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Introduction of Cryptococcosis

Cryptococcosis is an acute, subacute, or chronic pulmonary meningeal mycosis caused by Cryptococcus neoformans. It is a soil saprophyte and is particularly abundant in the feces of pigeons( pigeon’s dropping). It does not appear to infect birds, probably because of their high body temperature and infection throughout the world.

Classification of Cryptococcus

• Kingdom: Fung
• Phylum: Basidiomycota
• Class: Tremellomycetes
• Order: Tremellales
• Family: Tremellaceae
• Genus: Cryptococcus
• Species: C. neoformans

Other species are-

• C. albidus ( nitrate positive)
• C. laurentii ( melibiose fermentation)
• C. gattii ( Trehalose positive)
• C. neoformans: All are negative
• C. neoformans var. grubii ( serptype A)- world-wide distribution
• C. neoformans var. neoformans  (serotype D)-Restricted distribution and prevalent in France, Italy, and Denmark.

Morphology of Cryptococcus

It is a yeast characterized by wide polysaccharide capsule and budding, found both in culture and tissue fluid. It is a true yeast and is Gram-positive. The capsule may be identified by India ink or nigrosin preparation.

Size: 5-15 µm

Antigenicity

Based on carbohydrate antigen, there are 4 serotypes e.g. A, B, C, and D. Infection due to serotypes A and B are common.

Virulence markers

1. Polysaccharide capsule
2. Phenol oxidase enzyme
3. Growth rate at 37°C

Source of infection

Infection is usually acquired by inhalation of dust-containing yeast cells.

Pathogenesis

The disease is usually seen in immunosuppressed hosts and most infections are asymptomatic. Pulmonary cryptococcosis may lead to mild pneumonitis. Cryptococcal meningitis happens by hematogenous spread and is often seen in AIDS patients. Skin, lymph nodes, bones, and other organs may be involved when the dissemination of infection takes place. Cutaneous cryptococcosis varies from small to ulcers to large granuloma. Visceral forms of cryptococcal infection stimulate tuberculosis and cancer clinically.

Laboratory Diagnosis 

Specimens:

• CSF
• Sputum
• pus
• Brain tissue

Direct Microscopy

India ink preparation

positive due to having capsule appearing as a clear halo around the yeast cells as shown above figure. Gram stain: Gram-positive yeast cells as shown above figure. The histopathological examination of tissue can be done by staining with H/E, PAS, and mucicarmine stains.

Culture

Growth on Sabouraud  Dextrose Agar (SDA) shows smooth, mucoid, and cream-colored colonies as shown above figure.

Lactophenol cotton blue mount shows budding yeast cells as shown below.

Birdseed agar (BSA): It is a selective medium for Cryptococcus. C. neoformans produces brown colonies within a week at 30°C, such property is not shown by other yeasts. Cryptococcus neoformans produces phenoloxidase, which oxidizes the caffeic acid in the niger seed(resemble sunflower seeds in shape but are smaller in size and black that contains proteins, oil, and soluble sugars, botanical name-Guizotia abyssinica) into melanin.

i.e.   Phenol oxidase

                         ↓

Caffeic acid…………Brown pigment

Latex agglutnation test

Capsular polysaccharide antigen may be detected in CSF, serum, or even in urine by this latex agglutination test.

Animal Inoculation test

C. neoformans inoculation into mice through intracerebral or intraperitoneal routes creates a fatal infection. Encapsulated budding yeast cells can be demonstrated in the brain of the infected mice.

Urea Hydrlolization Test

It shows urea hydrolyzation test positive.

Sugar fermentation and assimilation test

They do not ferment carbohydrates.

Molecular Test

Detection of C.  neoformans DNA in tissue samples by Nested and Real-Time PCR Assays

Treatment of Cryptococcosis

• Amphotericin B
• Flucytosine
• Ketoconazole

Prevention and Control

1. People having Immunocompromized or Immunosuppressed status should avoid contact with birds and avoid digging and dusty activities in areas heavily contaminated with bird droppings.
2. Avoid the area where availability of dried pigeon feces.
3. Wear masks to prevent the inhalation of C. neoformans.

Keynotes on Cryptococcus

1. All fungi are gram-positive.
2. For Cryptococcus spp. identification other physiological tests like sugar  (glucose, galactose, sucrose, maltose, lactose, trehalose) fermentation and sugar assimilation tests are used.
3. For the rapid formation of melanin by C. neoformans , it is important that glucose constitute no more than 0.1% of the medium since the phenoloxidase of this organism is repressed by glucose.

Further Readings

1. Medical Mycology. Editors:  Emmons and Binford, 2nd ed 1970, Publisher Lea and Febiger, Philadelphia.
2. Rippon’s JW: Medical Microbiology. The pathogenic fungi and the Pathogenic Actinomycetes. 3^rd ed 1988 Publisher WB Saunder co, Philadelphia.
3. Clinical Microbiology Procedure Handbook, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
4. A Text-Book of Medical Mycology. Editor: Jagdish Chander.  Publication Mehata, India.
5.  Practical Laboratory Mycology. Editors: Koneman E.W. and G.D. Roberts, 3rd ed 1985, Publisher Williams and Wilkins, Baltimore.
6. Topley & Wilsons Medical Mycology. Editors: M.T. Parker & L.H. Collier, 8th ed 1990, Publisher Edward Arnold publication, London.
7. Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
8. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
9. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
10. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC119960