PHTH1012 007 Mycology Introduction.pdf

INTRODUCTION TO MYCOLOGY Did you know… Bread would be flat without fungus Fungus is also crucial in making many wines and cheeses! Many antibiotics wouldn’t exist without fungus? Mushrooms that we enjoy on pizza are fungi! MYCOLOGY FUNGI: - Large group of organisms with varied forms and habits - Som...

INTRODUCTION TO MYCOLOGY Did you know… Bread would be flat without fungus Fungus is also crucial in making many wines and cheeses! Many antibiotics wouldn’t exist without fungus? Mushrooms that we enjoy on pizza are fungi! MYCOLOGY FUNGI: - Large group of organisms with varied forms and habits - Some are pathogenic for man, animals and plants - others are saprophytic and inhabit the soil breaking down dead organic matter. - Some saprophytic species are used in the production of substances of commercial importance e.g. antibiotics. - may be unicellular or multi-cellular. Fungi Have several features that distinguish them from other organisms. -They have a filamentous branching system of cells, with apical growth, lateral branching and a heterotrophic nutrition. - They are characterized by a life cycle that begins with germination from a spore or resting structure, followed by a period of growth as a substrate is exploited to produce biomass. - Finally there is a period of sporulation, where propagules (a unit that can give rise to another organism) are formed that can be disseminated from the parent mycelium. Some terminologies: Aerial hyphae: - hyphae above the agar surface Vegetative hyphae: - hyphae below the surface of the agar or substrate Arthroconidium: -An asexual propagule (unit that gives rise to another organism) formed by the breaking up of a hypha Asexual: - reproduction by division or redistribution of cells without nuclear fusion Assimilation: - ability to use a carbon or nitrogen source for growth Blastoconidium: - a conidium formed by budding along a hypha, pseudohypha, or single cell, as in yeast Chlamydoconidium: - An enlarged rounded conidium that is thick walled. May be located at the end of the hypha (terminal) or along the hypha (intercalary), singly or in chains. Cluster of blastoconidia single terminal chlamydospores, Some terminologies cont’d: Budding: - A process of asexual reproduction in which the new cell develops as a similar outgrowth from the older parent cell (yeast or yeast-like fungi) Columella: - Enlarged dome shaped structure at the tip of a sporongiophore that extend into the sporangium A cup-shaped columella (long 720 × 540 - 86k Conidium: - An asexual propagule that forms on the side or the end of the hypha or conidiophore Some terminologies con’td: Dematiaceous (means bundled): - Having structures that are brown to black (due to melanotic pigment in the cell wall) – proper name is Phaeohyphomycoses- prefix phaeo means dark pigment Dimorphic: - two distinct morphological forms. A mould phase at 25-300C and yeast phase when cultured at 35-370C (Candida is not truly dimorphic). Germ tube: (characteristic of Candida albicans) - Tubelike outgrowth from a conidium or spore; beginning of a hypha Hyaline: - Clear, transparent, colourless Phaeo - prefix meaning dark (brownish or blackish) Phialide - A cell that produces and extrudes conidia. Shaped like a flask or vase Rhizoid: - Rootlike, branched hypha extending into the medium Septate: - Having cross walls Spherule: - Large round, thick-walled structure containing spores Spore: - Propagule that develops by sexual reproduction ( ascospore, basidiospore, or zygospore) or by asexual means within a sporangium (sporangiospore) Thallus: - vegetative body of a fungus Vesicle: - Enlarged structure at the end of a conidiophore or sporangiospore FUNGI Con’td: - fungi pathogenic to man and animals may show dimorphism - cell wall is composed of chitin. - can assimilate CO2 from the air as do plants - utilize preformed compounds as a source of carbon. - classified by their type (sexual or asexual) and arrangement of spores (ascus, basidium, conidium etc.). CLASSIFICATION Taxonomic classification - based on spores that result from sexual reproduction (nuclear fusion and meiosis) - species may be defined on the basis of its asexual (imperfect or anamorphic) state CLASSIFICATION THALLOPHYTA Simple vegetative structure not divided into roots, stems or leaves Algae Fungi (Contains Chlorophyll) (Do not contain Chlorophyll) Eumycetes (True Fungi) 1) Zygomycetes/Phycomycetes (Water Molds) A. Rhizopus B. Mucor C. Abasidia CLASSIFICATION CON’TD 2). Ascomycetes A. True yeasts B. Highly organised forms producing ascospores in culture (sexual reproduction involves a sac or ascus) Eg. Some dematiaceous fungi Asexual reproduction – via conidia. Molds with septate hyphae. Eg. Blastomyces, histoplasma, microsporum, trichophyton. Yeast genera including saccharomyces CLASSIFICATION CON’TD 3) Basidiomycetes - Mushrooms 4). Deuteromycetes (Fungi Imperfecti) - Fungi lacking a complete life cycle reproducing by asexual spores (conidia) Eg. C. immitis, C. albicans Classification of fungal agents con’td 2) Morphological - Mold - Yeast 3) Clinical - Superficial Mycosis 1. Pityriasis versicolor 2. Tinea nigra - Cutaneous Mycosis 1. Dermatophytosis 2. Candidiasis Classification of fungal agents con’td - Subcutaneous Mycosis 1. Chromomycosis (Proper name Chromoblastomycosis) 2. Phaeohyphomycosis 3. Sporotrichosis 4. Maduromycosis - Systemic Mycosis 1. Histoplasmosis 2. Blastomycosis 3. Coccidioidomycosis 4. Paracoccidioidmycosis 5. *Penicilliosis (P. marneffei) *Sporotrichosis Opportunistic Mycoses - Caused by endogenous or ubiquitous organisms of low inherent virulence that cause infection in debilitated, compromised patients Caused most commonly by: Candida, Cryptococcus, Aspergillus, Rhizopus, Mucor and Absidia Actinomycetes (true bacteria): - Diverse group of Gram positive bacilli - Form chains of filaments - Most are saprophytes that live in soil - Related to corynebacteria, mycobacteria, streptomycetes - Responsible for 3 human infections: Actinomycosis, nocardiosis and actinomycetoma Hyaline fungi which could either be contaminants or implicating fungi in infection: Hyalohyphomycetes comprising several fungi with hyaline hyphae: -Aspergillus -Fusarium -Penicillium -Paecilomyces Phaeohyphomycetes: Include the melanized or dematiacious fungi (darkly pigmented fungi). Species of: Exophiala Phialophora Cladosporium Curvularia Alternaria Hypha (ae): - Filaments originating from a germinating fungus spore or branching from other filaments. Aseptate (no cross walls) (e.g. Phycomycetes ) or Septate (cross walls) Mycelium: A mat of hyphae, characteristic of the culture SPORES - Spores may be sexual or asexual. Spores germinate to produce new colonies. SEXUAL SPORES: - Develop from specialized cells in the mycelium as a result of nuclear fusion.. ASEXUAL SPORES: - Spores which develop without nuclear fusion. - The following are types of thallospores: A. Blastospores: - Spores produced by budding from vegetative cells; as with some yeast sp. e.g. Candida, Cryptococcus and Saccharomyces. B. Chlamydospores - Spores which develop by concentration of protoplasm into a hyphal cell, with thickening of the cell wall. May be found in all fungi. C. Arthrospores - Spores which develop by fragmentation of hyphae into short oblong rounded elements as in Geotrichum and Coccidioides.. Conidiospores - Spores which develop on a specialized supporting structure of the mycelium. The supporting structure is called Conidiophore Occurs in FUNGI IMPERFECTI. Macroconidium: - Large, multicellular thin or thick walled, spindle shaped conidiospore. Microconidium: - Small, unicellular conidiospores, usually formed along the hyphae singly or in clusters. Macroconidia and microconidia are characteristic of the genera Microsporum , Trichophyton and Epidermophyton (No microconidia).. SPORANGIOSPORES - Spores which develop endogenously within a swollen structure - Sporangium on the end of a hypha. Occurs in Phycomycetes. The Epidemiology of Fungal Infections Infections divided into 2 categories: 1. Nosocomial: > acquired in a healthcare setting > almost always opportunistic 2. Community Acquired: > opportunistic mycoses > endemic mycoses - acquired by living in a geographic area constituting the natural habitat of a pathogenic fungus Epidemiology Cont’d: Seventh most common cause of infectious diseases over the past two and a half decades Factors contributing to increase fungal infections - immunosupressed or immunocompromised pts - a mobile and aging population with an increased prevalence of chronic medical conditions - use of new and & aggressive medical & surgical therapeutic strategies. Risk Factors: - Broad spectrum antibiotic - Cytotoxic drugs - Organ transplantation - Indwelling catheter - Extensive surgery or burns - Neutropenia - ICU stay - Haemodialysis - Malnutrition PATHOGENIC FUNGI FOR MAN A) Superficial Mycoses 1) Pityriasis versicolor 2) Dermatophytes 3) Candidiasis 1) Pityriasis versicolor (Liver spots) Aetiology – Malassezia furfur 2) Dermatophyes (Ringworm fungi) - Affect skin, nail and hair - structures with an abundance of keratin. These fungi are: Microsporum – skin and hair Trichophyton - skin, nail and hair Epidermophyton – skin and nail Dermatophytes Continued Diseases include: Tinea capitis - ringworm of scalp Tinea barbae - chronic folliculitis of beard, neck or face Tinea corporis - infection of glabrous skin Tinea manus - involves interdigital spaces and palmer surfaces of hand Tinea unguium - infection of nails Tinea cruris - infection of groin ( commonly called jock itch) Tinea pedis - infection of feet (commonly called athlete’s foot) 3) Candidiasis: - Pathogenic Yeast-like fungi, Endogenous infection - Include skin infections, nail infection, thrush, oesophagitis, enteritis, pneumonia, septicemia B) Subcutaneous Mycosis Diseases: - Sporotrichosis – Sporothrix schenckii - Maduromycosis (mandura foot) – caused by a variety of fungi ( P. boydii - most frequent agent) and bacteria (including species of Streptomyces, Actinomadurae, and Nocardia) - Chromoblastomycosis (granulomatous infection of skin) - cause by dematiaceous fungi Isolation and identification of pathogenic fungi cont’d. 3) Subcutaneous (Lab diagnosis) Sporotrichosis (Sporothrichum schenckii) > Microscopy - Organisms difficult to find - Asteroid bodies in tissues are considered diagnostic. > Culture - Dimorphic - 25C (mould); at 37C (yeast), tissues (yeast) > LFCB stain - Conidia in a typical rosette pattern Sporothrix schenckii – narrow septate hyphae and branching with tearshaped conidia (left 25-300C) , rosettelike cluster in young culture - Round, oval and fusiform budding cells of various sizes (right 37 0C, cigar bodies) 3) Subcutaneous cont’d (Lab. Diagnosis) Chromomycosis (several dematiaceous fungi) > Microscopy - Brown pigmented spherical sclerotic bodies (scrapings treated with KOH and in histological preparations) > Culture - Slow growing dematiaceous fungi - Organisms are identified according to the pattern and type of sporulation exhibited by the isolate. Brown-pigmented, round to polyhedral, thick walled sclerotic bodies KOH preparation Fonsecaea pedrosoi Subcutaneous Treatment SPOROTRICHOSIS - Localized subcutaneous infection responds to orally administered potassium iodide. - Systemic manifestations of sporotrichosis may require itraconazole therapy. CHROMOMYCOSIS - Surgery and 5-fluorocytosine (5FC) are employed. - Results generally are unsatisfactory, particularly in advanced chronic disease. Subcutaneous Treatment MADUROMYCOSIS (Mycetoma) a) Actinomycotic Mycetoma Streptomycin or amikacin, co-trimoxazole or dapsome, or ciprofloxacin b) Eumycotic Mycetoma Prolonged antifungal therapy with itraconazole (10 months) can improve early lesions Antifungal chemotherapy with Amphotericin B for late, mutilating lesions. Surgical excision with amputation as a last resort. Isolation and identification of Systemic Mycoses 4) Systemic i) Histoplasmosis (Histoplasma capsulatum) > Direct microscopy (sputum, mucocutaneous lesions, biopsy, RES) Giemsa stain - intracellular yeasts with halo (macrophages) > Culture - dimorphic, tuberculate macroconidia (25 0C) > Serology - C.F.T, I.D. > Histoplasmin skin test ; +ve indicates exposure only Histoplasma capsulatum – septate hyphae and large round thick walled macroconidia (left 25 0C, tuberculate, projections) - Small round/oval budding cells (right, 370C) Isolation and identification cont’d. 4) Systemic cont’d ii) Coccidiomycosis (Coccidiodes immitis) > Microscopy - spherules with endospores (sputum) > Culture - arthrospores Confirmation - identification of arthrospores and their conversion to spherule in TC or in animals > Serology - Skin test, C.F.T., I.D. Coccidiodes immitis – Spherules with endospores Isolation and identification cont’d. 4) Systemic cont’d iii) Blastomycosis (Blastomyces dermatitidis) > Microscopy - Typical thick walled, broad based, single budding yeast in sputum (eg. Stains-H&E; PAS; Mucicarmine; GMS) > Culture - Saboraud’s & Mycobiotic agar- dimorphism demonstrated Systemic cont’d Treatment Histoplasmosis - 10 pulmonary histoplasmosis and localized lung lesions do not require treatment Amphoteracin B > - progressive pulmonary disease and disseminated histoplasmosis Coccidiodomycosis - 10 coccidiomycosis resolves without treatment - Chronic pulmonary and disseminated coccidiodomycosis require Amphotericin B Systemic cont’d Treatment: Blastomycosis Amphotericin B – serious pulmonary disease Uncomplicated pulmonary disease – respond to fluconazole? P. Brasiliensis - thermally dimorphic fungus distributed in Brazil and South America. Lesions due to Paracoccidioidomycosis on the face of a Brazilian child LABORATORY DIAGNOSIS 1) Specimens The specimens for laboratory diagnosis of fungus infections will depend upon the type of infection and may include: Skin scraping infected hair infected nail Dermatophytes Biopsy material Exudate (granulous or ulcerative lesions) Subcutaneous Sputum biopsy tissue Blood films bone marrow CSF Swabs Other exudates Systemic mycoses Cryptococcus Monilia Microscopic Examination Direct Microscopy Wet preparation with saline (sputum, pus ) - Look for budding yeast cells, hyphae & pseudohyphae, conidia, granules, sperules 10% KOH preparation ( skin, hair, nail eg. Dermatophytes) - digest tissue elements (epithelial cells, leucocytes, debris) India Ink preparation - Negative staining to observe capsule around yeast eg. Cryptococcus neoformans Stained preparations. Gram stain - any fungal forms may be observed (eg. Yeast, C. albicans) Acid fast stain - eg. Nocardia – partially acid fast Lactophenol cotton blue - phenol in stain kills any organism while lactic acid preserves fungal structures - cotton blue stains the chitin in fungal cell wall (Slides prepared from culture) Stained preparations con’td. Gomori methenamine silver stain (GMS) Periodic acid-Schiff (PAS) Haematoxyllin & Eosin (H&E) CULTURE Blood agar Sabouraud’s Dextrose Agar (SDA) ( RT or at 370C) Mycobiotic ( RT or at 370C) Brain Heart Infusion Agar Thioglycolate Broth Others Microsporum canis – septate hyphae with spingle shaped, rough and thick walled macroconidia (usually > 6 cells). Few microconidia Trichophyton mentagrophytes – Cigar shaped macroconidia ( not always present), septate and coiled hyphae, microconidia clustered on branched conidiophores. Epidermaphyton floccosum – septate hyphae with smooth, club shaped macroconidia (singly/clusters, 2-6 cells). No microconidia. Serological Examination Largely performed in specialised centres e.g. C.D.C in Atlanta for the diagnosis of certain systemic mycoses. Examples of serological tests include: Immunodiffusion (ID)- precipitation Tube Agglutination – known killed antigen + serum Complement Fixation (CFT) Indirect Latex Fixation (ILA) Indirect fluorescent antibody (IFA)