Fungal Infections Flashcards
What do fungal PAMPs activate?
TLRs
- esp. TLR 2 and 4 for dermatophytes
NOD-like
C-type lectins (ex. mannose binding lectin)
- Dectin-1 binds to b-glucans and triggers IL-22/23
- Dectin-2 bind to α-mannans
- Dectin is on on mast cells too
Scavenger receptors
- DC-SIGN binds mannose
Malassezia cell wall carbohydrates are IgE binding epitopes with AD
- also have IgG and IgA produced (increased with more organisms)
What type of immune response is most important for destroying established fungal infections?
Th1
Th17 - esp for dermatophytosis and Malassezia
no correlation between circulating Abs and protection for dermatophytes
- believed cell-mediated immune response is most important
What are the predominant fungal genera identified on the skin surface of dogs?
Alternaria
Cladosporium
Epicoccum
variable proportions of Malassezia spp
- Malassezia globosa
- Malassezia restricta
- Malassezia pachydermatis (overgrows in inflamed skin)
What are the predominant fungal genera identified on the skin surface of cats?
Cladosporium
Alternaria
Epicoccum
What are the predominant fungal genera identified on the skin surface of horses?
Alternaria
Cladosporium
Fusarium
Aspergillus
Penicillium
Scopulariopsis
(many represent repeated transient contamination from the saprophytes)
Which breeds of cats have higher carriage rates of Malassezia spp, even in the absence of skin lesions?
Devon Rex and Sphynx cats
What is thought to be a primary reason for the increased number of fungal infections, especially opportunistic infections, in veterinary medicine?
higher number of immunosuppressed individuals being treated for cutaneous or systemic immune-mediated diseases or receiving chemotherapy treatment
What are the two stains most commonly used to identify fungus?
periodic acid-Schiff (PAS) reaction
- bright magenta hue
- will only identify living fungus
Grocott’s methenamine silver (GMS) stain
- black color
- more sensitive and will identify more things (including nonviable)
(Fungal cell walls contain polysaccharides that form aldehyde groups upon oxidation and can be detected by both stains)
If a fungal organism is cultured from the skin, what is important to confirm the pathogenic significance of the fungi?
fungal elements identified in the tissue
What are the typical histopathologic morphologic features of Malassezia?
3–8 μm peanut-shaped yeast
Located in superficial keratin and follicular ostia
PAS can be helpful
What are the typical histopathologic morphologic features of Candida?
can grow as yeasts, hyphae, and pseudohyphae simultaneously
3–4 μm ovoid yeast with single budding
Ellipsoid pseudohyphae with constriction at septae
Parallel-walled hyphae with regular septation
PAS can be helpful
What are the typical histopathologic morphologic features of dermatophytes?
2–3 μm wavy septate hyphae with parallel walls
2 μm arthrospores
Endothrix (inside) or ectothrix (outside/degrade cuticle)
- most geophiles and zoophiles are ectothrix
- most anthropophiles are endothrix
Can cause deep pseudomycetomas in Persian cats
PAS or GMS can be helpful
What are the typical histopathologic morphologic features of white-grain eumycotic mycetoma?
2–7 μm septate and nonpigmented hyphae
Infrequent dichotomous branching
Frequent 2–7 μm terminal cystic dilations
What are the typical histopathologic morphologic features of black-grain eumycotic mycetoma?
2–7 μm septate and pigmented hyphae
Infrequent dichotomous branching
Frequent 2–7 μm terminal cystic dilations
What are the typical histopathologic morphologic features of hyalohyphomycosis?
2–6 μm septate hyphae with parallel walls, occasional acute-angle branching
Up to 10 μm terminal bulbous swellings
4–12 μm yeast-like cells may be the only form
What are the typical histopathologic morphologic features of Aspergillus spp?
2–6 μm septate hyphae with parallel walls
Frequent acute-angle branching
What are the typical histopathologic morphologic features of mucorales?
Sparsely septate, 8–20 μm hyphae
Nonparallel walls and irregular branching
What are the typical histopathologic morphologic features of entomophthoromycosis?
6–20 μm (Basidiobolus), 5–12 μm (Conidiobolus) hyphae, sparsely septate
Nonparallel walls, rare irregular branching, terminal bulbous dilations
Eosinophilic sleeve around hyphae
What are the typical histopathologic morphologic features of phaeohyphomycosis?
2–10 μm pigmented hyphae ± branching with beaded appearance
4–15 or rarely 30–50 μm yeast-like forms
Fontana-Masson may highlight melanin
What are the typical histopathologic morphologic features of chromoblastomycosis?
10–20 μm pigmented Medlar bodies with transverse and longitudinal septation
What are the typical histopathologic morphologic features of oomycosis?
2–7 μm (Pythium) or 8–25 μm (Lagenidium)
Nonparallel walls, irregular branching, rare septae
Eosinophilic sleeve around hyphae
Will not stain with PAS
What are the typical histopathologic morphologic features of Blastomyces spp?
5–20 μm yeast
Double contoured wall
Broad-based budding
Yeasts are typically sparse
What are the typical histopathologic morphologic features of Histoplasma spp?
2–4 μm, round yeast
Clear halo, thin cell wall
Numerous yeasts within macrophages
GMS preferred over PAS
What are the typical histopathologic morphologic features of Cryptococcus neoformans and Cryptococcus gattii?
3.5–20 μm, oval yeast
Wide clear capsule resembling a “soap bubble”
Cutaneous lesions common in facial/nasal region (especially in cats)
Yeasts are typically numerous
Capsule highlighted by mucicarmine (or Alcian blue)
What are the typical histopathologic morphologic features of Coccidioides immitis and Coccidioides posadasii?
20–80 μm spherules containing 2–5 μm endospores
Double contoured wall
Lesions typically distributed over bone infection sites
Spherules may be sparse
What are the typical histopathologic morphologic features of Sporothrix spp?
3–10 μm long and 1–2 μm diameter, elongated or “cigar-shaped” yeasts
Yeasts are numerous and commonly in macrophages
Lesions typically on head/neck (especially in cats)
What is the ecological niche of Malassezia species?
lipophilic fungi
- have a low carbohydrate-degrading capacity
- reduction of glycosyl hydrolase-encoding genes
- lack of a fatty acid synthase gene (can’t make long chain fatty acids)
- concomitant expansion of lipid hydrolysing enzymes
live almost exclusively on skin/mucosal sites of warm-blooded vertebrates
What phylum does Malassezia belong to?
Basidiomycota
What species of Malassezia are most relevant for medical dermatology?
M. pachydermatis
M. globosa
M. sympodialis
M.restricta
M. furfur
M. nana - most common in cats esp ear canal
M. slooffiae - common in cats esp claw folds
What enzymes allow Malassezia yeasts to collect and use fatty acids from the skin or mucosal surfaces of their hosts?
lipases
phospholipases
acid sphingomyelinases
Which culture media is best for Malassezia?
lipid-enriched media such as modified Dixon’s agar
M. pachydermatis can grow on Sabouraud’s dextrose agar
- uniquely able to utilize lipid fractions in it
- doesn’t prefer it though
Using cytologic techniques, which anatomical locations in healthy (non-Basset hound) dogs are reported to have the highest numbers of Malassezia yeasts?
peri-oral/ lip region
interdigital skin
perianal skin and anal mucosa
(less common in axillae, inguinal, and dorsum)
(Basset hounds have more pretty much everywhere)
What are some virulence attributes of Malassezia species?
Cell wall morphology
- thick
- spiral
- composition
Adherence to corneocytes
- small family of proteins anchored to plasma membrane
- can modulate cytokines and chemokines
Wide expansion of lipase and phospholipase gene
- might act in synergism with biofilm formation
Biofilm formation
Malassezia antigens appear to be resistant to lysis by NK cells
What dog breeds are at increased risk of Malassezia dermatitis?
West Highland white terriers
English setters
shih tzus
basset hounds
American cocker spaniels
boxers
dachshunds
poodles
Australian silky terriers
Newfoundland
What is a risk factor for Malassezia dermatitis in dogs?
presence of skin folds
hypersensitivity disorders
cornification defects
endocrinopathies
What is a risk factor for Malassezia dermatitis in cats?
hypersensitivity disorder
visceral neoplasia
other serious internal disease
Which molecular techniques allow for accurate identification of fungal species and recognition of genotypes that may have relevance for host-adaptation and virulence?
sequencing of:
- D1/D2 domain of the large subunit of the rRNA gene
- internal transcribed spacer (ITS)
- intergenic spacer (IGS)
- chitin synthase 2 (CHS2) gene
- ß-tubulin genes or regions
Multi-plex PCR (skin samples)
MALDI-TOF MS (from culture samples)
next generation sequencing
What hampers testing for resistance in Malassezia species in vitro?
unsuitability of the current CLSI and EUCAST reference methods
lack of an agreed modified protocol optimized for M. pachydermatis
absence of clinical breakpoints for either systemic or topical therapies
Which medications are recommended for treatment of Malassezia
Systemics
Azoles
- fluconazole may be varied
- resistance is reported
Terbinafine
- resistance has also been reported
Topicals
So many ones including essential oils and honey
Nystatin
- Wild type seens susceptible
- MIC might be increased in diseased states
3+% chlorhex (based in vivo)
topicals with azoles
What mutation in M. pachydermatis has been associated with azole resistance?
ERG11
gene that encodes lanosterol 14 –alpha-demethylase
target site for antifungal azoles
What are dermatophytes?
a distinct group of keratinolytic and keratinophilic fungi
possess a unique ability to digest keratin
allows them to colonize and infect keratinized tissues
What phylum do dermatophytes belong to?
Ascomycota
characterized by the formation of ascospores within specialized sac-like structures called asci
Malassezia overgrowth has been reported many species including cats, horses, cattle, and goats. What is the unifying feature of this condition in all species?
pruritus
What anatomical locations are most commonly affected with Malassezia dermatitis in cats and dogs?
ventral neck or abdomen, intertriginous regions, pinnae, feet, and skin folds
What anatomical locations are most commonly affected with Malassezia dermatitis in horses?
intertriginous
What are the histopathologic findings associated with Malassezia dermatitis?
moderate to marked epidermal hyperplasia
- involving the stratum spinosum
- to around 10 layers thick
- extending to the follicular infundibula
- scalloping/ “festooning” of the epidermal margin (not in cats)
mixed parakeratotic hyperkeratosis with mounds of keratin/crust
mixed dermatitis
- eosinophils and lymphocytes
- lymphocytic exocytosis of predominantly T lymphocytes
low sensitivity of histology compared with cytology
- approximately 30% of biopsies are falsely negative
- PAS can be helpful
Malassezia
Where are Candida spp frequently found in healthy animals?
gastrointestinal and urogenital tracts of both humans and domestic animals
What are infections with Candida spp associated with in domestic animals?
usually opportunistic and secondary to:
- immunosuppressive drug therapy
- systemic disease (ex. diabetes/Cushing’s)
- prolonged antibiotic treatment
- young immunocompromised animals (esp pig or foals) = “thrush”
Vulvovaginal candidiasis due to synthetic progestogen in horses
What is are the clinical signs of a Candida spp infection?
pale, pseudomembranous plaques with ulcers on the tongue and gingiva
- Erosions/ulcers frequently covered by gray, malodorous exudate
can also affect oral mucocutaneous junction, distal extremities, perineum, vulva/scrotum, external ear canal, and intertriginous sites
pruritus is variable
What are the histopathologic findings associated with candidiasis?
large areas of superficial pustulation
- containing predominantly neutrophils
serocellular crust formation, spongiosis, and erosion to ulceration
organisms present in crusts or pustules
- yeasts, hyphae, and pseudohyphae
What order do dermatophytes belong in?
Onygenales
includes fungi that primarily infect keratinous substrates
What are the 7 currently accepted dermatophyte genera?
Trichophyton
Microsporum
Nannizzia
Epidermophyton
Paraphyton
Lophophyton
Arthroderma
What is the sexual form of a dermatophyte called?
teleomorph
produce ascospores which are more resistant in environment
*less common, mostly found in soil
What is the asexual form of a dermatophyte called?
anamorph
form spores such as (macro/micro)conidia or arthrospores
*most common
What are the 3 groups that dermatophytes are categorized into based on their environment?
anthropophilic (primarily living on humans)
- ex. T. rubrum
zoophilic (primarily living on animals)
- M. canis in dogs and cats
- T. verrucosum in livestock except pigs
- T. equinum in horses
geophilic (primarily living in the environment)
- ex M. gypseum/N. gypsea
What happens in the anamorph stage of dermatophytes?
introduction of spores to host –>
spores germinate and invade tissue –>
hyphae proliferate and produce enzymes to digest keratin –>
lesions form –>
produce spores through budding or hyphal fragmentation –>
spores are shed into the environment
Which animals are more prone to dermatophytosis?
Young animals with immature immune system
Geriatric animals with weakened immune response
Immunocompromised animals
What environmental factors can contribute to the development of dermatophytosis?
Overcrowding
Poor sanitation
Warm and humid environments
How are dermatophyte infections spread?
direct contact
- M. canis prefers this
fomites (including ectoparasites, environment, etc)
microtrauma significantly increases risk of development
- such as can be caused by ectoparasites
- laboratory settings need skin abrasion and moisture to cause infection
What is a defense mechanism against dermatophyte infections in cats?
normal grooming
What happens in the adherence step of the dermatophyte infection process?
occurs between 2–6 h after contact in M. canis
1) Arthroconidia adhere to keratinocytes through adhesins
- Subtilisin (Sub3) binds to host glycoproteins
- T. mentagrophytes protrudes fibrillar projections
2) Hydrophobic interactions enhance ability to stick
3) Protein-protein interactions occur
- Sub3 interacts with keratin and collagen
4) Enzymatic degradation
- Sub3, Sub4, and metalloprotease MEP3 degrade keratin
5) Signal transduction pathways in host and fungus
6) Cytoskeletal rearrangements in keratinocytes to allow closer contact
What does subtilisin (Sub3) in M. canis do?
is a serine protease
binds to glycoprotein receptors on the host’s keratinocytes
- facilitates initial attachment
degrade surface glycoproteins
- exposes more binding sites
interacts with host proteins like keratin and collagen
degrades keratin and other structural proteins
What happens during the germination stage of a dermatophyte infection?
starts penetrating into skin
happens within 4 hours for T. mentagrophytes
arthroconidia increase in size and produce germ tubes
- extend horizontally and penetrate through the SC
leads to formation of hyphae and tissue invasion
secures the fungal structure and facilitates infection
What happens during the invasion stage of a dermatophyte infection?
hyphae penetrate the SC and grow in various directions
fungi begin producing arthrohyphae and invade keratinized tissues
form arthroconidia within seven days
How long does it typically take for lesions associated with dermatophyte infections to occur after exposure?
one to three weeks
How do dermatophytes degrade keratin?
produce sulfites to cleave cysteine bonds in keratin
- but high environmental cysteine levels are toxic
convert cysteine to sulfite via the enzyme cysteine dioxygenase (Cdo1)
sulfite efflux pump (Ssu1) helps secrete sulfite to keep cycle going
*both Cdo1 and Ssu1 are needed for dermatophytes to invade properly
What the primary enzymes (virulence factors) that dermatophytes have to degrade keratin?
Endoproteases (keratin to large peptides via peptide bonds)
- Subtilisins (Sub)
- Fungalysins-type metalloproteases (Mep)
Exoproteases (large peptides into smaller ones/amino acids)
- Leucine aminopeptidases (Lap)
- Dipeptidyl peptidases (Dpp)
What happens to the pH of skin during a dermatophyte infection?
amino acid metabolism causes an alkaline shift
What is the optimal pH for dermatophyte keratinases to be active at?
Early Infection Stages: slightly acidic environment
Later Infection Stages: higher, more alkaline pH levels
mediated by the conserved PacC/Pal signal pathway
How is the NLRP3 inflammasome activated in dermatophyte infections?
microbial binding to CLRs or TLRs –>
induces pro-IL-1β synthesis and NLRP3 transduction –>
- via NF-κB-dependent activation
various factors (cathepsin, K+ efflux, ROS) NLRP3 activation –>
leads to assembly of a complex with NLRP3, ASC, and pro-caspase-1
- facilitates pro-caspase-1 activation and processing of mature IL-1β
How do dermatophytes evade the immune system?
1) Secretion of Immunosuppressive Substances
- ex. mannans
2) Induction of Non-Effective Antibody Responses
- stimulate the production of unhelpful antibodies (ex. IgE)
3) Delayed-Type Hypersensitivity (DTH) Evasion
4) Activation of Host Signaling Pathways (allow spread/divert attention)
- ex. activate MAPK pathways in keratinocytes
5) Modulate Th17 Cells and C-type Lectin Receptors
6) keratin degradation enzymes
7) cysteine dioxygenze
8) cell wall components affecting complement inhibition
Which breed of cat and which breed of dog are predisposed to dermatophyte infections?
Persian cats
- may have worse infections
Yorkshire terriers
hunting dogs may be predisposed to geophilic infections
What is the difference between a kerion and a pseudomycetoma/mycetoma?
Kerion: Presents as a single or multiple erythematous, alopecic, dome-shaped, exudative nodule(s) with granuloma or pyogranuloma formation, often containing fungal spores
- usually have a good prognosis in dogs
- typically more superficial
Pseudomycetoma and Mycetoma: Characterized by nodules that fistulate, ulcerate, and drain serous to purulent debris and may contain grains
- prognosis is usually bad for cats and dogs
Why is green fluorescence under the Wood’s lamp (320 and 400 nm wavelength) observed in M. canis infections?
a chemical metabolite in the cortex or medulla of the hair
- pteridine
can develop as early as 5-7 days post-infection (usually 10-14)
sensitivity ~71% and specificity ~ 92%
other species like M. distorum, M. audouinii, and T. schoenleinii do it too
- N. gypsea occasionally can
- T. verrucosum can in cattle
What culture media is used for grow dermatophytes?
dermatophyte test media (DTM)
- contains phenol red that changes color when pH increases
- gross colony and microscopic morphology can be altered
Sabouraud dextrose agar
- better colony morphology
- may not produce macroconidia –> switch to sporulation media
*usually contain cycloheximide to slow growth of contaminants
- interferes with the translocation step in protein synthesis
What species of dermatophyte is most common in cows, goats, sheep, and camelids?
T. verrucosum
What species of dermatophyte is most common in pigs?
Microsporum nanum (now Nannizzia nana)
What is a hypha?
A single vegetative filament of a fungus
What is a mycelium?
a mass of hyphae
What is the difference between septate, pauciseptate, and sparely septate?
septate if they have divisions between cells
- pauciseptate if hyphae have few septations
sparsely septate if they have many nuclei within a cell
What species of dermatophyte is most common in horses?
Trichophyton equinum
What are the pros and cons of culturing for dermatophytes?
Pros
- Low cost
- Easy to perform
- Can distinguish between species (with limitations)
Cons
- Requires expertise to determine species
- Can be contaminated by saprophytes
- takes days–weeks
What are the pros and cons of PCR for dermatophytes?
Pros
- Highly sensitive
- Can distinguish between species
- takes hours–days
Cons
- Unable to distinguish dead and alive fungi
*Negative PCR in a treated cat is compatible with cure
What are the pros and cons of ELISA for dermatophytes?
Pros
- Highly specific
- takes hours–days
Cons
- False positives due to past infections
What is the colony and conidia morphology of M. canis?
Colony: white to cream with reverse yellow pigment, cottony to wooly
Macroconidia: thick walled, spindle-shaped with 5-15 cells
Which common dermatophyte is this consistent with?
M. canis
Which common dermatophyte is this consistent with?
M. canis
Which common dermatophyte is this consistent with?
N. gypsea (M. gypseum)
What is the colony and conidia morphology of N. gypsea/M. gypseum?
Colony: cream to buff, yellowish reverse, suede-like to granular
Macroconidia: ellipsoidal, thin-walled, verrucose, four to six-celled
Which common dermatophyte is this consistent with?
N. gypsea (M. gypseum)
Which common dermatophyte is this consistent with?
T. mentagrophytes
What is the colony and conidia morphology of T. mentagrophytes?
Colony: white to cream, brown reverse, powdery to granular
Microconidia: numerous, hyaline, smooth-walled, spherical to pyriform
Less commonly can have
- Macroconidia: smooth, thin-walled, clavate-shaped
- spherical chlamydospores
- spiral hyphae
Which common dermatophyte is this consistent with?
T. mentagrophytes
Dermatophytic kerion
Dermatophyte (inset is PAS)
Dermatophytic kerion (inset is PAS)
Dermatophyte with furunculosis
Dermatophyte with minimal folliculitis
Piedra
White-grain mycetoma
Black-grain mycetoma
phaeohyphomycosis
fungal melanin appears as greenish with Romanowsky stain
Oomycete
Background is India ink.
Cryptococcus
Cryptococcus
second inset is a mucicarmine stain highlighting the capsule
Histoplasma
Blastomyces
Blastomyces
Coccidiomycosis
Coccidiomycosis
Sporotrix
Sporotrix
