MycologyClass 1

http://129.215.156.68/Movies/hypha.htm

Claudio Cortes M. D.V.M., Ph.D

Medical Microbiology and ImmunologyThe University of Toledo3000 Arlington Avenue, mail stop 1021Toledo OH, 43614419.383.6386Email: claudio.cortes@utoledo.edu

Contact information

Medical Mycology

SUBJECT CLASS

Introduction: Fungal structure and growth 1Fungal physiology and antifungal 2Subcutaneous pathogens 3Cutaneous pathogens (Dermatophytes) 4Opportunists- Mycoses I 5Opportunists- Mycoses II 6

“Septate hyphae, intercalary and terminal chlamydospores with rare microconida along hyphae branches were identified. Multiple small budding yeast around a big one was found in BAL sample. Diagnostic: Dimorphic fungi Paracoccidiodes brasiliensis”

OBJECTIVES To impart sufficient basic knowledge about

the growth of Fungi. Terminology

Distinguish structures/components of several forms of fungi

Assist you in diagnosing mycotic diseases.

Topics

1. Fungi characteristics

2. Fungal growth

3. Structures (naming fungi)

4. Fungi and disease

What are fungi?

1. Characteristics of fungi

HIV

16S rRNA

Fungi closer to animals than to plants

Pneumocystis carinii (renamed Pneumocystis jiroveci)

Eukaryotes

Fungi are heterotrophic (non-photosynthetic) Do not contain chlorophyll

Fungal cell membrane have a unique sterol. Ergosterol

Cell wall similar in structure to plants. Differs in chemical compositions Chitins biosynthesis

Characteristics of fungi cont.

Fungi digest then ingest Exoenzymes

Vegetative body may be unicellular (yeasts)

or composed of microscopic thread called

Hyphae (singular Hypha)

Reproduce by means of spores, usually

wind-disseminated Sexual (meiotic) and asexual (mitotic) spores may be

produced.

Characteristics of fungi

Viruses 0.08 mBacilli 4-6 mCocci 0.8 mSpirochetes 8 - 10 mProtozoa 15 mFungi 5 – 15 mNematodes 10 mm

SIZE COMPARISON OF PATHOGENS

Why study fungi?

EnvironmentSaprophytic role (decomposer)

Complex enzymatic machineryPlant and Fungi (mutualism)

Food Truffles, morels, mushroom, puffballs Cheese (Penicillium species) Soy products (Aspegillus, Rizhopus) Alcohol (Saccharomyces)

Bioremediation

Economy

Industry (pharmaceutical) Antibiotic/drugs

Penicillin (Penicillium notatum). Alexander Fleming. Cephalosporins (Acremonium = Cephalosporium) Cyclosporins -> immunosuppressant.

Medicine Mycotoxicosis (toxic fungi)

Mushroom poisoning Pre-formed toxin

Mycotoxins Production of toxin

Fungal toxins produced during infection Ethanol Oxalic acid

Allergies, Hypersensitivities and Chronic lung disease (often occupational)

FARMER’S LUNG – Moldy hay MALT WORKER’S DISEASE – Moldy barley CHEESE WASHER’S LUNG – Moldy cheese WOOD TRIMMER’S DISEASE – Moldy wood

Infection

2. Fungal growth

A B

The basic units of growing fungi

Yeasts Hyphae (mold)

Yeasts Single cells dividing usually by budding (single nucleus) Reproduce by budding (or by fission in some groups) Found where there is plenty of moisture (spread in water

films and by turbulence). Yeast cannot push into substrates as do hyphae. Growth in the form of yeasts is quite common for many

human pathogens. Growth of the organism within phagocytes and for easy

hematogenous spread. Cause cutaneuos candidiasis (AIDS), systemic mycosis

(hospitalized patients), and vaginal yeast infection

A B

Mold growth Long filaments growing at apex branching. (sing. Hypha): Mold Apical growth

Hyphae branch to form mycelium Mycelium (plural mycelia) Push into substrates

Common in cutaneous and subcutaneous infection Ringworm-> Dermatophytes

www.myhome.cortes.comMYCELIUM

www.rhodes.edu/biology/hill/hill/ResearchBackground.html

Classification based on cell division(hyphae)

Septate (with septa) Aspergillus and many other species have septate

hyphae. Septate hyphae with acute-angle branching important in pulmonary disease caused by Aspergillus spp.

Aseptate or coenocytic (without septa) Non-septate hyphae are associated with Mucor, some

zygomycetes, and other fungi.

Septate Aseptate

Classification based on fungal growth

Yeast

Mold

DIMORPHIC FUNGI

Growing both in the form of a yeast and a mold Common in systemic fungal infection The environment determines their morphology.

Temperature, CO2, nutrients This conversion is associated with a change in cell

wall composition. Complete reversal of a morphological change follows

return of the fungus to the initial environment.

37°C25°C

DimorphicYeast Mold Uncertain

Histoplasmosis

Coccidioidomycosis (Valley fever)

Blastomycosis (N. American blastomycosis)

Paracoccidioidiomycosis (S. American

blastomycosis)

Penicilliosis

Sporotrichosis(Rose-thorn or rose-gardeners)

Classification based on growth

Candida spp Dermatophytes

Aspergillus

Zygomycetes

Fusarium

Dematiaceous fungal infections (Trauma) - Cromoblastomycosis (Dermatitis verrucosa) - Phaeohyphomycosis

Mycetomata (Madura foot) (tumor like presentation)

Pneumocystis jiroveci (AID): Cysts

Lacazia loboiCryptococcus neoformans

3. Structures of Fungi

Naming fungi

Naming fungi Hyphae and Septa

Little variation Sporulation structures and spores

Variable and are basis of most identifications

Increasingly rRNA gene sequencing is being applied

Spores Many forms of spore production -> allow fungal identification Spores allow fungi to spread to novel sites.

Conidiospores/conidia (conidiophores)

Blastic conidiogenesis

Aspergillus, Penicillium, candida

Thallic conidiogenesis

Dermatophytes (Microsporum)

Coccidioides immitis (arthospores)

Naming fungi cont.

Ascospores (Ascus)Basiodiospores (Basidium) Zygospores (Zygosporangium)

Sporangiospores (sporangium; sporangiophore)

Zygomycetes

Asexual forms(Anamorphs)

Sexual forms (Teleomorph)

Conidiospores (conidia) Blastic conidiogenesis

where the spore is already evident before it separates from the conidiogenic hypha

Blastoconidia are pushed out of conidiophore (specialized structure). New wall is formed as the conidium balloons out.

Penicillium, Aspergillus, Fusarium, Stachybotrys, Trichoderma, Candida albicans, etchttp://www.mycolog.com/CHAP4a.htm

Asexual forms(anamorphs)

Conidiophore

ConidiaConidiospore

Conidiospores (conidia)

Blastic conidiogenesisi.e Penicillium Marneffei

Thallic conidiogenesis (Spores produced by modifying an existing hypha. Wall is remodeled.)

Dermatophytes (Microsporum canis)

Systemic mycosis Dimorphic;Coccidioides immitis

Asexual forms(anamorphs)

Macroconidium

arthospores

Sporangiospores (sporangium; sporangiophore) Spores produced following cytoplasmic cleavage within a

sporangium. Zygomycetes -> zygomycosis

sporangiophore

sporangiosporessporangium

Asexual forms(anamorphs)

• Ascospores -> ascomycetes (ascus sing = asci plural)

• Basidiospores produced Basidiomycetes

• Zygospores produced by zygomycetes**

Basidium

Sexually produced spores (teleomorph)

Sporangium

Zygosporangium

Sporangiophore

Sexual reproduction(Zygospores)

Asexual reproduction by spores(Sporangiospore)

Meiotic during

Germination

Fertilization

Life cycle of black bread mold (Rhizopus stolonifer)

zygomycetes

Biology of plant 5th edition. Raven PH. 1992

Naming yeasts Often little morphologic differences

Yeasts grouped by morphology,

pigment and how they divide, then

put into distinct species on basis of

a metabolic profile.

Most features of metabolic profile

associated with different patterns

of sugars and forms of nitrogen

(nitrate, nitrite, ammonium etc)

used

DNA sequencing coming on fast

Spores Many forms of spore production -> allow fungal identification Spores allow fungi to spread to novel sites.

Coccidioides

Conidiospores/conidia (conidiophores)

Blastic conidiogenesis

Aspergillus, Penicillium,

Thallic conidiogenesis

Dermatophytes (Microsporum)

Coccidioides immitis (arthospores)

Naming fungi cont.

Ascospores (Ascus)Basiodiospores (Basidium) Zygospores (Zygosporangium)

Sporangiospores (sporangium; sporangiophore)

Zygomycetes

Asexual forms(Anamorphs)

Sexual forms (Teleomorph)

Fungi and disease

Species of Fungi

>1,000,000 species

~400 pathogenic

Subcutaneous Mycoses

Chromomycosis

(Dermatitis verrucosa)

Mycetoma (actinomycetes)

(Madura foot)

Sporotrichosis*

(rose-thorn or rose-

gardeners' disease)

Cutaneous Mycoses

Systemic Mycoses

Opportunistic fungi

Dermatophytes

Athlete’s foot Jock itch Ringworm diseases

Cutaneous candiadiasis*

Candida: patients with AIDS

Histoplasmosis (Darling’s

diseases)Coccidioidomycosis

(Valley fever)Blastomycosis

(North American blastomycosis)

Paracoccidioidiomycosis (South

American blastomycosis)

Penicilliosis

Sporotrichosis *

Candidiasis *

Aspergillosis,

Pneumocystosis,

Zigomycosis

Cryptococcosis.

Classification Mycosis diseases

Skin, hair and nails.

Rarely invade deeper tissue

Subcutaneous tissue

Rarely spread systemically

May become widely disseminated.

Predilection for specific organs

Ubiquitous saprophytes

Predisposing diseases/condition

Involve skin and deep viscera

Cutaneous Mycoses

Skin, hair and nails Rarely invade deeper tissue

Dermatophytes Athlete’s foot or tinea pedis Jock itch or tinea cruris Ringworm diseases (tinea corpora, faciei, capitatis,

manuum, unguium) Cutaneous candiadiasis

Skin and mucosal; Oral thrush and Candida esophagitis, are extremely common in patients with AIDS.

Subcutaneous Mycoses

Subcutaneous tissue and rarely spread systemically. The causative agents are soil organisms introduced into the extremities by trauma

Chromomycosis (dermatitis verrucosa)

Mycetoma (Madura foot)

Sporotrichosis (rose-thorn or rose-gardeners' disease)

Systemic Mycoses Involve skin and deep viscera May become widely disseminated Predilection for specific organs

Histoplasmosis (Darling’s diseases) Coccidioidomycosis (valley fever, San Joaquin Valley fever,

California valley fever, and desert fever) Blastomycosis (North American blastomycosis) Paracoccidioidiomycosis (South American blastomycosis” and

"Paracoccidioidal granuloma”) Penicilliosis (south Asia) Systemic candidiasis (Hospital)

Opportunistic fungi Ubiquitous saprophytes and occasional

pathogens that invade the tissues of those patients who have: Predisposing diseases:

Diabetes, cancer, leukemia. Predisposing conditions:

Genetic disorders (cystic fibrosis), Agammaglobulinemia, steroid, contraceptive pills or antibiotic therapy, transplantation.

Aspergillosis, Candidiasis, Pneumocystosis, Zigomycosis, Cryptococcosis.

Diagnosis of fungal infections

Clinical recognition

Case HistoryPhysical examinationOrgan Imaging

Laboratory SpecimenSkin and nail scraping, urine, sputum, BAL, blood Pleural fluid, Peritoneal fluid, tissue biopsies.

Direct MicroscopyHistopathology

Culture and identification

Serodiagnostic testWet Mount (10 % KOH)

Biopsy

DNA probesPCR

Skin test (Dermal Hypersensitivity)

Dimorphic fungi

(systemic)

Histoplasmosis (Histoplasma capsulatum) Lungs, mucocutaneous, pericarditis. Grows in soil and material contaminated with bird or bat

droppings. The Central River Valleys in the midwestern and south

central United States are endemic for histoplasmosis. Latin America, part of Asia, Europe, Middle East, Africa.

Inhaled conidia (spores) small, intracellular, budding yeast

Tuberculate conidia(240C)

Yeast(370C)

YeastTissue

Penicillium Marneffei Prominent mycotic pathogen among HIV-infected individual. Southeast Asia. Imported cases in USA and Europe. Lung, lymphadenopathy, hematogenous dissemination. Intracellular (like Hisptoplasmosis). Inhaled conidia convert to yeast form with transverse

septa.

Mold: Conidia and red pigments

Yeast transverse septa.

Coccidioidomycosis (Coccidioides immitis) California valley fever

Desert soil. Southewestern USA, Mexico, region of Central and South America

Lung. CNS, bones and joins. Cutaneous infection Inhaled arthroconidia convert to spherules (contains

endospores).

arthroconidia Spherules with endospores

Blastomycosis (Blastomyces dermatitidis) “North American blastomycosis”

Lung and skin Soil and organic debris USA OHIO-Mississippi River Valley. North-America Inhaled conidia convert to large broad-based, budding

yeast.

Yeast (budding broad-based)Conidia

Paracoccidioidomycosis

(Paracoccidioides brasiliensis) South American blastomycosis," and "Paracoccidioidal

granuloma” Chronic single or multiples organs involvement Lungs, mouth, nose, lymph nodes Inhaled conidia convert to multipolar budding yeast

Multiple small budding yeast around a big one(Pilot wheel) (370C)

Septate hyphae, and terminal chlamydospores (240C)

Subcutaneous mycosis

“Rose-thorn or rose-gardeners” Lungs, joints, bones, and even the brain. Nodules and ulcers along lymphatics at site of

inoculation (Lymphatic nodules) Peru, tropical places, USA. Zoonotic Inhaled conidia covert to yeast Systemic mycosis (rare)

Swollen conidiophores, tear shaped, round conidia, sleeve shapes, rosette form

(240C)

Yeast/cigar shape(370C)

Sporothrichosis (Sporothrix schenkii)

Opportunistic fungi

Aspergillosis(A. fumigatus, A. niger, A. flavus and A. clavatus )

Ubiquitous molds found in organic matter Lungs Immunocompromised-> Systemic Inhaled conidia Hyphae (in lungs) England, USA Neutropenia Acute angle branching (<45oC)

methenamine-silver–stained tissue section of lung. (Figure courtesy of The Geraldine Kaminski Medical Mycology Library,

produced by David Ellis and Roland Hermanis, Doctorfungus Corporation. http://www.doctorfungus.org/imageban/index_enlarge.pl.)

Zygomycosis Ubiquitous molds

Lungs, skin Immunocompromised-> Systemic Inhaled conidia Hyphae England, USA Aseptate hyphae Diabetes Sinus involvement

Cutaneous mycosis

Epidermophyton

Microsporum

CandidaTrichophyton

Check figure 73-1. Medical microbiology.

Arthoconidia (thallic)

chlamydospores

Tuberculate conidia

microconidia

END

Overview of medically important fungiGroup (criteria

based on Morphology and

disease Disease Etiologic agentDiagnostic in vivo form

Diagnostic in vitro form Natural habitat

1. Molds Chromoblastomycosis Cladosporium Chestnut brown Branching chains of Woody plantBlack fungi carrionii thick-walled single-celled conidia material

Unifomr cells 10 um in diameter

Fonsecaea Same Series of single- Samepedrosoi celled conidia

giving rise to seriesof secondary conidia

Phaeohyphomycosis Exophiala Hyaline to brown Single-celled conidia Woody plant materialjeanselmei yeast-like cells, in balls at the apices

filaments, septate of annellideshyphae, in variouscombinations

Wangiella Same Single-celled conidia Soil and similar ambientsdermatitidis in balls a thte apices

of phialidesand annellides

Xylohypha Brown septate Sparsely branched, bantiana Hypahe long chains of

single-celled conidia

Overview of medically important fungiGroup (criteria

based on Morphology and

disease Disease Etiologic agentDiagnostic in vivo form

Diagnostic in vitro form Natural habitat

1. Molds Chromoblastomycosis Cladosporium Chestnut brown Branching chains of Woody plantBlack fungi carrionii thick-walled single-celled conidia material

Unifomr cells 10 um in diameter

Fonsecaea Same Series of single- Samepedrosoi celled conidia

giving rise to seriesof secondary conidia

Phaeohyphomycosis Exophiala Hyaline to brown Single-celled conidia Woody plant materialjeanselmei yeast-like cells, in balls at the apices

filaments, septate of annellideshyphae, in variouscombinations

Wangiella Same Single-celled conidia Soil and similar ambientsdermatitidis in balls a thte apices

of phialidesand annellides

Xylohypha Brown septate Sparsely branched, bantiana Hypahe long chains of

single-celled conidia

Overview of medically important fungi (cont.)Group (criteria

based on Morphology and

disease Disease Etiologic agentDiagnostic in vivo

formDiagnostic in

vitro form Natural habitat3. Dimorphic Blastomycosis Blastomyces Round to oval Small, round, smooth Woody plants

dermatiditis yeast s 8-15 um in diameter single-celled conidia materialhaving broad-based budded daugther cells

Coccidioidomycosis Coccidiodes Spherules 30-60 um Alternating barrel Soilimmitis containing single-celled shaped arthroconidia

endospores 2-5 um in diameter2.5 - 4 by 3-6 um

Histoplasmosis Histoplasma Oval, intracellular yeasts Tuberculate macro- Soil enriched by bat, starling capsulatum 2-5 - 3.5 um in diameter microconidiaconidia and smooth- or chicken droppings

walled

Paracoccidioidomycosis Paracoccidioides Multiples budding yeast, roundTypically sterile Probably woody plantsbrasiliensis budded cells

2-10 um attached to mature cells30-60 um in diameter

Sporotrichosis Sporothrix Round to oval yeast 3-5 Conidia develpoing Woody plant materialschenckii um in diameter from sympodial

conidiophores and fromthe hyphae

4 Oportunistic Aspergillosis Aspergillus Septate, dichotomously branchingChain of conidia Sameinfections favus hypahe 2.5 - 3.5 um in from phialides

diameter Same

Aspergillus Samefumigatus Usually sterile, some

isolate from phialidesMycetoma Madurella granules in tissue and draining

mycetomatis sinuses

Overview of medically important fungi (cont.)Group (criteria

based on Morphology and

disease Disease Etiologic agentDiagnostic in vivo form

Diagnostic in vitro form Natural habitat

2. Dermatophytes Tinea capitis Microsporum Hyphae in scaly Macro- and micro- Animals (zoophilic)canis erythematous conidia Kittens/dogs

lesions, arthroconidia within and around hair(ectothrix)

Thrichophyton Hyphae in scaly Conidia of varios Human (anthropophilic)tonsurans erythematous sizes and shapes

lesions, arthroconidia within hair(endothrix)

Tinea corporis Microsporum Hyphae in stratum Macro- and micro- Soil (geophilic)gypseum corneum conidia

Trichophyton Same Same Humans and othermentagrophytes animals (rodent, rabbits,etc)

Trichophyton Same Same Humansrubrum

Tinea cruri/pedis Epidermophyton Hyphae in stratum Club-shaped conidia Humansflloccosum corneum

Tinea cruri/pedis/ungium Trichophyton Same Macro- and micro- Humans and otherinterdigitale conidia animals(mentagrophites)

Tinea cruri/pedis/ungium Trichophyton Same Same Humansrubrum

Cell wall composition and taxonomic classification of representative medically important fungi

Principal cell wall polymer Taxonomic Group Examples

Chitin-chitosan Zygomycetes Rhizopus arrhizus

Chitin-glucan Ascomycetes (mycelial) Pseudallescheria boydii

Basidiomycetes (mycelial) Schizophyllum commune

Glucanmannan Ascomycetes (yeast) Sacchamomyces cerivisea

Chitin-mannan Fungi imperfecti Candida albicans

Basidiomycetes (yeast) Filobasidiella neoformans

Hair Perforation Test

Urease Test

Growth at 37°C

Macro-conidia Micro-conidia Distinguishing Characteristics

Trichophyton rubrum

Negative Negative Positive Pencil shaped/cigar shaped

Club shaped to pyriform, along the sides of the hyphae

Red reverse pigmentHair perf. test neg.

Club shaped microconidia

Trichophyton mentagrophytes

Positive Positive Positive Club shaped when present

NumerousUnicellular to round in grape like clusters

Round microconidia in grape like clusters Spiral

hyphae

Trichophyton tonsurans

Usually (-)Occasionally +

Positive Positive Cylindrical to cigar shaped and sinuous,

if present

Numerous, varying in shape and size, club shaped to balloon

shaped

Microconidia varying in shape and size

Growth enhanced by thiamine

Trichophyton verrucosum

Negative Negative Positive “Rat-tailed” if present

Rare or Absent Chlamydospores in

chains typically seen

Chlamydospores in chainsGrowth better on media

with thiamine and inositol

Trichophyton terrestre

Positive Positive Negative 2-8 celled borne at right angles to

hyphae

Club shaped with squared-off base on

pedicels

Microconidia with squared-off base on short pedicels

Epidermophyton floccosum

Negative Positive Positive Club shaped, often in clusters

Absent Khaki colored colony with brown reverse

Microconidia absent

Microsporum canis

Positive Positive NA Fusoid, thick, rough walled with

recurved apex

Typically absentClub shaped if

present

Fusoid, rough walled macroconidia with

recurved apex

Microsporum gypseum

Positive Positive NA Ellipsoidal to fusiform, thin, Rough walled

Moderately abundant Club shaped

Thin walled macroconidiaTawny-buff granular

colony

Microsporum nanum

Positive Positive NA Typically 2 celled Pear or egg shaped

Rough walled

Clavate when present 2 celled pear shaped macroconidia

Pathogen Fungal ligand(s) Phagocytic receptor(s)

Aspergillus fumigatus Mannans, B-glucans DC-SIGN, dectin-1

Blastomyces dermatitidis BAD1 CR3, CD14

Candida albicans Mannans, B-glucans DC-SIGN, dectin-1

Coccidiodes posadasii Mannans, B-glucans DC-SIGN, dectin-1

Cryptococcus neoformans Glucoronoxylomannan TLR2, TLR4, CD14, CD18, FcyRII

Histoplasma capsulatum HSP60 CD18, VLA-5

Pneumocystis jiroveci Mannans, B-glucans DC-SIGN, dectin-1

Definitions and Nomenclature http://labmed.ucsf.edu/education/residency/fung_morph/fungal_site/

page1.01.html#cleistothecia

Anamorph

Asexual or "imperfect" form of a fungus; for example, Scedosporium apiospermum is the anamorphic form of the teleomorph Pseudallescheria boydii

Arthroconidia

Conidia arising from pre-existing cells in the mycelium; adjacent cells collapse to release the mature form; see, for example, Geotrichum and Coccidioidomycosis

AscosporeSexual spore produced in a sac-like structure called an ascus

BlastoconidiaOne of three types of vegetative "spore" arising directly from the vegetative mycelium; budding form, e.g. seen in yeasts

ChlamydoconidiaConidia arising from pre-existent cells in the hyphae, which thicken and enlarge; may be intercalary, sessile, or terminal

ColumellaThe swollen, dome-shaped tip of a sporangiophore that extends into the sporangium

Conidia(singular conidium) Asexual "spores" of fungus

ConidiophoreSpecialized hyphal element bearing conidia

HolomorphTaxonomic name including teleomorphic and anamorphic forms of a fungus; the name of the teleomorph also serves as the name of the holomorph

Hyphae(singular hypha) The fundamental, threadlike structure of molds

Metula(plural metulae) Structure below the phialide in some Penicillium and Aspergillus species; see for example Aspergillus terreus

Mycelium(plural mycelia) The mass of filaments that constitutes the body of a mold; may be vegetative or aerial (reproductive)

Phialide

A conidiogenous cell that produces conidia from within its apex, which does not increase in width or length during conidiogenesis

Rhizoid

Root-like, branched hyphae which usually extend into growth medium; found especially in Zygomycetes. See, for example, Rhizopus

Sporangia

A fruiting body which forms a closed sac; see, for example, Absidia , Rhizopus

SporangiophoreA specialized hyphal element that bears the sporangium

StolonHorizontal hyphae growing along the surface of growth medium; runner

TeleomorphSexual or "perfect" form of a fungus; see Anamorph and Holomorph, above