Mycology

Pathogenic Fungus

no endotoxins or exotoxins, just colonize humans

increased incidence since mid-1980s (especially among immunocompromized, such as AIDS and cancer patients)

40% of all hospital deaths are from Candida, a fungus (more than bacteria or viruses)
increase caused by (1) excessive IV and catheter use, and (2) overuse of antibiotics (which kill the competition)
progression accelerated in patients with granulocytopenia and HIV; bone marrow and solid organ transplants, etc.

larger and more complex than bacteria, non-motile with rigid cell wall (chitin), classified as yeast or mold

yeasts – oval, spherical, or elongated cells which reproduce by budding and form moist or mucoid colonies
molds – branching tubular hyphae divided by septa – intermingled hyphae form mycelium (visible dry colonies)

Staining properties – stained by silver stain (Gomori methenamine silver)

also visible on PAS, Wright’s and Giemsa stains, even Gram stain (Candida only)

Culturing Fungus – requires Sabouraud's medium (agar containing glucose and peptone) – bacteria don’t grow

also requires uncoagulated protein (e.g., blood) for cultivating the yeast phase of dimorphic fungi
fungi will grow and reproduce indefinitely if proper food source is available

Cultural Recognition – stain with lactophenol cotton blue then look at hyphal size, septae, spores, and chlamydospores

asexual spores (used to reproduce) are useful for identification, since they are quite distinctive – four types:

(1) blastospores - simple buds of single yeast cells
(2) arthrospores - spores resulting from hyphae fragmentation
(3) conidiophores - spores developing from a specialized projection growing out of the hypha, the conidiophore
(4) sporangiospore (endospores) - multiple spores developing in a sac at the end of a stalk (sporangiophore)

chlamydospores (used to survive unfavorable conditions) are large with a double wall
some fungi cease spore formation in culture (pleomorphism)

Drugs that kill them – three catagories, defined in terms of their targets in the organism:

(1) polyene macrolides (e.g., Amphotericin B, nystatin) bind ergosterols in cytoplasmic membrane (strong but toxic)
(2) azoles (e.g., fluconazole) inhibit cytochrome P450-dependent enzymes and block ergosterol synthesis (safer)
(3) flucytosine interferes with pyrimidine metabolism and is used as an adjunct to amphotericin B

Opportunistic Fungal Infections – Found Everywhere, but Only Cause Disease in Immunocomrpomised Patients

(1) Yeast-Like (single ovoid cells):

(A) Candida – colonizes skin and mouth in absence of intact integument and bacterial flora – e.g., burns, IV catheters, antibiotics

killed by complement, neutrophil phagocytosis, and CD4 cells (AIDS patients get it)
if disseminated, can be found in blood, urine, liver, spleen, mucous membranes, even tongue (in AIDS) and eye
subspecies:

C. albicans – most common – 10% of all nocosomial infections – does not survive outside of humans

resistant to ketoconazole and fluconazole (one study – 50% AIDS patients have fluconazole-resistant strains)

C. glabrata – high complication/mortality rate – need high dose of fluconazole to kill

C. lusitaniae, C. tropicalis, C. parapsilosis, and Trichosporon beigelii – resistant to amphotericin B

C. krusei, Cryptococcus neoformans – resistant to fluconazole

(B) Cryptococcus neoformans – grows in pigeon feces and soil (although pigeon exposure does not increase risk)

conidia spores are inhaled, causing subacute or chronic infection, usually involving CNS – produces meningitis
not a problem before AIDS outbreak
infects lung, CSF, blood, urine, skin/mucous membrane, multiple systemic sites
two variant strains – neoformans and gatti – both found worldwide
huge anti-phagocytic polysaccharide capsule – excludes India ink stain
killed by T-lymphocyte dependent macrophage activation – extracellular killing (since large fungus)
cryptococcal meningitis is the most frequent CNS infection in AIDS
fungistatic therapy does not kill fungus, so frequent relapse – requires lifelong therapy
can be given to mice (experimental model)

(2) Mold-Like (have hyphae):

(A) Aspergillus – ubiquitous – usually colonizes existing cysts and cavities to form mycetomas (fungus balls)

airborne spread, infects 4%-38% upon contact; mortality in infected patients is 85% – early treatment is critical
infects lung, nasal sinus, skin, CNS, ear, eye, urine, multiple systemic sites
allergic bronchopulmonary aspergillosis (Farmer’s lung) can occur in patients with chronic bronchial asthma
also causes mycotic keratits, otomycosis, and nasal sinus infection
pulmonary disease and dissemination can occur in patients without enough neutrophils (neutropenia)

disseminates to become systemic

most common pathogens: A. fumigatus, A. flavis, A. niger, A. terreus

(B) Zygomycetes – fruit and vegetable mold – cause vascular invasion and infarction

ketoacidosis accellerates growth – rhinocerebral disease (nasal cavity, paranasal sinuses, orbit, brain)
pulmonary lesions and dissemination also can occur in immunosuppressed patients

Primary Fungal Infections – Less Common, but Can Infect Anyone (fundi = endemic fungi)

Thermal Dimorphic fungi – hyphae and spread by arthrospores at room temp, but become yeast-like when they infect, inducing granulomas

(1) Histoplasma – mostly asymptomatic (50% skin test reactivity in some areas) – histoplasmosis looks like TB

H. capsulatum grows in soil of Ohio, Mississippi, St. Lawrence rivers – grow in bird and bat droppings

(2) Coccidioides – mostly asymptomatic, can cause fever, pulmonary nodule (thin-walled cavity), meningitis, TB-like lesions

C. immitis grows in desert areas of the southwestern U.S. – spread by airborne arthroconidia
the appearance of spherules in tissue establishes the diagnosis

(3) Blastomyces – pneumonia or granulomas – also heaped hyperkeratotic cutaneous lesion surrounded by microabsesses

Blastomycosis is slowly progressive – causes fulminant disease in debilitated, elderly patients
B. dermatitidis natural reservoir is unknown, but not person-to-person (soil?) – usually southeastern US

(4) Sporothrix – lymphocutaneous disease – ulcerating nodules at inoculation and along draining lymphatics

acute and granulomatous inflammation – thin hyphae with daisy-like arrangement of spores are diagnostic
this fungus is an occupational hazard for florists and gardeners

Phospholipases of C. Albicans increase virulence

phospholipases have been identified as an important virulence factor in Candida

phospholipases and proteases allow penetration of endothelial cells to allow infection
phospholipase activity has been observed in pathogenic fungi (Aspergillus fumigatus, Cryptococcus neoformans)

also, less pathogenic fungi have lower phospholipase activity

to prove phospholipases are associated with virulence – three tests (modified Koch postulates)

(1) associated with virulent species

more phospholipase in disease-causing form than in commensals
more virulent strains have more phospholipase – it has the closest correlation to virulence

(2) gene can be isolated – disrupting it should decrease virulence; adding it should increase virulence

these were verified experimentally

(3) virulent strain secretes it during infectious process

verified experimentally

therefore, phospholipase is a virulence factor for C. albicans, and potentially many other fungi as well