General Mycology and Overview of Fungal Infections Learning

General Mycology and Overview of Fungal Infections

Learning objectives At the end of the session, the students will be able to understand: ▰Classify fungi and fungal infections ▰ Describe clinical presentation of common fungal infections ▰ Describe laboratory diagnosis of fungal infections ▰ Describe treatment of fungal infections Essentials of Medical Microbiology 2

GENERAL MYCOLOGY Essentials of Medical Microbiology 3

GENERAL MYCOLOGY ▰ Medical mycology - branch of medical science thatdeals with the study of medically important fungi ▰ ‘fungus’ is derived from Greek ‘mykes’ meaning mushroom (a type of edible fungus) Essentials of Medical Microbiology 4

GENERAL MYCOLOGY (Cont. . ) Fungi differ from bacteria and other eukaryotes in many ways: ▰ Eukaryotic and possess eukaryotic cell organelles. ▰ Possess a rigid cell wall, composed of chitin, β-glucans and other polysaccharides. ▰ Cell membrane contains ergosterol instead of cholesterol. ▰ May be unicellular or multicellular. ▰ Lack chlorophyll and divide by asexual and/or sexual means by producing spores. Essentials of Medical Microbiology 5

CLASSIFICATION OF FUNGI Essentials of Medical Microbiology 6

Morphological Classification 1. Yeast: Round to oval cells that reproduce by budding - Cryptococcus neoformans (pathogenic) - Saccharomyces cerevisiae (non-pathogenic) 2. Yeast-like: Yeasts forming pseudohyphae (e. g. Candida) - Differentiated from true hyphae as they have constrictions at septa 3. Molds: long branching filaments called hyphae - Hyphae - septate or nonseptate Essentials of Medical Microbiology 7

Morphological Classification (Cont. . ) Based on the growth pattern in culture medium ▰ Aerial mycelium: It is the part of the mycelium which projects above the surface of culture medium ▰ Vegetative mycelium: It is the part of the mycelium that grows on the surface of the culture medium Essentials of Medical Microbiology 8

Morphological Classification (Cont. . ) 4. Dimorphic fungi: exist as molds in the environment at ambient temperature (25°C) and as yeasts in human tissues at body temperature (37°C). Ø Histoplasma capsulatum Ø Blastomyces dermatitidis Ø Coccidioides immitis Ø Paracoccidioides brasiliensis Ø Penicillium marneffei Ø Sporothrix schenckii. Essentials of Medical Microbiology 9

Morphological forms of fungi Essentials of Medical Microbiology 10

Taxonomical Classification ▰ Based on the production of sexual spores 1. Phylum zygomycota: sexual spores – zygospores, and possess aseptate hyphae, e. g. Rhizopus and Mucor. 2. Phylum ascomycota: Sexual spores - ascospores and possess septate hyphae, e. g. Aspergillus. 3. Phylum basidiomycota: Sexual spores - basidiospore e. g. Cryptococcus 4. Phylum deuteromycota (Fungi imperfecti): sexual state is either absent or unidentified yet Essentials of Medical Microbiology 11

Types of fungal spores Sexual Spore Observed in- Zygospores Zygomycetes Ascospores Aspergillus Basidiospores Cryptococcus Asexual Spore Observed in Vegetative asexual spore Arthrospore Coccidioides Trichosporon Blastospore Candida Chlamydospore Candida albicans Essentials of Medical Microbiology 12

Types of fungal spores (Cont. . ) Aerial asexual spore Conidiospore or conidia Aspergillus Sporangiospore Zygomycetes Microconidia Dermatophytes Macroconidia Dermatophytes Essentials of Medical Microbiology 13

LABORATORY DIAGNOSIS OF FUNGAL DISEASES Essentials of Medical Microbiology 14

Specimen Collection ▰ Depends on site of infection - skin scraping, hair, nail, sputum, etc. ▰ Systemic mycoses - blood sample, Cerebrospinal fluid (CSF), etc Essentials of Medical Microbiology 15

Microscopy ▰ Fungal elements can be detected in the clinical specimens by direct microscopic examination of material from the lesion. Essentials of Medical Microbiology 16

Microscopy (Cont. . ) Potassium hydroxide (KOH) preparation ▰ Keratinized tissue specimens treated with 10% KOH digests keratin fungal hyphae clearly seen ▰ 20– 40% KOH - nail & hair Essentials of Medical Microbiology 17

Microscopy (Cont. . ) Potassium hydroxide (KOH) preparation (Cont. . ): ▰ Biopsy specimens - dissolved in 10% KOH in a test tube and examined after overnight incubation ▰ Glycerol (10%) can be added to prevent drying ▰ DMSO (dimethyl sulfoxide) - help in tissue digestion Essentials of Medical Microbiology 18

Microscopy (Cont. . ) Gram stain: ▰ Useful in identifying the yeasts (e. g. Cryptococcus) and yeast like fungi (e. g. Candida). ▰ They appear as gram-positive budding yeast cells India ink and nigrosin stains: ▰ Used as negative stains for demonstration of capsule of Cryptococcus neoformans Essentials of Medical Microbiology 19

Microscopy (Cont. . ) Calcofluor white stain: ▰ More sensitive than other stains. ▰ Binds to cellulose and chitin of fungal cell wall and fluoresce under UV light Essentials of Medical Microbiology 20

Microscopy (Cont. . ) Histopathological stains ▰ For demonstrating fungal elements from biopsy tissues ▰ Periodic acid schiff (PAS) stain: Ø PAS positive fungi appear magenta/deep pink, whereas the nuclei stain blue Ø PAS which stains only the live fungi Essentials of Medical Microbiology 21

Microscopy (Cont. . ) Histopathological stains (Cont. . ) ▰ Masson fontana stain: for pigmented (or pheoid) fungi ▰ Hematoxylin and Eosin stain ▰ Mucicarmine stain: for staining the carminophilic cell wall of Cryptococcus and Rhinosporidium Essentials of Medical Microbiology 22

Microscopy (Cont. . ) Histopathological stains (Cont. . ) ▰ Gomori methenamine silver (GMS) stain: Alternative to PAS Ø Stains both live and dead fungi Ø Stains the polysaccharide component of the cell wall Ø Fungi appear black & background pale green color Essentials of Medical Microbiology 23

Microscopy (Cont. . ) Lactophenol cotton blue (LPCB): It is ▰ Used to study the microscopic appearance of the fungal isolates grown in culture. It contains: ▰ Phenol acts as disinfectant ▰ Lactic acid preserves the morphology of fungi ▰ Glycerol prevents drying ▰ Cotton blue stains the fungal elements blue. Essentials of Medical Microbiology 24

Culture ▰ Fungal culture is frequently performed for isolation and correct identification of the fungi. Essentials of Medical Microbiology 25

Culture Media ▰ Sabouraud’s dextrose agar (SDA): Ø Most commonly used medium Ø Contains peptone (1%), dextrose (4%) and p. H of 5. 6 Ø May not support some pathogenic fungi ▰ Neutral SDA (Emmons’ modification): Ø Differs from original SDA in having neopeptone (1%) and dextrose (2%) and p. H of 7. 2 Essentials of Medical Microbiology 26

Culture Media (Cont. . ) ▰ Corn meal agar and rice starch agar: Nutritionally deficient media used for stimulation of chlamydospore production ▰ Brain heart infusion (BHI) agar and blood agar: Enriched media, used for growing fastidious fungi like Cryptococcus and Histoplasma Essentials of Medical Microbiology 27

Culture Media (Cont. . ) ▰ Niger seed agar and bird seed agar: selective growth of Cryptococcus ▰ CHROMagar Candida medium: selective as well as differential medium for speciation of Candida. Essentials of Medical Microbiology 28

Culture Condition Temperature: Most of the fungi grow well at 25– 30°C except the dimorphic fungi that grow at both 25°C and 37°C ▰ BOD incubators (biological oxygen demand): capable of maintaining low temperature ▰ Incubation time: 2– 3 weeks ▰ Antibiotics - cycloheximide (actidione), chloramphenicol and gentamicin - added to the culture media to inhibit bacterial growth ▰ Essentials of Medical Microbiology 29

Culture Identification ▰ Correct identification of the fungus is based on the macroscopic appearance of the colonies grown on culture and microscopic appearance (LPCB mount of colonies). Essentials of Medical Microbiology 30

Macroscopic Appearance of the Colony Rate of growth: ▰ Rapid growth (<5 days): seen in saprophytes, yeasts and agents of opportunistic mycoses ▰ Slow growth (1– 4 weeks): observed in dermatophytes, agents of subcutaneous and systemic mycoses. Essentials of Medical Microbiology 31

Macroscopic Appearance of the Colony (Cont. . ) ▰ Pigmentation: seen on the reverse side of the culture media ▰ Texture: refers to how the colony would have felt if allowed to touch. It may be of various types such as - glabrous (waxy/leathery), velvety, yeast like, cottony or granular/powdery. ▰ Colony topography Essentials of Medical Microbiology 32

Microscopic Appearance of Fungi ▰ Teased mount: Identification is based on : Ø Nature of hyphae (septate or aseptate, hyaline or phaeoid, narrow or wide) Ø Type of sporulation (conidia or sporangia) ▰ Slide culture: in situ microscopic appearance of the fungal colony Essentials of Medical Microbiology 33

Microscopic Appearance of Fungi (Cont. . ) Essentials of Medical Microbiology 34

Microscopic Appearance of Fungi (Cont. . ) ▰ Cellophane tape mount: Ø Impressions taken by placing the cellophane tape on the colonies LPCB mount is made Ø Easy to perform than slide culture and in-situ fungal morphology maintained Essentials of Medical Microbiology 35

Other Methods of Identification ▰ For Candida: Germ tube test, Dalmau plate culture, carbohydrate fermentation and carbohydrate assimilation tests are done ▰ For dermatophytes: Hair perforation test, dermatophyte test medium and dermatophyte identification medium are used ▰ Urease test can be done for the fungi that produce urease enzyme, e. g. Cryptococcus Essentials of Medical Microbiology 36

Immunological Methods ▰ Antibody detection - ELISA, immunodiffusion test, agglutination test, and complement fixation test (CFT) ▰ Antigen detection - latex agglutination test for cryptococcal antigen from CSF ▰ Immunohistochemistry: detecting antigens (e. g. proteins) on cells of a tissue section Essentials of Medical Microbiology 37

Automations ▰ Automated identification system such as MALDI-TOF and VITEK are revolutionary in accurate identification of yeasts and to some extent molds. Essentials of Medical Microbiology 38

Molecular Methods ▰ Polymerase chain reaction (PCR) and its modifications - multiplex PCR, nested PCR and the most advanced real time PCR ▰ DNA sequencing methods. Essentials of Medical Microbiology 39

TREATMENT OF FUNGAL INFECTIONS Essentials of Medical Microbiology 40

TREATMENT OF FUNGAL INFECTIONS Class Drug Use Mechanisms Antifungal Antibiotics Amphotericin B (AMB) Systemic mycoses Nystatin, Hamycin Topical use (skin infection) Echinocandins Caspofungin, micafungin Systemic mycoses Inhibits β-glucan synthesis in fungal cell wall Benzofurans Griseofulvin Dermatophytoses Disrupts mitotic spindle by binding to fungal cell tubulin Polyenes Essentials of Medical Microbiology Bind to ergosterol and disrupts fungal cell membranes 41

TREATMENT OF FUNGAL INFECTIONS (Cont. . ) Class Drug Synthetic antifungal agents Imidazoles: Clotrimazole, Azoles miconazole, ketoconazole Triazoles Use Mechanisms Topical use Inhibits ergosterol synthesis of fungi Systemic mycoses (invasive fungal infections) Fluconazole Cryptococcus and Candida Itraconazole Sporothrix, chromoblastomycosis, dermatophytes, Cryptococcus, Candida (except C. krusei, C. glabrata) and Trichosporon Voriconazole All invasive fungal infections except mucormycosis Posaconazole and isavuconazole All invasive fungal infections including mucormycosis Essentials of Medical Microbiology 42

TREATMENT OF FUNGAL INFECTIONS (Cont. . ) Class Drug Use Mechanisms Antimetabolite Flucytosine (5 -FC) Systemic mycoses Used in combination with amphotericin B. Fluorouracil inhibits thymidylate synthetase, thus inhibiting DNA synthesis Allylamines Terbinafine Topical use Inhibits ergosterol synthesis Essentials of Medical Microbiology 43

OVERVIEW OF FUNGAL INFECTIONS Essentials of Medical Microbiology 44

SUPERFICIAL MYCOSES Essentials of Medical Microbiology 45

SUPERFICIAL MYCOSES Tinea versicolor: ▰ Caused by lipophilic fungus Malassezia furfur - characterized by flat-round scaly hypopigmented to hyperpigmented patches of skin. Tinea nigra: ▰ Characterized by painless, black, nonscaly patches present on palm and sole; caused by - black-colored yeast like fungus Hortaea werneckii. Essentials of Medical Microbiology 46

SUPERFICIAL MYCOSES (Cont. . ) Piedra: ▰ Characterized by nodule formation on hair shaft - either black in color (caused by Trichosporon beigelii) or white in color (caused by Piedraia hortae). Essentials of Medical Microbiology 47

SUPERFICIAL MYCOSES (Cont. . ) Dermatophytoses (or tinea or ringworm): ▰ Most common superficial mycoses affecting skin, hair and nail - caused by a group of related fungi (called dermatophytes). These include: ▰ Trichophyton species: Infect skin, hair and nail ▰ Microsporum species: Infect skin and hair ▰ Epidermophyton species: Infect skin and nail. Essentials of Medical Microbiology 48

SUBCUTANEOUS MYCOSES Essentials of Medical Microbiology 49

SUBCUTANEOUS MYCOSES Mycetoma: ▰ Also known as Maduramycosis or Madura foot - chronic, slowly progressive granulomatous infection of the skin and subcutaneous tissues ▰ Clinically manifested - triad - swelling, discharging sinuses and presence of granules in the discharge. ▰ Two types – Eumycetoma and Actinomycetoma Essentials of Medical Microbiology 50

SUBCUTANEOUS MYCOSES (Cont. . ) Sporotrichosis : ▰ Also known as Rose Gardner’s disease - caused by a thermally dimorphic fungus, Sporothrix schenckii. ▰ Presents as subcutaneous noduloulcerative lesions. Essentials of Medical Microbiology 51

SUBCUTANEOUS MYCOSES (Cont. . ) Chromoblastomycosis: ▰ Refers to slow growing chronic subcutaneous lesions caused by group of dematiaceous or phaeoid fungi (i. e. darkly pigmented fungi). ▰ Produce a characteristic morphology called sclerotic body. ▰ Agents include: Fonsecaea, Phialophora Cladosporium and Rhinocladiella. Essentials of Medical Microbiology 52

SUBCUTANEOUS MYCOSES (Cont. . ) Rhinosporidiosis: ▰ Caused by Rhinosporidium seeberi. ▰ Produces large friable polyps in the nose (most common site), conjunctiva and rarely in other sites. Essentials of Medical Microbiology 53

SYSTEMIC MYCOSES Essentials of Medical Microbiology 54

SYSTEMIC MYCOSES ▰ Caused by the saprophytic fungi, which spread by inhalation of spores leading to pulmonary infection. ▰ These agents are thermally dimorphic, include: Ø Histoplasma capsulatum Ø Blastomyces dermatitidis Ø Coccidioides immitis Ø Paracoccidioides brasiliensis Essentials of Medical Microbiology 55

OPPORTUNISTIC MYCOSES Essentials of Medical Microbiology 56

OPPORTUNISTIC MYCOSES ▰ Caused by the fungi that are normally found as human commensals or in environment; but can act as human pathogen in presence of opportunities such as low immunity. Essentials of Medical Microbiology 57

OPPORTUNISTIC MYCOSES (Cont. . ) Candidiasis: ▰ Most common fungal disease in humans, affecting the skin, mucosa, and various internal organs. ▰ Caused by Candida, a yeast like fungus that produces pseudohyphae. ▰ Various species - Candida albicans, C. tropicalis, C. parapsilosis etc. ▰ Common in presence of various predisposing factors - patients on steroid or immunosuppressive drugs, post-transplantation, malignancy, HIV infection. Essentials of Medical Microbiology 58

OPPORTUNISTIC MYCOSES (Cont. . ) Cryptococcosis: ▰ Caused by a capsulated yeast - Cryptococcus neoformans - capable of producing potentially fatal meningitis in HIV infected people. ▰ Negative staining of CSF - performed to demonstrate the capsule - appears as refractile delineated clear space surrounding the round budding yeast cells against the black background. Essentials of Medical Microbiology 59

OPPORTUNISTIC MYCOSES (Cont. . ) Zygomycosis: ▰ Group of life-threatening infections caused by aseptate fungi; belonging to the phylum Zygomycota. ▰ Include - Rhizopus, Mucor, Rhizomucor, Lichtheimia, etc. ▰ Risk factors: diabetic ketoacidosis. ▰ Clinical forms: Rhinocerebral mucormycosis. Essentials of Medical Microbiology 60

OPPORTUNISTIC MYCOSES (Cont. . ) Zygomycosis (Cont. . ): Laboratory diagnosis: ▰ Culture on SDA - white cottony woolly colonies and LPCB mount of the colonies showing typical broad aseptate hyaline hyphae. ▰ Rhizopus shows a unique root like growth arising from hyphae called rhizoid. Essentials of Medical Microbiology 61

OPPORTUNISTIC MYCOSES (Cont. . ) Aspergillosis: ▰ Refers to the invasive and allergic diseases caused by a hyaline mold named Aspergillus. ▰ Important species - A. fumigatus, A. flavus and A. niger. ▰ Risk factors: Glucocorticoid use and profound neutropenia Essentials of Medical Microbiology 62

OPPORTUNISTIC MYCOSES (Cont. . ) Aspergillosis (Cont. . ): Clinical forms: ▰ Pulmonary aspergillosis – most common form. ▰ Other forms - invasive sinusitis, cardiac, cerebral, ocular aspergillosis. Laboratory diagnosis: ▰ SDA reveals typical narrow septate hyaline hyphae. Essentials of Medical Microbiology 63

OPPORTUNISTIC MYCOSES (Cont. . ) Penicilliosis: ▰ Penicillium species produce various opportunistic infections - endophthalmitis, otomycosis, keratitis and onychomycosis. ▰ Penicillium marneffei - only dimorphic species of Penicillium, which causes opportunistic infection (wart-like skin lesions) in HIV-infected patients. Essentials of Medical Microbiology 64

OPPORTUNISTIC MYCOSES (Cont. . ) Pneumocystosis: ▰ Refers to plasma cell pneumonia due to Pneumocystis jirovecii, which typically occurs in HIV infected patients. Essentials of Medical Microbiology 65

MYCOTOXICOSES Essentials of Medical Microbiology 66

MYCOTOXICOSES Mycotoxicoses: ▰ Occurs following consumption of food contaminated by toxins liberated by certain fungi; e. g. aflatoxin by Aspergillus flavus which causes carcinoma liver. Essentials of Medical Microbiology 67

MYCOTOXICOSES (Cont. . ) Mycetism: ▰ Refers to toxic effects produced by, eating poisonous fleshy fungi; usually different types of mushrooms. Essentials of Medical Microbiology 68

MICROSPORIDIOSI S Essentials of Medical Microbiology 69

MICROSPORIDIOSIS ▰ Previously placed under parasites. ▰ Microsporidia are now taxonomically re-classified under fungi. ▰ They comprise of several species (e. g. Encephalitozoon cuniculi and others), which cause gastrointestinal, musculoskeletal and ocular infections. Essentials of Medical Microbiology 70

Questions: ▰ Q 1. All are yeast or yeast like fungi except: a. Candida b. Trichosporon c. Cryptococcus d. Trichophyton Essentials of Medical Microbiology 71

Questions: ▰ Q 2. Organisms that does not affect the nail: a. Trichophyton b. Epidermophyton c. Microsporum d. Candida Essentials of Medical Microbiology 72

Questions: ▰ Q 3. Asteroid bodies is observed in: a. Sporotrichosis b. Histoplasmosis c. Candidiasis d. Chromoblastomycosis Essentials of Medical Microbiology 73

Questions: ▰ Q 4. Tinea versicolor is caused by: a. Trichophyton rubrum b. Trichophyton violaceum c. Malasezzia furfur d. Candida albicans Essentials of Medical Microbiology 74

Questions: ▰ Q 5. Azole active against mucormycoses: a. Voriconazole b. Fluconazole c. Itraconazole d. Posaconazole Essentials of Medical Microbiology 75