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Opportunistic Fungal Infections

Candida

Susan RichardsonJanuary 11, 2010

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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.

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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

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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

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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

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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

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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)

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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)

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Cell wall Candida albicans

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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)

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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)

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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

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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

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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.

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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

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Candida

Human commensal (endogenous)– 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

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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

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Oral thrush

Vaginal candidiasis

Cutaneous candidiasis

19BalanitisDiaper dermatitis

Cutaneous candidiasis

20Onychomycosis and paronychia Chronic mucocutaneous candidiasis

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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

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Central catheter

Parenteral nutrition

Broad-spectrum antibiotics

Neutropenia

Very low birth weight

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Disseminated candidiasis

Endophthalmitis Disseminated skin lesion

Disseminated candidiasis

24Hypo-echoic splenic lesions

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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

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Candida species

Top: Calcofluor White x400: Yeast and

pseudohyphae

Bottom: Gram stain x1000: Yeast and

pseudohyphae

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Pathology of disseminated candidiasis

Yeast-like cells and septate hyphaeGMS

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Pathology of disseminated candidiasis

Esophagus, vascular invasion, blastoconidia and pseudohyphae, PAS

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Pathology of disseminated candidiasis

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

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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

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Yeast Identification

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Candida species

Candida albicansSabouraud 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

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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

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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

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Yeast identification

C. parapsilosisShort, curved pseudohyphae

C. lusitaniaeSlender, branched, curved pseudohyphaeshort chains of blastoconidia

C. guilliermondiiFew, short pseudohyphaeClusters of blastoconidia at septae

C. lipolyticaElongated blastoconidia in short chainsarthroconidia

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Yeast identification

C. dubliniensisTerminal chlamydospores

C. tropicalisGraceful long pseudohyphaeSingle/small groups blastoconidia along pseudohyphae

C. kruseiElongate blastoconidiaCross-matchsticks, tree-like

C. glabrataNo pseudohyphae, small blastoconidia

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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

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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)

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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

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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

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Candida antifungal susceptibility testing

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