Mycology Flashcards
1
Q
Mycology Laboratory Methods
A
Direct examination
- Calcofluor white
- fluorochrome
- binds to cellulose and chitin in fungal cell wall
- highlights fungi, prototheca, and cysts of acanthamoeba
- India ink
- Fontana Mason stains capsule of cryptococcus
- GMS or PAS in tissue
Fungal culture
- Brain heart infusion (BHI) agar
- Sabouraud dextrose agar
- Inhibitory mold agar
- above agars incubated at 25-30 degrees for 4-6 weeks
- Special media:
- Cottonseed agar converts mold phase of blastomyces to yeast form
- cornmeal or potato dextrose agar (latter shows pigment form Trichophyton rubrum)
- cornmeal or rice agar with Tween 80
- Sabouraud dextrose agar, Dixon medium, or Leeming-Notman medium overlaid with sterile olive oil
- Trichophyton agars
- Bird seed (niger seed) agar
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Q
Sabouraud dextrose agar (SDA)
- principle
- purpose
A
- Principle
- Acid pH and high dextrose concentration inhibits bacterial growth but permits growth of fungi; some also contain abx
- Purpose
- many fungi
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Q
Inhibitory mold agar (IMA)
- principle
- purpose
A
- Principle
- chloramphenicol and sometimes gentamicin or ciprofloxacin to suppress bacteria
- Purpose
- selective isolation of fungi from specimens that may contain commensal bacterial flora
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Q
Brain heart infusion agar (BHI)
- principle
- purpose
A
- Principle
- brain/meat infusion, peptone and dextrose
- Chloramphenicol and gentamicin can be added
- Principle
- nonselective formulation is general purpose medium used for bacteria, yeasts, and molds
- when containing abx, it prevents commensal bacterial from growing
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Q
Cyclohexamide containing media
- principle
- purpose
A
- Principle
- inhibits growth of many saprophytic fungi, while permitting growth of most pathogenic fungi
- chloramphenicol and gentamicin can be added
- Purpose
- Selective isolation of slow growing pathogenic fungi that may be overgrown by rapidly growing saprophytic fungi
- cycloheximide also inhibits growth of C neoformans, Candida, Aspergillus, and other zygomycetes
- targets dermatophytes or thermally dimorphic fungi
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Q
Olive oil overlay on SDA, Dixon medium, or leeming-notman medium
use
A
isolate malassezia
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Q
Trichophyton agars
A
differentiate between species of trichophyton
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Q
Bird seed agar
A
demonstrate pheno oxidase activity of cryptococcus neoformans
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Q
Mold morphology
A
- Hyaline septate molds
- surface of colonies may be white or colored
- reverse side of plate is usually light
- hyphae with frequent septations
- Dematiaceous molds: septate molds that make melanin
- surface and reverse side of plate are both dark
- Zygomycetes: aseptate or pauciseptate
- lid lifters
- may be pigmented, but do not make melanin (reverse side is light)
- Dimorphic fungi: septate hyphae at 25-30 degrees; yeast when reincubated at 37 degrees
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Q
Yeast gross morphology and classification
A
- creamy or mucoid (cryptococcus) colonies; molds make fuzzy colonies
- candida albicans form “feet” starlike projections
- further classified by MALDI or by special yeast agar
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Q
Yeasts with blastoconidia only
A
Cryptococcus
Candida glabrata
Rhodotorula
Malassezia
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Q
Yeasts with blastoconidia and pseudohyphae
A
Candida (except glabrata)
Saccharomyces cerevisiae
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Q
Yeast with arthroconidia
A
Trichosporon
Geotrichum
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Q
Hyaline molds
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Hyphae and other structures are nonmelanized
- aspergillus
- penicillium
- fusarium
- dermatophytes
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Q
Dematiaceous molds
A
Alternaria
P boydii/S boydii
Scedosporium prolificans
Curvularia
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Q
Mold with pauciseptate hyphae
A
Zygomycetes (rhizopus, mucor, cunninghamella, rhizomucor)
17
Q
Thermally dimorphic fungi
A
Histoplasma
Blastomyces
Coccidioides
paracoccidioides
sporothrix
penicillium marnefii
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Q
Histoplasma capsulatum
- diagnosis
- culture
- geographic distribution
- transmission
- appearance in tissue
A
Diagnosis and culture
- 2-4 um ovoid yeast with narrow based budding
- in histiocytes
- slow growing cottony white mold
- septated hyaline hyphae with intermittent microconidia and large spiny macroconidia
- undergo yeast conversion at 37 degrees
Ohio and Missippi river valleys, Latin America, and Spain
Primary infection is pulmonary, can be disseminated (reticuloendothelial); sclerosing mediastinitus
Transmitted via droppings from chickens or bats
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Q
Histoplasma duboisii
- geography
- disease
- culture
- tissue
A
- Africa
- local or disseminated (skin, soft tissue, bone)
- Colony and microscopic morphology indistinguishable from var capsulatum
- often intraceullar within giant cells
- round to oval, thick walled yeast, narrow based budding (unlike B dermatitidis which is a similar size)
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Q
Blastomyces dermatitidis
- diagnosis
- culture
- disease
- similar morphology to ___
- geographic distribution
- transmission
A
- uniform, large yeasts (8-15 um) with broad based budding
- thick wall and double contoured yeast
- Slow growing mold with cottony white surface that darkens to tan with age at 25-30 degrees
- septate hyaline hyphae with short, unbranched conidiophores producing single, pyriform to round, smooth conidia (lollipops)
- Morphology similar to nondimorphic mold Chrysosporium
- Conversion to yeast at 37 degrees
- Mississipppi and Ohio River valleys; also in southeastern US and around Great Lakes
- soil, inhaled
- pulmonary primary infection
- disseminated infection affects skin, mucous membranes, and bone
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Q
Coccidioides immitis/posadasii
- morphology
- resemble ___
- yeast conversion?
- geographic distribution
- transmission
- disease
A
- large spherules with thick walls that contain hundreds of endospores
- 25-30 degrees grows as moist gray colonies that become white and cottony as they mature
- thin, hyaline septate hyphae and arthroconidia; mature arthroconidia are barrel shaped, and alternate with empty cells
- Immature arthroconidia resemble Malbranchea
- difficult yeast conversion
- San Joaquin Valley California (C posadasii in southwest)
- soil inhaled
- Can be easily inhaled by lab personel (be careful!)
- primary infection is pulmonary
- disseminated infection affects skin and bone and joints
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Q
paracoccidioides brasiliensis
- morphology
- gross
- yeast conversion
- geographic distribution
- disease
A
- round, large yeast with circumferential budding
- 25-30 degrees: slow growing mold with a white to tan surface
- hyaline septate hyphae with terminal and intercalary chlamydospores and infrequent pear shaped microconidia arranged along hyphae
- converts to yeast at 37 degrees
- Central and South American rain forest
- primary infection is pulmonary
- Can disseminate: skin, mucosa, reticuloendothelial
- Behaves like blastomyces
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Q
Sporothrix schenkii
- morphology
- geographic distribution
- transmission
- disease
A
- 4-6 um, cigar shaped yeast with narrow based budding
- 25-30 degrees: grows rapidly into moist, white to pale orange colonies that turn brown with age
- delicate hyaline septate hyphae producing conidiophores topped by clusters of microconidia (rosettes or daisies)
- converts to yeast at 37 degrees (cigars with narrow based buds)
- worldwide distribution
- acquired by penetrating injury from contaminated plant (rose garden)
- Alcoholics known to become infected through inhalation
- lymphocutaneous infection with nodular, ulcerative lesions following lymphatics
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Q
Penicillium marneffei
- appearance of clinical sample
- culture
- disease; geographic distribution
A
- 3-5 um yeast divide by fission in clinical sample
- Culture
- 25-30 degrees: rapidly growing tan colonies initially powdery or velvety on the surface, and become colored with maturity (blue/green centrally)
- red pigment diffuses into agar around colonies
- hyaline septate hyphae with conidiophores and metulae producing brushlike clusters of phialides
- converts to yeast at 37 degrees (3-5 um), oval
- penicilliosis: endemic in southeast Asia
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Aspergillus species
- clinical specimens appearance
- culture appearance
* clinical
* hyaline, **septate hyphae** with **45 degree** branching
* **fruiting heads** in air filled tissue pockets
* Culture:
* rapidly growing; identified by colony morphology and microscopic appearance of **swollen vesicle** at ends of conidiophores
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Culture appearance of aspergillus fumigatus
* blue green with white apron and light reverse
* conidiophores terminate in a swollen vesicle having a single row of phialides that cover only the top **2/3 of the vesicle**
* each phialide gives rise to chain of small conidia
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Culture appearance of aspergillus flavus
* yellow green to **olive with light** reverse; lateral striations
* **circumferential phialides**
* some strains **uniseriate** (single row of phialides) or **biseriate**
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Aspergillus niger morphology
* Dark brown to black colonies with light reverse
* vesicles with 2 rows of phialides (biseriate) covering the entire vesicle
* phialides produce chains of rough, round, dark conidia
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Aspergillus terreus morphology
* colonies are **cinnamon brown** on the surface with a **yellow or orange** reverse
* phialides cover top **2/3 of vesicle** , but unlike fumigatus are **biseriate** and have **longer chains of conidia**
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Diagnosis of aspergillus
* Culture
* ELISA for serum marker **galactomannan** and/or **1-3-beta-D-glucan**
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Aspergillosis
- transmission
- clinical disease
- clinical features of A flavus, A niger, and A terreus
* Ubiquitous in **soil** and on **decaying vegetables**
* **Respiratory tract** affected
* Immunocompetent host will have cavitary lung disease: **aspergilloma** (fungus ball); most commonly caused by **A fumigatus**
* Atopic host: allergic bronchopulmonary aspergillosis (**ABPA**) or allergic sinonasal aspergillosis (**allergic fungal sinusitis**)
* Immunosuppressed host: **invasive bronchopulmonary aspergillosis** (IBPA) or **invasive fungal sinusitis**, with angioinvasive growth (Aspergillus species and zygomycetes have capacity to invade vessel walls)
* **A flavus** produces aflatoxins in food, carcinogens for HCC
* **A niger** pulmonary infection associated with oxalosis (calcium oxalate tissue deposition); most common aspergillus associated with otitis externa
* **A terreus** resistant to amphotericin B
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Fusarium infections
similar to aspergillus + fungal keratitis and opportunistic infection in burn wounds
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Pseudallescheria boydii/Scedosporium boydii disease
- transmission
- diseases
- treatment
* most common cause of eumycotic mycetoma after penetrating trauma
* fungal keratitis
* pneumonia after near drowning accidents
* P boydii/S boydii resistant to amphotericin B
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Conidia arranged in clusters
**Fusarium**
* canoe shaped
* multicellular macroconidia with 3-6 cells each
**Acremonium**
* long, threadlike, unbranched phialides bearing clusters of single celled microconidia
**Gliocladium**
* branching conidiophores bearing flask shaped phialides, resembling Penicillium; microconidia cluster in a ball (golfball at end of finger tip)
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Conidia arising in chains
**Penicillium**
* brushlike arrangement of flask shaped phialides that give rise to unbranching chains of microconidia
**Paecilomyces**
* Branching conidiophores with elongated, flask shaped phialides arranged in pairs or brushlike groups
* long chains of oval or spindle shaped microconidia bend away from the phialides
* colonies are yellowish brown or pinkish mauve, white, or yellow-green, but are never bright green or blue-green
**Scopulariopsis**
* single or branched conidiophores that give rise to annellides (similar to phialides)
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Conidia arranged singly
**Chrysosporium**
* single, cutoff microconidia directly on hyphae or on the tips of simple conidiophores
* Looks like **blastomyces**
**Sepedonium**
* singly borne, hyaline conidia at the ends of branched or unbranched conidiophores
* conidia are large, thick walled and echinulate (spiny), resembling macroconidia of mold form of **H capsulatum**
**Beuveria**
* single, small, round or oval microconidia emerging from each inflection point in a zigzagging (geniculate) flask shaped conidiophore
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Dermatophytes are grown on what medium?
cycloheximide
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Diagnosis of dermatophytes
1. **KOH prep or calcofluor white prep of skin scrapings**
2. Species ID by **colony morphology** (trichophyton rubrum produce red pigment on reverse)
3. Species ID by **microscopic examination of conidia**
* **Trichophyton rubrum**:
* Tear shaped microconidia along hyphae: "birds on a telephone wire"
* **Trichophyton mentagrophytes**:
* microconidia are arranged in clusters
* spiral hyphae
* cigar shaped macroconidia
* can penetrate hair shafts, unlike T rubrum
* **Trichophyton tonsurans**: microconidia have marked variability in size and shape; scalp ringworm
* **Microsporum canis**:
* Macroconidia are spindle shaped and rough (echinulate) and pointed
* Macroconidia contain \> 6 cells separated by transverse septae
* **Microsporum gypseum**:
* Macroconidia are oval with **rounded ends unlike M canis** and have transverse septae
* Macroconidia contain less than 6 cells
* Microconidia also present
* **Epidermophyton floccosum**:
* Macroconidia are smooth, **club shaped** found singly or in clusters
* Macroconidia contains **2-6 cells**, separated by **transverse septae**
* No microconidia
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Microsporum canis
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Trichophyton mentagrophytes
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trichophyton tonsurans
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microsporum gypseum
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epidermophyton floccosum
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Why are dematiaceous molds pigmented on surface and reverse of plate?
both hyphae and conidia are melanized (however, some only melanize one or the other)
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Fast growing dematiaceous molds with conidia with transverse septae
**Bipolaris**
oval **dark conidia** (**3-5** septations) arising from **bent** conidiophores; **germ tubes at both ends**
**Dreschslera**
similar to bipolaris but lacks bipolar germ tubes in saline incubation; instead **germ tubes are along sides** of the conidia
**Excerohilum**
resembles bipolaris but its **conidia are longer** and thinner and have **5-12 septations**
**Helminthosporum**
bottle brushlike microscopic appearance, with side by side conidia arranged in whorls along conidiophores
**Curvularia**
Transverely septated conidia curve distinctly when mature
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Dematiaceous molds- fast growers: conidia with transverse and longitudinal septae
**Alternaria**
chains of muriform (brick wall) conidia that have alternating blunt and pointed ends
**Ulocladium**
oval muriform conidia borne singly on bent conidiophores
**Stemphilium**
Resembles ulocladium but borne upon straight condiophores
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bipolaris
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drechslera
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excerohilum
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Helminthosporium
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Curvularia
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Alternaria
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ulocladium
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Stemphilium
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Slow growing dematiaceous molds
- early growth is yeast like becoming mold like with age
- always look mold like
**Early yeast like, mold like later**
* exophiala
* wangiella
* hortaea
**Always mold like**
* pseudoallescheria boydii/scedosporium boydii complex
* scedosporium prolificans
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Pseudallescheria boydii/ scedosporium boydii
- colonies
- sexual versus asexual forms
- treatment
* **light gray or brown surface**, owing to melanization of its oval, truncated microconidia
* **hyphae are hyaline** (nonmelanized) creating a light reverse
* produces large dark **cleistothecia** in sexual state
* asexual form lacks cleistothecia
* alternative asexual form (**graphium**) has mats of long conidiophores stuck together side by side, resembling the bristles of a broom
* resistant to **amphotericin B**, but susceptible to **triazoles**
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Scedosporium prolificans
- colonies
- differences from P boydii
- micro
- treatment
* **gray or black** surface and reverse (dematiaceous)
* unlike P boydii its growth is **inhibited by cycloheximide** and it has **no sexual state**
* **microconidia** are oval and truncated, forming clusters at the end of annellides (conidiogenous cells)
* **annellides** have swollen bases and thin necks
* resistant to amphotericin B, azoles and echinocandins (so **antifungal therapy is ineffective**)
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Clinical features of dematiaceous molds
1. **Chromoblastomycosis**
* **subcutaneous** mycosis associated with prominent **pseudoepitheliomatous hyperplasia**
* tissue shows **pigmented hyphae** and **sclerotic (muriform) bodies**
* Organisms:
* **fonsecaea pedroi**
* **phialophora verrucosa**
* **cladophialophora carrionii**
* **tropical and subtropical**
* gain entrance through **puncture wounds**, lower extremities
2. **Mycetoma** (Madura foot or maduromycosis)
* **subcutaneous** infection with draining **sinus tracts**
* can be caused by **bacteria** (actinomycotic mycetoma) or **molds** (eumycotic mycetoma)
* subcutaneous nodule contains **granules** (mold with a proteinaceous matrix)
* usually from **puncture** wound
* Organisms:
* **Madurella**
* **exophiala**
* **wangiella**
* **P boydii/S boydii**
* actinomycotic: aerobic actinomycetes (**nocardia**)
3. **Phaeohyphomycosis**: do not fit into the above categories
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Zygomycetes
- diagnosis in clinical specimens
- culture (growth rate, list species)
- define zygophore
* clinical specimens have broad, hyphae with few septations and few branches; nondichotomous
* Rapid growth in culture
* Zygophore is a diploid reproductive stage in the life cycle; defining zygomycetes
* Species
* Rhizopus
* Mucor
* Lichtheimia (formerly Absidia)
* Cunninghamella
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Characteristics of rhizopus organisms
* **rhizoids** and **unbranched sporangiophores** that arise directly from rhizoids
* sporangia are spherical and full of spores that collapse when mature (like a collapsed umbrella)
* sporangiophores **lack an apophysis**
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Mucor species
* do not produce rhizoids
* sporangiophores are branched or unbranched, but lack apophysis
* sporangia are spherical and fall apart, releasing spores
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Lichtheimia species
* has rhizoids but sporangiophores arise at points between rhizoids rather than over rhizoids
* sporangiophores are branched and form a conical **apophysis** at the top
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Cunninghamella species
* **branched sporangiophores** topped by large **vesicles**
* vesicles covered by spines (**denticles**), which supports a **single spore** contained within a round **sporaniolum**
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Clinical features of zygomycetes
* several forms of invasive infection
* rhinocerebral
* pulmonary
* GI
* cutaneous
* hosts often immunocompromised
* risk factors
* DM (esp ketoacidosis)
* stem cell or solid organ transplant
* neutropenia
* corticosteroid therapy
* severe burns
* invade vessel walls (like aspergillus)
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cunninghamella
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Chromagar
agar plates that are selective for yeast allowing for some differentiation between some Candida species on basis of color
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Germ tube test
* yeast incubated in **serum at 37 degrees** for up to **3 hours**
* **wet mount** is made and examined for germ tubes (**hyphae with no constriction at the juncture with the yeast cell**)
* **C albicans** and **dubliniensis** make true hyphae without constriction (germ tubes) (most candida species do not make germ tubes)
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Rapid treholase assimilation
Presumptively identify **C glabrata**
* **yeast** isolate is inoculated into broth containing **treholase** and a **pH indicator**
* after **3 hour** incubation at **42** degrees **fermentation** of the substrate causes a **color change**
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Urease test, fungi
Presumptively identify **Cryptococcus**
* urease activity distinquishes **ascomycetous** yeasts (no urease activity) from **basidiomycetous** yeasts (urease activity)
* **urease disks** detect urease activity by a color change within a few hours; somewhat longer testing needed for **urea agar slants**
* common basidiomycetous yeasts: **rhodotorula and trichosporon**
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Phenol oxidase test, fungi
Presumptive identification of **C neoformans**
* C neoformans **oxidizes diphenolic compounds** such as **caffeic acid**, **dopamine**, and **dopa** to produce darkly pigmented **melanin** or melanin precursors
* culture yeast on **bird seed agar** (which has caffeic acid)...if brown colonies grow, it is C neoformans
* Rapid method involves use of **caffeic acid** **disks** inoculated with yeast isolate and can demonstrate brown pigment production within a few hours
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Definitive identification of yeasts
1. biochemical tests
2. MALDI-TOF
3. demonstration of specific morphology with specialized media
4. rice or cornmeal agar supplemented with Tween 80 will induce formation of blastoconidia, pseudohyphae, true hyphae, arthroconidia or chlamydospores
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C albicans
- clinical significance
- morphology (micro and colonies)
- tests
- treatment
* most common candida species isolated from humans
* **3-5 um budding yeast** with **pseudohyphae** and occasional true hyphae
* have **feet** on agar (not seen in non-albicans species)
* when cultured on **cornmeal or rice** agar with Tween it forms **pseudohyphae** with clusters of **blastoconidia** at septations, as well as **terminal** **chlamydospores**
* positive **germ tube test**
* forms **green colonies** on chromogenic agar
* most susceptible to azoles, echinocandins, and amphotericin B
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C glabrata
- clinical specimens
- morphology
- growth rate
- culture characterstics
- other tests
- treatment
* isolated from **blood** and **urine**
* **2-4 um** **budding yeast (blastoconidia)**, without pseudohyphae
* grow more **slowly** than other candida species
* yeast morphology medium does not induce structures other than budding yeast
* positive **trehalose** test
* reduced susceptibilty to **azoles** but most are susceptible to echinocandins and amphotericin B
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Cryptococcus species
- human pathogens
- geographic distribution
- size
- transmission
- morphology
- tests
* **C neoformans** and **C gatii** are the most common pathogens in the US and tropical zones/northwest, respectively
* Inhalation of soil
* C neoformans: soil containing **bird excreta** (pigeon, chicken)
* C gatti: **eucalyptus trees**
* **3-15 um** narrow budding yeast
* **encapsulated**
* cell walls contain **melanin**
* Colonies often **mucoid**
* **Never forms other structures** like pseudohyphae
* **Phenol oxidase test**
* **Cryptococcal capsular polysaccharide antigen** can be detected in either serum or body fluids (CSF)
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Pneumocystis jiroveci
- culture
- diagnosis
- diseases
- serum markers
- stains
* **cannot culture in vitro**
* diagnosis depends on direct examination of clinical specimen
* disease in HIV with **CD4 \< 200 cells/ml**
* disease also occurs in **SCID, chemo** (esp lymphoma), **transplanted or collagen vascular disease** patients
* **LDH** high
* serology not helpful
* **Respiratory sample: exudate**
* **Giemsa, PAS, GMS stain orgs**
* **Immunofluorescent** staining wtih antibodies also work
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Colony colors:
Coccidioides
Blastomycosis
Paracoccidioides
Sporothrix
Penicillium marnefii
A flavus
A fumigatus
A terreus
Trichophyton rubrum
* Coccidioides: moist gray turns cottony white
* Blastomycosis: Cottony white turns tan
* Paracoccidioides: white-tan
* Sporothrix: pale orange turns brown
* Penicillium marnefii: powdery tan turns blue/green centrally with surrounding red
* A flavus: yellow-green
* A fumigatus: dark blue-green with white apron
* A terreus: cinnamon brown with yellow-orange reverse
* Trichophyton rubrum: red reverse
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Ulocladium
| (usually a contaminant)
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Scedosporium prolificans
- invasive infection with arthritis or osteomyelitis
- growth inhibited by cyclohexamide
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Bipolaris
- fast growing dematiaceous mold
- allergic sinusitis
- brain lesions in IC
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Exserohilum
- fast growing dematiaceous mold
- phaeohyphomycosis in nasal sinuses, skin, subcutis, cornea
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Helminthosporium
- fast growing dematiceous mold
- contaminant
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Curvularia
- fast growing dematiaceous mold
- cornea and sinus opportunistic infection
- mycetoma
- phaeohyphomycosis
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Alternaria
- fast growing dematiaceous mold
- usually a contaminant
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Stemphylium
- contaminant
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Chrysosporium
causes adiaspiromycosis, a granulomatous infection with large thick-walled spherules
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Sepedonium
- contaminant
- differs from H capsulatum by
1. not forming yeast at 37 degrees
2. not forming microconidia
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Beauveria bassiana
- contaminant
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Fusarium
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Gliocladium
- contaminant
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Acremonium
- white grain mycetoma
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Penicillium
- contaminant
- corneal, cutaneous, external ear, respiratory, and UTIs, and endocarditis with prosthesis
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Paecilomyces
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Scopulariopsis
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Microsporum canis
- scalp and skin infections, occasionally nails
- kids acquire infection from dogs and cats
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Microsporum gypseum
- scalp and skin
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Trichophyton rubrum
- skin and nails and only rarely hair
- one of the more common causes of onychomycosis
- most frequently isolated dermatophyte infecting humans
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Trichophyton mentagrophytes
- invades all parts of body surface
- athlete's foot
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Trichophyton tonsurans
- scalp ringworm
- skin and nails infected also
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Epidermophyton floccosum
- skin and nail infection (not hair)
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Penicillium marneffei PDA
Penicillium marneffei SDA
