Climatic Influences on Cryptococcus gattii [corrected] Populations, Vancouver Island, Canada, 2002-2004.

Vancouver Island, Canada, reports the world's highest incidence of Cryptococcus gattii infection among humans and animals. To identify key biophysical factors modulating environmental concentrations, we evaluated monthly concentrations of C. gatti in air, soil, and trees over a 3-year period. The 2 study datasets were repeatedly measured plots and newly sampled plots. We used hierarchical generalized linear and mixed effect models to determine associations. Climate systematically influenced C. gattii concentrations in all environmental media tested; in soil and on trees, concentrations decreased when temperatures were warmer. Wind may be a key process that transferred C. gattii from soil into air and onto trees. C. gattii results for tree and air samples were more likely to be positive during periods of higher solar radiation. These results improve the understanding of the places and periods with the greatest C. gattii colonization. Refined risk projections may help susceptible persons avoid activities that disturb the topsoil during relatively cool summer days.

Detection of Cryptococcus gattii in selected urban parks of the Willamette Valley, Oregon.

Human and animal infections of the fungus Cryptococcus gattii have been recognized in Oregon since 2006. Transmission is primarily via airborne environmental spores and now thought to be locally acquired due to infection in non-migratory animals and humans with no travel history. Previous published efforts to detect C. gattii from tree swabs and soil samples in Oregon have been unsuccessful. This study was conducted to determine the presence of C. gattii in selected urban parks of Oregon cities within the Willamette Valley where both human and animal cases of C. gattii have been diagnosed. Urban parks were sampled due to spatial and temporal overlap of humans, companion animals and wildlife. Two of 64 parks had positive samples for C. gattii. One park had a positive tree and the other park, 60 miles away, had positive bark mulch samples from a walkway. Genotypic subtypes identified included C. gattii VGIIa and VGIIc, both considered highly virulent in murine host models.

A decade of experience: Cryptococcus gattii in British Columbia.

It has been over a decade since Cryptococcus gattii was first recognized as the causative organism of an outbreak of cryptococcosis on Vancouver Island, British Columbia. A number of novel observations have been associated with the study of this emergent pathogen. A novel genotype of C. gattii, VGIIa was described as the major genotype associated with clinical disease. Minor genotypes, VGIIb and VGI, are also responsible for disease in British Columbians, in both human and animal populations. The clinical major genotype VGIIa and minor genotype VGIIb are identical to C. gattii isolated from the environment of Vancouver Island. There is more heterogeneity in VGI, and a clear association with the environment is not apparent. Between 1999 and 2010, there have been 281 cases of C. gattii cryptococcosis. Risk factors for infection are reported to be age greater than 50 years, history of smoking, corticosteroid use, HIV infection, and history of cancer or chronic lung disease. The major C. gattii genotype VGIIa is as virulent in mice as the model Cryptococcus, H99 C. neoformans, although the outbreak strain produces a less protective inflammatory response in C57BL/6 mice. The minor genotype VGIIb is significantly less virulent in mouse models. Cryptococcus gattii is found associated with native trees and soil on Vancouver Island. Transiently positive isolations have been made from air and water. An ecological niche for this organism is associated within a limited biogeoclimatic zone characterized by daily average winter temperatures above freezing.

Surveillance for Cryptococcus gattii in horses of Vancouver Island, British Columbia, Canada.

In the last decade Cryptococcus gattii has emerged as an important human and animal pathogen in southwestern British Columbia (BC), Canada. When the disease initially emerged it was identified in humans and multiple animal species on the east coast of Vancouver Island. From fall 2003 until summer 2004, active surveillance was initiated to look for horses exposed to or infected with the organism by performing nasal cultures and serum antigen testing in horses residing within 10 km of known areas of environmental reservoirs of the fungus. Surveillance efforts were facilitated by local equine practitioners who were also encouraged to report clinical cases. Nasal colonization was identified in four of the 260 horses tested but none had a serum cryptococcal antigen titer. All positive horses were from the same geographic area near Duncan, BC. During the study period, a single horse was diagnosed with systemic cryptococcosis and euthanized; clinical and post mortem information is described. As this organism continues to disseminate in the Pacific Northwest it is important for veterinarians to be familiar with the disease as early diagnosis may enable more effective treatment.

Respiratory health of two cohorts of terminal grain elevator workers studied 30 years apart.

INTRODUCTION: We evaluated the respiratory health of two cohorts of grain terminal elevator workers who participated in one of either respiratory health surveys undertaken in 1978 and 2008. METHODS: Questionnaire and spirometry data from 584 workers from the 1978 survey and 215 workers from the 2008 survey were compared using logistic regression and general linear modeling. RESULTS: The geometric means of area samples of grain dust averaged 8.28 mg/m(3) in 1978 and 2.06 mg/m(3) in 2008. Workers in the 1978 survey had a significantly higher prevalence of respiratory symptoms (with the largest adjusted odds ratio of 3.78, 95% CI 2.07-7.25, for shortness of breath), a lower prevalence of atopic conditions and lower mean lung function. CONCLUSION: Current grain workers had a lower risk of respiratory health consequences and a greater prevalence of atopic conditions than workers surveyed 30 years prior, most likely associated with reduced exposure to grain dust in the terminal elevator environment.

In vitro antifungal susceptibilities and amplified fragment length polymorphism genotyping of a worldwide collection of 350 clinical, veterinary, and environmental Cryptococcus gattii isolates.

The in vitro susceptibilities of a worldwide collection of 350 Cryptococcus gattii isolates to seven antifungal drugs, including the new triazole isavuconazole, were tested. With amplified fragment length polymorphism (AFLP) fingerprinting, human, veterinary, and environmental C. gattii isolates were subdivided into seven AFLP genotypes, including the interspecies hybrids AFLP8 and AFLP9. The majority of clinical isolates (n = 215) comprised genotypes AFLP4 (n = 76) and AFLP6 (n = 103). The clinical AFLP6 isolates had significantly higher geometric mean MICs for flucytosine and fluconazole than the clinical AFLP4 isolates. Of the seven antifungal compounds examined in this study, isavuconazole had the lowest MIC(90) (0.125 µg/ml) for all C. gattii isolates, followed by a 1 log(2) dilution step increase (MIC(90), 0.25 µg/ml) for itraconazole, voriconazole, and posaconazole. Amphotericin B had an acceptable MIC(90) of 0.5 µg/ml, but fluconazole and flucytosine had relatively high MIC(90)s of 8 µg/ml.

Spread of Cryptococcus gattii into Pacific Northwest region of the United States.

Cryptococcus gattii has emerged as a human and animal pathogen in the Pacific Northwest. First recognized on Vancouver Island, British Columbia, Canada, it now involves mainland British Columbia, and Washington and Oregon in the United States. In Canada, the incidence of disease has been one of the highest worldwide. In the United States, lack of cryptococcal species identification and case surveillance limit our knowledge of C. gattii epidemiology. Infections in the Pacific Northwest are caused by multiple genotypes, but the major strain is genetically novel and may have emerged recently in association with unique mating or environmental changes. C. gattii disease affects immunocompromised and immunocompetent persons, causing substantial illness and death. Successful management requires an aggressive medical and surgical approach and consideration of potentially variable antifungal drug susceptibilities. We summarize the study results of a group of investigators and review current knowledge with the goal of increasing awareness and highlighting areas where further knowledge is required.

Biomarkers of airway acidity and oxidative stress in exhaled breath condensate from grain workers.

RATIONALE: Grain workers report adverse respiratory symptoms due to exposures to grain dust and endotoxin. Studies have shown that biomarkers in exhaled breath condensate (EBC) vary with the severity of airway inflammation. OBJECTIVES: The purpose of the study was to evaluate biomarkers of airway acidity (pH and ammonium [NH(4)(+)]) and oxidative stress (8-isoprostane) in the EBC of grain workers. METHODS: A total of 75 workers from 5 terminal elevators participated. In addition to EBC sampling, exposure monitoring for inhalable grain dust and endotoxin was performed; spirometry, allergy testing, and a respiratory questionnaire derived from that of the American Thoracic Society were administered. MEASUREMENTS AND MAIN RESULTS: Dust and endotoxin levels ranged from 0.010 to 13 mg/m(3) (median, 1.0) and 8.1 to 11,000 endotoxin units/m(3) (median, 610) respectively. EBC pH values varied from 4.3 to 8.2 (median, 7.9); NH(4)(+) values from 22 to 2,400 microM (median, 420); and 8-isoprostane values from 1.3 to 45 pg/ml (median, 11). Univariate and multivariable analyses revealed a consistent effect of cumulative smoking and obesity with decreased pH and NH(4)(+), and intensity of grain dust and endotoxin with increased 8-isoprostane. Duration of work on the test day was associated with decreased pH and NH(4)(+), whereas duration of employment in the industry was associated with decreased 8-isoprostane. CONCLUSIONS: Chronic exposures are associated with airway acidity, whereas acute exposures are more closely associated with oxidative stress. These results suggest that the collection of EBC may contribute to predicting the pathological state of the airways of workers exposed to acute and chronic factors.

Within- and between-person variability of exhaled breath condensate pH and NH4+ in never and current smokers.

Recent studies have suggested that the collection of exhaled breath condensate (EBC) may be a viable method in occupational field studies to sample secretions of the lower airway because it is simple to perform and non-invasive. However, there are unresolved questions about whether certain laboratory conditions may influence the analysis of EBC biomarker measurements. A total of 12 subjects performed 116 EBC tests. The effect of short and long-term sample storage and sample volume on two biomarkers of acid stress, pH and NH4+, in EBC were investigated and did not significantly influence either marker measurement after argon deaeration. We also investigated the variability and the effect of smoking on the biomarkers by collecting six samples each from five adult never smokers and five adult current smokers over a period of 1 month (n=60 total). For pH, the within-person and between-person variability was larger in current smokers compared to never smokers. Similar results were found for NH4+. Cigarette packs smoked per day now was also associated with both pH (p=0.01) and NH4+ (p=0.04) using mixed effects regression analysis. The variability and smoking results suggest that repeated measurements of EBC pH and NH4+ from the same individual may accurately predict the biological state of the airways of current smokers when compared to never smokers.

Effect of heat-treatment and the role of phospholipases on Fungizone-induced cytotoxicity within human kidney proximal tubular (HK-2) cells and Aspergillus fumigatus.

The objectives of this study were to determine the effects of heat-treatment on Fungizone (FZ)-induced cytotoxicity in human kidney (HK-2) cells and fungal isolates of Aspergillus fumigatus, and to determine the possible role of phospholipases (PLA2 and PLC) on heat-treated FZ (HFZ)-associated renal cell toxicity. HK-2 cells were grown at 37 degrees C in T75 flasks and seeded in 96-well plates at 20,000 cells/well. FZ and HFZ concentrations of 10, 25 and 50 microg/mL of AmpB were prepared. Snake venom PLA2 and PLC (2.15 U/mL) were pre-incubated with HFZ for 1h prior to addition to the cells. After 18 h of incubation, an MTS assay was performed to assess cell viability through mitochondrial respiration. A spore suspension of A. fumigatus was prepared and 96-well plates were seeded at 500,000 spores/well. HFZ and FZ were prepared as above and incubated with the fungi at 35 degrees C. After 72 h, the minimum inhibitory concentration (MIC) was determined as the lowest concentration of drug that inhibited visible growth. Student-Newman-Keuls multiple comparisons tests were conducted to determine statistical significance. FZ-induced cytotoxicity was significantly greater than for HFZ in HK-2 cells at amphotericin B (AmpB) concentrations between 10 and 50 microg AmpB/mL (n = 5-9, p < 0.05). HFZ and FZ were found to have similar minimum inhibitory concentration (MIC) ranges for A. fumigatus (0.225-0.25 microg) AmpB/mL; (n = 6). The addition of PLA2 and PLC to 50 microg heat-treated AmpB/mL significantly enhanced the cytotoxicity compared to controls (n = 6, p < 0.05). The presence of the phospholipases did not alter FZ-associated renal cell toxicity. Taken together, these findings suggest heat-treatment significantly decreased FZ-induced cytotoxicity in HK-2 cells without altering toxicity against a reference strain of A. fumigatus. In addition, PLA2 and PLC enhanced the renal toxicity associated with HFZ, but not that of FZ.

Reduction in the bactericidal activity of selected cathelicidin peptides by bovine calf serum or exogenous endotoxin.

Synthetic cathelicidin peptides exhibit enhanced antimicrobial action and avid binding to LPS, thereby detoxifying the action of endotoxin released from degrading bacteria. A series of cathelicidin antimicrobial peptide (CAP) and sheep myeloid antimicrobial peptide (SMAP) congeners were examined to determine whether LPS-binding could predict other beneficial characteristics of the peptides. The peptides were challenged in complex media with bovine calf serum or LPS, and their ability to kill the Gram negative pathogens Klebsiella pneumoniae (ATCC 43816) or Pseudomonas aeruginosa (PA103) was then assessed. LPS-binding efficiency was not correlated with antimicrobial activity in complex media. Additionally, LPS- and serum-binding may interfere with the antimicrobial activity of peptides in complex media.

Beta(1-->3)-glucan exposure levels among workers in four British Columbia sawmills.

Beta(1-->3)-glucans were extracted from wood dust samples taken during the summer of 1997 at four British Columbia sawmills. Personal dust samples were collected using a GSP-sampler for inhalable dust and the sampling strategy targeted all production and maintenance jobs at least once at each mill. Potential exposure determinants data were documented concurrently, including weather conditions, log storage methods, wood conditions, species, production level, jobs and tasks. beta(1-->3)-glucans were measured by enzyme inhibition immunoassay (EIA). A total of 223 personal beta(1-->3)-glucan samples were analyzed. 45.7% were below the limit of detection (LOD). Geometric mean concentration ranged from 3.5 to 18.9 micro g/m(3) across the four mills. The highest levels were measured at the Interior mills, particularly in the log processing and sawmill areas. Multivariate regression models indicated that land-based log storage, clean-up jobs, high wood dust concentration, lumber yard department and the interaction between land-based log storage method and log processing department were associated with increased beta(1-->3)-glucan concentration.

Clinicopathologic features of an unusual outbreak of cryptococcosis in dogs, cats, ferrets, and a bird: 38 cases (January to July 2003).

OBJECTIVE: To determine clinical and pathologic findings associated with an outbreak of cryptococcosis in an unusual geographic location (British Columbia, Canada). DESIGN: Retrospective study. ANIMALS: 1 pink-fronted cockatoo, 2 ferrets, 20 cats, and 15 dogs. PROCEDURE: A presumptive diagnosis of cryptococcosis was made on the basis of serologic, histopathologic, or cytologic findings, and a definitive diagnosis was made on the basis of culture or immunohistochemical staining. RESULTS: No breed or sex predilections were detected in affected dogs or cats. Eleven cats had neurologic signs, 7 had skin lesions, and 5 had respiratory tract signs. None of 17 cats tested serologically for FeLV yielded positive results; 1 of 17 cats yielded positive results for FIV (western blot). Nine of 15 dogs had neurologic signs, 2 had periorbital swellings, and only 3 had respiratory tract signs initially. Microbiologic culture in 15 cases yielded 2 isolates of Cryptococcus neoformans var grubii (serotype A) and 13 isolates of C. neoformans var gattii (serotype B); all organisms were susceptible to amphotericin B and ketoconazole. Serologic testing had sensitivity of 92% and specificity of 98%. CONCLUSIONS AND CLINICAL RELEVANCE: Serologic titers were beneficial in identifying infection in animals with nonspecific signs, but routine serum biochemical or hematologic parameters were of little value in diagnosis. Most animals had nonspecific CNS signs and represented a diagnostic challenge. Animals that travel to or live in this region and have nonspecific malaise or unusual neurologic signs should be evaluated for cryptococcosis.

Phenotypic antibiotic resistance of Escherichia coli and E. coli O157 isolated from water, sediment and biofilms in an agricultural watershed in British Columbia.

This study examined the distribution of antibiotic resistant Escherichia coli and E. coli O157 isolated from water, sediment and biofilms in an intensive agricultural watershed (Elk Creek, British Columbia) between 2005 and 2007. It also examined physical and chemical water parameters associated with antibiotic resistance. Broth microdilution techniques were used to determine minimum inhibitory concentrations (MIC) for E. coli (n=214) and E. coli O157 (n=27) recovered isolates for ampicillin, cefotaxime, ciprofloxacin, nalidixic acid, streptomycin and tetracycline. Both E. coli and E. coli O157 isolates showed highest frequency of resistance to tetracycline, ampicillin, streptomycin and nalidixic acid; respectively. For E. coli, the highest frequency of resistance was observed at the most agriculturally-impacted site, while the lowest frequency of resistance was found at the headwaters. Sediment and river rock biofilms were the most likely to be associated with resistant E. coli, while water was the least likely. While seasonality (wet versus dry) had no relationship with resistance frequency, length of biofilm colonization of the substratum in the aquatic environment only affected resistance frequency to nalidixic acid and tetracycline. Multivariate logistic regressions showed that water depth, nutrient concentrations, temperature, dissolved oxygen and salinity had statistically significant associations with frequency of E. coli resistance to nalidixic acid, streptomycin, ampicillin and tetracycline. The results indicate that antibiotic resistant E. coli and E. coli O157 were prevalent in an agricultural stream. Since E. coli is adept at horizontal gene transfer and prevalent in biofilms and sediment, where ample opportunities for genetic exchange with potential environmental pathogens present themselves, resistant isolates may present a risk to ecosystem, wildlife and public health.

Ancient dispersal of the human fungal pathogen Cryptococcus gattii from the Amazon rainforest.

Over the past two decades, several fungal outbreaks have occurred, including the high-profile 'Vancouver Island' and 'Pacific Northwest' outbreaks, caused by Cryptococcus gattii, which has affected hundreds of otherwise healthy humans and animals. Over the same time period, C. gattii was the cause of several additional case clusters at localities outside of the tropical and subtropical climate zones where the species normally occurs. In every case, the causative agent belongs to a previously rare genotype of C. gattii called AFLP6/VGII, but the origin of the outbreak clades remains enigmatic. Here we used phylogenetic and recombination analyses, based on AFLP and multiple MLST datasets, and coalescence gene genealogy to demonstrate that these outbreaks have arisen from a highly-recombining C. gattii population in the native rainforest of Northern Brazil. Thus the modern virulent C. gattii AFLP6/VGII outbreak lineages derived from mating events in South America and then dispersed to temperate regions where they cause serious infections in humans and animals.

Campylobacter spp. distribution in biofilms on different surfaces in an agricultural watershed (Elk Creek, British Columbia): using biofilms to monitor for Campylobacter.

Despite its relevance to public health, presence and concentrations of Campylobacter spp. in biofilms in natural aquatic environments has not been investigated. This study examined the occurrence of Campylobacter spp. in biofilms on a variety of surfaces (river rock, slate rock, wood, Lexan™, sandpaper, and sediment) and in water from December 2005 to December 2006 to find a substratum that facilitated campylobacters detection in natural aquatic environments. Samples were collected at four sites in an agricultural watershed (Elk Creek, British Columbia). Campylobacter spp. presence was determined using culturing methods. Correlations between chemical, physical and microbiological water quality parameters and Campylobacter spp. distribution on different surface types were also investigated. Campylobacter spp. had a prevalence of 13% in the wet season, but was not recovered in the dry season. Its prevalence was highest in sediment (27%), followed by slate rock (22%), Lexan and wood (13%), river rock (9%) and water (8%), respectively. No Campylobacter spp. was found in sandpaper biofilms. Several other criteria were used to assess substrata effectiveness, such as correlation amongst Campylobacter spp., indicator bacteria and water quality parameters, cost and availability of substratum, potential for standardizing substratum, ease of biofilm removal and probability of substratum loss in situ. Results show that sediment, slate rock or wood could be used as substrata for Campylobacter spp. monitoring. The study also highlights the potential use of nitrates and enterococci as faecal contamination indicators to protect public health.

Cryptococcus gattii: an emerging fungal pathogen infecting humans and animals.

Infectious fungi are among a broad group of microbial pathogens that has and continues to emerge concomitantly due to the global AIDS pandemic as well as an overall increase of patients with compromised immune systems. In addition, many pathogens have been emerging and re-emerging, causing disease in both individuals who have an identifiable immune defect and those who do not. The fungal pathogen Cryptococcus gattii can infect individuals with and without an identifiable immune defect, with a broad geographic range including both endemic areas and emerging outbreak regions. Infections in patients and animals can be severe and often fatal if untreated. We review the molecular epidemiology, population structure, clinical manifestations, and ecological niche of this emerging pathogen.

Cryptococcosis: update and emergence of Cryptococcus gattii.

Cryptococcosis is a fungal disease that occurs throughout the world. Recent reclassification of Cryptococcus species along with a change in the distribution pattern has prompted reevaluation of the organism and the diseases caused by this pathogen. This review highlights the emergence of Cryptococcus gattii as a primary pathogen in North America and summarizes our current understanding of the disease in mammals and birds.

Preventing airborne disease transmission: review of methods for ventilation design in health care facilities.

Health care facility ventilation design greatly affects disease transmission by aerosols. The desire to control infection in hospitals and at the same time to reduce their carbon footprint motivates the use of unconventional solutions for building design and associated control measures. This paper considers indoor sources and types of infectious aerosols, and pathogen viability and infectivity behaviors in response to environmental conditions. Aerosol dispersion, heat and mass transfer, deposition in the respiratory tract, and infection mechanisms are discussed, with an emphasis on experimental and modeling approaches. Key building design parameters are described that include types of ventilation systems (mixing, displacement, natural and hybrid), air exchange rate, temperature and relative humidity, air flow distribution structure, occupancy, engineered disinfection of air (filtration and UV radiation), and architectural programming (source and activity management) for health care facilities. The paper describes major findings and suggests future research needs in methods for ventilation design of health care facilities to prevent airborne infection risk.

Cryptococcus gattii: Emergence in Western North America: Exploitation of a Novel Ecological Niche.

The relatively uncommon fungal pathogen Cryptococcus gattii recently emerged as a significant cause of cryptococcal disease in human and animals in the Pacific Northwest of North America. Although genetic studies indicated its possible presence in the Pacific Northwest for more than 30 years, C. gattii as an etiological agent was largely unknown in this region prior to 1999. The recent emergence may have been encouraged by changing conditions of climate or land use and/or host susceptibility, and predictive ecological niche modeling indicates a potentially wider spread. C. gattii can survive wide climatic variations and colonize the environment in tropical, subtropical, temperate, and dry climates. Long-term climate changes, such as the significantly elevated global temperature in the last 100 years, influence patterns of disease among plants and animals and create niche microclimates habitable by emerging pathogens. C. gattii may have exploited such a hitherto unrecognized but clement environment in the Pacific Northwest to provide a wider exposure and risk of infection to human and animal populations.