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Mycology Update 2017

  1. 1. MYCOLOGY Margie Morgan, PhD D(ABMM) Just the basics – Updated 2017 Mycology
  2. 2. Starting point Yeast are: • unicellular / produce budding daughter cells • colony on solid media are usually white to beige and appear much like bacterial colonies • some genera produce mucoid colonies (Cryptococcus)
  3. 3. Starting point Molds are: • Produce filaments or hyphae • Produce conidia [spores] • Colonies on solid agar are downy, fluffy, cottony • Most mold colonies produce pigment which aid in identification hyphae spores
  4. 4. Specimen collection and Transport • Fungi are very hardy organisms • No requirement for special transport media • Sterile containers required to prevent bacterial contamination • Numerous anatomic sites are appropriate for culture • Respiratory specimens – sputum, bronchial lavage, brushings, nasal sinuses • Tissue biopsies • Cutaneous - Skin scrapings, material from lesions • Ocular • Sterile body fluids including CSF • Blood, bone marrow
  5. 5. Fungal Culture Media • Sabouraud’s glucose agar (SABS) All purpose Fungal media – No antibiotics Best used for subculture of fungi for identification workup Contains 2% glucose, pH @7.0 • Inhibitory mold agar (IMA) Selective and enriched agar with chloramphenicol and gentamicin to inhibit bacteria Good for the primary recovery of pathogenic fungi Saprophytic fungi and dermatophytes somewhat inhibited
  6. 6. Fungal Culture Media • Mycosel/Mycobiotic agar • Selective SABS agar with chloramphenicol and cycloheximide • Used for selective culture of dermatophytes – fungi that cause skin, hair and nail infections • Brain heart infusion agar • Primary recovery of all fungi • Can inhibit bacterial growth by adding chloramphenicol and cycloheximide • Can add blood to agar to nurture systemic fungi • Fungal cultures are incubated for 4 weeks at 30˚C • Lower temperature than bacterial culture incubation [35˚C] • If agar plates are used for fungal cultures the plates must be sealed with air permeable tape to prevent spore dissemination from plates
  7. 7. What effect does Cycloheximide have when added to media? • Prevents rapidly growing environmental molds from overgrowing pathogenic fungal species • This is the good aspect of cycloheximide in media • Beware: it is not all good, it can suppress important fungi from growing. Inhibited fungi include: • Trichosporon beigelii • Candida tropicalis • Cryptococcus neoformans • Yeast phase of Blastomyces • Yeast phase of Histoplasma
  8. 8. • Inoculate fungal media with specimen • Seal plates with tape to prevent culture contamination and escape of fungal spores • Incubate at 30˚C for 4 wk • If growth occurs - perform proper identification methods: Yeast identification methods Manual and automated biochemical reactions capable of identifying most but not all yeast species. Newer methods (1) Mass spectrometry – MALDI-TOF and (2) 16 sRNA sequencing are more precise methods with the expanding taxonomy of yeast species. Processing of Fungal Cultures
  9. 9. Lactophenol cotton blue [LCB] adhesive tape preparation is standard method used for mold identification. The LCB mounting medium consists of phenol, lactic acid, glycerol and aniline cotton blue dye. Clear adhesive tape touches a mold colony, picking up fungal hyphae and pressed into one drop of LCB on a microscope slide. Newer (better methods) for unusual molds: MALDI-TOF 16sRNA sequencing Mold Identification methods
  10. 10. Safety in the Mycology Laboratory • If a culture is growing a mold, it cannot be opened on the bench top • All mold work must be performed in a BSL-2 biosafety cabinet with Hepa filtration • Yeast identification can be performed on the bench top
  11. 11. Direct Exams used to identify fungi directly from patient specimens • Gram stain – Most specimen types can be Gram stained. Reliable detection of yeast • KOH preparation – Skin, Hair or Nails examined for both yeast and/or hyphae • Calcofluor white stain – Most specimen types can be stained and examined for yeast and/or hyphae • India ink – Primarily used on CSF for the detection of Cryptococcus neoformans and C. gattii
  12. 12. • Yeast cells stain blue [Gram positive]. • Examine stain for budding cells to confirm that it is a yeast cell and not an artifact. • You can also detect pseudo-hyphae on Gram stain. • Mold can be difficult to detect on a Gram stain. pseudohyphae mold pseudohyphae Gram Stain X100 oil immersion
  13. 13. •Detect yeast and/or pseudo-hyphae in skin, hair and nail specimens using 40X light microscopy. •KOH dissolves keratin found in cellular material and freeing fungal hyphae and yeast cells. •KOH exams can be difficult to interpret! KOH – potassium hydroxide prep
  14. 14. • Yeast, pseudo-hyphae, and mycelial fungi will bind with the Calcofluor white stain and fluoresce. • Read using a fluorescence microscope (40X) • More Sensitive and Specific than KOH preparation. Calcofluor white stain
  15. 15. One drop of black ink is placed into one drop of CSF On microscope slide - examined using light microscope (40X) It is a “negative” stain – which stains the background not the yeast cell. The clearing around the yeast is due to the polysaccharide capsule of Cryptococcus neoformans and C. gattii. India Ink
  16. 16. Grocott’s Methenamine Silver Stain [GMS] – yeast and hyphae stain grey to black. Will stain living and dead fungi • Observe the width of the hyphae, presence of septations and angle of branching. • Observe the size and budding pattern of yeast. • Observe on later slides how these oservations can assist in identification. Examination of fungi in fixed tissue
  17. 17. Positive staining hyphae will appear magenta – will also stain structures containing a high proportion of carbohydrate macromolecules (glycogen, glycoprotein, proteoglycans) Stains living fungi. Periodic Acid Schiff [PAS]
  18. 18. Mucicarmine stains the polysaccharide capsule pink for Cryptococcus neoformans and C. gatti. Also stains mucin in fixed tissue. Mucicarmine [Mucin] stain
  19. 19. Great for cellularity, not a specific stain for fungi. Hematoxylin and Eosin Stain
  20. 20. DIMORPHIC FUNGI Important systemic pathogens with some unique characteristics
  21. 21. What does Dimorphic mean? • Depending on temperature and conditions of environment one fungi demonstrates two forms • Mycelial form - Hyphae and conidia (spores) • Free living form in nature and laboratory temperature <=30˚C • Yeast or yeast like form • Parasitic phase found in human tissue or in the lab >= 35˚C Histoplasma capsulatum – mold Incubated at 30˚C Histoplasma capsulatum – yeast from tissue and 35˚C culture
  22. 22. Dimorphic Fungi capable of causing systemic infection – most common • Histoplasma capsulatum • Blastomyces dermatitidis • Coccidioides immitis • Paracoccidioides brasiliensis • Sporothrix schenckii • Penicillium marneffei (Talaromyces marneffei)
  23. 23. Histoplasma capsulatum • World wide distribution / In USA considered endemic in Ohio, Missouri, and Mississippi River valleys • Environmental source: Bat guano (Spelunker = cave explorers) and bird droppings
  24. 24. Histoplasmosis Disease • 95% of infections are subclinical • 5% infections: • Progressive pulmonary • Chronic systemic infection with dissemination to the RES system including bone marrow • Acute fulminating systemic disease (fatal) • Reactivation disease can occur in elderly and immunosuppressed (AIDS is a good example) • Bone marrow exam is useful in diagnosing disseminated infections • Mucocutaneous lesions are a unique & common site of dissemination
  25. 25. Histoplasma capsulatum - Yeast Intracellular within macrophages Small 2 – 4 um, regular in size, and oval to round. Do not have a capsule, it is a staining artifact. H & E stain GMS
  26. 26. H & E PAS Gram Wright’s Histoplasma will stain with a variety of stains
  27. 27. Histoplasma rapid diagnosis • Antigen detection in urine • Quantitative Enzyme immunoassay • Random urine specimen • Most sensitive for disseminated infection and chronic pulmonary disease • Antigen is detectable in >=85% of these infections • Good for diagnosis of immune suppressed patients that do not produce a detectable antibody response • Antibody tests are available but have been replaced mostly by Antigen detection
  28. 28. Histoplasma capsulatum • Culture incubated at 30˚C • Requires 2 – 8 weeks to grow • Colony - white to brown and cottony • Microscopic appearance – tuberculated macroconidia that are large and round (8 – 16 µM) and small microconidia (2 - 4µM) • Microconidia are the infectious particle growing in nature and capable of penetrating deep into the lung • DNA probe necessary to confirm identification • Sepedonium species looks somewhat like Histoplasma usually a contaminate. It is not dimorphic.
  29. 29. Histoplasm capsulatum
  30. 30. Histoplasma capsulatum in tissue • Granulomas are usually produced – caseating or non-caseating • Infection usually begins by breathing in the microconidia and infecting the Lung • Infection disseminates to organs of the Reticuloendothelial System (RES) – with high % of dissemination to the Bone Marrow • Intracellular budding yeast (2 – 4 µM) are seen in all tissues
  31. 31. Leishmania species Note small round kinetoplast next to nucleus Toxoplasma Histoplasma capsulatum Beware of look alike organisms in tissue specimens!!
  32. 32. The mold phase of H capsulatum var. duboisii is identical to H. capsulatum. The difference is the size of the yeast cells: Note yeast cell is 8 – 10 uM which is 2X the size of H. capsulatum yeast cell. H. capsulatum var duboisii disease is found in Central Africa – differs from H. capsulatum with primary infection in skin and bone Unusual variant of H. capsulatum
  33. 33. Blastomyces dermatitidis • Epidemiology • Ohio and Mississippi River valleys • No association with specific animal or activity • Forrest and river banks? • Primarily a pulmonary infection which may disseminate primarily to the skin and bone • Well demarcated skin lesions are often seen in disseminated Blastomycosis. • Skin lesions are full of yeast cells.
  34. 34. Blastomyces dermatitidis • Culture at 30˚C • Grows in 2- 3 weeks • Fluffy white – buff colored mold, prickly • Pear shaped conidia at the end of supporting hyphae – looks like lollipops • Look alike fungus – Chrysosporium species • Do DNA probe test to confirm identification Blastomyces Chrysosporium
  35. 35. • Slow growing yeast colony (4 weeks to form a colony) • Yeast cell is 8 – 20 um in size • Unique Broad Based Budding pattern and the double contoured wall. Blastomyces culture at 35° C Broad based bud
  36. 36. Blastomyces dermatitidis histopathology • Mixed pyogenic and granulomatous inflammation is observed in tissue • Broad based budding yeast cells
  37. 37. Coccidioides immitis • Endemic in SW USA, Mexico, South America, in areas known as the Sonoran life zone/ warm and desert sands • Infection is from inhalation of fungal particles found in the sand • New: Coccidioides posadasii is genetically related to C. immitis. These two species are located in different endemic regions, but produce the same disease process Coccidioidomycosis
  38. 38. Coccidioidomycosis • 95% of infections are asymptomatic or with limited symptoms • The remaining 5% are focal pulmonary, progressive pulmonary or disseminated infections. • Dissemination to the central nervous system carries a high fatality rate. • Higher incidence of dissemination occurs in patients with: • defects in cell mediated immunity (HIV), • darker skinned ethnic groups, • pregnancy
  39. 39. Coccidioides immitis [posadasii] • Culture at 30˚C • Requires 2 – 3 days to grow, colony starts waxy and becomes wooly in around 7 – 10 days • Under the microscope (40X) one observes foci with septate hyphae with thick walled barrel shaped arthroconidia - clear spaces in-between. The clear spaces are dead arthroconidia. • Arthroconidia are the infectious particle in nature • Culture is very infectious to laboratory personnel
  40. 40. Coccidioides 30°C Culture • Malbranchea species mimics C. immitis/posadasii under the microscope • Because of look-a-like fungi, one needs to confirm identification of Coccidioides immitis/posadasii with DNA probe (molecular) or similar method to be accurate Coccidioides Malbranchea
  41. 41. Culture at 30*C and 35*C produce hyphae with barrel shaped alternating arthroconidia. There is no yeast phase for C. immitis [posadasii] No yeast phase with Coccidioides!
  42. 42. Coccidioides Histopathology • Thick walled spherules (10 – 80 uM) with endospores are seen in tissue. This is the second form of Cocci. No yeast cells are produced in tissue. • Spherules are in all stages of development- fragmented spherules to well formed with endospores • Granulomatous inflammation with caseation is usually observed
  43. 43. Development of Cocci spherules from the inhalation of Arthroconidia from nature
  44. 44. Rhinosporidium seeberi (aquatic parasite) forms spherules but much larger than those of Cocci. Rhinosporidium spherules are usually > 80 uM in size. Also R. seeberi usually cause only oral or nasal mass lesions Oral or nasal mass lesions of Rhinosporidium seeberi Coccidioides spherules Coccidioides is not the only spherule forming organism!
  45. 45. Paracoccidioides brasiliensis • South American Blastomycosis – endemic area Brazil, Venezuela, Columbia • Inhale infectious particle from soil • >95% of infections in males, possibly due to estrogen inhibition of mycelial to yeast transformation • Disease presentation: 1. Pneumonia 2. Disseminated infection to 2 or more organs 3. Extrapulmonary lesions on the face and oral mucosa
  46. 46. Paracoccidioides • Cultures at 30°C for mycelial phase are usually not performed - due to slow growth and nonspecific sporulation • Culture @ 37˚C • Slow growing yeast • Large yeast (10 – 30uM) with multiple daughter buds (2 – 10 uM) in size • Unique budding yeast cell known as the Mariner’s wheel or Pilot’s wheel yeast
  47. 47. Tissue Exam of Paracoccidioides brasiliensis • If more than 2 buds off mother yeast cell – High likelihood it is Paracoccidioides • Granulomatous inflammation with Giant Cells formed in tissue
  48. 48. Sporothrix schenckii • Sporotrichosis • Cutaneous inoculation of fungus from penetrating injury with a spore or thorn (rose bush) • Begins as single skin lesion w/wo ulceration • Lesion can progress to many lesions and lymphocutaneous spread, possibly to bone and other organs • Pulmonary and CNS infections are rare but reported
  49. 49. Sporothrix schenckii • 30*˚C culture grows in 3 -5 days as mold • Beige colony/ turns brown to black over time • Septate hyphae with conidia in daisy wheel pattern • 37˚C culture small oval yeast cells, elongated 2 – 5 µM, described as cigar bodies
  50. 50. Sporothrix schenckii Histology • Pyogenic to granulomatous inflammation • Hard to find yeast cells in human tissue • More commonly an Asteroid body known as Splendore- Hoeppli phenomenon can be seen, but this is not specific to Sporothrix, also seen in: • Zygomycetes (Mucorales) • Aspergillus • Blastomycosis • Candida
  51. 51. Daisy like spore arrangement Sporothrix schenckii Cigar shaped yeast cell Splendore-hoeppli
  52. 52. Green colony with red diffusable pigment grows in 2 – 3 days at 30 C° Uncommon dimorphic fungus – yeast like cells produced in tissue Only dimorphic species of Penicillium Diseases: skin lesions in tropics and Pneumonia or systemic disease in advanced HIV Penicillium (Talaromyces) marneffei
  53. 53. Penicillium (Talaromyces) marneffei
  54. 54. SUBCUTANEOUS FUNGAL INFECTIONS Very unique structures in tissue!
  55. 55. Subcutaneous Fungal Infections Most common will be described • Mycetoma [2 types] • Actinomycotic – caused by higher bacteria • Eumycotic – caused by dark pigmented molds • Chromomycosis [Chromoblastomycosis] • Phaeohyphomycosis • Sporotrichosis
  56. 56. Mycetoma • First observed in India and known as Madura Foot or Maduromycosis • Found in the hot temperate parts of the world • Three criteria define Mycetoma: -Traumatic lesion leads to swollen extremity -Draining sinuses evolve -Sulfur granules observed in tissue and found in the weeping drainage from sinus tract • Fungus grows on organic debris in soil • Implanted into subcutaneous tissue from trauma
  57. 57. Swollen extremity with draining sinuses that drain sulfur granules Sulfur Granule Mycetoma
  58. 58. Actinomycotic Mycetoma There are two types of Mycetoma: 1. Actinomycotic – caused by higher bacteria species 2. Eumycotic – caused by black molds • Actinomycotic Mycetoma • 98% of cases • Nocardia species most common agent • Sulfur granules formed in tissue. The granules vary in color based on Nocardia species and contain a matrix of the filamentous bacteria
  59. 59. Edge of granule has thin filamentous bacteria: How can you tell if it is Nocardia? Nocardia is modified acid fast [PAF] positive and is an aerobic bacteria. Actinomyces is PAF negative and grows anaerobically. Beware! Sulfur granule caused by Actinomyces israelii looks identical to one formed by Nocardia. Actinomycotic sulfur granule
  60. 60. Gram stain = filamentous Gram positive bacilli can be poorly staining and appear speckled. Positive [red] on the Modified Kinyoun acid fast stain. Modified acid-fast stain [modified Kinyoun stain] Gram stain of sputum Nocardia
  61. 61. Grows in 3 – 5 days on agar media [Sabs, 5% Sheep’s blood agar] Colony is dry/crumbly - Musty smell Total of 85 species: Nocardia asteroides most common Therapy: Trimethoprim/Sulfa Identification by Maldi-TOF or 16s rRNA gene sequencing Nocardia species – Mycetoma, Pulmonary and Brain infections
  62. 62. Thermoactinomycetes Gram positive filamentous rods – related to Nocardia • Thermoactinomyces and Saccharopolyspora • Flourish in areas of high humidity and high temperatures (40-60*C) • Grow in agricultural environments – like stacks of hay • Farmer’s lung – hypersensitivity pneumonitis or extrinsic allergic alveolitis, occupational disease • Immunologically medicated inflammatory disease of the lung • Inhalation exposure to the thermophilic actinomycetes • Diagnosis • Detailed environmental history • Serology testing
  63. 63. Eumycotic mycetoma Caused by pigmented/black fungi due to melanin -Numerous species found in soil -2% of cases of mycetoma -Traumatic implantation implants the mold into the subcutaneous tissue -Most common black molds include: Cladophialophora (Cladosporium) carrionii Cladophialophora bantiana Phialophora verrucosa Fonsecaea pedrosoi Exophiala species Wangiella species
  64. 64. Eumycotic sulfur granule – the granule is composed of a matrix of thick fungal hyphae
  65. 65. Chromomycosis/Chromoblastomycosis • Three characteristics: • Wart like lesions in subcutaneous tissue • Sclerotic bodies observed in tissue • Growth of dark/pigmented fungi • Black mold naturally found in the soil cause infection through abrasion/ implantation • Black molds that can cause Chromomycosis: • Cladophialophora [Cladosporium] carrionii • C. bantiana • Phialophora verrucosa • Fonsecaea pedrosoi • Exophiala species • Wangiella species
  66. 66. Chromomycosis/Chromoblastomycosis Wart like/Verrucous lesions In subcutaneous tissue Sclerotic Body/Medlar Body/ Copper Penny is the unique structure found in tissue
  67. 67. Prototheca wickerhamii – the cause of Protothecosis • Algae without chlorophyll • Causes skin lesions & nodules • Most common in patients with suppressed immune system • Compare morula of Protothecosis to sclerotic body of Chromomycoses
  68. 68. Phaeohyphomycosis Traumatic implantation of dark fungi into subcutaneous tissue • Variety of infections but nodules/lesions most common with/without dissemination • Dark hyphae observed in tissue
  69. 69. Black mold also known as Dematiaceous mold -Black colored colonies and the reverse [underside of colony] is also black -Natural-colored brown hyphae and spores (melanin) -One of the common molds that grow due to water damage!
  70. 70. Black Molds – Dematiaceous fungi • Black colonies • Brown hyphae and spores (melanin) • Numerous species • Difficult to identify • All have one of four types of sporulation • Rhinocladiella-like • Cladosporium-like • Phialophora-like • Acrotheca-like Rhinocladiella Cladosporium Phialophora Acrotheca
  71. 71. Exophiala species Black Molds cause Mycetoma/Chromomycosis/Phaeohyphomycosis plus sinusitis, pulmonary, brain, and disseminated infections. These fungi are difficult to identify using only morphology – require sequencing/MALDI for speciation Cladophialophora bantiana - Brain infections
  72. 72. Wangiella dermatitidis Phialophora verrucosa
  73. 73. Alternaria species– Opportunistic fungus - sinusitis in normal host, dissemination in immune suppressed. Other black molds of importance:
  74. 74. Bipolaris australiensis Disseminated fungal infection in both normal and immune suppressed hosts: Skin, nasal sinuses, bone brain
  75. 75. Curvularia lunata Center cell is the largest Opportunistic fungal pathogen Reported in Bone Marrow Transplantation and Aplastic anemia Can cause unusual erythematous skin lesions
  76. 76. Exserohilum rostrum • Outbreak associated with compounded pharmaceutical [steroid] products contaminated with dust/dirt • Used for infections into lumbar spine and knee joints for pain management and led to infections: • Meningitis • Spinal abscess • Synovial infections
  77. 77. Scedosporium apiospermum/Pseudallescheria boydii Cat fur-like colony/growth in 3 – 5 days, lollipop like spores Opportunistic pulmonary pathogen. Can be difficult to distinguish from Aspergillus in fixed tissue stains.
  78. 78. IMPORTANT YEAST CAUSING HUMAN INFECTION Candida species Cryptococcus neoformans & Cryptococcus gattii
  79. 79. Candida species • Candida species (@ 10 infect humans) • Yeast are normal flora in GI, GU and skin • Opportunistic pathogen involving skin or mucous membranes from excessive exposure to moisture, antibiotics, or immune suppression • Thrush, vaginitis, skin lesions, nail, diaper rash • Also can cause more serious infections such as fungemia, endocarditis, and systemic tissue infection.
  80. 80. Candida species • Candida albicans – most common species causing @ 60% of human yeast infections • Candida glabrata, C. krusei, and C. tropicalis are causing higher % of infections than in the past • Fluconazole is a very useful oral and IV anti-fungal • These 3 species are more likely to be resistant to Fluconazole • New species Candida auris – outbreaks in hospitals, intrinsically resistant to most anti-fungals • Candida parapsilosis has emerged as a pathogen of children and common in IV line infections (forms biofilms on plastics) • C.parapsilosis is susceptible to fluconazole.
  81. 81. Candida species • Grow in 24 – 48 hours 30-35*C • Many agars: SABS, IMA, Blood agar • Bacteria-like colony – pasty white • Budding yeast – oval @ 7-8 um in size** • Form pseudohyphae not true hyphae (Yeast do not detach and form elongated hyhae) • Identify using biochemical rxn, MALDI, or Sequencing • Exception: **Candida glabrata is only Candida species @ 4 µM in size and does NOT form pseudohyphae – this species breaks all the Candida rules pseudohyphae
  82. 82. Candida albicans Identification • Germ tube formation • Incubate small amount of yeast in serum for 3-4hrs at 35 ˚C • If incubate >4 hrs – C. tropicalis will also form germ tubes • C. dubliniensis is also positive • Specific ID from biochemical reactions, MALDI-TOF or Sequencing • Chlamydospore formation • Growth on cornmeal agar at 48 hrs • Rudimentary structures unique to C. albicans chlamydospore
  83. 83. ChromAgar for the identification of Candida using chromogenic substrates. The unique colors can identify 4 yeast species. Yeast with pseudohyphae
  84. 84. Candida Histopathology • Pyogenic to granulomatous • Usually observe yeast cells (8 um), pseudohyphae and/or hyphae- like structures • Candida glabrata = smaller yeast cells (4 um) and absence of pseudohyphae GMS stain of Candida glabrataCandida species not glabrata
  85. 85. Cryptococcus neoformans • In nature forms a 2um non-encapsulated yeast cell. • Associated with bird droppings (esp. pigeon). C neoformans is enriched by the nitrogen in the heaped droppings. • Non-encapsulated yeast cells are inhaled – travel through the pulmonary system with hematogenous spread to brain and meninges • Tropism to the meninges • Infects mostly compromised hosts – one of the major AIDS defining infections
  86. 86. Cryptococcus gattii – a close relative of C. neoformans • Isolated from forested area of the Pacific Northwest (British Columbia, Washington, Oregon and California) found in soil debris and tree species • Infection of normal and immune suppressed hosts • Primarily a pulmonary disease [Cryptococcoma] but can develop meningitis like C. neoformans • Culture, biochemical & staining identical to C. neoformans • Defining reactions – • L Canavanine glycine bromthymol blue medium – C. gatti = blue C. neoformans = colorless MALDI-TOF will also ID both species
  87. 87. Cryptococcus neoformans and C. gattii • Irregularly sized yeast cells (2 – 20 um) • Polysaccharide capsule is the primary virulence factor • Polysaccharide capsule is detected in diagnostic testing: • India ink exam- CSF placed in one drop of black ink. It is a negative staining method – background is stained not the capsule • Sensitive test for AIDS patients (90% sensitive) • Cryptococcal antigen test – Titer of capsular polysaccharide detected in either CSF or serum • More sensitive than India ink for all patient groups • Test for diagnosis or can follow recovery with falling titer • Grows on mycologic agars / sensitive to cycloheximide – • Mucoid colonies due to capsule polysaccharide formation • Urease enzyme + Inositol assimilation + • Brown colonies produced on bird seed agar
  88. 88. Both C. Neoformans & C. gattii produce brown colonies on Birdseed agar
  89. 89. Positive India Ink Urea medium demonstrating urease enzyme activity of Cryptococcus Observe Budding cells Variability in size Positive Mucoid colonies of C. neoformans and C. gattii
  90. 90. Mucicarmin stains the capsular polysaccharide of Cryptococcus Pneumocystis jiroveci (yeast like fungus) could be confused with C. neoformans – Careful! Central nuclear staining in pneumocystis C. neoformans/ C. gattii – no nuclear staining Cryptococcus vs. Pneumocystis Pneumocystis froth on H & E
  91. 91. Trichosporon • T. asahii most common – 9 species • Commonly inhabit the soil. Colonize the skin/ gastrointestinal tract of humans. • Rectangular shaped yeast cells • Cause of superficial infection - white piedra, a distal infection of the hair shaft, • Second most commonly reported cause of disseminated yeast infections in humans • Rare, even among patients with impaired host defenses: • Corticosteroid use, solid tumors, HIV/AIDS, and intravascular devices, including catheters and prosthetic heart valves • Mortality 50 – 80%
  92. 92. CUTANEOUS AND SUPERFICIAL MYCOSES Malassezia furfur Dermatophytes
  93. 93. Malassezia furfur • Most superficial of the dermatomycoses • Normal flora on the skin, • More common on oily skin or high use of skin oils • Diseases: • Skin: macules, papules, patches, plaques on chest back and shoulders with either hypo or hyper pigmentation – does not invade into deeper tissues – known as pityriasis versicolor • Fungemia: caused by skin flora tunneling in with IV lipid feeding lines (parenteral nutrition) – in neonates or rarely adults
  94. 94. Malassezia furfur • Lipophilic yeast – oil required for growth • Media for isolation must contain oil or us an oil overlay • Small budding yeast 2 – 4 µM with collarette (appears like necklace at junction of mother and daughter yeast cell) • In tissue described as “Spaghetti and Meatballs” Spaghetti and meatballs
  95. 95. Size range for Yeast • Candida glabrata/Histoplasma capsulatum/ M. furfur • 2 – 4 um • Candida species • 8 – 10 um plus pseudohyphae • Cryptococcus neoformans/gattii • 2 – 20 um • Blastomyces dermatitidis • 8-15 um
  96. 96. Dermatophytes – Ringworm infections • Hair, skin and nail infections • 3 genera of fungi • Microsporum species (many) • Epidermophyton floccosum • Trichophyton species (many) • Disease described by area of the body infected: For example: tinea capitis (head), t. pedis (foot) • Usually a clinical diagnosis not requiring culture • KOH prep or Calcofluor white prep can be used to visualize fungal hyphae from skin scrapings
  97. 97. Positive KOH prep Showing thin septate fungal hyphae Calcofluor white stain with fluorescence – thin fungal hyphae
  98. 98. Microsporum canis Ringworm acquired from dog and cat White colony/ yellow on backside of colony Tuberculate thick walled macroconidia [spiny projections] Few if any microconidia
  99. 99. Microsporum gypseum – skin infection from exposure to contaminated soil, sandy colored colony, large macroconidia are produced.
  100. 100. Trichophyton rubrum White colony with red diffusable pigment Pencil shaped macroconidia many micro- conidia Skin infection from fomites Red diffusible pigment
  101. 101. Trichophyton Tonsurans White colony grows in @ 5 days No macroconidia Ballooning microconidia Primary cause of epidemic scalp ringworm in children
  102. 102. Epidermophyton floccosum Beaver tail large macroconidia – no microconidia Khaki green colored colony – most often cause infections in skin and nails.
  103. 103. Opportunistic Fungal Pathogens Infections in the immune suppressed host or special circumstances Hyaline molds
  104. 104. Opportunistic Fungi - hyaline • Hyaline – no color to the hyphae • Regular septations (walls) in the hyphae • Branching of hyphae in stains of fixed tissue – angle of branching can be helpful in identification • Usually grow in 3 – 5 days at 35 or 30˚C • ??? of species in the thousands– taxonomy changing daily
  105. 105. Aspergillus species • Hyphae - Hyaline with septations • Numerous round conidia produced • In tissue - Branching at 45 degree angle • Usually pulmonary infection but can disseminate • Invade vessels, cause thrombosis & infarctions Septation
  106. 106. Aspergillus Structure
  107. 107. Aspergillus species Four species most common in human infections: 1. Aspergillus fumigatus 2. Aspergillus flavus 3. Aspergillus niger 4. Aspergillus terreus – unique and important – intrinsic resistance to Amphotericin B Aspergillus Galactomannan Enzyme immunoassay – detects circulating Aspergillus antigen in the blood and/or bronchial lavage fluid Low sensitivity and specificity False positive reaction from therapy with Piperacillin/Tazobactam, infection with Histoplasma capsulatum
  108. 108. Aspergillus fumigatus Blue/Green colony grows in 3 – 5 days Phialids with colorless spores are directed upward
  109. 109. Aspergillus flavus Green/yellow colony Orange colored sporeson phialids that surround the vessicle
  110. 110. Aspergillus niger Black colony – visible black heads Black spores supported by phialids surround the vesicle
  111. 111. Aspergillus terreus Sandy colored colony Colorless conidia supported by phialids headed upward Aleurioconidia produced Intrinsic resistance to Amphotericin B
  112. 112. Aspergillus – fruiting head seldom seen in fixed tissue usually only dichotomous (continuous) branching septate hyphae which branch at a 45° angle Can appear much like that of Pseudallescheria boydii (Scedosporium) Growth in culture can differentiate the two fungi.
  113. 113. Fusarium species – Common in nature/plants Disease related to immune status of host Infections reported: Disseminated in bone marrow transplants Corneal infections in contact lens wearers
  114. 114. Scopulariopsis species –found in soil and plants Infections: Nail, skin, sinusitis, pulmonary and disseminated
  115. 115. Penicillium species – one of the most common mold in the environment , cause of bread mold, uncommon cause of human disease
  116. 116. MUCORMYCOSIS/ ZYGOMYCOSIS Fungi in the Order Mucorales Hyaline Broad hyphae without septations
  117. 117. Mucormycosis/Zygomycosis • Rhinocerebral mucormycosis • Infections in diabetics, the elevated glucose enriches fungal growth • Sinus – orbit of eye - brain • Sinus and pulmonary infection in the immune suppressed host • Broad, hyaline, aseptate hyphae produced • Cultures grow in 24 hrs, producing coarse aerial hyphae • Mince infected tissue and place on agar • Do not grind tissue – grinding will kill the aseptate hyphae
  118. 118. Order Mucorales – coarse, aerial hyphae after 24 hours on SABS agar at 30˚C
  119. 119. Rhizopus Absidia Distant rhizoids Mucor No rhizoids Rhizoids Order Mucorales
  120. 120. 90˚ angle branching, aseptate, ribbon like hyphae Invades vessels and can cause infarcts and thrombi Zygomycetes (Mucorales)