Mycology - Scanlan
What hyline molds form clusters from conidiophores?
- Acremonium - Tusarium - Gliocladium - Trichoderma spp
What are 3 types of invasive rhinosinusitis?
- Acute necrotizing - Chronic invasive - Granulomatous invasive
What is the role of the laboratorian?
- Advise physician about specimen collection - Culture and identify - Critical for correct treatment
Describe a non-selective culture media
- Brain-heart infusion agar (BHI) w/wo sabouraud's dextrose agar (SABHI), potato flake agar - Add cycloheximide and chloramphenicol - Selective recovery or dimorphic molds
Describe the morphology and microscopic details of Aspergillus flavus
- Granular to wooly - Shades of yellow, yellow-green, or yellow-brown - "Daisy-petal like" arrangement of chains of conidia - Single of double row of phialides cover entire spherical vesicle - Conidiophore is long (500-800um): distinct roughening of the wall near the vesicle - Conidia have a smooth spherical shape with yellow-brown pigmentation
Describe the morphology and microscopic details of Aspergillus terreus
- Granular, radially rugose (wrinkled or creased) - Cinnamon colored, brown, or orange-brown - Similar to A. fumigatus - Sporulation from top half of club shaped vesicle - Smaller and phialides are longer with a double row (hard to see double row) - Long chains of smooth spherical conidia - Key feature: Tease mount show mycelium with smooth spherical microconidia (attached laterally from vegetative hyphae)
Who are risks for hyline mold and hyalophomycosis infections?
- Immunocompromised - Bone marrow and organ transplant patients - Hematologic malignancies - Nosocomial infections in hospitals
What is a septate?
- Individuals cells separated by transverse walls called septate
Explain the clinical correlation of Allergic Bronchopulmonary Aspergillus
- Inflammation, eosinophils, Charcot-Layden crystals - Increase in serum levels of IgG and IgE to Aspergillus - Most common A. flavus and A. fumigatus
How is an infection contracted by hyaline molds?
- Inhalation of spore contaminated dust (sinusitis or bronchopulmonary disease)
Explain the clinical correlation of Disseminated Aspergillosis
- Invades all tissues and CNS - High mortality rate 94% in bone marrow transplants with treatment
What is a mycelium?
- Mat of growth
What are filamentous mycelium?
- Molds
Describe the Absidia species
- More delicate than Rhizoids - Internodal (originating from hyphae between the conidiophores) - Sporangiophores may branch - Funnel like structure of terminal conidiophore (apophysis)
What are the steps included in the Microslide technique?
- Mostly used to create permanent slides - Grow fungus in plug or cornmeal or potato dextrose agar with cover slip on top - Lactophenol cotton blue stain the cover slip after growth - Preserve slide with clear nail polish around cover slip
Describe what a mold looks like
- Multicellular - Cotton-like, velvet-like, wooly, pigmentation, granular - Hyphae 2-10um in diameter - Spores
What is a arial mycelium (reproductive mycelium)?
- Mycelium that projects from the substate - Contains spores of fruiting bodies
Describe selective culture media
- Mycosel/mycobiotic agar (primary recovery low dermatophytes) - BHI with penicillin, streptomycin, gentamicin, or chlorampheicol (inhibits bacteria)
Describe the Mucor species
- Other species ruled out before this one - No rhizoids - Branched or unbranched sporangiophores - Ends of sporangiophores are slightly bulbous columella extending with a spherical smooth walled sporangium - Sporangiophores are spherical or ellipsoid, hyaline or yellow brown pigmented
What hyline molds form chains from phialides?
- Penicillium - Paecilomyces - Scopulariopsis spp.
Describe how the stains look for the Aspergillus spp.
- Poorly in H&E stains - Well outlined in PAS and GMS stains
Describe Chlamydoconcidia
- Pre-existing cells in the hyphae become thickened or enlarged - Found within (intercalary), along the side (sessile) or at the tip (terminal) of the hyphae
Histopathology Infectious of Zygomycetes
- Ribbon-like broad Aseptate hyphae (hyphae range from 3-25um in width and nonparallel walls) - Often break into small fragments - Poor staining (PAS and GMS) - Purulent (contains neutrophils along with hyphae) - Can form fungus balls in body cavities - Treatment includes Amphotericin B and surgical debridement
Secondary workup on selective media for Aspergillus species
- Sabouraud's dextrose agar
Describe Superficial Fungal Infections
- Skin infections usually located in a specific infection site
Where are Zygomyces species found?
- Soil - Dung - Vegetative matter
What are some general types of reproduction?
- Vegetative sporulation - Aerial sporulation - Sexual sporulation
Describe Opportunistic Fungal Infections
- "Nonpathogenic fungi" - Usually infections as a result of other condition (HIV, IV drug use, immunosuppressed, and prosthetic devices)
What is a vegetative mycelium?
- "roots" mycelium extending into the media
How long do you incubate the fungal sample before discarding as negative?
- 30 days because colonies sometimes grow on contaminants
What temperature is required to culture fungi?
- 30 degrees Celsius
Explain the clinical correlation of Invasive pulmonary Aspergillosis
- Almost exclusively in immunocompromised - Presents as pneumonia (cough, fever, signs of respiratory distress) - Invades blood vessels and can disseminate
What is a Dimorphic fungi?
- Alternate between mold and yeast form - Based on environmental conditions - Temperature, CO2, nutrients (Susceptible to change)
Describe Arthroconidia
- Are formed from preexisting cells in the hyphae, which become thickened and enlarged - Conidia are rebased by lysis of adjacent cells
What are the majority of Aspergillus infections?
- Aspergillus fumigatus - Aspergillus flavis (serious) - Aspergillus niger - Aspergillus terreus
What species of fungi does hyaline molds and hyalohyphomycosis include?
- Aspergillus species and aspergillosis
What are 3 types of vegetative reproduction?
- Blastoconidia - Chlamydoconidia (Chlamydospores) - Arthroconidia
What morphological classifications do dimorphic fungi have?
- Blastomycosis - Coccidioidomycosis - Histoplasmosis - Sporotrichosis - Paracoccidioidomycosis - Phaeohyphomycosis
Describe the morphology and microscopic details of Aspergillus fumigatus
- Blue-green, green-gray, or green-brown pigmentation -Single row of phialides over the top of a clubbed shaped vesicle - Conidia are spherical, smooth, and arranged in long chains (may bend inward and present in a yellow-green or green pigment) - Conidiophores derived from a foot cell
Describe Blastoconidia
- Budding forms produced by yeast
What morphological classifications do yeast have?
- Candida species - Cryptococcus - Others
Describe the thick walls of fungi
- Chitin, mannans, sometimes cellulose - Allow absorption of specialized dye used in identification
What class is an ascospore in?
- Class Ascomycetes
What is cleistothecium?
- Closed bag-like structure containing smaller bag-like structures called asci - Smallest component of an ascus
Describe the fungus balls of Aspergillus spp.
- Colony growing in preexisting cavity such as nasal sinus, or lung cyst - Amorphous hyphae poorly stained - Well formed chains of conidia may be seen in cavities exposed to air
Describe Phialides
- Conidia producing segments on metulae
Describe Metulea
- Conidiaphore secondary segments
Describe Asci
- Contains four ascospores
Describe the morphology and microscopic details of Aspergillus niger
- Covered by dense aggregate of jet-black conidia - Peppered effect - Reverse of colony is buff or yellow grey in color - Profuse sporulation with dense aggregates of jet-black conidia covering the surface of the vesicle - Mature conidia become roughened (echinulate) - "Dandelion-Like"
What do construction zones do to hyline molds?
- Disrupt spores - Contaminate air filters
Describe the Circinella species
- Distinctive background curve of the sporangiophore - Terminate in globose sporangium (brown staining sporangiospores - No rhizoids
What additional tests can be performed and what do they do?
- ELISA - Sandwich ELISA - Detects circulating galactomannan (GM) - Low cost with rapid results - Useful in high risk patients - Less invasive - PCR - Used to validate patients and controls with invasive pulmonary Aspergillus
What does Basipedal condition mean?
- Each new conidium formed from the tip of the phialide pushing the other conidia ahead of it; older conidia are larger and more deeply staining
What is a blastoconidia?
- Elongation of budding yeast cells, sausage like constrictions
Describe the basics of mycology
- Eukaryotic - Thick cell walls - Single cell fungus - Filamentous mycelium - Reproduction by spores asexually or sexually
Describe the appearance of Zygomyces species and Zygomycosis
- Fast growing - 2-3 days usually - Wooly - Extends boarder to boarder of plate "lid lifter" - Broad - Irregular width - Ribbon-like - Aseptate hyphae - Sporangia
Describe characteristics of Hyalohyphomycetes
- Filamentous hyline molds - Grow in 3-5 days - Pale or pastel surface colors - Typically cover entire plate with outer margin (except Gliocladium and Trichoderma (they form a green or yellow lawn) - Appear granular - Opportunistic fungal infections (uncommon agents: Fusarium, Acremonium, and others)
Describe Sporangia
- Fruiting bodies forming closed sacs containing sporangiospores
What is a Hypha, Hypae (plural)
- Fundamental unit consisting of threadlike structure
What are 2 types of non-invasive rhinosinusitis?
- Fungal ball - Allergic fungal
What is an Oospore?
- Fusion from two different cells - Often from different hyphae
What is a zygospore?
- Fusion of morphologically identical cells - Often from same hyphae (homothalic) - Zygomycetes
Describe the morphology and microscopic details of Penicillium spp.
- Granular - Various shades of green - Sometimes yellow or yellow-brown - Branching of conidiophores - Primary medullae and secondary phialides - Tips are chains of conidia producing a Penicillus (brush) structure - Phialides are blunt and box-like - Conidia are spherical, evenly stained, and are acropetal (acropetal-identical conidia produced in sequence)
Describe the Cunninghamella species
- Has sporangiola (spherical spores from a large globose columella, no encasement in sporangium, and is attached by a tiny hair-like denticle)
Describe Deep-seated or systemic Fungal infections
- Highly virulent, invades deep tissues and can disseminate throughout the body
Describe the morphology of Aspergillus spp.
- Hyline, septate, parallel walls 3-6 um in diameter - Regular dichotomous branching at 45 degree angles
Pathogenicity of Zygomyces species
- Hyphae invasion of blood vessel walls - Dissemination of mycotic thrombi and metastatic foci in many organs
Describe Conidiophore
- Hyphae segment that supports a conidia-bearing fruiting head
Describe Sporangiophore
- Hyphae segment that supports a sporangium
Who is at risk for fungal infections?
- Immunocompromised - Transplant patients - Diabetics with neuropathy - Low PMN count - Systemic lupus erythematous (SLE) collagen disorders - Dysgammaglobulinemia - Alcohol and IV drug abuse - Steroids (suppress immune system), cytotoxic agents, prolonged antibiotic therapy - Work place exposure-animals and mold spores
How do you prepare a Transparency tape "cellophane tape"?
- Press cellophane tape gentle but firmly to surface to the colony - Press one slide of the tape to slide with drop of lactophenol cotton blue - Stretch tape across slide lowering it into the stain - Pull taught and affix other side of tape avoiding air bubbles - Might be difficult to use 100x oil emersion
Describe the Rhizopus species
- Production of distinct root-like rhizoids - Nodal (derived from hyphae immediately adjacent to sporangiophores) - Umbrella-like collapse of post mature sporangium
What can be seen in sputum samples that contain Aspergillus spp.
- Provisional diagnosis of Aspergillus - Contain various white cells with eosinophilia and hyphal fragments - Typical 45 degree branching hyphal fragments - Charcot - Leyden crystals elongated double pyramids formed from eosinophils
Describe the colony morphology of Aspergillus
- Rapid growth (3-5 days) - Distinct outer margin with white apron at advancing area of growth - Early colonies have cottony appearance later become sugary or granular
Describe the morphology and microscopic details of Aspergillus nidulans and Aspergillus glaucus
- Rarely encountered in clinical laboratories - Produces sexual (telomorphic) reproductive structures (cleistothecia and ascospores) - White, gray-white, cottony or granular and may have radial rugae - Dark stippling effect - Cleistothecia ("burnt nuts" appearance, dark soccer ball and thin walled spherical hyline "Hulle" cells
What is the role of a Primary care physician?
- Recognize sign and symptoms of fungal infection - Collect specimens and order - Treat patient based on identification
How do you prepare a Tease mount?
- Remove part of colony - Mount in drop of lactophenol aniline (cotton) blue - Tease apart colony (dissecting needle) and overlay with cover slip apply gentle pressure to disperse - Examine at 10x then try 40x or 100x
What is sexual sporulation?
- Requires the merging and recombination of two specialized fertile cells
What are 6 important genera of Zygomyces?
- Rhizopus spp. - Absidia spp. - Syncephalastrum spp. - Circinella spp. - Cunninghamella spp. - Mucor spp.
What hyline molds are borne singly and directly from the hyphae?
- Scedosporium - Chrysosporium - Sepedonium - Beauveria spp
Describe a Teleomorph
- Sexual reproductive form of a fungus; "perfect" fungus (known sexual spore)
Describe the Syncephalastrum species
- Shape and arrangement of sporangia differ - Merosporangia are cylindrical and arranged like "daisy petals" around a small spherical columella - Examine hyphae and colony morphology to not confuse this with Aspergillus flavus
Describe the morphology and microscopic details of Paecilomyces spp.
- Similar to Penicillium spp - Lighter pastel in color - Yellow-brown and brown variants are more frequently encountered - Granular surface - Tips of phialides are long and tapered terminating in a point - Conidia are oval to elliptical, irregular size, and uneven in staining - Basipedal condition
Describe what a yeast looks like
- Single cells - Resemble bacteria usually pasty like colonies - Oval to round - Budding cells
Describe the tissue of the Aspergillus spp.
- Sometimes purulent first changing to granulomatous (often no cellular response) - Sometimes produce oxalic acids (deposits of birefringent crystals (calcium oxalate))
How do we collect specimen and transport it?
- Specimen processing includes aspirated material or tissue biopsy recommended and its generally not swabs - Portion of the specimen is moved to a fungal culture media - Direct wet mounts or smears may provide presumptive diagnosis and may aid in the selection of appropriate culture media
What are 5 types of aerial reproduction
- Sporangia - Sporangiophore - Conidiophore - Metulae - Phialides
What are Fungas Ball infections?
- Tangled mass of hyphae cavities
Describe some general microscopic features of Aspergillus
- Uniform 4-6 um in diameter hyaline, septate hyphae with parallel walls - 45 degree angle dichotomous branching - Foot cell (base of origin of the conidiophore) - Conidiophores terminate in a swollen vesicle with uniseriate or two biseriate rows of phialides - Contain chains of pigmented conidia - Telomorphic cleistothecia containing ascospores also differentiate some species
Where are sites of infection of Zygomyces species?
- Upper respiratory infection (Inhalation of spores) - Primary gastrointestinal disease (ingestion of contaminated food) - Direct inoculation of skin and mucous membranes (inoculation of broken areas of skin)
How can we prevent dehydration of the fungal sample?
- Use oxygen permeable tape - Use breathable bags - Dedicated incubator with pan of sterile water
Identify signs of a fungal infection
- Vague or atypical symptoms - Low grade fever, night sweats, weight loss, fatigue, cough, and chest pain - Systemic disease usually begin with mucocutaneous lesions (ulcerative lesions of the intestine, larynx, pharynx, genitals, and tongue & pustular lesions of the skin or granulomas of the oral cavity) - Nonspecific findings: Accelerated SED rate, increase in C reactive protein, high gamma-globulin or elevation of neutrophils and monocytes
Describe a Anamorph
- Various asexual forms or structures produce by an "imperfect" fungus (no sexual spore known)
How do fungi asexually and sexually reproduce?
- Vegetative mycelium (arthrospores, chlamydospores, blastospores) - Aerial fruiting bodies (conidia)
How do we diagnose fungal infections?
- Visual examination of colony morphology - Microscopic observations in direct mount (typically just genus) - Biochemical tests - Genera and species differentiation (moving to nucleic acid probes)
Where are hyaline molds and hyalophomycosis located?
- Widely distributed in nature - Soil - Decaying vegetation - Variety of organic matter
What is a aseptate?
- Without walls between cells
What are single cell fungus?
- Yeast
What morphological classifications do molds have?
- Zygomyces - Hyaline molds - Hyalohyphomycetes - Dermatophytes
Can you use tubes or plates?
YES! - Consider space - Easier for transport - But tougher to get direct preps and test
