August 2016 Case 2

A 30 year old male with headache

Nariman A. Nawar, MD. Rocco LaSala, MD.


A 30-year-old male patient with a known history of Chiari I malformation, HIV and amphetamine abuse presented to the outpatient clinic with a two month history of occipital headache. The headache was progressively worsening and associated with nausea, vomiting and anorexia.

Physical exam was negative for fever and focal neurological deficit. Brain MRI showed abnormal signals in the deep gray nuclei. The radiologist expressed concern for infectious encephalitis. Lumbar puncture was then performed.

Gross Description

Laboratory Findings - The cerebrospinal fluid cell count showed lymphocytosis and microscopic examination demonstrated the organism depicted in Figures 1 and 2.

Figure 1: Cerebrospinal fluid stained with Giemsa stain showing intracellular (A) and extracellular organisms (B).
Figure 2: Mucicarmine stain.
Figure 3: India ink showing spherical yeasts with surrounding clear halo. The halo is produced by capsular polysaccharide that displaces the ink.


The morphology of the organisms is most characteristic of:


Please select an answer above.


Cryptococcus neoformans is an ubiquitous, pathogenic and encapsulated yeast that lives in the environment.  Infection with C. neoformans is acquired through inhalation of soil or dusts contaminated with yeast cells. The incidence of cryptococcal infection is as high as 15% in Sub-Saharan Africa due largely to the high incidence of HIV infection in this region.1  While cellular immunodeficiency is a known predisposing risk factor for cryptococcosis, immunocompetent individuals may also sustain serious infection.

Virulence of C. neoformans is attributed primarily to its capsule. C. neoformans’ capsule consists of glucuronoxylomannan (GXM) polysaccharide. Synthesis of the capsule is mediated by several genes, of which CAP64 is required. 2 The capsule permits the organism to avoid phagocytosis by white blood cells.  Other virulence factors include melanin production and extracellular enzymes secretion. C. neoformans synthesizes melanin by converting 3,4 dihydroxyphenylalanine to dopaquinone, which later polymerizes to melanin. This conversion is catalyzed by a phenoloxidase enzyme. Melanin is postulated to protect the organism from phagocytosis through an anti-opsonization effect. 2-3 Extracellular enzymes, including proteases and phospholipases contribute to tissue destruction by breaking down lipids and proteins while superoxide dismutase protects the organism from oxygen free radical damage. 3-4

Four main types of cryptococcal infections have been most commonly described: meningitis, pneumonia, cutaneous disease and disseminated cryptococcosis. Rare cases linking C. neoformans to urinary tract infection, prostatitis and tenosynovitis have also been reported. 1

As mentioned, cryptococcal meningitis frequently occurs in patients with HIV infection or with other forms of cellular immunodeficiency. Infection typically presents with headache, fever and signs of meningism. Cerebrospinal fluid analysis typically demonstrates lymphocytosis, low or normal glucose level and high protein content.  Elevated opening pressure on lumbar puncture is another common finding, yet these signs are not specific for Cryptococcus infection and may be observed in other types of fungal meningitis. 1

Culture is perhaps the easiest and most definitive means of laboratory confirmation of cryptococcosis. On blood agar, C. neoformans classically forms white, mucoid colonies. The mucoid appearance derives from production of its GXM polysaccharide capsule. In birdseed agar, yeast colonies demonstrate a brown to red appearance due to the production of caffeic acid, a characteristic that delineates C. neoformans from most other Cryptococcus species.1  Microscopic features of the organism are those of budding spherical yeast-like cells, which span a very wide size range (approximately 2 - 20 µm) – a feature not generally observed with other pathogenic yeasts.

In cases where the identification of C. neoformans capsule in body fluid is not visualized by routine Giemsa stain, use of India ink stain may be helpful. A slide stained with India ink demonstrates a pitch black back ground in which the organism will appear as a light bulb in a dark room owing to displacement of the ink by the thick capsule (Figure 3). While the sensitivity of India ink is not high, its ease and rapidity still lends it some utility.   Mucicarmine staining, which should highlight the capsular material in a bright pink color, may also be used for the same purpose in tissue sections or in fluid prepared for cytology examination.

Immunoassays are available for direct detection of cryptococcal capsular polysaccharide antigen from CSF or serum matrices.  Available formats include latex agglutination, ELISA and immunochromatography. This method employs anticryptococcal antibodies with specificity for capsular antigen.  Serum or cerebrospinal fluid containing cryptococcal antigen will react; and the sensitivity (up to 90%) is much better than India ink.5-6

Although cryptococcosis has traditionally been associated with C. neoformans, the infection has also been recognized more recently in connection with a different etiology, specifically C. gattii. First identified in 1999, more than 96 cases of cryptococcosis due to C. gattii were reported to CDC between 2004 and 2011. 7-8 C. gattii shares morphological and phenotypic features with C. neoformans and distinguishing between these two species may be difficult.  A canavanine, glycine and bromothymol blue (CGB) medium has been proposed as a useful tool to differentiate them.  Using this agar, C. gattii (but not C. neoformans) will convert substrate to ammonia, producing alkaline pH and a shift in medium color from yellow to cobalt blue. 9

In summary, Cryptococcus neoformans is a pathogenic yeast that is known for its CNS tropism. The morphological hallmark of C. neoformans is the thick polysaccharide capsule. Rapid diagnosis of cryptococcal infection can be made by immunoassay or direct microscopic examination. Cryptococcus gattii is a related species that may also produce disease.


We like to thank Dr. Melina Flanagan and Dr. Patrick Bacaj for their educational support in this case. 


  1. Winn WC, Koneman EW. Koneman's color atlas and textbook of diagnostic microbiology. Philadelphia: Lippincott Williams & Wilkins; 2006. Accessed July 1, 2016.
  2. Chang YC, Penoyer LA, Kwon-Chung KJ The second capsule gene of cryptococcus neoformans, CAP64, is essential for virulence.Infect Immun. 1996;64(6):1977-83.
  3. Buchanan KL, Murphy JW. What makes cryptococcus neoformans a pathogen? Emerging infectious diseases. 1998;4(1):71-83.
  4. Mayo Medical Laboratories. Hot topic - A guide to serologic testing for select fungi - mayo medical laboratories.
  5. Center for Disease Control and Prevention. Cryptococcal screening program training modules for laboratory workers. site. Accessed July 1, 2016.
  6. Dominic RS, Prashanth HV, Shenoy S, Baliga S. Diagnostic value of latex agglutination in cryptococcal meningitis. J Lab Physicians. 2009;1(2):67-68. Accessed July 1, 2016. doi: 10.4103/0974-2727.59702.
  7. Centers for Disease Control and Prevention. C. gattii infection statistics | fungal disease | CDC.
  8. Center for Disease Control and Prevention. Definition of C. gattii infections | fungal disease | CDC.
  9. K. R. Klein, L. Hall, S. M. Deml, J. M. Rysavy, S. L. Wohlfiel, N. L. Wengenack. Identification of cryptococcus gattii by use of l-canavanine glycine bromothymol blue medium and DNA sequencing. Journal of Clinical Microbiology. 2009;47(11):3669-3672. doi: 10.1128/JCM.01072-09.