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Occupational exposure to Cryptococcus gattii : evaluation of exposure to Cryptococcus gattii among arborists… Hingston, Adrian Owen 2006

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OCCUPATIONAL EXPOSURE TO CRYPTOCOCCUS GATTII: Evaluation of Exposure to Cryptococcus gattii among Arborists and CRD Watershed Employees on Vancouver Island by ADRIAN OWEN HINGSTON B.Sc, Simon Fraser University, 2003 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Occupational and Environmental Hygiene) THE UNIVERSITY OF BRITISH COLUMBIA April, 2006 © Adrian Owen Hingston, 2006 ABSTRACT Cryptococcus gattii (C. gattii) has emerged as an infectious pathogen on Vancouver Island, B . C . The goal of this thesis is to describe exposure to C. gattii among workers who work outdoors under the canopies of trees on Vancouver Island, specifically arbor workers and Capital Regional District (CRD) Watershed maintenance workers. Environmental sampling of trees, soil, water and air was used to characterize the geographical distribution of the organism on Vancouver Island and the Southern Gulf Islands. Air sampling was performed in the breathing zone of workers carrying out woodland tasks. Task analysis and questionnaire data were used to characterize the work tasks performed by arbor workers and C R D Watershed Maintenance workers. This organism was found in varying concentrations on the east coast of Vancouver Island from Victoria in the south to Courtenay in the north. The highest concentrations of positive samples were found in Parksville (i.e. 35% of all samples obtained were positive for C. gattii), Duncan (24%), and Salt Spring Island (20%). A wide range of tree species have been colonized by the organism including economically important varieties such as Douglas fir, Red Cedar, and Alder. The highest levels of C. gattii occurred in the drier months of the year, with very few positive air samples occurring between the months of November and March. Relative humidity was significantly (negatively) associated with airborne C. gattii in non-working conditions, and the location where the sample was taken as well as the month in which it was taken were significant determinants of airborne C. gattii concentration. The highest levels of airborne C. gattii occurred under working conditions. With work tasks such as chain sawing and chipping C gattii can become airborne in smaller particle sizes that are able to enter deeper portions of the human respiratory tract, which may enhance its ability to initiate disease. There is a risk of occupational exposure to C. gattii to workers performing woodland tasks on Vancouver Island. The risk of exposure is higher in the drier months of the year and when the worker performs tasks that create aerosols. Workers should be made aware of the existence of the pathogen as well as the signs and symptoms of cryptococcosis. ii TABLE OF CONTENTS A B S T R A C T II T A B L E OF CONTENTS ". HI LIST OF T A B L E S V LIST OF FIGURES VIII ABBREVIATIONS IX A C K N O W L E D G M E N T S X 1 - INTRODUCTION i 1.1 - STUDY SETTING 3 1.2 - B A C K G R O U N D A N D REVIEW OF THE LITERATURE 5 1.2.1 - Background 5 1.2.2 - Where Cryptococcus gattii has been found 6 1.2.3 - Cryptococcal Disease U 1.2.4 - Laboratory Identification 16 1.2.5 - Air Sampling for Cryptococcus gattii 17 1.2.6 - Determinants of Exposure 17 1.3 - R E S E A R C H QUESTIONS 19 2 - MATERIALS A N D METHODS 2 0 2.1 - STUDY OBJECTIVES A N D DESIGN 20 2.2 - STUDY P O P U L A T I O N 22 2.3 - D A T A C O L L E C T I O N 22 2.3.1 - Environmental Sampling to Determine the Presence of Cryptococcus gattii 23 2.3.2 - Occupational Sampling for Cryptococcus gattii 28 2.3.3 - Questionnaire 31 2.3.4 - Task Analysis 32 2.4 - D A T A A N A L Y S I S 33 2.4.1 - Environmental Sampling to Determine the Presence of Cryptococcus gattii 33 2.4.2 - Occupational Sampling for Cryptococcus gattii 33 2.4.3 - Questionnaire 34 2.4.4 - Task Analysis 34 3 - RESULTS 35 3.1 - FACTORS R E L A T E D TO CRYPTOCOCCUS GATTII C O N T A M I N A T I O N IN N O N -O C C U P A T I O N A L SETTINGS 35 3.1.1 - Geographical Distribution of Cryptococcus gattii 35 3.1.2-Tree Type 42 3.1.3 - Patterns of Contamination 43 3.1.4 - Determinants of Airborne Contamination 47 3.2 - FAC T O R S I N F L U E N C I N G T H E O C C U P A T I O N A L G E N E R A T I O N OF A I R B O R N E CRYPTOCOCCUS GATTII 50 3.2.1 - Occupational Measurement Data 50 3.2.2 - Questionnaire Data 61 3.2.3 - Task Analysis 67 3.3 - DEMOGRAPHICS A N D H E A L T H OF THE STUDY P O P U L A T I O N 69 4 -DISCUSSION 75 4.1 - STUDY QUESTION 1 - C A N ARBORIST/CRD W O R K E R S B E P O T E N T I A L L Y EXPOSED TO CR YPTOCOCCUS GA TTII1 75 iii 4.2 - STUDY QUESTION 2- W H A T CONDITIONS A F F E C T AIRBORNE CRYPTOCOCCUS GATTII CONCENTRATION? 78 4.3 - STUDY QUESTION 3- A R E ARBORISTS/CRD W O R K E R S A T A N I N C R E A S E D RISK OF EXPOSURE TO CRYPTOCOCCUS GATTIP. 83 4.4 - S T U D Y QUESTION 4- W H A T A R E THE ARBORISTS/CAPITAL R E G I O N A L DISTRICT WORKERS P O P U L A T I O N DEMOGRAPHICS A N D H E A L T H STATUS? 86 4.5 - STRENGTHS A N D LIMITATIONS 88 4.5.1 - Strengths 88 4.5.2 - Limitations • 88 5 - CONCLUSIONS A N D RECOMMENDATIONS 91 REFERENCES 93 APPENDICES 101 APPENDIX A - E T H I C A L A P P R O V A L 101 APPENDIX B - INTRODUCTORY LETTER 102 APPENDIX C - CONSENT F O R M 103 APPENDIX D - QUESTIONNAIRE : 105 iv LIST OF TABLES Table Title Page # 1 Environmental associations of C. gattii as published by Malik et al. in 2003 10 2 Sample taking procedure for tree, soil, air, and water samples 24 3 Ingredients used to make Staib agar for C. gattii culturing 24 4 Methods used to plate samples on Staib agar 25 5 Ingredients used to make Canavanine-Glycine-Bromothymol (CGB) agar to 26 differentiate C. gattii from C. neoformans 6 Variables included in the First Sample database 27 7 Variables from occupational sampling 30 8 Total number, number positive for C. gattii, and percent positive for C. gattii in 36 each sample type from the first sample database 9 Descriptive statistics for airborne C. gattii measurements from the first sample 36 database 10 Descriptive statistics for airborne C. gattii measurements from the first sample 36 database 11 Number and percent positive for C. gattii for tree, soil, air and water 38 measurements in cities/ towns on Vancouver Island and the Gulf Islands from the first sample database (locations listed from south to north of Vancouver Island) 12 Number of measurements, number of positive measurements and percent of 42 measurements that yielded positive results for C. gattii from all tree types from which greater than 50 samples were taken 13 Number of measurements, number of positive measurements, and percent of 43 measurements that yielded positive results for C. gattii from evergreen, deciduous, and unknown tree types 14 Relationship of relative humidity with airborne C. gattii concentration under non 46 occupational settings 15 Relationship of temperature and airborne C. gattii concentration under non- 46 occupational conditions 16 Descriptive statistics for transformed airborne C. gattii concentration stratified by 47 meteorological sampling condition in non-occupational settings 17 Variables in the regression equation that describes airborne C. gattii 48 concentration (In cfu/m3) under non-occupational conditions 18 Occupational sample types and percent positive by sampling location 50 19 Air sampling results for airborne C. gattii from occupational sampling sites on 50 Vancouver Island v 20 Air sampling results for all positive samples from occupational sampling 51 sites on Vancouver Island 21 Number of distinct sites, geometric mean and standard deviation for 52 occupational air measurements for C. gattii 22 Number of occupational sampling sites and the number and percent 53 positive of environmental samples in 100m, 500m, and 1000m buffer zones 23 Number of samples, percent positive, geometric mean, and geometric 56 standard deviation for occupational airborne C. gattii samples in each month 24 Number, percent positive, geometric mean, and geometric standard 58 deviation of transformed airborne C. gattii measurements in air while workers performed work tasks 25 Variables in the regression equation that describes airborne C. gattii 59 concentration in occupational sampling conditions 26 Size distribution from Andersen 6-stage sampling results in conditions 60 where work was not being performed 27 Size distribution from Andersen 6-stage sampling results in conditions 61 where work was being performed 28 Duration of arbor workers and CRD workers at their current job 62 29 Mean (%) and standard deviation (%) of work spent outdoors in current job 63 for arbor workers and CRD Workers 30 Task comparison of arbor workers and CRD workers 64 31 Reported dust exposure comparison of arbor workers and CRD workers 65 32a Relative ranking of the species of trees worked with for each work task 66 involving cutting 32b Relative ranking of the species of trees worked with for each work task not 66 involving cutting 33 Percentage of work shift that arbor laborers and Arborist/Owners spend 68 performing specific tasks 34 Mean, minimum, maximum and standard deviation of the age of arbor 69 workers and CRD workers 35 City of residence for arbor workers and CRD workers 70 36 Duration of arbor workers and CRD workers at current residence 70 37 Leisure time spent outdoors for arbor workers and CRD workers 71 38 Presence of C. gattii in the park followed by percentage of arbor workers 71 and CRD workers having visited the listed park vi 39 Percentage of arbor workgroup and CRD workgroup traveling to listed area 72 a minimum of once in the past year 40 Percentage of the arbor workgroup and CRD workgroup reporting 73 respiratory symptoms or other symptoms lasting over a month in the previous 5 years vu LIST OF FIGURES Figure # Title Page # 1 B iogeoc l imat i c z o n e s of V a n c o u v e r Island, Brit ish C o l u m b i a 4 2 T h e s i s overv iew. E a c h level represents a s tage of this project f rom the 21 p lanning of da ta col lect ion to the results of da ta ana lys is . 3 Distr ibut ion of s a m p l e types from the first sample da tabase 35 4 Loca t ions of C i t ies and T o w n s in wh ich greater than 100 m e a s u r e m e n t s 39 have been taken (for key, s e e table B) 5 Loca t ions of posi t ive and negat ive s a m p l e s for C. gattii in the C R D 41 wa te rshed 6 Pe rcen t posi t ive of a i rborne m e a s u r e m e n t s for C. gattii in e a c h month 44 7 Ar i thmet ic m e a n s of a i rborne C. gattii concent ra t ion data by month in 44 co lony forming units per meter c u b e d 8 G e o m e t r i c m e a n s of a i rborne C. gattii concent ra t ion data by month in 45 co lony forming units per meter c u b e d 9 Occupa t i ona l samp l ing s i tes and sur round ing env i ronmenta l samp l ing 53 locat ions in Parksv i l le 10 Occupa t i ona l samp l ing s i tes and sur round ing env i ronmenta l s a m p l e s in 54 C o o m b s 11 Occupa t i ona l samp l ing s i tes and sur round ing env i ronmenta l s a m p l e s in 54 V ic tor ia 12 Occupa t i ona l samp l ing site and sur round ing env i ronmenta l samp l ing 55 locat ions in N a n a i m o 13 Occupa t i ona l samp l ing s i tes and sur round ing env i ronmenta l samp l ing 55 locat ions in Q u a l i c u m B e a c h v i i i ABBREVIATIONS C. neoformans Cryptococcus neoformans C. gattii Cryptococcus gattii B . C . British Columbia C R D Capital Regional District cfu / m 3 Colony Forming Units per Cubic Meter °C Degrees Celsius R A P D Random Amplification of Polymorphic D N A ix ACKNOWLEDGMENTS This project would not have been possible without the funding support that was provided by the Workers' Compensation Board of Brit ish Columbia. M y thesis committee, for their support and patience over the duration of the study: Thesis Supervisor - K a r e n Bartlett, PhD Assistant Professor, University of Bri t ish Columbia Committee - Hugh Davies, PhD Assistant Professor, University of Bri t ish Columbia Helen W a r d , PhD Associate Professor, University o f Bri t ish Columbia Also, the subjects who took part in this study were integral to its success. The C R D Watershed management and workers were accommodating and flexible with regard to the study. As wel l , the arborist companies who allowed investigators to accompany them to their work sites were important in helping this study achieve success. Sunny Mak at the Brit ish Columbia Centre for Disease Control, in addition to keeping the C. gattii database up to date, gave technical assistance in accessing the data and constructed the sampling maps that are found on pages 54 through 56 of this thesis. Brad Johnson of Tree Frog Gardening and Landscaping was cooperative and accommodating in regards to helping investigators by performing arborist tasks in particular areas of interest. Finally, Charles and Gale Hingston, for their support of this study throughout the study period, accommodation of the investigator at their home, provision of tools and help with simulated work activities, and helping with environmental sampling on Salt Spring Island. x 1 - INTRODUCTION Cryptococcus gattii (C. gattii) has recently emerged as an infectious pathogen on the east coast of Vancouver Island, British Columbia (B.C.).' C. gattii is a yeast that belongs to the taxonomic division of fungi Basidiomycota, which is characterized primarily by the production of sexual spores on a structure called a basidium. The presence of C. gattii in tree, water, soil, and air has been established through environmental sampling by Dr. Bartlett at the University of British Columbia. In 2001 a chart review by epidemiologists at the British Columbia Centre of Disease Control gave evidence of cases of cryptococcosis due to C. gattii emerging in 1999. Prior to 1999 this organism had never been encountered as an environmental pathogen in Canada. The first positive environmental culture in B.C. was identified in March, 2002, and was found in a Douglas fir in a treed area of Parksville, B.C., in the environs of clinical cases. Since then many trees on Vancouver Island have tested positive for the presence of the organism. In May 2002, quantitative air samples were taken under a positive tree and a significant number of airborne C. gattii were recovered (1080 cfu/m3). This suggests potential exposure of workers in the forestry sector to C. gattii by creating aerosols when cutting wood, or by disturbing soil that is contaminated with the organism. In B.C. there were 139 confirmed human cases of cryptococcosis between 1999 and June 2005, including four fatalities. This translates to a rate of approximately 36 cases per million population per year on Vancouver Island. The route of entry of the organism is through the lungs. Systemic spread of the organism through the body via the circulatory system to the brain and meninges is possible. C. gattii is primarily found in tropical and subtropical climates and, prior to its discovery in B.C., was never before described in a temperate climate. Therefore, there is no evidence-base upon which to draw to determine the health risks to forestry workers working on Vancouver Island. The range of tree hosts colonized by this organism includes economically important trees harvested for lumber (e.g. Douglas fir) or chipped for animal bedding (e.g. Alder). The unique relationship of this organism with economically important tree species has prompted questions from workers in the forestry sector regarding the risk of disease due to exposure to C. gattii. Occupational exposure 1 to workers who spend their time outdoors under the canopies of trees had not yet been studied. The goal of this thesis was to describe exposure to C. gattii among specific groups of workers who work outdoors under the canopies of trees along the southeast coast of Vancouver Island. This research will be used to direct future investigations into the risk of disease due to C. gattii amongst workers on Vancouver Island and surrounding areas. 2 1.1 -STUDY SETTING Vancouver Island is situated at the south-west corner of Canada. It has an area of 32, 284km 2 and a population of approximately 750,000, two-thirds of whom live in the southern part of the island.2 The most inhabited areas on the island are situated on the south-east coast and include Victoria in the south (population 311,000), Nanaimo (86,000), Duncan (40,000), Parksville (25,000), Courtenay (47,000), and Campbell River (34,000)2. Vancouver Island has a temperate climate with a mean annual temperature of approximately 3.3 °C in the winter and 17.0 °C in the summer.3 The average precipitation is 167 mm per month in the winter and 34 mm per month in the summer. Vancouver Island has three main biogeoclimatic zones including the predominant Coastal Western Hemlock zone, the Coastal Douglas Fir zone which is found along the south east coast of the island and the Mountain Hemlock zone which is restricted to higher elevations. Figure 1 shows the biogeoclimatic zones on Vancouver Island. Since 2002 when the first positive results for C. gattii were cultured from environmental samples taken in Rathtrevor Beach Park in Parksville, British Columbia, positive samples have been collected in a variety of locations on Vancouver Island, the Gulf Islands, and more recently British Columbia's lower mainland. There have been greater than 9,300 samples taken from more than 4,500 distinct sites in south-western British Columbia. The main areas of study have been population centres along the central/southern part of the east coast of Vancouver Island including Victoria, Duncan, Nanaimo, Parksville, Courtenay and Comox because positive samples for C. gattii have been found in these areas. The scope of this study includes Vancouver Island and the Southern Gulf Islands. 3 B i o g e o c l i m a t i c Z o n e s o f V a n c o u v e r I s l a n d a n d t h e S o u t h e r n G u l f I s l a n d s [Mountain Hemlock Coastal Douglas Fir Coastal Western Hemlock Figure 1 - Biogeoclimatic zones of Vancouver Island, British Columbia1 Within the study area, special emphasis was placed on the Capital Regional District (CRD) Watershed which is located in the Langford area in the south of Vancouver Island. The watershed is situated near Victoria, an area close to where cases of cyptococcosis have occurred, but which had not been examined for the presence of C. gattii. The watershed provides water to greater Victoria and encompasses 14,000 hectares of land. It is well serviced by roads, and is very well characterized with respect to flora and fauna, as well as soil and climate (6 meteorological stations are located within the watershed). 4 1.2 - BACKGROUND AND REVIEW OF THE LITERATURE 1.2.1 - Background There are over 1.5 million species of fungus in the world but only 270 species have been shown to cause disease in humans.4 One such species, C. gattii was first recognized in 1970 by Vanbreuseghem and Takashio.5 The first environmental isolation of the organism was made by Ellis and Pfeiffer, in November of 1989, in the Barossa Valley of Southern Australia.6 Five years after it was first recognized, the sexual state (teleomorph) of C. gattii was identified by Kwon-Chung and placed in the genus Filobasidiella.7 There are at least 38 species of Cryptococcus; however it is only Cryptococcus neoformans (C. neoformans) and C. gatti that can consistently grow at mammalian body temperatures and are thus human health risks.4 C. gattii is among the fungal family basidiomycetes.8 The name Cryptococcus gattii is still being debated. Originally, in 1970 the name Cryptococcus neoformans var. gattii was proposed by Vanbreuseghem and Takashio.5 According to the International Code of Botanical Nomenclature, however, the correct nomenclature for Cryptococcus neoformans, serotypes B and C, is F. neoformans var. bacillispora Kwon Chung9, Cryptococcus gattii is currently the term in common use. C. neoformans and C. gattii are encapsulated yeasts which may cause life-threatening meningoencephalitis.9 C. neoformans has a world-wide distribution9 and appears to cause infections in immunocompromised individuals 1 0" in urban areas12, whereas C. gattii occurs predominantly in immunocompetent individuals 1 3" 1 5 living in tropical or sub-tropical regions. 9 1 6 In 1978 Kwon-Chung et al. found that the two varieties differed considerably, both in their physiology and in their pathogenicity for mice.9 Cryptococcus has been separated into C. gattii and C. neoformans based upon physiological, ecological, and serological differences.1 7'1 8 Five serotypes have been identified on the basis of antigenic differences in capsular polysaccharide.19 These serotypes include A , D and A D (C. neoformans) and serotypes B and C (C. gattii)}9 Xu et al. indicated that C. neoformans (serotypes A and D) and C. gattii (serotypes B and C) are estimated to have diverged from each other 37 million years ago, with serotype A and 20 D diverging 18.5 million years ago and serotypes B and C, 9.5 million years ago. 5 Sorrel, in a review of the literature, concluded that serotypes B and C are more closely related to one another than serotypes A and D. 8 There continues to be a controversy, however, over whether C. gattii should be designated as a separate species form C. 21 20 neoformans with authors arguing for , and against. C. gattii can be further divided into three phylogenetic groups: I, II, and III. Phylogenetic group I contains serotype B and C strains, whereas groups II and III include serotype B strains only. 2 2 1.2.2 - Where Cryptococcus gattii has been found Global Distribution Historically C. gattii appears to have had a more restricted geographical distribution than C. neoformans. Isolations of C. gattii, from both environmental sampling and clinical cases have been restricted almost entirely to tropical and sub-tropical areas of the globe.8' 9 Since the first environmental isolation of C. gattii in 19896, there has been a concentrated effort by researchers to determine the ecological niche and the global distribution of the organism. In 1990 and 1991 respectively, Ellis and Pfeiffer reported environmental isolations of C. gattii from Australia6 and Southern California. 2 3 It was hypothesized that C. gattii was restricted to areas in which Eucalyptus camaldulensis (River redgum) was growing (Hawaii, southern California, Mexico, Brazil, parts of Africa, Southeast Asia, Australia, and Papua New Guinea).6 An association between the distribution of disease caused by C. gattii and areas in which Eucalyptus camaldulensis had been planted was 6 23 • emphasized by Ellis and Pfeiffer , and subsequently confirmed in the United States in 1991, B raz i l 1 2 ' 2 4 in 1995, Mexico 2 5 in 1996, and Italy26 in 1997. Studies of samples taken from Eucalyptus camaldulensis around the world have had mixed results. Studies in Italy27, and India 2 8 in 1997, and Braz i l 2 9 in 2000, have reported positive environmental isolations. These studies supported Ellis and Pfeiffer's earlier observation that C. gattii had been exported from Australia on contaminated seeds or 6 30 31 * 32 seedlings of Eucalyptus camaldulensis. ' Studies from Central Africa in 1994, India and Spain 3 3 in 1997, and Italy3 4 in 2003, however, of the association of C. gattii with Eucalyptus camaldulensis and/or Eucalyptus tereticornis (Forest redgum) proved 6 negative. In a 2002 review of the global distribution of the organism, Campisi et al. indicated that in general, the association of C. gattii serotype B with Eucalyptus camaldulensis and Eucalyptus tereticornis is rare where Eucalyptus camaldulensis trees' had been introduced.34 C. gattii was isolated from bat guano in Brazi l 3 5 in 1993, and more recently from debris of almond trees in Columbia 3 6 in 1998. In 2001, Fortes et al. reported the first isolation of C. gattii from decaying wood in a hollow of a native jungle tree,- Guettarda acreana, in Brazi l 3 7 . Although C. gattii has almost exclusively been limited to tropical and subtropical climates, there was a report from temperate areas of Uruguay where it had been environmentally isolated from fragments of a wasp's nest in 1993.3 8 The areas in which clinical cases have been reported have also tended to be restricted to tropical and subtropical regions.9 Kwong-Chung and Bennett, in 1984, analyzed 96 clinical isolates of C. gattii and found that isolates had not been found in Europe (excluding the United Kingdom) and Japan; infrequently in North America (excluding Southern California), the United Kingdom, New Zealand, and Argentina; and were predominantly found in regions with tropical to subtropical climates such as South East Asia, Southern California, Brazil, Mid to South Pacific, and the central part of Africa. 9 Eight cases of cryptococcosis caused by C. gattii in Brazil were described by Rozehbaum in 1992.3 9 A large number of cases were reported in Papua New Guinea in 1997, though no environmental isolates could be found, even after extensive sampling.4 0 The same year, Poonwan et al. described 6 clinical isolates of C. gattii from Thailand. 4 1 In 1998, Baro et al. described 5 outbreaks of disease caused by C. gattii in goats in Spain. 4 2 In a paper the following year, Baro and colleagues indicated that after environmental sampling an environmental source had not been located.43 Two human infections due to C. gattii were reported in both Greece 4 4 in 2001 and Singapore45 in 2002. Other cases have been reported from Italy2 6 as well as Kenya, and Zaire. 4 6 Similar to environmental isolations, clinical cases have tended to have occurred in tropical and subtropical climates. There was a report from Argentina, however, where several human clinical cases have occurred.4 7 7 It is interesting to note that Kwong-Chung and Bennett suggested that, in order to find C. gattii in its natural environment, ecologists should focus their attention on tropical and subtropical regions and the habitats that exist there.9 It has also been pointed out that, due to the geographical endemicity of C. gattii infections in tropical and subtropical regions, cases in temperate areas represent patients who were initially infected in endemic regions and later presented with cryptococcosis elsewhere.16 The current outbreak on the east coast of Vancouver Island is the first time that C. gattii has been described from clinical and environmental samples in a temperate climate. C. gattii abundance in the environment and the cases of disease due to this fungus appear to be higher than anywhere else in the world. 1 6 In British Columbia, Canada, the B.C. Centre for Disease Control, the Vancouver Island Health Authority, the University of British Columbia, and animal health specialists are continuing their investigation into the distribution of the fungus in the environment and 48 the infections in humans and animals due to exposure. A recent statement issued by the B.C. Centre for Disease Control indicated that most people are likely to inhale the fungal spores at some point in their lifetime without developing an infection.4 8 On the east coast of Vancouver Island, C. gattii has been found in tree bark, soil, and water as well as in the air throughout the region between Victoria and Courtenay and on the Southern Gulf Islands. Recently, several air samples from the lower mainland of British Columbia's south west corner have been found to be positive for the fungus.49 A recent report indicated that all isolates from Vancouver Island have been serotype B with the majority exhibiting a random amplification of polymorphic D N A (RAPD) profile VGII (93%), with the rest being VGI (7%).' Ecological Niche Ellis and Pfeiffer, in 1990 provided the first great advance in understanding the ecological niche of C. gattii when they observed a specific association between C. gattii and two closely related species of the red gum group of eucalyptus trees: the river red gum, Eucalyptus camaldulensis6'310 and the forest red gum, Eucalyptus tereticornis50 Eucalyptus camaldulensis is indigenous to all mainland Australian states growing mainly along streams and rivers, whereas Eucalyptus tereticornis is common along the eastern coast of Australia as well as certain parts of Papua New Guinea. 5 1 When their observations were first published in 1992, Ell is and Pfeiffer indicated that these were the 8 only ecological niches of C. gattii, and that the distribution of human cryptococcosis corresponded to the distribution of these trees.6'5 0 A study in 1997 in India looked at almost 700 air samples taken from under trees which the origin could be traced to Australia. Five positive air samples were found under three Eucalyptus camaldulensis trees. The authors of this study pointed out that the distribution of Eucalyptus camaldulensis correlated with the distribution of human cases of cryptococcosis caused by C. gattii in northern India.2 8 A 1996 analysis of Australian isolates of clinical and eucalypt origin by R A P D revealed that all eucalypt isolates (n=45) and 92% of clinical isolates (n=48) exhibited a single major R A P D profile, designated VGI . 5 2 Serotyping indicated that all isolates were type B. 5 3 In 1996, however, there were four VGII isolates found that had originated in an area of Western Australia where none of the known eucalypt hosts existed, indicating the existence of at least one unrecognized natural source of C. gattii.53 At this time only VGI isolates had been cultured from the two known eucalypt sources.50 It was noted however, that a different species of the red gum group, Eucalyptus rudis, exists in Western Australia, where the VGII isolates had been found. 5 2 It is interesting to note that since this time Eucalyptus rudis has been shown to harbour C. gattii serotype B. 5 4 In 1997, Chen et al. reported that 7 of 9 clinical isolates from the Northern Territory were assigned to profile VGII with the remaining 2 assigned to VGI. The results of this study, Chen et al. suggested in 1997, strongly supported the existence of an alternative environmental niche of C. gattii.55 A study from Malaysia also indicated that the fungus was present in an area where none of the known eucalypt hosts existed.5 6 Eucalyptus trees have been exported commercially from Australia to other continents and have been established outside of Australia.8 Positive samples for C. gattii have been 23 28 27 29 obtained from eucalyptus trees in California , India , Italy and Brazil. It is interesting to note that all isolates of C. gattii from eucalypts have been serotype B. A literature review published by Malik et al. in 2003 found that there were several more environmental associations for serotype B that have been located in Australia, along with several others in South America. 5 4 This review also indicated that only one environmental association had been confirmed for serotype C. The findings from this study are presented in table 1. 9 Table 1 - Environmental associat ions of C. gattii as published by Malik et al . in 2003 Environmental Associations of Cryptococcus gattii serotype B: Australia Eucalyptus camaldulensis (River redgum) Eucalyptus rudis (Flooded gum) Eucalyptus gomphocephala(Tuart) Eucalyptus minita (Darwin Woollybuti) Eucalyptus microcorys (Tallowood) Eucalyptus largiflorens (Blackbox) Koala enclosures South America Moquilea tometose (pottery tree) Adenanthera pavonine (Circassian seed tree) Eryttrina velutina (coral tree) Eucalyptus tereticornis (Forest redgum) Eucalyptus tetrodonta (Darwin stringyback) Eucalyptus blakelyi (Blakely's redgum) Eucalyptus grandis (Flooded gum) Angophora costata (Smoot-barked apple) Syncarpia glomulifera (turpentine gum) Cassia grandis (pink shower tree) Ficus microcarpa (fig tree) Guettarda acreana (Guettarda) Environmental associations of C. gattii (C. bacillisporus) serotype C: Columbia Terminalia catappa (Almond tree) A study in 2000 looked at the existence of C. gattii in the north-eastern region.of Brazi l . ' 2 This study observed that six out of thirty-two tree hollows that were tested were positive for either C. neoformans or C. gattii and that 2 of the tree hollows were positive for both varieties of Cryptococcus. Long lasting positivity (19-36 months) and a significant number of colony forming units per gram (0.15-21.7 x 103 cfu/g) indicated that the trees had been colonized by the fungi. This was the first time that these two varieties of Cryptococcus were found sharing the same ecological niche. 1 2 Ellis and Pfeiffer concluded that plant debris, especially debris from eucalyptus trees, appears to be the natural reservoir for C. gattii.6 Although there have been many studies looking at the association between tree species and C. gattii, the role that trees play in the life-cycle of the fungus is not well known. 8 ' 5 6 Sorrell and Ellis, in a review of the ecology of C. neoformans and C. gattii, explained that the highest concentrations of C. gattii are found in woody debris within hollows of aged trees, where the fungus is protected from sunlight and from desiccation.5 6 Sorrell and Ell is explain that these hollows are appropriate for colonization and growth due to 10 the high concentrations of polyphenol compounds and lignin which can be used by C. gattii as a substrate for growth, due to its phenoloxidase activity.5 6 Several studies have looked into the question regarding how C. gattii circulates in the environment. Ellis and Pfeiffer in 1990 analyzed results from periodic air sampling and found that the dispersal of basidiospores was concomitant with the flowering period of the trees. 6 j 0 However, human cases of disease in Australia do not appear to be seasonal which led Sorrell and Bennett to suggest that there may be a primary association between the fungus and bark.5 6 The observation of high levels of C. gattii in woody debris in hollow trees has led to the suggestion that wood-inhabiting animals, such as tunnel-excavating caterpillars and beetles, act as vectors for the fungus.8 As an example Sorrell and Bennett suggested that cryptococcal basidiospores may be transported to this niche by animals living within a restricted range, or across greater distances by wind or birds.5 6 It is interesting to note that there is evidence that suggests that koalas appear to be capable of amplifying the number of cryptococci in certain environments.54 Lazera et al. suggested that simple disturbances at tree cavity entrances either by wind or the movement of small animals may be capable of dispersing C. gattii propagules into the air.' 2 In their paper, Lazera et al., also suggested that fallen trees and deforestation activities in endemic areas may be related to propagation and human infection. In summary, in 1997, almost 30 years had passed since C. gattii was first discovered and the ecology of the fungus remained poorly understood.56 1.2.3 - Cryptococcal Disease Cryptococcus has a world-wide distribution and, in addition to infecting people, it has caused disease in domestic and native animals, including cats, dogs, ferrets, horses, goats, sheep, cattle, birds, koalas and other marsupials.54 Infection by C. neoformans is well characterized and usually occurs in immunocompromised patients, whereas less is known about infections due to C. gattii, which usually produces disease in individuals with competent immune systems. 5 4 '" ' 1 4 C. gattii has also been characterized as more pathogenic than C. neoformans}4 The nature of the infectious propagule is uncertain but based upon size (less than 3 microns in diameter), it is possible that it is the basidiospore or desiccated yeast cells. 5 6 11 The demonstration of haploid fruiting under laboratory conditions and the observation of basidiospore-like cells in eucalypt debris3 0 lends credence to the hypothesis that the basidiospore is the form of Cryptococcus which is pathogenic for man and animals.5 6 Also, encapsulated yeasts display poor viability under conditions of nutrient deprivation and low moisture57, whereas, basidiospores are resistant to drying, are small (less than 2 microns in diameter), readily become airborne, develop into yeasts on culture, and are pathogenic for mice. 5 8 Kwon-Chung and Bennett explained in their 1992 paper that the best-characterized, major, virulence determinants in both C. neoformans and C. gattii are the polysaccharide capsule, the products of the laccase enzyme pathway, and the ability to grow at physiological temperatures, which all contribute to its ability to survive in the host.58 Odom et al. showed that it is the calcineurin gene that is essential for high temperature growth of the organism and its pathogenic action. 5 9 Cryptococcal infection is usually acquired by inhalation, although inoculation into damaged skin has been reported in the literature.60 Inhalation will typically result in clinical manifestation of pulmonary infection6 1, which may be followed by dissemination to other body sites including the central nervous system 6 2, skin 6 3 , and bone.6 4 Central nervous system complications such as nerve palsies and hydrocephalus occur more frequently and require surgical intervention more often after infection by C. gattii compared to infection with C. neoformans}3 It has been shown that clinical disease due to infection with C. gattii often results in focal pulmonary or cerebral mass lesions and is associated with high morbidity. 1 3 ' 6 5 It does not appear that the disease is capable of being transmitted from one affected animal to another thus cryptococcosis is not a contagious or anthropozoonotic disease.54 C. gattii and C. neoformans have the ability to grow at 37°C, which may in part explain its pathogenicity in mammals, because other members of the genus grow poorly at this temperature.54 C. gattii appear round and yeast-like, with a variably-sized polysaccharide capsule as its distinguishing feature54 This capsule, the authors explain, provides protection from environmental insults and the phagocytic response of the host. The fungus, when imbedded in tissues, can reproduce by forming one or two daughter cells, 12 or buds, that are connected to the parent cell by a narrow isthmus. The buds can break off throughout their growth and thus the cell population during infection can vary in size. 5 4 Until the recent increase in cases in British Columbia,,there had been no reported outbreaks of cryptococcal disease in the medical literature.66 Disease due to infection by C. neoformans is the most common cause of fungal meningitis worldwide and the fourth most common life-threatening infection in individuals with A IDS 5 8 , whereas infections due to C. gattii in AIDS patients remains rare.4 4 Fungal infections are not notifiable diseases so there are few precise estimates of incidence of infections due to C. gattii available.6 7 This being the case, it has been suggested that the incidence of isolations and infections associated with unusual yeasts is significantly underreported.67 In British Columbia there is a central veterinary laboratory that services both B.C. and Alberta. Typically this laboratory will diagnose 4 to 6 animal cases of cryptococcosis every year, but by August 2001 it had diagnosed 12 cases of cryptococcosis in animals on Vancouver Island alone. The B.C. Centre for Disease Control became aware of a concomitant increase in human cases in the same geographic location.6 8 The resultant report, by Craig Stephen and colleagues was the first report of a large-scale outbreak of cryptococcosis that involved humans, terrestrial animals, and marine mammals.6 8 The vast majority of clinical and veterinary infections were caused by isolates of the molecular type VGII, but two were caused by VGI . 1 Humans Kwon-Chung and Bennett suggested that the incidence of cryptococcosis was higher in areas with warm summer and winter temperatures and those with higher average maximum rainfall. 1 6 Similar to the distribution of environmental isolations of the pathogen, cases of cryptococcosis due to C. gattii have generally been reported in countries with tropical and subtropical climates such as India 6 9, New Guinea 7 0, Brazil 7 1 , Singapore72, Zaire 7 3, Australia 7 4, and southern California in the United States.75 Ellis and Pfeiffer made the observation in the early 1990's that the distribution of human disease generally corresponded to the distribution of host eucalypts.6'4 6 Cryptococcal disease may develop in humans following environmental exposure and inhalation of the infectious propagule.8 Infections caused by the fungus can be effectively 13 treated. Ghannouin indicated that more drugs are being approved by the FDA for treating cryptococcal disease along with the development of new strategies for treatment.76 It has been suggested in several papers that C. gattii be considered a primary pathogen.14' I 5 ' 6 5 Sorrell points out that disease caused by C. gattii is different from that caused by C. neoformans because it results in a higher incidence of cryptococcomas in lung and brain, increased neurological morbidity, and a slower response to antifungal therapy.8 Several studies are currently looking into the mechanism of how Cryptococcus passes the blood brain barrier and enters into the central nervous system. 7 7 ' 7 8 In an information fact sheet regarding cryptococcal disease48, the B.C. Centre for Disease Control explained that symptoms of the disease appear 2-9 months or longer following exposure and include: Prolonged cough Sharp chest pain Fever Unexplained shortness of breath Night sweats Severe headache Weight loss A chronic cell mediated immunity defect has been identified in a small number of apparently normal individuals which appears to predispose them to disseminated infection.7 9 Cryptococcal meningitis due to C. gattii, however, is not always found coupled with a chronic cell mediated immunity defect. Cryptococcal disease due to C. gattii has been associated with either transient immunosuppression preceding infection, or inhalation of a heavy inoculum of airborne propagules in the environs of flowering eucalyptus trees.79 In British Columbia, reported cases of cryptococcal disease due to C. gattii began in 1999. A report by the B.C. Centre for Disease Control indicated that there had been 59 cases of disease including 7 cases in 1999 with one death, 17 cases in 2000, 16 cases in 2001, and 19 cases in just the first 8 months of 20 02. 4 8 Before 1999 there was an average of 2 to 3 cases reported annually in B.C. which were caused by C. neoformans.48 A study by Fyfe et al. indicated that the 38 patients with cryptococcal disease in British Columbia between 1999 and 2001 ranged from 20 to 82 years of age. 8 0 About one quarter of these 14 38 cases presented with meningitis whereas the rest presented with pulmonary infection. Fyfe et al., in this report that has yet to be published, indicated that the most common self-reported symptoms were cough (57%), shortness of breath (54%), night sweats (54%) and fever (54%). Chest x-rays at the time of presentation showed the following: single or multiple pulmonary nodules in 26 (68%), infiltrate in 6 (16%), pneumothorax in 1 (3%) and normal x-ray in 5 (13%). Five cases with normal chest x-rays all presented with meningitis.80 The use of oral systemic steroids increased the odds of disease 8.11 times (1.74 - 37.80). Three outdoor activities, Fyfe et al. explained, chopping wood, pruning, and branch clean-up, were protective against infection. The authors suggested that low levels of exposure to C. gattii may result in immunity and prevent symptomatic infection if exposed to higher doses. Cases and environmental isolates all shared a common molecular genotype.80 This report by Fyfe et al. has been met with some controversy and has yet to be replicated. The B.C. Centre for Disease Control indicated that the occurrence of the fungus on Vancouver Island means that physicians and the 48 public should be alert for symptoms and seek early diagnosis and treatment. Animals Not only is it important to study cryptococcosis in animals to assess the risk of disease in the animal population, but animal data can also be used in assessing the implications of cryptococcal exposure in humans. Sorrell and colleagues indicate that the location in which human exposure occurs is rarely identifiable because of their propensity to travel widely and the relatively long incubation period of the disease.53 They explain that it is important to study animal disease because animals generally reside within a restricted territorial range.53 Animal cases of cryptococcosis have been described in the literature in cats6, dogs6, koalas 6 ' 8 1, goats42, a cheetah82, ferrets80, horses8 3, birds 8 4 ' 8 5, and porpoises. 8 0 ' 8 6 The first veterinary case of cryptococcal disease from New Zealand occurred in a North Island kiwi and was described in 1995.8 7 The kiwi has a lower body temperature than mammals, which the authors suggest, could increase the animals susceptibility to the organism. It is interesting to note that several months prior to clinical infection a mulch of Eucalyptus • • 87 has been spread throughout the kiwi enclosure. 15 In B.C., veterinarians were asked in 2003 to help monitor the progress of animal cases of cryptococcosis along the east coast of Vancouver Island so that animals could work as sentinels for human infection.4 9 The majority of the initial animal cases were identified through a single private veterinary laboratory that serves both British Columbia and Alberta as described earlier in this review.6 8 A study of the 34 cases of cryptococcosis that were identified in animals in 2000 and 2001 reported that none of the 17 cats, 13 dogs, 2 ferrets, and 2 porpoises were known to be immunocomprom ised. 8 0 By the-end of March 2002 Fyfe et al. reported that a total of 45 lab-confirmed animal cases had been identified.8 0 These authors indicated that no seasonal pattern of disease could be identified and that the date of exposure and the reported onset of symptoms ranged from 2 days to over two months. Nine of the animal owners that had been interviewed in this study had no travel history to the central part of Vancouver Island, but only 3 of these failed to identify any travel history or residence on the east coast of Vancouver Island.6 8 Lester et al., in a retrospective study of animal cases in B.C., analyzed the results of cryptococcal disease in a pink-fronted cockatoo, two ferrets, 20 cats, and 15 dogs. 8 8 The authors reported that there were no breed or sex differences found in infected dogs or cats. In February 2006 there had been over 200 reported animal cases since the outbreak began, though this estimate is surely low due to non-reporting of animal cases.4 9 1.2.4 - Laboratory Identification Kwon-Chung and Bennett noted that the basidiospore of C. gattii is dry and non-capsulated, but when put on agar medium, they swell and develop into encapsulated, budding yeasts within a few hours.58 The first stage in the identification process is to culture the specimen on Staib (bird seed) agar.89 Malik et al. pointed out that using Staib agar containing antibiotics can be useful especially when sampling sites are expected to be heavily contaminated.54 The antibiotics suppress growth of bacterial contaminants, while identifying colonies of Cryptococcus from other yeasts and filamentous fungi is achieved by the brown-colour-effect they produce on the agar 5 4; when cultured on Staib agar plates at 30°C cryptococcal colonies exhibit a brown pigment due to phenoloxidase production.9 0 Malik et al. observed that the main difference between C. gattii and C. neoformans when cultured onto Staib agar is the fact that C. gattii colonies are typically much more mucoid than C. neoformans.54 16 The next step in the identification process differentiates C. gattii from C. neoformans by culturing the brown colonies onto canavanine-glycine-bromothymol blue agar. Kwon-Chung and colleagues have indicated that isolates of C. gattii assimilate D-proline, D-tryptophan and L-malic acid and can utilize creatinine as a source of nitrogen and glycine as a source of carbon and nitrogen.19 It is resistant to cycloheximide (1.6 ug/ml) and to canavanine (which inhibits the growth of C. neoformans by disrupting protein and R N A synthesis). Use is made of these differences for biotyping on canavanine-glycine-bromothymol blue agar.19 It was shown that the canavanine-glycine-bromothymol blue agar gives a clearer and more accurate distinction between C. neoformans and C. gattii than creatinine-dextrose-bromothymol blue and glycine-cycloheximide-phenol red j . 19 media. 1.2.5 - Air Sampling for Cryptococcus gattii There are several different types of bioaerosol monitoring equipment. A study by Staib in 1985 was a preliminary investigation of the usefulness of the RCS air sampler for epidemiological studies of cryptococcosis.9' Staib explained that the RCS has a great advantage over the Andersen sampler due to the fact that it is handy and independent of electric main. It was found that the RCS appeared expedient due to observations from two separate examinations in the cage of a Palm Cockatoo.9' Further studies must show if there is a difference in the quantitative detection of cfu of this fungus between the RCS and the Andersen sampler system. 9 2 ' 9 3 1.2.6 - Determinants of Exposure Individuals working under the canopies of trees may be exposed to aerosolized matter due to contamination of the areas in which they are working and the materials that they are working with, and also as a result of the tasks that they do. There is very limited evidence in the literature surrounding arborists and watershed maintenance staff and what it is that they do during the course of their work. No literature was found regarding worker exposures to C. gattii. Liao et al. indicated that seasonal patterns should be considered whenever exposures to fungal spores are being investigated.94 This study, as well as several others has found that higher airborne fungal concentrations are found in the drier summer months, in comparison to winter months.9 4"9 6 Other studies, however, have shown the opposite, that 17 airborne fungi concentrations are higher in the wetter, winter months. Specifically related to C. gattii, Ellis and Pfeiffer suggested that the dispersal of basidiospores occurred during the flowering period of the eucalypts with which it had been associated.6'30 Although there were no studies that explained C. gattii exposures under working conditions, there have been several studies that have investigated bioaerosol exposures amongst sawmill workers. Duchaine and Meriaux, in their study of workers' exposure to airborne microfungi, concluded that working in sawmills is associated with bioaerosol exposure and respiratory health problems.97 In this study there were fifty work sites that were sampled, including debarking, sawing, planing and sorting. Over fifteen hundred strains of microfungi were isolated. Another study of the respiratory health impacts of working in Canadian sawmills measured respirable dust, bacteria, endotoxins, and molds from 17 sawmills. 9 8 The investigators had workers complete respiratory health questionnaires, as well as take part in lung function testing, skin-prick tests, and venous blood sampling for specific immunoglobulins against molds found in the sawmills. The authors found that the workers had normal lung functions, and most respiratory symptoms could be attributed to the worker's smoking history. High levels of specific antibodies were seen in some of the workers.9 8 A study of Norwegian sawmill workers by Eduard et al. measured the personal exposure of wood trimmers to mold spores and wood dust.99 The authors explained that antibody levels were higher and symptoms suggestive of mucous membrane irritation, chronic nonspecific lung disease, allergic alveolitis, and organic dust toxic syndrome were more frequently reported by wood trimmers than by planing operators. 18 1.3 - RESEARCH QUESTIONS This research has been designed to answer the following four research questions: 1. Are arbor workers and Capital Regional District Watershed workers on Vancouver Island and the Gulf Islands potentially exposed to C. gattii? A priori hypothesis: These workers are at risk of exposure whenever they work in areas in which C. gattii has colonized trees 2. What conditions affect airborne C. gattii concentration? A priori hypotheses: • Meteorological conditions will affect airborne C. gattii concentration • Summer months wil l lead to higher C. gattii concentrations • Rainy conditions will decrease concentrations of viable airborne C. gattii due to surface tension inhibiting the fungus from being aerosolized • Clear conditions wil l decrease concentrations of viable airborne C. gattii due to desiccation and ultra-violet radiation • High relative humidity will lead to lower airborne C. gattii 3. Are arbor workers and Capital Regional District Watershed workers on Vancouver Island and the Gulf Islands at an increased risk of exposure to C. gattii compared to the general population or other work populations due to the work that they do? A priori hypothesis: That the work task wil l have an affect on airborne C. gattii concentrations • Cutting tasks will lead to higher C. gattii concentrations 4. What are the arbor worker and Capital Regional District Watershed worker population demographics? 19 2 - MATERIALS AND METHODS 2.1 - STUDY OBJECTIVES AND DESIGN This was a cross sectional field study that was comprised of: • Environmental sampling for C. gattii • Occupational sampling for C. gattii • Task analysis of arborists and arbor-care workers • Pilot questionnaire data collection Environmental sampling investigated the area under study for the presence of C. gattii. Occupational sampling consisted of sampling the air while workers completed their work tasks in addition to environmental sampling of the work site area. Subsequent to sampling, lab work was necessary to culture the samples in order to determine the presence of C. gattii and to quantify the potential exposure to the organism. Task analysis was performed on arbor workers in order to determine the typical work tasks that this particular worker population does over the course of their work. This task analysis was also done so that the results of the occupational sampling could be of use when attempting to compare arbor workers to other occupations that may be exposed to C. gattii due to similar working conditions and tasks. Questionnaire data including work history, health status, and travel history were completed by a number of arbor workers as well as the group of C R D workers. 20 Figure 2 outlines the collection of data and how it relates to the major goals of this research. Each number represents a stage in this project. The arrows from each level to the next indicate how one level of information has been used to describe the next level. It is important to note that there are two distinct types of data that have been used in this project. The first, called 'Distribution' in figure 2, relates to non-occupational sampling for C. gattii, including tree, soil, air and water samples. The second, called 'Occupational Sampling', represents all of the occupational data that was collected over the course of this study and includes air sampling, task analysis and questionnaire data. Data Types Data Collection Ana ita lysis DISTRIBUTION OF C. GATTII W A T E R S A M P L E S SOIL S A M P L E S G E O G R A P H I C A L DISTRIBUTION OCCUPATIONAL SAMPLING FOR C. GATTII T R E E S A M P L E S AIR S A M P L E S AIR S A M P L E S T R E E T Y P E S E A S O N A L P A T T E R N S OF C O N T A M I N A T I O N T A S K A N A L Y S I S Q U E S T I O N N A I R E W O R K T A S K Research Goals DETERMINANTS OF AIR CONTAMINATION DETERMINANTS OF EXPOSURE WORK POPULATION CHARACTERISTICS Figure 2 - Thesis overview. Each level represents a stage of this project from the planning of data collection to the results of data analysis. 2.2 - STUDY POPULATION There are two groups of workers who were chosen for this study. They were: (1) arbor workers and (2) C R D Watershed workers. These workers were targeted due to the nature of the work they do and the areas in which they work. There is the potential that both of these occupational groups are exposed to C. gattii during their work. There is also the potential that the levels of exposure that these workers experience may exceed exposures to the general public due to the time spent under and around the canopies of trees and the work tasks being done which create aerosols of wood dust and C. gattii propagules. Arbor workers were selected as an appropriate group of workers to take part in this study due to the nature of their work and the large number of employees working in the field along the east coast of the island. These workers perform work for private homes, business, and municipalities (cities and parks). They carry out a variety of tasks that include, but are not limited to: tree pruning, tree clearing, debris removal, stump grinding, and tree removal. The group of C R D Watershed workers was chosen due to their broad range of work activities, the area in which they work, as well as their willingness to participate in this study. The C R D Water Management maintains and operates the Sooke watershed which is located close to Goldstream Provincial Park on the Island Highway between Victoria and Duncan, an area in which C. gattii had not been found prior to this study but which is in relatively close proximity to several highly contaminated areas. This group was also chosen because the C R D watershed is located in the Mountain Hemlock biogeoclimatic zone, where there have been limited discoveries of C. gattii but which is close to areas of the Coastal Douglas Fir biogeoclimatic zone where positive samples have been found regularly. There were 25 workers in this group, most of whom perform their work tasks, including tasks such as danger-tree removal, road building and fire suppression, outdoors during upkeep and maintenance of the C R D Watershed. 2.3 - DATA COLLECTION Ethical approval for the occupational portion of this study including the introductory letter, the consent form, and the questionnaire was obtained from the University of British Columbia Behavioural Research Ethics Board on July 14 t h 2005 (appendix A). 22 2.3.1 - Environmental Sampling to Determine the Presence of Cryptococcus gattii Environmental sampling for C. gattii has been ongoing since 2001. The environmental sampling was largely convenience sampling with sample locations at the sides of roads, in public parks, and on private properties of individuals who have a special interest in C. gattii (e.g. homes of animal cases, homes of human cases and relatives and friends of research staff from the School of Occupational and Environmental Hygiene). Regions of interest were selected due to locations of cases of human or animal illness, though much of the time the sample locations were randomly selected by researchers. Tree samples were largely restricted to trees that are immediately adjacent to walking paths, roads, or property lines. Upon locating an area of interest, (either at random or due to cases of cryptococcosis), an Etrek GPS device was used to record the latitude, longitude and spatial accuracy for the location of each sample. Where tree cover limited the use of the GPS device a street address and/or an area description was recorded on the field sampling sheet. There are four general categories of samples that were taken throughout this study. They are: • Tree • Soil • A i r • Water The specific sample location was carefully described so that re-sampling of the location remained a possibility in the case of future studies. Each sample was given a distinct field sample code. The sampling date was recorded along with weather conditions including temperature, and relative humidity. Table 2 presents a brief description of how each sample type was collected. 23 Table 2 - Sample taking procedure for tree, soil, air and water samples. Tree Trees were sampled using Starswab II swabs. The initial swabbing of a tree was called a composite swab and entailed sampling cracks and crevasses on all four sides of the tree. Samples were stored in the Starswab II™ casing in clear Amies media and refrigerated until being brought back to the laboratory for culturing. Soil Soil samples were taken using a Ziploc bag. The investigator pushed their hand into the bag from the bottom and then gathered a soil sample with the bag. Alternatively a small trowel (sterilized by wiping with alcohol) was used in some instances to collect soil into the Ziploc bag. The bags were then stored in a cooler until brought to the lab. Air Air samples were taken using a Reuter Centrifugal Sampling (RCS) impaction device, Andersen N-6, or the Andersen 6-stage sampler. The devices were wiped down using alcohol wipes before use. The RCS device was loaded with a strip filled with Staib agar media that had been stored in individual casing in a cooled environment. Two minute samples were then taken using the RCS sampler. The strip was then re-inserted into its casing and put into a cooler to restrict growth until the sample was brought back to the lab. Andersen sampling devices were loaded with Petri dishes filled with Staib agar. Ten minute samples were taken using the Andersen samplers. Following the sample being taken the Petri dishes were labelled, sealed, and then put in a cooler for transport to the lab. Water Water samples were collected from the surface of a body of water with a sterile plastic container. Water samples were stored in a cooler until they were transported to the lab. After taking a sample it was transported to the laboratory at the School of Occupational and Environmental Hygiene at the University of British Columbia for culturing. Each sample was given a Sample Identifier Code; a reference code that referred to the distinct tree the sample was associated with. Culturing of the samples was first done on Staib agar {table 3) to determine the presence of Cryptococcus. Table 3 - Ingredients used to make Staib agar for C. gattii culturing (amount per litre of agar) Ground Niger Seed 23.33 g Potassium Phosphate 0.17 g Creatinine 0.13 g Agar Powder 2.89 g Glucose 0.17g Chloramphenicol 0.05 g Distilled Water 1000 mL Alcohol 1.33 mL 24 Once plated, following the procedures described in table 4, the culture was allowed to incubate for 48-96 hours at 30°C. Throughout this period the investigator observed each plate to determine the presence of brown yeast. Table 4 - Methods used to plate samples on Staib agar Tree Tree samples were plated onto Petri dishes filled with Staib agar in order to facilitate a semi-quantitative measure of contamination. The swab was swept across the left half of the Petri dish. The upper left quarter of the Petri dish was then swept with a heat-sterilized platinum loop in order to streak the upper right quarter. After re-sterilizing the loop the upper right quarter was used to streak the lower right quarter. The following code was used to quantify the growth: 1 + - Growth occurred in the lower left corner only 2+ - Growth occurred in both the lower and upper left quarters 3+ - Growth occurred on the left side and in the upper right quarter 4+ - Growth occurred in all 4 quarters of the Petri dish Soil Approximately 2 grams of the soil was transferred from the Ziploc bag into a 50 ml vortex tube. The lab code and weight were written on the side of the tube. Ten millilitres of sterile water was then added to the soil using a 10 ml disposable pipette and auto pipette. After water has been added to the sample it was vortexed for 10 seconds. A pipette was then used to pull one hundred microlitres out of the solution. This solution was then released onto a Petri dish with Staib agar and spread with a heat sterilized glass rod. Brown yeast colonies were counted following 48 hours of incubation. Air RCS strips or Andersen Petri dishes were brought back to the lab in a chilled cooler. The air sample was put directly into the incubator. Incubation for approximately 48 hours was followed by the investigator reading the plate for brown yeast. Colonies of brown yeast were counted. Water Water samples were brought into the lab in plastic containers. Two hundred millilitres of the water sample was pulled through a Fisherbrand sterile 0.45 micron mixed cellulose esters filter using a vacuum pump. The filter was removed from the apparatus using sterilized tweezers and placed on a Petri dish filled with Staib agar. The Petri dish was then placed in the incubator. Brown yeast colonies were counted following 48 hours of incubation. Positive samples were streaked onto a new Petri dish filled with Staib agar until a pure culture was obtained. Pure cultures were then streaked on Canavanine-Glycine-Bromonthymol (CGB) agar to differentiate between C. gattii and C. neoformans. C. 25 gattii cultures turn blue within one to five days. A description of CGB agar is found in table 5. Table 5 - Ingredients used to make Canavanine-Glycine-Bromothymol (CGB) agar to diferentiate C. gat/V from C. neoformans™ (amount per litre of agar) Glycine 10.00 g Sodium bromothymol blue 0.05 g Potassium Phosphate 1.00 g Thiamine- HCI 1.00 mg Magnesium Sulphate 1.00 g L- canavanine sulphate 30.00 mg Agar 15.00 g Distiled Water 776.5 mL Data accrued from the field samples that were taken during this study were then added to a Microsoft Access™ database that contained all of the past environmental samples that had been accumulated by Dr. Bartlett and her team at the School of Occupational and Environmental Hygiene at the University of British Columbia. This database has been maintained by Dr. Bartlett and Sunny Mak, a GIS technician at the B.C. Centre for Disease Control. This thesis reports on data in this database up to November 18th, 2006. A Microsoft Excel™ spreadsheet was created from the Microsoft Access™ database including 9,306 environmental samples. Site specific information regarding sample type (tree, soil, air, or water) and location (latitude/ longitude and city) were included in this spreadsheet. This database also included information indicating whether samples were positive or not, and the concentration (cfu of C. gattii) of each sample. For this thesis the database contained only samples that were taken on Vancouver Island or the Southern Gulf Islands. Due to concerns surrounding over-sampling being one in various geographic locations the database was divided to contain only the first sample of tree, soil, and water sample types for each distinct tree (Sample Identifier Code). Air samples, on the other hand, were taken to contain only the first sample for each Sample Identifier Code in each month. Table 6 shows the variables that were included in this 'First Sample' database. The First Sample' database was subsequently imported into SPSS (Version 13) for analysis. 26 Table 6 - Variables included in the First Sample database Identifier Variables Tree ID Distinct Sample Identifier Code (normally a distinct tree) Result Variables Pos/Neg Whether sample was positive or negative for C. gattii Concentration Concentration of C. gattii in the sample Tree - Quantitative assessment of growth on agar (1-4) Soil - Colony Forming Units per Gram Air - Colony Forming Units per Cubic Meter Water - Colony Forming Units per Litre Descriptor Variables City City or township from which the sample was taken Date Day, Month, and Year on which the sample was taken Type Sample Type: Tree, Soil, Air, or Water Air Sampler Type of air sampling equipment used (RCS, Andersen N-6, Andersen 6 stage) Tree Species Species of tree from which the sample was taken. Alder Alnus rubra Arbutus Arbutus menziesil Red Cedar Thuja plicala Douglas Fir Pseudotsuga menziesii Hemlock Tsuga heterophylia Maple Acer macrophilium Oak Quercus garryana Pine Pinus Ever/Decid* Evergreen or Deciduous tree species Environmental Condition Variables Temperature* Temperature at the time the sample was taken (Celsius) RhP Relative Humidity at the time the sample was taken (%) Weather' Qualitative Weather Description Clear Overcast Rain * - Tree type was recoded into evergreen and deciduous. All stumps, downed logs, shrubs, fence posts, and other surfaces for which the tree type was unknown were recorded as 'unknown', t -Temperature, relative humidity, and a weather description were not available from the field data for all of the records. This information was subsequently located from the historical archives on the Environment Canada website. 1 0 0 27 Air sampling data was lognormally distributed and therefore air concentration data was log transformed (base e) prior to analysis. A l l air samples that were negative for C. gattii were assigned the value equal to one half of the limit of detection for the sampler that was used. These values were 0.89 cfu/m 3 for samples taken with the RCS and 1.77 cfu/m 3 for samples taken with the Andersen sampler. Relative humidity values were adjusted for regression analysis so that the baseline value represented the lowest measured relative humidity when sampling. This was done by subtracting 27% from each relative humidity measurement. 2.3.2 - Occupational Sampling for Cryptococcus gattii The C R D Watershed management were contacted directly and asked to participate in this study. Management was interested in having environmental samples taken because no samples had previously been taken from the watershed, and the management wanted to be prepared for questions from the public. Environmental samples in the watershed were first taken to determine the presence of C. gattii in the area. Due to a lack of C. gattii in the watershed no occupational air sampling was performed on C R D watershed staff. Contact information for arbor workers was obtained through www.mytelus.com and www.canada411 .com as well as regional phone books. An introductory letter (appendix B) was written to explain the study and to invite workers to participate. Letters were then followed up by telephone contact during which workers were invited to participate in the study and their initial questions were answered. Workers in areas where environmental presence of the organism had been discovered were asked to take part in the sampling portion of the study whereas the workers in other areas were asked to fill out questionnaires (over the phone). There were 172 companies that were initially targeted by this research. Arbor workers were asked to participate in the sampling procedure in which the investigator accompanied them to their worksite and sampled at the area where work was being done. When consent to the study was given it was noted in the occupational sampling database. When a sampling trip was planned the investigator travelled to a specific region and called the workers to set up the sampling visit the night before a sampling day. This approach appeared to be appropriate due to the nature of arbor work, 28 and based upon worker suggestions, however it led to logistical problems as many of the sampling days were missed because workers in the area were not able to help out on the specific days for a variety of reasons (i.e. doing appraisals, not working, in an area where they were uncomfortable bringing investigators). When a company was able to have the investigators join them the workers were first asked to fill out a consent form (appendix C). Once consent was obtained the investigators attempted to take area samples (tree and soil) as well as air samples prior to any work being done. Air samples around the worker were then taken during their normal work activities using similar sampling methods as those described earlier. Because air sampling was done using area samplers (Andersen N-6, Andersen Six-Stage, or the RCS) none of the air samples could be considered personal samples. In addition, it was impossible to sample the arbor workers' breathing space when they climbed trees. With these limitations, occupational sampling was done as close as the investigator could safely get to the worker without endangering themselves or their sampling equipment in an attempt to get air samples that were most representative of the air in the workers' actual breathing zone. The location of the work site, including city/town, street address, and GPS location were recorded. A description of the location of the tree being worked on was also noted. Worker activities and weather conditions were recorded throughout the sampling procedure whenever possible. Also, as a subset of occupational exposure sampling, several contaminated logs were used when performing simulated tasks in order to sample the potential for exposure to C. gattii under working conditions. Experienced individuals were recruited to perform specific tasks on the contaminated logs. Work on these contaminated logs was done in an area from which C. gattii had been cultured on numerous occasions. The workers and the areas under study were protected from exposure and contamination by using N-95 respirators and tarps, respectively. A Microsoft Excel™ database was constructed that contained all of the occupational sampling data. The simulated work sampling was included in this dataset, although the sample location that was recorded was the location from which the wood was taken, not where the work was done. The data was cleaned and then imported into SPSS (version 29 13) for analysis. The following variables were included in the occupational sampling database: Table 7 - Var iables from occupational sampling Identifier Variable Tree ID Distinct Identifier of Sample Location Result Variables Pos/Neg Whether sample was positive or negative for Cryptococcus gattii Concentration Concentration of Cryptococcus gattii in the sample Tree Quantitative assessment of growth on agar (1-4) Soil Colony Forming Units per Gram Air Colony Forming Units per Cub ic Meter Descriptor Variables City City or township from which the sample was taken Site Distinct work site code Date Day, Month, and Year on which the sample was taken Type Sample Type: Tree, Soi l , Air Air Sampler Type of air sampling equipment used ( R C S , Andersen N-6, Andersen 6 stage) Meteorological Variables Temperature* Temperature at the time the sample was taken (Celsius) RH* Relative Humidity at the time the sample was taken (%) Weather* Qualitative Weather Description Clear, Overcast .Rain Work Specific Variables No Work Prior to any work being done Worker, while up the tree, used a chain saw to cut branches off of the tree Worker, while on the ground, used a chain saw to cut the tree Worker used a mechanical chipper to cut branches and debris into a mulch-like consistency Worker cut rounds of wood with axe into p ieces for burning Use of a large portable machine with which the stump was chipped to about 6 inches under ground After all work was finished at the site Limbing Chain Sawing Chipping Bucking Stump Grinding No Work- Post Work Other Undefined tasks * - Temperature, relative humidity, and weather description data was obtained from the Environment Canada website to complete the records historical data a rch i ves . 1 0 0 30 Again, air sampling data was lognormally distributed and therefore air concentration data was log transformed (base e) prior to analysis. A l l air samples that were negative for C. gattii were assigned the value equal to one half of the limit of detection for the sampler that was used. These values were 0.89 cfu/m 3 for samples taken with the RCS and 1.77 cfu/m 3 for samples taken with the Andersen sampler. Relative humidity values were adjusted for regression analysis so that the baseline value represented the lowest measured relative humidity when sampling in occupational settings. This was done by subtracting 30% from each relative humidity measurement. 2.3.3 - Questionnaire The entire population of 172 arbor workers, as well as the group of workers in the C R D Watershed, were given an introductory letter. A l l participants who were contacted were asked to complete interviewer administered questionnaires in person. It was decided later that workers would be asked to fill out questionnaires during a phone interview due to the difficulty setting up appointments for personal interviews. The questionnaire used can be found in appendix D. This questionnaire was developed by Dr. Hugh Davies and Dr. Karen Bartlett of the School of Occupational and Environmental Hygiene and is an adaptation of the questionnaire used by McDougall and Fyfe at the B.C. Centre for Disease Control in a previous study.68 The questionnaire questions related to smoking are those found in the American Thoracic Society's questionnaire.101 The questionnaire contained information about personal characteristics, work history, travel history, as well as recent health and smoking status. Questions relating to work tasks were found on pages 2-5 of the questionnaire. A questionnaire dictionary was created that outlined the number of characters that each variable contained, variable names and a description of what the variable name represented. This questionnaire dictionary was used to allow accurate data transferal into electronic form. Completed questionnaires were cleaned for accuracy and coded for data entry purposes. The questionnaires were then delivered to a data entry company who entered the data 31 into electronic format. Once entered into a spreadsheet the data was checked for errors and variable names were assigned to the columns of data. The data was imported into SPSS (version 13) for analysis. 2.3.4 - Task Analysis Task analysis was done, whenever there was adequate time, while the occupational sampling of the arbor workgroup was performed. When task analysis was performed the information regarding task and time of day was recorded for all of the workers at a given work site. Work tasks were recorded at 15 minute intervals and categorized into the following: Dragging This task entailed dragging branches and tree debris from the area where it was cut to the area where the chipper was located. Chipping Operation of a mechanized chipper included managing the controls and feeding branches and debris into the device which chipped wood pieces, with diameters up to approximately 10 inches, to a mulch-like consistency. On-looking Surveying the site, watching other workers, discussing job, and breaks. Chain Sawing on Ground Felling trees or cutting logs with worker on the ground. Limbing on Ground Removal of branches from tree after it has been felled. Limbing on Tree Removal of branches from tree while the tree was upright. The worker either climbed the tree or used a mechanical device to access branches. Bucking Using an axe to chop rounds of wood into smaller, firewood size pieces. Time at each task was computed based upon the intervals that were recorded (15min intervals). This information was imputed into an excel spreadsheet and then imported into SPSS (version 13) for analysis. 32 based upon their job title. These were: Each crew would have one worker falling into this group. This worker would typically be the more experienced worker in the crew and be in charge of the work being done at the specific work site. The younger and less experienced members of the work crew. 2.4 - DATA ANALYSIS Al l data analysis was performed using SPSS version 13. 2.4.1 - Environmental Sampling to Determine the Presence of Cryptococcus gattii Variables were investigated using descriptive analysis. Chi-squared values were calculated to determine differences in the proportions of positive samples for C. gattii for categorical variables, such as area, tree species, and weather descriptor. One way Analysis of Variance (ANOVA) was used to determine whether there were significant differences in the mean concentrations of C. gattii in air for the same categorical variables. When significant differences were found between groups Tukey's Post Hoc test was used to determine where the differences existed. Pearson correlation coefficients were used to observe relationships between the airborne concentrations of C. gattii and continuous variables. Linear regression was used to describe relationships between the concentrations of C. gattii and continuous variables. A correlation matrix was constructed to observe whether the continuous independent variables were significantly related to one another. The exclusion criteria for a multiple linear regression model was determined to be a Pearson correlation coefficient greater than 0.7. Backward stepwise linear regression was used to create a determinants of airborne contamination model. Removal criteria for the model was set to p > 0.05. Workers were categorized Arborist/Owner Arbor Laborer 33 2.4.2 - Occupational Sampling for Cryptococcus gattii Similar statistical methods as those used in describing the environmental sampling data were used to describe the occupational sampling data. This included the same exclusion criteria for the correlation matrix as well as the same removal criteria for the multiple linear regression model that was used to construct a determinants of exposure model. 2.4.3 - Questionnaire Descriptive statistics were performed on the questionnaire data and can be found in the results section of this report. The dataset was split by the work group variable prior to analysis in order to facilitate a comparison of the two work groups. 2.4.4 - Task Analysis Al l of the recorded time intervals in which workers performed specific tasks throughout their shifts were first calculated as percent of the work shift. The dataset was divided in order to allow comparisons between the two work groups: Arbor Laborer and Arborist/Owner. Descriptive analysis allowed calculation and comparison of the group mean and standard deviation of the percent of the work shift performing specific work tasks. T-tests were used to test for differences in work tasks between the two groups of workers. 34 3 - R E S U L T S 3.1 - FACTORS RELATED TO CRYPTOCOCCUS GATTII CONTAMINATION IN NON-OCCUPATIONAL SETTINGS The following section reports the results of sampling for C. gattii that has been done on Vancouver Island and the Southern Gulf Islands since 2001. The 'first sample' database was used to describe the distribution of C. gattii in the study area and C. gattii in air from sampling in non-occupational settings. 3.1.1 - Geographical Distribution of Cryptococcus gattii Water, soil, tree, and air samples were taken from locations on Vancouver Island and the southern Gulf Islands to determine the geographical distribution of cryptococcal contamination. Sampling from the Capital Regional District Watershed, where a higher concentration of samples were taken, is also described. Vancouver Island and Southern Gulf Islands A total of 4,787 samples were included in the geographical 'First Sample' database. These samples were taken between October 2001 and November 2005. The environmental samples included 2,561 (53.5%) tree samples, 1,464 (30.6%) soil samples, 676 (14.1%) air samples, and 86 (1.8%) water samples (figure 3). Water Swab Figure 3 - Distribution of sample types from the first sample database 35 Data was collected since 2001 in an effort to locate and describe the ecosystem of C. gattii which was previously unknown for B.C. Tree samples were taken, and are the majority of samples, because C. gattii was known to colonize trees. As the ecosystem became clearer, more sample types were introduced in order to get a more complete understanding regarding C. gattii in the B.C. environment. Table 8 shows the number of positive samples for Cryptococcus gattii and the percent positive for each sample type. Table 8 - Total number, number positive for C. gattii, and percent positive for C. gattii in each sample type from the first sample database Total Number Number Positive Percent Positive Tree 2,561 247 9.6 Soil 1,464 160 10.9 Air 676 158 23.4 Water 79 7 . 8.1 Total 4,787 565 11.8 A description of air sampling results are given in table 9. Table 9 - Descriptive statistics for airborne C. gattii measurements from the first sample database Number of Air Mean Standard Range Geometric Geometric Measurements (cfu/m3) Deviation Mean Standard , Min Max , (cfu/m ) , , (cfu/m ) Deviation (cfu/m3) (cfu/m3) 676 46G 258.0 61) 3,750.0 Z I 57 Table 10 presents the results from the 158 air samples that were positive for C. gattii. Table 10 - Descriptive statistics for all positive airborne C. gattii measurements from the first sample database Number of Mean Positive Air (cfu/m3) Measurements 158 196 509 2 3,750 33 6.0 Standard Range Geometric Geometric Deviation Mean Standard Min Max (cfu/m ) (cfu/m ) Deviation (cfu/m3) (cfu/m3) 36 Table 11 presents a list of the cities and towns where more than 100 measurements of C. gattii (any type) were taken. The table indicates how many measurements were taken and the percent positive for each measurement type, as well as the total number of measurements and overall percent positive for each location. Cities are ordered in table 11 based upon their geographic location with the southern most town listed first and the northern most town listed last. Table 11 demonstrates the large differences in the distribution of C. gattii on Vancouver Island and Salt Spring Island, a Southern Gulf Island. The majority of the areas listed in Table 11 are located on the Southern and Central Eastern coast of Vancouver Island. Figure 4 indicates where each of the above cities and towns are located in B.C., Canada. Based upon the data Parksville is the most colonized area with approximately one third (34%) of all samples being positive for C. gattii. Other towns of interest include Duncan (20.9%), Salt Spring Island (20.3%), Courtenay (11.5%), and Errington (10.6%). It is also interesting to note that towns that are geographically very close to one another can have very substantial differences in contamination with C. gattii. An example of this is evident when Parksville (35%) is compared to surrounding towns of Errington (10.6%), Coombs (2.9%) and Qualicum Beach (0.8%). Another interesting finding that is evidenced in table 11 is the fact that there are several cities in which there was a low overall percentage of positive samples but a high percentage of positive air samples. This is most visible in the case of Qualicum where 29% of air samples were positive (n = 90) but only 2% and 0% of soil (n = 45) and tree (n = 75) samples were positive. 37 Table 11 - Number and percent positive for C. gattii for tree, soil, air and water measurements in areas on Vancouver Island and the Gulf Islands from the first sample database (locations listed from south to north of Vancouver Island) Area Area Tree Soil Air Water Total IDa N % N % N % N % N % positive positive positive positive positive 1 Victoria 287 5.2 154 4.5 90 27.7 0 531 8.9 2 Langford 185 0.5 87 1.1 5 0.0 12 0.0 289 '0 .7 3 Saltspring 139 16.5 136 25.0 6 0.0 5 20.0 286 20.3 Island 4 Duncan 124 14.5 79 24.1 68 42.6 4 25.0 275 24.4 5 Nanaimo 280 1.1 136 5.9 17 17.6 1 0.0 434 3.2 6 Parksville 365 34.8 128 35.2 198 33.8 6 33.3 697 34.6 7 Qualicum 75 0.0 45 2.2 21 28.6 0 141 5.0 Beach 8 Errington 88 9.1 51 15.7 8 0.0 4 0.0 151 10.6 9 Coombs 82 3.7 45 2.2 11 0.0 0 138 2.9 10 Cameron 53 5.7 29 0.0 6 0.0 14 0.0 102 2.9 Lake 11 Port Alberni 58 5.2 40 2.5 10 0.0 8 0.0 111 3.6 12 Hornby 49 0.0 48 2.1 18 0.0 1 0.0 116 0.9 Island 13 Courtenay 143 10.5 91 12.1 54 14.8 5 0.0 293 11.6 14 Comox 74 0.0 58 1.7 17 5.9 2 50.0 151 2.0 TOTAL* 2,002 10.9 1,127 12.2 529 26.3 62 8.1 3,714 13.5 TOTAL b 2,561 9.6 1,464 10.9 676 23.4 86 8.1 4,787 11.8 "- City ID used to show geographical locations in figure2 *- Total for all cities with greater than 100 samples. b -Total for all cities (not just those with more than 100 samples) 38 Figure 4 - Locations of Cities and Towns in which greater than 100 measurements have been taken (for key, see table B) 39 The Capital Regional District Watershed Subset The C R D watershed was sampled on six separate occasions between October 2003 and April 2005. On the initial site visit in October 2003, four samples were taken, all of which were negative. The subsequent visit in May 2004 included 2 full days of sampling which resulted in a total of 74 samples taken throughout the watershed. A l l 74 samples were negative for C. gattii. There were no samples taken in the watershed over the summer of 2004. Follow-up investigations of the CRD Watershed were undertaken on two separate days in February of 2005 during which a combined total of 138 samples were taken. Two of the samples resulted in positive laboratory identification for the presence of C. gattii. One of these two samples was a tree sample of a Western Red Cedar and the other was a soil sample from one of the many roads throughout the watershed. A follow-up visit in April 2005 yielded no positive results from a total of 43 samples that were taken in the areas where positive results had been observed in February. A l l samples from the C R D watershed were taken from the environmental sampling database and analyzed separately. In total, 259 samples were taken from the C R D Watershed including 160 tree, 81 soil, 13 water, and 5 air samples. Two of the 259 measurements were positive. Thus, less than 1% of the measurements that were taken from this geographical area resulted in positive results. The two samples that were positive are depicted in figure 5 with large black circles. Both positive samples were taken from a region in the central-west of the watershed on a major road that logging traffic frequents. The small grey dots represent negative measurements. 40 Figure 5 - Locations of positive and negative samples for C . gattii in the C R D watershed 4 1 3.1.2-Tree Type Table 12 presents a list of tree types from which more than 50 measurements were taken. Table 12 - Number of measurements, number of positive measurements and percent of measurements in the first sample database that yielded positive results for C. gattii from al tree types from which greater than 50 samples were taken Tree Type Number of Number Percent Samples Positive Positive Garry Oak 127 18 14.2% Red Cedar 344 47 13.7% Alder 234 28 12.0% Arbutus 142 16 11.3% Douglas Fir 1,017 103 10.1% Maple 205 14 6.8% Pine 83 4 4.8% Hemlock 52 0 0.0% Total 2,004 230 11.4% Combined Total* 2,561 247 9.6% *- All trees, including those with fewer than 50 measurements Significant differences were seen between groups in table 12 (Chi-square 32.62; p < 0.005). There has yet to be a sample from Hemlock that is positive for C. gattii. Maple and Pine were also substantially lower than the mean percent positive for all tree types. A l l of the other tree species have been positive for the organism between 10% and 15% of times they were sampled. A similar analysis was performed using evergreen, deciduous, or unknown tree categories. Table 13 presents the number of measurements taken, the number of positive measurements, and the percent positive for the evergreen, deciduous, and unknown tree type headings. It is important to note that the unknown tree-type heading included shrubs and wood that was degraded and could not be identified as well as other surfaces. 42 Table 13 - Number of measurements, number of positive measurements, and percent of measurements that yielded positive results for C. gattii from evergreen, deciduous, and unknown tree types Tree Category Number of Number % Positive Samples Positive Evergreen 1703 172 10.1% Deciduous 678 65 9.6% Unknown 180 10 5.6%, Total 2,561 247 9.6% There were no significant differences observed between the groups in table 13. The percent of positive samples for C. gattii taken from evergreen tree types is very similar to the percent of positive samples for the organism taken from deciduous tree types. The unknown tree type heading is substantially lower, compared to the two tree type categories. 3.1.3 - Patterns of Contamination Air sampling data was used to describe the influence of season on the propagation of airborne C. gattii propagules. The following two sections report how time of year and weather conditions influenced measurements of airborne C. gattii. Time of Year The air sample database was analyzed to determine whether the time of year had an effect on the presence of C. gattii found in air measurements, and on the concentration of C. gattii propagules found in air. Figures 6, 7, and 8 show the percent positive and concentration of positive measurements taken in each month from the entire dataset. 43 50 4 5 4 0 35 30 % Positive 25 20 15 10 5 0 Wbrrth Number of Samples January 24 February - 34 March 35 April 16 May 49 June 102 July 88 August 168 September - 52 October 57 November - 25 December - 26 Figure 6 - Percent positive of airborne measurements for C. gattii in each month (N = 676) CFU/m' 180 160 140 120 100 80 60 40 20 0 I — Month Standard Deviation (cfu / m 3) January - 2.6 February - 4.4 March - 0.1 April - 19.2 May - 155.3 June -291.3 July - 559.0 August - 179.9 September - 47.5 October - 116.5 November - 2.6 December - 4.7 Figure 7 - Arithmetic means of airborne C. gattii concentration data by month in colony forming units per meter cubed (N = 676) 44 Geometric Standard C. gattii (cfu/m3) Month Deviation January February -March April May June July August September -October November -December -1.9 2.2 1.1 4.5 4.6 9.1 12.4 3.8 5.2 4.7 1.9 2.6 Figure 8 - Geometric means of airborne C. gattii concentration data by month in colony forming units per meter cubed (N = 6 7 6 ) As seen in figures 6, 7, and 8 there a higher prevalence of a sample being positive for C. gattii and also there is a higher mean concentration of C. gattii in air samples in the warmer months of the year. Sampling in the months between November and March has led to very few positive air samples for C. gattii. Weather Patterns The possible effects of weather patterns, more specifically the effects of relative humidity and temperature, were investigated, along with a categorical description of the weather condition on the day when the measurement was taken. The effect of relative humidity was investigated with respect to the transformed (base e) concentration of C. gattii in air. The mean relative humidity measurement was 61% (Standard Deviation 17%) with a minimum of 27% and a maximum of 100%. There was a significant negative relationship between percent relative humidity and the transformed C. gattii concentration (r = -0.203, p < 0.05) Table 14 gives the results of linear regression showing the relationship between percent relative humidity and the transformed C. gattii concentration. 45 Table 14 - Relationship of relative humidity with transformed (base e) airborne C. gattii concentration under non occupational settings B T Sig Constant 1.476 9.798 0.00 Relative Humidity -0.21 -5.385 0.00 Table 14 shows that, without considering the other variables, as relative humidity increases from the lowest relative humidity measured (27%) the transformed C. gattii concentration decreases 0.21 In cfu/m 3 for every percent that relative humidity increases. With this model, relative humidity explains only 4.1% of the variation in transformed C. gattii air concentration. The effect of temperature was also investigated with respect to the transformed air concentration of C. gattii. The mean temperature when measurements were taken was 17.3°C (Standard Deviation 6.5°C) with a minimum of 0.0°C and a maximum of 34.0°C. There was a significant positive relationship between temperature and the transformed C. gattii concentration (r = 0.141, p < 0.05) Table 15 gives the results of simple linear regression showing the relationship between percent relative humidity and the transformed C. gattii concentration. Table 15 - Relationship of temperature and airborne C. gattii concentration under non-occupational conditions B t Sig Constant 0.092 0.483 0.629 Temperature 0.038 3.691 0.000 Without considering the other variables, as temperature increases from zero the transformed C. gattii concentration increases by 0.038 In cfu/m 3 for every unit increase in degrees Celsius. With this model, temperature explains just 2.0% of the variation in transformed C. gattii air concentration. Weather condition was either coded as Clear, Overcast, or Rain. Table 16 describes the transformed (base e) concentration data for each of the three weather conditions. 46 Table 16 - Descriptive statistics for transformed (base e) airborne C. gattii concentration stratified by meteorological sampling condition in non-occupational setings N Geometric Mean (cfu/m3) Geometric SD Min (cfu/m3) Max (cfu/m3) Clear 348 2.49 6.45 0.89 3,752 Overcast 224 2.29 6.32 0.89 1,495 Rain* 104 1.02 1.67 0.89 19 All Samples 676 2.11 5.75 0.89 , 3,752 *- Significantly diferent mean (p < 0.05) Significant differences were found between Rain and either the Clear and Overcast conditions. Both the Clear and Overcast stratification have a geometric mean that is approximately 2 times greater than the geometric means that are measured when sampling was performed in the Rain condition. 3.1.4 - Determinants of Airborne Contamination Regression Model There were significant differences (p < 0.05) in transformed airborne C. gattii concentration and each of the categorical variables in the dataset (Weather Description, Month, City and Tree Type). Correlation between relative humidity and temperature, the two continuous variables under investigation was -0.6 (r = -0.587) which was within 0.7 to -0.7 which was the inclusion criteria for a multiple linear regression model. A backwards stepwise linear regression model was performed with airborne C. gattii (In cfu/m3) as the dependent variable was constructed. The following categorical variables were used: „-Weather Description Reference Clear Degrees of Freedom 2 Month Reference January Degrees of Freedom 11 47 City Reference Victoria Degrees of Freedom 32 Tree Reference Fir Degrees of Freedom 9 Relative humidity and temperature were two continuous variables that were used in the model. The resultant determinants model explained 28.8% of the variability in transformed airborne C. gattii results. Table 17 shows the coefficients obtained for the variables that were used in the model (n = 676) Table 17 - Variables in the regression equation that describes airborne C. gattii concentration (In cfu/m3) under non-occupational conditions B t sig Lower 95% CI Upper 95% CI Intercept .715 3.998 .000 .364 1.067 Alder .554 2.518 .012 .122 .986 Oak .780 4.083 .000 .405 1.155 Spruce 1.730 4.912. .000 1.038 2.421 March -.681 -2.514 .012 -1.212 -.149 June .815 4.499 .000 .460 1.171 July .732 3.792 .000 .353 1.111 RH% -.023 -5.183 .000 -.032 -.014 Rain -.424 -2.348 .019 -.780 -.069 Coombs -1.107 -2.340 .020 -2.036 -.178 Duncan 1.431 7.111 .000 1.036 1.827 Little Qualicum 1.140 3.373 .001 .476 1.803 Falls Park Mill Bay -1.204 -2.446 .015 -2.170 -.237 Parksville 1.321 9.092 .000 1.035 1.606 The model shows that Alder, Oak and Spruce tree types lead to significantly higher estimates of C. gattii in air compared to all of the other tree types. The months of June and July led to higher estimates of airborne C. gattii, whereas predictions of air samples 48 taken in March are lower than any other month of the year. Using this model, predictions of airborne C. gattii taken from Coombs, and Mi l l Bay will be lower, and those taken from Duncan, Little Qualicum Falls Park, and Parksville higher, than the other cities entered into the model. A l l variables considered, temperature did not remain in the model, whereas relative humidity did and leads to predictions of lower airborne C. gattii for every increase in percent relative humidity above 27%. Under baseline conditions the model predicts that the transformed airborne concentration would be equal to the intercept from table 17, 0.715 log cfu/m 3. Taking the antilog of this value gives a concentration of 2.04 cfu/m 3. The beta values in table 17 indicate how much the variable affects the model's predictions from the baseline value. For categorical variables the beta value equals the amount that the baseline is changed when the specific condition is true. The most important categorical variables are Spruce, Duncan, and Parksville which are all predictive of higher values than the baseline, and Mi l l Bay and Coombs which are predictive of lower values than the baseline. What is not clear from an initial glance at table 17 is the fact that relative humidity, the variable with the lowest beta value can have large implications on the prediction due to the fact that it is a continuous variable and the beta value is multiplied by the relative humidity condition used in the prediction. For example, the beta value in conditions where the relative humidity is 80% would be negative 1.219. The following equation would be used as the prediction model to estimate the predicted concentration of C. gattii in air. Log cfu/m3 = 0.715 + (0.554) Alder + (0.780) Garry Oak + (1.730) Spruce + (-0.681) March + (0.815) June + (0.732) July + (-0.023) RH + (-0.424) Rain + (-1.107) Coombs + (1.431) Duncan + (1.140) Little Qualicum Falls Park + (-1.204) Mill Bay + (1.321) Parksville 49 3.2 - FACTORS INFLUENCING THE OCCUPATIONAL GENERATION OF AIRBORNE CRYPTOCOCCUS GATTII A description of the work of arbor workers and C R D Watershed workers as well as how these tasks affect the potential for exposure to C. gattii was undertaken. 3.2.1 - Occupational Measurement Data There were a total of 907 samples taken over the course of the occupational sampling portion of this study. Samples were taken at several locations along the east coast of Vancouver Island. Table 18 gives a general description of all of the samples that were taken at occupational sites. Table 18 - Occupational sample types and percent positive by sampling location Tree Soil Air All Samples N % N /o N % N % Positive Positive Positive Positive Victoria 130 9.2 69 26.1 142 3.5 341 10.6 Nanaimo 23 4.3 22 0.0 19 5.3 64 3.1 Parksville 77 15.6 62 8.1 144 13.2 283 12.7 Coombs 7 0.0 ' 6 0.0 12 0.0 25 0.0 Qualicum 62 3.2 51 3.9 81 6.2 194 4.6 Overall 299 9.0 210 11.9 398 7.5 907 9.2 Significant differences were seen in all samples between towns in table 18 (Chi-Square 15.2; sig. < 0.004). Of the 398 air samples there were 30 (7.5%) which were positive. Descriptive results of the occupational air samples for C. gattii are presented in table 19. Table 19 - Air sampling results for airborne C. gattii from occupational sampling sites on Vancouver Island Number of Air Mean Standard Range Geometric Geometric Measurements (cfu/m3) Deviation Mean Standard Min Max (cfu/m3) (cfu/m3) Deviation (cfu/m3) (cfu/m3) 398 296.4 3075.3 0.9 53,125.0 2.1 4.7 50 Clearly, the results from table 19 indicate that there is a very large range in C. gattii colony forming units per cubic meter that has been found in occupational sampling data. It is evident, however, that this data is largely affected by several very heavily concentrated samples that were taken at occupational sampling sites. Table 20 shows data for only the 30 positive air samples taken under occupational conditions. Table 20 - Air sampling results for all positive samples from occupational sampling sites on Vancouver Island Number of Air Mean Standard Range Geometric Geometric Measurements (cfu/m3) Deviation Mean Standard Min Max (cfu/m3) (cfu/m3) Deviation (cfu/m3) (cfu/m3) 30 3,914 10,716 4 53,125 200 17 The results from table 20 indicate that air samples taken under occupational settings that are positive for C. gattii have a geometric mean that is greater than those taken under non-occupational sampling settings (table 10) by a factor of 6 (200 cfu/m 3 and 33 cfu/m 3 respectively). Comparing the means between transformed occupational and non-occupational positive sampling results indicated that the means are significantly different (p < 0.05) from one another. Table 21 describes from which areas occupational air measurements were taken, the number of samples taken from the area, and the geometric mean and geometric standard deviation of the concentration of C. gattii found in air. It is clear from table 21 that there are large differences in. the proportion of positive samples between geographic locations with Parksville standing out as the most contaminated. 51 Table 21 - Number of air samples, geometric mean and standard deviation for occupational air measurements for C. gattii AREA N Geometric Mean (cfu/m3) Geometric SD Victoria 142 1.60 1.78 Nanaimo 19 1.87 1.27 Parksville* 144 3.39 10.93 Coombs 12 1.77 1,00 Qualicum 81 . 1.50 1.77 Overall 398 2.09 4.68 *- Parksville was significantly different from Victoria and Qualicum (p < 0.05) Parksville was found to be the more highly contaminated area that was sampled under occupational conditions. There were significant (p < 0.05) differences in transformed airborne C. gattii concentration between the cities in which occupational sampling was done. These significant differences existed between Parksville and Victoria, as well as between Parksville and Qualicum with Parksville being the most contaminated. Figures 9 through 13 are maps of occupational sampling sites in Parksville, Coombs, Victoria, Nanaimo, and Qualicum Beach respectively. These maps, which were created by Sunny Mak at the B.C. Centre for Disease Control, show occupational sampling sites with surrounding 100m, 500m and 1000m buffer zones. Table 22 presents a summary of the environmental sampling that has been done within the buffer zones of the occupational sampling sites for each of the cities. The color of the single dot on figures 8 through 12 at occupational sampling sites indicate whether there were positive samples taken from the occupational sampling site or not. In the figures a black dot represents a positive environmental sample for C. gattii and a grey dot represents a negative sample for C. gattii. 52 Table 22 - Number of occupational sampling sites and the number and percent positive of environmental samples in 100m, 500m, and 1000m buffer zones City Number of 100m Buffer 500m Buffer 1000m Buffer Occupational Zone Zone Zone Sampling Sites Number % Positive Number % Positive Number % Positive Parksville 5 13 23.1 43 16.3 68 16.2 Coombs 1 . 3 0.0 10 0.0 10 0.0 Victoria 5 38 2.6 51 2.0 88 2.3 Nanaimo 1 17 5.9 17 5.9 25 4.0 Qualicum 6 33 3.0 33 3.0 58 1.7 Beach The following five figures show how occupational sampling sites may be negative although environmental sampling sites in close proximity may be positive. Alternatively, there were occupational sampling sites at which there were positive samples, though all of the immediately surrounding environmental samples are negative. Cryptococcus gattii 500 M Buffer — - Expressway Local road Residential | Industrial • Positive §SipS°C,na' Highway Tra.l Commercial BUI Recreational • Not Positive 1000 M Buffer Major Road Ferry Route WKKM Institutional Open Area Figure 9 - Occupational sampling sites and surrounding environmental sampling locations in Parksville 53 0 0 5 1 KM 2 t i l ) I i ,,t, I J Cryptococcus gattii • Positive » Not Positive Occupational Sample 1000 M Buffer Expressway Highway Major Road Local road Trail Ferry Route Residential Commercial Institutional industrial Recreational Open Area Figure 10 - Occupational sampling sites and surrounding environmental samples in Coombs Cryptococcus gattii • Positive • Not Positive 500 M Buffer Occupational Sample 1000 It Buffer Expressway Highway Major Road Local road Trail Ferry Route Residential Commercial Institutional Industrial Recreational Open Area Figure 11 - Occupational sampling sites and surrounding environmental samples in Victoria 54 Cryptococcus g&tti) • Positive * Not Positive V , -U&x 500 M Buffer Occupational Sample 1000 M Buffer Expressway Highway Major Road Local road Tr3il Ferry Route Residential Commercial Institutional Industrial Recreational Open Area Figure 12 - Occupational sampling site and surrounding environmental sampling locations in Nanaimo # 7 ' / Cryptococcus gattii 500 M Buffer Expressway Local road Residential industrial • Positive Occupational Sample — Highway Trail Commercial WKM Recreational • Not Positive 1000 M Buffer —— Major Road Ferry Route Him Institutional Open Area Figure 13 - Occupational sampling sites and surrounding environmental sampling locations in Qualicum Beach 55 Environmental Conditions There were several variables that have been used to describe the weather and season over the occupational sampling periods. These were Month, Temperature, percent Relative Humidity, and Weather Description. The affects of each of these four variables on air samples for C. gattii is described individually below. Month The month in which the sample was taken was investigated to determine whether it could be considered a significant determinant of airborne C. gattii in occupational settings. Table 23 describes the number of samples, the percent positive and the geometric mean and standard deviation of the air concentration data for samples taken in each month under occupational conditions. Table 23 - Number of samples, percent positive, geometric mean, and geometric standard deviation for occupational airborne C. gaff/7 samples in each month Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec N 0 22 49 0 0 81 49 55 59 19 63 0 % Positive - 4.5 0.0 - - 6.2 0.0 32.7 0.0 5.3 7.9 Geometric - 1.46 1.26 - - 1.50 - 14.71* 1.50 2.01 1.50 Mean (cfu/m3) Geometric SD 1.58 1.42 - - 1.77 - 27.58 1.34 1.72 2.27 * - Statistically significant difference in geometric mean C. gattii concentration (p < 0.05) From table 23 it is clear that samples taken in August had greater airborne C. gattii concentration than other months (p < 0.05). These samples represent several sampling sites but are driven by an extremely high group of positive samples for C. gattii that were taken in Rathtrevor Beach Park. Temperature The average temperature for occupational sampling was 16.6 °C (SD 6.7 °C; min 3 °C; max 27 °C). Temperature alone was positively related, significant (p < 0.05) and explained 3.1 % of the variability in the occupational C. gattii concentration measurements by linear regression. 56 Relative Humidity The average percent relative humidity on measurement days was 64.5% (SD 16.4%; min 30%; max 95%). Relative humidity was also a significant factor (p < 0.05) and was negatively related to C. gattii concentration, explaining 3.6% of the variability in the occupational C. gattii concentration measurements by linear regression. Weather Description A categorical weather description of Clear, Overcast, or Rain was given to each sample based upon the weather conditions the day in which sampling was taking place. There were a total of 191 air samples taken on Clear days (Geometric mean 3.01 cfu/m 3; Geometric Standard Deviation 7.95), 125 when the skies were overcast (1.43 cfu/m 3; 1.66), and 82 on rainy days (1.61 cfu/m 3; 2.17). There were significant differences in C. gattii concentrations between the Clear and the Overcast and the Rain sampling conditions. Work Task Work tasks were chosen based on observations of the actual work behaviors that these workers perform. Table 24 indicates how many measurements were taken during each of the aforementioned work tasks, as well as the percent of samples that were positive, the geometric mean, and the geometric standard deviation of the concentration distribution. 57 Table 24 - Number, percent positive, geometric mean, and geometric standard deviation of transformed airborne C. gattii measurements in air while workers performed work tasks N Percent Geometric Geometric Positive Mean (cfu/m3) SD No Work- Prior 60 13.6 2.19 4.02 Limbing 76 10.5 3.02 7.62 Chain Sawing 140 5.0 1.68 3.43 Chipping 79 7.4 2.43 6.69 Bucking 6 0.0 1.41 1.43 Stump Grinding 9 0.0 1.52 1.35 No Work- Post 20 5.0 0.65 1.77 Other 8 0.0 1.77 1.00 There were no significant differences (p < 0.05) in transformed airborne C. gattii concentration between any of the 8 work tasks. Determinants of Occupational Exposure to Airborne Cryptococcus gattii Regression Model The 'occupational sampling' database was analyzed using backwards stepwise linear regression to create a model using the following categorical variables: Weather Description Reference Clear Degrees of Freedom 2 Month Reference June Degrees of Freedom 7 City Reference Victoria Degrees of Freedom 4 Work Task 58 Reference Pre Degrees of Freedom 7 Relative Humidity and Temperature were the two continuous variables that were used in the model. The resultant determinants model explained 86.2% of the variability in transformed airborne C. gattii results. Table 25 shows the variables that were retained in the final model. Table 25 - Variables in the regression equation that describes airborne C. gattii concentration in occupational sampling conditions (n = 398) B t sig Lower 95% Upper 95% CI CI Intercept .429 9.148 .000 .336 .521 Limbing -.152 -1.951 .052 -.305 .001 March -7.091 -36.592 .000 -7.472 . -6.710 July .180 1.902 .058 -.006 .365 August .231 2.207 .028 .025 .437 September -6.887 -36.256 .000 -7.260 -6.513 October -6.629 -30.142 .000 -7.062 -6.197 Parksville 6.897 40.937 .000 6.565 7.228 Estimates of airborne C. gattii taken in July and August led to higher estimates, whereas predictions of samples taken in March, September, and October will be lower than the other months of the year. Using this model, predictions of airborne C. gattii in Parksville will be higher, than the other cities that were entered. A l l variables considered, temperature and relative humidity did not remain in the model. Under baseline conditions the model predicts that the transformed airborne concentration would be equal to the intercept from table 20, 0.429 log cfu/m 3. Taking the antilog of this value gives a concentration of 1.536 cfu/m 3. The beta values in table 25 indicate how much the variable affects the model's predictions from the reference value. For categorical variables the beta value is the amount that the baseline is changed when the condition is true. The most important categorical variables is Parksville which is a strong predictor of higher concentrations than the baseline, and the months of October, March and September which are strong predictors of lower concentrations than the baseline. 59 Limbing tasks are also significant in predicting lower exposure values of airborne C. gattii than the baseline. This model explains the factors that are significant in changing the transformed airborne C. gattii concentration prediction under occupational settings from the reference value of 1.390 cfu/m3.The following equation would be used to calculate the prediction. Log cfu/m3 = 0.429 + (-0.152) Limbing + (-7.091) March + (0.180) July + (0.231) August + (-6.887) September + (-6.629) October + (7.379) Parksville Size Distribution of Airborne Propagules A separate database was created which contained all of the positive C. gattii Andersen 6-stage measurements from both the environmental sampling database and the occupational sampling database. Of the 212 Andersen 6-stage measurements that have been taken since May 2002, 67 (31%) were positive. Table 26 shows the size distribution from positive C. gattii samples that were taken in conditions where work was not being performed. Table 27 shows the size distribution from positive C. gattii Andersen 6-stage measurements when chain sawing or chipping tasks were being performed. Table 26 - Size distribution from Andersen 6-stage sampling results in conditions where chain work was not being performed (N = 59) Stage Mean % of Total SD (cfu/m 3) (cfu/m 3) 1 62 33.3% 157 2 50 27.0% 149 3 58 31.2% 180 4 14 7.5% 52 5 1 0.5% • 2 6 1 0.5% 2 Total 186 100% 522 60 Table 27 - Size distribution from Andersen 6-stage sampling results in conditions where chain sawing and chipping was being performed (N = 8) Stage Mean % of Total SD (cfu/m 3) (cfu/m3) 1 705 38.8% 806 2 373 20.5% 535 3 350 19.3% 559 4 315 17.3% 523 5 66 3.6% 144 6 8 0.5% 6 Total 1817 100% 2091 The following list illustrates the aerodynamic particle size separation that the Andersen 6-stage sampler allows: Stage l >7microns Stage 2 4.7-7 microns Stage 3 3.3-4.7 microns Stage 4 2.1-3.3 microns Stage 5 1.1-2.1 microns Stage 6 0.65-1.1 microns This separation allows direct comparison to human lung deposition with the most important particle sizes, with respect to C. gattii, being found in stages 4, 5 and 6. The results from tables 26 and 27 indicate that there is an increase in the percentage of viable, small particles (< 3 microns) under working conditions. Also, it is interesting to note that the measured mean concentration of C. gattii in air from positive samples was approximately 10 times higher on average in the occupational settings when sampling with the Andersen 6-stage sampler. 3.2.2 - Questionnaire Data Invitations to participate in the study were mailed out in August 2004 to 172 companies which were selected based upon having sufficient mailing information and a business name indicating arborist-type work tasks. A total of twenty-two letters were returned due to incorrect mailing information. A l l 150 remaining companies that were sent invitations 61 were phoned at least once. Calling companies did not result in contact on most occasions as these companies tend to rely on answering machines to take messages from potential clients. Repeat calls were concentrated on companies that were located in Parksville and surrounding areas. In total, 33 companies were contacted. Three of the 33 no longer worked in the industry, and another three refused to participate. The remaining 27 companies all agreed in principle to participate in the study. Of the 27, only 9 companies were able to take the time (in person or over the phone) to complete the questionnaire. This resulted in a total of 12 questionnaires being filled out by arbor workers. It is clear that the population that has been sampled may not be a representative sample due to selection bias. First of all, more effort was made to obtain contact, consent, and questionnaire information from arbor workers in areas where knowledge of more contamination existed. Also, with so few companies being available to fill out questionnaires due to the fact that they were either unavailable for contact or too busy to participate, it may be the case that only those companies who had a genuine interest in the research or were at greatest risk of exposure due to the areas in which they worked participated in this study. In addition to the 12 questionnaires that were filled out by arbor workers, there were 17 questionnaires completed by C R D Watershed workers (17 of 18 workers). Table 28 gives the mean and standard deviation for the duration at the current job for both the Arborist/Arbor Care workers and the C R D Watershed Maintenance workers/ Table 28 - Duration of arbor workers and CRD workers at their current job (years) Mean SD (years) (years) Arbor Work 10.3 10.8 CRD 10.5 11.2 No significant difference was found between groups in the duration that the workers had spent in their current job. 62 Workers were asked a variety of questions pertaining to their current job characteristics. Table 29 shows the mean percentage of time that these two groups of workers indicated that they spend outdoors in their job for each season as well as over the course of the entire year. Table 29 - Mean (%) and standard deviation (%) of work spent outdoors in current job for arbor workers and CRD Workers Winter Spring Summer Fall Overall Mean % SD % Mean % SD % Mean % SD % Mean % SD % Mean % SD % Arbor Work* 85.0 29.5 92.9 16.3 85.8 28.1 92.5 17.6 89.1 22.1 CRD 36.6 38.5 44.5 38.0 44.9 39.3 42.6 37.5 42.2 37.5 * - All of the Means for the arbor workgroup are significantly higher than the CRD workgroup (p < 0.05) Table 29 indicates that the arbor workgroup reported that they spend almost 90% of their work outdoors whereas the C R D Watershed workgroup indicated that they spend on average just over 40% of their time at work outdoors. Participants were also asked questions regarding the following work tasks: • Digging • Deck Mowing • Planting • Thinning • Pruning • Felling • Yarding • Chipping • Burning • Clearing of Rotting Wood Workers were asked whether they performed the aforementioned tasks in their current job, and if so, in what season they performed them. Also, when the participants indicated that they did perform the task, they were asked to estimate the number of hours in an average week that they would perform the task each season. The results are reported in table 30. 63 Table 30 - Task comparison of arbor workers (n = 12) and CRD workers (n = 17) from questionnaire data (% of participants replying yes and mean hrs/ week for each season and any season) Winter Spring Summer Fall Any Season % Yes hrs/wk % Yes hrs/wk % Yes Hrs/wk % Yes hrs/wk % Yes hrs/wk DIGGING Arbor 25 2.3 25 6.0 25 2.3 25 7.7 25 4.6 CRD 35 9.0 29 14.4 41 6.3 29 9.0 47 9.7 DECK MOWING Arbor 0 - 0 - 0 - 0 - 0 -CRD 12 6.0 12 6.0 6 4.0 6 4.0 12 5.0 PLANTING Arbor 8 2.0 17 10.5 8 2.0 17 10.5 17 6.3 CRD 24 13.3 24 37.5* 12 10.0 18 28.3 29 22.3 THINNING Arbor 83 9.4 75 9.4 75 9.4 75 8.9 83 9.3 CRD 12 14.0 12 14.0 6 15.0 12 14.0 18 14.3 PRUNING Arbor 100 8.8 75 11.7 67 9.4 67 11.8 100 10.4 CRD 29 19.2 18 16.0 18 7.7 18 9.3 29 13.1 FELLING Arbor 92 10.1 92 10.1 75 10.1 67 10.4 92 10.2 CRD 0 - 12 32.0 0 - 12 22.0 12 13.5 YARDING Arbor 33 6.0 33 6.0 33 6.0 33 6.0 33 6.0 CRD 12 10.0 18 8.0 6 12.0 12 8.0 18 9.5 CHIPPING Arbor 100 14.2 100 14.3 100 14.2 100 14.3 100 14.3 CRD 0 - 0 - 0 - 0 - 0 -BURNING Arbor 17 9.0 8 4.0 .0 - 8 8.0 17 5.3 CRD 12 2.0 18 6.0 6 1.0 29 5.3 35 3.6 C L E A R ROTTING WOOD Arbor 58 5.1 58 5.1 58 5.1 58 5.1 58 5.1 CRD 6 2.0 6 2.0 0 - 0 - 6 1.0 * - Significant differences between groups (p < 0.05) Table 30 illustrates that arbor workers were more likely to perform tasks that involve the removal of trees or parts of trees than the C R D Watershed workers. Chipping is the task 64 in which there is the clearest difference when the two groups of workers are compared. Chipping is commonly done by the arbor workers; the entire sample of the population indicated that they did some chipping in every season of the year. There were no C R D Watershed workers who indicated that they did chipping in any season. Felling, pruning, thinning, and clearing of rotten wood are other tasks that many of the arbor workers indicated that they performed whereas the C R D Watershed workers did little or none of these tasks. The participants were asked if they were exposed to dust (wood dust and 'other' dust were asked separately), and they were asked to estimate the number of hours in an average week that they are exposed to dust. Both of the questions (exposed and hours/week) were asked on a seasonal basis. Table 31 reports the results of the dust exposure questioning. Road dust was given as an example of 'other dust' when administering the questionnaire. Table 31 - Reported dust exposure comparison of arbor workers (n = 12) and CRD workers (n = 17) from questionnaire data (% reporting yes and mean hrs/ week in each season and all seasons) Winter Spring Summer Fall All Seasons % Yes hrs/wk % Yes hrs/wk % Yes hrs/wk %Yes ' hrs/wk % Yes hrs/wk WOOD DUST Arbor Work 83 16.1 83 18.9 83 26.7* 83 25.6 83. 21.8 CRD 24 12.5 29 14.4 24 5.3 29 10.0 35 10.6 OTHER DUST Arbor Work 33 5.7 42 5.8 58 6.5 58 6.5 70 6.1 CRD 41 10.9 53 9.6 71 9.1 59 8.3 77 9.5 - Significant differences between groups (p < 0.05) Table 31 indicates that arbor workers are much more likely to be exposed to wood dust, and that they spend almost two times as much time exposed to wood dust in comparison to C R D Watershed workers. With respect to 'other dust' both groups had approximately three quarters of workers who indicated that they were exposed to 'other dust' in any season, although C R D Watershed workers indicated that they spend about 33% more time in conditions where they are exposed to 'other dust' in comparison to arbor workers. 65 Workers were asked to give information regarding the species of trees with which they worked. For each of the tasks indicated previously the participant was asked to rank the species of tree with the numbers 1 through 9, with 1 being the tree that is most commonly worked with 9 the least common. The rankings were summed and then re-ranked for each entire work group. The results are reported for tasks involving* cutting in table 32a and those not involving cutting in table 32b. Table 32a - Relative ranking of the species of trees worked with for each work task involving cutting (1-most frequent to 9-least frequent) Chipping Thinning Pruning Felling Arbor CRD Arbor CRD Arbor CRD Arbor CRD Douglas Fir 1 - 1 1 1 3 1 1 Arbutus 9 - 8 - 9 1 7 7 Garry Oak 6 - 7 - 7 1 9 -Alder 5 - 6 3 5 4 5 5 Grand Fir 3 - 3 - 3 7 3 4 Maple 4 - 5 - 4 8 4 7 Red Cedar 2 - 2 2 2 4 2 3 Hemlock 6 - 4 5 6 4 6 2 Pine 8 - 5 3 8 9 8 6 Table 32b - Relative ranking of the species of trees worked with for each work task not involving cutting (1-most frequent to 9-least frequent) Yarding Planting Burning Clear Rotting Wood Arbor CRD Arbor CRD Arbor CRD Arbor CRD Douglas Fir ' 1 1 - • 4 1 1 1 -Arbutus 5 4 - - 5 4 9 -Garry Oak 7 - - 1 7 - 6 -Alder 4 5 - 5 4 5 5 -Grand Fir 2 8 - 2 2 8 3 -Maple 6 7 1 2 6 7 4 -Red Cedar 3 2 1 6 3 2 2 -Hemlock 7 3 - - 7 3 6 -Pine 9 6 3 7 9 6 8 -66 From table 32a and 32b it is clear that there are differences in the tree species that these two groups work with. Table 32a illustrates tasks that involve the cutting of tree materials whereas table 32b shows the tasks that do not involve any cutting. From the cutting tasks Arbor workers have indicated that they typically work with Douglas fir the most and Red Cedar second most. C R D Watershed staff indicated that these same two species ranked highly when doing cutting tasks but they also indicated that when pruning it was Garry Oak and Arbutus that were the most important and that Hemlock ranked number two when felling tasks were undertaken. 3.2.3 - Task Analysis For the arbor workgroup task analysis was performed on 26 individual workers over 60 to 360 minute periods of their work days. C R D workers were not included in the task analysis portion of this study. The mean duration of task analysis per shift was 154.6 minutes. The work tasks that the worker performed was recorded every 15 minutes over the duration of the task analysis period. The work task was recorded according to the following list: • Dragging • Limbing on Tree • Chipping • Limbing on Ground • Chain Sawing on Ground • On Looking • Bucking The workers were split into two job title groups. These were: I - Arborist/ Owner 2- Arbor Laborer Table 33 gives the mean percentage and standard deviation of each work shift for both work groups. 67 Table 33 - Percentage of work shift that arbor laborers and Arborist/Owners spend performing specific tasks Arbor Laborer (n=17) Arborist/Owner (n=9) Mean SD Mean SD Dragging 27.0%* 21.1% 6.7% 9.3% Chipping 17.6%* 17.5% . 4.2% 6.3% On looking 17.7% 11.8% 25.1% 14.0% Chain sawing on Ground 11.5% 11.4% 9.3% 10.5% Limbing on Ground 3.0% 6.7% 4.6% 8.5% Limbing on Tree 2.9% 9.4% 41.2%* 15.6% Bucking 1.8% 7.6% 0.0% 0.0% * - Significant differences between groups (p < 0.05) Table 33 shows that arbor laborers spent much more of their shift performing dragging and chipping tasks compared to workers in the Arborist/Owner group. On the other hand, workers in the Arborist/Owner group spent over 40% of their work shift limbing while up trees, in comparison to just 3% of the arbor laborer's time being spent doing the same task. Individuals worked in work crews that had between 2 and 4 workers. In each work group there was one Arborist/Owner working with 1 to 3 arbor laborers. None of the workers wore any form of respiratory protection during any of the observed work shifts, although the use of face shields and hearing protection was common amongst all workers using chain saws. 6 8 3.3 - DEMOGRAPHICS AND HEALTH OF THE STUDY POPULATION The following is a description of the 29 workers who were able to complete questionnaire. The information is described for the two work groups: Arbor workers and C R D Watershed workers. Gender and Race Al l of the workers in both of the groups indicated that they were of White/Caucasian ethnicity. A l l 12 of the arbor work workers were males, whereas two of the 17 C R D workers were female. Age The average age of arbor workers and C R D workers, as described in table 34, was 39.2 years and 47.3 years, respectively. Table 34 - Mean, minimum, maximum and standard deviation of the age of arbor workers and CRD workers N Mean Min Max SD (years) (years) (years) (years) Arbor Work 12 39.2 16.2 55.9 15.2 CRD 17 47.3* 32.0 58.2 8.8 * - Significant differences between groups (p < 0.05) Residence These workers all resided on Vancouver Island, with the C R D workers largely restricted to Victoria and surrounding areas. Table 35 indicates the cities in which these workers reside and table 36 gives the duration of time that arbor workers and C R D workers have been at their current residence. 69 Table35 - City of residence for arbor workers and CRD workers City Arbor Work CRD Victoria s 1 13 Sooke 1 Cobble Hill 1 Mill Bay 1 Duncan 1 Shawnigan Lake 1 Nanaimo 1 Parksville 2 Qualicum 4 Coombs/ Errington 1 Comox 1 Campbell River 1 Table 36 - Duration of arbor workers and CRD workers at current residence Mean SD (years) (years) Arbor Work 10.5 8.5 CRD 11.6 10.1 No significant (p < 0.05) differences were observed in the duration living at the current residence between the two workgroups. The C R D workers all live in the south of Vancouver Island in relatively close proximity to the C R D watershed. The arbor workgroup was spread out along the island and most of them were located in the central east portion of the island. This geographical distribution of the residences of arbor workers is unlikely representative of the actual distribution; rather it displays the recruitment and selection bias of this study. The geographic bias is due to the recruitment strategy that placed more emphasis on recruiting arbor workers from areas from which a high proportion of samples were positive for C. gattii. Leisure Participants were also asked about the percentage of their leisure time that they spend outdoors in each season (table 37). Arbor workers were higher than the C R D workers in every season. 70 Table 37 - Leisure time spent outdoors for arbor workers and CRD workers (Mean% and SD%) Winter Spring Summer Fall Mean % SD % Mean % SD % Mean % SD % Mean % SD% Arbor Work 40 27 54 27 60 30 54 27 CRD 28 25 38 24 48 26 37 23 No significant (p < 0.05) differences were observed in the percentage of leisure time that the two groups spend outdoors although it appears as though there is a trend indicating that arbor workers spend more of their leisure time spent outdoors. Travel Questions related to travel were also asked. Table 38 indicates how these two groups of workers differ in relation to the percentage of each group indicating that they had ever traveled to the major parks on Vancouver Island. Table 38 - Presence of C. gattii in the park followed by percentage of arbor workers and CRD workers having ever visited the listed park Presence of C. Arbor Work CRD Watershed gattii (%) (%) Cathedral Grove Yes 92 82 Pacific Rim No 67 77 Strathcona No 50 73 Miracle Beach No 67 47 Helliwell No 18 27 Boyle Point No 9 20 Englishman River Falls Yes 67 73 Little Qualicum Falls Yes 67 53 Rathtrevor Beach Yes 75 80 Beban No 58 33 Piper's Lagoon No 27 0 Shawnigan Lake No 42 80 Bamberton No 17 53 Mount Douglas No 17 87 Beacon Hill No 42 0 Goldstream No 50 100 Table 38 shows that the majority of all arbor workers and C R D workers have visited the four parks (Cathedral Grove, Englishman River Falls, Little Qualicum Falls, and 71 Rathtrevor Beach) in which there has been found evidence of C. gattii through environmental sampling for the organism. Table 39 shows the percentage of workers from each group who indicated that they had traveled to the Gulf Islands, Other Parts of B.C. (not Gulf Islands or Vancouver Island), Other Provinces or Territories in Canada, the United States, or Other Countries (not Canada or the United States). Table 39 - Percentage of arbor workgroup and CRD workgroup traveling to listed area a minimum of once in the past year N Gulf Other Parts Other United Other Islands of BC Province or Territory States Country Arbor Work 12 50 67 25 8 8 CRD 17 29 59 18 24 35 Table 39 indicates that a greater portion of the arbor workers indicated they traveled more locally compared with CRD Watershed workers, whereas more of the C R D Watershed workers indicated that they traveled out of the country over the course of the last year. Smoking Smoking status was given by all subjects in the arbor workgroup, although three of the C R D workers refused to answer smoking related questions. Two (17%) of workers from the arbor work group smoked, whereas three (18%) of C R D workers were current smokers. Of those who answered smoking questions and were not current smokers, 60% and 46% of arbor workers and C R D workers respectively had previously smoked but had since quit. Health None of the workers indicated that they had been diagnosed with cryptococcal disease in the past. Participants were asked whether they had been diagnosed with any of the following illnesses over the past year and within the past five years. 72 • Tuberculosis • Cancer • Bronchitis • Pneumonia • Anemia One of the arbor workers indicated that he had been diagnosed with bronchitis in the past year, and four responded that they had been diagnosed with bronchitis in the past five years. There were no other diseases in the past year or past five years reported by the arbor workers. One worker from the C R D watershed indicated that he had been diagnosed with bronchitis in the previous 5 years. Similarly, there was one other worker from the C R D workgroup who responded that he had been diagnosed with pneumonia in the last 5 years. Questions related to whether the worker had experienced respiratory or other symptoms which lasted over one month in the preceding 5 years were asked. Table 40 indicates the symptoms that were included in the questioning and how each of the two work groups responded to the questions. Table 40 - Percentage of the arbor workgroup and CRD workgroup reporting respiratory symptoms or other symptoms lasting over a month in the previous 5 years Respiratory Symptoms Arbor CRD Other Symptoms Arbor CRD Cough 33.3 17.6 Neck Stiffness 8.3 17.6 Cough with Blood 0.0 5.9 Fever 16.7 11.8 Cough with Phlegm 33.3 17.6 Chills 16.7 5.9 Shortness of Breath 0.0 0.0 Night Sweats 8.3 ' 5.9 Chest Pain 16.7 11.8 Unexplained Weight Loss 0.0 5.9 Loss of Appetite 0.0 11.9 Nausea 8.3 11.8 Nodules 0.0 11.8 Headache 16.7 5.9 Muscle/ Joint Pain 8.3 11.8 At Least One Respiratory Symptom 50.0 17.6 At Least One Other Symptom 16.7 35.3 73 Table 40 shows the percentage of the workgroup reporting respiratory symptoms was approximately three times greater in the arbor workgroup in comparison to the C R D Watershed workgroup. The percentage of workers in the C R D Watershed workgroup reporting other symptoms over the last five years was double the percentage of the arbor workgroup. 74 4 - DISCUSSION Knowledge of the hazards in the environment is an important step in determining one's risk of exposure to, and disease from the given hazards. This study was an attempt to discern whether there is the possibility of workers being exposed to C. gattii while working under or around the canopies of trees, as well as investigating the variables that are determinants of exposure. There is no way of visibly telling if a tree is positive for C. gattii in any given area, therefore this study has been an effort to gain the knowledge of the factors that determine the presence and concentration of C. gattii in air under non-working and working conditions. 4.1 - STUDY QUESTION 1 - CAN ARBORIST/CRD WORKERS BE POTENTIALLY EXPOSED TO CRYPTOCOCCUS GATTII? Exposure to C. gattii can occur when an individual is in an outdoor environment where the pathogen exists. Considerations regarding whether the two work groups, arbor workers and C R D Watershed workers, can potentially be exposed to C. gattii include the geographical distribution of the organism with respect to the workers as well as the time during their work that these workers spent outdoors in environments where C. gattii exists. Geographical Distribution The 4,787 sites from which measurements were taken since 2001 were not uniformly distributed in the study area. The sampling results do, however indicate that contamination was widespread on Vancouver Island and the surrounding Southern Gulf Islands with the highest levels of contamination being found in the central east coast area of Vancouver Island, in areas such as Parksville and Duncan. The more populated areas on Vancouver Island and the Southern Gulf Islands have been investigated and there has been some C. gattii recovered from all of them. The convenience sampling strategy that has been used since 2001 in collecting this data, as well as the knowledge of the ecological relationship between C. gattii and trees, the simplicity of sampling techniques used, and lack of equipment needs has resulted in a predominantly high proportion of tree and soil samples (53.5% and 30.6% respectively) versus air and water (14.1% and 1.8% respectively). A i r measurements resulted in a 75 higher proportion of positive C. gattii samples (23.4% of air samples were positive) compared to tree (9.6%), soil (10.9%), and water samples (8.1%). This difference was due to the fact that most air samples were taken in the vicinity of culture positive trees, whereas tree, soil, and water samples were taken as a screening tool to map the presence of colonized sources of C. gattii. Al l of the areas that were well sampled (i.e. those with greater than 100 samples) have had some colonization of C. gattii and in many cases widespread colonization. Sampling to this point, however, has largely been driven by history of either human or animal cases of cryptococcosis or prior positive measurements. Because it was not random sampling, it was not appropriate to conclude that C. gattii is pervasive throughout the entire study area. It is evident, however, that specific areas on the south-central east coast of Vancouver Island including Salt Spring Island, Duncan, and Parksville are more highly colonized than others. Although there are very high levels of colonization in these areas, several other, nearby locations were found to have low levels of colonization. A good example of this was seen in and around Parksville where 34.6% of samples from Parksville itself were positive and all of the surrounding towns had a much lower proportion of positive results (Qualicum Beach 5.0%, Coombs 2.9%, and Errington 10.6%). It is unclear why this is the case but it may be a reflection of the sampling strategy, or of the differences in micro-climates within the study area. Although C. gattii was found in most populated areas on the east coast of Vancouver Island, small 'pockets' of positive environmental samples were generally evident rather than a widespread occurrence of single positive samples. Return sampling to an area in which a tree had been found initially to be positive for C. gattii could have led to a high proportion of positive samples in colonized areas. The colonization of an organism occurs when it is able to find a beneficial ecosystem that supplies sufficient nutrients. It is still unclear what characteristics support C. gattii colonizing trees on Vancouver Island. An issue that must be considered is that of persistent colonization versus transient positivity. Measurement from a tree could have initially been positive even though true colonization of the organism may not have taken place. This situation could have led to negative measurements at a previously positive location on future dates. This was not however captured, due to the use of a 'first sample' 76 database. Alternatively, a tree which initially resulted in a negative sample for C. gattii may have subsequently have had C. gattii become established in concentrations above the limit of detection of the measurement method. The observation of transient positivity was highlighted in the C R D Watershed. The C R D watershed was sampled on 6 occasions for a total of 259 measurements. Two of these measurements were positive initially, but upon returning to the area no positive samples were recovered. The samples that were positive initially were taken from an area within the watershed that was open to travel by logging trucks before and during the sampling period. Watershed staff indicated that the logging trucks using the area originated on the central east part of Vancouver Island and would travel through both Parksville and Duncan prior to their travel through the Watershed. Although transient positivity was observed in the Watershed, no evidence of colonization was found. Though none of the trucks themselves were investigated for the presence of C. gattii, it is likely that the organism was being introduced to the area by external vectors which were under human control. The categorical variable Parksville was significant in both the environmental and occupational determinants models with a positive beta-value in each. The variables for Duncan and Little Qualicum Falls Park both remained in the environmental determinants of contamination model with coefficients greater than one. Alternatively Coombs and Mill Bay were in the environmental determinants of airborne contamination model with similarly large, but negative coefficient values. Victoria was selected as the baseline for both of the determinants models. For workers it is clear that the area variable is the most important determinant of exposure because workers who are performing their work in areas in which there is no background'level of contamination are'not at r iskof exposure to C. gattii. Time Spent Outdoors .- 7 Workers in both of the occupational groups investigated in this study spend a large portion of their work time outdoors (Arbor workers 89%, C R D Watershed workers 42%). The more time that these workers spend outdoors in colonized areas, the more likely they are to be exposed to C. gattii. Conclusion 11 Workers from both of the work groups studied are potentially exposed to C. gattii due to the area in which they work and the fact that they do their work outdoors under the canopies of trees. A l l areas that have been well-sampled have had at least some samples that were positive for C. gattii, thus workers in any area are at some risk of exposure when working outdoors. Workers in areas where there is not much evidence of C. gattii colonization, such as the CRD, are at a much lower risk of exposure than those workers, such as many arbor workers, who work outdoors in areas where there is greater colonization. 4.2 - STUDY QUESTION 2- WHAT CONDITIONS AFFECT AIRBORNE CRYPTOCOCCUS GATTII CONCENTRATION? Without the resources and technology to perform widespread and extensive personal sampling, there was no definitive way in which to accurately describe personal exposures to airborne fungi, such as C. gattii, in a large area such as Vancouver Island and the Southern Gulf Islands. This study was initially intended to create a Job Exposure Matrix that would have been used to describe which, of several woodland occupations, have potential exposure to C. gattii. Job Exposure Matrices are typically designed to indicate the presence, intensity, frequency, and/or probability of exposure to a specific agent in a specific job. 1 0 2 Another option for answering the study questions was to create a determinants of exposure model. In a 2002 study, Teschke et al. reviewed the literature to determine which methods were best at explaining exposures in work populations. Teschke found that most authors concluded that the Job Exposure Matrices were not sensitive and had only slight to fair agreements with techniques in which the authors had more confidence.1 0 2 There is a growing body of literature on determinants of exposure in a wide variety of industries. These determinants of exposure models are good tools to predict exposures when measurements are not available. 1 0 2 Also, determinants of exposure models can be useful in creating questionnaires.102 Thus, the determinants of exposure models created in this study should be useful in directing adjustments to the current questionnaire for future studies of occupational exposures to C. gattii in British Columbia. Another important consideration when deciding between constructing a Job Exposure Matrix and a Determinants of Exposure model was the fact that there existed a sizeable 78 amount of sampling information that has been collected by Dr. Bartlett since 2001. This database contained information related to environmental conditions such as sampling date, temperature, and relative humidity. The information found in this database was more appropriately used in a Determinants of Exposure model than attempting to use it to create a Job Exposure Matrix. Tree Type Recoding tree types into either deciduous or evergreen groups was done due to the abundance of literature linking C. gattii to Eucalypts (evergreen trees with deciduous bark). Tree samples indicated that the percent of positive samples for C. gattii taken from evergreen and deciduous trees is very similar. It is interesting to note though, that the trees that were considered 'unknown' had a significantly lower proportion of positive samples as compared to either the deciduous or evergreen groups. There may clearly be ecological differences between the unknown tree locations and the known locations. Within the unknown group there were stumps and logs where the tree type was not discernable due to degradation. In locations like this, there may be a lack of appropriate nutrients for the organism to grow due to the degradation of the medium or over-competition by other organisms. Also, the unknown group contains other species of trees (shrubs) and other surfaces, and therefore may have lacked the nutrients or other factors that allow C. gattii to grow. It is interesting to note that no samples of Eucalypts (n=28), the most commonly cited natural reservoir for C. gattii in the literature6, were positive for the organism. Arbutus, similar to Eucalyptus in that it is an evergreen with deciduous bark, is relatively common on Vancouver Island and the Gulf Islands. Arbutus had a slightly higher proportion of positive results for C. gattii than Douglas fir, one of the most common species of trees on Vancouver Island. 1 0 3 Hemlock is the only tree type that was extensively sampled that did not give any positive results. Hemlock is not commonly found in the Coastal Douglas fir Biogeoclimatic zone, an area from which the majority of positive samples for C. gattii have come. Maple and pine were both well sampled but each had a low percent of samples that were positive for C. gattii, at 6.8 and 4.8 percent respectively. Douglas fir, red cedar, and alder, three of the most common tree types found on Vancouver Island, were among the most commonly positive tree species. Garry Oak was the tree type that had the highest proportion of positive samples (14.2%). A l l variables considered, the 79 Alder, Garry Oak, and Spruce tree types were significant in the environmental model, all with a probability of higher airborne concentrations than the Douglas fir tree type and the other tree types that were entered into the model. Douglas fir was the tree type that both the arbor workgroup and the C R D workgroup indicated was the most common for almost all of the work tasks that they perform. Alder and Garry Oak, two of the significant tree types in the environmental determinants model were among the middle of the rankings that both of the work groups indicated for the majority of work tasks. Garry Oak was the top ranked tree type for C R D workers performing Planting, Pruning, Burning, and Clearing of Rotting Wood. Seasonal Patterns Liao and Luo, in a 2002 study from Taiwan, found that the distribution of airborne fungi appeared to be associated with seasonal changes.94 They concluded that in order to best document the actual environmental exposures to fungal spores, the effects of seasonality and temporality on the distribution of microorganisms should be considered. This group of researchers, as well as several others have found that airborne concentrations of fungi were higher in the summer in comparison to the winter.9 4"9 6 Other studies, however, indicate the opposite findings, that higher concentrations of airborne fungi are found in winter months in comparison to summer. 1 0 4 1 0 5 This thesis of occupational exposure to C. gattii observed both a higher proportion of positive air samples as well as a higher concentration of airborne fungi in summer months. In several studies, there is a link between airborne fungal concentrations and environmental conditions that support microbiological growth. 9 6 1 0 6 In B.C., however, with respect to C. gattii it appears that there is very little difference in soil and tree concentrations over the course of the year whereas there are large differences in airborne concentrations between months. This would indicate that airborne fungal concentrations are more driven by conditions supporting aerosolization as compared to those which support fungal growth and proliferation. In this study, using January as the reference month for the environmental contamination model, there were significantly higher estimates of C. gattii concentrations for the months of June and July, and significantly lower estimates of C. gattii concentrations for March. In the occupational determinants of exposure model, March, September, and October all remained in the model with large, negative coefficients. 80 Temperature and relative humidity were also investigated and the results, contrary to what several studies have shown 9 4 1 0 6 , indicate that airborne C. gattii concentration increases with decreasing relative humidity. Liao and Luo observed that fungal concentrations increased linearly in both summer and winter with relative humidity.9 4 Similarly, Bartlett et al., in a study of airborne organisms in B.C. classrooms showed a positive correlation between airborne fungal contamination and relative humidity. 1 0 6 Again, the findings by Liao and Luo, and by Bartlett et al. may be attributed to conditions that better facilitate proliferation of the organism that were being investigated. In this current study of C. gattii however, relative humidity was significant in the non-occupational determinants model with a negative coefficient indicating that higher relative humidity in fact leads to a decrease in airborne C. gattii. Liao and Luo, in the report indicated above, explained that hygroscopic growth, which means the increase of the particle diameter by condensation or water absorption, influences the deposition of aerosol.9 4 Several studies performed at a variety of humidity levels concluded that airborne fungal spores are, in fact, hygroscopic in nature. 9 4 1 0 7 The hygroscopic nature of fungal spores, Liao and Luo indicate, leads to larger aerodynamic equivalent diameters and thus changes in environmental deposition and activity in air, and the deposition in an individual's respiratory tract.94 This may be one explanation regarding why higher relative humidity resulted in lower concentrations of airborne C. gattii. In the study by Bartlett et al., which was done on the lower mainland of B.C., an area with similar environmental conditions as the current study, the authors reported that there were no significant relationships between temperature and airborne fungi concentrations.105 which is similar to the results of the current study. Weather conditions were also investigated, being coded into rain, overcast, and clear. This was done for several reasons. First, the a priori hypothesis assumed that rainy conditions led to less C. gattii becoming aerosolized. Also the C. gattii that is aerosolized ought to fall out of the air more rapidly due to the wet, more humid conditions.9 4 In addition, Clear and Overcast conditions were separated because it has been noted in the literature that solar radiation and direct sunlight has a marked effect on airborne fungi. 1 0 8 Thus, the a priori hypothesis was that the Overcast condition would be the condition in which we saw the highest concentrations of viable airborne propagules. Rain was, in fact, significant in the environmental determinants model with a negative coefficient 81 indicating that rainy conditions do led to reduced airborne C. gattii compared to the other weather categories. Conclusion There were several factors in this study that appear to play a role in determining airborne C. gattii concentrations. Important considerations include the tree type under which air samples were taken as well as the weather conditions at the time in which the sample was taken. A l l trees, other than Hemlock, have been shown to harbor C. gattii at detectable levels. The most important trees with respect to high C. gattii concentrations were Alder, Garry Oak, and Spruce, though more common trees such as Douglas fir and Red Cedar are capable of being colonized by the organism. Relative humidity was significant in the non-occupational determinants models with lower average airborne C. gattii concentrations being predicted as relative humidity increases. Rainy conditions also appear to inhibit C. gattii from becoming airborne. 82 4.3 - STUDY QUESTION 3- ARE ARBORISTS/CRD WORKERS AT AN INCREASED RISK OF EXPOSURE TO CRYPTOCOCCUS GATTII? There are several things that need to be taken into account when considering whether these workers are at an increased risk of exposure to C. gattii. Factors such as work task, particle size, physical requirements of the job, time spent outdoors, and area in which work is being done have all been considered in this investigation. Work Tasks It was presumed that work tasks that create aerosols would increase C. gattii found in the air, as well as the concentration of C. gattii in the air. Wickman et al. showed that mechanical and/or human activity changed airborne fungal concentrations.109 The authors report that large numbers of people or abundant activity stirs up dust and settled spores and intensifies air currents, delaying deposition by gravity. In the current study, this phenomenon was not seen in the occupational determinants of exposure model. The only work task that remained in the model was limbing, with a small, negative coefficient. There are two major reasons that may explain the lack of evidence regarding whether tasks increase or decrease aerosolized C. gattii concentrations. First, much of the occupational sampling that was performed was done in areas in which no environmental samples have been positive for C. gattii. This suggests that there was not a background level of contamination for much of the occupational sampling and therefore tasks could not have possibly affected airborne C. gattii at the work site. Secondly, personal sampling for C. gattii was not performed due to a lack of appropriate personal sampling techniques, and therefore all sampling that was done was area sampling. Although sampling was attempted as close as was safe to the worker, much of the time the sampling equipment needed to be situated at quite some distance from where work was being performed due to both equipment safety as well as the safety of the investigator. 83 Task analysis was performed in an effort to discern differences within the arbor workgroup. It is clear that there are differences in the percentage of the work shift spent on certain tasks between the two groups of workers within the arbor workgroup. However, the percentage of the work shift performing tasks that involve cutting wood and potentially creating large amounts of aerosolized matter appear similar between the two groups. Comparisons of the arbor workgroup and the C R D workgroup clearly indicated that arbor workers spend much more of their time doing work tasks that directly involve trees and the cutting of trees. C R D workers indicated that they did more digging and driving in their work than the arbor workers. The two questions pertaining to dust exposures gave more credence to this fact in that arbor workers indicated that they were exposed to wood dust more than twice as much as C R D workers, and C R D workers indicated that they were exposed to other dusts, such as road dust, a third more than arbor workers. Particle Size Aerodynamic equivalent diameter particle size is an important consideration in any study regarding airborne pathogens because it plays a role in both exposure to the pathogen and the risk of infection from exposure. According to Vincent, interest in size specific sampling began in the early 1900's when it became apparent that penetration into and deposition in the respiratory tract was dependent on particle s ize . " 0 Particle size also determines particle motion in the environment which includes settling under gravity, re-suspension, and transport by air movement.94 From the results it appears that work tasks increase the percentage of viable small particles containing C. gattii (< 3 microns); sampling with the Andersen 6-stage sampler resulted in approximately 13% more viable particles being impacted on stages 4, 5, and 6 under working conditions as compared to non-working conditions. Vincent suggests that approximately one half of particles that have an aerodynamic equivalent diameter of 3 microns wil l deposit in the alveolar region of the lungs. 1 1 0 Time Outdoors As indicated in section 4.2 of this thesis these workers spend a large portion of their work time outdoors. If a worker is in an area where there is C. gattii in the environment the 84 chances of being exposed and the amount that the worker is exposed increases with the duration of time spent outdoors. Area If there is no C. gattii contamination in the area in which work is being done these workers will not be at an increased risk of being exposed to the pathogen. Thus, for arbor workers or C R D workers to be at any risk of being exposed to C. gattii they must be in an area where the organism exists. Conclusion When workers are performing woodland tasks in areas where C. gattii exists they are at an increased risk of exposure to the organism in comparison to the general public or to workers who are not performing woodland tasks. The air samples that have been by far the most concentrated with C. gattii have come from occupational sites when cutting tasks were performed (53,125 cfu/m 3 compared to 3,750 cfu/m3). Unfortunately, the majority of occupational sampling that was performed in this study appears to have been done at work sites where there has not been background contamination. Comparison of the positive air samples for C. gattii under non-occupational and occupational sampling settings indicate that those taken under occupational settings are significantly higher (p < 0.05) than those taken under non-occupational settings. The geometric mean in occupational settings was higher than those taken in non-occupational settings by a factor of six. Also, under occupational settings workers appear to be exposed to more small particulate carrying C. gattii. Arbor workers spend a large portion of their days outdoors under physical working conditions. Much of their work involves cutting tasks, which, in contaminated areas can lead to high concentrations of C. gattii. C R D workers, on the other hand, do not appear to be in an area in which the colonization of C. gattii has occurred but who may be exposed to C. gattii basidiospores when the organism is transported into the C R D watershed. 85 4.4 - STUDY QUESTION 4- WHAT ARE THE ARBORISTS/CAPITAL REGIONAL DISTRICT WORKERS POPULATION DEMOGRAPHICS AND HEALTH STATUS? Since the outbreak began in 1999, until June 2005, there were 139 confirmed cases of cryptococcosis due to C. gattii in B.C., none of which were observed in workers from the arbor work or C R D work populations. Interestingly, there have been no cases seen in any workers performing woodland tasks on Vancouver Island. This may support Fyfe et al.'s earlier observation that woodland tasks resulting in low levels of exposure may be protective against infection.8 0 Half of the arbor workgroup indicated that they had experienced adverse respiratory health symptoms lasting over one month in the preceding five years in comparison to 18% of the C R D watershed workers. This may be due to the fact that arbor workers appear to be spending more time on cutting tasks, and being exposed to greater levels of small particulate in their work in comparison to CRD workers. Alternatively, 35% of the C R D workgroup reported other symptoms in comparison to 17% of the arbor workgroup, which might simply be a reflection of the difference in age between the two workgroups. Both of the work groups that were investigated over the course of this study were predominantly male workers. The arbor worker average age of 39.2 years old was essentially the same as the average age of the B.C. workforce. 1" The CRD workgroup, on the other hand, had an average age 8 years older than the arbor workgroup, and the provincial average. In the arbor workgroup the standard deviation in age was almost double that seen in the C R D work group. The large standard deviation in the age of the arbor workgroup was due to the age split between the arborist/ owners and the arbor laborers. Just under one fifth of the workers in both occupational groups reported that they were current smokers. This is very close to, but slightly lower than the overall Canadian average for males which was reported by Statistics Canada at 23% in 2003, and similar to the average reported for B C the same year. 1 1 2 86 These two groups of workers reported that they spent a large portion of their leisure time outdoors and that a good portion of both groups had visited most of the major parks found on Vancouver Island. It is interesting to note that Rathtrevor Beach Park, the location in which the first samples that were positive for C. gattii were discovered in 2001, was among the parks that were visited by the greatest number of workers in both work groups. Workers in the arbor workgroup reported that, in the past year, they traveled much more within Canada than CRD workers, whereas the CRD workers were much more likely to travel to the United States and other countries. Most of the workers from both groups had traveled to the parks on Vancouver Island in which C. gattii has been found (Cathedral Grove, Englishman River Falls, Little Qualicum Falls, and Rathtrevor Beach). Questionnaire The questionnaire took too much time to complete for these workers. Workers were resistant to filling out the questionnaires because of its length and investigators found that, in general, the amount of time spent on the questionnaire was not appreciated by the workers. Several sections, such as those on travel history took up long periods of time to fill out with limited only information being gained. Also, questions that asked for information regarding events greater than five years in the past introduced recall bias and generally added to the duration of the process. In future studies the questionnaire ought to be shortened so that the workers are able to complete it in under 15 minutes. In order to achieve this the questionnaire ought to be changed and the travel history portions of the questionnaire should be updated to contain only the information that is most pertinent to the study. Also, portions of the questionnaire that asked the worker to break down their work tasks by season should be streamlined in future studies. These two changes should enable the questionnaire to be administered in much less time without losing too much useful information. 87 4.5 - STRENGTHS AND LIMITATIONS 4.5.1 - Strengths Sampling Data A major strength of this thesis is that there was a large database of sampling information that had been compiled for this organism on Vancouver Island since 2 0 0 1 . This prior sampling was useful in directing the occupational sampling to areas which were more likely to have positive background levels for the organism. The large sample size was also useful in adding confidence to the determinants of airborne contamination model. 4.5.2 - Limitations Environmental Sampling Bias A major limiting factor in this study is that all of the samples collected for C. gattii since 2 0 0 1 have been taken in a non-random nature. Samples have been taken primarily in areas from which positive cases of cryptococcal disease have emerged or in areas where the organism has been previously located. Also, due to the convenience sampling strategy that has been used there it is not known whether C. gattii exists in non-populated areas of Vancouver Island or the Southern Gulf Islands, away from roads and parks. In an effort to offset the sampling bias that was evident in the environmental sampling database, all return sampling was removed for tree, soil, and water sampling types. Ai r samples, because of the prior knowledge that seasonal variations affect airborne C. gattii, were removed from the database when there were duplicate samples in any given month at a distinct sampling site. This first sample database was created to try to get a better sense of the actual distribution of C. gattii on Vancouver Island and the Southern Gulf Islands. Sampling Devices No personal sampling devices were used over the course of this study and thus, personal exposures could not be accurately measured. Area samples were the best estimate of personal exposure. This was problematic because it was often very difficult to take samples in the workers breathing space, and at times it was not possible to get within 88 close proximity of the worker performing their tasks. This was especially evident when arbor workers climbed the tree in order to limb the upper portions of the tree. In order to deal with this limitation the investigators made an effort to take air samples as close as possible to the breathing space of the workers while they performed their work tasks. Tree Types There is no explicit knowledge of the number of trees of each type in the study area and therefore it is difficult to determine whether the distribution of C. gattii is specifically related to the distribution of certain types of tree. Since 2001 there has been an effort to sample trees of all types on Vancouver Island. This has resulted in a database that contains information on a variety of tree species from most of the well-sampled locations on Vancouver Island. Occupational Sampling Bias The arbor workgroup may not be representative of the overall arbor work population due to a low level of participation. Selection bias was introduced due to the preference of choosing companies who were in areas in which the investigators knew there was some contamination of C. gattii. This was a concern because it limited the number of companies that were approached. Also, because this work group is very busy there is the chance that only those workers who were interested in the study took part. This might be a concern because these workers, being more interested and aware of health hazards, may do more to protect themselves from these concerns. An effort was made to contact as many of the workers in the work groups as possible. When occupational sampling and task analysis took place the workers were asked to try to go about their normal work activities as if the investigators were not there. Pre Sampling Weather Data Weather data in the weeks preceding sampling was not recorded. The Weather Canada historical archives were investigated but they were not deemed appropriately location specific and took substantial time to calculate from the database. In future studies the 89 weather conditions and precipitation data should be calculated throughout the study for use in the determinants o f exposure model calculation. Task Titles The tasks used in the occupational air sampling, the task analysis and the questionnaire did not all correspond. This did not al low direct comparison between the three data sets. In future studies all task lists should contain the same work task titles. Repeated Measures within Specific Areas Due to the non-random sampling strategy and the repeated measures at specific areas under varying conditions a random effects model should have been used. This would have allowed for a more thorough statistical description o f the varitation in the baseline concentrations o f C. gattii both between sampling areas as wel l as within sampling areas. 90 5 - CONCLUSIONS AND RECOMMENDATIONS Arbor workers on Vancouver Island and the Southern Gulf Islands are being potentially exposed to C. gattii at varying levels. Although the a priori hypothesis that specific work tasks such as chain sawing would increase exposure to C. gattii was not substantiated in this study, there were higher airborne concentrations of C. gattii seen under working conditions compared to non-working conditions. Workers on Vancouver Island and" the Southern Gulf Islands who are performing arbor work tasks on contaminated wood have the potential of being exposed to very high concentrations of C. gattii, and will be exposed to smaller particulate containing the organism compared to if work tasks were not being performed. An important finding was that a wide range of tree species have been colonized by the C. gattii including economically important varieties such as Douglas fir, Red Cedar, and Alder. Air sampling suggests that the highest levels of airborne C. gattii occur in the drier months of the year with very few positive air samples being found between the months of November and March. Relative humidity and Rainy conditions were significantly associated with airborne C. gattii in non-working conditions. The location where the sample was taken and the month in which it was taken were significant predictors of airborne C. gattii concentrations in both occupational and non-occupational settings. Further study of the transport and distribution of C. gattii in British Columbia would give researchers more insight regarding why the pathogen has become so widespread on Vancouver Island. A transect study of the major roads through an area such as the Sooke Watershed could lead to important discoveries regarding the conditions from which manifestation of persistent colonization occurs. It would be interesting and useful to study the transport and dispersal of C. gattii by arbor work activities such as the sale of wood chips for animal bedding or park paths. Future studies should concentrate on the risk factors that lead to clinical manifestation of cryptococcal disease in British Columbia. Determination of the risk of exposure is just one step in assessing the risk of contracting the disease. Other risk factors, such as biological susceptibility to the organism, should be researched in order to be able to adequately assess the risk of disease due to C. gattii. 91 Those who are living and working on Vancouver Island or the Southern Gulf Islands should be made aware of the existence of the pathogen and the signs and symptoms of disease. This is especially important for arbor workers who may be exposed to high concentrations of small C. gattii propagules. Workers who are aerosolizing wood dust and other matter due to the tasks that they do are encouraged to don an N-95 respirator in accordance with W C B regulation 8.32.' 1 3 If workers are aware that they are working in a contaminated area it would be prudent to wear a fit-tested N95 respirator because they may be exposed to high concentrations of small particulate matter carrying biologically viable C. gattii. 92 R E F E R E N C E S 1. Kidd SE, Hagen F, Tscharke RL, et al. A rare genotype of Cryptococcus gattii caused the cryptococcosis outbreak on Vancouver Island (British Columbia, Canada). Proc Natl Acad Sci USA. 2004; 101:17258-63. 2. Statistics Canada. Community Profiles. Available at: http://wwwl2.statcan.ca/english/Profil01/CP01/Index.cfm?Lang=E. Accessed 03/12, 2006. 3. Environment Canada. Canadian Climate Normals or Averages 1971-2000. Available at: http://www.climate.weatheroffice.ec.gc.ca/climate_normals/index_e.html. Accessed 02/22, 2006. 4. Perfect J. 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Health-related aerosol measurement: A review of existing sampling criteria and proposals for new ones, [review] [72 refs]. J Environ Monit. 2005 Nov;7:1037-1053. 111. Pacwill Environmental. Available at: http://pacwill.ca/6stage.htm. Accessed 03/02, 2006. 112. Statistics Canada. 2001 Census of Canada. Available at: http://www.12.statcan.ca/english/census01/home/index.cfm. Accessed 02/26, 2006. 113. Worksafe BC . OHS Regulation. Available at: http://www.worksafebc.com/default.asp. Accessed 03/20, 2006. 100 APPENDIX D - QUESTIONNAIRE UBC Questionnaire - Page 1 University of British Columbia School of Occupational and Environmental Hygiene Investigation of Occupational Exposures to Forestry Workers from Environmental Cryptococcus neoformans var. gattii. Principal Investigator: Dr. Karen H Bartlett Objectives statement: We're undertaking this study to examine the potential hazard faced by outdoor workers who may come into contact with Cryptococcus neoformans var. gattii a pathogenic yeast strain that has been isolated in several recent respiratory disease cases. We know that this organism makes its home in tree bark and can become airborne, particularly when disturbed. The answers you give will help us understand what risks are faced by those working in the forest industry on Vancouver Island. Confidentiality statement: The information you provide to us will be held in strictest confidence. This top sheet will be removed from the rest of the questionnaire, and all further analyses will be conducted using an anonymous numeric code. Your personal information will be securely filed and only the Principal Investigator will have access. This section will be completed by UBC ; , Access code [_ ] ' , Site code [ | • , , ' r V Date \ I I ] f \ [1-4] 1 „• • - [S-6] . i " . * V dd mm yy. .[7-1*2] ^_ Part A . Note: To preserve confidentiality, Researchers will remove this page from the questionnaire and it will be stored confidentially in a secure location. Demographics: What is your full name? (a) Last [ ] (b) First [ ] (c) M t [ ] [13] What is your home telephone number? [ - - ] [11-23] Gender? (a) Male [_] (b) Female [_] [24] Ethnicity: (a) Caucasian [ ] (b) First Nations [_] (c) Oriental [ ] [25-26] (d) Indo-Canadian [ ] (e) Afro-Canadian [ ] (f) Other[ ] (please specify) [ ] (g) Doh't know [_] (h) Refused [_] Date of Birth [__/__/____] P 7 " 3 2 l dd nun yy We would like to biow where you have lived since 1999: Home address: (a) [ ] (33] (b) \ 1 [3-1-35] (c) [ : ] [36-41] Street address City Postal code How long have you been at this address? (d) [ years] 142-43] If you have lived at above address less than 5 years: Prior address: (a) [_ ] [44] (b) [ ] [45-46] (c) [ _] [47-52] Street addi ess City Postal code How long were you at this address? (d) [_ years] [53-54] If you have lived at either address listed above for a total of less than 5 years: Prior address: (a) [ 1 [55] (b) \ 1 .[56-57] (c) [__ ] [58-63] Street address City Postal code How long were you at this address? (d) [ years] [£4-65] 105 Questionnaire - Page 2 Access code [ ] Site code[ ] Date [ / / ] dd nun yy Part B. Occupation Current Occupation? [ ] [66-67] Employer's name [ ] [68-69] Note: For reference only. We will NOT contact your employer without your permission Employer's Location (a)[ ] [70] (b) [ ] [71-72] (c) [ ] [73-7S Address City postal code What percentage of your work day is spent outdoors in this job? (a) Winter [ %] [79-81] (b) Spring [ %] [82-84] (c) Summer [ %] [85-87] (d) Fall [ %] [ss-90j Please indicate below if you do any of these activities as part of your job. Please also estimate the approximate number of hours you spend at each task per week in hours, during each season: Task Winter Yes No hours Spring Yes No hours Summer Yes No hours Fall Yes No hours (a) Digging/ [ _ J [ _ J [_] L J L J LJ LJ LJ LJ 1 11 ! LJ moving earth [91] [92-93] [95-96] [97] [98-99] [100] [101-102] (b) Deck-Mowing L J L J LJ LJ LJ LJ L J L J L_] L J L J L_] [103] [104-105] [106] [107-108] [109] [110-111] [112] [113-114] (c) Planting L J L J LJ L J L J LJ L J L J [_] LJ LJ [_] f.! 151 [116-117] [118] [119-120] [121] [122-123] [124] [125-126] (d) Thinning L J L J [ ] LJ LJ L_] LJ LJ L_] L J L J LJ [127] [128-129] [130] [131-132] [133] [134-135] [136] [137-138! (e) Pruning L J L J LJ L J L J LJ L J L J LJ LJ LJ [_] [139] [140-141] f!42] [143-144] 1145] [146-147] [148] [149-150] (f) Felling LJ [_] LJ L J L J LJ L J L J L_] L J L J LJ [151] [152-153] (154] [155-156] [157] [158-159] [160] [161-162] (g) Yarding L J L J LJ L J L J LJ L J L J LJ LJ LJ LJ [163] [164-165] [166] [167-168] [169] [170-1711 [172] [173-174] (i) Flay-mowing L J L J LJ L J L J [_] L J L J LJ L J L J LJ [175] [176-177] [178] [179-180] [181] [182-183] [184] [185-1861 0) Chipping L J L J [_] L J L J l_J L J L J LJ L J L J [_] [187] [188-189] [190] [191-192] [193] [194-195] [196] [197-198] (k) Burning L J L J LJ L J L J [_1 L J L J LJ L J L J L_] [199] [200-201] [202] [203-204] [205] [206-207] [208] [209-210] (1) Clear rotting L J L J [_] L J L J LJ L J L J LJ' L J L J LJ wood [211] [212-213] [214] [215-216] [217] [218-219] [220] [221-222] Other: (describe) (m) L J L J L_] L J L J [_] L J L J LJ L J L J LJ [223] [224] [225-226] [227] [228-229] [230] [231-232] [233] [234-235] (n) L J L J [_] [ ] [ ] LJ L J L J [_J .[_][_] LJ [236] [237] [238-239] [240] [241-242] [243] [244-245] [246] [247-248] (0) L J L J LJ L J L J LJ L J L J LJ L J L J [_J [249] [250] [251-252] [253] [254-255] [256] [257-258] [259] [260-261] (q) Exposed to L J L J [_] L J L J LJ L J L J [_] L J L J [_] wood dust? [262] [263-264] [265] [266-267] [268] [269-270] [271] [272-273] (r) Exposed to L J L J LJ L J L J LJ L J L J LJ [_] LJ LJ other dust? (e.g.. [274] [275-276] [277] [278-279] [280] [281-282] [283] [284-285] road) How long have you been at this job? [ ] [286-287] [ _ J [288-289] Months Years 106 Questionnaire - Page 3 Access code [ ] Site code[ ] Date [_ / / _J dd' mm yy Previous Occupation? [ 1 [290-291 Start Date: [ ] [292-293] [ ] [294-295] End date: [ ] [296-297.1 [ ] [298-299] mm yy mm yy Employer's name [ . _ _ _ _ ] poo) Note: For reference only. We will NOT contact your employer without your permission Employer's Location (a) [ ] poi] (b) [ ] [302-303] (c) [ ] [304-309.] Address City postal code What percentage of your work day was spent outdoors in this job? (a)Winter [ %] 1310-3123 (b)Spring [ %] 1313-315] (c)Summer [ _%] [316-318] (d)Fall [ %] [319-321] Please indicate below i f you did any of these activities as part of your job. Please also estimate the approximate number of hours you spend at each task per week in hours, during each season: Task Winter Spring Summer Fall Yes No hours Yes No hours Yes No hours Yes No hours (a) Digging/ L J L J [_] L J L J [_J L_l [_] L I L I L_J L J moving earth 1322] [323-324] [325] [326-327] [328] [329-330] [331] [332-333] (b) Deck-Mowing L J L J L J L J L J L J L J L J L J L J L J [ ] [334] [335-336] [337] [338-3391 [340] [341-342] [343J [344-345] (c) Planting L i [_l L J L J L J ' L J [ J L J [_] L J L J L J [346] [347-348] [349] [350-351] [352] [353-3541 1355] [356-357] (d) Thinning L J L J [_] [_] L I [_1 [_] L J L J L J L J L J [358] [359-360] [361] [362] [363-364] [365-366J [367] [368-369] (e) Pruning L J L J L J L J L J L J L J L. J L J L J L J L J (370] [371-372] [373] [374-375] [376] [377-3781 [379] [380-381] (1) Felling L J L J [_] L J L J L J L_] L I L J L J L J L J [382] [383-384] [385] [386-387] [388] [389-390] [391] [392-393] (g) Yarding L J L J [_] L J L J [_] L J L J L J L J L J L I [3941 [395-3961 [397] [398-399] [400] [401-402] [403] [404^405] (i) Flay-mowing L J L J [_J L J L J [_] L I U L J L J L J L J [406] [407-408] [409] [410-411] [412] [413-414] [415] [416-417] (j) Chipping L J L J L J L J L J L J L I U L I L J L J L J [418] [419-120] [421] [422-123) [424] [425-4261 [427] [428-429] (k) Burning L J [_J L J L J L J L J L J L J [_] L J L J L J [430] [431-432] [433] [434-435] [436] [437-4381 [439] [440-441] (1) Clear rotting L J L J [_J L J L J L J [_] L I L J L J L J L J wood "4421 [443-444] [445] [446-447] [448] [449-450] [451] [452-453] Other: (describe) Cm) L J L J L J L J L J L J L J L J L J L J L I L J [454] [455] [456-457] [458] [459-4601 [461] [462-463] [464] [465-466] (n) L J L J L I L J L J L J L J L J L J L J L J L J [467] [468] [469-470] [471] [472-473] [474] [475-476] [477] [478-179] (0) L J L J [_] L J L I L J L J L J [_] U U L J [480] [481] [482-483] [484] [485-486] [487] [488-489] [490] [491-492] (q) Exposed to L J L J [_] L J L J [_] L J L J L_J L J L J L I wood dust? [493] [494-495] [496] [497-198] [499] [500-501] [502] [503-50-4] (r) Exposed to L J L J [_] L J L J L J L J L J [_T L J L J • L J other dust? (e.g. [505] [506-507] [508] [509-510] [511] [512-513] [514] [515-516] road) How long were you at this job? [ ] [517-518] I" 1 [519-5201 Months Years If you have had other employment in the last 5 years„please ask for more pages. 107 Questionnaire - Page 4 ;esscode[ ] ' Site code[ ] -Date [ _ _ / _ __ /__ J dd mm yy What percentage of your leisure time is spent outdoors? a) Winter [ %] [522-S23] b) Spring [ %] [524-526] c) Summer [ %] [527-529] d) Fall [ %] [530-532] For each task below, please rank (1 = most common, 2 = next most common, etc.) the tree species you most often worked with IN T H E P A S T Y E A R , then the next most common, etc. Place a zero against any tree species you never or rarely work with: T A S K Douglas Arbutus Garry Alder Grand Maple Cedar Hemlock Pine Fir Oak Fir (a) Planting L J [_] [_] L J L J L J L J L J [_J [533] [534] [5351 [536] [537] [538] [539] [540] [541] (b)Thinning [_] [_J L J L I [_] L J L J L J L J [542] [543] [544] [545] [546] [547] [548] [549] [550] (c) Pruning L J L J L J L J [_] L J L J L J L J 1551] [552] [553] [554] [555] [556] L557] [558] [559] (d)Felling [_] L J L J L J [_] L J L J L J L J [560] [561] (562] [563] [564] [565] (566) [567] [568] (e) Yarding L J L_J L J L J L J L J L J L J L J [569] [570] [571] [572] [573] [574] |575] [576] [5771 (f) Flay-mowing [_J L J [_] L J L J L J L J L J L J [578] [579] [580] [581] [582] [5S3] (584] [5S51 [586] (g) Chipping L J L J L J L J L J L J L J L J L I [587] [588] [589] [590] [591] [592] [593] [594] [595] (h) Burning L J [_] L J L J L J L J L J L J L J [596] [597] [598] [599] [600] [601] [602] |603] [6041 (i) Clear rotting L J [_] L J L J L I L J L I L J L J wood [605] [606] [607] [608] (609] [610] [611] [612] [613] Other forestry/grounds tasks (describe) (i) [_] L J L J L J L J [ ] [ ] L J L J [614] [615] [616] [617] [6is] [619] [620] • [621] [622] [6231 (k) L J L J L J L J L J • L J L J L J L J [625] [626] [627] [628] [629] [630] [631] [632] [633] [634] (1) L J L J L J L J L J L J L J [_] L J [635] [636] [637] [368] [369] [370] [371] [372] (373) [374] (m) work at home? L I L J L J L J L J L J L J L J L J [635] [636] 1637] [638] (6391 [640] [641] [642] 1643] 108 Questionnaire - Page 5 Access code [ ] Site code[ ] Date [_ / _ / _ J , dd mm yy Now please rank the tasks/trees again, this time thinking about the T H E P A S T 5 Y E A R s . Place a zero against any tree species you never or rarely work with: T A S K Douglas Arbutus Garry Alder Grand Maple Cedar Hemlock Pine Fir Oak Fir (a) Planting [_] L J [_J [_] [ ] L J L I [ 1 L I [644] [645] [646] [M7J [648] [649] [650J [651] [652J (b)Thinning L J L J L J L J L J 1 ! [_J ' L J L J [653] [654] [655] [656] [657] [658] [659] [660] [661] (c) Pruning L J L J L J L J L I L ] [_] L J L J [662] [663] [664] [665] [666] [667] [668] [669] [670] (d)Felling L J L J L J L J L J L ) L J L J [_J [671] [672] [673] [674] [675] [676] [677] [678] [679] (e) Yarding L J [_] L J L I L J [_] L J L I L J [680] [681] [682] [683] [684] [685] [686] [6871 [688] (0 Flay-mowing [_] L J L J L J • L J [.__] L J L J L J [6S9J [690] [691] [692] [693] [694] [695] [696] [697] (g) Chipping [_] L J [_J L J [_] L J L J L J L J [698] [699] [700] [701] [702] [703] [704] [705] [706] (h) Burning L J L J L J L J L J L J [_] [_] L J [707] [70S] [709] [710] [711] [712] [713] [714] [715] (i) Clear rotting L_] [_] L I [ ] [_] L J L I [_] L J wood [716] [717] [718] [719] [720] [721] [722] [723] [724] Other forestry/grounds tasks (describe) (i) L J [_] L J L ] L J L J . L I [_] [_] [725] [726] [727] [728J [729] [730] [731] [732J [733] 1.734] 00 L J L_] L J L J L J L J L J L J L J [735] [736] [737] [738] [739] [740] [741] [742] [743] [744] (1) L J L _ l L J L J L I L J L J L J L J [745J [746] [747] [748] [749] [750] [751] [752] [753] [754] (m) work at home? L J L J [_J L J L J L J L J L J L J [7551 [7561 (7571 [758] [7591 [760] [7611 [762] |763) 109 Questionnaire - Page 6 Access code [ J Site code[ ] Date [_ _ / _ _ / _ J ckl mm yy Part C. Travel Please indicate on the map the number of days spent in each of the different areas of Vancouver Island indicated (indicate days working, and days leisure separately): Elk Falls Park ,——1 ^ L L J CAMPBELL Mirade "• Beach Park COURTENAY MacMillan Park Sproat Lake Park S PORT ALBERNI Little Qualicum Falls Paik Lcrg Beach Rathtravor Beach Park <./ 9 Eriglishnar Rjver Falls Park f t NANAIMO . • . Pacific Rim Naiiona! Park <^ ^ Cowichan River Perk @ Reserve West 0 0 3 5 1 Trail ^=a=^ fiamberton Park i Goldstrearn Park S Location Last year Last 5 years Work (days) Leisure (days) Work (days) Leisure (days) A [764-765] [766-768] 1769-771] [772-774] [775-776] B [777-778] [779-781] [782-784] [785-787] [788-790] C [791-792] [793-795] [796-798] [799-801] [802-804] D [805-806] [807-809] [810-811] [812-814] [815-816] E [817-818] [819-821] [822-824] [825-827] [828-830] F [831-832] [S33-835] [836-838] [839-S41] [ 12 is-: I G [845-846] [847-849] [85(l-8.->2] [853-855] [856-858] H [859-860] [861-863] [864-866] [867-869] [870-872] I [S73-874] [875-877] [S78-880] 1881-883] [884 886] J [887-888] [889-8911 [892-894] [895-897] [898-900] K 1901-902] [903-905] [906-908] [909-911] [912-914] L [915-916] [917-919] [920-922] [923-925] [926-927] M [928-929] [930-932] [933-935] [936-938] [939-941] 1 1 0 Questionnaire - Page 7 Access code [ ] Site code[ ] Date [_ / / dd mm Please indicate where else you have trave ed in the past year: Location (i) Name Places visited (ii) Date (month/year) (iii) U of days spent there? (iv) Working (Y/N) (a) Gulf Islands [942] [943-946] [947-949] [950) [951] [952-955] [956-95S] [959] [960] [961-964] [965-967] [958] [9691 [970-973] [974-976] [977] (b) Another part of BC [978] [979-982] [983-985] [9S6] [987] [988-991] [992-994] [995] [996] [997-1000] [1001-1003] [1004] [1005] [1006-1009] [1010-1012] [1013] (c) Another province or territory in Canada [1014] [1015-1018] [1019-1021] [1022] [1023] [1024-1027] [1028-1030] [1031] [1032] [1033-1036] [1037-1039] [1040] (d) United States [1041] [1042-1045] [1046-1047] [1048] [1049] [1050-1053] [1054-1056] [1057] [1058] [1059-1062] [1063-1065] [1066] (e) Another Country [1067] [1068-1071] [1072-1074] [1075] [1076] [1077-1080] [1081-1083] [1084] [1085] [1086-1089] [1090-1092] [1093] [1094] [1095-1098] [1099-1101] [1102] 111 Questionnaire- Page 8 xess code [ ] Site code[ ] Date [___/_ I dd mm yy Have you ever visited any of the fol lowing Vancouver Island Parks: Location Yes/No (i) First year visited (ii) Most recent year visited (iii) # of trips (iv) Ever worked in this park? (a) Cathedral Grove [1103] [1104-5] [1106-7] [1108-09] [1110] (b) Pacif ic R im Park [1111] [1112-3] [1114-5] [1116-17] [1118] (c) Strathcona Park [1119] [1120-21] [1 122-23] [1124-25] [1126] (d) Miracle Beach [1127] [1128-29] [1130-31] [1132-33] [1134] (e) Hel l iwel l Provincial Park, Hornby Isl. [11351 [1136-37] [1138-39] [1140-41] [1142] (f) Boyle Point, Denman Island [1143] [1144-45] [1146-47] 11148-49] [1150] (g) Englishman River Falls Park [1151] [1152-53] [1154-55] [1156-57] [1158] (h) Little Qualicum River Park [1159] [1160-61] [1 162-63] [1164-65] [1166] ( i ) Rathtrevor Beach Park [1167] [1168-69] [1170-71] [1172-73] [1174] (j) Beban Park [1175] [1176-77] [1178-79] [1180-81] [1182] (k) Piper's Lagoon [11831 [1184-85] [1186-87] [1188-89] (1190] (1) Shawnigan Lake [1191] [1192-93] [1194-95] [1196-97] [1198] (m) Bamberton Park [1199] [1200-01] [1202-03] [1204-05] [1206] (n) Mount Douglas Park [1207] [1208-09] [1210-11] [1212-13] [1214] (o) Beacon H i l l Park [1215] [1216-17] [1218-19] [1220-21] [1222] (p) Coldstream Park [1223] [1224-25] [1226-27] [1228-29] [1230] (q) Other parks: (please list) [1231] [1232-33] [1234-35] [1236-37] [1238] [1238] [1239] [124041] [1242^13] [1244-45] [1246] [1247] [1248] [1249-50] [1251-52] [1253-54] [1255] 112 Questionnaire - Page 9 Date r d d m m yy A^s^elZZZLT Si^efZl] D a t e f — 1 PartD. Health Have you ever been diagnosed with cryptococcal disease? (a) Yes [_J (b) No [_J [1*6 c. If yes, what treatment did you receive? [ . . J [1257-58] If yes. Did you receive steroid treatment? [_ ~e. If yes, did you receive x-rays? ] [1259-61] ] [1262-64] Have you had any ofthe following in the past year? M Bronchitis [ 1 [1265] (b) Pneumonia L J WW ( c) Tuberculosis [ ] \m,\ (d)SuUi W Ane-n» L I ! « « . (0 Other? _ J (g) P^ase specify [1271] Have you had any of the following in the past 5 years? a) Bronchitis [ ] [1272] (b) Pneumonia [ ] [1273] (c) Tuberculosis | ] [1274J (d) Cancer [ ] [12751(e) Anemia [ ] [i276](f) Other? [ ] [12771(g) please specify [ ] [127S] If you had any illnesses in the last 5 years, do you remember having had any of the following symptoms WHICH L A S T E D FOR O V E R O N E M O N T H during the course of your illness; (a) Cough [__] [1279] (b) Cough with blood [ ] [12SO] (c) Cough with phlegm [ ] [issi] (d) Shortness of breath [ ] [12S2] (e) Chest pain [ ] [1283] (f) Headache [ ] [i2R4j (g) Neck Stiffness [_J [1285] (h) Fever [ ] [1286] (i) Chills [ ] [i287] (j) Night sweats [ ] [1288] (k) Unexplained weight loss [ ] [i289] (1) Loss of appetite [ ] [12901 (rn) Nausea [ ] [1291] (n) Muscle/joint pain [ ] [1292] (0) Skin rash [ ] [1293] (p) Nodules [ ] [1294] (q) Any other symptoms, [1295] please specify [ J [1296-98] Do you smoke tobacco? (a) Yes f ] (b) No [ ] (c) Refuse [ ] [1299] (ifYESJ: d. Daily [ ] [1300] Occasionally [ ] [1301] Number of cigarettes per day [ ] [13023 e. Age started smoking [ ] [1303-4] |lf NO~|: Have you ever smoked? (f) Yes [ ] (g) No [ ] [1305] h. At what age did you start smoking [ ] [i306-7] i. At what age did you quit? [ ] [1308-9.1 j . Have you started and stopped smoking more than once? Yes [ ] No [ ] fisio] May we contact you again should the need arise (a)Yes [_] (b) No [_J [ i3U) If Y E S : (c) Telephone number [ ] L 1 1 " W A r e a code telephone 113 

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