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Clinical Microbiology > Intro to Mycology > Flashcards

Intro to Mycology Flashcards

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1
Q

What are bacteria?

A

Bacteria are prokaryotes- they do not have a true nucleus. They are part of kingdom bacteria, which includes eubacteria and archaea (extremophiles). Eubacteria were discussed in previous units

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2
Q

How are bacteria different from eukaryotes?

A

They have different protein synthesis than eukaryotes and their cell wall has peptidoglycan. Both of these factors are good drug targets

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3
Q

Bacterial genome

A

Bacteria have circular DNA and 1-3 chromosomes. They only have exons- 95% of the DNA codes for genes

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4
Q

What are fungi?

A

Eukaryotes- have a true nucleus and many chromosomes. They have DNA introns and exons, and protein synthesis is similar to other eukaryotes. They do not have peptidoglycan

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5
Q

Kingdom fungi

A

Includes slime molds, mushrooms, smuts, rusts, mildews, molds, stinkhorns, puffballs, truffles and yeasts.

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6
Q

Why are fungi classified in kingdom fungi? (5)

A
  1. Absorb food directly through their cell walls
  2. Reproduce sexually and asexually
  3. None conduct photosynthesis- only plants conduct photosynthesis
  4. Absorptive Heterotrophs
  5. Saprobic, parasitic, or mutualistic
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7
Q

Absorptive heterotrophs

A

The organism utilizes complex organic material from the environment for energy source.
Fungi are good recyclers-they decompose dead organisms and waste. They also decompose cellulose, lignin and keratin. Digestive enzymes are secreted outside of the cell(s) to break down large molecules in the environment. The smaller molecules are then taken into, and used by, the fungal cell

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8
Q

Saprobes

A

Absorb nutrients from dead organic matter

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9
Q

Parasites

A

Absorb nutrients from living hosts

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10
Q

Mutualists

A

Derive nutrients from other organisms in a way that benefits both partners.

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11
Q

Mycology

A

Study of fungi (yeasts AND molds). Fungi are also called Thallophytes - considered lower plants

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12
Q

Structure of fungi

A

One cell or a relatively undifferentiated mass of cells called a thallus, instead of having an organized plant body (stem, root, and leaf).

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13
Q

Characteristics of fungi (6)

A
  1. Aerobic
  2. Eukaryotic with membrane-bound nucleus
  3. Cell wall of chitin
  4. Ergosterol cell membrane
  5. Requires carbon source
  6. Either multicellular (mold or mushroom) or unicellular yeast
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14
Q

Fungi cell wall

A

Made of chitin, 5-10% protein with 50-60% carbohydrate polymer. The cell wall is responsible for alkali resistance and helps to resist osmotic pressure. It also provides strength

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15
Q

Fungi cell membrane

A

Made of ergosterol (provitamin D2) or zymosterol (unsaturated sterol, resembles ergosterol, intermediate in the synthesis of cholesterol)

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16
Q

Yeast morphology

A

Yeast are unicellular and have oval to round cells. They bud to form daughter cells (blastoconidia or blastospores). Following mitosis, one daughter nucleus is sequestered in a small bleb outgrowth of cytoplasm that is isolated from the parent cell by the formation of a new wall.

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17
Q

Blastoconidia (blastospores)

A

Yeast daughter cells

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18
Q

Mold morphology

A

They are multicellular and made of hyphae, or can be made of pseudohyphae. They rapidly grow, and may be able to produce spores in fruiting structures

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19
Q

Pseudohyphae

A

Also called false hyphae, found in molds. They are elongated blastoconidia, constricted at their point of attachment, true hyphae are not constricted

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20
Q

Hyphae

A

Long strands of cells found in molds. Septate hyphae have crosswalls, hyphae without crosswalls are called aseptate/nonseptate or coenocytic

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21
Q

Mycelium

A

Mass/group of hyphae

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22
Q

Dimorphic fungi

A

Fungi with a yeast phase (tissue phase) at 35-37 degrees. They can also have a mold (hyphal) phase at 25-30 degrees.

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23
Q

Fungal morphology

A

Have spores- single celled, reproductive structures. They can reproduce sexually or asexually. Spores are extremely small and easily spread, and they may cause infection upon inhalation or entry into skin abrasion

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24
Q

Fungi sexual reproduction

A

Fusion of two compatible haploid nuclei to form a zygote

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25
Q

Fungi asexual reproduction

A

Nuclear and cytoplasmic division or mitosis to produce two or more identical cells.

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26
Q

Risk factors contributing to rising incidence of fungal infections (4)

A
  1. Transplant recipients
  2. Immunosuppression
  3. Underlying medical conditions, especially diabetes mellitus
  4. Previous antimicrobial therapy
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27
Q

Modes of transmission for fungi (4)

A
  1. Inhalation of conidia- must work with molds in type 2 BSC & wear gloves
  2. Traumatic Inoculation - contact with soil
  3. Medical Devices (e.g., catheter)
  4. Person to person (yeasts)
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28
Q

Intoxication

A

Accidental or recreational ingestion of fungal metabolites: alkaloids, psychotropic chemicals, aflatoxin, other toxic substances. 2 types- mycotoxicosis and mycetismus

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29
Q

Mycotoxicosis

A

Ingestion of mycotoxins produced from fungus, not be ingestion of fungus itself

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30
Q

Mycetismus

A

Mushroom poisoning resulting from eating mycotoxins found within the intact mushroom.

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31
Q

Types of fungal diseases (4)

A
  1. Intoxication
  2. Hypersensitivity disease
  3. Colonization
  4. Infection
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32
Q

Hypersensitivity disease

A

Allergic disease. Type 1 hypersensitivity is fungal spore exposure. Inhalation can cause asthma symptoms, skin exposure can cause eczema. Nasal or mucous membrane exposure can cause rhinitis or hay fever

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33
Q

Colonization of fungi

A

Yeast isolates are commonly identified as normal microbiota of skin and mucous membranes

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34
Q

Fungal infection

A

Ranges from mild and self-limiting or severe life threatening. Opportunistic or true-pathogenic isolates regardless of immune status

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35
Q

4 categories of fungal infection

A
  1. Superficial and cutaneous
  2. Subcutaneous mycoses
  3. Systemic mycosis
  4. Opportunistic
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36
Q

Superficial and cutaneous infections

A

Example- dermatophytes (Trichophyton). Affects superficial layers of skin, hair, and nails. Cause little or no inflammation

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37
Q

Subcutaneous mycoses

A

Example- sporothrix schenckii. Includes localized infections of subcutaneous tissue following the traumatic implantation of the aetiologic agent. Results in significant inflammation

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38
Q

Systemic mycosis

A

Examples- Blastomyces, Cryptococcus. Deep tissue and organ infection resulting from dissemination of from other area (lung, skin, traumatic injury)

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39
Q

Opportunistic mycosis

A

Examples- Aspergillus, Zygomycetes , Candida. Occurs in immunocompromised individuals

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40
Q

Level 1 laboratory service

A

Direct examination of clinical samples for fungal elements

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41
Q

Level 2 laboratory service

A

Level 1 plus identification of yeasts using commercially available systems (API 20C, Uni-Yeast Tek, etc.). Also includes latex agglutination for detection of cryptococcal antigen and performance of commercial fungal immunodiffusion tests

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42
Q

Level 3 laboratory service

A

Level 2 plus identification of filamentous fungi

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43
Q

Level 4 laboratory service

A

Level 3 plus identification of all fungi. Includes performance of all fungal serological tests and performance of fungal susceptibility tests

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44
Q

Types of fungal specimens (11)

A
  1. Blood
  2. Bone marrow (intracellular pathogens)
  3. Sterile body fluids
  4. Tissue
  5. Respiratory tract
  6. Urine
  7. Hair, skin, or nails- Dermatophyte
  8. Oropharyngeal samples
  9. Vaginal secretions
  10. External eye
  11. Exudates, pus, and drainage
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45
Q

Collection of blood specimens

A

Uses lysis centrifugation- Wampole Isolator System. This method must be used for H. capsulatum, and is plated onto media absent of cycloheximide. Special media is used- a BACTEC MYCO/F lytic bottle with 20-30 ml blood volume. Continuous monitoring systems can also be used

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46
Q

Collection of bone marrow specimens

A

Heparinized marrow is inoculated into isolator tubes. .5 ml sample minimum is necessary. Blood culture bottles are not recommended in this situation

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47
Q

Collection of sterile body fluid specimens

A

<2mls direct to media, >2mls centrifuge, plate pellet. All but CSF may be refrigerated before plating

48
Q

Collection of tissue samples

A

Ground and homogenize the sample and direct inoculation onto media. If Zygomycete is suspected, mince/slice sample

49
Q

Collection of respiratory tract samples

A

The first morning specimen should be collected. Plate flecks of blood or pus, and decontaminate/liquify as you would for Mycobacterium

50
Q

Collection of urine samples

A

A 10-50 ml sample should be put through a centrifuge, plate the sediment with and without cycloheximide. You can also plate for quantification with calibrated loop (un-centrifuged sample). Interpretation of CFU not established with clinical picture as for bacteria

51
Q

How to collect hair, skin, and nail samples

A

Plate directly onto media. 10-12 affected hairs should be removed with forceps and you can check for fluorescence with Wood’s lamp. For skin, scrape the surface of the skin at an active margin. There are multiple methods of collection for nails

52
Q

Collecting nail samples (3)

A
  1. Collect debris from under the nail, or
  2. Scrape outer surface of nail and discard. Collect scrapings from the deeper, diseased areas of the nail and place in envelope.
  3. Nails cut into small pieces, pushed into agar have best recovery
53
Q

How to collect oropharyngeal samples

A

Thrush can be diagnosed by microscopic examination of material. Use CHROMAgar

54
Q

How to collect vaginal secretions

A

Diagnosed by clinical symptoms and microscopic examination. 20% of healthy females are colonized with yeast as normal flora, so culture identification is meaningless without clinical information

55
Q

How to collect samples from the external eye

A

Surgically obtained corneal scraping for mycotic keratitis is plated bedside directly onto non-inhibitory media

56
Q

How to collect exudates, pus, and drainage

A

Examine for granules, plate directly onto media. Crush granules, examine for hyphae, then plate

57
Q

Transport and storage of fungal specimens

A

Process and plate within 2 hours of collection. Leave at room temperature- avoid excessive heat or cold. Biopsy specimens should be kept moist. Skin, hair, and nail samples should be kept in a clean, dry envelope.

58
Q

Which fungal specimens should not be left at room temperature?

A

The only exceptions to this principle are if there is a delay in processing of CNS samples- store at 30 degrees in this case. Also, if urine or respiratory specimens are contaminated with bacteria, they should be stored at 4 degrees Celsius

59
Q

Processing of fungal specimens (2 steps)

A
  1. Direct smears
  2. Cultures for ID
60
Q

Direct microscopic examinations of specimens for fungal culture (9)

A
  1. Saline or wet preparation
  2. Lactophenol cotton blue (LPCB)
  3. 10-40% KOH
  4. Gram stain
  5. Modified Kinyoun acid fast smear
  6. India Ink
  7. Periodic acid Schiff (PAS)
  8. Grocott-Gomori’s methenamine silver stain (GMS)
  9. Calcofluor white
61
Q

Saline or wet preparation of fungal specimens

A

Detect fungal elements: hyphae, pseudohyphae, Blastoconidia

62
Q

Lactophenol cotton blue (LPCB)

A

Lactic acid preserves fungal structures, and phenol kills organisms. Cotton blue stains chitin in fungal cell walls which helps visualize fungal elements

63
Q

10-40% KOH

A

Dissolves keratin in tissue cells. Fungal cells survive because chitin resists alkali

64
Q

Gram stain of fungal specimens

A

Yeasts stain purple, but they are not considered gram positive because they do not contain peptidoglycan. They are 2-4 times larger than bacteria. The hyphae often stain in a granular pattern

65
Q

Modified Kinyoun acid fast smear

A

A basic fuchsin/carbol fuchsin stain and methylene blue counterstain. The organisms stain red against a blue background. Yeast ascospores stain red, Blastoconidia stain blue, and other bacteria and fungi stain blue

66
Q

India ink

A

Stains fungal organisms that have capsules: Cryptococcus, Rhodotorula, Sporobolomyces, and Trichosporon beigelii.
Prototheca (an algae) also has a capsule

67
Q

Periodic acid Schiff (PAS)

A

Stain reaction with polysaccharides found in the cellulose and chitin present in fungal cell wall. Magenta organisms against a light pink, green or blue background

68
Q

Grocott-Gomori’s methenamine silver stain (GMS)

A

Stain reaction with fungal cell wall forms aldehydes, methenamine silver reacts forming brown/black ppt and color against greenish background. Better stain contrast than PAS.

69
Q

Calcofluor white

A

Fluorochrome dye concentrates in fungal cell walls. The organisms will fluoresce blue-white or green using UV microscope

70
Q

In which specimens are fungi clinically important?

A
  1. Sterile body fluid – CSF, blood, pleural fluid, synovial fluid
  2. Immunocompromised patient – any specimen site
  3. A routine culture growing large quantities of yeast/mold-sputum growing 4+ yeast or filamentous fungus
  4. A fungal isolate repeatedly growing from same clinical source, like sputum growing as much Aspergillus sp. as normal flora. This is especially true if no other pathogenic isolate is found an symptoms persist.
71
Q

Sterile body fluids (4)

A

CSF, blood, pleural fluid, synovial fluid

72
Q

Media used for fungal isolates (5)

A
  1. Sabouraud dextrose agar (SAB)
  2. SAB with cycloheximide and chloramphenicol (mycosel agar) pH 7.0
  3. Brain-heart infusion (BHI)
  4. Bird seed agar (niger seed agar)
  5. Cornmeal with Tween 80 agar
73
Q

When is SAB with cyclohexamide and chloramphenicol used?

A

Growth of dermatophytes and other pathogens

74
Q

When is Brain-heart infusion (BHI) used?

A

For recovery of systemic mycoses

75
Q

When is Bird seed agar used?

A

For Cryptococcus neoformans

76
Q

Cornmeal with Tween 80 agar

A

Used for differentiation of Candida sp. Look for chlamydospores, hyphae, pseudohyphae, arthroconidia, etc

77
Q

Temperature of fungal growth

A

22-30 degrees C and sometimes 35-37 degrees C

78
Q

How long do fungi take to incubate?

A

2-4 weeks, but systemic fungi may take up to 12 weeks. Yeasts grow faster than molds

79
Q

Growth rates of fungi (3)

A
  1. Rapid, <24 hr to 5 days-zygomycetes “contaminants”
  2. Intermediate (1-5 days) - hyaline and dematiaceous molds “contaminants”
  3. Slow growers (1-4 weeks) - systemic pathogens
80
Q

Fungal colony morphology

A

Classified by texture, due to height of aerial hyphae

81
Q

Methods for identification of fungal isolates (3)

A
  1. Biochemical/assimilation panels
  2. MALDI-TOF
  3. PCR
82
Q

Methods of molecular testing (3)

A
  1. Real-time PCR
  2. NASBA
  3. PCR
83
Q

Fungal species identified by PCR (5)

A
  1. Aspergillus sp.
  2. Candida sp.
  3. Cryptococcus
  4. Zygomyces
  5. Dimorphic fungi
84
Q

Serological tests (4)

A
  1. Complement fixation
  2. Immunodiffusion
  3. Agglutination
  4. Indirect fluorescent antibody (IFA)
85
Q

When should serological tests be used?

A

Best used in immunocompetent individuals

86
Q

Antigen detection of dimorphic fungi

A

Cross reaction amongst dimorphic fungi. Blastomyces, Paracoccidioimycosis, P.marneffei

87
Q

Antigen detection of Aspergillus

A

Galactomannan (GM), polysaccharide cell wall components

88
Q

Antigen detection of Candida

A

Difficult to distinguish colonization vs. disease. Mannan and mannoproteins = cell wall component

89
Q

Antigen detection of Cryptococcus neoformans

A

Glucuronoxylomannan=capsular polysaccharide. 5 serotypes = A,B,C,D,AD

90
Q

(1-3) Beta-D-glucan PANFUNGAl detection

A

Diagnostic marker invasive fungal infection
Polysaccharide cell wall component in most fungi. Except Zygomycetes.

91
Q

Candida metabolites (2)

A

Fungal polyols- DA can be detected in the urine or serum. Produced by all Candida except C. krusei. Used along with antigen detection methods to determine presence of fungal infection.
1. D-arabinitol (DA)-increased in renal dysfunction, report ratio using creatinine
2. D-mannitol (DM)-not produced by mammalian metabolism

92
Q

Antifungal Therapies

A

There are less antifungal agents available compared to antibacterial options. There are toxicity issues as fungal cells are similar to human cells- both eukaryotic organisms. Best options target unique fungal characteristics- fungal cell wall and membrane. Almost all fungus isolates share cell wall constituents providing broad antifungal activity

93
Q

Fungistatic drugs

A

Stops organism growth but does not kill isolate, requires functioning immune response for organism elimination

94
Q

Fungicidal drugs

A

Kills organism

95
Q

Delivery of antifungals

A

Can be topical, oral, or intravenous. Infection location and patient history considered.

96
Q

Antifungal drugs (6)

A
  1. Amphotericin B
  2. Nystatin
  3. Flucytosine
  4. Azoles
  5. Echinocandins
  6. Terbinafine
97
Q

Antifungal susceptibility testing (6)

A
  1. CLSI M27-A guidelines for Candida & Cryptococcus sp.
  2. Macrobroth, microtiter, agar dilution
  3. Endpoint 80% inhibition of fungal growth with azoles
  4. Endpoint 100% inhibition of growth with amphotericin B
  5. Etest (AB Biodisk)
  6. Alamar/Sensititre
98
Q

Alamar/Sensititre

A

A blue solution is added to microtiter wells. Fungal growth reduces media - turns pink endpoint - lowest dilution that remains blue

99
Q

Antifungal drug classes (4)

A
  1. Polyene antifungal class- Amphotericin B, Nystatin
  2. Anti β(1,3) D-glucan synthase- echinocandins
  3. Allylamine class- Terbinafine
  4. Azole class
100
Q

Amphotericin B (fungizone)

A

Often chosen as 1st line treatment, I.V. administration.
Fungistatic or fungicidal depending upon concentration. Mode of action – Binding to ergosterol in cell membrane resulting in pore formation, causing leakage of potassium and other cell components resulting in cell death. Found to bind to cholesterol in mammalian cell membranes. Can be immunostimulatory inducing cytokine production IL-1B, TNF. Issues- shock can occur upon administration, renal (nephro) toxicity in 80% of patients within 2 weeks of therapy.

101
Q

Amphotericin B target organisms (5)

A

Broad usage – Candida sp. Cryptococcus, all dimorphic isolates, Aspergillus and some activity against protozoa.
In general resistance is unusual in most isolates. AIDS patient associated with resistant yeast isolates – Candida, Cryptococcus

102
Q

Which organisms have intrinsic resistance against Amphotericin B (4)

A

Scedosporium, Fusarium, Candida lusitaniae, guilliermondii

103
Q

Nystatin (mycostatin, nilstat, nystex)

A

Most commonly used as a topical agent to treat yeast infections, oral treatment for thrush or yeast induced gastroenteritis. Fungistatic or fungicidal depending upon concentration. Irreversible interaction with sterols, including ergosterol in plasma membrane resulting in pore formation resulting in leakage of cellular contents. Although very similar to Amphotericin B, not commonly IV delivered due to toxicity. Problems - renal (nephro) toxicity

104
Q

Nystatin target organisms (4)

A

Broad usage especially useful for systemic Candida sp. infections, Cryptococcus, all dimorphic isolates, Aspergillus

105
Q

Echinocandins

A

Fungicidal for Candida sp. AND fungistatic for Aspergillus sp.
Mode of action – inhibitor of β(1,3) D-glucan synthase, a needed enzyme for cell wall β(1,3) D-glucan polysaccharide synthesis. Problems – well tolerated, rare problems with fever, rash, GI upset.

106
Q

Echinocandins target organisms

A

Broad spectrum against Candida sp. and Aspergillus sp. however not active against Zygomycetes, Cryptococcus neoformans, or Fusarium sp

107
Q

Terbinafine (Lamisil)

A

Used to treat skin infections with yeast, can have oral or topical administration. Fungicidal for dermatophytes, molds, dimorphic fungi AND fungistatic for Candida albicans. Mode of action – disruption of ergosterol synthesis by inhibiting squalene epoxidase allowing an accumulation of the product and lack of ergosterol production. Problems – rare reports of hepatotoxicity, exacerbation of lupus erythematosus, GI upset

108
Q

Terbinafine target organisms (3)

A

Candida sp., dermatophytes Trichophyton sp.

109
Q

Flucytosine (5-fluorocytosine)

A

Oral agent, limited use as a single agent therapy, since resistance is problematic. Fungistatic drug. Mode of action – Taken up by fungal cells by using a fungal-specific cytosine permease it then inhibits protein synthesis and DNA synthesis. Problems – rapid resistance, hepatic toxicity rarely reported.

110
Q

Flucytosine (5-fluorocytosine) target organisms

A

Cryptococcus sp. resistance problems encountered, utilized for non-invasive Candidia sp.(urinary)

111
Q

Griseofulvin

A

Oral agent, common for dermatophyte infections. Fungistatic drug. Mode of action – arrests metaphase by disrupting mitotic structures, stopping cell division. Problems – none currently documented, resistance not detected

112
Q

Griseofulvin target organisms

A

Dermatophytes – fungal infections found in the skin, hair and nails.

113
Q

Azoles

A

Oral drugs for yeast and molds. For yeasts, they are fungistatic- ketoconazole, fluconazole, clotrimazole, miconazole, voriconazole. For molds, they are fungicidal- 2nd generation drugs - posaconazole, voriconazole, ravuconazole. Mode of action – Inhibition of fungal cytochrome P-450 enzyme needed for the synthesis of ergosterol in the cell membrane

114
Q

Azoles target organisms

A

Candida sp. and 2nd generation drugs can be utilized for the treatment of molds. There are some resistance issues- Candida krusei has intrinsic resistance to fluconazole. There are increased findings of resistance in Candida sp. isolated from immunocompromised hosts.

115
Q

Problems with azole drugs

A

Rash, rare GI disruption, rare report of hepatic disease, some optic concerns with voriconazole (30% patient report blurry vision, photophobia)

116
Q

Ketoconazole mechanism

A

Fungistatic, disrupts cell membrane integrity

Clinical Microbiology (19 decks)

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