Cryptococcus neoformans complex meningitis
Introduction to Cryptococcus infections Cryptococcus neoformans is a yeast-like saprophytic fungus that can cause severe infections, often in individuals with impaired cell-mediated immunity (e.g. AIDS) or other underlying medical conditions, such as end stage organ disease, which was present in the current case.1,2 The overall incidence of infection in the United States is about 1 per 100,000 with about a 12% mortality rate.3 The incidence in those with AIDS is significantly greater than the general population. Between 0.2 to 2.9% of those with AIDS have detectable levels of C. neoformans complex.3 Primary C. neoformans complex infections often occur in the lungs, and dissemination to the central nervous system (CNS) is possible. Infections can be indolent, and signs and symptoms can include headache and cranial nerve palsies, as was present in the current case. Exposure is classically associated with aerosolization of droppings from birds (e.g. pigeons) or from bats (e.g. in caves).
Identification of Cryptococcus neoformans complex by the clinical laboratory
The Cryptococcus fungal genus is comprised of multiple species. The most commonly identified human pathogens in the genus are C. neoformans and C. gattii, which appear similar when cultured in the laboratory and can be clinically reported as a single taxonomic complex (i.e. Cryptococcus neoformans complex).
C. gattii is classically associated with infections in the immune competent host, whereas the C. neoformans species is classically associated with infections of the immunocompromised host. The taxa within the complex include C. neoformans var. neoformans, C. neoformans var. grubii, and C. gattii.4,5 The complex can be further delineated into eight major molecular types.6
There are several means by which the laboratory can help to diagnose a patient with a Cryptococcus neoformans complex infection: direct observation of C. neoformans in a patient sample, direct detection of C. neoformans nucleic acid in a patient sample, direct immunologic detection of C. neoformans in a patient sample, or culture and subsequent characterization of C. neoformans by phenotypic, genetic, or proteomic identification.
Identification by visual examination of the patient sample
In this case, a presumptive identification of C. neoformans was made directly from the patient's cerebrospinal fluid (CSF). Differentiating these organisms from other yeasts by Gram stain is very difficult (Figure 1). Classically, cryptococci in CSF can be identified with high specificity using an India ink preparation, which was done in this case (Figure 2). The India ink does not penetrate the cryptococcal carbohydrate capsule. This capsule is comprised primarily of glucuronoxylomannan, which is a polysaccharide of mannose, xylose, and glucuronic acid.7 The lack of India ink penetration of the capsule allows the yeast cells and their capsules to be highlighted in high contrast to the inky background. Typically, the size of the yeast cells is variable, but they are often 4-8 μm in diameter. Narrow-based budding can be present, as was seen in this case (Figure 2).
Identification by immunoassay of the patient sample
Immunoassays are commonly used to identify organisms in the C. neoformans complex in a patient's CSF or serum, and these tests have the advantage of being both rapid and reliable.8, 9 These tests do not test for patient antibodies, but these assays test for the presence of the cryptococcal glucuronoxylomannan antigen in the patient's body fluid. Clinically useful immunological tests include latex agglutination, enzyme immunoassay, and later flow assays.8 Cross-reactivity between Aspergillus, Trichosporon, and C. neoformans complex has been rarely reported in immunoassays, and can potentially cause false positive results.10-12 Capsule-deficient forms of C. neoformans complex have been reported to cause false-negative results.13 Titers can be performed with these immunoassays to estimate the infection load in the sample, and this titer can be followed over time to monitor response to therapy.8
Identification by nucleic acid detection directly from the patient sample
Methods have been developed to detect C. neoformans nucleic acid directly from patient samples by using PCR14,15 or FISH.16 These methods are not commonly used in the clinical laboratory, likely because of the accepted and established use of immunoassays. In the future, nucleic acid detection of C. neoformans may become more common if it is incorporated into a molecular testing panel that is designed to detect multiple causative agents of meningitis.17
Identification by analysis of cultured isolates
Cryptococci can also be cultured in the microbiology laboratory and subsequently identified using phenotypic, genetic, or proteomic approaches. Classic phenotypic findings of Cryptococcus spp. that help to differentiate them from other yeast organisms include the presence of a capsule (e.g. mucoid yeast colonies), assimilation of inositol, production of urease, and failure to ferment glucose (or any other carbohydrate).18
A unique feature of the C. neoformans complex that helps to differentiate it from other Cryptococcus spp. is the production of phenol oxidase, which can metabolize caffeic acid to melanin. Melanin is a virulence factor that protects the fungus from oxidation from white blood cells.19,20 One medium that contains caffeic acid is birdseed agar, so colonies of C. neoformans complex that are grown on birdseed agar develop a brown color.18,19 Alternatively, filter paper containing caffeic acid can be inoculated with a cryptococcal colony and observed for the development of brown pigment.
If desired, the C. neoformans complex can be further characterized to species using phenotypic methods. C. gattii can be differentiated from C. neoformans phenotypically by its ability to use glycine as a carbon source. Canavanine-glycine-bromothymol blue (CGB) agar can be used to detect this difference.21,22 If the organism (i.e. C. gattii) is inoculated onto CGB, it will oxidize the glycine in the medium, which results in ammonia production. The ammonia alkalinizes the CGB agar medium. This increase in pH causes the bromothymol blue in CGB to shift from yellow to blue in color, which is the indication that the organism (i.e. C. gattii) has oxidized of glycine. So, when C. neoformans var. neoformans is in grown on CGB agar, the agar remains yellow; but when C. gattii is grown on CGB agar, the agar turns blue.22
Further classification of a strain to one of the eight major molecular types is not routinely performed in clinical microbiology laboratories, but type identification may have some clinical benefit because it has the potential to influence the interpretation of antifungal susceptibility testing.23 It has recently been demonstrated that these eight types can be resolved using proteomic MALDI-TOF mass spectrometry analysis,24 although currently, organisms are usually only identified to the species or complex level when using MALDI-TOF mass spectrometry for organisms identification in the clinical laboratory.25
Individuals infected with C. neoformans complex require antifungal therapy, and they may require serial therapeutic lumbar punctures or a CSF drain to ameliorate elevated intracranial pressure.2 A common antifungal regimen includes the use of amphotericin B for induction therapy and fluconazole for consolidation and maintenance therapies.23,26
The C. neoformans complex organism was also detected by CSF culture and blood culture. Initial immunoassay results from CSF and serum identified the antigen at a titer ≥1:1024. The patient was started on antifungal therapies and intracranial pressure was managed. Four weeks later, the serum titer decreased to 1:512 and lumbar puncture opening pressure had decreased to within normal limits.
Contributed by Daniel D. Rhoads, MD; A. William Pasculle, ScD