Cryptococcus gattii expands its territory

Case study

A 44-year-old male presented to a hospital in Georgia complaining of nausea, vomiting, and headache with onset two days before admission. The patient denied any vision changes, numbness or tingling, or seizure activity. Prior medical history was significant for hypertension, hyperlipidemia, and pulmonary sarcoidosis. Initial laboratory results revealed a slightly elevated white blood cell count with neutrophilia. A lumbar puncture produced clear and colorless CSF containing 77% macrophages. Admission laboratory results are summarized in Table 1. Microbiology specimen testing included CSF, blood, sputum, bronchial lavage, lung biopsy, lymph node biopsy, and urine cultures. Direct microscopic examination of the gram stained CSF revealed no WBCs but many yeasts. The cryptococcal antigen titer was positive at 1:2048. Blood cultures were positive for the growth of yeast and subcultured onto chocolate, sheep blood, brain heart infusion, and inhibitory mold agar. The CSF cultures also produced a heavy growth of yeast. A yeast identification kit identified the pathogen as C. neoformans. An India ink preparation contained encapsulated yeasts, but the microbiologist noted the yeasts looked slightly more oval and larger as compared to the positive C. neoformans control and suspected the pathogen was C. gattii. The specimen was sent to a reference laboratory and identified as C. gattii. Fungal cultures of lung biopsy, lymph node, sputum, and bronchial lavage specimens were negative. Urine and acid fast bacilli cultures were also negative. The patient was placed on an initial therapy of high-dose amphotericin B and flucytosine, followed with maintenance therapy using fluconazole. Four weeks after admission, the patient was discharged from the hospital.

Apathogenic cryptococcal species is now on the radar of the Centers for Disease Control and Prevention (CDC). Cryptococcus gattii, formerly known as Cryptococcus neoformans var gattii, is being investigated because of its quick emergence, expanding ecological niche in the United States, and increased virulence. C. gattii is indistinguishable from C. neoformans by most routine microbiological testing but is differentiated by growth requirements and molecular analysis. This distinction is significant as it has implications for clinical management and treatment.

C. neoformans and C. gattii

Cryptococcus is a yeast-like fungus. The genus contains two pathogenic species responsible for most human cryptococcal infections, C. neoformans and C. gattii. While C. neoformans has a worldwide distribution and is often found in the soil, C. gattii has historically been limited to tropical and subtropical regions. Recently this niche has expanded to include the United States. Scientists have correlated the distribution of C. gattii with different species of eucalyptus trees that are native to Australia. The export of eucalyptus species from Australia may have contributed to the rapid spread of C. gattii to the West Coast of the United States.¹

C. gattii is an encapsulated yeast of the phylum basidiomycota. It is germ tube test negative, hydrolyzes urea, produces melanin, and does not grow on cycloheximide media. C. gattii shares many characteristics with C. neoformans, making differentiation using commercially available kits difficult. The organisms are distinguished by microscopic observations, growth media, amino acid utilization, serotyping, and genotyping. 

In cryptococcal meningitis the cerebrospinal fluid examinations show an increased white blood cell count with a predominance of lymphocytes or macrophages. An India ink preparation demonstrates the larger, more oval capsules associated with C. gattii. On routine fungal media, this yeast produces a smooth, cream-colored colony that does not have psuedohyphae and grows to maturity at 37°C in about three days. The medium of choice for differentiation is canavanine-glycine-bromothymol blue agar. C. gattii grows in the presence of L-canavanine and utilizes glycine as its sole carbon source, resulting in an agar color change from yellow to blue. C. neoformans will not grow in the presence of L-canavanine, and the agar will remain yellow.² A recent report suggests that utilization of the amino acids D-alanine, L-phenylalanine and L-tryptophan can also distinguish C. gattii from C. neoformans.³

Cryptococcal polysaccharide capsular antigens in cerebrospinal fluid (CSF) may be detected using latex agglutination kits or enzyme immunoassay. These antigens are also used to subclassify cryptococci into serotypes. C. neoformans produces serotypes A and D while C. gattii exhibits serotypes B and C.² C. gattii isolates from most parts of the world have been identified as serotype B, whereas serotype C is less common.⁴ A variety of molecular methods have been employed to genotype cryptococcal species. From these techniques, eight genotypes have been identified. C. neoformans comprises four genotypes, VNI–VNIV, and C. gattii contains genotypes VGI–VGIV. Genotype VGII is further subtyped into VGIIa, VGIIb, and VGIIc.^1,5 

Disease overview

Cryptococcosis is a life-threatening fungal infection. When fungal spores are inhaled, they are deposited into the pulmonary alveoli. The cryptococcal polysaccharide capsule allows C. gattii to evade the host immune response through inhibition of phagocytosis. Pulmonary infection may be followed by meningitis, which is often the first indication of infection.⁶ 

Individuals with AIDS are more susceptible to infection due to their immunocompromised state, and C. neoformans is a common fungal infection in these patients. However, studies reveal that this is not the case for C. gattii. While C. gattii is capable of infecting patients with HIV, the species has been found more often than C. neoformans in otherwise healthy individuals. However, patients with histories of lung conditions such as sarcoidosis, chronic obstructive pulmonary disease, and emphysema, along with invasive cancers such as leukemias and lymphomas, are more susceptible to infection with C. gattii as compared to the general population.⁷

In the United States, C. gattii serotype B, molecular subtype VGIIa, is largely responsible for clinical disease. The VGIIa subtype was responsible for outbreaks in Canada, which spread into the Pacific Northwest of the United States.⁴ A summary from the CDC indicates that from 2004 to 2010, 60 cases of C. gattii infection were identified: 43 cases from Oregon, 15 from Washington, one from California, and one from Idaho.⁸ Slightly more than half of these patients were immunocompromised. Ninety-two percent of the isolates were VGII, 5% were VGI, and 3% were VGIII.⁸ In 2007, the first case of C. gattii in North Carolina was reported, indicating an expansion of the pathogen’s ecological niche. The North Carolina isolate was molecular type VGI, and it was identical to the isolates found in California and Australia.⁵

Symptoms associated with C. gattii infection include fever, cough, fatigue, headaches, and shortness of breath. Fungal dissemination to the bloodstream and central nervous system can result in meningoencephalitis, meningitis, and formation of cryptococcomas (localized, solid, tumor-like masses) in the brain, skin, lungs, and other organs. Cryptococcomas are more often associated with infections due to C. gattii than C. neoformans.⁴

Antifungal therapies for C. gattii are similar to those recommended for C. neoformans: an initial two-to-four-week therapy of amphotericin B and flucytosine followed by fluconazole or itraconazole for two to 12 months.⁹ 

Conclusion

Cryptococcosis is most often due to infection with C. neoformans. However, C. gattii is emerging in the United States as an important fungal pathogen of significant virulence, capable of infecting both immunocompetent and immunocompromised hosts. Identification is important as there may be distinctions in clinical features, antifungal susceptibilities, and patient management. Differentiation between the two species using conventional laboratory methods is difficult; therefore laboratories should consider implementation of testing procedures such as growth on CGB media, amino acid utilization, and molecular typing.