1. Staib (1962) reported that C
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. SHORT COMMUNICATION M. REFAI, M. TAHA, S. A. SELIM,
Sabouraudia (1983) 21,
@ 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

6. No. of yeasts with BCE on different media
Birds
Pal's
medium
Eucalyptus leaves medium
Modified tobacco agar medium 3
Canaries 2
Parrots 5
Total

7. Hala Abd-El Hakeem Saleh Ali
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. Throat swabs (71)
Vaginal swabs (50)
Sources
Yeasts %
No. of +ve samples
9.8 7 16 8
C. albicans - 4
Other Candida sp. 24 12
Total Candida sp.
4.2 3
C. neoformans
5.6
Rhodotorula rubra
19.7 14 32
Total
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

11. Total
Chicken
Animal %
No. of isolates
No.of isolates
48.59
260
48.90
156
48.14
104
C. albicans
21.49
115
25.39 81
15.74 34
Cr.
neoformans
5.98 32
7.84 25
3.24 7
Other Candida
3.55 19
0.63 2
7.87 17
G. candidum
20.37
109
17.24 55
25.00 54
Mixed yeast
100.0
535
319
216

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 states of 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. (C)
(D)
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 by enzyme immunoassay Biotin 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. neoformans calcineurin 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.

51. Effects the capsule on Cytokine Production
* Since the capsule blocks phagocytosis, any cytokines induced by the phagocytic process would not be induced by the encapsulated C. neoformans cells.

52. Cryptococcal Products  
GXM is the major cryptococcal component in body fluids,
It is highly probable that the organism also sheds galactoxylomannan (GalXM) and mannoproteins (MP) in vivo.
Cryptococcal antigens in serum or spinal fluid are diagnostic for cryptococcosis

53. Melanin Synthesis
One characteristic that differentiates pathogenic isolates of C. neoformans from nonpathogenic isolates and other Cryptococcus species is the organism's ability to form a brown to black pigment on a medium (such as birdseed or caffeic acid agar) that contains diphenolic compounds

54. Melanin Synthesis
This pigment, first described by Staib, is a melanin-like compound produced by C. neoformans isolates with phenoloxidase activity
It is reported that naturally occurring C. neoformans mutants lacking melanin were less virulent in mice than melanin-producing strains

55. Mannitol Production
* Accumulating evidence suggests that production of the hexitol D-mannitol may contribute to survival of C. neoformans in the host.

56. Other Potential Virulence Factors
Proteases
C. neoformans grown in culture produces a protease that could digest human plasma proteins, and casein.
Supernatants of broth cultures liquefied gelatin in vitro indicating that this cryptococcal strain secreted a collagenase-like protein
Thus, C. neoformans proteases possibly serve as virulence mechanisms by initiating invasion of host tissues;.

57. Other Potential Virulence Factors
Phospholipase, lysophospholipas, lysophospholipase-transacylase
Correlation between phospholipase activity and virulence in BALB/c mice
Extracellular phospholipase activity produced by C. neoformans may disrupt mammalian cell membranes and allow the yeast cells to penetrate into host tissues

58. Other Potential Virulence Factors
Cryptococcal Phenoloxidase diphenoloxidase has been purified (75 Kda containing copper)
Disruption of the gene CNLAC1 encoding this enzyme resulted in loss of virulence of C. neoformans

59. Regulation of Virulence
Regulation of virulence factors such as capsule production and melanin formation is not well understood
The gene GPA1, which encodes a G-protein -subunit, is involved in the regulation of these virulence factors as well as in C. neoformans mating

60. Regulation of Virulence
Disruption of GPA1 resulted in
Defects in mating in response to nitrogen starvation,
Defects in capsule production in response to iron limitation,
Defects in melanin synthesis in response to glucose starvation  
gpa1 mutants were much less virulent in a rabbit model of cryptococcal meningitis

61. Regulation of Virulence
Reconstitution of the gpa1 mutant with wild-type GPA1 restored mating, capsule production, melanin synthesis, and virulence.
Addition of cyclic AMP also restored these phenotypes, which suggests that C. neoformans GPA1 regulates these factors by sensing the nutritional signals of the environment and regulating cyclic AMP metabolism in the organism

62. Conclusions Virulent isolates of C. neoformans
must be able to produce small particles that can get into the alveolar spaces,
must be able to grow at 37oC at a pH of 7.3 to 7.4 in an atmosphere of approximately 5% CO2,
must have an intact calcineurin pathway.

63. Conclusions
The ability to produce a large capsule and shed great amounts of capsular material into the body fluids makes the organism highly virulent.
Other factors, such as melanin, mannitol, superoxide dismutase, protease, and phospholipase production, may enhance the pathogenicity of C. neoformans.
The effectiveness of many of these cryptococcal virulence factors depends on the status of the host's defensive

64. شكرا على حسن انتباهكم
ا. د. محمد كمال محمد رفاعى
كلية الطب اليبطرى
جامعة القاهرة