with the use of longer observation periods extending further from the original sampling. Because the effect of endotoxin levels on ill- ness episodes was highly significant for a 2-year period, it would be even more signiﬁ- cant for shorter observation periods. Discussion Air endotoxin was positively associated with an increase in episodes of respiratory illness among children during their ﬁrst 2 years of life despite adjustment for many host and envi- ronmental factors, including indicators of fun- gal exposure. The method of endotoxin collection is unique compared with previous Table 4. Association between illness episodes (dependent variable) and natural logarithm of endo- toxin concentrations: multiple linear regression analysis. Independent variable β Unadjusted (model R 2 = 0.01) Intercept 6.99 Ln(endotoxin) 0.24 Adjusted (model R 2 = 0.18) Intercept 7.74 Ln(endotoxin) 0.46 Year tested 1998 –0.77 1999 –1.23 2000 –0.37 2001 1.18 2002 –0.90 2003 Reference Temperature –0.21 Age 0.01 Breast-feeding 0.94 Child care 0.04 Exposure to smoke Low –0.70 Medium 0.17 High Reference Income < $30,000 –0.57 $30,000–49,999 –0.93 ≥ $50,000 Reference Independent variable β Unadjusted (model R 2 = 0.01) Intercept 60.7 Ln(endotoxin) 3.09 Adjusted (model R 2 = 0.15) Intercept 47.3 Ln(endotoxin) 4.68 Year tested 1998 –1.50 1999 –4.88 2000 4.77 2001 13.2 2002 –7.77 2003 Reference Temperature –2.73 Age 0.16 Breast-feeding 0.70 Child care 18.3 Sex (male) 7.36 SE p-Value 0.18 < 0.0001 0.12 0.0556 1.92 < 0.0001 0.13 0.0003 0.0001 0.55 0.1645 0.55 0.0269 0.56 0.5019 0.55 0.0321 0.52 0.0881 SE p-Value 2.28 < 0.0001 1.59 0.0533 0.06 0.004 0.58 0.02 0.39 0.37 0.41 0.33 24.7 1.66 6.90 7.01 7.08 7.17 6.78 0.85 0.05 0.22 7.45 3.79 0.0012 0.0038 0.1031 0.0200 0.0483 0.0757 0.6358 0.0204 0.1614 0.0056 0.0565 0.0050 0.8279 0.4863 0.5003 0.0674 0.2528 0.0014 0.0029 0.0013 0.0147 0.0533 studies of indoor air, most of which sampled floor dust rather than airborne dust, which may be better correlated with inhalation expo- sure. Air sampling, done infrequently in previ- ous studies, usually consisted of a grab sample (up to 30 min), whereas our 5-day collection period would be expected to provide a more stable average estimate of exposure. Another unique feature of exposure is that endotoxin sampling was done during the cold season when the ground is frozen and usually snow covered. This makes it less likely that the meas- ured endotoxin in air is simply a reﬂection of what was present outdoors at the time of the sampling. Rylander (2002) suggested that fun- gal products, and specifically (1–3)-β-D-glu- can, may coexist with endotoxins and thus may confound the association. In the present study, ergosterol, a marker of fungal growth in Prince Edward Island homes, did not influence the illness–endotoxin association. Michel et al. (1991) measured endotoxin concentrations in the house dust of 28 adult subjects with chronic stable asthma. Exposure to higher levels (> 5.6 ng lipopolysaccha- ride/mL) was associated with poorer asthma control measured by symptoms, medication use, and lung function. In a subsequent study, Michel et al. (1996) refined their previous ﬁndings in a group of 69 adults with asthma who were sensitized to house dust mites. Asthma control was related to house dust endotoxin, averaging 2 ng/mg dust in those exposed to Der p 1 levels > 10 µg/g dust, but 1.2 1.1 1.0 0.9 0.8 ize 0.7 ts 0.6 fec 0.5 fE 0.4 0.3 0.2 0.1 0.0 11 10 9 8 izse 67 tffceE 54 3 2 1 0 A B Endotoxin and respiratory illness not in those exposed to lower levels of the major dust mite antigen. Rizzo et al. (1997) and Douwes et al. (2000) reported adverse effects of settled dust endotoxin in school-age children with asthma, atopy, or asthma symp- toms but not in those without an atopic his- tory. Park et al. (2001a) and Gehring et al. (2001a) reported increased wheezing in infants living in homes with increased levels of settled dust endotoxin, consistent with the findings of the present study in which airborne endo- toxin was measured and fungal burden was accounted for using airborne ergosterol. Indoor fungus therefore is not likely to have con- founded the observed relation, although fun- gus has been associated with respiratory symptoms and may be associated with indoor endotoxin (Gehring et al. 2001b; Verhoeff and Burge 2004). Compared with occupational settings, the indoor air endotoxin concen- trations in our study were low (on average, < 2 EU/m3), yet associations with adverse health effects were observed. The relatively large sample size with daily symptom monitor- ing over 2 years in each subject appeared to give us the power to detect these effects. Further support from health effects at low levels comes from Park et al. (2000), who reported airborne endotoxin in Boston homes during the warm season to be generally < 1 EU/m3, and corresponding ﬂoor dust was < 100 EU/mg (estimated values taken from a log–scaled graph). Before our study, Park et al. (2001a) also found associations between settled dust endotoxin and wheeze. Endotoxins are also postulated to confer health beneﬁts. Lipopolysaccharide, the main component of endotoxin, may shift the cytokine response toward a Th1 response and away from a Th2 response, thereby reducing the chance of developing atopy (Lapa e Silva et al. 2000). Consistent with this theory is the observation that children growing up on farms, where endotoxin exposure is higher than in urban areas, have less atopy (Von Ehrenstion et al. 2000; Von Mutius et al. 2000). Evidence thus far suggests that early childhood exposure to endotoxin may protect against future asthma, but later in life endotoxin appears to exacerbate asthma (Reed and Milton 2001). The present study indicates that very early exposure is not benign but associated with increased illness episodes. Litonjua et al. (2002) reported on 226 children between the ages of 1 and 5 years with a parental history of atopy who were followed for 4 years. House dust endotoxin was associated with reported wheez- ing that decreased with increasing duration of follow-up. This observation suggests that early exposure offers future protection, or that the initial endotoxin measure became less represen- tative of ongoing endotoxin exposure over time. The present study was somewhat differ- ent: beginning at birth, including all children 613 Table 5. Association between illness days (depen- dent variable) and natural logarithm of endotoxin concentrations: multiple linear regression analysis. 90 180 270 360 450 540 630 720 999 Window (days) 90 180 270 360 450 540 630 720 999 Window (days) Figure 1. The effect size over time between illness and bedroom endotoxin measured at birth. (A) Annualized illness episodes. (B) Annualized illness days. Effect size is represented as the β-coefﬁcient for the effect of the natural logarithm of endotoxin on illness along with its 95% conﬁdence interval. Environmental Health Perspectives • VOLUME 114 | NUMBER 4 | April 2006