Intro to Mycology Flashcards
What are bacteria?
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
How are bacteria different from eukaryotes?
They have different protein synthesis than eukaryotes and their cell wall has peptidoglycan. Both of these factors are good drug targets
Bacterial genome
Bacteria have circular DNA and 1-3 chromosomes. They only have exons- 95% of the DNA codes for genes
What are fungi?
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
Kingdom fungi
Includes slime molds, mushrooms, smuts, rusts, mildews, molds, stinkhorns, puffballs, truffles and yeasts.
Why are fungi classified in kingdom fungi? (5)
- Absorb food directly through their cell walls
- Reproduce sexually and asexually
- None conduct photosynthesis- only plants conduct photosynthesis
- Absorptive Heterotrophs
- Saprobic, parasitic, or mutualistic
Absorptive heterotrophs
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
Saprobes
Absorb nutrients from dead organic matter
Parasites
Absorb nutrients from living hosts
Mutualists
Derive nutrients from other organisms in a way that benefits both partners.
Mycology
Study of fungi (yeasts AND molds). Fungi are also called Thallophytes - considered lower plants
Structure of fungi
One cell or a relatively undifferentiated mass of cells called a thallus, instead of having an organized plant body (stem, root, and leaf).
Characteristics of fungi (6)
- Aerobic
- Eukaryotic with membrane-bound nucleus
- Cell wall of chitin
- Ergosterol cell membrane
- Requires carbon source
- Either multicellular (mold or mushroom) or unicellular yeast
Fungi cell wall
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
Fungi cell membrane
Made of ergosterol (provitamin D2) or zymosterol (unsaturated sterol, resembles ergosterol, intermediate in the synthesis of cholesterol)
Yeast morphology
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.
Blastoconidia (blastospores)
Yeast daughter cells
Mold morphology
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
Pseudohyphae
Also called false hyphae, found in molds. They are elongated blastoconidia, constricted at their point of attachment, true hyphae are not constricted
Hyphae
Long strands of cells found in molds. Septate hyphae have crosswalls, hyphae without crosswalls are called aseptate/nonseptate or coenocytic
Mycelium
Mass/group of hyphae
Dimorphic fungi
Fungi with a yeast phase (tissue phase) at 35-37 degrees. They can also have a mold (hyphal) phase at 25-30 degrees.
Fungal morphology
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
Fungi sexual reproduction
Fusion of two compatible haploid nuclei to form a zygote
Fungi asexual reproduction
Nuclear and cytoplasmic division or mitosis to produce two or more identical cells.
Risk factors contributing to rising incidence of fungal infections (4)
- Transplant recipients
- Immunosuppression
- Underlying medical conditions, especially diabetes mellitus
- Previous antimicrobial therapy
Modes of transmission for fungi (4)
- Inhalation of conidia- must work with molds in type 2 BSC & wear gloves
- Traumatic Inoculation - contact with soil
- Medical Devices (e.g., catheter)
- Person to person (yeasts)
Intoxication
Accidental or recreational ingestion of fungal metabolites: alkaloids, psychotropic chemicals, aflatoxin, other toxic substances. 2 types- mycotoxicosis and mycetismus
Mycotoxicosis
Ingestion of mycotoxins produced from fungus, not be ingestion of fungus itself
Mycetismus
Mushroom poisoning resulting from eating mycotoxins found within the intact mushroom.
Types of fungal diseases (4)
- Intoxication
- Hypersensitivity disease
- Colonization
- Infection
Hypersensitivity disease
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
Colonization of fungi
Yeast isolates are commonly identified as normal microbiota of skin and mucous membranes
Fungal infection
Ranges from mild and self-limiting or severe life threatening. Opportunistic or true-pathogenic isolates regardless of immune status
4 categories of fungal infection
- Superficial and cutaneous
- Subcutaneous mycoses
- Systemic mycosis
- Opportunistic
Superficial and cutaneous infections
Example- dermatophytes (Trichophyton). Affects superficial layers of skin, hair, and nails. Cause little or no inflammation
Subcutaneous mycoses
Example- sporothrix schenckii. Includes localized infections of subcutaneous tissue following the traumatic implantation of the aetiologic agent. Results in significant inflammation
Systemic mycosis
Examples- Blastomyces, Cryptococcus. Deep tissue and organ infection resulting from dissemination of from other area (lung, skin, traumatic injury)
Opportunistic mycosis
Examples- Aspergillus, Zygomycetes , Candida. Occurs in immunocompromised individuals
Level 1 laboratory service
Direct examination of clinical samples for fungal elements
Level 2 laboratory service
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
Level 3 laboratory service
Level 2 plus identification of filamentous fungi
Level 4 laboratory service
Level 3 plus identification of all fungi. Includes performance of all fungal serological tests and performance of fungal susceptibility tests
Types of fungal specimens (11)
- Blood
- Bone marrow (intracellular pathogens)
- Sterile body fluids
- Tissue
- Respiratory tract
- Urine
- Hair, skin, or nails- Dermatophyte
- Oropharyngeal samples
- Vaginal secretions
- External eye
- Exudates, pus, and drainage
Collection of blood specimens
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
Collection of bone marrow specimens
Heparinized marrow is inoculated into isolator tubes. .5 ml sample minimum is necessary. Blood culture bottles are not recommended in this situation
Collection of sterile body fluid specimens
<2mls direct to media, >2mls centrifuge, plate pellet. All but CSF may be refrigerated before plating
Collection of tissue samples
Ground and homogenize the sample and direct inoculation onto media. If Zygomycete is suspected, mince/slice sample
Collection of respiratory tract samples
The first morning specimen should be collected. Plate flecks of blood or pus, and decontaminate/liquify as you would for Mycobacterium
Collection of urine samples
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
How to collect hair, skin, and nail samples
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
Collecting nail samples (3)
- Collect debris from under the nail, or
- Scrape outer surface of nail and discard. Collect scrapings from the deeper, diseased areas of the nail and place in envelope.
- Nails cut into small pieces, pushed into agar have best recovery
How to collect oropharyngeal samples
Thrush can be diagnosed by microscopic examination of material. Use CHROMAgar
How to collect vaginal secretions
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
How to collect samples from the external eye
Surgically obtained corneal scraping for mycotic keratitis is plated bedside directly onto non-inhibitory media
How to collect exudates, pus, and drainage
Examine for granules, plate directly onto media. Crush granules, examine for hyphae, then plate
Transport and storage of fungal specimens
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.
Which fungal specimens should not be left at room temperature?
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
Processing of fungal specimens (2 steps)
- Direct smears
- Cultures for ID
Direct microscopic examinations of specimens for fungal culture (9)
- Saline or wet preparation
- Lactophenol cotton blue (LPCB)
- 10-40% KOH
- Gram stain
- Modified Kinyoun acid fast smear
- India Ink
- Periodic acid Schiff (PAS)
- Grocott-Gomori’s methenamine silver stain (GMS)
- Calcofluor white
Saline or wet preparation of fungal specimens
Detect fungal elements: hyphae, pseudohyphae, Blastoconidia
Lactophenol cotton blue (LPCB)
Lactic acid preserves fungal structures, and phenol kills organisms. Cotton blue stains chitin in fungal cell walls which helps visualize fungal elements
10-40% KOH
Dissolves keratin in tissue cells. Fungal cells survive because chitin resists alkali
Gram stain of fungal specimens
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
Modified Kinyoun acid fast smear
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
India ink
Stains fungal organisms that have capsules: Cryptococcus, Rhodotorula, Sporobolomyces, and Trichosporon beigelii.
Prototheca (an algae) also has a capsule
Periodic acid Schiff (PAS)
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
Grocott-Gomori’s methenamine silver stain (GMS)
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.
Calcofluor white
Fluorochrome dye concentrates in fungal cell walls. The organisms will fluoresce blue-white or green using UV microscope
In which specimens are fungi clinically important?
- Sterile body fluid – CSF, blood, pleural fluid, synovial fluid
- Immunocompromised patient – any specimen site
- A routine culture growing large quantities of yeast/mold-sputum growing 4+ yeast or filamentous fungus
- 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.
Sterile body fluids (4)
CSF, blood, pleural fluid, synovial fluid
Media used for fungal isolates (5)
- Sabouraud dextrose agar (SAB)
- SAB with cycloheximide and chloramphenicol (mycosel agar) pH 7.0
- Brain-heart infusion (BHI)
- Bird seed agar (niger seed agar)
- Cornmeal with Tween 80 agar
When is SAB with cyclohexamide and chloramphenicol used?
Growth of dermatophytes and other pathogens
When is Brain-heart infusion (BHI) used?
For recovery of systemic mycoses
When is Bird seed agar used?
For Cryptococcus neoformans
Cornmeal with Tween 80 agar
Used for differentiation of Candida sp. Look for chlamydospores, hyphae, pseudohyphae, arthroconidia, etc
Temperature of fungal growth
22-30 degrees C and sometimes 35-37 degrees C
How long do fungi take to incubate?
2-4 weeks, but systemic fungi may take up to 12 weeks. Yeasts grow faster than molds
Growth rates of fungi (3)
- Rapid, <24 hr to 5 days-zygomycetes “contaminants”
- Intermediate (1-5 days) - hyaline and dematiaceous molds “contaminants”
- Slow growers (1-4 weeks) - systemic pathogens
Fungal colony morphology
Classified by texture, due to height of aerial hyphae
Methods for identification of fungal isolates (3)
- Biochemical/assimilation panels
- MALDI-TOF
- PCR
Methods of molecular testing (3)
- Real-time PCR
- NASBA
- PCR
Fungal species identified by PCR (5)
- Aspergillus sp.
- Candida sp.
- Cryptococcus
- Zygomyces
- Dimorphic fungi
Serological tests (4)
- Complement fixation
- Immunodiffusion
- Agglutination
- Indirect fluorescent antibody (IFA)
When should serological tests be used?
Best used in immunocompetent individuals
Antigen detection of dimorphic fungi
Cross reaction amongst dimorphic fungi. Blastomyces, Paracoccidioimycosis, P.marneffei
Antigen detection of Aspergillus
Galactomannan (GM), polysaccharide cell wall components
Antigen detection of Candida
Difficult to distinguish colonization vs. disease. Mannan and mannoproteins = cell wall component
Antigen detection of Cryptococcus neoformans
Glucuronoxylomannan=capsular polysaccharide. 5 serotypes = A,B,C,D,AD
(1-3) Beta-D-glucan PANFUNGAl detection
Diagnostic marker invasive fungal infection
Polysaccharide cell wall component in most fungi. Except Zygomycetes.
Candida metabolites (2)
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
Antifungal Therapies
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
Fungistatic drugs
Stops organism growth but does not kill isolate, requires functioning immune response for organism elimination
Fungicidal drugs
Kills organism
Delivery of antifungals
Can be topical, oral, or intravenous. Infection location and patient history considered.
Antifungal drugs (6)
- Amphotericin B
- Nystatin
- Flucytosine
- Azoles
- Echinocandins
- Terbinafine
Antifungal susceptibility testing (6)
- CLSI M27-A guidelines for Candida & Cryptococcus sp.
- Macrobroth, microtiter, agar dilution
- Endpoint 80% inhibition of fungal growth with azoles
- Endpoint 100% inhibition of growth with amphotericin B
- Etest (AB Biodisk)
- Alamar/Sensititre
Alamar/Sensititre
A blue solution is added to microtiter wells. Fungal growth reduces media - turns pink endpoint - lowest dilution that remains blue
Antifungal drug classes (4)
- Polyene antifungal class- Amphotericin B, Nystatin
- Anti β(1,3) D-glucan synthase- echinocandins
- Allylamine class- Terbinafine
- Azole class
Amphotericin B (fungizone)
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.
Amphotericin B target organisms (5)
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
Which organisms have intrinsic resistance against Amphotericin B (4)
Scedosporium, Fusarium, Candida lusitaniae, guilliermondii
Nystatin (mycostatin, nilstat, nystex)
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
Nystatin target organisms (4)
Broad usage especially useful for systemic Candida sp. infections, Cryptococcus, all dimorphic isolates, Aspergillus
Echinocandins
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.
Echinocandins target organisms
Broad spectrum against Candida sp. and Aspergillus sp. however not active against Zygomycetes, Cryptococcus neoformans, or Fusarium sp
Terbinafine (Lamisil)
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
Terbinafine target organisms (3)
Candida sp., dermatophytes Trichophyton sp.
Flucytosine (5-fluorocytosine)
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.
Flucytosine (5-fluorocytosine) target organisms
Cryptococcus sp. resistance problems encountered, utilized for non-invasive Candidia sp.(urinary)
Griseofulvin
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
Griseofulvin target organisms
Dermatophytes – fungal infections found in the skin, hair and nails.
Azoles
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
Azoles target organisms
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.
Problems with azole drugs
Rash, rare GI disruption, rare report of hepatic disease, some optic concerns with voriconazole (30% patient report blurry vision, photophobia)
Ketoconazole mechanism
Fungistatic, disrupts cell membrane integrity
