Structure,Pathogenesis, Lab Diagnosis of Cryptococcus species

Cryptococcus species

It is a ubiquitous saprophyte that is found wide in nature and grows abundantly in soil containing birds dropping (especially pigeon`s dropping) or eucalyptus trees and decaying woodThe birds are not infected .Human infection results from inhalation of the organism.

• A characteristic polysaccharide capsule of C. species is of variable thickness (1-30µm) surrounds these yeasts.
• the capsule is thinner and the yeast smaller, while thicker capsules tend to be found from infected tissues.
• The capsules stain pink by mucicarmine technique.

Habitat

• Cryptococcus is encapsulated yeast that is found in soil contaminated with pigeon droppings .
• Cryptococcus neoformans grows easily in pigeon feces, however, the birds are not naturally infected

Speciation

• The genus Cryptococcus includes around 37 species.
• Among these, Cryptococcus neoformans is the only species that is pathogenic to humans.
• Other species are as follows:

1. Cryptococcus albidus,
2. Cryptococcus laurentii,
3. Cryptococcus terreus,
4. Cryptococcus uniguttulatus
5. , Cryptococcus luteolus
6. Cryptococcus gastricus.

• It has four serotypes A,B,C,D.
• Serotyping is based on capsular agglutination reactions.
• It includes three varieties; var. gattii, var. neoformans and var. grubii.
• Cryptococcus neoformans serotype A is responsible for the majority of human cases.

Morphology

It is oval or spherical yeast. The size of a cell varies and may range 2-10um in diameter. It possesses a prominent polysaccharide capsule. It reproduces by budding but unlike Candida albicans it doesn’t produce pseudohyphae like others yeast cells, it is also gm positive, however, a capsule may interfere with staining of this fungus.

Encapsulated pathogenic yeast fungus (Cryptococcus neoformans). Cryptococcus is a yeast-like fungus that reproduces by budding. An acidic mucopolysaccharide capsule completely encloses the fungus. It can cause the disease cryptococcosis; especially in an immunodeficient patient, such as patients with HIV or AIDS. The infection can cause meningitis in the lungs, skin or other body regions. The most common clinical form is meningoencephalitis. It is caused by inhalation of fungus found in soil that has been contaminated by pigeon droppings.

Virulence factors and pathogenesis

1. Capsule

Cryptococcus neoformanis the only pathogen known to have a polysaccharide capsule and the capsule is the major virulence factors . The capsule protects the yeast from desiccation under drying condition. The cell size reduction resulting from the capsule collapse place the yeast in the ideal range for alveolar deposition.

In addition, the polysaccharide capsule contains compounds that are not recognized by phagocytes and hence escape phagocytic activity.

2. Phenol oxidase enzyme

Phenol oxidase enzyme found inC.neoformansis responsible for melanin production . Melanin production can decrease lymphocyte proliferation and TNF production, making the organisms resistant to lymphocyte attack.

Disease caused byCryptococcus neoformans

Diseases caused byC.neoformansis commonly called cryptococcosis . It usually affects the lungs,brain, and meninges and occasionally other parts of the body .C. neoformansinfections cause an estimated 1 million cases of cryptococcal meningitis per year among people with HIV/AIDS. C. gattii infections are more common in tropical and subtropical regions.

Cryptococcosis is a fungal infection caused by numerous species, two of which cause the majority of cryptococcal infections in humans and animals they areCryptococcus neoformansandCryptococcus gattii.Both species can be found in soil throughout the world and cause infection once they are inhaled, particularly in people who have weak immune systems.

1. Lung infection

Pulmonary diseases or infection of lungs is common inC.neoformansinfection lung infection is often asymptomatic or may produces pneumonia . Diseases occur mainly in patients with reduced CMI, especially AIDS patients or patients associated with diseases of the reticuloendothelial system (RES).

2. Cryptococcal meningitis

In a small portion of the population , hematogenous spread of the organisms from lungs to meninges and brain tissues may result in cryptococcal meningitis it is or meningo-encephalitis.

3. Skin and other infection

Though the organism mainly affects lung tissues , it may sometimes cause infection of skin , lymph nodes and bone.

Clinical types

• Cryptococcosis commonly starts following inhalation of the organism
• Cryptococcosis is considered opportunistic infections as it affects mainly immunosuppress individuals
• The polysaccharide capsule, the phenoloxidase enzyme, the organism's ability to grow at 37°C and phospholipase enzymes are its major virulence factors. It produces urease. The clinical types of cryptococcosis are pulmonary, CNS, visceral, osseous, cutaneous and mucocutaneous. The course of the infection is usually subacute or chronic. In healthy individuals, the disease is primarily pulmonary and is asymptomatic. CNS cryptococcosis such as meningitis and brain abscess are commonly seen in acute settings. Cutaneous and mucocutaneous cryptococcosis is the result of a disseminated disease. In osseous cryptococcosis, cranial bones and vertebrae are commonly affected. Visceral cryptococcosis follows dissemination of fungus to any organ or tissue of the body, most commonly heart, testis, prostate, and eye. Patients with AIDS, carcinoma, leukemia, collagen vascular disease, Hodgkin’s disease, sarcoidosis and those undergoing immunosuppressive therapy are at increased risk of cryptococcosis. The occurrence of disseminated forms of cryptococcal diseases is clearly related to immune deficiencies of the T-cells. Before the AIDS epidemic, most cases of cryptococcosis were related to cancer, especially lymphoreticular malignancies

Lab diagnosis

Specimen:blood , CSF, Sputum, biopsy materials etc

• The specimens collected are sputum, skin biopsy, CSF etc.
• In most clinical materials, such as CSF and pulmonary tissue mounted in 10% KOH.
• A capsule may or may not be present.
• The capsule is best visible in India ink preparations.
• Aspirates and tissues are inoculated onto Sabouraud dextrose agar
• . Colonies of C.neoformans are fast growing, soft, glistening, smooth, usually mucoid, and cream to yellowish brown in color.
• It usually takes 48 to 72 hours.
• It grows well at 25°C as well as 37°C.
• Ability to grow at 37°C is one of the features that differentiates C.neoformans from other Cryptococcus spp.
• Detection of cryptococcal antigens, particularly polysaccharide capsular antigen by latex agglutination is helpful in diagnosis.

1. Direct microscopy (India ink preparation)

In spinal fluid mixed with India ink, the yeast cell is seen microscopically surrounded by a wide unstained capsule .

2.Culture

C.neoformansis easily cultured on routine fungal culture media . It may grow on bacterial culture media like BA. On BA or sabouraud`s agar ,C.neoformansproduces moist creamy white colored mucoid colonies usually after 2-3 days of incubation .When examined microscopically , the yeast cell appears capsulated but the capsules are often smaller than seen in specimens and occasionally capsule may be absent.

3. Urease test

The rapid urease test is a most useful tool for screening of urease producing yeast recovered from respiratory secretions and other clinical specimensCryptococcus neoformans isurease positive.

Besides, rapid urease test, nitrate reduction test and detection of phenol –oxidase production are also useful in identifyingC.neoformans.SomeC.neoformansmay not produce phenol oxidase enzyme.

Prevention

There is no vaccine against cryptococcal infection, in fact, there are no any specific means of infection.

Treatment

Combined treatment with Amphotericin B and flucytosine is generally needed in case of meningitis and other disseminated diseases. Fluconazole is used in AIDS patient for long-term suppression of cryptococcal meningitis .

REFERENCE

Cheesbrough, M. Medical Laboratory Manual for Tropical Countries. Vol. Vol 2. ELBS London, 2007.

Tille, P. Diagnostic Microbiology. 13th. Elsevier, 2014.