[Beta](1 [right arrow] 3)-Glucan in House Dust of German Homes: Housing Characteristics, Occupant Behavior, and Relations with Endotoxins, Allergens, and Molds.

[Beta](1 [right arrow] 3)-Glucans are potent proinflammatory agents that have been suggested to play a role in indoor-related respiratory health effects. The aim of this study was to assess whether [Beta](1 [right arrow] 3)-glucan concentrations in house dust are correlated with levels of endotoxins, allergens, and culturable mold spore counts in house dust. Further, the associations of [Beta](1 [right arrow] 3)-glucan with housing characteristics and occupant behavior were assessed. [Beta](1 [right arrow] 3)-Glucan was measured in settled house dust from living room floors of 395 homes of two German cities, Erfurt and Hamburg, with a specific enzyme immunoassay. Concentrations ranged from below the limit of detection to 19,013 [[micro]g/[m.sup.2] (22,588 [micro]/g dust). Concentrations per square meter were found to be correlated with endotoxins, mite and cat allergens, and culturable mold spores. Correlations were weaker when concentrations were expressed per gram of dust, indicating that variance in concentrations of all factors is largely determined by the amount of dust sampled. Associations between [Beta](1 [right arrow])-3)glucan, housing characteristics, and occupant behavior were found for concentrations per square meter but not for concentrations per gram of dust. The following characteristics were associated with a significant increase in [Beta](1 [right arrow] 3)-glucan levels: carpets in the living room [means ratio (MR) = 1.9-2.1], keeping a dog inside (MR = 1.4), use of the home by four or more persons (MR = 1.4), use of the living room for [is greater than] 180 hr/week (MR = 2.1), lower frequency of vacuum cleaning (MR = 1.6-3.0) and dust cleaning (MR = 1.2 and 1.4, respectively), and presence of mold spots during the past 12 months (MR = 1.4). We conclude that that the amount of dust sampled can be used as a proxy for hygiene and that [Beta](1 [right arrow] 3)-glucan concentrations per square meter are related to the amount of dust sampled. Key words: [Beta](1 [right arrow] 3)-glucan, allergens, endotoxins, house dust, housing characteristics, indoor factors, molds, occupant behavior. Environ Health Perspect 109:139-144 (2001). [Online 19 January 2001] http://ehpnet1.niehs.nih.gov/docs/2001/109p139-144gehring/abstract.html

There is increasing evidence for the impact of dampness and mold growth in homes on respiratory symptoms in children and adults (1-4). Microorganisms and microbial components such as bacterial endotoxins and mold glucans present in house dust are believed to be important (5,6), but it is not yet clear which components primarily account for the observed effects.

[Beta](1 [right arrow] 3)-Glucans are glucose polymers with various molecular weights and degrees of branching that can elicit inflammatory reactions. They are present in the cell walls of most fungi and yeasts, some bacteria, most higher plants, and many lower plants. Rylander et al. demonstrated dose-response relationships between levels of airborne [Beta](1 [right arrow] 3)-glucan and eye and throat irritation, dry cough, and itching skin (7) as well as irritation of the nose, hoarseness, and tiredness (8). In a subsequent study, [Beta](1 [right arrow] 3)-glucans were found to be associated with an increased prevalence of atopy and a decrease in forced expiratory volume in 1 sec ([FEV.sub.1]) over the number of years living in the building (9). Douwes et al. (10) demonstrated an association between [Beta](1 [right arrow] 3)-glucan levels on living room floors and peak flow variability in symptomatic children, and in particular in atopic children with asthmatic symptoms.

[Beta](1 [right arrow] 3)-Glucan concentrations in settled house dust from 395 German homes were studied as part of a collaborative study on Indoor Factors and Genetics in Asthma (INGA). This is the first large-scale study in which relations between glucan levels and a large number of housing characteristics and occupant behavior have been assessed. Because the health effects described for [Beta]-glucans may also be caused by many other house dust-associated agents such as allergens, endotoxins, and molds, probably sharing (at least partially) the same determinants such as home dampness, for example, the first aim of this study was to assess the association between [Beta](1 [right arrow] 3)-glucan concentrations in house dust and levels of endotoxins, allergens, and culturable mold spores. The second aim of the study was to assess associations between [Beta]-glucan concentrations and housing characteristics and occupant behavior.

Materials and Methods

Study population. A total of 405 homes (204 in Erfurt and 201 in Hamburg) were visited within the framework of the INGA study. The study population consisted of a subset of the subjects who had participated in the European Respiratory Health Survey (11). Details of the selection of the study population are described elsewhere (12). The homes were visited from June 1995 to November 1996 by trained personnel. Every subject willing to participate asnwered a detailed questionnaire on building and housing characteristics and occupant behavior.

Dust sampling and extraction. In each apartment a dust sample was taken from the living room floor according to a standardized protocol following the recommendations of an international workshop on dust mites (13). All dust samples were taken using the same type of vacuum cleaner (Flusterjet Vitall 371; Philips, Hamburg, germany) by vacuuming an area of 1 [m.sup2] for 2 min. After sampling in 100 homes, the vacuum cleaners were replaced by new ones to avoid a decrease of air flow during sampling. Ninety-five percent of living room floors had carpets, and 78% of these had wall-to-wall carpeting. Dust was collected on cellulose filters using ALK sampling nozzles (ALK Laboratories, Horsholm, Denmark). The samples were stored at -20 [degrees] C until extraction. Gravimetric measurements of filters were performed before and after vacuuming. Allergens and endotoxin were extracted from the whole dust sample including filter paper as described earlier (14). The dust was not sieved. A subsequent heat extraction to dissolve [Beta](1 [right arrow] 3)-glucan was performed on pellets after the first extraction (15). After extraction of dust samples, the extracts were stored in pyrogen-free glassware at -20 [degrees] C until analysis.

[Beta](1 [right arrow] 3)-Glucan analysis. We assayed [Beta](1 [right arrow] 3)-glucan with a specific enzyme immunoassay, which was developed and described by Douwes et al. (15). Samples were analyzed in duplicate on different days, using one aliquot of each sample per analysis. The mean of the duplicates was used in the further analyses. The interassay variability, expressed as mean coefficient of variation, was 17%, which is similar to what is usually found for many immunoassays. The intraassay variability was considerably lower, but the exact variability is unknown because in this study no intra-assay duplicates were included.

Blank paper filters used for dust sampling contained [Beta](1 [right arrow] 3)-glucan (mean concentration 830 [micro]g/filter, SD 89 [micro]g/filter, n = 10), which was, on average, 42% (range 4-86%) of the total amount of [Beta](1 [right arrow] 3)-glucan detected in each dust sample. Thus, a correction was applied by subtracting the mean concentration detected in the blank filters from the absolute [Beta](1 [right arrow] 3)-glucan level measured in the whole sample (including dust and filter). We do not expect this to bias our results, but using glass fiber filters for future studies will circumvent these problems with blank filters. The limit of detection, defined as 3 x the SD of the blanks, was 267 [micro]g/filter. [Beta](1 [right arrow] 3)-Glucan concentrations were expressed per square meter and per gram of house dust.

Allergen and endotoxin content in house dust. Endotoxin content was quantified using a chromogenic kinetic Limulus amoebocyte lysate test (16). Escherichia coli endotoxin (lot no 5L570, Bio Whittaker, Walkersville, UK) was used as standard endotoxin. The endotoxin potency of this standard was 14.5 EU/g. Concentrations were expressed as nanogram per gram of dust.

Analysis of dust mite allergens Dermatophagoides pteronyssinus (Der p 1) and Dermatophagoides farinae (Der f 1) and cat allergens (Fel d 1) was done using a monoclonal enzyme-linked immunosorbent assay (ELISA) (14) with standards UVA 93/03, UVA 93/02, and UVA 94/01 (Indoor Biotechnologies, Clwyd, UK). The allergen concentration was expressed as nanograms per gram of dust. The lower limit of detection derived from calibration curves was 1 ng/mL for both Der p 1 and Der f 1, and 1.5 ng/mL for Fel d 1.

Culturable mold spore counts. We collected separate dust samples to measure molds. Sampling was performed as described for [Beta](1 [right arrow] 3)-glucan. Samples were stored at room temperature until analysis. Mold spore counts in house dust were performed as described by Koch et al. (17). We analyzed a total of 30 mg of sieved housed dust (500 [micro]m) for identification and quantification of viable fungi. Dilutions of dust in 0.9% NaCl were plated on DG18 (dichloran-18% glycerol) agar. The plates were incubated at 25 [degrees] C for 10 days. Mold spores were counted by naked eye and expressed as colony forming units (CFU) per gram of dust. The lower limit of detection of this method is 1,000 CFU/gram of dust. The following identified genera were counted separately among the cultured colonies: Alternaria, Aspergillus, Cladosporium, and Penicillium. Determination of the genus was performed by high-powered light microscopy (Carl Zeiss Jena, Ergaval, Germany).

Indoor climate. Relative humidity and temperature were measured with Tinytalk II Data Loggers, (Gemini Data Loggers Ltd., Chisester, UK) for 7 days starting on the day dust samples were taken. The data loggers were placed in the living room, roughly 2 m above the floor. We used the median relative humidity and median temperature of each measurement series in further analysis. We assume that these measurements are reasonable proxy variables for temperature and humidity for the time of dust sampling, but are not representative for a longer period.

Building characteristics and occupant behavior. A checklist on building characteristics and occupant behavior was developed, and a trained interviewer completed the checklist in each dwelling. From this checklist, housing characteristics and occupant behavior were assessed for further evaluation.

The heating system was classified either as central or noncentral heating; the latter category included gas heating, fireplace heating, and other noncontinuous heating systems such as coal or wood burning.

Ventilation was recorded during the week that humidity and temperature measurements were taken and classified as good, medium, and poor according to the number of hours per week the window was fully and/or partially opened. Hours were summed (number of hours the window was fully opened + 0.15 x number of hours the window was partially opened), and tertiles were used as cutoffs.

Presence in the living room expressed as hours per day was recorded for up to four persons during the week when humidity and temperature measurements were taken. Presence was recorded for weekdays and weekends separately. Hours were summed (5 x weekday + 2 x weekend), and use of the of the living room by the inhabitants was classified according to the total number of hours the living room was used. Cutoffs were set arbitrarily at 25 and 180 hr/week, which approximately corresponds to one person using the living room for 1 whole day and one person using the living room for 7 days, respectively.

Statistical analysis. Because [Beta](1 [right arrow] 3)-glucan levels in living room floor dust expressed as micrograms per square meter and as micrograms per gram of dust were best described by a lognormal distribution (skewness 3.8 and 7.1, respectively), [Beta](1 [right arrow] 3)-glucan concentrations were natural log transformed. We therefore expressed mean concentrations as geometric means (GM) with a geometric standard deviation (GSD). [Beta](1 [right arrow] 3)-Glucan concentrations below the limit of detection calculated from the field blanks (see "[Beta](1 [right arrow] 3)-Glucan Analysis") were assigned a value of one-half of the detection limit.

We used the Spearman rank-order correlation coefficient to analyze correlations between [Beta](1 [right arrow] 3)-glucan concentrations and indoor climate, the amount of dust sampled, and other agents in the dust (i.e., endotoxins, allergens, and fungi). Associations between [Beta](1 [right arrow] 3)-glucan concentrations on the one hand and housing characteristics and occupant behavior on the other hand were analyzed by means of t-tests based on In-transformed data comparing the category of study with the respective reference category.

To select the most important factors with regard to their impact on [Beta](1 [right arrow] 3)-glucan concentrations, we performed stepwise multiple linear regression on [Beta](1 [right arrow] 3)-glucan concentrations per square meter and per gram of dust with variables that were found to be at least marginally associated (p [is less than] 0.10) with [Beta](1 [right arrow] 3)-glucans. Forward selection was done. A significance level of 0.10 was used for entry into the model. Finally, we used the same model for glucan concentrations per gram of dust and per square meter for comparability purposes, including any factor selected for either glucan per gram of dust or for glucan per square meter.

Associations between [Beta](1 [right arrow] 3)-glucan levels and explanatory variables are presented as the means ratio (MR), which is the ratio of the geometric mean in the category of study versus the reference category. Statistical significance was set at a conventional 5% level.

All analyses were performed using the SAS software, version 6.12 (SAS Institute Cary, NC, USA).

Results

[Beta](1 [right arrow] 3)-Glucan concentrations. [Beta](1 [right arrow] 3)-Glucan concentrations in living room floor dust were available for 395 of the 405 homes visited. Ten samples were not available because of insufficient handling in the laboratory. [Beta](1 [right arrow] 3)-Glucan concentrations in living room floor dust from 24 homes were below the limit of detection, and [Beta](1 [right arrow] 3)-glucan levels in living room floor dust from the remaining homes ranged from 271 to 19,013 [micro]g/[m.sup.2] and from 415 to 22,58 [micro]g/g dust, respectively. Geometric means, GSDs, and percentiles are presented in Table 1.

Table 1. Distribution of [Beta](1 [right arrow] 3)-glucans in living
room floor dust expressed per square meter and per gram of dust.

                                           Percentile

[Beta]-Glucans     GM (GSD)     5th   25th    50th    75th     95th

[micro]g/         1,197 (2.5)   134     742   1,249   2,183   10,817
 [m.sup.2]

[micro]g/g dust   1,711 (1.9)   489   1,295   1,830   2,519    4,065

Concentrations less than the detection limit were assigned a value of
one-half of the detection limit.

Dust weight, endotoxin and allergen measurements, and culturable molds. The average amount of dust sampled from the 395 living room floors was 0.94 [+ or -] 0.83 g/[m.sup.2], ranging from 0.02 to 8.0 g/[m.sup.2]. Endotoxin concentrations varied between 10 and 120,700 ng/g dust (median 2,200 ng/g dust). Der fl levels in living room floor dust were higher than Der p 1 levels (median 415 vs. 156 ng/g dust), ranging from nondetectable to 1,255,751 and 344,806 ng/g dust, respectively. Fel d 1 concentrations ranged from below the limit of detection to 5,274 ng/g dust, with a median of 244 ng/g dust. Total culturable mold spore counts varied between 5,000 and 2,600,000 CFU/g dust with a median of 95,000 CFU/g dust.

Detailed presentations of the results for mite allergens and endotoxins are given by Gross et al. (18) and Bischof et al. (16). Results for Fel d 1 and culturable mold spore counts will be presented in forthcoming publications.

[Beta](1 [right arrow] 3)-Glucan, dust weight, endotoxins, allergens, and culturable molds. Strong positive correlations were found between levels of [Beta](1 [right arrow] 3)-glucan, endotoxin, mite and cat allergens, and culturable molds per square meter on the one hand and the amount of dust sampled on the other hand. Correlations with the amount of dust sampled were stronger for [Beta](1 [right arrow] 3)-glucan, endotoxin, mite allergens Der p 1 and Der f 1, and total culturable molds (r = 0.78, 0.63, 0.52, 0.47, and 0.56, respectively) than for Fel d 1, Alternaria, Aspergillus, Cladosporium, and Penicillium (r = 0.34, 0.23, 0.22, 0.32, and 0.19, respectively), but all were highly significant (p [is less than] 0.01).

Correlations between [Beta](1 [right arrow] 3)-glucan levels and concentrations of endotoxin, mite and cat allergens, and culturable molds are presented in Table 2. When all concentrations were expressed per square meter, strong correlations were found between [Beta](1 [right arrow] 3)-glucan levels and concentrations of endotoxins, mite allergens Der p 1 and Der f 1, and total culturable molds. Weaker but also statistically significant correlations were found between [Beta](1 [right arrow] 3)-glucan concentrations and levels of Fel d 1 and culturable mold spore counts of the various identified genera.

Table 2. Spearman rank-order correlations of [Beta]-glucans and
endotoxins, indoor allergens, and fungi in living room floor dust,
and indoor climate in the living room.

                               [Beta](1[right arrow]3)-Glucan

                             [micro]g/[m.sup.2]   [micro]g/g dust

                               r     p-Value        r     p-Value

Endotoxins                    0.55   < 0.01(*)     0.08     0.10
Indoor allergens
 Der p 1                      0.46   < 0.01(*)     0.14   < 0.01(*)
 Der f 1                      0.42   < 0.01(*)     0.11     0.03(*)
 Fel d 1                      0.28   < 0.01(*)     0.06     0.26
Fungi
 Total fungi                  0.50   < 0.01(*)     0.02     0.62
 Alternaria                   0.29   < 0.01(*)     0.13     0.01(*)
 Aspergillus                  0.18   < 0.01(*)     0.05     0.36
 Penicillium                  0.26   < 0.01(*)     0.03     0.57
 Cladosporium                 0.24   < 0.01(*)     0.18   < 0.01(*)
Indoor climate
 Temperature ([degrees] C)   -0.09     0.09       -0.06     0.28
 Relative humidity (%)        0.04     0.43        0.12     0.02(*)

(*) Statistically significant.

When all concentrations were expressed as micrograms per gram of dust instead of micrograms per square meter, no statistically significant correlation was found between [Beta](1 [right arrow] 3)-glucan levels and concentrations of endotoxins and Fel d 1, respectively. Correlations between [Beta](1 [right arrow] 3)-glucan concentrations and levels of Der p 1 and Der f 1 in living room floor dust were still statistically significant, but much weaker. Additionally, there was a statistically significant correlation between [Beta](1 [right arrow] 3)-glucan levels and culturable mold spores counts from the two genera Alternaria and Cladosporium. Correlations between [Beta](1 [right arrow] 3)-glucan levels and total culturable mold spore counts and culturable mold spore counts from the other genera were not found when concentrations were expressed per gram of dust.

[Beta](1 [right arrow] 3)-Glucan concentrations and indoor climate. We found a marginal statistically significant negative correlation (p = 0.09) between [Beta](1 [right arrow] 3)-glucan concentrations per square meter and indoor temperature (Table 2). Additionally, a weak but statistically significant positive correlation between [Beta](1 [right arrow] 3)-glucan levels and relative humidity was found when [Beta](1 [right arrow] 3)-glucan concentrations were expressed per gram of dust (Table 2). Average indoor relative humidity ranged from 11% to 73%, with a median of 45%. Average room temperature ranged from 9 [degrees] C to 28 [degrees] C, with a median of 22 [degrees] C. The average room temperature of 9 [degrees] C was measured in December in an apartment that was being renovated, so the heating was turned off. All analyses were performed with and without this apartment. As our results were not influenced by this observation, we decided not to exclude it from the analysis. Average room temperature for the remaining apartments was at least 15 [degrees] C.

[Beta](1 [right arrow] 3)-Glucan concentrations, season, housing characteristics, and occupant behavior. Geometric means and GSDs of [Beta](1 [right arrow] 3)-glucan concentrations in living room floor dust expressed as micrograms per square meter and micrograms per gram of dust, respectively, related to place of residence, season, housing characteristics, and occupant behavior are presented in Table 3.

Table 3. [Beta](1 [right arrow] 3)-Glucan in living room floor dust
related to potential influencing factors: GM (GSD).

                                   [Beta](1 [right arrow] 3)-Glucan
                                             GM (GSD)

                                    [micro]g/
Factors                      No.    [m.sup.2]         [micro]g/g

Place of residence
 Erfurt                      201   1,117 (2.4)       1,578 (1.8)
 Hamburg                     194   1,287 (2.6)       1,862 (1.9)(**)
Season
 Summer                      204   1,173 (2.6)       1,681 (1.9)
 Winter                      191   1,224 (2.4)       1,744 (1.8)
Building characteristics
 Building material
  Brick/clinker              217   1,202 (2.4)       1,843 (1.9)
  Concrete                   157   1,161 (2.6)       1,588 (1.8)(**)
  Framework/clay              13   1,647 (1.9)       1,518 (1.4)(*)
 Type of building
  One-/two-family house      114   1,381 (2.5)       1,781 (1.9)
  Multiple-family dwelling   214   1,116 (2.5)(**)   1,696 (1.9)
  Concrete slab house         67   1,174 (2.3)       1,646 (1.8)
Characteristics of living
room
 Carpets
  None                        19     719 (2.9)       1,741 (2.7)
  Carpets < 5 years old      164   1,036 (2.5)       1,528 (1.9)
  Carpets 5-10 years old     116   1,430 (2.3)(**)   1,913 (1.8)
  Carpets > 10 years old      93   1,385 (2.5)(**)   1,791 (1.6)
 Indoor plants
  [is less than or equal
  to] 10                     201   1,199 (2.5)       1,887 (1.8)
  11-20                      121   1,055 (2.6)       1,511 (2.0)(**)
  > 20                        73   1,471 (2.4)(*)    1,609 (1.7)(**)
Use characteristics
 No. of persons living in
 the home
  1-3                        261   1,087 (2.5)       1,714 (1.8)
  [is greater than or
  equal to] 4                134   1,444 (2.5)(**)   1,706 (1.9)
 Use of the living room
 during sampling
  [is less than or equal
  to] 25 hr/week              23   1,075 (2.5)       1,596 (2.2)
  25-180 hr/week             330   1,155 (2.5)       1,725 (1.9)
  > 180 hr/week               42   1,687 (2.3)(**)   1,671 (1.5)
 Residence time
  [is less than or equal
  to] 5 years                 64     885 (2.6)       1,538 (1.9)
  > 5 years                  331   1,269 (2.5)(**)   1,747 (1.8)
 Ventilation
  Poor                       116   1,122 (2.3)       1,675 (1.9)
  Medium                     146   1,323 (2.3)       1,735 (1.8)
  Good                       133   1,135 (2.8)       1,717 (1.9)
 Dog indoors
  No                         356   1,156 (2.5)       1,724 (1.9)
  Yes                         34   1,685 (2.8)(**)   1,534 (1.7)
Cleaning habits
 Vacuum cleaning of
 carpets
  Daily                       33     761 (2.6)       1,327 (2.3)
  Several times a week       170   1,170 (2.5)(**)   1,744 (1.9)(*)
  Once per week              158   1,280 (2.4)(**)   1,738 (1.7)(*)
  Once per month or less      29   1,766 (2.5)(**)   1,834 (1.9)(*)
 Mopping of smooth floors
  Daily/several times a
  week                       104     983 (2.7)       1,557 (2.0)
  Once a week                196   1,283 (2.3)(**)   1,717 (1.7)
  Once a month or less        67   1,498 (2.6)(**)   1,987 (1.8)(**)
 Dusting (dry)
  Daily/several times a
  week                       100     977 (2.5)       1,557 (1.8)
  Once a week                138   1,172 (2.3)       1,682 (1.9)
  Once a month or less       156   1,395 (2.6)(**)   1,839 (1.8)(**)
 Dusting (wet)
  Daily-once a week          130   1,065 (2.3)       1,713 (1.8)
  Once a month or less       264   1,270 (2.6)(*)    1,706 (1.9)
 Mold spots during past 12
 months
  No                         349   1,164 (2.5)       1,688 (1.9)
  Yes                         42   1,557 (2.5)(*)    1,940 (1.9)

(a) Concentrations less than the detection limit were assigned a
value of one-half of the detection limit. (*) p [is less than]
0.10, (**) p [is less than] 0.05 refers to comparisons of the
category of study with the first category of the respective factor
by means of a t-test based on In-transformed data.

When [Beta](1 [right arrow] 3)-glucan levels were expressed per square meter, concentrations in Erfurt were only slightly lower than in Hamburg. We found [Beta](1 [right arrow] 3)-glucan concentrations marginally associated with type of the building, but not with any of the other building or housing characteristics. Elevated [Beta](1 [right arrow] 3)-glucan levels were observed in living rooms that had carpets and [is greater than] 20 indoor plants. Furthermore, keeping a dog inside, use of the apartment, and cleaning habits were important for [Beta](1 [right arrow] 3)-glucan concentrations. Additionally, [Beta](1 [right arrow] 3)-glucan concentrations were slightly elevated in homes with mold spots during the past 12 months.

When [Beta](1 [right arrow] 3)-glucan levels were expressed per gram of dust, concentrations in Hamburg were significantly higher than in Erfurt. Furthermore, levels of [Beta](1 [right arrow] 3)-glucan were associated with building material and the number of indoor plants. Dose--response relationships were found between levels of [Beta](1 [right arrow] 3)-glucan and frequencies of vacuuming carpets, mopping of smooth floors, and dusting. No other building characteristic and occupant behavior was found to be of importance.

Neither concentrations expressed per square meter nor concentrations expressed per gram of dust were associated with season of dust sampling, year of construction of the building, story and size of the dwelling, type of heating (central vs. noncentral), ventilation, keeping a cat indoors, use of humidifiers (active and passive), water damage, visible mold or mildew, and visible wet spots during the past 12 months (data not shown).

Multiple linear regression analysis. Means ratios and 95% confidence intervals for the resulting models for [Beta](1 [right arrow] 3)-glucan levels expressed as micrograms per square meter and as micrograms per gram of dust are presented in Table 4. Nineteen percent and 9%, respectively, of the variance of the [Beta](1 [right arrow] 3)-glucan concentrations were explained by the variables in the regression analysis.

Table 4. Means ratios (MR) and 95% confidence intervals (CI) as
determined by multiple linear regression (n = 378, [R.sup.2] = 19%,
9%), based on In-transformed data.

                                         [Beta](1 [right arrow] 3)-
                                         Glucan([micro]g/[m.sup.2])

Factor                           No.    MR      95% CI      p-Value(a)

Place of residence
 Erfurt                          193   1
 Hamburg                         185   1.16   (0.94-1.43)     0.1554
Building characteristics
 Type of building
  One-/two-family house          111   1
  Multiple-family dwelling       202   0.84   (0.69-1.03)     0.1035
  Concrete slab house             65   1.05   (0.78-1.41)     0.7353
Characteristics of living room
 Carpets
  None                            15   1
  Carpets < 5 years old          158   1.85   (1.17-2.95)     0.0094
  Carpets 5-10 years old         114   2.36   (1.47-3.78)     0.0004
  Carpets > 10 years old          91   2.09   (1.30-3.34)     0.0023
Indoor plants
 [is less than or equal to] 10   191   1
 11-20                           117   0.85   (0.70-1.03)     0.1043
 > 20                             70   1.19   (0.94-1.50)     0.1558
Dog indoors
 No                              344   1
 Yes                              34   1.43   (1.05-1.94)     0.0247
Use characteristics
 No. of persons living in the
 home
  1-3 persons                    248   1
  [is greater than or equal
  to] 4 persons                  130   1.35   (1.12-1.64)     0.0022
 Use in person-hours per week
  [is less than or equal to]
  25 hr/week                      23   1
  25-180 hr/week                 313   1.34   (0.92-1.95)     0.1319
  > 180 hr/week                   42   2.06   (1.29-3.29)     0.0027
Cleaning habits
 Vacuum cleaning the carpets
  Daily                           31   1
  Several times a week           166   1.64   (1.17-2.29)     0.0040
  Once a week                    154   1.84   (1.30-2.63)     0.0008
  Once a month or less            27   3.04   (1.88-4.93)    <0.0001
 Dusting (dry)
  Daily/several times a week      97   1
  Once a week                     35   1.22   (0.96-1.55)     0.1043
  Once a month or less           146   1.35   (1.05-1.73)     0.0203
Mold spots during past 12
months
 No                              337   1
 Yes                              41   1.35   (1.02-1.78)     0.0353

                                   [Beta](1 [right arrow] 3)-
                                      Glucan ([micro]g/g)

Factor                            MR      95% CI      p-Value(a)

Place of residence
 Erfurt                          1
 Hamburg                         1.18   (1.01-1.37)     0.0333
Building characteristics
 Type of building
  One-/two-family house          1
  Multiple-family dwelling       0.96   (0.83-1.12)     0.6371
  Concrete slab house            1.06   (0.85-1.31)     0.6107
Characteristics of living room
 Carpets
  None                           1
  Carpets < 5 years old          0.98   (0.70-1.36)     0.8875
  Carpets 5-10 years old         1.19   (0.85-1.68)     0.3120
  Carpets > 10 years old         1.08   (0.77-1.52)     0.6553
Indoor plants
 [is less than or equal to] 10   1
 11-20                           0.81   (0.70-0.94)     0.0045
 > 20                            0.87   (0.73-1.03)     0.1033
Dog indoors
 No                              1
 Yes                             0.92   (0.74-1.15)     0.4645
Use characteristics
 No. of persons living in the
 home
  1-3 persons
  [is greater than or equal
  to] 4 persons                  1.03   (0.90-1.18)     0.6904
 Use in person-hours per week
  [is less than or equal to]
  25 hr/week                     1
  25-180 hr/week                 1.17   (0.89-1.54)     0.2612
  > 180 hr/week                  1.23   (0.87-1.72)     0.2389
Cleaning habits
 Vacuum cleaning the carpets
  Daily                          1
  Several times a week           1.23   (0.96-1.56)     0.0962
  Once a week                    1.19   (0.92-1.53)     0.1871
  Once a month or less           1.28   (0.90-1.81)     0.1666
 Dusting (dry)
  Daily/several times a week     1
  Once a week                    1.09   (0.91-1.29)     0.3531
  Once a month or less           1.09   (0.91-1.30)     0.3626
Mold spots during past 12
months
 No                              1
 Yes                             1.17   (0.96-1.43)     0.1277

Concentrations less than the detection limit were assigned a value of
one-half of the detection limit.

(a) Comparisons of the category of study with the first category of
the respective factor by means of a t-test based on In-transformed
data.

[Beta](1 [right arrow] 3)-Glucan levels in Hamburg were somewhat higher than in Erfurt. The difference between the two cities was not significant when concentrations were expressed per square meter but was statistically significant when concentrations were expressed per gram of dust.

Carpets in the living room were associated with a statistically significant increase in [Beta](1 [right arrow] 3)-glucan concentrations per square meter, [Beta](1 [right arrow] 3)-Glucan concentrations measured in living rooms with a carpet were higher compared to those in living rooms without a carpet, with a tendency toward higher concentrations in carpets older than 5 years, [Beta](1 [right arrow] 3)-Glucan levels per gram of dust were not associated with carpets in the living room. Furthermore, there was an association between [Beta](1 [right arrow] 3)-glucan concentrations and the number of indoor plants in the living room. The highest [Beta](1 [right arrow] 3)-glucan levels per square meter were measured in living rooms with [is greater than] 20 indoor plants, whereas concentrations per gram of dust were lower in dust samples from living rooms with [is greater than] 10 plants compared to living rooms with [is less than or equal to] 10 plants. In addition, keeping a dog indoors, use of the apartment by four or more persons, and use of the living room for [is greater than] 180 person-hours per week was associated with elevated levels of [Beta](1 [right arrow] 3)-glucan per square meter.

Cleaning habits were of major importance for [Beta](1 [right arrow] 3)-glucan in living room floor dust expressed per square meter. Lower frequencies of vacuum cleaning and dusting were associated with elevated absolute [Beta](1 [right arrow] 3)-glucan concentrations (per square meter). Vacuum cleaning only a few times a week was associated with a statistically significant increase in [Beta](1 [right arrow] 3)-glucan levels per square meter compared to daily vacuum cleaning. Vacuum cleaning only once per week and once per month or less was associated with an even higher increase in [Beta](1 [right arrow] 3)-glucan levels compared to daily vacuum cleaning. [Beta](1 [right arrow] 3)-Glucan levels expressed as micrograms per gram of dust were not associated with cleaning habits. In homes with mold spots during the past 12 months, statistically significant higher levels of [Beta](1 [right arrow] 3)-glucan per square meter were measured than in homes without mold spots, whereas concentrations per gram of dust were only slightly elevated in homes with mold spots.

To evaluate the effect of concentrations below the limit of detection on the results of the regression analyses, the final multiple linear regression models were recalculated excluding [Beta](1 [right arrow] 3)-glucan concentrations below the limit of detection. Effect sizes tended to be a bit smaller, but effects did not disappear, and no additional effects were found.

Discussion

The aim of this study was to assess whether [Beta]-glucan concentrations in house dust are correlated with endotoxin, allergen, and culturable mold spore levels in house dust, and whether they are associated with housing characteristics and occupant behavior. When concentrations were expressed per square meter, [Beta](1 [right arrow] 3)-glucan levels were highly correlated with concentrations of endotoxin, mite allergens Der p 1 and Der f 1, cat allergen Fel d 1, and culturable mold spore counts. When concentrations were expressed per gram of dust, correlations were weakened. Associations between [Beta](1 [right arrow] 3)-glucan levels and housing characteristics and occupant behavior were mainly found when concentrations were expressed per square meter and not when concentrations were expressed per gram of dust. The following housing characteristics and occupant behavior were of major importance for [Beta](1 [right arrow] 3)-glucan levels per square meter: carpets in the living room, keeping a dog indoors, use of the apartment by four or more persons, and use of the living room for [is greater than] 180 hr/week, lower frequencies of vacuum cleaning and dusting, and presence of mold spots during the past 12 months.

[Beta](1 [right arrow] 3)-Glucan levels. Mean [Beta](1 [right arrow] 3)-glucan concentrations measured in the present study are in accordance with mean [Beta](1 [right arrow] 3)-glucan concentrations in living room floor dust from the pilot study reported previously (19). [Beta](1 [right arrow] 3)-Glucan concentrations in settled house dust from approximately 125 homes were also reported by Douwes et al. (10). Mean [Beta](1 [right arrow] 3)-glucan concentrations measured by Douwes et al. (10) were much lower than mean [Beta](1 [right arrow] 3)-glucan concentrations measured in the present study. One reason that the concentrations were lower in that study is that they included many homes with smooth floors not covered with wall-to-wall carpet, whereas 95% of the living room floors in this study had carpets, of which 78% had wall-to-wall carpeting. This makes a substantial difference, particularly for concentrations expressed per square meter. No other studies on [Beta](1 [right arrow] 3)-glucan concentrations in settled house dust were found in the literature. Rylander and colleagues (6,7,9,20,21) conducted studies on airborne [Beta](1 [right arrow] 3)-glucan concentrations, but a comparison of [Beta](1 [right arrow] 3)-glucan concentrations measured in settled house dust and airborne [Beta](1 [right arrow] 3)-glucan concentrations has not yet been done.

[Beta](1 [right arrow] 3)-Glucan concentrations and endotoxins, allergens, and culturable molds. When levels were expressed as micrograms per square meter, strong correlations were found between [Beta](1 [right arrow] 3)-glucan levels and endotoxins, mite allergens, and total culturable mold spore counts. Weaker but also statistically significant correlations were found between [Beta](1 [right arrow] 3)-glucan levels and cat allergens and culturable mold spore counts of the identified genera of Aspergillus, Alternaria, Cladosporium, and Penicillium. When levels were expressed as micrograms per gram of dust, correlations were much weaker. Statistically significant correlations with [Beta](1 [right arrow] 3)-glucan concentrations were only found for levels of mite allergens Der p 1 and Der f 1 and mold spore counts of the two genera Alternaria and Cladosporium. The strong correlations among concentrations per square meter and the weak correlations and the lack of correlation, respectively, when concentrations were expressed as micrograms per gram of dust indicate that variance in levels per square meter of all the indoor factors considered was largely determined by the amount of dust sampled. Actually, correlations between the amount of dust sampled and glucans, endotoxins, mite allergens, and total culturable molds were very high. From the weak correlations between concentrations per gram of dust, we conclude that [Beta](1 [right arrow] 3)-glucan, endotoxin, mite and cat allergen levels, and culturable molds are (at least partially) determined by different characteristics.

We did not find an association between total culturable mold spore counts and [Beta](1 [right arrow] 3)-glucan concentrations expressed as micrograms per gram of dust. This is in accordance with previous findings (19). Possible reasons for this are a) only culturable molds were counted, whereas [Beta](1 [right arrow] 3)-glucan most likely represents total mold biomass, including nonculturable propagules; and b) [Beta](1 [right arrow] 3)-glucan is not completely specific as a marker for molds because it may also originate from plants and some bacteria. Interestingly, in our previous pilot study we also found a weak but statistically significant correlation between [Beta](1 [right arrow] 3)-glucan levels and culturable mold spore counts from Alternaria per gram of dust. The reason for this is not clear. Additionally, in this study [Beta](1 [right arrow] 3)-glucan was correlated with culturable mold spore counts from Cladosporium per gram of dust. The reason for this also is not clear.

[Beta](1 [right arrow] 3)-Glucan concentrations and housing characteristics and occupant behavior. There are no studies reported in the literature that identify building characteristics and occupant behavior associated with the level of [Beta](1 [right arrow] 3)-glucan concentrations in homes. We found [Beta](1 [right arrow] 3)-glucan concentrations per square meter in one- and two-family houses and in concrete slab houses to be higher than in multiple-family dwellings. The reason for this is not clear. Beyond this, carpets, presence of a dog, use of the apartment, and cleaning habits were of major importance for concentrations per square meter but not for concentrations per gram of dust. Because [Beta](1 [right arrow] 3)-glucan concentrations per square meter as well as the housing characteristics and occupant behavior (carpets, presence of a dog, use of the apartment, cleaning habits) are associated with the amount of dust, the associations with concentrations per square meter and the lack of association when concentrations were expressed per gram of dust are plausible. With these results, the amount of dust sampled can be used as a proxy for hygiene, and [Beta](1 [right arrow] 3)-glucan concentrations expressed per square meter are related to the amount of dust sampled.

The association between [Beta](1 [right arrow] 3)-glucan concentrations and mold spots during the past 12 months is in accordance with the results of other studies where [Beta](1 [right arrow] 3)-glucan concentrations were found to be higher in buildings with problems of mold growth than in buildings without those problems (6,9,20,21).

[Beta](1 [right arrow] 3)- Glucan concentrations and indoor climate. When [Beta](1 [right arrow] 3)-glucan concentrations were expressed per square meter, an association between indoor climate in terms of temperature and relative humidity was lacking. When concentrations were expressed per gram of dust, a weak positive correlation was found with relative humidity, indicating that house dust from damper rooms contains higher [Beta](1 [right arrow] 3)-glucan levels. This weak correlation and the lack of correlation, respectively, may be due to the short period of only 1 week in which temperature and humidity measurements were taken. Temperature and humidity may not be representative for a longer period.

Conclusion

In conclusion, levels of [Beta](1 [right arrow] 3)-glucan, endotoxin, mite and cat allergens, and culturable mold expressed per square meter are largely determined by the amount of dust sampled. [Beta](1 [right arrow] 3)-Glucan concentrations per square meter can be determined by housing characteristics and occupant behavior, whereas [Beta](1 [right arrow] 3)-glucan concentrations per gram of dust cannot. Our results indicate that the amount of dust sampled can be used as a proxy for hygiene and that [Beta](1 [right arrow] 3)-glucan concentrations expressed per square meter are related to the amount of dust sampled. Further investigations on health effects are needed to determine whether it is sufficient to simply weigh the dust sampled (which would be more cost effective) instead determining [Beta](1 [right arrow] 3)-glucan concentrations in house dust.

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Address correspondence to U. Gehring, GSF-National Research Center for Environment and Health, Ingolstadter Landstrabe 1, 85764 Neuherberg, Germany. Telephone: [phone]. Fax: [phone]. E-mail: gehring@ gsf.de

This study was supported by the Federal Ministry for Education, Science, Research, and Technology, grant 01 EE 93016.

Received 18 May 2000; accepted 11 September 2000.

Ulrike Gehring,(1,2) Jeroen Douwes,(3) Gert Doekes,(3) Andrea Koch,(4) Wolfgang Bischof,(4) Barbel Fahlbusch,(5) Kai Richter,(6) H.-Erich Wichmann,(1,2) and Joachim Heinrich(1) for the INGA Study Group

(1) GSF - National Research Center for Environment and Health, Institute of Epidemiology, Neuherberg, Germany; (2) Ludwig-Maximilians-University of Munich, Institute of Medical Data Management, Biometrics and Epidemiology, Department of Epidemiology, Munich, Germany; (3) Utrecht University, Environmental and Occupational Health Group, Utrecht, The Netherlands; (4) Friedrich-Schiller University of Jena, Institute of Occupational, Social and Environmental Medicine, Erfurt, Germany; (5) Friedrich-Schiller University of Jena, Institute of Clinical Immunology, Jena, Germany; (6) Grosshansdorf Hospital, Hamburg, Germany