First reported case of Cryptococcus gattii in the Southeastern USA: implications for travel-associated acquisition of an emerging pathogen

Abstract

In 2007, the first confirmed case of Cryptococcus gattii was reported in the state of North Carolina, USA. An otherwise healthy HIV negative male patient presented with a large upper thigh cryptococcoma in February, which was surgically removed and the patient was started on long-term high-dose fluconazole treatment. In May of 2007, the patient presented to the Duke University hospital emergency room with seizures. Magnetic resonance imaging revealed two large CNS lesions found to be cryptococcomas based on brain biopsy. Prior chest CT imaging had revealed small lung nodules indicating that C. gattii spores or desiccated yeast were likely inhaled into the lungs and dissemination occurred to both the leg and CNS. The patient's travel history included a visit throughout the San Francisco, CA region in September through October of 2006, consistent with acquisition during this time period. Cultures from both the leg and brain biopsies were subjected to analysis. Based on phenotypic and molecular methods, both isolates were C. gattii, VGI molecular type, and distinct from the Vancouver Island outbreak isolates. Based on multilocus sequence typing of coding and noncoding regions and virulence in a heterologous host model, the leg and brain isolates are identical, but the two differed in mating fertility. Two clinical isolates, one from a transplant recipient in San Francisco and the other from Australia, were identical to the North Carolina clinical isolate at all markers tested. Closely related isolates that differ at only one or a few noncoding markers are present in the Australian environment. Taken together, these findings support a model in which C. gattii VGI was transferred from Australia to California, possibly though an association with its common host plant E. camaldulensis, and the patient was exposed in San Francisco and returned to present with disease in North Carolina.

Figure 1. Imaging from diagnosis through recovery depicting the clinical course of C. gattii infection.

A) MRI imaging of the upper thigh cryptococcoma. B) CT imaging of a pulmonary nodule, likely to be a cryptococcal granuloma. C–D) MRI imaging of brain cryptococcomas after seizure presentation at the emergency room. E–F) MRI imaging of brain cryptococcomas after long-term fluconazole treatment, with reduced mass.

Figure 2. Clinical isolates exhibit mating differences.

All mating cultures were incubated at room temperature in the dark, for 10 to 14 days in dry conditions using the mating type a tester isolate B4546 as a partner on Mirashige and Skoog Media. The brain biopsy isolate exhibits reduced fertility as evidenced by a marked delay and paucity in hyphal growth (A) whereas hyphal growth, basidia, and basidiospore formation indicative of sexual reproduction is evident in matings with the leg biopsy isolate (B). All mating experiments were repeated and representative images are shown here.

Figure 3. Molecular typing and phylogeny of the North Carolina clinical isolates with global isolates.

A) MLST reveals the leg and brain isolates are identical with each other, and also identical with a distinct genotype predominantly from Australia, and in a single clinical case from California, USA. B) Neighbor joining phylogenetic analysis based on sequences from seven MLST loci in panel A (SXI idiomorphs not included) illustrates discrimination between four molecular types (VGI-VGIV), and displays the relationship of the clinical case presented in this study to global genotypes observed, including all VGI genotypes thus far reported (including the Vancouver Island VGI genotypes), and the Vancouver Island/Pacific NW outbreak genotypes VGIIa/major, VGIIb/minor, and VGIIc. Note that not all sequence types and strains in (B) are represented in (A).

Figure 4. DNA sequence alignment of VNTR15 among 18 VGI isolates of C. gattii.

The noncoding VNTR marker divides the isolates with an identical MLST profile into two groups: one group contains two NC clinical isolates which are identical to one another, the other clinical isolates, and five of the ten Renmark isolates, and the second group includes the remaining five Renmark isolates. A 40 bp deletion was observed in the following isolates: E307, E554, E360, E296, and E278. In addition, a single nucleotide polymorphism (SNP) (66A → 66G) discriminated the VGI type strain isolate WM276 from the other 17 VGI isolates (see red circle).

Figure 5. Molecular typing reveals that the Australian environmental isolates are distinct from clinical isolates.

A) PCR product size differences at locus VNTR-MS1 reveals that environmental isolates from Renmark harbor a deletion compared to the clinical isolates with the same MLST genotype. Agarose gel electrophoresis of the VNTR-MS1 marker, illustrating an ∼100 bp difference between the larger (EJB1-L, EJB2-B, PAT12ISO1, B4496, WM276*), and smaller (E278, E549, E280, E306, E307, E569, E286, E296, E310, E554) PCR products. * Denotes that the sequenced type strain WM276 is not identical to the other clinical isolates. The bold circles represent four clinical isolates (from three cases) that type as identical through all genotypic tests conducted. B) A linear representation of the progressive genotypic analysis. As the number of markers increased, along with the genetic variability, the number of isolates typing as identical to the North Carolina clinical case decreased. At the end of the genotypic studies only two clinical isolates, previously identified from Australia and California, typed as identical with the present NC clinical case.

Figure 6. North Carolina clinical isolates are pathogenic in a heterologous host.

Groups of 12 to 19 larvae of Galleria mellonella were each infected with an infectious inoculum of 1.0×10⁵ cells of isolates EJB1-L, EJB2-B, WM276, and E296. Survival was monitored and plotted daily for 16 days. All isolates were significantly virulent (p<0.005) in comparison with the mock control (sterile PBS) infection. The experiment was replicated in duplicate with similar results in each replicate, and representative results are shown here.

Figure 7. Proposed model for the emergence of C. gattii in the United States.

The original source is postulated to be Australia, where identical clinical and closely related environmental isolates have been reported. Given that human-human transmission other than introgenic is unknown, the most parsimonious model for geographically dispersed clinical isolates is that isolates identical to the clinical cases are present in the environments in Australia and California, USA. In this model, the patient from the Southeastern United States traveled to the endemic area of CA, was exposed to the pathogen by inhalation, and ultimately returned to present with disseminated disease in North Carolina, USA.