1. CANDIDA & CRYTOCOCCUS Prepared by: Miss Norzawani Jaffar Bsc (Hons) Biomedical Sciences, UKM Lecture 15

2. Learning Outcomes At the end of learning session, student must be able to ; Understand and describe the general morphology for Candida spp. and Cryptococcus spp. Explain the disease that cause by Candida spp. and Cryptococcus spp. Explain and understand the laboratory test for species identification and confirmation.

3. Pathogenic Yeasts (Candida and Cryptococcus) Two genera of yeasts stand out as human pathogens, one being Cryptococcus sp. and the other Candida sp.

4. YEAST The term "" is not always strictly defined; generally single- cell fungi. Other, more rigorous definitions may be applied. Yeasts are normal components of gastrointestinal and genitourinary tracts Most common fungal isolate from patients Unicellular, eukaryotic, generally round to oval Multiply principally by blastoconidia (buds) While pseudohyphae are the rule, true septate hyphae may be formed by some yeasts under certain conditions While morphology may contribute to identification, biochemical tests are usually necessary for definitive diagnosis Growth at 37°, germ tube test, carbohydrate assimilation, fermentation, urease test, and phenol oxidase test may be employed to speciate

5. Candida Species Generally grow at 37°, ferment glucose and may ferment other carbohydrates, and form pseudo- or true hyphae Harbored by the gastrointestinal tract Candida albicans Germ tube positive Creamy colonies, as other yeasts May display pseudohyphae and true hyphae Most commonly isolated candidiasis Virulence factors include rapid germination within tissue, protease production, surface integrin-like molecules for binding extracellular matrix, complement protein binding receptor, phenotypic switching, and surface variation and hydrophobicity

6. Candida albicans, showing pseudohyphae with clamydospores, this fungus usually takes yeast

7. Candida albicans on blood agar; moist, opaque colonies are characteristic of yeast.

8. Candida albicans incubated in rabbit serum at 37° (germ tube test). Germ tubes are indicated by arrows and are the beginnings of true hyphae: no constriction is at the origin of the germ tube and the parent cell.

9. Candidiasis Candidiasis, caused by Candida albicans, or lesser by C. tropicalis and C. glabrata. The species is commonly present in human mucosa, which becomes invasive when immunity weakens. In newborns natural resistance is low, candidiasis can develop within a few days. Disseminated Cadidiasis can be fatal when untreated.

10. Candidiasis encompasses infections that range from superficial, such as oral thrush and vaginitis, to systemic and potentially life-threatening diseases. Candida infections of the latter category are also referred to as candidemia and are usually confined to severely immunocompromised persons, such as cancer, transplant, and AIDS patients as well as non-trauma emergency surgery patient

11. Candida tropicalis May cause infection, especially of the immunocompromised; may also be isolated without evidence of disease Creamy colonies with mycelial fringe Blastoconidia arise singly or in small groups anywhere along long pseudohyphae

12. Candida tropicalis on a Dalmau plate.

13. Candida parapsilosis Relatively frequent cause of candidal endocarditis Creamy colonies; sometime with lacy appearance Blastoconidia arise singly or in small groups anywhere along pseudohyphae; at low power has characteristic shaggy star appearance At high power, crooked or curved, short pseudohyphae and occasional large hyphal elements (termed "giant cells") can be seen

14. Low power appearance of Candida parapsilosis on Dalmau plate. Note the characteristic "shaggy star" morphology.

15. Cryptococcus spp. Morphology Do not form pseudo- or true hyphae, except for Cryptococcus neoformans, which rarely forms them Urease positive Cryptococcus neoformans is the only common, medically important yeast to produce phenol oxidase, colonies grown on birdseed agar (which contains niger or thistle seeds) turn dark brown in 2-5 day of incubation

16. Cryptococcus neoformans Common infection in immunocompromised patients CNS infection most common, but may cause other disease, especially of lungs Capsule is seen in classic India ink preparations; but given the appearance of poorly encapsulated strains, this technique is becoming less useful Mucicarmine stains and Gomori methenamine silver (GMS) stain are useful on histologic sections, see figures under Direct Examination, above

17. Cryptococcus neoformans 1000x stained with Calcofluor.

18. Cryptococcus neoformans in blood culture; gram stain.

19. Cryptococcus laurentii Colonies are white or greenish on birdseed agar; cream to yellow, pink, orange, or tan on routine growth media Like C. neoformans, may grow at 37° C on Sabouraud. dextrose agar Like other Cryptococcus species, urease positive Used within the laboratory as a control organism to test purity of sugars within certain media Yeast ovoid to elongate, as single cells or in pairs or occasionally in chains of 3 or more cells

20. Cryptococcus laurentii on Dalmau plate. Note the absence of hyphae.

21. Cryptococcosis Cryptococcosis, caused by yeast Cryptococcus neoformans invading tissues when immunity weakens, which could lead to fungal meningitis. Yeast Cryptococcus neoformans usually associates with bird dropping from pigeons. The potential danger is that the sexual spores of this yeast could be carried by wind.

24. Candida Species: Direct Examination Hyphae show distinct points of constriction resembling sausage links Budding yeast forms (blastospores) often seen

25. Cryptococcus Species: Direct Examination Cells spherical and irregular in size, ranging from 5-20μ in diameter Classically have thick polysaccharide capsule, however, poorly encapsulated strains are becoming more common One or more budding forms are attached by a narrow constriction

26. Candida ID is chromogenic medium for the culture and isolation of yeast, the identification of Candida albicans, and the separation of other yeast into two group. Most filamentous fungi will also grow. Bacteria are inhibited. It can be very useful in detecting mixed yeast population in clinical specimen. The plates are incubated in ambient air for 48 hr at 35-37°C; some species of Candida require 30°C for optimal growth.

27. The colony colors indicate the following; BLUE: C. albicans or C. dubliniensis (some strain of Trichosporon can be blue, but the colony morphology is very different from that of the Candida spp.) PINK: C. tropicalis, C. guilliermondii, C. kefyr or C. lusitaniae (Cryptococcus spp., may also pink) WHITE or CREAM: other yeast and some filamentaous moulds. * Notes; Plates must always be stored and incubated in the dark. Some bacteria (notably Enterococcus faecalis) may grow on the medium and have a pink or blue color.

28. END Q & A