On-Line Supplement. Endotoxin Exposure: Predictors and Prevalence of Associated Asthma Outcomes in the U.S.

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1 On-Line Supplement Endotoxin Exposure: Predictors and Prevalence of Associated Asthma Outcomes in the U.S. Peter S. Thorne, PhD, a * Angelico Mendy, MD, MPH, a Nervana Metwali, PhD, a Päivi Salo, PhD, b Caroll Co, MS, c Renee Jaramillo, MStat, c Kathryn M. Rose, PhD, c Darryl C. Zeldin, MD b a Department of Occupational and Environmental Health, University of Iowa, Iowa City, Iowa b Division of Intramural Research, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina c Social & Scientific Systems, Inc., Durham, North Carolina Running Title: Endotoxin and NHANES Respiratory Disease *Corresponding Author: Peter S. Thorne, PhD Department of Occupational and Environmental Health The University of Iowa, College of Public Health 145 N. Riverside Dr., S341A CPHB Iowa City, IA USA Tel: (319) peter-thorne@uiowa.edu Funding: Sample extraction and endotoxin analysis work at the University of Iowa was funded by CDC/NCHS ( NCE1). Data analysis was funded through a grant to the University of Iowa, Environmental Health Sciences Research Center (NIH P30 ES005605) and through a contract to Social & Scientific Systems, Inc. (HMSN ). This work was also funded, in part, by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences (NIH Z01 ES025041).

2 SUPPLEMENTAL METHODS Study Framework We used data from the NHANES conducted from 2005 to 2006 by National Center for Health Statistics (NCHS). NHANES is a continuous cross-sectional survey of the US noninstitutionalized civilian population. It uses a complex multistage design to derive a representative sample and oversamples people below poverty level, aged 12 to 19 years, aged 60 years or older, pregnant women, African Americans, as well as Mexican Americans to ensure adequate subgroup analyses. The NHANES study design and protocol was established by the NCHS and approved by the NCHS Ethics Review board. Written informed consent was obtained from all participants enrolled. Details on NHANES protocols and procedures are available at Endotoxin Analysis Combined bed and bedroom floor dust samples were collected at each participant s home using a Sanitaire Model 3683 vacuum cleaner and a Mitest Dust Collector (Indoor Biotechnologies, Inc., Charlottesville, VA). A 0.84 m 2 (1 square yard) surface on both bed and adjacent floor was independently vacuumed for 2 min. Sieved, frozen house dust samples generally weighing between 20 and 50 mg were shipped to our laboratory for extraction and analysis of endotoxin employing multiple levels of quality assurance. The sieved dust was extracted with 1 ml of sterile pyrogen-free LAL water plus 0.05% Tween-20 to a final concentration of 50 mg/ml. Dust was shaken for 1 hr at 22 C, then centrifuged at 4ºC and 600 g to remove large insoluble particles and the supernatant was assayed. Endotoxin was measured using a kinetic chromogenic Limulus amebocyte lysate assay. A number of quality assurance measures were adopted. These included rigorous chain of custody verification, E2

3 internal and external audits, bar coding of samples, use of a single lot of assay reagents for the entire study, blind repeats of NHANES dust samples (n=665), use of a single microplate reader for all assays and application of Westgard quality control rules to accept or reject a run. Reservoir house dust collected from households enrolled in two prior studies were taken from cryostorage, sieved, blended and homogenized to create one high endotoxin and one low endotoxin sample each weighed into 450 vials with 50 mg/vial. These low and high control standards (n=722) were analyzed at four dilutions on each of 398 microplates used for assay of NHANES samples and were tracked over the course of the study. Control standard endotoxin from E. coli 055:B5 was used to develop 12-point standard curves and absorbance was read at 405 nm with 181 reads and 0.31 min interval time. Samples were assayed at four dilutions increasing four-fold from 1:400 to 1:25,600. The lower limit of detection was Endotoxin Units (EU)/mg house dust. A detailed description of our laboratory methods for the endotoxin assay is available at: Assessment of respiratory and allergic diseases Respiratory health and allergy-related outcomes were assessed using a questionnaire and Computer-Assisted Personal Interviewing (interviewer administered) system. Questionnaires were constructed using prior NHANES surveys and validated survey instruments. Wheeze and wheezing-related outcomes in the past 12 months included any wheeze, sleep disturbance because of wheezing, exercise-induced wheezing, doctor/er visit(s) for wheezing attack, prescriptions for wheezing, limitations of usual activities because of wheezing, work or school missed due to wheezing (age 6 to 69 years). Asthma and asthma related outcomes were defined by an ever diagnosis of asthma, current asthma, asthma attack in the past 12 months, and use of asthma medication in past 30 days. Allergy-related outcomes and symptoms included current hay fever, diagnosed allergies, allergy symptoms, sneezing or runny or blocked E3

4 nose, allergy medication in past 30 days, itchy rash for at least 6 months, itchy rash in the past 12 months, diagnosed eczema, any eczema medications in past 30 days, and diagnosed sinus infection. Dust allergens, Socio-demographics and allergic sensitization Dust sample extracts from the University of Iowa were also analyzed for a panel of allergens (cockroach: Bla g 2; dog: Can f 1; cat: Fel d 1; dust mites: Der p 1 and Der f 1; mouse: Mus m 1; and rat: Rat n 1) using the ELISA MARIA Multiplex Array assay (Indoor Biotechnologies). Bla g 1 was assayed using enzyme-linked immunosorbent assay (ELISA) test kit (Indoor Biotechnologies). The Aspergillus fumigatus assay was performed at Air Quality Sciences, Inc. using a custom-prepared ELISA from Greer Laboratories (Lenoir, NC). Given the high number of samples with a concentration below the detection limit (5,451 out of a total of 6,957), we decided to exclude Alternaria from our analysis. IgE specific to 15 aeroallergens (Alternaria alternata, Aspergillus fumigatus, Bermuda grass, birch, cat dander, cockroach, dog dander, dust mites [Der p 1 and Der f 1), mouse urine proteins, oak, ragweed, rat urine proteins, Russian thistle, rye grass) and 4 food allergens (egg white, cow milk, peanut and shrimp) were measured using the ThermoScientific ImmunoCAP Specific IgE System (Kalamazoo, Michigan). For children ages 1 to 5 years, 9 specific IgEs were tested. Sensitization status was defined as specific IgE against any measured aeroallergen 0.35 ku/l. Data on socio-demographics, family income, household size, home characteristics, environmental tobacco smoke, and the presence of pets or cockroaches in homes were collected using questionnaires. Generation of Smoothed Plots E4

5 The relationship between endotoxin and respiratory outcomes was illustrated graphically, using smoothed plots to exhibit trends in outcome prevalence across a range of concentrations. Relationships were first modeled using unweighted generalized additive model (GAM), adjusting for age, race/ethnicity, gender, PIR, and endotoxin as a loess function. The 95% confidence intervals were estimated using bootstrapping with 1000 replicates. The logit of rate of disease outcome was expressed as a continuous function of the log-transformed endotoxin level, obtained using a locally weighted regression smoother. The smoothing parameter for each model was selected based on Akaike s information criterion, which uses iterative smoothing span optimization to prevent over-smoothing. A total of 10 iterations are considered for choosing the span. The smooth plots were generated with R version with the modeling done using the GAM (version 1.0.9) package. E5

6 Table E1. Endotoxin predictors considered in forward stepwise regression Season of home visit (2 season) Age (categorical) Home owned, bought, rented, other Family income Family poverty income ratio Race/ethnicity Household reference person educational level How many years family lived in home Type of home When was home built Has home had mildew of musty smell Household size Animals in home now Dog in home now Cat in home now Small furry animal in home now Has participant seen cockroaches in the home Does anyone smoke in the home Floor surface carpeted Room temperature Room relative humidity Impermeable mattress cover Impermeable pillow cover E6

7 Table E2. Geometric mean (GM) and distribution of endotoxin concentration in EU/mg for NHANES samples and the quality control samples included in each assay. Endotoxin values are raw, unweighted data. GSE is geometric standard error and CI is confidence interval. Sample Source Number GM (GSE) 95% CI 5 th Percentile 95 th Percentile NHANES (0.31) Low control samples (0.22) High control samples (1.03) E7

8 Table E3. Odds Ratios and 95% confidence intervals from logistic regression models for the presence of adverse respiratory outcomes using endotoxin exposure levels above and below the median, stratified by sensitization status Unadjusted Model Adjusted Model* Health Outcome Overall Sensitized Not Sensitized Overall Sensitized Not Sensitized n OR 95% CI OR 95% CI OR 95% CI n OR 95% CI OR 95% CI OR 95% CI Wheezing-Related Outcomes in the Past 12 Months Any wheeze , , , , , , 1.76 Exercise-induced wheeze , , , , , , 3.18 Prescription medication , , , , , , 1.84 for wheeze Doctor/ER visit for , , , , , , 1.98 wheeze Asthma-Related Outcomes Diagnosed asthma , , , , , , 1.77 Current asthma , , , , , , 1.84 Asthma attack in past , , , , , , 2.50 Months Any asthma medication in , , , , , , 1.87 past 30 days Combined Outcomes Current asthma and any , , , , , , 2.53 wheeze Current asthma and exercise-induced wheeze in past 12 months , , , , , , 1.89 * Adjusted for age, gender, race/ethnicity, and PIR. Overall excludes participants missing sensitization status. E8

9 Table E4. Adjusted odds ratios* showing the effect of increased endotoxin exposure (above vs. below the median endotoxin value) on asthma outcomes stratified by poverty-income ratio. PIR < 1.85 PIR >= 1.85 Health Outcome OR (95%CI) OR (95%CI) Any wheeze 1.45 (1.10, 1.91) 1.13 (0.77, 1.66) Exercise-induced wheeze 1.39 (0.87, 2.22) 1.48 (0.98, 2.23) Prescription medication for wheeze 1.50 (1.20, 1.88) 1.04 (0.82, 1.32) Doctor/ER visit for wheeze 1.85 (1.12, 3.05) 1.07 (0.80, 1.44) Diagnosed asthma 1.22 (0.85, 1.74) 1.09 (0.85, 1.40) Current asthma 1.25 (0.84, 1.84) 1.24 (0.91, 1.68) Asthma attack in past 12 months 1.40 (0.82, 2.38) 1.05 (0.67, 1.63) Any asthma medication in past 30 days 1.14 (0.82, 1.59) 1.36 (1.02, 1.80) Current asthma and any wheeze 1.29 (0.92, 1.83) 1.26 (0.93, 1.71) Current asthma and exercise-induced wheeze *Adjusted for age, gender, and race/ethnicity 1.07 (0.76, 1.51) 1.18 (0.76, 1.83) E9

10 Table E5: P-value for effect modification by age, environmental exposure, and specific sensitization of the association between a 10- fold increase in endotoxin and current asthma, wheeze in the 12 months, and current asthma and wheeze. Covariates Current Asthma Wheeze in past 12 months Current Asthma & wheeze Age Environmental exposures Mildew or musty smell Cockroaches in home Pets in home Sensitization to specific allergens Cat Dog Mouse Rat Der f Der p Cockroach Alternaria Aspergillus Total IgE * Models adjusted for age, gender, race/ethnicity, and PIR E10

11 Table E6: Change in odds of current asthma and wheeze ( odds) with corresponding P-value associated with a 10-fold increase in endotoxin for each value of specific IgE held constant*. Specific IgE Current asthma Wheeze in past 12 months Current asthma & wheeze Mouse allergen Rat allergen Dog allergen Rat allergen Rat allergen odds P-value odds P-value odds P-value odds P-value odds P-value * Adjusted for age, gender, race/ethnicity, and PIR E11

12 Figure E1. Stratified analysis for wheezing in the past 12 months associated with a 10-fold increase in endotoxin. Each strata shown represents a separate unadjusted logistic regression model. PIR is poverty income ratio. E12

Documentation, Codebook, and Frequencies

Documentation, Codebook, and Frequencies Laboratory Component: Allergen Specific IgE(s) and Total IgE in Serum Survey Years: 2005 to 2006 SAS Export File: AL_IGE_D.XPT First Published: June 2008 Last Revised: