Cryptococcus neoformans

One hundred years ago, Cryptococcus neoformans was discovered when this encapsulated yeast was isolated independently from peach juice in Italy and from the tibial lesion of a patient in Germany

it had been given a variety of names,

including Saccharomyces neoformans, Blastomyces neoformans,

Cryptococcus hominis

•Reports of infection due to other species of Cryptococcus (C. albidus and C.laurentii) are extremely rare

•C. neoformans var. neoformans has long been isolatable from soil and avian habitats.

•Birds do not become infected, probably because their relatively high body temperature is inimical to C. neoformans.

•However, birds are likely to distribute the yeasts in nature.

•There are two varieties of C. neoformans and four major serotypes, based on capsular epitopes.

• Nearly all infections of patients with AIDS involve strains of a single variety and serotype, although such strains vary at the phenotypic and genotypic levels

•The 19 species within the genus Cryptococcus are characterized as variously encapsulated budding yeasts.

•Despite reports of other pathogenic species, the major pathogen is the species C. neoformans, an encapsulated, usually

spherical yeast of which two varieties are recognized, C. neoformans var. neoformans and C. neoformans var. gattii.

•By using cross-absorbed rabbit polyclonal antisera, the capsular serotypes of C. neoformans were defined as serotypes A

through D and serotype AD.•Rare isolates lack a capsule or

possess a capsule but cannot be serotyped.

•On routine laboratory media, colonies of C. neoformans develop within 36 to 72 h.

•They are white to cream colored and opaque and may be several millimeters in diameter.

• Colonies may also develop sectors that differ in pigmentation.

•The colonies are typically mucoid, and the amount of capsule can be judged from the degree of colonial mucosity.

•Highly encapsulated colonies may coalesce and slowly trickle down a slant to puddle in the bottom of the tube or drip off the medium of inverted plates.

•Microscopically, most clinical isolates appear as spherical, budding, encapsulated yeast cells in both tissue and culture.

•Rarely, short hyphal forms are seen, and filamentous variants have been isolated

•Capsule size or thickness is determined by the genetics of the strain as well as by conditions of growth.

•For a given strain, capsule production in vitro can be optimized by cultivation on

•solid or liquid medium in the presence of glucose, thiamine, glutamate, neutral pH,

•temperature of 37 0 C, elevated carbon dioxide, or decreased concentration of iron.

•As judged from the ability to readily isolate the major pathogenic variety of C. neoformans from soil and avian habitats, C. neoformans is ubiquitous in nature, yet the incidence of cryptococcosis

is relatively low. •Therefore, it is likely that many more

people inhale the yeast cells (or basidiospores) than become ill.

•Indeed, an intact cell-mediated immunity (CMI) and competent phagocytes provide strong defenses against cryptococcosis.

• limited serological studies indicate that most healthy individuals possess antibodies to C. neoformans

Growth and Physiology

•C. neoformans has minimal growth requirements of simple carbon and nitrogen sources, and even vitamin supplementation with thiamine may not be required for growth.

•C. neoformans generally does not grow as rapidly as yeasts such as Saccharomyces cerevisiae and Candida albicans under similar conditions.

• Unlike other species of Cryptococcus, the ability of C. neoformans to use low-

molecular-weight nitrogenous compounds such as creatinine may partially explain its ecological niche in avian guano.

•It is inhibited by alkaline pH and high temperature.

•Unlike C. neoformans, most other species

of Cryptococcus are unable to grow at 370C and are nonpathogenic.

•Indeed, the growth of C. neoformans is tightly regulated by temperature, since in vitro temperatures of 39 to 400C will

significantly slow its growth rate, and at this temperature, the yeasts undergo intracellular vacuolization and produce aberrant budding patterns and pseudohyphal structures.

•All species of Cryptococcus are nonfermentative, hydrolyze starch, assimilate inositol, and produce urease.

•These characteristics, as well as the morphological features of the capsule

and the rare presence of pseudohyphae, distinguish them from other clinically important yeasts.

•most strains readily excrete large amounts of urease, and its detection in vitro has led to a rapid urease test for the identification of C. neoformans

•A unique biochemical feature of C neoformans is its ability to produce diphenol oxidases , which may

• function as antioxidants and enhance survival of the yeast in the host .

•The importance of temperature to pathobiology is further emphasized by temperature- sensitive mutants of C. neoformans, which do not grow at 370C and are avirulent in animals regardless of their ability to form capsules or produce melanin

•The presence of this enzyme, which oxidizes a number of diphenolic substrates and leads to the production

•of melanin, has been used for identification .

Virulence Factors

•Pathogenicity requires the production of phenol oxidase, growth at 370C, and the presence of a capsule.

•The basis for the neurotropism of C. neoformans is unclear but may

involve selective evasion of host defenses or enrichment in the target tissues.

(e.g., utilization of catecholamines, which are excellent substrates for phenol oxidases and are abundant in the central nervous system [CNS] and the adrenal glands, another target organ).

•The capsule or CPS potentiates infection, depresses inflammation, inhibit phagocytosis and suppresses both cellular and humoral immunity

Cryptococcosis

•Cryptococcosis is a chronic, subacute to acute pulmonary, systemic or meningitic disease, initiated by the inhalation of basidiospores and/or desiccated yeast cells of Cryptococcus neoformans.

•Primary pulmonary infections have no diagnostic symptoms and are usually subclinical.

• On dissemination, the fungus usually shows a predilection for the central nervous system, however skin, bones and other visceral organs may also become involved.

•Although C. neoformans is regarded as the principle pathogenic species, C. albidus and C. laurentii have on occasion also been implicated in human infection.

•Pulmonary Cryptococcosis:

•Asymptomatic carriage of Cryptococcus has been reported from the respiratory tract.

•patients with chronic lung disease, such as bronchitis and bronchiectasis, may also have asymptomatic colonization, with Cryptococcus being isolated from their sputum over many years

•Subclinical cryptococcosis may result of environmental exposure, normal individuals may experience a self-limiting pneumonia with accompanying sensitization.

•Most primary infections of this type have no diagnostic symptoms and are usually discovered only by routine chest x-ray. When present, symptoms include cough, low-grade fever and pleuritic pain.

•Chronic pulmonary cryptococcosis also increases the risk of dissemination to the central nervous system.

Central Nervous System:

•Dissemination to the brain and meninges is the most common clinical manifestation of cryptococcosis and includes meningitis, meningoencephalitis or expanding cryptococcoma.

•Meningitis is the most common clinical form.

•Cryptococcoma is a rare entity.

•Cutaneous Cryptococcosis:

•Primary cutaneous cryptococcosis in the form of ulcerated lesions or cellulitis occasionally occurs, especially in immunosuppressed patients.

•These lesions may resolve spontaneously or with systemic antifungal treatment. However, all patients with skin lesions should be monitored carefully for possible dissemination to the central nervous system.

Cryptococcosis of Bone: •Osseous cryptococcosis occurs in up

to 10% of disseminated cases and may involve bony prominences, cranial bones and vertebrae.

• The lesions are lytic without periosteal reaction and symptoms of dull pain on movement are reported.

• Occasional cases of arthritis have also been reported, mostly involving the knee joint.

Ocular Cryptococcosis:

•Ocular manifestations of cryptococcosis most commonly include papilledema and optic atrophy, due to raised intracranial pressure.

• Other ocular signs of cryptococcosis are uncommon and usually occur as a result of dissemination.

Laboratory diagnosis:

1. Clinical material: Cerebrospinal fluid (CSF), biopsy

tissue, sputum, bronchial washings, pus, blood and urine.

2. Direct Microscopy: (a) For exudates and body fluids make

a thin wet film under a coverslip using India ink to demonstrate encapsulated yeast cells. Sputum and pus may need to be digested with 10% KOH prior to India ink staining.

•(b) For tissue sections use PAS digest, GMS and H&E, mucicarmine stain is also useful to demonstrate the polysaccharide capsule. Examine for globose to ovoid, budding yeast cells surrounded by wide gelatinous capsules. Note, non-encapsulated variants, although rare, may also occur.

•The demonstration of encapsulated yeast cells in CSF, biopsy tissue, blood or urine should be considered significant, even in the absence of clinical symptoms.

• Positive sputum specimens should be considered potentially significant, even though Cryptococcus may also occur in respiratory secretions as a saprophyte.

• Basically, all patients with a positive microscopy for cryptococci, from any site should be investigated for disseminated disease, especially by culture and antigen detection

India ink preparation of CSF showing a typical yeast cell of C. neoformans surrounded by a characteristic wide gelatinous capsule.

Tissue section showing from a skin biopsy showing typical yeast cells of C. neoformans.

Tissue section of lung showing showing atypical non-encapsulated yeast cells of C. neoformans.

3. Culture: Inoculate specimens onto primary

isolation media, like Sabouraud's dextrose agar. Look for translucent, smooth gelatinous colonies, later becoming very mucoid and cream in color.

•When nonsterile specimens are to be cultured, media containing cycloheximide should not be used because the organism is susceptible to this agent.

•Phenol oxidase leads to the formation of melanin, which can be demonstrated by the acquisition of a brown to black pigment when C. neoformans is grown on Staib’s birdseed agar or caffeic acid medium

Bird seed agar plate showing the typical brown colour effect seen with C. neoformans.

Mucoid colonies of C. neoformans.

4. Serology: • The detection of cryptococcal

capsular polysaccharide antigen in spinal fluid is now the method of choice for diagnosing patients with cryptococcal meningitis.

• In AIDS patients, cryptococcal antigen can be detected in the serum in nearly 100% of cases.

•However, in non-AIDS patients antigen detection in serum is less sensitive with only about 60% of patients with cryptococcosis reported as being positive.

5. Identification:• The genus Cryptococcus is

characterized by globose to elongate yeast-like cells or blastoconidia that reproduce by multilateral budding. Pseudohyphae are absent or rudimentary.

• On solid media the cultures are generally mucoid or slimy in appearance. Red, orange or yellow carotenoid pigments may be produced, but young colonies of most species are usually non-pigmented, and are cream in color.

•Most strains have encapsulated cells with the extent of capsule formation depending on the medium. Under certain conditions of growth the capsule may contain starch-like compounds which are released into the medium by many strains.

•Within the genus Cryptococcus, fermentation of sugars is negative, assimilation of nitrate is variable and assimilation of inositol is positive.