1. Opportunistic Fungal Infections
Candida
Susan Richardson
January 11, 2010

2. 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.

3. 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

4. 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

5. 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

6. 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

7. 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)

8. 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)

9. Cell wall Candida albicans

10. 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)

11. 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)

12. 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

13. 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

14. 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.

15. 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

16. 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

17. 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

18. Mucosal candidiasis
Oral thrush
Vaginal candidiasis

19. Cutaneous candidiasis
Diaper dermatitis
Balanitis

20. Cutaneous candidiasis
Onychomycosis and paronychia
Chronic mucocutaneous candidiasis

21. 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

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

23. Disseminated candidiasis
Endophthalmitis
Disseminated skin lesion

24. Disseminated candidiasis
Hypo-echoic splenic lesions

25. 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

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

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

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

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

30. 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

31. Yeast Identification

34. 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

35. 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

36. 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

37. 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

38. 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

39. 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

40. 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)

41. 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

42. 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

43. Candida antifungal susceptibility testing

44. Candida antifungal susceptibility testing