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).
Professor Dr. med. Dr. med. vet. Friedrich Staib, 1925-2011
SHORT COMMUNICATION M. REFAI, M. TAHA, S. A. SELIM, 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
On Bird seed agar brown colonies of C. neoformans and white colonies of Candida albicans.
Application of Molecular Genetic Methods for Diagnosis of Fungi Thesis presented by Mahmoud Dardiri El-Hariri 2009
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
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
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.
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
Some Mycological and Molecular biological Studies on Mixed Yeast Infection Thesis presented by Shimaa Yousef Abou-Elmagd 2011
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
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.
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
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.
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)
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.
Basidiospores of C. neoformans var. neoformans.
On Sabouraud's dextrose agar Colonies : cream colored, smooth, mucoid
India Ink Preparation: Positive - distinct, wide gelatinous capsules surrounding the yeast cells are present.
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
Physiological Tests: Germ Tube test is Negative Hydrolysis of Urea is Positive Growth on Cycloheximide medium is Negative Growth at 37C is Positive
Fermentation Reactions: Negative: Glucose; Sucrose; Lactose; Galactose; Maltose; Trehalose.
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.
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.
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
Molecular typing of Cryptococcus neoformans URA5 gene nucleotide sequencing URA5 gene sequences of the reference strains are deposited in GenBank
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
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.
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.
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.
India Ink Preparation: Positive - distinct, wide gelatinous capsules surrounding the yeast cells are present.
(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).
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.
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.
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.
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.
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
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 .
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
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.
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
Basic Characteristics for Pathogenicity Inside the lungs, the yeast cells (blastospores or basidiospores) become rehydrated and acquire the characteristic polysaccharide capsule
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
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.
Virulence factors of Cryptococcus neoformans · Capsule, Cryptococcal products: · melanin production, · mannitol production, · superoxide dismutase, · proteases, · phospholipase B, and · lysophospholipase.
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.
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
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
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.
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.
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
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
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
Mannitol Production * Accumulating evidence suggests that production of the hexitol D-mannitol may contribute to survival of C. neoformans in the host.
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;.
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
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
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
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
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
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.
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
شكرا على حسن انتباهكم ا. د. محمد كمال محمد رفاعى كلية الطب اليبطرى جامعة القاهرة