Mycology

Mycology - the study of fungi Fungi - includes molds and yeasts. Molds - exhibit filamentous type of growth. Yeasts - exhibit pasty or mucoid form of fungal growth. 50,000 + valid species Fungi stain gram positive, and require oxygen to survive. Fungi are eukaryotic, containing a nucleus bound by a membrane, an endoplasmic reticulum, and mitochondria. (Bacteria are prokaryotes and do not contain these) Fungi are heterotrophic like animals and most bacteria; requiring organic nutrients as a source of energy. (Plants are autotrophic)

Mycology
Fungi are dependent upon enzyme systems to derive energy from organic substrates. Saprophytes - live on dead organic matter. parasites - live on living organisms. Fungi are essential in recycling of elements, especially carbon.

Mycology
Role of fungi in the economy: Industrial uses of fungi Mushrooms. (Class Basidiomycetes) Truffles. (Class Ascomycetes) Natural food supply for wild animals. Yeast as food supplement, supplies vitamins. Penicillium - ripens cheese, adds flavor (roquefort, etc.). Fungi used to alter texture, improve flavor of natural and processed foods.

Mycology
Fermentation Fruit juices (ethyl alcohol). Saccharomyces cerevisiae - brewer's and baker's yeast. Fermentation of industrial alcohol, fats, proteins, acids, etc. Antibiotics First observed by Fleming; noted suppression of bacteria by a contaminating fungus of a culture plate.

Mycology
Plant pathology Most plant diseases are caused by fungi. Medical importance 50-100 species are recognized human pathogens. Most prefer to be free-living saprophytes; and only accidentally become pathogens. To be pathogenic, they must tolerate the temperature of the host site and possess an enzymatic system that allows them to utilitize animal tissues. Increased incidence of fungal infections in recent times.

Mycology
Importance of Medical Mycology During the time period between 1941 - 1973, the number of reported deaths in the U.S. due to scarlet fever, typhoid, whooping cough, diphtheria, dysentery and malaria decreased from 10,165 cases to 107; but reported deaths due to mycoses during the same time period, increased from 324 to 530. Increased mobility - We can travel to a geographical area where a fungus exists as part of the commensal flora of the local population, or is endemic to the area. We have an aging population.

Mycology
The Immunosuppressed Patient: AIDS Drugs - immunosuppressant drugs used in organ transplant patients, cancer and leukemia patients.

Mycology
Immunology of the Mycoses Antibody mediated immunity (B-cell, humoral) Antibodies are often produced in response to a fungal infection, but do not confer immunity. Serological tests for identification of fungal diseases detect these antibodies. Cellular mediated immunity (T-cell) T-cell immunity is effective in resistance to fungal infections.

Mycology
Basic terms as they relate to mycology: Hypha (hyphae plural) - fundamental tubelike structural units of fungi.

Septate - divided by cross walls. Aseptate - lacking cross walls.

Mycelium - a mass (mat) of hyphae forming the vegetative portion of the fungus.

Aerial - growing or existing in the air. Vegetative - absorbs nutrients. Fertile - bears conidia (spores) for reproduction.

Mycology
Basic Terms (continued) Sporulation & Spores - preferred terms when there is a merging of nuclear material. Self-fertile are termed homothallic. Mating types are termed heterothallic. Sexual spores - exhibit fusion of nuclei. Ascospore - spore formed in a sac-like cell known as an ascus. Often eight (8) spores formed. (Ascomycetes) Basidiospore - sexual spore produced on a specialized club-shaped structure, called a basidium. (Basidiomycetes) Zygospore - a thick-walled spore formed during sexual reproduction (Phycomycetes)

Mycology
Sporulation & Spores (continued) Asexual spores - most common type.

Conidia - asexual fungal spores borne externally in various ways from a conidiophore; often referred to a macroconidia (multicellular) and microconidia (unicellular). Arthroconidium (Arthrospore) - special type of asexual spore formed by disarticulation of the mycelium. Blastoconidia (Blastospore) - asexual spore formed from a budding process along the mycelium or from another blastospore.

Mycology

Asexual Spores (continued) Chlamydospore - thick-walled asexual spore formed by direct differentiation of the mycelium (concentration of protoplasm and nutrients). Sporangiospore - an asexual spore contained in a sporangium at the end of a sporangoiphore. Thallospore - asexual spore produced on a thallus (hypha).

Mycology
Miscellanous terms: Ascus - sac-like structure containing (usually eight) ascospores developed during sexual reproduction in the Ascomycetes. Conidiophore - a specialized branch of hypha on which conidia are developed. Dematiaceous - pigmented, dark in color, usually gray to black. Hyaline - colorless, clear. Dermatophyte - fungus that causes superficial mycoses.

Mycology
Miscellanous terms: (continued) Diphasic (dimorphic) - the ability of some fungi to grow as either yeast or filamentous stages, depending on conditions. Ectothrixic - ability of the fungus to grow on the outside of a hair shaft. Endothrixic - ability of the fungus to grow and penetrate into the hair shaft. Germ Tube - small projections which arise from cells of certain yeasts; indicates the onset of hyphal formation.

Mycology
Miscellanous terms: (continued) Pseudohyphae - a chain of elongated budding cells that have failed to detach (not true hyphae). Rhizoids - root-like structures. Sporangiophore - a special aerial hypha or stalk bearing a sporangium. Sporangium - a sac or cell containing spores produced asexually. Sterigmata - a specialized structure that arises from a basidium and supports basidiospores.

Mycology
Classifications of Fungi: Geographic grouping - where they exist. Epidemiologic grouping - how organism is transmitted. Taxonomy grouping - according to morphologic and cultural characteristics. Topographic Grouping - type of mycosis produced.

Mycology
Topographic Grouping of Fungi: (most often used) Superficial - Confined to the outermost layers of the skin and hair. No host cellular or inflammatory response due to organisms being remote from living tissue. Essentially no pathology; the disease is recognized purely on cosmetic basis. Cutaneous - in the keratin of the skin, nails, and hair. These organisms prefer non-living cornified layers. The disease is called a dermatophytosis or dermatomycosis. Host response is patchy scaling or eczema eruptions. They are classified according to the area of the body that is involved.

Mycology
Topographic Grouping of Fungi: (continued) Subcutaneous - Involve the deeper layers of skin and often muscle tissue. Man is an accidental host following inoculation of fungal spores via some form of trauma. This type of infection is often identified by the presence of a characteristic tissue reaction or granule. Systemic - Attack the deep tissues and organ systems; often creating symptoms that resemble other diseases.

Mycology
Categories of systemic disease: Those caused by truly pathogenic fungi with the ability to cause disease in the normal human host when the inoculum is of sufficient size (Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, Paracoccidioides braziliensis). Those caused by opportunistic fungi, low virulence organisms, which require the patient's defenses to be lowered before the infection is established (Aspergillus spp. Candida albicans, Cryptococcus neoformans).

Mycology
Saprophytes: Contaminates or opportunistic pathogens? Most are inhibited by cycloheximide. Grouped by type of mycelia produced.

Septate vs. aseptate Hyaline vs. dematiaceous

Mycology
Saprophytes: Hyaline Members Aspergillus spp. Growth rate varies, colors vary, surface velvety to cottony. Mycelium - septate and hyaline with unbranched condiophores (compare to Syncephalastrum, which appears similar, but is aseptate). A. fumigatus is considered a potential pathogen, especially if from a pulmonary source.

Mycology
Saprophytes: Hyaline Members Paecilomyces spp. Rapid grower, colors vary. Brush-like conidiophores. Long, tapered sterigmata.

Mycology
Saprophytes: Hyaline Members Penicillium spp. Commonly rapid growing; white to bluish-green. Conidiophores characteristically form a brush-shaped structure. Sterigmata are flask shaped.

Mycology
Saprophytes: Hyaline Members Scopulariopsis spp. Moderately slow growing. White turning brown with age. Branched or unbranched conidiophores. Sterigmata are coarsely roughened.

Mycology
Saprophytes: Hyaline Members Trichoderma spp. Moderately rapid growth. Flask-shaped conidiophores. Conidia are clustered.

Mycology
Saprophytes: Hyaline Members Fusarium spp. Rapid growth, white colonies may be come brightly colored. Short conidiophores often branched, have macro- and microconidia, which are oval to sickleshaped. Has been reported to cause eye infections.

Mycology
Saprophytes: Dematiaceous Members Alternaria spp. Rapid growth; colonies become very dark with age; may become overgrown with looser white to gray aerial mycelium. Conidiophores bear single or branched chains of large, brown conidia.

Mycology
Saprophytes: Dematiaceous Members Curvalaria spp. Rapid growth. Velvety colonies vary in color from grayish-brown to black. Spirally arranged brown conidia are borne at the tips. Brown, septate, unbranched conidiophores.

Mycology
Saprophytes: Dematiaceous Members Cladosporium spp. Rapid growth. Green colonies, reverse is black. Septate, dematiaceous mycelium. Conidia are borne in chains.

Mycology
Saprophytes: Aseptate Members All are susceptible to cycloheximide. Rapid growers. Some have root-like structures termed rhizoids. Spore bearing structures are called sporangiophores.

Mycology
Saprophytes: Aseptate Members Mucor spp. Very rapid growth; can fill a culture tube in one day. Unbranched sporangiophores. No rhizoids

Mycology
Saprophytes: Aseptate Members Rhizopus spp. Rhizoids are present. Sporangiophores nodal in origin.

Mycology
Saprophytes: Aseptate Members Syncephalastrum spp. Very rapid growth. White to dark gray colonies with dense, cottony, aerial mycelium. Aseptate, hyaline mycelium, with short, branched sporangiophores, terminating into tips. Many tubular sporangia containing chains of spores.

Mycology
Yeasts: Candidia albicans Cutaneous infections such as oral thrush or vaginitis, but can become systemic. ID by positive germ tube test, or production of chlamydospore on cornmeal agar.

Mycology
Yeasts: Cryptococcus neoformans Associated with pigeon feces. Has thick capsule. Grows at 37 degrees & produces melanin-like pigment on caffeic acid agar. ID in CSF by India ink stain for capsule.

Mycology
Yeasts: Geotrichum spp. Commonly present in GI tract. Implicated in respiratory infections; frequently as a secondary invader. Causes disease in the immunosuppressed. Produces arthrospores.

Mycology
Bacteria - Like Fungi: Some produce sulfur granules (Actinomyces israeli) Often require special media, stains, & conditions for growth, i.e. anaerobic (Actinomycetes spp). Some are partially acid-fast (Nocardia spp.)

Mycology
Dimorphic Fungi: Sporothrix schenkii Rose fever, gardeners often affected. Infection via traumatic implantation.

Mycology
Dimorphic Fungi: Coccidioides immitis San Joaquin valley fever. Mycelial phase present in culture. Yeast phase in tissue.

Mycology
Dimorphic Fungi:
Histoplasma capsulatum Mississippi Valley fever. Associated with bird droppings. Disease mimics tuberculosis. Found frequently in reticuloendothelial cells as extracellular inclusions.

H. capsulatum mold phase

H. capsulatum yeast phase

Mycology
Dimorphic Fungi: Blastomyces dermatitis North American Blastomycosis (Gilcrest disease). Large yeast cells with single bud.

Mycology
Dimorphic Fungi: Paracoccidioides braziliensis South American Blastomycosis. Large yeast cells with multiple buds (mariners wheel).

Mycology
Superficial mycoses: Malassezia furfur Cause of pityriasis versicolor (tinea versicolor). Phaeoannellomyces werneckii Cause of superficial phaeohyphomycosis (tinea nigra).

Mycology
Superficial mycoses: Trichosporon beigelii Cause of white piedra.
Piedraia hortae Cause of black piedra.

Mycology
Cutaneous mycoses: Microsporum spp. - in hair, skin, rarely nails; frequently in children, rarely in adults; often spontaneous remission occurs (ringworm). Trichophyton spp. - hair, skin & nails; in both children & adults (athletes foot). Epidermophyton spp. - skin, nails, rarely hair; in adults, rarely in children (ringworm).

Mycology
Cutaneous mycoses: Microsporum spp. Macroconidia are attached singly. Thick walled. Mature forms are echinulate (spiny).

Mycology
Cutaneous mycoses: Trichophyton spp. Macroconidia are attached singly. Have smooth walls.

Mycology
Cutaneous mycoses: Epidermophyton spp. Macroconidia are attached in multiples. Smooth walls (beaver tails). Grows slowly.

Mycology
Subcutaneous mycoses:
Chronic, supperative or granulomatous infections of the subcutaneous tissues, usually on an extremity (hands, feet); can extend through the lymphatics or form sinus tracts. Caused by a variety of fungi and bacteria-like fungi that live in the soil. Chromoblastomycosis: Non-contagious skin diseases characterized by the development of a warty lesion that has a cauliflower appearance. Occurrence is usually on the legs or feet. Caused by Fonsecaea pedrosoi, Cladosporium spp., and Phialophora spp. Fonsecaea pedrosoi is the most common etiologic agent of chromoblastomycosis

Mycology
Subcutaneous mycoses: Mycetoma: Localized, tumorous lesions in cutaneous and subcutaneous tissues usually the foot. Nodules are formed, and a collection of pus and formation of sinuses results. Actinomycotic mycetomas must be differentiated from Eumycotic (true fungi) mycetomas and as they have greatly differing treatments.

Mycology
Subcutaneous mycoses: Mycetoma: Actinomycotic mycetoma

Lumpy-jaw - The initial nodules formed are firm and described as "woody" or "lumpy. Sulfur granules - Bread crumb-like aggregates of microorganisms and cellular debris which take on the appearance of a sulfur particle. Caused by Nocardia spp. & Streptomyces spp. Advanced cases often require amputation due to physical impairment.

Mycology
Subcutaneous mycoses: Mycetoma: Eumycotic Mycetoma

Clinical picture similar to actinomycotic mycetomas. Disease is slowly progressive, with patients frequently dying of secondary infections. Treatment consists of excising early, localized lesions (amputation may be required for advanced infections). Antifungal drugs have little effect. Body tries to wall off the offending fungus. Invasion of deeper tissue may follow, with bone involvement, draining sinus tracts, or progression through the lymphatics.

Mycology
Systemic mycoses:
Dissemination of any fungal agent, yeast, or bacterialike fungus to involve any tissue or organ. Agent must be dimorphic. Histoplasma capsulatum Coccidioides immitis Paracoccidioides brasiliensis Blastomyces dermatitidis

Mycology
Systemic mycoses: Histoplasma capsulatum

Histoplasmosis - an infection of the reticuloendothelial system resulting in patchy bronchopneumonia containing yeast-laden phagocytic cells within alveolar spaces. Worldwide in distribution, it is endemic in the Mississippi, Missouri, St Lawrence, and Ohio river valleys. Strong association with bird and bat droppings.

Mycology
Systemic mycoses: Coccidioides immitis

Coccidioidomycosis - usually an asymptomatic or mildly symptomatic self-limiting upper respiratory tract infection, but may become disseminated and fatal. San Joaquin Fever, or Valley Fever - it is endemic in southwestern US, esp. Arizona and California. Caution must be taken with cultures; organism produces arthroconidia which are very easily inhaled.

Mycology
Systemic mycoses: Paracoccidioides brasiliensis

Paracoccidioidomycosis - also called South American blastomycosis. Blastomycosis - also called North American blastomycosis or Gilcrest disease.

Blastomyces dermatitidis

Laboratory Methods in Medical Mycology

Collection, Handling and Processing of Specimens

Skin - cleaned with 70% alcohol to remove dirt, oil and surface saprophytes Nails - cleaned same as for skin. Usually clipped; need to be finely minced before inoculating to media Hair - obtained from edge of infected area of scalp. Use a Wood's lamp (fluorescence) to help locate infected hair. Hair can be obtained by plucking, brushing, or with a sticky tape Body fluids - normal sterile collection procedures

Laboratory Methods in Medical Mycology

Preparation of Specimens for Transport to Laboratory Hair & nails sent in a dry envelope, inside proper container Other specimens are usually sent frozen or on dry ice Packaging - biohazard regulations. Any growing cultures must be on tube media (not plates). Aluminum screwcapped inner tube with outer cardboard mailing tube Inside labeling information: Patient ID, specimen source, suspected organism Outside labeling information - must state: WARNING: POTENTIAL PATHOGEN

Laboratory Methods in Medical Mycology

Appropriate Processing of Specimen to Recover Fungus Skin, nails, & hair - direct exam following KOH preparation CSF - centrifuged; examine sediment microscopically, inoculate media Pleural fluid, sputum, and bronchial aspiration - specimen must be fresh as saprophytes would overgrow pathogens such as H. capsulatum. Specimens may be refrigerated up to 2 hours Gastric washings - same as for pleural fluids Genito-urinary specimens - first morning specimen preferred

Laboratory Methods in Medical Mycology

Appropriate processing of specimen to recover fungus (continued) Blood/bone marrow - generally inoculated directly to BHI broth and BHI slant. Wound abscess or drainage - should be cultured anaerobically, especially if actinomycosis is suspected Tissue specimens - examine for pus, caseous material or granules; mince aseptically, can use small amount of sterile saline and the supernatant inoculated

Laboratory Methods in Medical Mycology

Direct Examination of Specimens Direct microscopic exam is required on any material sent to lab for fungus culture. Look for spores, hyphae, mycelial elements, budding yeast, mycotic granules Wet mount prep - good for yeast; examination is done in natural environment, so loss of fragile structure is minimized KOH prep - done on skin scrapings, hail, nails, sputum, vaginal specimens, etc. The KOH clears the specimens tissue cells, mucous, etc., so fungal elements can be seen

Laboratory Methods in Medical Mycology

Stains Used in Mycology Lactophenol Cotton Blue (LPCB) - very popular for quick evaluation of fungal structures; will stain the chitin in cell walls of fungi Periodic Acid - Schiff Stain (PAS) - stains certain polysaccharide in the cell walls of fungi. Fungi stain pink-red with blue nuclei Gomori Methenamine Silver Stain - outlines fungi in black due to the silver precipitating on the fungi cell wall. Internal structures are deep rose to black; background is light green Gridley Stain - Hyphae and yeast stain dark blue or rose. Tissues stain deep blue and background is yellow Mayer Mucicarmine Stain - will stain capsules of Cryptococcus neoformans deep rose

Laboratory Methods in Medical Mycology

Stains Used in Mycology Fluorescent Antibody Stain - simple, sensitive, and specific. Applications for many different fungal organisms Papanicolaou Stain - good for initial differentiation of dimorphic fungi. Works well on sputum smears Gram Stain - most fungi are gram positive; Actinomyces and Nocardia are gram variable Modified Acid-Fast Stain - differentiates acid-fast Nocardia from other aerobic Actinomyces Giemsa Stain use on blood and bone marrow specimens. India Ink - demonstrates the capsule of Cryptococcus neoformans in CSF specimens

Laboratory Methods in Medical Mycology

Fungal Culturing Tubed media is used rather than plated media because:

there is less chance for spore release into the environment less chance for dehydration ease of storage

The agar in a tube is inoculated in a straight line. The agar on plates is Inoculated like a large "S" so that rapid growing fungi can removed Incubation should be aerobic (and anaerobic if Actinomycetes are suspected) Incubate cultures at room temperature & also at 37C if dimorphic fungi are suspected Cultures are kept for 4 weeks & should be examined every other day Systemic pathogens often require 10 days to 2 weeks

Laboratory Methods in Medical Mycology

Media Used for Isolation of Fungi Sabouraud's dextrose agar (Sab-Dex) - classic medium, recommended for most studies Sabouraud's dextrose agar with chloramphenicol - chloramphenicol inhibits bacterial growth Mycosel agar - commercially available. Contains chloramphenicol to inhibit bacterial growth; cycloheximide to inhibit saprophytic fungi and some yeasts (including C. neoformans) Brain heart infusion slant (BHI) - more enriched than Sab-Dex. Used to recover H. capsulatum Potato-dextrose agar (PDA) and Corn-meal agar - used in slide cultures; these induce spore formation, which greatly aids in identification

Laboratory Methods in Medical Mycology

Special Applications Media Caffeic Acid Agar - Cryptococcus neoformans will produce melanin resulting in black colonies (protect media from light during incubation) Birdseed Agar - used to isolate Cryptococcus neoformans from contaminated cultures KT Medium & Kelley Agar - used to convert dimorphic fungus Blastomyces dermatitidis from mycelial to yeast form Modified Converse Liquid Medium (Levine's) - used to promote spherule production by Coccidioides immitis Christensen's urea agar - urea is hydrolyzed by some yeast to form ammonia. The pH increases turning the media from yellow to dark pink

Laboratory Methods in Medical Mycology

Colony Morphology (macroscopic features) Surface topography - some fungal colonies may cover the entire surface of agar; others grow in a more restricted manner Surface texture examples: cottony or wooly (floccose), granular, chalky, velvety, powdery, silky, glabrous (smooth, creamy), waxy Pigmentation - fungi may be colorless or brightly colored. Color may be on fungus itself, on its sporulating apparatus, on the agar, or on the bottom of the colony (reverse pigmentation). Pigment color is due to the color of the sporulating apparatus. Pigment can diffuse into the agar. It is important to note the top pigment (obverse); the underside pigment (reverse); and any discoloration of the medium

Laboratory Methods in Medical Mycology

Microscopic Evaluation of Fungi Teased Preparations part of the colony is removed to a slide with inoculating needle. Lactophenol cotton blue stain often used Slide Culture Techniques - provides a view of undisturbed microscopic morphology Transparent Tape Preparation clear cellophane tape used to lift parts of a colony to a microscope slide

Laboratory Methods in Medical Mycology

Biochemical Studies Used to ID Yeast and Yeast-like Organisms Carbohydrate assimilation & fermentation

Measures utilization of a carbohydrate under anaerobic conditions as determined by acid and gas production Specimen is inoculated beneath broth. Bromcresol purple is the indicator. Acid production turns purple broth to yellow. Gas is detected by appearance of bubbles trapped in a fermentation tube. Observe every 48 hours for 14 days Utilizes 3 tubes with different sources of nitrogen. Bromthymol blue is the indicator (blue to yellow is positive).

Nitrogen assimilation

Laboratory Methods in Medical Mycology

Other Tests of Importance Germ tube - Candidia albicans & Candidia stellatoidea produce germ tubes when incubated in a protein medium Demonstration of chlamydospores - yeast is inoculated by jabbing the appropriate agar (Cornmeal agar with tween 80). Cultures are observed every 24 hours for 3 days looking for chlamydospore production

Laboratory Methods in Medical Mycology

Hypersensitivity: Skin Tests and Serological Tests Skin tests - demonstrate T-cell immunity (cellular) to a fungus Serological tests - demonstrate B-cell (humoral) immunity to a fungus (acute & convalescent specimens are required)

Complement fixation Agglutination tests Precipitin tests Immunofluorescence Immunodiffusion techniques Counterimmunoelectrophoresis