Opportunistic Fungal Infections Candida Susan Richardson January 11, 2010

Opportunistic Fungal Infections Require impairment of host immunity to cause serious infection Clinical infection - localized to severe systemic infection Yeasts: Candida spp. (albicans, tropicalis, parapsilosis, krusei, glabrata, lusitaniae, kefyr, guilliermondii etc.) Cryptococcus neoformans Filamentous fungi: Aspergillus spp. (fumigatus, niger, flavus) Zygomycetes (Rhizopus, Mucor, Rhizomucor, Absidia) Fusarium spp. Penicillium spp. (marneffei) Pseudallescheria boydii (Scedosporium apiospermium) Curvularia spp.

Predisposing Factors (Immunologic) Cancer (esp. hematological malignancy) Key defect: Neutropenia Organ Transplantation (bone marrow, liver, lung, kidney) Key defect: Neutropenia, Impaired T cell function Cellular Immune Dysfunction (AIDS, lymphoma, CMC) Key defect: Impaired T cell function

Predisposing Factors (Non-Immunologic) Chemotherapy (cytotoxic) - mucosal damage of GI, respiratory, GU tracts Antibiotics - Broad spectrum; loss of normal flora, esp. anaerobic Invasive devices - breach skin/mucosal defences, i.e. intravenous lines, urinary catheters, tracheostomies Invasive procedures - surgery, diagnostic biopsies

Transmission of Opportunistic Fungi Candida, Trichosporon, Malassezia ENDOGENOUS unique strain colonization precedes infection antibiotic suppression of normal flora, fungal overgrowth EXOGENOUS hand carriage health care worker

Transmission of Opportunistic Fungi Aspergillus, Zygomycetes, other filamentous fungi, Cryptococcus EXOGENOUS inhaled conidia ventilation systems, construction, heliports, plants, environment direct contact - dressings, arm boards, burns, wounds

Candida MOST COMMON invasive fungal infection in immunocompromised patients 4th most common cause of nosocomial blood stream infection Species implicated in human disease most often: C. albicans C. tropicalis C. parapsilosis C. krusei (fluconazole resistant) C. glabrata C. lusitaniae (amphotericin B resistant)

Candida Thick cell wall of mannan and glucan polysaccharides Unicellular, budding (asexual) reproduction (blastospores) Filament formation Pseudohyphae (buds stay attached, constricted, chains of elongated blastospores) Hyphae (buds germinate)

Cell wall Candida albicans

Candida - Pathogenicity / Virulence Factors C. albicans >>> virulent than other Candida species Rapid switching of expressed phenotype Enhanced ability to reassort and regulate genetic expression by chromosomal rearrangement and recombination phenotypic - nutrient stress produces different colony forms virulence factors (including antifungal resistance, e.g. C. lusitaniae vs. amphotericin B)

Candida - Pathogenicity / Virulence Factors Hyphal formation Hyphal formation is associated with tissue invasion ( yeast forms associated with epithelial colonization) spontaneous C.albicans non-hyphae-forming mutant shows decreased pathogenicity in a rat Candida vaginitis model Experimental renal infection - yeast and hyphae initiate renal lesions, but hyphae are essential for invasion of the renal pelvis. Hyphae adhere more readily to host epithelial surfaces than do yeast cells (50x more adherent)

Candida - Pathogenicity / Virulence Factors Contact sensing - growth of hyphae on filters or membranes (thigmotropism) When placed on agar medium grow through pores and along grooves. Tissue penetration may be aided by following surface discontinuities and microscopic breaks Surface hydrophobicity Hydrophobic C. albicans at 25 C >>virulent than more hydrophilic C. albicans at 37 C Hydrophobic CA show increased adherence and more rapid hyphal germ tube formation

Candida - Pathogenicity / Virulence Factors Surface virulence molecules (receptors, adhesins, pyrogens, and immunomodulators) Candida adhere to: epithelial cells (buccal, cervical, corneal, urinary, gastrointestinal mucosa), vascular endothelial cells, spermatozoa, plastics Candida form ligands to host components - C3d, iC3b, fibrinogen, laminin, fibronectin, fucose receptors, N-acetylglucosamine receptors

Candida Pathogenicity / Virulence Factors Molecular mimicry Surface coat of molecules that mimics host components (decreases recognizability) C. albicans cells in the bloodstream become rapidly coated with host platelets via the fibrinogen-binding ligand. Lytic enzymes Hydrolases with broad substrate specificities (proteinase, phospholipase(s), lipase(s), acid phosphomonoesterase). Aspartyl proteinase - most potent or thoroughly studied.

Candida Pathogenicity / Virulence Factors Growth rate and undemanding nutrient requirements Virulent strains have shorter doubling times than attenuated strains C. albicans not fastidious, but nutritionally deprived mutants (auxotrophs for adenine, lysine, serine, uracil and heme) show decreased virulence

Candida Human commensal (endogenous) Environmental (exogenous) skin, gastrointestinal, genitourinary tracts 5 - 15% carriage rate in normal people increased carriage with use of antibiotics Environmental (exogenous) much less common food, animals, soil hospital environment outbreaks have occurred

Candida - Clinical Mucous membrane infections Thrush (oropharyngeal) Esophagitis Vaginitis Cutaneous infections Paronychia (skin around nail bed) Onychomycosis (nails) Diaper rash Balanitis Chronic mucotaneous candidiasis children with T-cell abnormality

Mucosal candidiasis Oral thrush Vaginal candidiasis

Cutaneous candidiasis Diaper dermatitis Balanitis

Cutaneous candidiasis Onychomycosis and paronychia Chronic mucocutaneous candidiasis

Candida - Clinical Urinary tract infection Fungemia Disseminated (systemic, invasive) infection Immunocompromised patients Cancer/chemotherapy Neonatal candidiasis Endophthalmitis (eye) Liver and spleen Kidneys Skin Brain Lungs Bone

Clinical profile Central catheter Parenteral nutrition Broad-spectrum antibiotics Neutropenia Very low birth weight

Disseminated candidiasis Endophthalmitis Disseminated skin lesion

Disseminated candidiasis Hypo-echoic splenic lesions

Candida - Laboratory Diagnosis 1 Specimens - Blood, tissue (biopsy or autopsy), sterile fluid, urine, CSF, skin, respiratory secretions Microscopy (direct on specimen - except blood and urine) Gram stain, Calcofluor Histopathology (tissues) H & E - stain poorly GMS, PMS - stain well

Candida species Top: Calcofluor White x400: Yeast and pseudohyphae Bottom: Gram stain x1000: Yeast and

Pathology of disseminated candidiasis Yeast-like cells and septate hyphae GMS

Pathology of disseminated candidiasis Esophagus, vascular invasion, blastoconidia and pseudohyphae, PAS

Pathology of disseminated candidiasis Hematogenous renal candidiasis. Disseminated miliary abscesses, cortex and medulla. Necrotic papillae.

Candida - Laboratory Diagnosis 2 Culture (all specimens) Colony morphology White, smooth, creamy, sometimes wrinkled Laboratory identification Unique color on chromagar Chlamydospore production (terminal vesicle) Germ tube production (in horse serum) beginning of true hypha (no constriction) C. albicans - Germ tube positive Other Candida - Germ tube negative Carbohydrate assimilation and fermentation (API 20C, Vitek2, RapID and reference) Urea and nitrate Microscopic morphology on Cornmeal Tween 80

Yeast Identification

Candida species Candida albicans Sabouraud Agar Morphology: Creamy white yeast, may be dull, dry irregular and heaped up, glabrous and tough Chromagar producing green pigmented colonies on specially designed medium to speciate certain yeasts based on color they produce

Candida species Germ tube: inoculation of yeast in horse serum incubated at 370C for 2 to 3 hours Germ Tube: Positive Germ tube is a continuous filament germinating from the yeast cell without constriction at the point of attachment. e.g. C. albicans, C. dubliniensis Germ Tube: Negative Shows constriction at the attachment site e.g. other Candida species, esp. C. tropicalis

Candida species Candida albicans Oxgall Agar large round and thick walled chlamydospores Cornmeal Agar clusters of blastospores along pseudohyphae at regular intervals x400 x1000 x400 x1000

Yeast identification C. lusitaniae C. parapsilosis C. guilliermondii Slender, branched, curved pseudohyphae short chains of blastoconidia C. parapsilosis Short, curved pseudohyphae C. guilliermondii Few, short pseudohyphae Clusters of blastoconidia at septae C. lipolytica Elongated blastoconidia in short chains arthroconidia

Yeast identification C. tropicalis C. dubliniensis C. krusei Graceful long pseudohyphae Single/small groups blastoconidia along pseudohyphae C. dubliniensis Terminal chlamydospores C. krusei Elongate blastoconidia Cross-matchsticks, tree-like C. glabrata No pseudohyphae, small blastoconidia

Candida - Laboratory Diagnosis 3 Candida antigen, antibody and metabolite detection NOT useful in routine practice Low sensitivity and specificity Polymerase chain reaction No more sensitive than blood culture in studies to date

Candida - Treatment Remove infected intravenous lines Antifungal therapy for systemic infection Amphotericin B IV Azoles (fluconazole, itraconazole, voriconazole, posaconazole) orally, intravenous Flucytosine (only with Ampho B because of resistance) Echinocandins (caspofungin, micafungin)

Candida antifungal resistance Primary (inherent) resistance C. lusitaniae (amphotericin B) C. glabrata (fluconazole) C. krusei (fluconazole) Secondary (acquired) resistance Fluconazole, other azoles Amphotericin B 5-FC

Candida antifungal susceptibility testing Testing methodology Reference broth microdilution (CLSI) Commercial broth microdilution with alamar blue (Sensititre, YeastOne) E-test Disk diffusion (CLSI Vitek 2

Candida antifungal susceptibility testing

Candida antifungal susceptibility testing