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Mycology Class 1

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1 Mycology Class 1

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

3 Medical Mycology SUBJECT CLASS Introduction: Fungal structure and growth 1 Fungal physiology and antifungal2 Subcutaneous pathogens3 Cutaneous pathogens (Dermatophytes)4 Opportunists- Mycoses I5 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 1. Fungi characteristics 2. Fungal growth 3. Structures (naming fungi) 4. Fungi and disease

7 What are fungi? 1. Characteristics of fungi

8 HIV 16SrRNA Fungi closer to animals than to plants Pneumocystis carinii (renamed Pneumocystis jiroveci)

9 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.

10 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

11 Viruses 0.08 Bacilli 4-6 Cocci 0.8 Spirochetes Protozoa 15 Fungi 5 – 15 Nematodes10 mm SIZE COMPARISON OF PATHOGENS

12 Why study fungi?

13 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 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) FARMERS LUNG – Moldy hay MALT WORKERS DISEASE – Moldy barley CHEESE WASHERS LUNG – Moldy cheese WOOD TRIMMERS DISEASE – Moldy wood Infection

16 2. Fungal growth

17 The basic units of growing fungi YeastsHyphae (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 Growing both in the form of a yeast and a mold Common in systemic fungal infection The environment determines their morphology. Temperature, CO 2, 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°C 25°C

23 Dimorphic Yeast 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 loboi Cryptococcus neoformans

24 3. Structures of Fungi Naming fungi

25 Hyphae and Septa 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 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)

28 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 Asexual forms (anamorphs)

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

30 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

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

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

33 Sporangium Zygospo rangium 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 5 th 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 Spores Many forms of spore production -> allow fungal identification Spores allow fungi to spread to novel sites. Coccidioides

36 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)

37 Fungi and disease

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

39 Subcutaneous Mycoses Chromomycosis (Dermatitis verrucosa) Mycetoma (actinomycetes) (Madura foot) Sporotrichosis* (rose-thorn or rose- gardeners' disease) Cutaneous Mycoses Systemic Mycoses Opportunistic fungi Dermatophytes Athletes foot Jock itch Ringworm diseases Cutaneous candiadiasis* Candida: patients with AIDS Histoplasmosis (Darlings 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

40 Cutaneous Mycoses Skin, hair and nails Rarely invade deeper tissue Dermatophytes Athletes 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 (Darlings 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 Case History Physical examination Organ Imaging Laboratory Specimen Skin and nail scraping, urine, sputum, BAL, blood Pleural fluid, Peritoneal fluid, tissue biopsies. Direct Microscopy Histopathology Culture and identification Serodiagnostic test Wet Mount (10 % KOH) Biopsy DNA probes PCR Skin test (Dermal Hypersensitivity)

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 Tuberculate conidia (24 0 C) Yeast (37 0 C) Yeast Tissue

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. Mold: Conidia and red pigments Yeast transverse septa.

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. Yeast (budding broad-based) Conidia

50 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) (37 0 C) Septate hyphae, and terminal chlamydospores (24 0 C)

51 Subcutaneous mycosis

52 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 (24 0 C) Yeast/cigar shape (37 0 C) Sporothrichosis (Sporothrix schenkii)

53 Opportunistic fungi

54 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 (<45 o C) 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 CandidaTrichophyton

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

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64 Cell wall composition and taxonomic classification of representative medically important fungi Principal cell wall polymerTaxonomic GroupExamples Chitin-chitosanZygomycetesRhizopus arrhizus Chitin-glucanAscomycetes (mycelial)Pseudallescheria boydii Basidiomycetes (mycelial)Schizophyllum commune GlucanmannanAscomycetes (yeast)Sacchamomyces cerivisea Chitin-mannan Fungi imperfectiCandida albicans Basidiomycetes (yeast)Filobasidiella neoformans

65 Hair Perforation Test Urease Test Growth at 37°C Macro-conidiaMicro-conidiaDistinguishing 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 Positive Club shaped when present Numerous Unicellular to round in grape like clusters Round microconidia in grape like clusters Spiral hyphae Trichophyton tonsurans Usually (-) Occasionally + 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 Positive 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 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 NegativePositive Club shaped, often in clusters AbsentKhaki colored colony with brown reverse Microconidia absent Microsporum canis Positive NA Fusoid, thick, rough walled with recurved apex Typically absent Club shaped if present Fusoid, rough walled macroconidia with recurved apex Microsporum gypseum Positive NA Ellipsoidal to fusiform, thin, Rough walled Moderately abundant Club shaped Thin walled macroconidia Tawny-buff granular colony Microsporum nanum Positive NA Typically 2 celled Pear or egg shaped Rough walled Clavate when present2 celled pear shaped macroconidia

66 PathogenFungal ligand(s)Phagocytic receptor(s) Aspergillus fumigatusMannans, B-glucansDC-SIGN, dectin-1 Blastomyces dermatitidisBAD1CR3, CD14 Candida albicansMannans, B-glucansDC-SIGN, dectin-1 Coccidiodes posadasiiMannans, B-glucansDC-SIGN, dectin-1 Cryptococcus neoformansGlucoronoxylomannanTLR2, TLR4, CD14, CD18, FcyRII Histoplasma capsulatumHSP60CD18, VLA-5 Pneumocystis jiroveciMannans, B-glucansDC-SIGN, dectin-1

67 Definitions and Nomenclature othecia Anamorph Asexual or "imperfect" form of a fungus; for example, Scedosporium apiospermum is the anamorphic form of the teleomorph Pseudallescheria boydiiScedosporium apiospermumPseudallescheria boydii Arthroconidia Conidia arising from pre-existing cells in the mycelium; adjacent cells collapse to release the mature form; see, for example, Geotrichum and CoccidioidomycosisGeotrichum

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 terreusAspergillus 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, RhizopusRhizopus Sporangia A fruiting body which forms a closed sac; see, for example, Absidia, RhizopusAbsidia

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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