Murray Fyfe

Genome Variation in Cryptococcus gattii, an Emerging Pathogen of Immunocompetent Hosts

ABSTRACT Cryptococcus gattii recently emerged as the causative agent of cryptococcosis in healthy individuals in western North America, despite previous characterization of the fungus as a pathogen in tropical or subtropical regions. As a foundation to study the genetics of virulence in this pathogen, we sequenced the genomes of a strain (WM276) representing the predominant global molecular type (VGI) and a clinical strain (R265) of the major genotype (VGIIa) causing disease in North America. We compared these C. gattii genomes with each other and with the genomes of representative strains of the two varieties of Cryptococcus neoformans that generally cause disease in immunocompromised people. Our comparisons included chromosome alignments, analysis of gene content and gene family evolution, and comparative genome hybridization (CGH). These studies revealed that the genomes of the two representative C. gattii strains (genotypes VGI and VGIIa) are colinear for the majority of chromosomes, with some minor rearrangements. However, multiortholog phylogenetic analysis and an evaluation of gene/sequence conservation support the existence of speciation within the C. gattii complex. More extensive chromosome rearrangements were observed upon comparison of the C. gattii and the C. neoformans genomes. Finally, CGH revealed considerable variation in clinical and environmental isolates as well as changes in chromosome copy numbers in C. gattii isolates displaying fluconazole heteroresistance. IMPORTANCE Isolates of Cryptococcus gattii are currently causing an outbreak of cryptococcosis in western North America, and most of the cases occurred in the absence of coinfection with HIV. This pattern is therefore in stark contrast to the current global burden of one million annual cases of cryptococcosis, caused by the related species Cryptococcus neoformans, in the HIV/AIDS population. The genome sequences of two outbreak-associated major genotypes of C. gattii reported here provide insights into genome variation within and between cryptococcal species. These sequences also provide a resource to further evaluate the epidemiology of cryptococcal disease and to evaluate the role of pathogen genes in the differential interactions of C. gattii and C. neoformans with immunocompromised and immunocompetent hosts. Isolates of Cryptococcus gattii are currently causing an outbreak of cryptococcosis in western North America, and most of the cases occurred in the absence of coinfection with HIV. This pattern is therefore in stark contrast to the current global burden of one million annual cases of cryptococcosis, caused by the related species Cryptococcus neoformans, in the HIV/AIDS population. The genome sequences of two outbreak-associated major genotypes of C. gattii reported here provide insights into genome variation within and between cryptococcal species. These sequences also provide a resource to further evaluate the epidemiology of cryptococcal disease and to evaluate the role of pathogen genes in the differential interactions of C. gattii and C. neoformans with immunocompromised and immunocompetent hosts.

Population distribution and burden of acute gastrointestinal illness in British Columbia, Canada

BackgroundIn developed countries, gastrointestinal illness (GI) is typically mild and self-limiting, however, it has considerable economic impact due to high morbidity.MethodsThe magnitude and distribution of acute GI in British Columbia (BC), Canada was evaluated via a cross-sectional telephone survey of 4,612 randomly selected residents, conducted from June 2002 to June 2003. Respondents were asked if they had experienced vomiting or diarrhoea in the 28 days prior to the interview.ResultsA response rate of 44.3% was achieved. A monthly prevalence of 9.2% (95%CI 8.4 – 10.0), an incidence rate of 1.3 (95% CI 1.1–1.4) episodes of acute GI per person-year, and an average probability that an individual developed illness in the year of 71.6% (95% CI 68.0–74.8), weighted by population size were observed. The average duration of illness was 3.7 days, translating into 19.2 million days annually of acute GI in BC.ConclusionThe results corroborate those from previous Canadian and international studies, highlighting the substantial burden of acute GI.

Risk Factors for Cryptococcus gattii Infection, British Columbia, Canada

To determine whether particular environmental, medical, or behavioral risk factors existed among Cryptcoccus gattii–infected persons compared with the general population, we conducted a sex-matched case−control study on a subset of case-patients in British Columbia (1999–2001). Exposures and underlying medical conditions among all case-patients (1999–2007) were also compared with results of provincial population–based surveys and studies. In case−control analyses, oral steroids (matched odds ratio [MOR] 8.11, 95% confidence interval [CI] 1.74–37.80), pneumonia (MOR 2.71, 95% CI 1.05–6.98), and other lung conditions (MOR 3.21, 95% CI 1.08–9.52) were associated with infection. In population comparisons, case-patients were more likely to be >50 years of age (p<0.001), current smokers (p<0.001), infected with HIV (p<0.001), or have a history of invasive cancer (p<0.001). Although C. gattii is commonly believed to infect persons with apparently healthy immune systems, several immunosuppressive and pulmonary conditions seem to be risk factors.

Frozen chicken nuggets and strips--a newly identified risk factor for Salmonella Heidelberg infection in British Columbia, Canada.

Salmonella enterica var. Heidelberg was isolated from an unusual food source during routine case follow-up, prompting a case control investigation of frozen chicken nuggets and strips. Most frozen nuggets and strips are raw; however, par-frying lends a cooked appearance. As such, suitable food preparation precautions might not be undertaken by consumers. Cases were confirmed in the laboratory between 1 January and 1 April 2003. Controls were generated through forward-digit dialing and individually matched by age category. Telephone interviews were conducted, and limited sampling of unopened product was performed. Eighteen matched pairs were interviewed. The odds of infection were 11 times higher in individuals who had consumed frozen processed chicken nuggets and strips (95% confidence interval, 1.42 < odds ratio < 85.20). One-third of cases and controls considered frozen nuggets and strips to be precooked, and one quarter used the microwave, an ill-advised cooking method. Consumer misconceptions contributed to the risk of infection. Clear labels identifying nuggets and strips as raw poultry are needed.

Ecological niche modeling of Cryptococcus gattii in British Columbia, Canada.

Background Cryptococcus gattii emerged on Vancouver Island, British Columbia (BC), Canada, in 1999, causing human and animal illness. Environmental sampling for C. gattii in southwestern BC has isolated the fungal organism from native vegetation, soil, air, and water. Objectives Our aim was to help public health officials in BC delineate where C. gattii is currently established and forecast areas that could support C. gattii in the future. We also examined the utility of ecological niche modeling (ENM) based on human and animal C. gattii disease surveillance data. Methods We performed ENM using the Genetic Algorithm for Rule-set Prediction (GARP) to predict the optimal and potential ecological niche areas of C. gattii in BC. Human and animal surveillance and environmental sampling data were used to build and test the models based on 15 predictor environmental data layers. Results ENM provided very accurate predictions (> 98% accuracy, p-value < 0.001) for C. gattii in BC. The models identified optimal C. gattii ecological niche areas along the central and south eastern coast of Vancouver Island and within the Vancouver Lower Mainland. Elevation, biogeoclimatic zone, and January temperature were good predictors for identifying the ecological niche of C. gattii in BC. Conclusions The use of human and animal case data for ENM proved useful and effective in identifying the ecological niche of C. gattii in BC. These results are shared with public health to increase public and physician awareness of cryptococcal disease in regions at risk of environmental colonization of C. gattii.

Protective Behavior Survey, West Nile Virus, British Columbia

We investigated personal protective behaviors against West Nile virus infection. Barriers to adopting these behaviors were identified, including the perception that DEET (N,N-diethyl-m-toluamide and related compounds) is a health and environmental hazard. Televised public health messages and knowing that family or friends practiced protective behaviors were important cues to action.

Rare Cryptosporidium hominis Subtype Associated with Aquatic Center Use

To the Editor: Cryptosporidiosis is the most frequently reported gastrointestinal illness in outbreaks associated with treated (disinfected) recreational water venues in the United States (1). In 2003, an increased number of cryptosporidiosis cases occurred in the Tri-Cities area of the Lower Mainland region (near Vancouver), in British Columbia, Canada. Although all cases were associated with the use of a community aquatic center, their onset dates were spread over a 3-month period, and the link between cases was unclear. The aim of this study was to determine if the cases in this disease cluster were related. Although suitable molecular markers had yet to be defined at the time of the outbreak, recent reports on the use of the gp60 gene for subtyping in molecular epidemiologic studies (2,3) have enabled us to reanalyze the isolates and report these results. Fifteen laboratory-confirmed cases were identified from October 15 to December 5, 2003. This number was in excess of the anticipated incidence rate for this community, which averaged 5 reported cryptosporidiosis cases per year. During the period of investigation, an incident of fecal contamination at the aquatic center on October 10, 2003, was documented and remediation involved increasing the free chlorine concentration. Because the regional health authority was concerned about the increased number of cases, the facility closed voluntarily on December 5 for further remediation. However, recorded free chlorine concentrations did not exceed 2.0 ppm at any time during the investigative period (October 5–December 31). The health authority released a public advisory encouraging those who used the facility to submit fecal specimens for laboratory testing. The health authority also sent letters to family physicians in the area, informing them of the disease cluster and requesting that unpreserved stool specimens be collected, in addition to the formalin-fixed specimens, for routine diagnostic testing. Nine fecal specimens were collected from clinically symptomatic case-patients with histories of exposure to the implicated aquatic center. Five specimens were selected by using the criteria that they were from patients with laboratory-confirmed cryptosporidiosis cases from 5 separate households. The specimens were then coded for anonymity before subsequent molecular analysis. Genomic DNA was extracted from purified Cryptosporidium oocysts by freeze-thawing, and the species was determined by PCR amplification and sequencing of the 18S rRNA gene as described previously (4). The gp60 gene was also amplified by PCR by using primers described by Ong and Isaac-Renton (5). DNA sequences of amplicons were determined by cycle sequencing and assembled as described previously (4,5). The gp60 allele and subtype were identified by multiple sequence alignment with GenBank reference sequences and phylogenetic analysis that used ClustalX version 1.8 (www.clustal.org) as well as manual quantification of microsatellite repeats. The 18S rRNA and gp60 genes were amplified successfully from 4 specimens. On the basis of the 18S rRNA gene sequence, all case-patients were infected with Cryptosporidium hominis, a species associated primarily with human-to-human transmission. The gp60 sequences from all 4 case-patients were identical and were subtype IdA19, a rarely reported subtype of C. hominis. Globally, most reports of the gp60 Id allele, such as 9 reported cases from Australia, have identified the IdA15G1 subtype (3). Another subtype, IdA18, was isolated from 5 case-patients in a 1997 foodborne outbreak in Spokane, Washington (6). To date, the IdA19 subtype has been identified in only 1 sporadic case, in northern Ontario (7), and a subset of cases (seven 1dA19 and 2 mixed IdA19 and IbA10G2) in the 2001 waterborne outbreak in North Battleford, Saskatchewan (5,8). The IdA19 subtype is identical in sequence to the IdA18 subtype except for 1 extra TCA repeat in the microsatellite region. Neither subtype has been reported anywhere in the world except in Canada and the Pacific Northwest. Because cases from all previous C. hominis outbreaks of cryptosporidiosis in British Columbia have been caused by the IbA10G2 subtype, the most prevalent subtype in sporadic and outbreak cases around the world (2,5,9,10), our results indicate the presence of a new subpopulation of C. hominis parasites that could cause future disease outbreaks. The identification of the same subtype in all 4 case-patients with cryptosporidiosis associated with the use of a community aquatic center was consistent with their exposure history and confirmed that all cases were linked epidemiologically. However, the association between the single northern Ontario sporadic case and the larger number of Saskatchewan and British Columbia outbreak cases is uncertain. The association with the IdA18 subtype in the Washington foodborne outbreak is also unknown. Further research is needed to determine the distribution and prevalence of gp60 subtypes in Canada as well as other parts of the world before we can more clearly understand the transmission of the IdA19 subtype.

Applying open population capture–recapture models to estimate the abundance of injection drug users in Victoria, Canada

Abstract Background: Injection drug users (IDUs) are considered a hidden, hard to reach population that is difficult to measure. Multi-list recapture methods are commonly used to estimate IDU population sizes but do not allow inference on population dynamics. In Victoria, Canada, closed population capture–recapture methods have been used to estimate the abundance of IDUs. In this study, we make use of a newer sample of a survey of IDUs to relax the closure assumption. Methods: The I-Track survey of IDUs was carried out in Victoria on three occasions (2003, 2005 and 2009). Data from the three samples were linked using unique subject identifiers. A Jolly-Seber model was used to estimate the number of IDUs. Results: The results were very similar to a two-sample closed population analysis. However, when using open-population models, it is possible to get estimates for each time period of abundance and survival. The estimate of the number of the IDUs in Greater Victoria was relatively stable with fewer than 3000 individuals over the six-year study. Discussion: Improved estimates of population size and dynamics will assist in improving access to harm reduction services, which may reduce higher risk drug use practices.