cough, mucoid sputum. Pneumonia is visible on x-ray
Intestinal candidiasis
Follows antimicrobial therapy that suppresses normal flora. Stool culture has predominant yeast growth
Systemic candidiasis
Hematogenous spread of the fungus occurring in debilitated patients. Inoculation can occur by medical devices or break in the skin barrier. Complications of the infection include endocarditis, meningitis, brain abscess, sepsis. There is a 19-24% mortality rate
Candida albicans habitat
In is normal flora of the skin, mouth, vagina, and stool. It is also found in the environment in leaves, flowers, water, and soil
Clinical significance of Candida albicans
Most common cause of mycoses worldwide. Grows at 42-45 degrees Celsius and is germ tube positive
Macroscopic and microscopic features of Candida albicans
Macroscopic- colonies appear cream colored, pasty, smooth, colonies have feet. CHROMagar -light green to light bluish-green. Microscopic- single yeast, budding, pseudohyphae
How do candida albicans appear on cornmeal?
Pseudohyphae, clusters of Blastoconidia, thick-walled terminal, single chlamydospores
Clinical significance of candida glabrata
Cause bloodstream, urogenital, lung and other infections. Difficult to treat, resistant to azole, reduced susceptibility to Amphotericin B. Echinocandins can be used for treatment.
How does candida glabrata appear on cornmeal?
There is only a small, single, oval budding yeast
How does Candida glabrata appear macroscopically and microscopically?
Macroscopic- colonies are small, white to cream, pasty, and smooth
Microscopic- single budding yeast (condida), there are no pseudohyphae
Clinical significance of Candida tropicalis
Found in immunocompromised people, similar to all other Candida species. It is problematic in leukemia patients and other cancer patients
How does candida tropicalis appear macroscopically and microscopically?
Macroscopic- colonies are cream colored and the edges may be wrinkled. Appear metallic blue on CHROMagar.
Microscopic- round to oval yeast cells
How does candida tropicalis appear on cornmeal?
Oval blastospores may appear singly or in clusters located along the long pseudohyphae. Pseudohyphae branch abundantly, rare true hyphae
Clinical significance of candida dubliniensis
Found worldwide. It is frequently associated with HIV resulting infections and bloodstream infections in non-HIV immunocompromised patients. These fungi do not grow at 42-45 degrees Celsius and are germ tube positive
How does candida dubliniensis appear macroscopically and microscopically?
Macroscopic- colonies are white to cream, soft, and can be smooth or wrinkled. On CHROMagar, they appear dark green
Microscopic- only blastoconidia
How do candida dubliniensis appear on cornmeal?
Branched pseudohyphae & true hyphae with Blastoconidia are present. Abundant chlamydoconidia includes single, pairs, chains, and clusters.
Candida auris habitat
Hospital and nursing home acquired. Found in many body sites and fluids, including blood, urine, respiratory and abdominal specimens
Clinical significance of Candida auris
These infections are very serious, they can cause significant bloodstream or brain infections that may lead to death. They are difficult to identify on automated systems since they can’t be differentiated from other species- MALDI-TOF or DNA sequencing must be used for an accurate identification. Candida auris is germ tube negative and grows well at 40-42. It has multi-colored colonies on CHROMagar
Candida auris treatment
Treatment is difficult since these fungi are resistant to many antifungals. Resistant to azoles, possible amphotericin B, 5FC, echinocandin resistance
How does Candida auris appear macroscopically and microscopically?
Macroscopic- white to cream
Microscopic- Blastoconidia (budding yeast), almost never forms pseudohyphae
Candida parapsilosis morphology
Radiating pseudohyphae and scattered blastoconidia, producing what are known as “spider” colonies
Pichia kudriavzevii morphology
Formerly Candida krusei- long, wavy, branched pseudohyphae with elongated to ovoid Blastoconidia, budding off in branches.
C. neoformans habitat
Pigeon roosts, fruit, milk, plants. Aerosolization and inhalation of pigeon excrement is thought to be the infection route
C. gatti habitat
Tropical and subtropical regions. US/Canada (BC, Vancouver, Washington) – trees, soil, water, wood products. Caused by inhalation of spores from the environment
Clinical significance of C. neoformans
Inhibited by cyclohexamide, affects the immunocompromised especially HIV(meningitis). Causes CNS infections, lesions on the skin, in the lungs, and other organs. Primarily a pulmonary disease. Latex agglutination for detection of cryptococcal antigen simple, sensitive and reliable - detects 90% of all cryptococcal meningitis cases.
C. gattii clinical significance
Inhibited by cyclohexamide, recent pathogen, affects immunocompetent individuals. The primary site of infection is the lungs, where it causing pneumonia. Dissemination to other organs, notably the central nervous system (CNS), causes meningoencephalitis or brain cryptococcomas. Increasingly found in immunocompromising conditions.
How do cryptococcus species (neoformans, gattii) appear macroscopically?
Both strains are identical. Colonies are cream then turn a tan color as they age. They are flat or heaped, mucoid and have smooth edges. Phenol oxidase positive, form brown to black colonies on bird seed or caffeic acid
How do cryptococcus species (neoformans, gattii) appear microscopically?
The fungi are round, dark-walled, budding yeast. They produce capsules
How do cryptococcus species (neoformans, gattii) appear on cornmeal?
Round, dark-walled budding yeast- no hyphae
Saccharomyces sp habitat
Commonly isolated from humans, mammals, birds, wine, beer, fruits, trees, plants, olives, and soil. Common colonizer of mucosa surface, known as “baker’s” or “brewer’s” yeast
Saccharomyces sp clinical significance
Non-pathogenic for immunocompetent, but RARE report of fungemia & aortic graft infection in immunocompetent.
Severe immunosuppression causes mucosal and disseminated disease-pneumonia, endocarditis, liver abscess, fungemia, and sepsis
Saccharomyces sp macroscopic appearance
Rapid growth, mature in 3 days. Colonies - flat, smooth, moist, glistening or dull, cream to tannish color
Saccharomyces sp microscopic and cornmeal appearance
Blastoconidia - unicellular, ellipsoid to elongate, multilateral budding is typical. Pseudohyphae, if present, are rudimentary, hyphae are absent
Saccharomyces cerevisiae virulence factors
So far, none have been identified. In a case review of 3300 yeast infection of cancer patients, 19 were identified as S. cerevisiae. In another case review, of 138 systemic fungal infections over a 15 year period, only 2 identified as S. cerevisiae. It was found that strains recovered from infected immunocompromised hosts were more resistant to host oxidative stresses. Can cause mucosal and disseminated infection in significantly immunocompromised hosts.
Malassezia sp habitat
Lipophilic yeast found on skin and body surfaces of humans and animals
Malassezia sp clinical significance
Causes catheter-associated fungemia in neonates and adults receiving prolonged intravenous lipids. May also cause lung infections. Symptoms- tinea versicolor or pityriasis versicolor – multi-colored skin
Malassezia sp macroscopic appearance
Smooth, cream to yellowish brown, with age – dry, wrinkled and brittle
Malassezia sp microscopic appearance
These fungi do not bud. They form phialides with small collarettes. Thick round to oval yeast-like cells in clusters, spaghetti and meatballs morphology
Malassezia sp appearance on cornmeal
Hyphal elements rare, phialides
Malassezia sp virulence factors
Nutritional requirements leads to pathogenesis- the organisms are unable to synthesize medium and long chain fatty acids. Patients receiving central-line mediated total parenteral nutrition supplementation can be at increased risk of infection.
10% of newborns in non-NICU colonized. Additionally, 80% of newborns in the NICU are colonized. There is an increased association with prematurity, immunocompromised people, prolonged antimicrobial use and prolonged hospital stay. Infection causes fever, respiratory distress, sepsis, and it most commonly causes superficial skin infections
Growth of M. furfur
M. furfur requires lipid supplementation for in vitro growth. Add olive oil to culture
Rhodotorula sp habitat
Air, soil, lakes, ocean water, and dairy products. Colonizes plants, humans, other mammals
Rhodotorula sp. clinical significance
Opportunistic mycoses in AIDS or acute leukemia patients - meningitis, endocarditis, ventriculitis, peritonitis, endophthalmitis, central venous catheter-infections, fungemia
Rhodotorula sp. macroscopic appearance
Rapid grower, smooth, glistening, mucoid - cream to pink, coral red, orange or yellow colonies
Rhodotorula sp. microscopic and corneal appearance
Blastoconidia- unicellular, elongate in shape, may be encapsulated. Pseudohyphae and hyphae are absent
Geotrichum sp. habitat
Worldwide - soil, water, air, sewage, plants, cereals, and dairy products. They are normal human flora from sputum and feces, colonizer GI tract
Geotrichum sp. clinical significance
Opportunistic infections (inhalation or ingestion) = geotrichosis. Causes bronchial, pulmonary, disseminated infections, and infections from trauma
Geotrichum sp. macroscopic appearance
Rapidly growth, white, dry, powdery to cottony colony, resembling “ground glass.” When disturbed on the surface, the colony becomes yeast-like or slimy. The optimal growth 25°C, most strains = NG or weak growth at 37°C
Geotrichum sp. microscopic appearance
Arthroconidia and coarse true hyphae are observed. Formation of arthroconidia and absence of empty cells that fragment to release arthroconidia (“disjunctor cells”) are typical. Blastoconidia, conidiophores, pseudohyphae = absent.
Trichosporon sp. habitat
Found in soil, water samples, vegetables, mammals, and birds. They are normal flora of the mouth, skin and nails
Trichosporon sp. clinical significance
White Piedra – forms white nodules on the hair shaft. Causes superficial and deep disseminated infections in humans. At risk populations include neutropenic patients and immunocompromised individuals
Trichosporon sp. macroscopic appearance
Cream progresses to a yellow/gray color. Appears smooth at first, then wrinkled and powdery-like.
Trichosporon sp. microscopic appearance
Oblong yeast cells
Trichosporon sp. cornmeal appearance
True hyphae, pseudohyphae with Blastoconidia singly or in short chains. Arthroconidia on older cultures
Chlamydospore
Chlamydospores are produced by many fungi and represent enlarged, thick-walled vegetative cells with varied forms and condensed cytoplasm
Blastoconidia
A fungal cell produced by budding
Arthroconidia
A spore that is formed from the hyphae by fragmentation. Microscopically, mature arthroconidia appear square, rectangular, or barrel-shaped with thick walls.
