Lecture 8 Flashcards
MYCOLOGY
Myco=fungi ology=study of
- Eukaryotic organisms
- Have a true nucleus, nuclear membrane and organelles
- Are Heterotrophs-depend on organic matter for carbon
- yeasts
-Unicellular but some species can form hyphe or pseudohyphae
-White - asexual reproduction by budding
-Form smooth round colonies - Most yeast are facultative anaerobes some are strict aerobes
*molds
-Multicellular and are organized into hyphae
-different colors
-reproduction sexual or asexual by spores
-fuzzy
- Most fungi except yeast are obligate aerobes
FUNGAL CELL WALL
- Thick cell wall made of chitin, glucan
(polysaccharides) & mannan (protein) - Cell membrane contains ergosterol and zymosterol
- Human cell membrane has cholesterol
- Anti-fungals rely on this difference to target fungi & not affect human cells
Fungus are classified by the method of their sexual reproduction & type of sexual spore produced
FUNGAL ASEXUAL REPRODUCTION
Performed when conditions in environment are good – no genetic diversity
-mycelium form asexual spore forming bodies above the ground.
-haploid nucleus inside the fruiting body divides many times by mitosis forming many haploid spores that are genetically identical to the parent
- Use of a spore forming body
Sporaniophore→sporangium – produce sporangiospores
Conidiophore→phialides – produce conidia - Splitting of hyphe→ produce arthroconidia
- Budding → producing blastoconidia
* Cell swells and a bud breaks free
FUNGAL SEXUAL REPRODUCTION
Performed when environmental conditions are poor
-meiosis: results in daughter cells each with half the number of chromosomes of the parent cell- each cell is genetically different
3 Methods of sexual reproduction :
1. Mating of hyphae results in the creation of a zygospore
2. Production of an ascus sac that produces ascus spores
3. Basidium forming basidiospores
class Glomeromycota
sexual spore - zygospore (thick wall cell in between hyphae)
Asexual spore= sporangiospore in a sac like structure called
sporangium
class ASCOMYCOTA
Sexual spore- ascospores present in a Sac like structure called an ascus
Asexual spore- conidia on
conidiophor
class BASIDIOMYCETES
Sexual fusion- results in formation of a club shaped structure called basidium forming basidiospores
Asexual reproduction: occurs more rarely
* Budding
* Fragmentation
* Conidia formation
FUNGAL STRUCTURES
- Multicellular = filamentous molds
- Unicellular = microscopic yeasts
- Molds have “hair” called mycelia - fuzzy or woolly appearance
- Mycelia made up of hyphe
- Hyphae grow at the tip and divide creating long and branching chains
- Enzymes secreted from the hyphal tip -breakdown organic matter into smaller molecules which are used
as food.
ASEPTATE HYPHAE
- Initial hypha grows out from a spore
- Non-septate or aseptate hyphae form one long cell with many nuclei
- While they do not form septa between nuclei, they do form a septum at branch points that connect one
filament to another, preventing the entire network from being compromised if one hypha is injured.
SEPTATE HYPHAE
- Hyphae with dividers called septa between the cells
- Pores between the cells let cytoplasm and nutrients flow throughout the mycelium
- nucleus, can fit through the pores
- When new cells bud septum does not form immediately
- As the new cell matures, the cell wall grows down into the cytoplasm, forming the septum (singular form of septa)
HYPHAE MORPHOLOGY
Hyaline vs Phaeoid Hyphae
Hyaline- transparent no colour
Phaeoid (Dematiaceous)
Dark pigment due to melanin in the cell wall
* Pale brown to dark brown or black
FORMS FUNGI CAN EXIST IN
Monomorphic
* single type of growth
* ONLY yeast OR mold
Dimorphic
* Exists as a mold or a yeast or a spherule depending on the temperature
* Yeast in tissue 37 0C,
* Spherule 37 0C
* Mold phase (RT, O2)
Polymorphic
* Exists as yeast and mold at the same time
* Produces many forms, hyphae, pseudohyphae and budding yeast (called blastoconidia)
MYCOSES
superficial
cutaneous
subcutaneous
systemic
SUPERFICIAL MYCOSES
- Strictly confined to skin and hair
- no overt symptoms
- Fungus involved do not activate tissue responses or inflammatory reactions
- Usually cosmetic concern rather than medical
Example of common superficial mycoses seen in North America
Tinea versicolor – caused my Malassezia furfur
* Causes hyperpigmentation or depigmentation of skin
TINEA VERSICOLOR
- Malassezia furfur often normal flora on skin -opportunistic
- Causes patchy lesions, scaling or varied pigments on skin most often face, chest trunk and abdomen
- Can cause dandruff
- Needs fat/oil to grow – found in areas with sebaceous glands
- Can cause catheter acquired fungaemia in patients
undergoing lipid replacement therapy - Lab Diagnosis: Microscopic exam of skin specimens in 10%KOH shows “spaghetti & meatballs” = budding yeast & hyphae
- Shine a fluorescent woods lamp on skin and it will fluoresce
- Will not grow on culture media unless covered with oil – w/o oil cultures will appear negative
CUTANEOUS MYCOSES
- Caused most often by Dermatophytes – have only asexual reproduction
- Involve keratinized area on host –skin, hair and nails
- Causes a ring like lesion (ringworm) now named Tinea _location found
-Tinea pedis = feet - athletes foot, Tinea capitis = hair,
Tinea corporis = body skin Tinea unguium - Onychomycosis - deformed nails - Transmitted by direct/indirect contact with infected host
- Symptoms: itching, scaling, lesions
- Three genera of dermatophytes which infect different areas:
Microsporum infects skin and hair (not nails)
Epidermophyton infects skin and nails (not hair)
Trichophyton infects hair, skin & nails
LAB DIAGNOSIS OF DERMATOPHYTES
- risk 2 orgs work in level 2 lab with PPE and class 2 BSC
Specimens:
* Skin scraped from the margin of the lesion
* Hair plucked, not cut, from the edge of the lesion
* Nails scrapings are obtained from the nail bed
lab testing
1. Perform Microscopic examination:
In 10%KOH or Calcofluor white fluorescent stain
* Look for 2 sizes of asexual reproductive cells-each a distinct shape
Macroconidia & or Microconidia
- Culture the Specimen onto Agar Media:
* portion of the specimen pressed into Dermatophyte test medium (DTM)
* Basic agar with peptones, glucose, antibiotics & antifungals (chloramphenicol & Cycloheximide)
* RT for up to 21 days - Perform Macroscopic Exam of the mold growing on the plate (colonial morphology):
* Description of color and texture of front of colony
* Description of color only of back of colony - Perform a Lactophenol cotton blue of the colony (staining)
* Examine this preparation microscopically for
characteristic macroconidia and or microconidia
Microsporum Microscopic Structures
Microsporum canis make:
-spindles shaped macroconidia with tapered pointed knob ends. Rough walled with small spines. more than 6 cells
- Microconidia that are found singly on septate hyphae and are club shaped
Microsporum gypseum make:
- Macroconidia with rough thin walls and rounded ends. less than 6 cells
-Microconidia that are club shaped
Epidermophyton floccosum
Microscopic Structures
- Septate hyphae
- Makes Macroconidia that are smooth-walled, & “beaver tail” shaped
- Made up of 2-4 cells
- Macroconidia may be clustered at the end of a hyphae
- Microconidia are absent
Trichophyton rubrum Microscopic
Structures
- Septate hyphae
- Makes Microconidia that are teardrop shaped & Macroconidia may or may not be present
- If present are long narrow thin, smooth walled with 2- 8 cell
Trichophyton mentagrophytes
Microscopic Structures
- Septate hyphae
- Makes Microconidia that are round and clustered on branched conidiophores & Macroconidia that are
thin- or thick-walled, 1- 6 cells
SUBCUTANEOUS MYCOSES
- Chronic infection of deeper skin layers spreading to muscle and bone but not usually to blood or organs
- Fungus usually found in soil in tropical areas & enter through sites of skin trauma
- Causes progressive non healing ulcers, may also see draining sinus tracts & tissue destruction
- An example of a subcutaneous mycoses is Mycetoma
- Characterized by formulation of melanotic granules containing the fungus being discharged onto the skin
surface through multiple sinuses - You can express granules from draining lesions
SYSTEMIC MYCOSES
- A fungal infection that spreads throughout the body
- Can affect multiple organs, blood, CSF
- Can be fatal especially in immunocompromised individuals
- Usually caused by dimorphic fungus, Aspergillus or a yeast
DIMORPHIC FUNGI CAUSING
SYSTEMIC MYCOSES
- Histoplasma capsulatum – spores found in bat or bird droppings
- Coccidioides immitis - arthrospores found in soil
- Blastomyces dermatitidis - Spores found in soil and wet, decaying wood
- Paracoccidioides brasiliensis - Spores found in soil around coffee crops or armadillo dens
- Sporothrix schenkii – spores found in soil & decomposing plant material especially roses (Rose handler’s disease)
- Can also be carried by cats
DIMORPHIC SYSTEMIC
MYCOSES how to get
- Get infected by inhaling the spores, ingestion or by inoculation of spore into the skin
- Healthy individuals usually have a limited acute respiratory or skin infection – some are asymptomatic
- In immunocompromised the infection becomes chronic pulmonary infection or granulomatous skin lesions
- lead to fatal systemic infections that
affect the bones, internal organs, blood, CSF & lymphatic system
LAB DIAGNOSIS OF DIMORPHIC
FUNGUS
- Risk level 3 organisms in a Containment Level 3 Lab PPE & a class II bsc
- Antigen or Antibody detection: using serological or immunodiagnostic techniques
- Molecular tests like PCR: available for some dimorphs
- Direct Specimen Microscopy: for detection of the yeast form in the body
- Culture on fungal media at 25C & at 35C: may take up weeks to become positive
- Microscopy from the culture: to see dimorphic yeast or mold structures
- barrel shaped arthroconidia
-tissue spherical
-mariners wheel
-rosette cluster of microconidia
OPPORTUNISTIC SYSTEMIC
MYCOSES
- Saprophytic fungi (naturally found in soil) or as normal flora – sometimes dismissed as non-pathogenic
Most Common Opportunistic Fungus:
Aspergillus spp., Mucor & Yeast (Candida)
* often as lab media contaminants
* But increasing cases of immunocompromised patients (especially AIDs) being infected by these organisms
* In order to be a true infection, there must be repeated isolation or isolation of the same organism from multiple specimens
* Or demonstration of the organism directly in the tissue
Aspergillus spp.
- Saprophytic fungus – found in soil or decomposing material
- Conidia are inhaled.
-common cause of allergies to mold or ear infections in healthy people - In immunocompromised, causes various respiratory infections, in lungs can be acute or chronic pneumonia
- In the body the conidia continue to germinate hyphae causing fungal ball
- Hyphae can spread via blood to other organs including brain -High mortality rate in this group
- Organism is monomorphic → always a mold
Diagnosis:
* Direct specimen microscopy for characteristic mold structures
* Must have growth from multiple specimens
* Molecular testing
Aspergillus vs Penicillium spp
- Penicillium is not a pathogen
- Has conidiophores with secondary branches called metula.
- On metula are flask shaped phialides that bear the conidia – no vesicle
Aspergillus:
* Has conidiophores with a round vesicle at the end
* The vesicle has the metula & the phialides that bear the conidia
MUCORMYCOSIS
- Environmental fungus called Mucor causing infection in diabetics and immunocompromised
- Infection is through inhalation, ingestion, or entering skin
- Causes infections in the sinuses, eyes, lungs, skin & gastrointestinal tract
- Invasion of blood vessels and blood clots causing rapid tissue necrosis
- Disseminates throughout the body including the brain
- Microscopic identification of characteristic fungal structures right from tissue biopsy
- Growth from cultures from multiple specimens
- Serology to detect antibodies
- Molecular
YEASTS
- Found in the environment & as healthy normal flora of skin, genitals & intestine
- Causes opportunistic infection in immunocompromised, those having antibiotic use or hormonal imbalances & malnutrition,
- Candida is most common genus – C. albicans most common species of Candida – many more though
- C. auris new species of importance – R to antifungals
- Cryptococcus is another genus of yeast pathogenic to humans - C. neoformans causes serious infection in AIDs patients
Forms Some Yeast can exist in:
Budding: Bud referred to as blastoconidium
Pseudohyphae: chains of buds that stay attached to the mother cell and to each other - have a constriction between adjacent cell
True hyphae or Germ tubes: long tube with parallel sides
and no constrictions
Chlamydospores: thick -walled survival spore made when the yeast is in adverse environmental conditions
Candida albicans
- Primary cause of yeast infections called Candidiasis
- Causes 3 main types of infections
Muco-cutaneous :
* Oral (thrush), vaginal, upper respiratory, GI tract infections causing diarrhea
Cutaneous:
* Skin and nail infections
Systemic:
* Infections of inner organs and blood –serious & can be fatal
Presumptive identification of C.albicans:
* Is Polymorphic – exist as buds, true hyphae (germ tube), pseudohyphae & chlamydospores
* Chlamydospore & Germ tube test positive
* Urea test negative
Germ tube positive, grows well at 40– 42º C
C. albicans vs C. dubliniensis
*C. dubliniensis is a separate species of Candida
* Causes infection in AIDS patients
* incorrectly identified as C. albicans because both Germ tube, chlamydospore positive & urea negative
* Need to differentiate because C. dubliniensis can be resistant to fluconazole which is used to treat C. albicans
* Differentiate by growth at 42 C
* C. albicans grows at 42
* C. dubliniensis does not grow at 42 C
* Can also do MALDI , PCR or other genetic testing
Candida auris
- fungus that is emerging as a serious global health threat & an infection control organism of concern
- carried on the skin of colonized patients
- can cause serious invasive disease
- multidrug-resistant
- Has caused outbreaks in healthcare settings
- It is difficult to identify – easily misidentified as other Candida species when using traditional testing.
- MALDI-TOF & Molecular methods are best ID methods
- Must perform AST with antifungals to determine resistance
Germ tube negative, grows well at 40–42º C
Other Clinically Significant
Candida spp.
C. tropicalis
C. glabrata
C. parapsilosis
- All can cause outbreaks of nosocomial infections
- Are resistant to numerous antifungals
Cryptococcus neoformans
- Found in nature (soil with pigeon droppings) - Route of entry is inhalation of yeast
- Causes systemic infections meningitis, septicemia and pulmonary disease
- Cryptococcus gattii also causes disease in immunocompromised & immunocompetent hosts
- Important to distinguish two species since clinical course and treatment can be different
- Special media available to differentiate the two – Canavanin Bromothymol blue agar
- C. gattii uses glycine as a sole carbon and nitrogen source while C. neoformans does not
- C. gattii turns the media from yellow to blue
Cryptococcus Neoformans
Identification
- Yeast-like, budding cells but no pseudohyphae or true hyphae or chlamydospores
- mucoid colonies with capsule
- India ink positive – tests for presence of capsule
- Urea positive
- Chlamydospore negative
- Germ tube negative
Detection of cryptococcal capsular polysaccharide antigen
* Antigen detected directly from CSF or serum
* Method of choice for diagnosing patients with cryptococcal meningitis
* detected using latex agglutination, & lateral flow
Treatment: fungus and yeast
- Cell membrane contains ergosterol which some antifungals act upon
- Primary antifungal amphotericin B
(polyenes) & fluconazole (azole) –both broad-spectrum - Lethal to fungus but toxic to patient – now fungus have shown resistance
Histoplasma capsulatum
spores found in bat or bird droppings
Coccidioides immitis
arthrospores found in soil
Blastomyces dermatitidis
Spores found in soil and wet, decaying wood
Paracoccidioides brasiliensis
Spores found in soil around coffee crops or armadillo dens
Sporothrix schenkii
spores found in soil & decomposing plant material especially roses (Rose handler’s disease)
* Can also be carried by cats
