1. 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)

2. 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.

3. 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.

4. 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.

5. Mycology Plant pathology - Most plant diseases are caused by fungi.
Medical importance -
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.

6. Mycology Importance of Medical Mycology
During the time period between , 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.

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

8. 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.

9. Mycology Basic terms as they relate to mycology:
Hypha (hyphae plural) - fundamental tube-like 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.

10. 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)

11. 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.

12. 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).

13. 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.

14. 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.

15. 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.

16. 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.

17. 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.

18. 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.

19. 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).

20. Mycology Saprophytes: Contaminates or opportunistic pathogens?
Most are inhibited by cycloheximide.
Grouped by type of mycelia produced.
Septate vs. aseptate
Hyaline vs. dematiaceous

21. 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.

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

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

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

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

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

27. 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.

28. 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.

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

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

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

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

33. 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.

34. 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.

35. 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.

36. 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.

37. 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.)

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

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

40. 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

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

42. Mycology Dimorphic Fungi: Paracoccidioides braziliensis
South American Blastomycosis.
Large yeast cells with multiple buds (mariner’s wheel).

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

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

45. 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 (athlete’s foot).
Epidermophyton spp. - skin, nails, rarely hair; in adults, rarely in children (ringworm).

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

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

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

49. 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 worldwide.

50. 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.

51. 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.

52. 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). Anti-fungal 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.

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

54. 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.

55. 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.

56. Mycology Systemic mycoses: Paracoccidioides brasiliensis -
Paracoccidioidomycosis - also called South American blastomycosis.
Blastomyces dermatitidis -
Blastomycosis - also called North American blastomycosis or Gilcrest disease.

57. 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

58. 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 screw-capped inner tube with outer cardboard mailing tube
Inside labeling information: Patient ID, specimen source, suspected organism
Outside labeling information - must state:
WARNING: POTENTIAL PATHOGEN

59. 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

60. 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

61. 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 specimen’s tissue cells, mucous, etc., so fungal elements can be seen

62. 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

63. 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

64. 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

65. 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

66. 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

67. 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

68. 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

69. 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
Nitrogen assimilation
Utilizes 3 tubes with different sources of nitrogen. Bromthymol blue is the indicator (blue to yellow is positive).

70. 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

71. 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