Associates of Cape Cod Ltd., East Falmouth, MA, USA) assisted by sonication. Samples taken before April 2001 were analyzed by the Limulus amoebocyte lysate (LAL) assay gel clot method from Associates of Cape Cod. The detection limit was 0.25 EU/ﬁlter. All subse- quent analyses were done by the LAL chro- mogenic method, also from Associates of Cape Cod. The detection limit was 0.1 EU/filter. All analyses were performed by the same ana- lyst in the same laboratory according to the manufacturer’s instructions. Apart from differ- ences in the lower limit of detection, the two methods gave similar results. We analyzed ergosterol, an indicator of fun- gal biomass, by gas chromatography and mass spectroscopy as described by Foto et al. (2005). The volume of air sampled ranged from 10.7 to 25.4 m3. Ergosterol was determined using an Agilent model 5973 quadrupole mass spec- trometer (Agilent Technologies, Inc., Palo Alto, CA, USA) operating in the electron ionization mode at 70 eV. Compounds were separated on an Agilent 6890 series gas chromatograph equipped with a 30 m × 0.25 mm inner diameter × 0.25 µm ZB-5 capillary column. The concentration of ergosterol was determined against an authentic external standard in the selective ion mode using m/z 363 and 396. The detection limit in selective ion mode was 4.5 ± 0.6 ng/mL. Ergosterol standard (Sigma Chemical Company, St. Louis, MO, USA) was recrystalized, freeze-dried, and stored at 4°C. Deﬁnition of covariates other than ergos- terol. During the postnatal interviews, several characteristics were recorded every 2 weeks for a period of 2 years: the presence of furry or feathered pets in the house, the presence of smokers inside the house, whether the baby was breast-fed, and the number of hours per week the child was cared for outside the home. Based on this information, we created a sum- mary variable for each of the characteristics. For the ﬁrst three characteristics, we calculated the percentage of postnatal interviews where the characteristic was declared. For example, if 20 of 50 postnatal interviews mentioned the presence of pets inside the house for a particu- lar child, the value of the pet variable would be 0.4. The last characteristic, the number of hours per week that the baby was cared for outside the home, was averaged over the entire 2-year period to create the child-care variable. For presentation, we then categorized some summary variables. The pet variable was cate- gorized into “never declared pets,” “sometimes declared pets,” and “always declared pets,” which divided responses almost equally into thirds. The exposure-to-smoke variable was categorized into terciles. The breast-feeding and the child-care variables were kept as con- tinuous data. The age variable was deﬁned as the age of the child in the middle of the span of the follow-up period. “Mold surface area” Environmental Health Perspectives • VOLUME 114 | NUMBER 4 | April 2006 refers to the total surface area of the home with mold visible to trained home inspectors. Statistical analysis. We tested the associa- tion between the number of illness episodes per year and airborne bedroom endotoxin concentration using multiple linear regression analysis. Valid endotoxin results were obtained for 351 houses. A total of 19 homes were excluded from the analysis—15 because of missing temperature data and four because of missing income data—leaving 332 homes for analysis. Eleven babies of 332 (3.3%) exited the study before turning 2 years of age (mean age, 1.1 years), and 56 babies had not reached 2 years of age (mean 1.7 years) by the last day of data collection used for this analysis. This is an ongoing study with children entering and exiting at different times. The illness episodes and covariates were annualized and thus adjusted for duration of follow-up. To test the effect of the 11 babies who exited early, we repeated the analysis with and without them, and no differences were found in the illness–endotoxin association. Mold surface area was expressed as ranks from highest to lowest. Endotoxin values fol- lowed a log-normal distribution, so they were log-transformed. A multiple linear regression model with the number of respiratory illness episodes per year as the dependent variable and the natural logarithm of endotoxin concentra- tion as the primary independent variable of interest was built with the stepwise method. A categorical variable—the year of sample collec- tion—was added to the model to account for any seasonal variations in illness from year to year and also the change in the lower limit of detection of the endotoxin analytic technique Endotoxin and respiratory illness after 2001. Endotoxin and year of home sam- pling were held in the model along with any variables with p-values < 0.10, resulting in the ﬁnal model, with variables endotoxin, year of home sampling, temperature, age, mean hour per week that the baby was cared for more than 1 day a week outside the home, percent- age of postnatal interviews in which the baby was breast-fed, income, and categorized per- centage of postnatal interviews where smokers were declared in home. A potential confounder was defined as a variable (Tables 1 and 2) that, if added to the model, would change the parameter (β) of the natural logarithm of endotoxin by > 10%. No confounders were found for the illness episodes model. The residuals from the regression equa- tion were normally distributed (Shapiro-Wilk statistic = 0.9932, p = 0.1362). We also exam- ined the homogeneity of variance assumption, and the chart of residuals against predicted val- ues showed no particular pattern. Interactions biologically plausible were also tested, and none were found to be statistically signiﬁcant at the 5% level. We found no evidence of interaction between allergies or asthma in par- ents and endotoxin. Careful examination of each of the partial residual plots (i.e., the com- ponent-plus-residual plot) did not reveal any sign of nonlinearity in the relationship between illnesses and air endotoxin. Further, adding a square term for endotoxin did not improve sig- niﬁcantly the R 2 of the model. The endotoxin measurement was made only at the beginning of the 2-year follow-up. To determine the robustness of the endo- toxin–illness association, we measured it at sev- eral time points between the initial endotoxin p-Valuea 0.61 0.19 0.95 0.61 0.70 0.73 < 0.0001 611 Table 1. Characteristics of 332 children overall and stratiﬁed by terciles of endotoxin for categorical variables [no. (%)]. Characteristic Male sex Parent with asthma or allergiesb Parent with university education Family income < $30,000 $30,000–49,999 ≥ $50,000 Environment Furry or feathered pets Never Sometimes Always Exposure to smoke Low Medium High Year tested 1998 1999 2000 2001 2002 2003 Overall 167 (50.3) 174 (52.7) 197 (59.3) 67 (20.2) 114 (34.3) 151 (45.5) 86 (25.9) 126 (38.0) 120 (36.1) 114 (34.3) 110 (33.1) 108 (32.5) 52 (15.7) 53 (16.0) 45 (13.6) 58 (17.5) 58 (17.5) 66 (19.9) 1st tercile 53 (47.8) 64 (57.7) 66 (59.5) 19 (17.1) 39 (35.1) 53 (47.8) 33 (29.7) 42 (37.8) 36 (32.4) 33 (29.7) 40 (36.0) 38 (34.2) 9 (8.1) 8 (7.2) 21 (18.9) 36 (32.4) 24 (21.6) 13 (11.7) 2nd tercile 54 (49.1) 50 (45.9) 64 (58.2) 20 (18.2) 39 (35.5) 51 (46.4) 24 (21.8) 42 (38.2) 44 (40.0) 40 (36.4) 33 (30.0) 37 (33.6) 15 (13.6) 19 (17.3) 14 (12.7) 13 (11.8) 14 (12.7) 35 (31.8) 3rd tercile 60 (54.1) 60 (54.6) 67 (60.4) 28 (25.2) 36 (32.4) 47 (42.3) 29 (26.1) 42 (37.8) 40 (36.0) 41 (36.9) 37 (33.3) 33 (29.7) 28 (25.2) 26 (23.4) 10 (9.0) 9 (8.1) 20 (18.0) 18 (16.2) ap-Value of the Pearson chi-square test of association between the characteristic and endotoxin. bThere are two missing values for parent with asthma or allergies.