Medical Mycology Lecture Notes PDF

02/08/1444 By Abdulfatah albakkosh Outline • Introduction and General Characteristics • Culture and Isolation • Body Sites and Possible Fungal Pathogens • Yeasts • Opportunistic Fungi • Cutaneous and Superficial Fungi • Subcutaneous Fungi • Systemic Fungi 1 02/08/1444 I. INTRODUCTION AND GENER...

02/08/1444 By Abdulfatah albakkosh Outline • Introduction and General Characteristics • Culture and Isolation • Body Sites and Possible Fungal Pathogens • Yeasts • Opportunistic Fungi • Cutaneous and Superficial Fungi • Subcutaneous Fungi • Systemic Fungi 1 02/08/1444 I. INTRODUCTION AND GENERAL CHARACTERISTICS 1. Molds: Multicellular fungi 2. Yeasts: Single-cell fungi 3. Mycosis: Fungal infection 4. Systemic mycosis: Multiorgan infection caused by fungi 5. Opportunistic mycosis: Fungal disease that occur primarily in immunocompromised patients . 6. Dimorphic fungi: Fungi that show both a non molds (e.g., yeast) and molds phase 7. Saprobe: Organism capable of living on decaying organic material 2 02/08/1444 Mycology is the study of fungi (molds, yeasts, and mushrooms). All fungi are • Eukaryotic (e.g., true nucleus, 80S ribosomes, mitochondria, as are humans). • Complex carbohydrate cell walls: chitin, glucan, and mannan. • Ergosterol = Major membrane sterol Imidazole antifungals inhibit synthesis of ergosterol. Polyene antifungals bind more tightly to ergosterol than cholesterol. 3 02/08/1444 Fungal Structure Hyphae are long, branching filaments that come together to form the mycelium. There are two main types of hyphae. a. Septate hyphae have cellular separation or cross-walls. b. Sparsely septate (formerly aseptate) hyphae contain few if any cellular separations. c. Pseudohyphae are a chain of cells formed by budding that resemble true hyphae. Pseudohyphae differ from true hyphae in that they are constricted at the septa, form branches that begin with septation, and have terminal cells smaller than other cells. 4 02/08/1444 Fungal Structure Hyphae are classified as vegetative and aerial. a. Vegetative hyphae function in food absorption and are the portion that extends below the agar surface or nutrient substrate. b. Aerial hyphae extend above the agar or nutrient substrate, and their function is to support reproductive structures called conidia. 5 02/08/1444 6 02/08/1444 Fungal Structure 3. Conidia are spore like asexual reproductive structures not produced by cleavage, conjugation, or free-cell formation. Conidia are only formed by the imperfect fungi. a. Conidia morphology is important in fungal identification. b. Conidia classification is based on conidia morphologic development. c. Microconidia are single-celled, small conidia. d. Macroconidia are multicellular, large conidia. 7 02/08/1444 Types of conidia a. Arthroconidia are conidia resulting from the fragmentation of hyphae into individual cells. Some fungi will have arthroconidia separated by normal (disjuncture) cells. b. Blastoconidia: Conidia that form as the result of budding c. Chlamydoconidia result from terminal cells in the hyphae that enlarge and have thick walls. These conidia can survive adverse environmental conditions. Chlamydoconidia are found in moulds, whereas similar structures (chlamydospores) are found in hyphae produced by some yeasts. Arthroconidia 8 02/08/1444 d. Poroconidia: Conidia formed by being pushed through a small pore in the parent cell e. Phialoconidia: Tube-shaped conidia that can be branched f. Annelloconidia are vase-shaped conidia; the remaining parent outer cell wall takes on a saw-toothed appearance as the conidia are released. 9 02/08/1444 Annelloconidia Phialoconidia 1. Sexual reproduction a. The fungus needs special structures called spores to be made by the fungus. b. Fungi that reproduce sexually are called "perfect fungi." 10 02/08/1444 c. Types of spores 1) Ascospores: Spores contained in a saclike structure. 2) Basidiospores: Spores contained in a club-shaped structure. 3) Oospores: Spores resulting from the fusion of cells from two different Hyphae. 4) Zygospores: Spores resulting from the fusion of two identical hyphae 11 02/08/1444 2. Asexual reproduction a. Asexual reproduction only involves division of the nucleus and cytoplasm. b. Fungi that undergo asexual reproduction are termed imperfect fungi. c. Imperfect fungi are the only fungal group to produce conidia. II. CULTURE AND ISOLATION 12 02/08/1444 A. Types of Fungal Media 1. Sabouraud dextrose agar (SDA) a. General-purpose, nutritionally poor medium mildly selective for fungi, no longer commonly used; several different formulations available. b. In one formulation, the agar has an acidic pH (5.6) that inhibits most bacteria. Modified SDA (Emmons) has a neutral pH and better supports the growth of fungi but is less inhibitory for bacteria. A. Types of Fungal Media 2. Sabouraud-brain heart infusion agar (SABHI) a. A nonselective medium for isolation of all fungi b. Contains dextrose, peptone, and brain heart infusion c. Can be made selective for dimorphic fungi by the addition of cyclohexamide, chloramphenicol, and gentamicin 13 02/08/1444 3. Brain heart infusion agar with blood (BHIB) a. Used to grow most fungi, especially those from sterile body sites b. Contains brain heart infusion and sheep blood c. Can be made selective for dimorphic fungi by the addition of cyclohexamide, chloramphenicol, and gentamicin. Cyclohexamide inhibits the saprophytic fungi and chloramphenicol inhibits many grampositive and gram-negative bacteria, whereas gentamicin inhibits primarily gram-negative bacteria. A. Types of Fungal Media 4. Selective agars contain various antimicrobial agents that will enhance the growth of specific fungal pathogens and will inhibit bacteria and other undesired growth. 14 02/08/1444 a. Inhibitory mold agar (IMA) 1) IMA is used to grow most fungal pathogens; it is especially formulated to recover the cyclohexamide-sensitive Cryptococcus. 2) Contains gentamicin and chloramphenicol b. Dermatophye test medium (DTM) 1) Used to isolate the dermatophytes 2) DTM contains cyclohexamide and gentamicin and phenol red as a pH indicator. Differential agars 5. Differential agars are used to enhance pigment development, conidia production, and mold-to-yeast phase transition. 15 02/08/1444 Differential agars a. Potato dextrose agar (PDA) 1) Used to enhance conidia development 2) Enhances pigment development of Trichophyton rubrum Differential agars c. Corn meal agar with Tween 80: Used to differentiate Candida spp. d. Agars containing rice, casein, and other nutrients are used to differentiate Trichophyton spp. 16 02/08/1444 B. Culture Considerations 1. Fungal cultures are incubated at 30°C. 2. Growth requires from several days to several weeks. 3. Cultures should be maintained in a high-humidity environment. 4. Several techniques are used to obtain culture material for slide preparation. a. Tease mount method • A dissecting needle is used to separate a fungal colony that has been placed on a microscope slide. • Using this method may cause fungus, especially conidia, to be damaged. • It may take more than one attempt to obtain a sample containing whole conidia. 17 02/08/1444 b. Cellophane tape method • b. Cellophane tape method: Cellophane tape is used to transfer aerial hyphae from the colony to a microscope slide for examination. C. The Slide Culture Method c. The slide culture technique uses an agar block covered by a cover slip. On the side of the agar block, fungi are grown as colonies. The coverslip is removed and used for examination under the microscope. This method prevents structural damage to the fungus. 18 02/08/1444 C. Direct Examination Methods 19 02/08/1444 C. DIRECT EXAMINATION METHODS • 1. Saline wet mount is used to view fungal elements, such as hyphae, conidia, and budding yeasts. It has limited use and is most commonly applicable for vaginal secretions to diagnose vaginitis. C. DIRECT EXAMINATION METHODS 2. Lactophenol cotton blue wet mount is used to stain and preserve fungal elements in culture isolates. 3. Potassium hydroxide (KOH) is used to dissolve non-fungal materials in skin, nail, and hair samples. 20 02/08/1444 Potassium hydroxide (KOH) • KOH mount – KOH dissolves keratin and cellular material but does not affect fungi. • Specimen is placed on a slide, a drop of 10-20% KOH is added and covered with a coverslip, • left for 20 min in incubator at 37°C to digest keratin. Then examined microscopically. C. DIRECT EXAMINATION METHODS 4. Gram stain can be used to view yeasts. 5. India ink can be used to reveal capsules surrounding C. neoformans found in cerebrospinal fluid (CSF). However, due to low sensitivity, direct antigen detection assays have generally replaced the India ink wet mount. 21 02/08/1444 C. DIRECT EXAMINATION METHODS 6. Calcofluor white stain is a fluorochrome that stains chitin found in the cell wall of fungi. The stain is not absorbed by human tissue. The slide is viewed using an ultraviolet light. Fungi will appear white to blue to green depending on the wavelength of light. KOH can be added to clear the specimen of cellular debris. C. DIRECT EXAMINATION METHODS Fungal antigen detection uses known antibodies to identify circulating fungal antigens in a patient’s serum, CSF, or urine. Antibodies are available for Histoplasma and Cryptococcus. These tests are important when patients are compromised and antibodies may not be reliably detected. 22 02/08/1444 III. BODY SITES AND POSSIBLE FUNGAL PATHOGENS A. Blood: Candida spp., Blastomyces dermatitidis, Histoplasma capsulatum, and Cryptococcus neoformans B. Cerebrospinal Fluid: Cryptococcus neoformans, Candida spp., Histoplasma capsulatum, and Coccidioides immitis C. Hair: Microsporum and Trichophyton D. Nails: Aspergillus, Epidermophyton, and Trichophyton E. Skin: Candida, Microsporum, Trichophyton, Epidermophyton, and Blastomyces dermatitidi III. BODY SITES AND POSSIBLE FUNGAL PATHOGENS F. Lungs: Candida albicans, Aspergillus, Rhizopus, Penicillium, Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides immitis G. Throat: Candida albicans and Geotrichum candidum H. Urine: Candida albicans and Candida glabrata I. Genital Tract: Candida albicans 23