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The Environmental Reporter
  Volume 10 | Issue 3

I hope you're doing well and enjoying the start of summer. I also hope that you'll find the following article about Cryptococcus gattii by Dr. Michelle Seidl both interesting and useful.

With best wishes,
Dave Gallup




Cryptococcus gattii
By Dr. Michelle Seidl, EMLab P&K Senior Analyst

Introduction & Overview
For just over a decade, a potentially lethal, airborne fungal infection has been slowly spreading through the Pacific Northwest (PNW) region of North America, following an outbreak in 1999 (Fyfe et al., 2008; MacDougall 2007). The culprit, known as Cryptococcus gattii1, affects humans and animals. This fungus, along with C. neoformans2, is one of two pathogenic Cryptococcus species responsible for the disease known as cryptococcosis or cryptococcal disease. The recent outbreak in the PNW was also the first incidence of cryptococcosis affecting wild and domestic animals as well as humans. Most reported cases of cryptococcosis are caused by C. neoformans, which is known to be one of the defining infections for many AIDS patients. Additional information on C. neoformans can be found elsewhere, including previous Environmental Reporter articles such as 4(2), 4(5) and 9(5).

Worldwide, C. gattii has been reported from many areas, known primarily from subtropical and tropical arid environments (Bartlett et al., 2008). Native to the tropics, it is thought to have originated in Australia (Ellis 1987), and is also known from other areas including European Mediterranean regions, the Netherlands and South America. The native habitat of this fungus appears to be Eucalyptus trees (Ellis & Pfeiffer 1990). It is not associated with birds or bird droppings.

In 1999, C. gattii infection emerged as an "epidemic" in the temperate rainforest climate of Vancouver Island, British Columbia (BC), Canada. The disease outbreak and expansion of the fungus in the PNW has been studied by a number of scientists during the past 12 years, including the BC Centre for Disease Control and Prevention, state laboratories and the Cryptococcus gattii Public Health Working Group (Center for Disease Control & Prevention 2010). Since 1999, C. gattii has been causing disease in humans and animals in BC, and Washington and Oregon in the USA. It is considered to be rare, yet the fungus has been implicated in over 200 cases, including 24 human deaths from severe brain and lung infections (Byrnes et al., 2009; Datta et al., 2009; Kidd et al., 2007a, 2007b).

Prior to 1999, C. gattii infections were relatively unknown from the PNW. In western North America, there was one report from Seattle, WA in 1971, and another from San Francisco, CA in 1990. It appears the fungus was uncommon in this region prior to 1999 (Perfect 2007). Also referred to as Cryptococcus non neoformans by many laboratories, a recent EPA report listed the reemergence of Cryptococcus gattii on Vancouver Island and its expansion into the PNW mainland, as an example of a serious and emerging outdoor environmental risk that may extend to the indoor environment through human carriage (Morey 2010).

In mild temperate climates, this fungus thrives in soil and grows on tree bark, particularly Douglas-fir (Pseudotsuga menziesii). According to Kidd et al. (2005 & 2007b), the fungus occurs in the top 15 cm of the soil, on trees, wood chips, mulch and other "natural reservoirs". As with many other fungi, such as Aspergillus fumigatus3, its spores are readily aerosolized by soil disturbances.

Geographic distribution of Pacific Northwest Cryptococcus gattii documented reports

Figure 1: Geographic distribution of Pacific Northwest Cryptococcus gattii documented reports (MacDougall et al., 2007).

Taxonomy, Morphology & Risk Factors
Cryptococcus gattii is a haploid, basidiomycete yeast. This parasitic fungus primarily occurs in a yeast phase, which is the asexual (or anamorphic) life cycle phase. This particular yeast is specifically known as an encapsulated yeast, meaning the yeast cell is completely enclosed, or encapsulated, by a mucopolysaccharide layer. The sexual (teleomorphic) life cycle phase was discovered by Kwong-Chung in 1975 as the parasitic, basidiomycete genus Filobasidiella, a member of the Tremellales group, broadly known as the jelly fungi. Recently, molecular studies confirmed that C. neoformans and C. gattii form a monophyletic lineage within the Tremellales (Findley et al., 2009). The history of nomenclature for Cryptococcus gattii is somewhat unstable. It was previously classified as a variety of C. neoformans and about a decade ago, gained species status (Kwon-Chung et al., 2002). Thus the two entities are now classified as separate species, Cryptococcus gattii and C. neoformans.

The basidiospores, as well as the yeast cells, are very small (<4 microns), making them ideal for inhalation and lung deposition. Once the basidiospores come into contact with a nutritious food source, they rapidly convert into the yeast phase (Perfect 2007). The yeast cells appear to be the main infectious particle that is inhaled into the mammalian host. The basidiomycete fruiting body has not been reported from nature, although genetic recombination has been demonstrated, indicating that it indeed exists, but remains elusive. Several environmental cues, such as low pH, appear to have an effect on the sexual cycle (Rodriguez-Carres et al., 2010).

Various factors can put humans and animals at risk for inhalation of spores and/or fungal fragments. These include disturbance of soil and vegetation caused by a variety of activities including hiking, digging, logging and construction. These activities can potentially increase aerial dispersal of infectious particles and direct contact with soil, tree cuttings and other vegetative debris. According to Washington State University plant pathologist Dr. Jack Rogers: "The fungus apparently lives on tree bark and other organic material and presents a hazard to humans and numerous animal species, especially perhaps those that live near a moist woodland or have hiked or camped in such" (Clark 2010). Thus, anyone spending time in the woods is potentially vulnerable, but due to its rarity, commonsense personal hygiene is the best protection.

Genetics
Within the past 12-13 years, C. gattii and its genetic variants expanded their range to include mainland BC, as well as Washington and Oregon. These outbreak strains were found to represent organisms with improved fitness and are thereby readily adapting to the "new" environment. The disease outbreak was caused predominantly by the C. gattii molecular type or strain known as VGII (Kidd et al., 2004 & 2005). A more highly virulent, genetically distinct strain of C. gattii recently emerged in Oregon (Figure 2). Although rare, this new strain, known as VGIIc, has been linked to 21 known cases in Oregon, 25% of which were fatal. This novel genotype has been far deadlier than strains from WA and BC. Despite its rarity, researchers report that VGIIc is especially alarming due to its virulence, and the fact it can infect humans as well as a variety of domestic and wild animal species such as dogs, cats, porpoises, alpacas, sheep and elk. Byrnes et al., (2010) studied how strains causing the disease are related to those on Vancouver Island and elsewhere globally. They also studied the highly virulent OR strain, and showed that VGIIc is also highly virulent in immune cell and whole animal experiments. Through intensive molecular epidemiology and population studies, VGIIc strain is known to be clonal and is hypothesized to have arisen relatively recently. Studies indicate that the global VGII population supports ongoing recombination (Byrnes et al. 2010; Fraser, et al. 2005). Kidd et al., (2007a) studied various populations and examined dispersal mechanisms in BC, in order to better predict where the disease may be headed. Two signs of emerging outbreaks are high clonality and the emergence of novel genotypes via recombination. These studies have been very informative in extending our understanding of how diseases emerge in new climates, how they adapt to different geographic regions, and ultimately how they cause disease.

Electron micrograph of Cryptococcus gattii, VGIIc

Figure 2: Electron micrograph of Cryptococcus gattii, VGIIc.
Image courtesy of Dr. Joseph Heitman, Duke Dept. of Molecular Genetics and Microbiology

The Disease
Cryptococcus gattii causes life-threatening disease in otherwise healthy hosts, and to a lesser extent in immunocompromised hosts, including organ transplant recipients and people with blood diseases. Cryptococcal disease is unusual for healthy individuals to acquire, particularly if secondary infections or conditions are not present. Individuals with C. gattii infection, generally do not have compromised immune systems and are not particularly associated with HIV infection. On the contrary, it is rare for individuals with Cryptococcus neoformans infection, to have fully functioning immune systems.

Exposure is by inhalation of spores or fungal cells wafting around in the air. Symptoms appear within many weeks to many months after exposure. The timeframe between exposure and onset of symptoms is estimated to be 2-11 months. Symptoms of cryptococcosis occur in the pulmonary and nervous systems of humans. In many cases, it is difficult to determine the precise location where the exposure originated. Although the disease mechanisms are unknown, symptoms include severe or prolonged cough, pneumonia-like illness, shortness of breath, sputum production, sinusitis, muscle soreness, blurred or double vision, seizures, headache, sharp chest pain, fever, weight loss, skin lesions, change in mental status and a general feeling of discomfort. Occasionally, the fungus is associated with soft tissue, lymph nodes, bone, and joint infections. Treatment is often possible with antifungal drugs. For serious cases, surgery may be necessary to remove tissue masses. More details on the disease can be accessed at various websites such as: CDC, WA state department of health and the OR state public health.

Differentiating between the 2 Cryptococcus species is key to a proper diagnosis. Cryptococcus neoformans has been studied more intensively, particularly since the onset of the AIDS epidemic in the 1980's. In contrast, C. gattii has been intensively studied only relatively recently, but the good news is that the disease is treatable, and as with many diseases, early diagnosis is critical. Medical treatment for C. gattii infection consists of prolonged intravenous therapy (for 6-8 weeks or longer) with anti-fungal drugs. In some cases, surgery may be necessary for removal of infected tissues. If cryptococcomas develop, more focused diagnostics using radiology is required, as well as additional follow-up examinations. If symptoms progress to the central nervous system and/or if the disease becomes disseminated, the treatment is the same for both species of Cryptococcus.

The highest incidence of C. gattii infection occurs in Papua New Guinea and northern Australia. Cases have also been reported from other regions, including Europe, India and Brazil (Byrnes et al., 2010). In western North America, the highest incidence has been in British Columbia, particularly Vancouver Island. The disease has expanded to mainland BC and the United States. Washington and Oregon states have a few hundred reports so far since the early 2000's, with no recent reports for California.

How a formerly restricted pathogen gets dispersed into new geographic locations is important to study for preventive purposes as well as epidemiologically. Dispersal of disease organisms can be caused by various things such as weather changes, weather events and patterns, as well as human activities. At this point in time, it is unknown exactly how C. gattii got to Vancouver Island, though there are several theories. One theory is that the initial establishment on Vancouver Island is thought to be associated with an El Niņo/Southern Oscillation warming event (Morey 1990). The spores and hyphal fragments have been found in seawater and freshwater, as well as on wheel wells, apparel and shoes of travelers to Vancouver Island (Galanis et al., 2009).

As previously stated, the fungus also infects animals, both wild and domestic. The number of reported animal cases is roughly 1.5 times the number of human cases. Cryptococcus gattii from the PNW is more virulent than that in Australia, where it infects about four people per million and is rarely fatal. On Vancouver Island, the rate was reported as 27 people per million, and is more often fatal (Struck 2007; Datta et al., 2009). Cryptococcal disease is not contagious and although it can spread to mammals, it does not jump from animal to animal or human to human.

Testing
Performing an air sample using MEA or PDA is not an appropriate sampling technique for Cryptococcus. Air sampling could determine what type of organism is present in a known location, but one would need to do surface sampling for testing purposes here. The differences between testing for C. neoformans and C. gattii are antigenic, so serotyping has been used for differentiation. Most physicians and clinicians are more familiar with C. neoformans identification, and more identification techniques are available. Once a Cryptococcus has been identified, growing the fungus on CGB agar appears to be the best way to differentiate between these two closely related species. Cryptococcus gattii grown on CGB agar will produce a blue color reaction indicating it can use glycine as a sole carbon source (Byrnes et al., 2010). EMLab P&K performs analysis and identification of Cryptococcus neoformans, which is also one of the 3 fungi included in the Avian Pathogen PCR Screen. As previously mentioned, Cryptococcus gattii requires specialized media for proper identification. If you have potential need for C. gattii testing, please contact the laboratory for possible testing options and information.

Since it is not routine for cryptococcal isolates to be tested for in a clinical setting, the actual incidence is not fully known and the natural history of C. gattii infection is not well understood. As expected, the current epidemiological understanding of this disease in WA and OR is incomplete and the fungus has been isolated from many substrates including a variety of plant species, soil, air, freshwater and seawater.

In conclusion, Cryptococcus gattii has become endemic to the PNW in recent years, reaching epidemic proportions in BC, primarily Vancouver Island. Currently in the state of Washington, a human or animal infection from C. gattii, is considered a reportable rare disease of public health significance. In Oregon, it has been a reportable disease since August 2011. Prior to this, reporting was voluntary so cases could have been missed. Though this environmental fungus is rare, it would be wise to educate individuals and health care providers in order to monitor trends and detect further disease emergence into additional geographic areas such as Northern California. This fungus which can cause disease in otherwise healthy hosts, and to a lesser extent, in immunocompromised hosts, seems to favor people with other health issues. Education and awareness is critical to our understanding of this organism and the full epidemiology of the disease as it changes and presumably spreads.

1 (Vanbreus. & Takashio) Kwon-Chung & Boekhout

2 (San Felice) Vuill.

3 Fresen.

References:
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Byrnes EJ III, R Bildfell, SA Frank, TG Mitchell, K Marr, J Heitman. 2009. Molecular evidence that the range of the Vancouver Island outbreak of Cryptococcus gattii infection has expanded into the Pacific Northwest in the United States. J Infect Dis. 199(7):1081-6.

Byrnes EJ III, W Li, Y Lewit, H Ma, K Voelz, et al. 2010. Emergence and pathogenicity of highly virulent Cryptococcus gattii genotypes in the Northwest United States. PLoS Pathog 6(4): e1000850. doi:10.1371/journal.ppat.1000850

CDC: Spread of Cryptococcus gattii into Pacific Northwest Region of the United States DOI: 10.3201/eid1508.081384

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KUOW (A service of the University of Washington): Fungi Attack!

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Rodriguez-Carres M, K Findley, S Sun, FS Dietrich, J Heitman. 2010. Morphological and genomic characterization of Filobasidiella depauperata: A homothallic sibling species of the pathogenic Cryptococcus species complex. PLoS ONE 5(3): e9620. doi:10.1371/journal.pone.0009620

Struck, Doug. 2007. Alien invasion: the fungus that came to Canada. The Washington Post, Washington Post Foreign Service. Sunday, April 8, 2007

Washington State Dept. of Health: Cryptococcus gattii



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