A 54-year old habitual pigeon breeder presented himself to the hospital with complaints of headache, stiffness of neck, nausea, vomiting, blurred vision, and confusion. He was not febrile. His companion described depression, agitation, and other behavioral changes in him. It was revealed that he was HIV seropositive since last few weeks.

What is your diagnosis?
This could be a case of subacute meningitis.

What is the specimen collected?
Approximately 3-5 ml of CSF is collected following lumbar puncture. Blood may also be collected for culture.

Which are the necessary investigations to be performed?
The CSF sample is divided into three; one part for cell type and cell count, second for protein and glucose analysis and the third part of microbiological examinations. If the CSF is not turbid, it should be centrifuged. Microbiological examinations include a Gram stained smear, wet India ink mount, bacterial and fungal culture and antigen detection. CSF was inoculated on to Blood agar and Sabouraud's dextrose agar or Birdseed agar and incubated at 37^oC for 1-2 days. Latex agglutination test to detect cryptococcal polysaccharide in CSF may also be performed.

What is your observation?
Gram stained smear of CSF sediment did not reveal any polymorphonucelar leucocytes, however Gram positive budding spherical yeast cells were seen. India ink wet mount was positive for spherical yeast cells with large capsules. Large, cream-coloured, mucoid colonies were obtained on Sabouraud's dextrose agar whereas brown colonies were seen on birdseed agar(e.g., Staib medium). Production of phenoloxidase by Cryptococcus neoformans in caffeic acid containing medium results in production of melanin pigments, which is incorporated into yeast wall resulting in brown colonies. Both the Gram stained smear and India ink wet mount of the colonies revealed yeast cells. The fungus is identified by positive urease test, negative nitrate reduction and various sugar assimilation tests as Cryptococcus neoformans.

Which are the various predisposing factors for this condition?
In healthy individuals, cryptococcosis is often asymptomatic. Resistance to cryptococcosis depends primarily on cell-mediated immunity. Most cases of cryptococcal meningitis occur in patients with conditions that weaken this system, such as acquired immunodeficiency syndrome (AIDS), reticuloendothelial malignancies, organ transplantation, or corticosteroid therapy and patients with sarcoidosis.

What is the pathogenesis of this condition?
Of the several species of Cryptococcus, C. neoformans is the common pathogen. The initial infection is acquired by inhalation of fungal cells from an environmental source. The major environmental sources of C. neoformans are either soil contaminated with pigeon guano (C. neoformans var. neoformans and var.grubii) or eucalyptus trees and decaying wood (C. gattii). In moist or desiccated pigeon excreta, C neoformans may remain viable for 2 years or longer. Cryptococcus can colonize the host respiratory tract without producing any disease. Infection is typically asymptomatic, and it can be either cleared or enter a dormant, latent form. Unencapsulated yeast are readily phagocytosed and destroyed, whereas encapsulated organisms are more resistant to phagocytosis. When host immunity is compromised, the dormant form can be reactivated and disseminate hematogenously to cause systemic infection. Why C. neoformans has a predilection for the CNS is still not resolved. Cryptococcal meningoencephalitis develops following hematogenous dissemination of C. neoformans from the lungs to the brain.

Which are the various varieties and serotypes of C. neoformans? (not for UGs)
Based on capsular agglutination reactions, there are five serotypes: A, B, C, D, and AD hybrid (hybrids between serotypes A and D). On the basis of biochemical tests, such as the ability to use glycine as the sole carbon and nitrogen source, resistance to canavanine, EDTA resistant urease, and the morphology of the sexual state, C. neoformans was originally contained two varieties: var. neoformans (serotypes A, D, and the AD hybrid) and var. gattii (serotypes B and C). More recently, C. neoformans var. gattii has been recognized to be a separate species, Cryptococcus gattii. Molecular studies and genome sequences have detected significant genetic variations between serotypes A and D, and recently serotype A has been distinguished as a new variety, var. grubii. Currently, this organism is classified into two varieties and a sibling species: C. neoformans var. neoformans (D), C. neoformans var. grubii (A), and C. gattii (B, C). Creatinine dextrose bromothymol blue thymine (CDBT) agar is the medium of choice for the differentiation of Cryptococcus neoformans var. neoformans and Cryptococcus neoformans var. grubii. Cryptococcus neoformans var. neoformans grows as bright red colonies, turning the medium a bright orange after 5 days. Canavanine-glycine-bromothymol blue (CGB) medium is used to identify Cryptococcus isolates. When inoculated in this medium and incubated at room temperature for 5 days C. neoformans var neoformans forms yellow coloured colonies whereas C. neoformans var gattii forms dark blue colonies. The perfect (ie, sexual, teleomorphic) form of C neoformans, is named Filobasidiella neoformans. F. neoformans var neoformans results from the mating of suitable strains of serotypes A and D. The perfect state of C neoformans var gattii is Filobasidiella bacillisporus and results from the mating of serotypes B and C.

How do you treat this condition?
Cryptococcus meningitis is invariably fatal without appropriate therapy; death may occur from 2 weeks to several years after symptom onset. Antifungal drugs such as amphotericin B, flucytosine, fluconazole must be promptly given. For cryptococcal infections in patients with concomitant HIV infection without a CD4 count of greater than 100 cells/μL, the therapeutic goal is to control the acute infection, followed by life-long suppression of C neoformans. For patients infected with HIV with a CD4 count of greater than 100-200 CD4 cells/μL, suppressive therapy may be safe to discontinue as long as their CD4 counts do not fall below 100 CD4 cells/μL.

Which are the other infections/diseases caused by this fungus?
Following pulmonary infection, cryptococci disseminate widely and may infect any organ. The organs most often involved include the CNS, bones, prostate, eyes, and skin. Other infections include myocarditis, chorioretinitis, hepatitis, peritonitis, renal abscess, prostatitis, myositis, and adrenal involvement. Prostatic foci may persist after therapy for CNS disease and may act as a reservoir for relapse in men with AIDS.