Mycology Update 2023 by Dr. Morgan

1. Mycology Update 2023
Margie Morgan, PhD, D(ABMM)
Mycormycetes Conidial Molds
Dematiacious Hyaline
Yeast
1

2. Starting points
Yeast are:
• Unicellular and bud to produce daughter cells
• Growth on solid media usually white to beige colonies and in the first 24-48
hours appear much like bacterial colonies
• A few genera produce pigment (ex. Rhodotorula = orange/red)
• Some genera produce mucoid colonies (ex. Cryptococcus)
• Taxonomic name updates will be presented in (parentheses)
Candida albicans
on blood agar
2

3. Starting points
Molds:
• Produce hyphae and conidia [spores]
• Growth on solid media usually produce downy, fluffy, or cottony colonies
• Growth time is variable (3 – 21 days) depending on the mold
• Most mold colonies are pigmented, which can assist with identification
• Taxonomic name updates will be presented in (parentheses)
hyphae
spores
3

4. Specimen Collection and Transport
• Fungi are hardy organisms
• Transport media usually not required
• Sterile containers used to prevent bacterial contamination /
room temperature transport is adequate
• 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
4

5. Fungal Culture Media / most common
• Sabouraud dextrose agar (SABS)
All purpose fungal medium – antibiotic free
Contains 2% glucose, pH @7.
Best use is for the subculture of fungi
for identification and susceptibility workup
• Inhibitory mold agar (IMA)
Selective and enriched medium - contains chloramphenicol
and gentamicin to inhibit bacterial growth
Good medium for the primary recovery of pathogenic fungi
5

6. Fungal Culture Media
• Mycosel (Mycobiotic) agar
• Highly selective SABs like medium containing chloramphenicol and cycloheximide
• Used primarily to culture dermatophytes – fungi that cause skin, hair and nail infections
• Should not be used as a primary plating medium for cycloheximide prevents growth of some
pathogenic fungi:
• Cycloheximide can suppress: Trichosporon species, Candida tropicalis and Cryptococcus
neoformans/gattii
• Brain heart infusion (BHI) medium /with or without blood
• Used for the primary recovery of fungi
• Inhibition of bacterial growth with addition of chloramphenicol and gentamicin
• Sheep’s blood can be added to the medium to nurture for fastidious systemic fungi such as
Histoplasma capsulatum
6

7. Mycology Laboratory Safety Precautions
• BSL-2 level safety precautions
• BSL-2 biosafety cabinet with HEPA filtration used for working with mold cultures
• Yeast procedures can be performed on the bench top
• PPE (personal protective equipment) include lab coats and gloves
• Biohazardous waste must be autoclaved
• All agar plates must be sealed to protect those working in the laboratory from
mold spores being released into the laboratory
7

8. • Using Biosafety Level 2 precautions, inoculate specimen onto medium and
streak for isolation
• Seal plates with oxygen permeable tape
• Tubes or vials can also be used for culture, but it is more difficult to
visualize fungal growth and perform testing compared to plates
• Incubate media at 30˚C for 4 weeks (extended time necessary for the
detection of slower growing systemic fungi ), screen periodically
• If growth occurs - perform appropriate identification tests
Processing of Fungal
Cultures
8

9. Yeast Identification Methods
• Biochemical reactions have been used for decades to identify yeast.
Recent taxonomic updates and new emerging yeast pathogens have
challenged the accuracy of biochemical identification methods.
• Newer methods with improved accuracy of identification include:
(1) MALDI-TOF (Matrix Assisted Laser Desorption/Ionization Time of Flight)
Colonies are smeared onto a target plate, superheated by laser and
converted into charged protein particles, the particles based on mass
and charge travel up a vacuum chamber to a detection plate to form
identification peaks that identify yeast to genus and species.
Most popular method/precise and inexpensive
(2) rRNA sequencing – particularly helpful for rare and difficult to identify
yeast species/ based on the detection of sequence differences
(polymorphisms) in the16S rRNA gene which is present in all yeast and
can be used to identify yeast to the genus/species level
9

10. • Lactophenol Cotton Blue [LCB] adhesive tape preparation has been
used for decades to identify molds by direct observation
• LCB mounting medium consists of phenol, lactic acid, glycerol and
aniline cotton blue dye.
• Clear adhesive tape is used to touch the mold colony, picking up fungal
hyphae/conidia, then pressed into one drop of LCB on a microscope
slide. Observe under light microscope using 40X.
• Can be used for identification to genus for most fungi.
Newer/ more definitive
identification methods include:
(1) MALDI-TOF – extraction
methods to prepare mold
(2) rRNA sequencing techniques
Mold Identification Methods
10

11. Gram stain
• Yeast cells stain Gram positive (blue) and can be well visualized
• Budding yeast cells can assist with confirmation that it is a yeast and
not a staining artifact
• Yeast pseudo-hyphae are well stained and quite visible
• Mold mycelia (true hyphae) can be difficult to visualize on Gram stain
pseudohyphae
Mold mycelia
Yeast
Pseudo-hyphae
Direct Examination of Specimens
X100 oil immersion
11

12. •KOH mount may be used to detect yeast, pseudo-hyphae, and hyphae in
skin, hair, and nail specimens
•KOH dissolves the keratin in cellular material and frees fungal hyphae
and yeast cells so they can be visualized in the KOH solution
•KOH prep lacks sensitivity and specificity, due to lack of staining, the
hyphae can be difficult to visualize and confused with cell borders
•Better methods are currently available
Potassium Hydroxide Prep / KOH
X40
Light microscopy
12

13. • Yeast, pseudo-hyphae, and mycelial fungi bind with the Calcofluor
white stain reagent and fluoresce a bluish color
• The stain is read using a fluorescence microscope (40X)
• More sensitive and specific method than a KOH preparation for
visualization of fungi
Calcofluor White Stain
13

14. One drop of black ink is placed into one drop of CSF
on a microscope slide, cover with coverslip – examine
using light microscope (40X)
“Negative” staining technique – stains the background
of prep not the yeast cell or capsule. The clearing
around the yeast cell is the polysaccharide capsule
produced by both Cryptococcus neoformans and C.
gattii.
India ink is less sensitive than the Cryptococcal
antigen test and Cryptococcal antigen is considered
the superior test for diagnosis of Cryptococcal
meningitis.
India Ink – Cryptococcus detection in CSF
14

15. Grocott’s Methenamine Silver Stain [GMS] – yeast and hyphae
stain grey to black and stains both living and dead fungi.
• Description of the yeast and hyphae can aid in identification
• Observe the width of the hyphae, the presence or absence of regular
septations, and the angle of branching
• Observe the size and budding pattern of yeast and presence of pseudo-hyphae
• Will explain on later slides how these observations can assist in identification
Examination of fungi in fixed tissue
15

16. PAS stains yeast and hyphae magenta. PAS is not specific to fungi,
will also stain structures containing carbohydrate macromolecules
(glycogen, glycoprotein, proteoglycans). PAS stains only living fungi.
Periodic Acid Schiff [PAS] Stain
16

17. Stains the polysaccharide capsule of Cryptococcus neoformans and
C. gatti pink. Will also stain mucin in fixed tissue.
Mucicarmine [Mucin] Stain
Fontana-Masson Silver Stain detects melanin, used in the rare
situation when capsule deficient cryptococcus organisms are encountered.
Cryptococcus stains brown.
17

18. Helpful for the description of cellularity in tissue
and morphologic assessment of size, septations
and branching pattern of molds and the size and
budding pattern of yeast. Can also visualize
structures such as sulfur granules.
Hematoxylin and Eosin Stain
18
Sulfur Granule
Aseptate fungal hyphae
Granuloma
Intracellular yeast

19. DIMORPHIC FUNGI
Important systemic pathogens with unique characteristics
19

20. What is a Dimorphic Fungus?
• Depending on temperature and conditions of environment, one fungi
demonstrates two forms:
• (1) Mycelial form - Hyphae with conidia (spores)
• Free living form in nature and growth in laboratory at temperatures <=30˚C
• (2) Yeast or yeast like form
• Parasitic phase found in human tissue and growth in the laboratory at temperatures >= 35˚C
• Exposure to increased body temperature transforms the mycelial form acquired in nature to
the yeast form that infects tissue
Histoplasma capsulatum –
Mycelial form in nature and growth at 30˚C
Histoplasma capsulatum –
Yeast form in tissue exam and growth at 35˚C
20

21. Most Common Dimorphic Fungi
• Histoplasma capsulatum
• Blastomyces dermatitidis
• Coccidioides immitis and C. posadasii
• Paracoccidioides brasiliensis complex
• Sporothrix schenckii complex
• Penicillium marneffei (Talaromyces marneffei)
21

22. Histoplasma capsulatum
• Epidemiology: Worldwide distribution,
in the USA primary endemic region Ohio, Missouri,
and Mississippi River valleys. Over time
has spread over a larger geographic area.
• Environmental sources: Aerosolized bat guano is rich in H. capsulatum
conidia, so infections occur in Spelunkers (cave explorers). Conidia found
in many species of Bird droppings, including chickens, making farmers at
risk.
22

23. Histoplasmosis
• Up to 90% of infections are asymptomatic or subclinical
• @ 10% acute pulmonary, chronic pulmonary or disseminated to
bone marrow, heart, CNS, gastrointestinal, skin & genitourinary
• Mucocutaneous lesions a unique dissemination site
• Who is most likely to have progressive Histoplasmosis?
• HIV/AIDS / both primary and reactivation disease
• Organ transplant / both primary and reactivation disease
• Taking medications such as corticosteroids or TNF-inhibitors
• Infants
• Adults aged 55 and older / both primary and reactivation disease
• Treatment for serious infections include : Itraconazole,
Fluconazole +/- Amphotericin B
23

24. Histoplasmosis Diagnosis
• Antigen detection in urine
• Quantitative enzyme immunoassay
• Used for screening for Histoplasmosis infection
• Performed on random collected urine specimen
• Most sensitive (>=85%) for the diagnosis of disseminated and chronic
pulmonary Histoplasmosis cases
• Useful test in immune suppressed patients that do not produce a
detectable antibody response and generally helpful due to the slow
growth of the organism
• H. capsulatum antibody tests are available (immunodiffusion and
complement fixation.) Antibody usually appears in the second month of
infection so not as useful for acute diagnosis as antigen detection in urine
24

25. H. capsulatum Culture
• Growth of Mold at 30*C
• Requires 2-8 weeks to grow
• Colony is white to brown and cottony
• Microscopic appearance on LCB tape mount
• Slender septate hyphae
• Tuberculate macroconidia, large & round (8 – 16 µM)
• Microconidia (2 - 4µM)
• Mold grows in nature, microconidia are inhaled /
penetrate deep into the lung causing infection
• MALDI-TOF, DNA hybridization probe, or 16S rRNA
sequencing necessary to confirm identification
• Confirmation is required due to look alike fungi, such as
Chrysosporium and Sepedonium species, which are usually
considered saprophytic fungi
25
Macroconidia have tubercles
(spike protrusions)

26. H. capsulatum Culture
• Growth of Yeast at 37*C
• Requires 4-8 weeks to grow
• Colony is white to beige with a creamy consistency
• Microscopic appearance
• 2-4 uM in size, oval to round, narrow-based budding
• Yeast is the form found in human tissue
• Increase in temperature in human induces the inhaled conidia/mycelia in nature to
undergo transformation in the human to the yeast phase
26

27. H. capsulatum in fixed tissue
• Granulomas produced in lung and other tissues can be either non-
caseating or caseating
• Primary infection usually in lung but can disseminate to organs of
the Reticuloendothelial System (RES) – high % with dissemination
to bone marrow
27

28. Histoplasma capsulatum – Yeast in fixed tissue
• Small yeast 2-4 uM, minimal size variation, narrow neck bud, oval to round.
• Yeast are often intracellular within macrophages
Possible confusion with C. glabrata, C.auris, pneumocystis, or Cryptococcus
due to size. Can perform Mucin stain to differentiate from Cryptococcus
Candida spp. are not usually found inside of macrophages
• Appear to be encapsulated due to staining artifact – which is caused by the
shrinkage of the yeast away from the cell wall
• Yeast stains well with a variety of stains
H & E
PAS
Gram Wrights
28
Appears to be
encapsulated

29. Leishmania donovani
amastigote
Note: kinetoplast (mitochondrial
DNA) next to nucleus, L. donovani
does not stain with GMS
Toxoplasma gondii
tachyzoites - Oval to
crescent in shape, no
capsule-like clearing around
tachyzoite
Beware of these Look-a-likes!
29
Histoplasma capsulatum
in aortic valve tissue – unlike in
other organs, Histoplasma can
produce variable size yeast cells
and hyphae forms in cases of
endocarditis.
This could be confused with other
molds.

30. Case History: Histoplasmosis
38 yr old male, Newly diagnosed with HIV/AIDS
HIV Viral Load =220,000 copies
CD4 count = 3
Anemia
Thrombocytopenia
Bilateral pulmonary infiltrates
Bone marrow aspiration procedure: Giemsa stain
and GMS stain of aspirate
Culture performed, examined with LPCB prep
30
Therapy: mild cases Itraconazole, serious or immune
suppressed add Amphotericin B.
Giemsa
stain
GMS stain
Lacto-phenol
cotton blue prep

31. The mold phase is identical in culture growth
and observance on adhesive tape preparation to
H. capsulatum.
Difference is the size of the yeast cell:
Note yeast cell of H. duboisii is 6-12 um,
which is 2X the size of H. capsulatum yeast cell.
Endemic area: central, east and west Africa
Primary infection: cutaneous with occasional
involvement of the bone
Unusual variant of Histoplasma –
Histoplasma duboisii
31

32. Blastomyces dermatitidis
• Epidemiology
• Ohio and Mississippi River valleys
• No association with any animal or specific activity
• Found in forested areas and riverbanks
• Inhalation of airborne conidia
• Primarily a pulmonary pathogen
• Small % of patients disseminate to skin and bone
• Well demarcated (raised border) skin lesions /
direct examination positive for yeast
• Dissemination most often in immune suppressed
patients
32

33. Blastomyces dermatitidis
• Mold grows in culture at 30˚C
• Growth in 2-3 weeks
• Fluffy white – buff colored mold, prickly
• Microscopic - pear shaped conidia at the end of supporting hyphae –
looks like a lollipop
• Look-alike fungus – Chrysosporium species and possibly others (?)
• Requirement to confirm identification: MALDI-TOF, DNA hybridization
probe or 16s RNA sequencing
Blastomyces Chrysosporium
33

34. • Yeast phase grows at 35*C
• Slow growing taking up to 4 weeks
• Large yeast cell with size variation, 8-15 um round to slightly oval
• Unique “Broad Based Budding” pattern and thick/double contoured wall.
Blastomyces dermatitidis
Broad based bud
Double contoured wall
34

35. Blastomyces dermatitidis Histopathology
• Mixed neutrophilic and granulomatous inflammation is observed in tissue
• Stains well with H&E, PAS and GMS. May stain weakly positive on
mucicarmine and cause confusion with C. neoformans and C.gattii
• Broad based budding yeast cell with thick wall is the classic structure
35
PAS stain
GMS Stain

36. Coccidioides immitis and C. posadasii
• These two species share close genetic relatedness
• Found in different geographic regions, C. immitis only in California
and both C. immitis and C. posadasii found outside CA
• Both species are thought to have the same infectious process, react
the same in serologic tests, and are morphologically identical
• Coccidioides endemic in SW USA (San Joaquin Valley), Mexico, and
South America, in areas known as the Sonoran life zone with warm
and desert sands, common in rodent burrows in the desert areas.
Global warming has led to an increased endemic area.
• Infection is from inhalation of fungal particles (arthroconidia) found in
the sandy soil in the Sonoran life zone areas
36

37. Coccidioidomycosis
• 65% of patients have asymptomatic infection to flu-like symptoms
• 30% of patients have progressive pulmonary disease, may include
erythema nodosum, may produce a coin lesion in the lung
• Usually <5% will experience disseminated infections
• Infection of skin, bones, joints, lymph nodes, adrenal glands, and CNS
• Tropism to the Central Nervous System (CNS) with high fatality rate
• Risk factors for severe or disseminated coccidioidomycosis include:
• Darker skinned individuals and those of Filipino descent
• HIV/AIDS
• Use of immunosuppressive medications
• Organ transplant
• Diabetes mellitus
• Pregnancy
37

38. Serologic diagnosis of Coccidioidomycosis
• Enzyme immunoassay (EIA): Sensitive and commonly used method
for diagnosing coccidioidomycosis, detects IgG and IgM antibodies
• Immunodiffusion (ID): detects IgM antibodies; positive early in the
course of infection,
• Not as specific as EIA detection of antibodies and can have “false” positive
results, must confirm infection with additional testing
• Complement Fixation (CF): detects IgG antibodies and allows for
assessment of disease severity.
38

39. Coccidioides Culture
• Culture at 30˚C grows the Mold phase
• Growth in 2-3 days as a waxy colony, becoming wooly in around 7–10 days
• Microscopically (40X) one observes areas with septate hyphae and thick-
walled alternating barrel shaped arthroconidia
• Barreled arthroconidia breakoff from hyphae and become the infectious
particle in nature and can be inhaled
• Beware! Mature culture is a potential biohazard in the laboratory
40X
2-3 days 7-10 days
39

40. Coccidioides Look-a-likes
• Malbranchea species similar to C. immitis and C. posadasii under
the microscope using adhesive tape prep
• Must confirm any fungi suspected to be Coccidioides using MALDI-
TOF, molecular DNA hybridization probe or 16S rRNA sequencing
Coccidioides Malbranchea
40

41. Coccidioides Histopathology
• No yeast cell produced in human tissue: Instead, a thick-walled spherule
variable in size (10 – 80 uM) with endospores is the structure observed.
• Spherules can be in all stages of development: fragmented spherules to
well formed mature cysts with endospores (2-5 uM)
• Hyphae may be observed in CNS infections or bronchus, but uncommon
• Granulomatous inflammation with caseation is usually observed
41
Position of Coccidioides
spherules can mimic the broad
based bud of Blastomyces

42. Case History:
Coccidiodoimycosis
75 year old male
Right pleural effusion
Lung nodule
Past medical history: Gout,
hypertension, anemia, obesity
Fungal serology testing:
Histoplasma urine Ag: negative
Cryptococcal Ag: negative
Coccidioides IgM: Indeterminate
Coccidioides IgG: Weak Positive
Titer 1:4
Epidemiology: Lives on ranch in Simi
Valley, California
Biopsy of lung
nodule stain/ GMS
Culture of lung
nodule incubated
at 30°C
42

43. • Rhinosporidium seeberi (an aquatic parasite) forms spherules that
are larger than those of Coccidioides
• Rhinosporidium spherules can be > 80 uM in size compared to those of
Coccidioides <=30 uM
• R. seeberi cause oral or nasal mass lesions
Oral /nasal mass lesions
Coccidioides is not the only spherule forming organism!
43
Prototheca wickerhamii
produce structures known
as morulas that mimic
spherules. Endospores
(2-20) are in a rosette
structure.

44. Paracoccidioides brasiliensis complex
• South American Blastomycosis – 80% of cases reported
from Brazil
• Most prevalent systemic fungal infection in Latin America
• Infection acquired from inhaling infectious particles from soil
and decaying vegetation or possibly trauma, leading to skin
infections.
• Disease presentation:
• Pneumonia and/or cutaneous involvement
• Disseminated infection can occur
• Extrapulmonary lesions on the face and oral mucosa are common site
of dissemination
44

45. Paracoccidioides
• Mold grows at 30*C – cultures usually not performed due to
slow growth and nonspecific sporulation
• Yeast grows at 35-37˚C
• Slow growing yeast – 3 weeks
• Large (10 – 30um), thick walled, with 2 or more tear drop daughter buds
(2–10 um)
• Unique multiply budding yeast cell known as the Mariner’s wheel or
Pilot’s wheel yeast
45

46. Tissue Exam of
Paracoccidioides brasiliensis complex
• Granulomatous non-necrotizing and
necrotizing or pyrogranulomatous response
• Mariner’s wheel yeast forms
GMS
GMS
46

47. Sporothrix schenckii complex
• Sporotrichosis
• Cutaneous inoculation from penetrating
injury, such as cut or scrape with contaminated plant thorns
• Known as “Rose gardener’s disease”
• Usually begins as skin lesion with or without ulceration and leads to a
progressive subcutaneous disease
• Lesion can progress with lymphocutaneous spread and possible
dissemination to bone and other organs
• Pulmonary and CNS infections occur, but rarely
47

48. Sporothrix schenckii complex
• Mold grows at 30˚C in 3 -5 days, starts
beige eventually turns brown/black
• Microscopic: thin septate hyphae with
conidia in daisy wheel or rosette pattern
• Yeast grows at 37˚C in 7 days as oval and
elongated yeast cells, 2 x 5 µm, described as cigar bodies
48

49. Sporothrix schenckii Histology
• Pyogenic to granulomatous inflammation
• Yeast may not be observed in human tissue
• If seen, elongated, pleomorphic described as cigar shaped
• More common structure seen in human tissue is an Asteroid body
known as Splendore-Hoeppli phenomenon (SHP)
• SHP are not unique to Sporotrichosis, also seen in infections with:
• Mucormycetes (Mucor, Rhizopus)
• Aspergillus
• Blastomyces
• Candida spp
49
Asteroid body
Pleomorphic yeast cell

50. • Disease: Cutaneous bumpy lesions (Talaromycosis) most common but
pneumonia or systemic infection can occur in immune deficient or HIV/AIDS
• Endemic in the soil in SE Asia, southern China and eastern India
• Mold grows in 2-3 days (<=30*C) / green mold with red diffusible pigment
• Microscopically looks like a typical Penicillium spp with branched
conidiophore and sporulation
• Small yeast like cells seen in fixed tissue / divide be transverse fission
Penicillium (Talaromyces) marneffei
50

51. 51
Diagnosis of Dimorphic infections based on appearance in Tissue

52. SUBCUTANEOUS FUNGAL
INFECTIONS
Mycetoma
Chromoblastomycoses
Phaeohyphomycosis
52

53. Mycetoma
• Chronic, granulomatous disease of the skin and
subcutaneous tissue, which sometimes involves muscle,
bone, and neighboring organs
• Occurs in hot temperate parts of the world –
• Trauma in nature, implants the fungi into the subcutaneous
tissue, farm workers are most at risk
• 3 criteria help to explain mycetoma:
-Swollen extremity from lesion progression
-Development of abscess and fistulas (sinus tracts)
-Sulfur granules drain from sinus tracts, observed in tissue
histology slides to help establish diagnosis
Two types of mycetoma:
1. Actinomycotic – infection with higher bacteria species
2. Eumycotic – usually infection with black molds,
occasionally hyaline molds
53

54. Actinomycotic Mycetoma
• 98% of all mycetoma infections are actinomycotic
• Nocardia species most common infectious agents
• Sulfur granules are formed in tissue. The granules vary in
color depending on the Nocardia species causing infection
• The granules are composed of a matrix of the filamentous
bacteria. Observe the edge of stained granule to observe
the size and morphology of the bacteria.
54

55. Edge of sulfur granule show thin filamentous
bacteria: How can you tell if infection is due to
Nocardia or Actinomyces?
Perform a PAF stain and culture:
• Nocardia species are modified (partial) acid
fast [PAF] stain positive and grow aerobically
• Actinomyces species are PAF stain negative
and grow best anaerobically
Careful! Sulfur granules created by infection
with Actinomyces species (an anaerobic
Gram positive bacilli) can look very similar to
ones formed by Nocardia species. This is a
distinct infection known as Actinomycosis.
Sulfur granules in Tissue
Actinomyces
55
Nocardia

56. Filamentous branching Gram positive bacilli which can stain poorly and
appear speckled.
Modified Kinyoun Acid-Fast Stain (PAF) = Stain red /Partial Acid-Fast
Modified (Partial)
Kinyoun acid-fast stain
Gram stain
Nocardia species
56

57. Besides Mycetoma, Nocardia spp can also cause
primary Pulmonary and Brain infection
Patients usually severely immune suppressed
Grows at 30 – 35*C in 3-5 days on many agars
including SABs and 5% Sheep’s blood agar
Colony is dry/crumbly with a musty odor and can
vary in color, usually white to coral
Total of 85 species of Nocardia with the members of
Nocardia asteroides complex most common
Identification: MALDI -TOF
16s rRNA gene sequencing
Nocardia species
57

58. Eumycotic Mycetoma
Infection with pigmented/black molds (dematiaceous molds)
-Numerous species of black molds found in soil and soil debris
-Infection by the implantation of the mold into the subcutaneous tissue,
usually a traumatic event occurring in environment
- Cause only @ 2% of mycetoma like infections
-Most common agents: Acremonium spp. and Scedosporium
apiospermum complex
Notice the thick
hyphae on the
edge of the granule
58
Dark grey black colony

59. Thermoactinomycetes
• Gram positive filamentous rods related to Nocardia species
• Thermoactinomyces and Saccaropolyspora species
• Flourish in areas of high humidity and high temperature (40*-60*C)
• Grow best in moist and hot agricultural environments, like stacks of hay
• Agents of Farmer’s lung – hypersensitivity pneumonitis or extrinsic allergic
alveolitis, occupational disease
• Immunologically mediated inflammatory disease of the lung
• Inhalation exposure to the thermophilic actinomycetes
• Pathology: Loose, non-necrotizing granulomas
• Diagnosis
• Detailed environmental history
• Serology testing
59

60. Chromoblastomycosis
• Three characteristics help in the diagnosis:
• Wart like lesions into subcutaneous tissue (scarred and nodular)
• Muriform bodies observed in fixed tissue slides
• Infection caused by black pigmented fungi (dematiaceous)
• Skin trauma causes implantation of fungi into tissue
Naturally brown in color, septate
structures known as sclerotic bodies,
or copper pennies are the unique
structure found in tissue – they break
up and pieces extend into the
surrounding subcutaneous tissue
Most common agents: Fonsecaea,
Cladophialophora, and Phialophora spp
60

61. Prototheca wickerhamii (Protothecosis)
• Algae without chlorophyll (not fungal organism)
• Usually related to outdoor penetrating injury
• Causes skin lesions that can become nodular, also olecranon bursa infection
• Most common in patients with suppressed immune system
• Compare morula of Protothecosis to muriform body of Chromoblastomycoses
• Morula of Protothecosis is NOT naturally brown in color and no septation
Prototheca
morula
Copper penny
61

62. Phaeohyphomycosis
Traumatic implantation of black pigmented fungi into subcutaneous tissue
• Infections usually nodular lesions or cysts on the skin, controlled by an
intact immune response
• May disseminate in lung, nasal sinus with extension to brain
• In fixed tissue, dark brown colored swollen hyphae and yeast like cells
• Most common agents: Alternaria, Curvularia, Exophiala and Phialophora
62

63. What are the Black molds /or the
Dematiaceous molds
• Black colored colonies; due to containing melanin or
melanin like pigments in the cell wall
• Commonly found in areas damaged by flooding
Most common black molds include:
Cladophialophora carrionii
Cladophialophora bantiana
Phialophora verrucosa
Fonsecaea pedrosoi
Exophiala species
Wangiella species
Rhinocladiella species
63

64. Black Molds / Dematiacious molds
• Numerous species
• Difficult to identify using microscopic morphology due
to shared sporulation characteristics, requires MALDI-
TOF or 16S rRNA sequencing to ID
• All have at lease one of these
four types of sporulation:
• Rhinocladiella-like
• Cladosporium-like
• Phialophora-like
• Acrotheca-like
Rhinocladiella
Cladosporium
Phialophora
Acrotheca
64

65. Exophiala species
A few common Black Molds
Cladophialophora
bantiana -
Associated with
dissemination infections
that can extend to brain
65
Wangiella
dermatitidis
Phialophora
verrucosa

66. Alternaria species
• Commonly found in nature in soil and plants
• Causative agent of phaeohyphomycosis, sinus, nail and skin
infection in the normal host
• Emerging pathogen in invasive disease in immune suppressed
• Grows in 3 – 5 days at 30*C
• Black colony producing multicellular conidia
66
Scotch tape exam

67. Curvularia lunata
Center cell is the largest
• Found on soil and plants in tropical and subtropical areas
• Phaeohyphomycoses, erythematous skin lesions, sinusitis, and
invasive infections including endocarditis.
• Emerging pathogen in the normal and immune suppressed
• Grows in 3-5 days at 30*C
67
Scotch Tape Exam

68. Exserohilum rostrum
• Causative fungus in an outbreak from a compound pharmaceutical [steroid]
product contaminated during manufacturing with dust containing Exserohilum
• Contaminated product injected into lumbar spine and knee joints for pain
management and led to serious and fatal infections:
• Meningitis
• Spinal abscess
• Synovial infections
68

69. Scedosporium apiospermum and S. prolificans
(old name: Pseudallescheria boydii complex)
• Found in soil
• Subcutaneous infections in normal host from trauma imbedding fungus into tissue
• Pulmonary pathogen with possible dissemination in immune suppressed
• Can invade vessels, particularly in the lung, and lead to infarcts
• Cat fur-like gray colony with growth in 3 – 5 days at 30 *C
• Microscopic appearance of colony: Lollipop like spore production with brown colored spores
• Difficult to distinguish from Aspergillus in fixed tissue stains – can branch at 45* angle
• Intrinsically resistant to Amphotericin B
69
PAS stain of fungal hyphae
Scotch tape prep

70. IMPORTANT YEAST CAUSING
HUMAN INFECTION
Candida species
Cryptococcus neoformans
Cryptococcus gattii
Trichosporon species
70

71. Candida species
• Greater than 200 species but @ 10 species infect humans
• Found as normal flora in GI, GU and skin
• Opportunistic pathogens involving skin, mucous membranes and invasive
sites from excessive exposure to moisture, excessive antibiotics, or
immune suppression
• C. albicans is the most commonly-isolated yeast in human disease (@60%)
• Implicated in both superficial and systemic disease.
• Nail, ear, endocarditis, and bloodstream most often
• Risk factors include age of >=65 years, immunosuppression prior to steroid use,
leukocytosis, intensive care unit stays, or presence of intravascular or urinary catheters.
71
Thrush

72. 72
Candida species Susceptibility to Azoles
(Fluconazole)
Unique features
C. albicans Susceptible Most common / @ 60%
C. parapsilosis Susceptible
C. glabrata (Nakaseomyces
glabrataa)
50% resistant Small yeast (2-4um) no
pseudohyphae produced
C. tropicalis 50-75% resistant
C. kruseii (Pitchia
kudriavzevi)
100% resistant
C. auris
Difficult to accurately identify,
MALDI-TOF is superior to
biochemical testing
100% resistant Emerging nosocomial pathogen,
resistant to Amphotericin B,
susceptible to the echinocandins.
Small yeast (2-4um) no
pseudohyphae produced

73. Candida species
• Growth in 24 – 48 hours at 30-35*C
• Growth on many agars SABS, IMA, Sheep’s Blood agar
• Bacteria-like colony – pasty white
• Oval shaped @ 7-8 um in size**
• Most species form pseudohyphae in human tissue. Not true hyphae, the
yeast do not detach, and the yeast cells elongate to form hyphae, they
have been described as “links of sausage”
• C. auris and C. glabrata are two common specie that do not produce
pseudohyphae in tissue
• Identify using biochemicals, MALDI-TOF, or 16 sRNA sequencing
pseudohyphae
73

74. Candida albicans
Identification
• Germ tube formation
• Incubate yeast in serum for 3-4 hrs at 35 ˚C
• Extension from yeast cell (tube) for C. albicans
• If incubate >4 hrs – C. tropicalis can produce a false
positive germ tube reaction
• Note: C. dubliniensis can also form germ tubes
• Chlamydospore formation
• Growth on cornmeal agar at 48 hrs
• Form rudimentary spore like structures unique to
C. albicans known as chlamydospore
chlamydospore
74
Starry appearing
Colony on BAP

75. Candida auris
• Usually infects critically ill patients who recently spent time in skilled nursing
or long-term care facilities and have lines and tubes in place
• Risk factors for C. auris infections are similar risk factors for other types
of Candida infections: recent surgery, diabetes, broad-spectrum antibiotic
and antifungal use.
• Infections in patients of all ages, from preterm infants to the elderly with
mortality 30 – 60%
• Very transmissible in healthcare settings through contact with contaminated
environmental surfaces or equipment, or from person to person
• Patients and healthcare workers can be colonized on skin, particularly axilla
and groin, and can persist for months
• Patient surveillance screening by PCR (most sensitive) and culture
• Intrinsic resistance to Fluconazole and Amphotericin B

76. ChromAgar for the preliminary identification of
Candida species:
When grown on this agar, a unique color is formed
for specific Candida species due to action on
chromogenic substrates .
Susceptibility testing of yeast is much like bacteria, using broth
dilution and Etest methods.
76

77. Candida Histopathology
• Pyogenic to granulomatous inflammation produced in tissue
• Most Candida species produce oval yeast cells (@ 8 um) and
pseudohyphae that will be visualized in tissue sections
• C. glabrata and C. auris form smaller yeast cells (4 um) and no
pseudohyphae is formed. Could be confused with H. capsulatum.
Candida glabrata or C. auris GMS stain
Candida species not glabrata or auris
77

78. Cryptococcus
neoformans
• In nature, a 2um non-encapsulated yeast cell
• Found in pigeon droppings and soil contaminated with droppings
• C neoformans is enriched by the nitrogen, humidity and warmth in the heaped
droppings
• The small, non-encapsulated yeast cells are inhaled – travel through the
pulmonary system with hematogenous spread to brain and meninges
(tropism). It is truly a neurotropic organism,
• Infects mostly compromised hosts – one of the major AIDS defining
infections, and less common in corticosteroid use and chemotherapy.
78

79. Cryptococcus gattii – close relative of C. neoformans
• Isolated from forested areas of the Pacific Northwest
(British Columbia, Washington, Oregon, and California
• Associated with soil debris, decaying tree bark, and eucalyptus trees
• Infection of both normal and immune suppressed hosts
• Primarily a pulmonary disease (cryptococcoma can be produced) but can disseminate to
the central nervous system
• Culture, biochemical & staining characteristics identical to C. neoformans
• One defining biochemical reaction for C. gattii
• L Canavanine glycine bromthymol blue medium –
C. gatti = blue C. neoformans = colorless
MALDI-TOF and 16sRNA sequencing will correctly ID both species
79

80. Cryptococcus neoformans
and C. gattii
• Variable sized yeast cells (2 – 20 um)
• Virulence factor: Polysaccharide capsule
• Polysaccharide capsule is basis of diagnostic testing:
• India ink exam- CSF placed in one drop of black ink. It is a
negative staining method – background stained but not the
polysaccharide capsule or yeast cell. Sensitivity >=50%
• Cryptococcal antigen test – Titer of capsular polysaccharide,
leached off yeast cell and detected in CSF or serum
• Best test for diagnosis with sensitivity >=99%, can also follow
patient recovery with decreasing titer of polysaccharide present
in the sample
80
Positive India Ink

81. Grows on SABS and IMA in
1-3 days at 30 – 35*C
Mucoid colonies due to production of
polysaccharide capsule
C. neoformans & C. gattii form
brown colonies on Birdseed agar due to
phenol oxidase activity
Gram stain /
stains as blue blobs due
to polysaccharide
capsule
Cryptococcus species have a Positive
urease enzyme reaction
Cryptococcus neoformans and C. gattii
Negative
81

82. Cryptococcus in tissue
82
Cryptococcus with
visible clearing around
yeast due to capsule/
Stain poorly with
H&E stain
Mucicarmine stains the capsular
polysaccharide of Cryptococcus
neoformans and C. gattii.
GMS stain –Notice the yeast are variable
in size.

83. Pneumocystis jiroveci
• Yeast like fungus / cannot be cultured
• Used to be named Pneumocystis carinii and considered a protozoan
parasite
• Causes pneumonia in the immunocompromised host (PCP) particularly
HIV/AIDS / alveoli become filled with the organisms
• Diagnosis: Staining of Bronchial lavage, lung biopsy tissue, induced
sputum using direct fluorescent antibody (DFA) or GMS stain.
DFA
83
GMS

84. Pneumocystis jiroveci (yeast like
fungus) could be confused with C.
neoformans – Careful! Central nuclear
staining in pneumocystis, collapsed
helmet forms.
C. neoformans/ C. gattii – no
nuclear staining
Cryptococcus vs.
Pneumocystis
Pneumocystis froth on H & E
84

85. Trichosporon spp
• T. asahii most common species
• Commonly inhabit soil, water, & vegetables
• Colonize the skin, mouth and nails.
• Grows within 24-48 hours, becomes wrinkled over time
• Hyphae, pseudohyphae, arthroconidia, & rectangular yeast cells (3 x 8 uM)
• Urease enzyme positive
• Cause of superficial infection of the hair shaft named White Piedra, nail
(onychomycosis), and cutaneous infections
• Uncommon cause of fungemia and disseminated disease in humans
• Most usually recovered from patients with corticosteroid use, solid tumors, HIV/AIDS, and
intravascular devices, including catheters and prosthetic heart valves
• Difficult to treat, elevated MICs to most anti-fungal drugs including echinocandins.
• Mortality 50 – 80%
85

86. CUTANEOUS AND SUPERFICIAL
MYCOSES
Malassezia furfur
Dermatophytes that cause Dermatophytoses
Microsporum species
Trichophyton species
Epidermophyton floccosum
86

87. Malassezia furfur
• Most superficial of the dermatomycoses
• M. furfur can be normal flora on human skin and can occur in
neonates
• More common on oily skin or patients with high use of skin oils
• Most common in warm and humid climates
• Diseases:
• Skin infection is most common: Pityriasis versicolor / Tinea versicolor
• macules, papules, patches, plaques on chest back and shoulders with either hypo
or hyper pigmentation – does not invade into deeper tissues
• Fungemia: caused by tunneling in with IV catheter lipid feeding lines
(parenteral nutrition/hyperalimentation) – in neonates or rarely adults
87

88. Malassezia furfur
• Lipophilic yeast – require long chain fatty acids for growth
• This is provided on media by a film of olive oil
• Small budding yeast like cells 2 – 4 µm with collarette (appears like
necklace at junction of mother and daughter cell)
• In skin and tissue preparations, described as “Spaghetti and Meatballs”
due to yeast and short hyphal fragments.
Spaghetti and meatballs
88
Spaghetti and meatballs with basket
weave morphology in the keratin
layer

89. Dermatophytes – Ringworm infections
• Hair, skin and nail infections
• 3 genera of fungi cause infections:
• Microsporum species (many)
• Epidermophyton floccosum
• Trichophyton species (many)
• Disease described by area of the body infected:
For example: tinea capitis (head), tinea pedis (foot)
• Usually, a clinical diagnosis not requiring culture
• KOH or Calcofluor white prep can be used to
visualize fungal hyphae from skin scrapings
89
Calcofluor white prep

90. Microsporum canis
• Skin or scalp ringworm infection acquired from an infected dog or cat
• Colony grows in 5-7 days at 30* C on mycosel or other fungal media/
White mold with yellow pigment on reverse side of colony
• Tuberculate thick-walled macroconidia [tiny spiny projections]
• Few if any microconidia (small spores)
90

91. Microsporum gypseum
• Mostly scalp or skin infection from exposure to contaminated soil
• Sandy colored colony that grows in 5-7 days, 30*C on mycosel or other
fungal media
• Large macroconidia are produced, no microconidia produced
91

92. Trichophyton rubrum
• Mostly infects skin and nails after
exposure to fomites, such as wet
towels
• Grows in 5 – 7 days at 30*C. on
mycosel and other fungal media.
• White mold, the back side of the
colony produces an intense red
colored pigment that diffuses into the
media
• Pencil shaped macroconidia
produced but tear shaped micro-
conidia are most common and can
appear like “birds on a wire”
morphology
Red diffusible pigment
92

93. Trichophyton tonsurans
• White colony, grows in 5-7 days at
30* C
• Light yellow reverse
• No macroconidia
• Only microconidia produced with
rare ballooning microconidia which
is the identification structure
• Primary cause of epidemic scalp
ringworm in children, nails &skin
93

94. Epidermophyton floccosum
• Infections in skin and
nails
• Yellow-Khaki green
colored colony that
grows in 5-7 days at
30*C on mycosel and
other fungal media
• Beaver tail shaped large
macroconidia and no
microconidia produced
94

95. Opportunistic Hyaline Molds
Two groups:
(1) Narrow uniform septate hyphae
(2) Broad non-septate ribbon like-
hyphae
Infections usually in the immune
suppressed host or special
circumstances
95

96. Hyaline molds with narrow septate hyphae
• Hyaline – no color to the hyphae
• Regular septations along hyphae
• Grow on a variety of agar media
in @ 3-5 days at 30˚C or 35°C
• Identification based on growth rate,
color and texture of colony, and
microscopic structures observed on LCB prep
• Definitive speciation: Using MALDI-TOF or 16sRNA sequencing
96

97. Aspergillus species
• Ubiquitous in nature with @ 200 species
• Hyaline hyphae produced with regular septations, occasionally
irregular bulbous forms can be seen
• Numerous round conidia produced at the ends of phialides
• Respiratory tract infection most common
• Can disseminate to other organs in immune suppressed
• Neutropenia can predispose to infection
• 45° angle branching of hyphae in fixed tissue
• Capable of invading vessels, causing thrombosis & infarctions
• Calcium oxylate crystals may be present in fixed tissue
Septation
97
Fungus ball
Invasion of vessels

98. Aspergillus Structure
98

99. Serology Tests Supportive not Conclusive for Diagnosis
Aspergillus Galactomannan Enzyme Immunoassay
• Detects circulating Aspergillus antigen in the blood and/or bronchial lavage,
• Most diagnostic in advanced disseminated Aspergillus infections
• Moderate sensitivity and specificity (PPV 68%/ NPV 96%)
• False positive reactions (10%) with patients on Piperacillin/Tazobactam therapy, infection with
H. capsulatum, and rice and pasta ingestion
(1,3)–Beta-D-glucan assay
• Detects1,3 Beta-D glucan in serum or BAL with the following pathogens: Candida spp.,
Acremonium, Aspergillus spp., Coccidioides spp, Fusarium spp., Histoplasma capsulatum,
Trichosporon spp, Sporothrix schenckii, Saccharomyces cerevisiae, and Pneumocystis
jiroveci.
• Does not detect Cryptococcus or Zygomycetes
• High values of product more meaningful, false positive reaction can occur from environmenal
contamination
99

100. Aspergillus fumigatus complex
• Ubiquitous airborne fungus in nature, infection starts
with inhaling conidia
• Most common Aspergillus species causing
pulmonary infection in immune suppressed:
• Aspergilloma, pulmonary nodules, cavitary formation and
invasive disease
• Allergic bronchopulmonary syndrome in cystic
fibrosis and asthma, accompanied by elevation in
eosinophil count, and IgE
• Blue/Green colored mold, turning grey with age
• Grows in 3 – 5 days at 30*C on multiple fungal media
• Phialides produce colorless conidia on upper third of
the swollen vesicle
100

101. Aspergillus flavus
• Normally isolated from cereals, grains,
legumes
• Same disease potential as A. fumigatus/
pulmonary infection with possible
dissemination, sinusitis and nail infections
• Brownish green –yellow mold growing in 3-5
days at 30*C
• Green/yellow hue to conidia produced on
phialides that surround the swollen vesicle
101

102. Aspergillus niger complex
• Contaminant in fruits and vegetables, also
found in soil and decaying vegetation
• Chronic otitis media, occasional
aspergilloma and dissemination in
immunosuppressed
• Black colony with white border – visible
black fruiting heads grows in 2 – 5 days at
30*C on many types of fungal media
• Dark grey to black conidia produced,
supported by phialides that surround the
vesicle
102

103. Aspergillus terreus complex
• Isolated from soil and decaying vegetation
• Infection primarily in the immune
compromised host
• Sandy colored mold grows in 3-5 days at
30*C on a variety of fungal media
• Colorless conidia supported by phialides,
headed upward
• Aleurioconidia can sometimes be seen
• Intrinsic resistance to Amphotericin B
103

104. Aspergillus Histology
– fruiting head seldom seen in fixed tissue
usually dichotomous (continuous)
branching septate hyphae which branch at
a 45* angle, occasional bulbous hyphae
Look a like fungus in fixed
tissue slides:
Scedosporium species
Growth in culture can help to
differentiate the two fungi.
104

105. Fusarium species
• Common in nature, grows on plants
• White to fuchsia colored mold grows in 3-5 days 30*C on fungal
media. It is inhibited by cycloheximide in media.
• Produces septate hyaline hyphae, banana boat shaped conidia
• Disease normal host: keratitis, nail and skin infections
• Disease immune compromised: sinusitis, pneumonia, deep
cutaneous lesions, and fungemia (unlike most fungi can be
isolated from blood culture)
• Random septate hyphae in fixed tissue, sometimes 45* angle
branching may be observed,
• Confirm identification with culture and identification
Banana boat shaped spore
105

106. Scopulariopsis species
• Found in soil and grows on plant
• Infections: Nail infections in normal host and skin, sinusitis,
pulmonary, brain abscess in immune suppressed host
• White to sandy colored mold growing in 3-5 days at 30*C on a
variety of fungal media
• Round, spiny conidia that chain
• Very resistant to antifungal agents including amphotericin
106

107. Penicillium species – > 300 species
• Common in air, frequent laboratory contaminate
• Cause of bread mold
• Uncommon cause of human disease
• Can appear as a culture contaminate
• Blue/green colony grows in 3-5 days 30*C on a
variety of fungal media
• Hyphae with branched phialides that produce
chains of conidia / appears like brush or bony hand
107

108. The Mucorales
Mucor, Rhizopus and related
molds
Fungi in the Order Mucorales
• Broad, Hyaline hyphae without
septation
• Spores produced in a sack-like
structure known as a sporangium
108

109. Mucorales
• Found in soil, rotten fruit and vegetables
• Acute Rhinocerebral Mucormycosis
• Occurs in diabetics for the elevated glucose enriches fungal growth,
usually caused by Mucor or Rhizopus species
• Infection starts in single sided nasal sinusitis then spreads to the
same side orbit of eye then brain / high fatality rate
• Pulmonary infection in immune suppressed: most
commonly diabetics, corticosteroids and burn patients
• Molds grow within 24-48 hr producing coarse aerial hyphae
• Microscopically, broad, hyaline hyphae without septation
seen with spores produced within a sporangium
• Mince, do not grind tissue submitted for culture, the
grinding will kill the tubular hyphal structures
109

110. Rhizopus species Absidia (Lichtheimia complex)
Mucor species
No
rhizoids
Rhizoids
Species within the Mucorales
Distant rhizoid
110
Cunninghamella species

111. • 90˚ angle branching, with rare to no septations, ribbon like hyphae, can
appear faintly staining particularly on GMS stain
• Distinguishing hyphae characteristic of Mucorales, from other hyphae in
quite important, for Mucorales are more resistant than many fungi to the
azoles and echinocandin antifungal agents
Invades vessels and
can cause infarcts
and thrombi
Order Mucorales in Tissue
111
90* angle branching

112. 112