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Mycology Class 1

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1 Mycology Class 1

2 Contact information Claudio Cortes M. D.V.M., Ph.D Medical Microbiology and Immunology The University of Toledo 3000 Arlington Avenue, mail stop 1021 Toledo OH, 43614

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

4 “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”

5 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.

6 Topics Fungi characteristics Fungal growth Structures (naming fungi)
Fungi and disease

7 1. Characteristics of fungi
What are fungi?

8 (renamed Pneumocystis jiroveci)
rRNA Fungi closer to animals than to plants Pneumocystis carinii (renamed Pneumocystis jiroveci) HIV

9 Characteristics of fungi cont.
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

10 Characteristics of fungi
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.

Viruses 0.08 m Bacilli 4-6 m Cocci 0.8 m Spirochetes m Protozoa 15 m Fungi 5 – 15 m Nematodes 10 mm

12 Why study fungi? 12

13 Saprophytic role (decomposer) Plant and Fungi (mutualism) Food
Environment Saprophytic role (decomposer) Complex enzymatic machinery Plant and Fungi (mutualism) Food Truffles, morels, mushroom, puffballs Cheese (Penicillium species) Soy products (Aspegillus, Rizhopus) Alcohol (Saccharomyces)

14 Industry (pharmaceutical)
Bioremediation Economy Industry (pharmaceutical) Antibiotic/drugs Penicillin (Penicillium notatum). Alexander Fleming. Cephalosporins (Acremonium = Cephalosporium) Cyclosporins -> immunosuppressant.

15 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

16 2. Fungal growth

17 The basic units of growing fungi
Yeasts Hyphae (mold)

18 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

19 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 MYCELIUM

20 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

21 Classification based on fungal growth
Yeast Mold

22 DIMORPHIC FUNGI 25°C 37°C 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.

23 Classification based on growth
Mold Yeast Dimorphic Uncertain Dermatophytes Aspergillus Zygomycetes Fusarium Dematiaceous fungal infections (Trauma) - Cromoblastomycosis (Dermatitis verrucosa) - Phaeohyphomycosis Mycetomata (Madura foot) (tumor like presentation) Candida spp Histoplasmosis Coccidioidomycosis (Valley fever) Blastomycosis (N. American blastomycosis) Paracoccidioidiomycosis (S. American blastomycosis) Penicilliosis Sporotrichosis (Rose-thorn or rose-gardeners) Pneumocystis jiroveci (AID): Cysts Lacazia loboi Cryptococcus neoformans

24 3. Structures of Fungi Naming fungi

25 Naming fungi Hyphae and Septa Sporulation structures and spores
Little variation Sporulation structures and spores Variable and are basis of most identifications Increasingly rRNA gene sequencing is being applied

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

27 Sexual forms (Teleomorph)
Naming fungi cont. Asexual forms (Anamorphs) Sexual forms (Teleomorph) Ascospores (Ascus) Basiodiospores (Basidium) Zygospores (Zygosporangium) Conidiospores/conidia (conidiophores) Blastic conidiogenesis Aspergillus, Penicillium, candida Thallic conidiogenesis Dermatophytes (Microsporum) Coccidioides immitis (arthospores) Sporangiospores (sporangium; sporangiophore) Zygomycetes

28 Conidiospores (conidia)
Asexual forms (anamorphs) 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, etc

29 Conidiospores (conidia)
Conidiophore i.e Penicillium Marneffei Blastic conidiogenesis

30 Asexual forms (anamorphs)
Macroconidium Thallic conidiogenesis (Spores produced by modifying an existing hypha. Wall is remodeled.) Systemic mycosis Dimorphic;Coccidioides immitis Dermatophytes (Microsporum canis) arthospores

31 Asexual forms (anamorphs)
Sporangiospores (sporangium; sporangiophore) Spores produced following cytoplasmic cleavage within a sporangium. Zygomycetes -> zygomycosis sporangiospores sporangium sporangiophore

32 Sexually produced spores (teleomorph)
Ascospores -> ascomycetes (ascus sing = asci plural) Basidiospores produced Basidiomycetes Zygospores produced by zygomycetes** Basidium

33 Life cycle of black bread mold (Rhizopus stolonifer) zygomycetes
Sporangium Sporangiophore Asexual reproduction by spores (Sporangiospore) Meiotic during Germination Sexual reproduction (Zygospores) Zygosporangium Fertilization Biology of plant 5th edition. Raven PH. 1992

34 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

35 Mycology: PF. Lehmann Spores October 8, 2008 Many forms of spore production -> allow fungal identification Spores allow fungi to spread to novel sites. Coccidioides

36 Sexual forms (Teleomorph)
Mycology: PF. Lehmann Naming fungi cont. October 8, 2008 Asexual forms (Anamorphs) Sexual forms (Teleomorph) Ascospores (Ascus) Basiodiospores (Basidium) Zygospores (Zygosporangium) Conidiospores/conidia (conidiophores) Blastic conidiogenesis Aspergillus, Penicillium, Thallic conidiogenesis Dermatophytes (Microsporum) Coccidioides immitis (arthospores) Sporangiospores (sporangium; sporangiophore) Zygomycetes

37 Fungi and disease

38 Species of Fungi >1,000,000 species ~400 pathogenic

39 Classification Mycosis diseases
Systemic Mycoses Cutaneous Mycoses Subcutaneous Mycoses Opportunistic fungi Involve skin and deep viscera 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 Dermatophytes Athlete’s foot Jock itch Ringworm diseases Cutaneous candiadiasis* Candida: patients with AIDS Chromomycosis (Dermatitis verrucosa) Mycetoma (actinomycetes) (Madura foot) Sporotrichosis* (rose-thorn or rose-gardeners' disease) Histoplasmosis (Darling’s diseases) Coccidioidomycosis (Valley fever) Blastomycosis (North American blastomycosis) Paracoccidioidiomycosis (South American blastomycosis) Penicilliosis Sporotrichosis * Candidiasis * Aspergillosis, Pneumocystosis, Zigomycosis Cryptococcosis.

40 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.

41 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)

42 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)

43 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.

44 Diagnosis of fungal infections
Clinical recognition Laboratory Specimen Skin and nail scraping, urine, sputum, BAL, blood Pleural fluid, Peritoneal fluid, tissue biopsies. Case History Physical examination Organ Imaging Skin test (Dermal Hypersensitivity) DNA probes PCR Culture and identification Direct Microscopy Histopathology Serodiagnostic test Wet Mount (10 % KOH) Biopsy

45 Dimorphic fungi (systemic)

46 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 Yeast (370C) Yeast Tissue Tuberculate conidia (240C)

47 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. Yeast transverse septa. Mold: Conidia and red pigments

48 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

49 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. Conidia Yeast (budding broad-based)

50 Multiple small budding yeast around a big one
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 Septate hyphae, and terminal chlamydospores (240C) Multiple small budding yeast around a big one (Pilot wheel) (370C)

51 Subcutaneous mycosis

52 Sporothrichosis (Sporothrix schenkii)
“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)

53 Opportunistic fungi

54 Aspergillosis Ubiquitous molds found in organic matter
(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.

55 Zygomycosis Ubiquitous molds Lungs, skin
Immunocompromised-> Systemic Inhaled conidia  Hyphae England, USA Aseptate hyphae Diabetes Sinus involvement

56 Cutaneous mycosis

57 Epidermophyton Microsporum Trichophyton Candida

58 Check figure 73-1. Medical microbiology.
microconidia Arthoconidia (thallic) Tuberculate conidia chlamydospores

59 END





64 Principal cell wall polymer
Mycology: PF. Lehmann October 8, 2008 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

65 Trichophyton mentagrophytes
Hair Perforation Test Urease Test Growth at 37°C Macro-conidia Micro-conidia Distinguishing Characteristics Trichophyton rubrum Negative Positive Pencil shaped/cigar shaped Club shaped to pyriform, along the sides of the hyphae Red reverse pigment Hair perf. test neg. Club shaped microconidia Trichophyton mentagrophytes Club shaped when present Numerous Unicellular to round in grape like clusters Round microconidia in grape like clusters Spiral hyphae Trichophyton tonsurans Usually (-) Occasionally + 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 “Rat-tailed” if present Rare or Absent Chlamydospores in chains typically seen Chlamydospores in chains Growth better on media with thiamine and inositol Trichophyton terrestre 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 Club shaped, often in clusters Absent Khaki colored colony with brown reverse Microconidia absent Microsporum canis NA Fusoid, thick, rough walled with recurved apex Typically absent Club shaped if present Fusoid, rough walled macroconidia with recurved apex Microsporum gypseum Ellipsoidal to fusiform, thin, Rough walled Moderately abundant Club shaped Thin walled macroconidia Tawny-buff granular colony Microsporum nanum Typically 2 celled Pear or egg shaped Rough walled Clavate when present 2 celled pear shaped macroconidia

66 Phagocytic receptor(s) Aspergillus fumigatus Mannans, B-glucans
Mycology: PF. Lehmann October 8, 2008 Pathogen Fungal ligand(s) Phagocytic receptor(s) Aspergillus fumigatus Mannans, B-glucans DC-SIGN, dectin-1 Blastomyces dermatitidis BAD1 CR3, CD14 Candida albicans Coccidiodes posadasii Cryptococcus neoformans Glucoronoxylomannan TLR2, TLR4, CD14, CD18, FcyRII Histoplasma capsulatum HSP60 CD18, VLA-5 Pneumocystis jiroveci

67 Definitions and Nomenclature http://labmed. ucsf
Definitions and Nomenclature othecia 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

68 Ascospore Sexual spore produced in a sac-like structure called an ascus Blastoconidia One of three types of vegetative "spore" arising directly from the vegetative mycelium; budding form, e.g. seen in yeasts Chlamydoconidia Conidia arising from pre-existent cells in the hyphae, which thicken and enlarge; may be intercalary, sessile, or terminal Columella The swollen, dome-shaped tip of a sporangiophore that extends into the sporangium

69 Conidia (singular conidium) Asexual "spores" of fungus Conidiophore Specialized hyphal element bearing conidia Holomorph Taxonomic 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

70 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

71 Sporangiophore A specialized hyphal element that bears the sporangium Stolon Horizontal hyphae growing along the surface of growth medium; runner Teleomorph Sexual or "perfect" form of a fungus; see Anamorph and Holomorph, above

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