Professor Dr. med. Dr. med. vet. Friedrich Staib, 1925-2011

1. Staib (1962) reported that C. neoformans is the only yeast that gives brown colonies on media containing the extract of Niger seeds (Guizotia abyssinica).

2. Professor Dr. med. Dr. med. vet. Friedrich Staib, 1925-2011

3. Sabouraudia (1983) 21, 163-165 @ 1983 International Society for Human and Animal Mycology SHORT COMMUNICATION ISOLATION OF CRYPTOCOCCUS NEOFORMANS, CANDIDA ALBICANS AND OTHER YEASTS FROM PIGEON DROPPINGS IN EGYPT M. REFAI, M. TAHA, S. A. SELIM, F. ELSHABOURII, H. H. YOUSSEFF This paper reports the first search for Cryptococcus neoformans in pigeon droppings in Egypt

4. On Bird seed agar brown colonies of C. neoformans and white colonies of Candida albicans.

5. Application of Molecular Genetic Methods for Diagnosis of Fungi Thesis presented by Mahmoud Dardiri El-Hariri 2009

7. Genotypic identification and characterization of yeasts with particular references to recent approaches for their control A Thesis Presented by Hala Abd-El Hakeem Saleh Ali 2010

8. Table ( 4 ) : Prevalence of yeast species in vaginal and throat swabs of human origin.

9. Molecular and biological studies on Cryptococcus neoformans A thesis presented by Randa Mohammed Ibrahem Alarousy M.V.S. (2004) Faculty of Veterinary Medicine – Cairo University • 2011

10. Some Mycological and Molecular biological Studies on Mixed Yeast Infection Thesis presented by Shimaa Yousef Abou-Elmagd 2011

12. The genus Cryptococcus • At least 24 species that exist in nature, of which Cr. neoformans is most pathogenic to man and animals. • Cryptococcus neoformans has a saprophytic nature as it is frequently isolated from soil and in association of birds droppings. • Other species isolated from clinical material include • Cr. albidus, • Cr. kutzongie, • Cr. laurentii and • Cr. luteolus.

13. Cryptococcus neoformans • C. neoformans has a predilection for the respiratory and nervous system of humans and animals. • Two varieties are distinguishable biochemically and by molecular techniques: ·    C. neoformans var. neoformans ·     C. neoformans var. gattii

14. Cryptococcus neoformans serotypes • The capsule of C. neoformans has 4 serotypes: *A and D: C. neoformans var. neoformans *B and C: C. neoformans var. gattii.

15. Genetic relationship between serotypes A and D • Serotypes A and D produced distinct RFLP patterns consistent with their separation into two major clusters • Consequently 3 varieties are suggested: * C. neoformans var grubii (ser. A) * C. neoformans var neoformans (ser. D) * C. neoformans var gattii (ser. B and C)

16. Sexual statesof Cryptococcus neoformans • Filobasidiella neoformans var. neoformans is the teleomorph of C. neoformans var. neoformans • Filobasidiella neoformans var. bacillispora is the teleomorph of C. neoformans var. gattii.

17. Basidiospores of C. neoformans var. neoformans.

18. On Sabouraud's dextrose agar • Colonies : cream colored, smooth, mucoid

19. India Ink Preparation: • Positive - distinct, wide gelatinous capsules surrounding the yeast cells are present.

20. On Canavanine-glycine-bromthymol blue (CGB) agar C.neoformans var. gattii isolates grows in the presence of L-canavanine and assimilates glycine as a sole carbon source.CGB agar turns blue

21. Physiological Tests: • Germ Tube test is Negative • Hydrolysis of Urea is Positive • Growth on Cycloheximide medium is Negative • Growth at 37C is Positive

22. Fermentation Reactions: • Negative: Glucose; Sucrose; Lactose; Galactose; Maltose; Trehalose.

23. Assimilation Tests Positive:Glucose; Galactose; Maltose; Sucrose; Trehalose; D-Xylose; Melezitose; Raffinose (weak); Cellobiose (weak); Inositol; L-Rhamnose; D-Arabinose; L-Arabinose (delayed); D-Ribose (weak); Galactitol (delayed); D-Mannitol; D-Glucitol. Variable:Soluble Starch; Glycerol; Succinic acid; Ribitol; L-Sorbose; Salicin; Citric acid; DL-Lactic acid. Negative:Potassium nitrate; Lactose; Melibiose; Erythritol.

24. Serotyping of C. neoformans • Slide agglutination test with antisera factors 1,5,6,7 and 8 * Serotype A + in factor sera 1,7 * Serotype B + in factor sera 1,5 * Serotype C + in factor sera 1,6 * Serotype D + in factor sera 1,8 • Indirect IF using Mab 13F1 descriminates between ser. A and D strains.

25. Molecular typing of Cryptococcus neoformans CNRE-1 RFLP pattern • Restriction fragment length polymerphism of genomic DNA • Probed with labeled Cryptococcus neoformans repetitive element 1 • Bands compared with standards

26. Molecular typing of Cryptococcus neoformans • URA5 gene nucleotide sequencing • URA5 gene sequences of the reference strains are deposited in GenBank

27. Human cryptococcosis • C. neoformans var. neoformans affects immunocompromised hosts predominantly • It is the commonest cause of fungal meningitis; worldwide, • 7-10% of patients with AIDS are affected. • AIDS associated cryptococcosis accounts for 50% of all cryptococcal infections reported annually

28. Human cryptococcosis • Meningitis is the predominant clinical presentation • Fever and headache are the most common symptoms. • Secondary cutaneous infections occur in up to 15% of patients with disseminated cryptococcosis and often indicate a poor prognosis. • Lesions usually begin as small papules that subsequently ulcerate, but may also present as abscesses, erythematous nodules, or cellulitis.

29. Animal cryptococcosis • Mastitis is the most common infection in cattle, which is characterized by hardness and swelling of affected quarters and grayish white secretions. • Pulmonary infection, nasal granuloma or meningoencephalitis were also reported in cats, dogs and horses. • In sheep and goats, septicaemic form with pneumonia encephalomeningitis have been recorded.

30. Laboratory diagnosis • a.Samples include spinal fluid, sputum, pus , skin scrapings, urine milk etc. • b.Microscopic examination: positive samples stained with India ink show round cells surrounded by unstained capsules.

31. India Ink Preparation: • Positive - distinct, wide gelatinous capsules surrounding the yeast cells are present.

32. (D) (C) Fig.(4): Brown Colour Effect (BCE) of Cr. neoformans isolates brown pigmented colonies and white colonies C. albicans. (A-B): Modified tobacco agar medium., (C): Eucalyptus leaves agar medium.,(D): Pal's agar medium. (White arrows refer to C. albicans white colonies).

33. Isolation and identification • c. Cultures on Sabouraud dextrose agar without cycloheximide, incubated at 37oC show creamy moist colonies. Brown coloured colonies are clear on Niger-seed medium. • d. Biochemical identification: Cr. neoformans does not ferment sugars, assimilates glucose, galactose,sucrose, maltose,trehalose, xylose and inositol and hydrolyses urea.

34. Serological diagnosis • Detection of antigen by agglutination • Detection of capsular material solubilized in the body during infection (antigen) • by a specific rabbit anti-Cr. neoformans antiserum using latex agglutination test. • A positive agglutination is diagnostic for cryptococcosis.

35. Detection of antigen byenzymeimmunoassayBiotin Amplified EIA for Antigen Detection * antigen detection in serum, and cerebrospinal fluid • Antibody is immobilized on the surface of microwells • Cryptococcal capsular polysaccharide is "captured" by the antibody. • The captured antigen is detected by "sandwiching" with biotin-labeled antibody and streptavidin-enzyme conjugate.

36. Serological diagnosis • Detection of antibodies • Antibodies to Cr. neoformans are usually detected at the late stages of infection or on recovery, • by indirect immunoflourescent technique, haemagglutination inhibition test or ELISA.

37. Pathogenesis • C. neoformans var. neoformans causes most cryptococcal infections in humans • C. neoformans var. neoformans is found worldwide; • Its main habitats are debris around pigeon roosts and soil contaminated with decaying pigeon or chicken droppings • Not part of the normal microbial flora of humans

38. Pathogenesis • It is generally accepted that the organism enters the host by the respiratory route in the form of a dehydrated haploid yeast or as basidiospores. • After some time in the lungs, the organism spreads hematogenously • It has a predilection for the brain, causing meningoencephalitis .

39. Pathogenesis • If untreated, cryptococcal meningoencephalitis is 100% fatal, and • Even when treated with the most effective antifungal drugs, cryptococcal infections can be fatal in case of unadequate T-cell­dependent immune function

40. Basic Characteristics for Pathogenicity The Infectious Particle* To enter the alveolar spaces of the lungs and establish pulmonary infection, an organism must produce viable forms smaller than 4 µm in diameter. * The typical vegetative form of C. neoformans is the yeast form with a cell diameter of 2.5 µm to 10 µm.

41. Basic Characteristics for Pathogenicity • The organism can also undergo sexual reproduction, and since it is a basidiomycete (Filobasidiella neoformans), it forms basidiospores. • The sexual spores (basidiospores) are approximately 1.8 µm to 3 µm in diameter

42. Basic Characteristics for Pathogenicity • Inside the lungs, the yeast cells (blastospores or basidiospores) become rehydrated and acquire the characteristic polysaccharide capsule

44. Basic Characteristics for Pathogenicity • To cause infection in humans, a C. neoformans isolate must grow at 37oC in an atmosphere of approximately 5% CO2 and at a pH of 7.3 to 7.4. • To survive at 37oC, the organism must have an intact gene that encodes the C. neoformanscalcineurin A catalytic subunit

45. Basic Characteristics for Pathogenicity • Calcineurin A mutant strains of C. neoformans can grow at 24oC, they cannot survive in vitro at 37oC, in 5% CO2, or at alkaline pH • Such mutants are not pathogenic for immunosuppressed rabbits • Calcineurin A appears to be a basic requirement for C. neoformans survival in the host and consequently is a necessary factor for the pathogenicity of the organism.

46. Virulence factors of Cryptococcus neoformans • · Capsule, • Cryptococcal products: ·        melanin production, ·        mannitol production, ·        superoxide dismutase, ·        proteases, ·        phospholipase B, and ·        lysophospholipase.

47. virulence factors of Cryptococcus neoformans • The polysaccharide capsule and the soluble extracellular constituents of C. neoformans (referred to here as cryptococcal products) are probably the dominant virulence factors.

48. The Capsule • High molecular weight polysaccharide of -1,3-D-mannopyranose units with single residues of D-xylopyranosyl and D-glucuronopyranosyl • Referred to as glucuronoxylomannan (GXM) and considered as the principal virulence factor • Acapsular mutants are typically avirulent

49. Effects of the capsule on phagocytosis * Encapsulated C. neoformans cells are not phagocytized or killed by neutrophils, monocytes, or macrophages to the same degree as acapsular mutants * Encapsulated C. neoformans have a stronger negatively charged surface than acapsular cells * The high negative charge could cause electrostatic repulsion between the organism and the negatively charged host effector cells and reduce intimate cell-cell interactions required for clearance of the cryptococci

50. Altered Antigen Presentation *The inability of macrophages to ingest the encapsulated organisms could also diminish antigen presentation to T cells and consequently reduce immune responses.