Articles 

Indoor Exposure to Molds and Allergic Sensitization 

Beate Jacob, 7"2 Beate Ritz,3 Ulrike Gehring, 1-2 Andrea Koch,4 Wolfgang Bischof,4 H. E. Wichmann, ~~z 

and Joachim Heinrich' for the INGA-Study Group 

IGSF-Institute of Epidemiology, Munich, Germany; Zlnstitute of Medical Data Management, Biometrics, and Epidemiology, LudwigMaximilians-University of Munich, Munich, Germany; 3Center for Occupational and Environmental Health, UCLA School of Public 

Health, Los Angeles, California, USA; 'Department of Indoor Climatology, University of Jena, Erfurt, Germany 

Evidence that indoor dampness and mold growth are associated with respiratory health has been 

accumulating, but few studies have been able to examine health risks in relation to measured levels of indoor mold exposure . In particular, little is known about the contribution of indoor molds 

to the development of allergic sensitization . As a part of an ongoing study examining the effects of 

ambient air pollutants on respiratory health and atopic diseases in German school children, we 

examined the relation between viable mold levels indoors and allergic sensitization in 272 children. We examined whether allergic sensitization in children is associated with higher fungal 

spore count in settled house dust sampled from living room floors . Adjusting for age, sex, parental 

education, region of residency, and parental history of atopy, we found that mold spore counts 

for Cladosporium and Aspergillus were associated with an increased risk of allergic sensitization . 

Sensitized children exposed to high levels of mold spores (> 90th percentile) were more likely to 

suffer from symptoms of rhinoconjunctivitis . We conclude that elevated indoor concentrations of 

molds in wintertime might play a role in increasing the risk of developing atopic symptoms and 

allergic sensitization not only to molds but also to other common, inhaled allergens. These effects 

were strongest in the group of children who had lived in the same home since birth. Key words: 

allergic sensitization, house dust, indoor allergen exposure, molds. Environ Health Perspect 

110:647-653 (2002) . [Online 24 May 20021 

http ://ehpnet] . niehs. nih .gov/docs/2002/I IOp647-653jacob/abstract.html 

The development of allergic sensitization 

and atopic disease in children is a function 

of a genetic predisposition to react to antigens and the timing and extent of exposure 

to allergenic agents . Evidence is accumulating that indoor allergen exposure early in life 

stimulates the development of allergic sensitization (1-,3) . Because most children and 

adults spend most of their time indoors, 

much attention has been directed to identifying indoor sources of allergens. House dust 

is a complex mixture of various biocontaminants and a major source of allergens in nonindustrial indoor environments . House dust 

contains allergens such as mites, epithels of 

pet dander, and molds (1-6), and threshold 

values at which exposure may cause sensitization have been proposed . Although some 

authors suggest sensitization to molds as a 

risk factor for allergic diseases and asthma 

(7,8), it is still unclear whether allergic sensitization is a risk factor for asthma (9) . 

Epidemiologic studies reported positive associations between respiratory symptoms and 

living in damp houses (10-m), a condition 

thought to permit mold growth . Yet little is 

known about the contribution of indoor 

mold levels to allergic sensitization rates 

(17,18). Some reasons for the lack of information are that environmental monitoring is 

time and cost intensive and requires a high 

level of subject cooperation. In the present 

study we were able to use actual measurement of mold spores instead of relying on 

self-reports of mold growth to examine the 

role viable mold spores contained in house 

dust play in causing allergic sensitization and 

asthmatic and allergic symptoms in children . 

Materials and Methods 

Study population and selection of homes. We 

conducted two crass-sectional surveys in 

1992-1993 and 1995-1996 to study the 

long-term health effects of ambient air pollution in German school children ages 5-14 

years living in three areas of Saxony-Anhalt 

[n = 2,470 children (89 .1%) and n = 2,814 

(74 .7%) participation rate, respectively, for 

each survey] . In both surveys, we elicited 

information about social and environmental 

factors (19) and asked parents to report allergic and respiratory symptoms and diseases 

for their children . In addition, our study 

physician examined all children and drew 

blood samples . For a select subgroup of children we were able to collect additional data, 

including samples of house dust and information concerning building and housing 

characteristics and living habits . 

Drawing from both survey populations, 

we selected affected (case) and unaffected 

(control) children ; cases were defined as children who could be classified as atopic 

according to at least one of the following 

three criteria : a positive skin prick test ; at 

least one positive specific IgE test (CAPRAST-FEIA; IgE > 0.35 kU/L) ; or physician 

diagnosis of asthma at any time before the 

survey . We used a stratified random sampling approach to select children in two age 

groups (5-7 and 8-10 years) from three residential areas ; 80 children each were selected 

in the younger age group and between 40 

and 50 in the older age group for a total of 

370 case and 370 control children . Parents 

of 231 selected case children (62%) agreed 

to participate . Control children had to be 

nonatopic and nonasthmatic (i .e ., they did 

not meet any of the above-mentioned criteria 

for cases . Parents of 223 selected control children (60°,%) agreed to participate . Overall, 

parents of 454 children allowed us to collect 

household dust samples . 

Trained personnel performed interviews 

to document housing characteristics and visited homes twice at an interval of approximately 6 months to collect two dust samples . 

All 454 homes were visited between 1996 

and 1998, but for the following analyses we 

considered only the homes of 340 children 

(178 case and 162 control children) who did 

not move between the medical examination 

and the home visit . For the following secondary analyses focusing on mold exposure 

and allergic sensitization, we excluded 20 

subjects with missing data for IgE sensitization and seven subjects missing other covariate data . We further restricted our case group 

to sensitized children only (i .e ., those children testing positive in at least one RAST 

test), excluding 41 children who qualified as 

Address correspondence to B . Jacob, GSF-Institure 

of Epidemiology, Ingolstadter Landstr . 1 D-85758 

Neuherberg, Germany . Telephone : +49-89-3187-

4150 . Fax: +49-89-3187-3380 . E-mail: beate.jacobCt 

t-online .de 

We thank G. Wdlke and B . Hollstein for coordinadng the home visits . The Federal Ministry for 

Education, Science, Research and Technology, 

grant EE 93016, supported this study. 

The Inga-Study Group: GSF-National Research 

Center for Environment and Health, Neuherberg, 

Institute of Epidemiology (H .E . Wichmann, J . 

Heinrich, P . Schneider, J . Cyrys, 1 . Grog, G . 

Wolke, G . Silbernagl, U. Gehring, B . Jacob, A . 

Houzer), Institute of Ecological Chemistry (I . 

Gebefiigi, G. Ldrinci, J . Schnelle) ; Friedrich Schiller 

University-Jena Institute of Occupational, Social 

and Environmental Medicine (W. Bischof, A. 

Koch, J . Vbitthauer, K .J . Heilemann), Institute of 

Clinical Immunology (L . Jager, B . Fahlbusch, G. 

Schlenvoigt) ; Gro(3hansdorf Hospital-Hamburg 

Center for Pneumology and Thoracic Surgery (H . 

Magnussen, K. Richter, R. Jorres) ; University of 

Utrecht-Division of Environmental and 

Occupational Health (B . Brunekreef, J . Douwes, 

G. Doekes) . 

Received 18 May 2001 ; accepted 17 December 

2001 . 

Environmental Health Perspectives " VOLUME 110 I NUMBER 7 I July 2002 647 
Articles " Jacob et al . 

cases only according to a positive skin-prick 

test . We considered the validity and reliability of the prick test results questionable 

because different test kits were used in each 

survey . Thus, 115 cases and 157 controls 

remained in the analyses . We chose to use 

only house dust samples taken in winter 

(November-April) to minimize the influence of seasonal variation (20) and to make 

our results comparable to previous studies 

using a similar restriction to winter sampling 

(8,10,11,21-24) . 

Approval of the study protocol was 

granted by the Ethics Committees of the 

University of Rostock and the University of 

Munich (LMU), and the study was performed in accordance with the institutional 

guidelines for the protection of human 

subjects . Written informed consent was 

obtained from the parents of all participating 

children . 

House dust sampling and mold identification. Dust sampling and extraction procedures were identical to those used in a 

parallel study of adults (25) and described in 

more detail elsewhere (20,26) . In each 

home, a dust sample was taken from the living room floor (97°'o were carpeted floors) 

by vacuuming an area of 1 mz for 2 min in a 

highly standardized manner using the same 

type of vacuum cleaner (Type Fliisterjet 

Vitall 371, 1,000 W; Phillips, Hamburg, 

Germany) and the same device (collector 

and filter ; ALK, Horsholm, Denmark) to 

collect dust on a paper filter (20) . In general, 

samples were obtained from carpets . The 

dust filters were weighed before and after 

vacuuming to analyze the settled dust gravimetrically. The dust samples were stored at 

room temperature and analyses were performed within 10 days after sampling . 

We analyzed 30 mg (500 pm) sieved 

house dust for identification and quantification of viable molds. Dusts were diluted in 

0.9% NaCI and plated on DG18 (dichloran18% glycerol agar) and 0.1 g/L chloramphenicol was added to prevent bacterial 

growth. Plates were incubated at 25°C for 

10 days (20,27), and all analyses were duplicated. The number of colony-forming units 

(CFU) was counted and expressed as CFU 

per gram of dust . Colonies were identified to 

genus level using high-powered microscopy 

(Ergaval ; Carl Zeiss, Jena, Germany) . 

The total number of CFUs may be of 

limited clinical and epidemiologic relevance 

because spores from different species have 

different allergenic potential (11) . Therefore, 

we studied both the total counts and the 

counts of selected mold genera separately . 

The detection limit for total molds was 

1,000 CFU/g dust, and, in some cases (high 

concentrations of total molds) for genusspecific CFU, 10,000 CFU/g dust . 

Allergic sensitization . Blood collection, 

centrifugation of blood, and serum storage 

followed the protocol of the European 

Community Respiratory Health Survey 

(ECRHS) (19,28). The serum samples 

were stored at -20°C. Specific IgE for 

Dermatophagoides pteronyssinus (d1), car allergens (el), Cladosporium (m2), mixed grasses 

(gG), and birch (t3) were measured by the 

CAP-FEIA method by Pharmacia Diagnostics 

(Freiburg, Germany) using identical batches 

of reagents for all assays (29) . The measurement range was 0 .35-100 kU/L, with a 

detection limit < 0.35 kU/L. Allergic sensitization was defined as testing positive for at 

least one specific IgE (>- 0 .35 kU/L). 

Statistical analysis. We performed statistical analyses using the statistical analysis 

package SAS for Windows version 6.12 (SAS 

Institute, Cary, NC, USA). We included in 

our multivariate analyses only those children 

for whom we had complete covariate data 

for potential risk factors for atopic diseases 

(age, sex, region of residency, educational 

level of the parents, and positive parental 

history of atopy) in addition to outcome and 

exposure information . Thus, we performed a 

complete-subject analysis for 272 children 

(115 sensitized cases and 157 controls) . 

Because of the log-normal distribution of 

mold spore counts, we present the median 

and the 25th and 90th percentile as measures of variation . We calculated the crude 

prevalence for sensitized cases and controls 

for binary response variables of allergic diseases or symptoms . We used the nonparametric Spearman rank-order coefficient (r,) 

to determine the relationships between the 

CFU of several genera of mold spores . 

Because established thresholds for mold 

genera are lacking, we classified subjects into 

three exposure categories (subjects exposed <_ 

25th, 25th-90th, and > 90th percentile) . 

Multiple logistic regression analyses allowed 

us to examine the effect of mold spore exposures on allergic sensitization, atopic symptoms, and atopic diseases adjusting for a fixed 

set of potential confounding variables (age, 

sex, region of residency, educational level of 

the parents, and positive parental history of 

atopy) by including them in the model. We 

report adjusted odds ratios (OR) and 95°,0 

confidence intervals (CI) for each allergic 

outcome and mold exposure category . 

Table 1 . Basic description of the study population of 272 children in eastern Germany. Characteristics 

Sensitized cases (n=115) Controls In=157) 

No . Percent No . Percent 

Demographic characteristic 

Place of residency 

Zerbst 42 

Bitterfeld 20 

Hettstedt 53 

Sex 

Boys 71 

Girls 44 

Age 

5-7 years 69 

8-10 years 46 

Parental education 

s 10 grades 58 

Z 12 grades 57 

Positive family history of atopy 29 

Dampness at home 29 

Living in the same apartment since birth 43 

Allergic sensitization 

z 1 BAST + 115 

BAST Derp1 + 48 

BAST cat + 30 

BAST birch + 39 

BAST gras + 84 

BAST Cladosporium+ 14 

Allergic symptoms' 

Sneezing attacks 16 

Red eyes 19 

Runny or stuffed nose 16 

Red eyes and runny or stuffed nose 5 

Persistent wheezing 8 

lItching rash 33 

Allergic diseases 

Asthmab-c 17 

Asthma attacks' 4 

Hay feverb 12 

Eczemab 23 

36.5 49 31 .2 

17 .4 32 20 .4 

46.1 76 48 .4 

61 .7 79 50 .3 

38 .3 78 497 

600 94 59 .9 

40 .0 63 40 1 

50 .4 83 52 .9 

49 .6 74 47 .1 

25 .2 27 17 .2 

25 .2 35 223 

37 .4 58 36 .9 

100 0 

41 .7 0 

26.1 0 

33,9 0 

73 .0 0 

12 .2 0 

13 .9 4 2.6 

16 .5 5 3.2 

13 .9 B 5.2 

4.4 0 0.0 

7.5 3 2.0 

29 .2 14 9.1 

14.8 0 0.0 

3.6 0 00 

10.4 1 0.7 

20.0 14 9.0 

eln past 12 months. bLifetime diagnosis. Toctor's diagnosis of asthma, asthmoid bronchitis, or spastic bronchitis. 

648 VOLUME 110 1 NUMBER 7 1 July 2002 " Environmental Health Perspectives 
Results 

Study population . Table 1 presents characteristics of the study group, including the 

distribution of allergic sensitization (RASTCAP >- 1), allergic symptoms, and diseases of 

sensitized cases and nonsensitized, nonasthmatic controls . We observed allergic sensitization [defined as testing positive for one 

specific IgE (> 0 .35 kU/L) , i .e . for Der p], 

cat, birch, grass, or Cladosporium] for 115 

children referred to as cases . Our study sample consists of 150 boys and 122 girls 

because more boys tested positive for specific 

IgE. Parents of children who were sensitized 

and thus belonged to the case group selfreported being atopic slightly more often 

(25 .2% vs. 17 .2%) than parents of nonsensitized children, but we observed no difference 

in parental reports concerning dampness of 

homes and residential mobility since birth of 

the child in both groups . 

Viable mold spore contamination Of 

homes. The average weight of house dust 

taken from living room floors was 0.90 t 

0.85 g/mZ and was similar for case and control homes (0 .87 t 1 .01 for cases; 0.92 t 

0.71 for controls), yet we observed a large 

variation in mold levels between households . 

Table 2 presents the distribution of number 

of CFUs obtained per gram of dust taken in 

winter for total (all species) mold spores and 

selected groups of the most common molds 

found in homes. Although CFU values varied greatly, we found only one sample with 

> 106 CFU/g dust (76 x 106 CFU/g dust) . 

Furthermore, no sample was free of molds. 

Restricting the period of analysis to winter 

samples only (November-April) meant that 

more than 60% of our samples were negative 

for Alternaria spores, and spore frequency 

was low in all samples positive for Alternaria 

(geometric mean 26; 95% CI, 16-43; 90th 

percentile, 10,000 CFU/g dust). Thus, we 

present results only for three genera of mold 

spores found most commonly in wintertime- Cladosporium, Perzicillium, and 

Aspergillus. Figure 1 shows the cumulative 

frequency of the mold concentrations in the 

homes of cases and controls . 

Cladosporiurn and Penicillium species 

were the most prevalent mold genera, but all 

three molds were positively correlated with 

and contributed to our total viable mold 

spore counts in the wintertime (r = 0 .52 for 

Chadosporium, 0.49 for Penicillium, and 0.43 

for Aspergillus) in both case and control 

homes (Table 3) . 

Furthermore, Cladosporium levels were 

not or were only weakly correlated with 

the indoor mold species Aspergillus and 

Penicilliurn in both seasons, suggesting that 

in the homes we studied two different patterns of mold growth contributed to overall 

high levels of mold spores ; in the first type of 

home we found the typical indoor species 

Aspergilhes and Penicillium commonly growing on foodstuffs and houseplants (30), and 

in the second type of home the dominant 

species was Cladosporium, an outdoor fungus 

that grows on textiles and foodstuffs when it 

gains access to the indoor environment. 

81,367 

70,562-93 827 

0(0%) 

50,000 

85,000 

210,000 

Table 2. Distribution of CFU per gram dust for total and selected genera of mold spores on living room 

floors of 272 households in eastern Germany. 

Molds 

Total molds 

Geometric mean 

95% CI 

n < LOD (%) 

25th Percentile 

Median 

90th Percentile 

Cladosporium 

Geometric mean 

95% CI 

n< LOD (%) 

25th Percentile 

Median 

90th Percentile 

Penicillium 

Geometric mean 

95% CI 

n<LODI%) 

25th Percentile 

Median 

90th Percentile 

Aspergillus 

Geometric mean 

95% CI 

n< LOD I%) 

25th Percentile 

Median 

90th Percentile 

LOD, Limit of detection 

100 

8 0 

E 

E 

60 

" 

40 

: 

1e 

t 

. 

; 

r.~ 

~? 20 

Sensitized cases (n= 115) 

" Controls 

I Cases 

2,997 

1,531-5,867 

19116,5%) 

5,000 

12,500 

40,000 

2,041 

942-4,425 

26(22.6%) 

5,000 

15,000 

55,000 

Allergic sensitization and mold spore 

counts in household dust. We examined the 

association between allergic sensitization of 

children and mold spore counts in household dust in multiple logistic regression 

models adjusting the odds ratios for sensitization (at least one RAST-CAP positive test) 

856 

399-1,836) 

31 (27 0°/Q) 

LOD 

5,000 

30,000 

1 oa 

> 

0 80 

Q 

~ Bo 

> 

~ 40 

E 

20 ta 

. "D . 

. 

a y 

0 50 100 150 200 250 300 350 400 

Total molds (CFO/g dust x 10-3 ) 

100 r . 

> 

Bu 

d 

;, so 

40 

E 

(i zo 

0 

s 

50 100 150 200 250 300 350 400 

PenicilliumICFO/g dust x 10-3 ) 

a 

lao 

d 80 

d 

m 

. 

E 

E 

60 

40 

20 

Controls (n= 157) 

71,118 

61,285-82,530 

0(0°/0) 

45,000 

75,000 

195,000 

1,501 

604-2,804 

35 (22 3%) 

5,000 

10,000 

30,000 

2,014 

1,049-3,866 

35(22.3%) 

5,000 

15,000 

50,000 

245 

117-513 

65 (41 .4%) 

LOD 

5,000 

25,000 

0 50 100 150 200 250 300 350 400 

Cladosporium(CFOlg dust x 10-3) 

" s 

4 

r 

0 

0 50 100 150 200 250 300 350 400 

Aspergillus(CFDIg dust x 10-3) 

Figure 1 . Cumulative frequency of the concentrations of molds in the homes of cases and controls . 

Environmental Health Perspectives " VOLUME 110 1 NUMBER 7 1 July 2002 649 
Articles " Jacob et al . 

by age, sex, residential region, parental education, and parental history of atopy. 

High levels of Cladosporium (35,000 

CFU/g dust or > 90th percentile) in wintertime household dust approximately tripled 

the risk of allergic sensitization in children 

(OR >- 90th percentile, 2.93; 95% CI, 

1 .17-7 .36) . Aspergillus spores increased the 

risk of allergic sensitization at a somewhat 

lower level [i .e ., when the spore count 

increased above 25,000 CFU/g dust (25th 

percentile ; OR 25th-90th percentile, 2.11 ; 

95°'o Cl, 1 .22-3 .65 ; OR z 90th percentile: 

1 .76 ; 95% CI, 0.73-4.28] (Table 4) . 

Sensitization of exposed children, however, 

was not limited to Cladosporiurn (specific 

IgE positive for Cladosporium) . Rather, children exposed to increased viable mold levels 

were more likely to be sensitized to other 

allergens as well, such as pollen, cat, or house 

dust mites, similar to what has been reported 

previously (17) . Considering mite allergen 

exposure as a potential confounder, we 

included also Der pl and Der fl levels into 

the model, but the results did not change 

(data not shown) . 

For I'enicillium and also for total molds 

counts, we found slightly increased sensitization risks with exposure at high levels of 

mold spores in winter, but our effect estimates were imprecise and included the null 

value . In summer, however, Penicillium was 

the most important indoor contributor to 

overall sensitization (OR a 90th percentile, 

2.83 ; 95% CI, 1 .25-6 .44 ; data not shown) . 

Our results suggest a positive trend for 

risk of general allergic sensitization-not just 

to mold allergens-when children are 

exposed to mold spores . When restricting the 

analyses to children who lived in the same 

apartment since birth (n = 101), odds ratios 

increased for Aspergillus counts and showed a 

dose-response pattern (OR 25th-90th percentile, 2 .21 ; 95% CI, 0 .83-5 .90 ; OR >-

90th percentile, 3 .14; 95% CI, 0.63-15 .7) . 

Effects were also observed for high levels of 

Cladosporium (OR ~o 90th percentile, 4.21 ; 

95% CI, 0 .72-24.7) and total molds counts 

(OR >- 90th percentile, 2 .53 ; 95% CI, 

0.52-12.4), but because of the loss in study 

subjects, the 95% confidence intervals 

included the null value . We did not observe 

consistent or strong associations with wintertime Penicillium spore counts in house dust 

(Figure 2) . 

Allergic symptoms and diseases and mold 

spore counts in bousebold dust. Sensitized 

cases exposed to high levels of viable mold 

spores (> 90th percentile) were more likely to 

suffer from symptoms of rhinoconjunctivitis, 

including pink eye and runny and/or congested nose (OR 10.8 for total molds, OR 

19 . 8 for Cladosporium, and OR 23.8 for 

Penicillium ; Table 5) . We did not have 

enough subjects to draw a conclusion about 

the occurrence of other atopic and allergic 

symptoms and diseases, but in general high 

mold spore counts of any type seemed to 

increase symptom prevalence to some degree . 

Discussion 

Distribution of molds. Studies from 

Germany (31), Sweden (9), Denmark, the 

Netherlands (22), the United Kingdom (32), 

and Michigan (USA) (24) reported that 

Penicilliurn was the most prevalent indoor 

mold genus, followed by Cladosporium, 

whereas AspeiRillus was the most commonly 

isolated indoor mold in Israel (17) . Although 

the molds Cladosporium spp . and Alternaria 

spp . are generally considered outdoor 

species, they are also commonly found 

indoors . Outdoor mold levels vary greatly 

with season, and these variations may also 

d N 

N 

.y 

d 

N 

d 

a 

100 

90 

80 

70 

SO 

50 

40 

30 

20 

10 

0 

100 

90 

e0 

70 

60 

50 

40 

30 

20 

10 

0 

d 

u 

d 

N 

.y 

m 

u 

`m a 

ges_25 ges25_ ges50_ ges75_ ges90_ 

Total molds 

pen 25 pen25_ pen50_ pen75_ pen90_ 

Penicillium 

contribute to variations in indoor levels of 

these molds . Therefore, we restricted our 

analyses of indoor dust samples to those 

taken in winter (November-April), when 

Cladosporium and Alternaria are less likely to 

grow outdoors . Aspergillus and Penicillium 

are the two most frequently encountered 

genera of indoor molds.The number of 

CFUs per gram of settled house dust is generally higher than the number measured in 

air samples because samples of settled dust 

probably reflect a cumulative measure of 

mold spores in homes . The number of 

CFUs per gram of dust found in our study 

was higher than those reported from other 

studies that used surface sampling methods 

(2,~ . Our geometric mean of CFUs per gram 

dust for total molds was 81,367 in the group 

of sensitized cases and 71,118 in controls . 

Verhoeff et al . (22) sampled settled dust from 

100 

N 

U 80 

N 

.y 

d N 

d 

d 

a 

60 

= 40 

20 

0 

d 

d N 

m 

u 

m a 

100 

90 

80 

70 

sg 

50 

40 

30 

20 

10 

g 

cla_25 c1a25_ cIa50_ 605 _ cIa90_ 

Cladosporium 

asp 25 asp25_ asp50_ asp75_ asp90_ 

Aspergillus 

Figure 2. Percentage of sensitized children in the quartiles of exposure to molds (cases and controls) . 

Table 3. Spearman correlation coefficient matrix of the concentration of the mold spores on living room 

floors in winter of 272 households of children in eastern Germany. 

Cases Total molds 

Total Molds 

r 1 .0 

~rValue 

Cladosporium 

r 0.55 

l}Value 00001 

Penicillium 

r 0.48 

IrValue 00001 

Aspergillus 

r 0 .44 

p-Value 0.0001 

Controls 

Cladosporium Penicillium Aspergillus 

0.47 0.49 0.37 

0 .0001 0.0001 00001 

1 .0 -0 .003 011 

0.975 0 254 

009 1 .0 0.27 

0 240 0 003 

0.19 0.25 1 .0 

0.019 0.002 

650 VOLUME 110 1 NUMBER 7 1 July 2002 " Environmental Health Perspectives 
Articles " Indoor exposure to molds and allergic sensitization 

mostly noncarpeted bedroom floors over 2 

months (October-November) and reported 

8,300 CFU/g dust in homes of children with 

respiratory symptoms and 9,940 CFU/g dust 

in control household samples . Wickmann (,'~ 

reported a mean of only 1,000 CFU/g dust 

sampled from living room floors in late winter 

(February-March) . Total numbers of CFUs 

per gram dust from carpets are significantly 

higher than for smooth floors W, and 97% 

of our samples were taken from carpeted 

floors, which may explain the differences . 

However, comparisons of quantitative 

and qualitative results from different studies 

are of limited value because studies not only 

used different sampling techniques for the 

same mold spores, but each study also 

focused on the identification of unique and 

different sets of mold spores (33,34) . 

Molds and allergic sensitization . 

Sensitization to molds is a risk factor for 

allergic diseases (8,9), and molds can be 

important indoor allergens (1/~ . Reports of 

prevalence of allergic sensitization to molds 

vary widely ranging from 2% to 30% in 

subjects with respiratory allergy (3S) . The 

great variability in reported prevalence could 

derive from differences in environmental 

conditions, such as the geoclimatic areas 

under investigation, differences in population 

sensitivity, and differences in the characteristics and properties of diagnostic tests used to 

assess allergen extracts (33) . The number of 

mold allergens for which reliable tests are 

available is small compared to other allergen 

extracts such as mites . Furthermore, isolation, purification, and standardization of 

allergens produced by molds are a major 

problem contributing to measurement error 

of unknown size in all studies . 

The likelihood of developing sensitivity 

to aeroallergens depends on the degree of 

atopic susceptibility, the concentration and 

potency of allergens one is exposed to, and 

adjuvant factors (36) . As did Garrett et al . 

(37), we found that winter exposure to high 

concentrations of mold spores such as 

Cladosporium increased allergic sensitization . Garrett et al . (37) also reported that 

atopy was significantly associated with 

Aspergillus. In our study, effects were most 

consistently observed for the species of 

Cladosporium and Aspergillus, where exposure above the 90th percentile increased the 

risk of allergic sensitization approximately 

2- or 3-fold (Cladosporium, OR, 2.93 ; 95% 

CI, 1 .17-7.36; Aspergillus, OR, 2 .11; 95% 

CI, 1 .22-3.65) . Exposure to high levels of 

Penicillium (> 55,000 CFU/g dust, > 90th 

percentile) elevated the risk for allergic 

Table 4. Associations between exposure to fungal spores and prevalence of allergic sensitization . 

Molds 

Sensitized 

(CFU/g dust) cases Controls 

Total molds 

s 25th Percentile s 48 .750 24 

> 25th/:s 90th Percentile 48 .750-200 .000 79 

> 90th Percentile > 200.000 12 

Cladosporium 

s 25th Percentile s5 .000 45 

> 25th/!. 90th Percentile 5.000-35.000 54 

> 90th Percentile > 35 .000 16 

Penicillium 

s 25th Percentile s 5 .000 40 

> 25th/:5 90th Percentile 5.000-55.000 64 

> 90th Percentile > 55 .000 11 

Aspergillus 

s 25th Percentile LOD 31 

> 25th/:s 90th Percentile LOD-25 .000 72 

> 90th Percentile > 25 .000 12 

Crude OR Adjusted OR' 

(95% C0 (95% C0 

44 1 0 1 .0 

100 1 .45 (0 .81-2 .58) 1 .56 (0.85-2.861 

13 1 .69 (0 67-4 29) 1 .67 (0.65-4.291 

72 1 0 1 .0 

76 1 14 (0 68-1 .89) 1 .15 (0.67-1 .95) 

9 284(1 .16-698) 293(1.17-7.36~ 

60 1 0 1 .0 

85 1 13 (0 .68-189) 1 .09 (0.64-1 .84) 

12 138 (0 55-3 42) 1 .38 (0.54-3.51/ 

65 1 0 1 .0 

77 2 06 (1 .16-3 35) 2.11 (1 .22-3.65) 

15 1 .68 (0 .70-4.01) 1 .76 (0.73-4.28) 

LOD, limit of detection . 

°Adjusted for age, sex, residential region, parental education, and parental atopy. 

Table 5. Adjusted odds ratios for atopic symptoms and diseases and indoor mold spore counts . 

Atopic symptoms 

OR for fungal exposure above the 90th percentilea 

Total molds Cladosporium Penicillium Aspergillus 

Sneezing attacksb 3 47 (1 .07-11 .3) 

Red eyes and runny 113 (1 23-103 1) 

or congested noseb 

Persistent wheezingb 0 82 (0 .10-7.13) 

Itching rash° 1 46 (0 .54-3.96) 

Asthmard 0 47 (0 .06-3.90) 

Hay feverF 2 14 (0 .39-11 .8) 

Eczema' 1 65 (0 .57-4.81) 

1 43 (0 .36-5.58) 

15 5 (2 .08-1154 0) 

1 18 (0 47-2 .94) 

1 18 (0 .41-3 39) 

0 52 (0 06-4 27) 

1 89 (0 .35-10 .3) 

0 54 (0 .12-2.44) 

2.12 (0 .52-8 63) NE 

17 .6 (1 69-183 4) NE 

2.55 (0 44-14 7) 2 15 (0 .41-11 4) 

0.62 (0 .17-2 24) 1 47 (0 55-3 .93) 

1 .74 (0 35-8,73) 1 29 (0 .27-6.18) 

2.57 (0 .54-12,3) NE 

1 .21 (0 .38-3 88) 2 16 (0 .80-2 .52) 

NE, not estimable . 

'Adjusted for age, sex, region of residency, parental education, and parental atopy . 

dAsthma, asthmoid bronchitis, or spastic bronchitis . 

bln past 12 months . 'Lifetime . 

sensitization in winter only slightly, but 

Penicillium was the dominant indoor mold 

allergen in summer. 

Although we conducted analyses stratifying for season (summer and winter), these 

analyses were not always informative because 

the sample was small . Summer total mold 

counts were dominated by high counts for 

the outdoor molds Cladosporium and 

Alternaria, and counts for these species correlated only weakly with the counts for the 

indoor molds Pencillium and Aspergillus (data 

not shown) . We also observed that indoor 

Cladosporium measures were much higher in 

summer than in winter (median of 35,000 

CFU/g in summer vs . 10,000 CFU/g in 

winter), while an opposite but weaker seasonal pattern was found for Aspergillus and 

Penicillium, supporting our notion that at 

least two different patterns of mold contamination of homes exist in the geographic area 

we studied. The latter two molds were more 

abundant in our winter samples . 

Our winter results did not change when 

we adjusted for summertime spore counts 

from the same households or when we 

adjusted for house dust mite allergens 

(results not shown), and our results were 

strengthened when we restricted the analyses 

to children living in the same home since 

birth, but sample size and statistical efficiency was limited for this and other types of 

subgroup analyses (e .g ., multiple logistic 

regression models examining sensistization 

to specific instead of all allergens ; data not 

shown). As in our study, Garrett et al . (37) 

reported an elevated risk of general sensitization to allergens such as dust mites and dog 

allergens when they found high levels of 

viable Cladosporium and Penicillium spores 

in the air of homes in wintertime . Also similar to our results, these associations weakened when Garrett and co-workers instead 

used spore samples collected in late spring 

(37) . This may be related to the known seasonal variability of mold spores in outdoor 

air (i .e ., in winter levels of viable mold spore 

contamination in homes depend mostly on 

indoor factors because it is unlikely that 

spores are carried in from outdoors) . 

Molds and allergic symptoms. High 

indoor mold exposure (> 90th percentile) 

seems to contribute to allergic symptoms 

and diseases in both sensitized and nonsensitized children ; however, because numbers in 

the nonsensitized group were small, effect 

estimates were imprecise or even nonestimable in this subgroup. This might suggest 

that both inflammatory allergic mechanisms, 

including type III allergy to mold-specific 

antigens and nonimmune inflammatory 

reactions to mold components, might be 

important (18) . It is not clear which inflammatory and/or allergic mechanisms primarily 

Environmental Health Perspectives " VOLUME 110 1 NUMBER 7 1 July 2002 651 
Articles " Jacob et al . 

account for the presumed pathogenic effects 

of mold exposure (18) . Nevertheless, allergic 

sensitization to mold spores plays a major 

role in atopy (14) . 

Existing studies suggest that exposure to 

allergens during a sensitive period in early 

life may enhance the risk of sensitization in 

genetically predisposed children (35), implying that for children with a positive family 

history, lower allergen concentrations may 

be sufficient to achieve sensitization (1) . We 

did not observe clear patterns for increased 

sensitization risk in children with a positive 

history of parental atopy (data not shown), 

but the number of children in this group was 

quite small (n = 56), and we sampled mold 

spores in homes only for children older than 

5 years of age. 

Sampling technique and identification 

methods. The presence of molds in indoor 

environments is generally assessed using air 

or surface samples (13) . Air sampling of 

viable mold particles usually is restricted to 

short periods of several hours and does not 

provide reliable data concerning the contamination by and growth of molds in nonindustrial indoor environments (34), especially 

because airborne samples are strongly influenced by outdoor levels of molds. High sampling variation has been observed for total 

airborne spore burden in repeated samples 

taken in the same home, possibly caused by 

domestic activity, cleaning, and ventilation 

(22) . Assessment of viable mold particles 

(mold propagules) in settled house dust 

might be a useful measure of longer-term 

and cumulative exposure to indoor molds 

and is less influenced by indoor activities and 

turbulence. We used a simple, settled-dust 

sampling technique of standardized vacuuming of floor dust in the living room to measure viable mold particles . 

To date, analysis of housedust samples to 

identify viable mold particles has not been 

standardized. Recently, a comparative study 

of 10 different analytic methods, however, 

showed that direct plating of dust onto DG 18 

agar was one of the more sensitive methods 

(27) . In fact, use of DG18 agar produced 

higher numbers of CFU for all mold spores. 

As an alternative to sampling mold 

spores, participants in previous studies of 

allergic and asthmatic diseases have often 

been asked to report dampness and odors as 

a surrogate for indoor mold exposures (14) . 

Awareness of the existence of such exposures, 

however, may have caused overreporting of 

symptoms in exposed subjects and thus may 

have led to response bias . Furthermore, air 

sampling and cultivation of spores from 

house dust samples show only a modest 

agreement with such self-reported exposures 

(11,31~. In the present study, relying on dust 

samples avoided reporting biases . But 

confounding bias may have occurred due to 

the fact that we included sensitized asthmatics in our case goup, and homes of asthmatics 

may be cleaned more rigorously to avoid 

symptoms . When we excluded from our 

analyses 17 children who were both asthmatics and sensitized, our results did not change. 

We standardized our method of house 

dust sampling. We also believe that settled 

dust may be the best proxy for long-term 

exposures to mold allergens in the home 

environment . Furthermore, approximately 

30% of all children for whom we collected 

samples had lived in the same home since 

birth and may have been exposed to high levels of mold throughout their Iives .To explore 

fully any exposure-response relation between 

allergic sensitization and exposure to indoor 

molds, it is necessary to recruit sufficient 

individuals with high and low exposure levels . Although we found a wide range of 

CFUs per gram of dust in the homes of our 

study subjects, our overall sample size was 

relatively small and further reduced when we 

restricted our analyses to dust samples taken 

during wintertime to minimize the effect of 

seasonal variability in molds. 

Conclusions 

Our results suggest that indoor mold spore 

exposure, mainly during winter, might 

increase the risk of sensitization to all allergens in children . These findings are limited 

by methodologic difficulties of quantifying 

molds and by the relatively small number of 

homes studied . For future research we 

encourage using a longitudinal study design 

with a larger number of cases to allow analyses for allergen-specific instead of total sensitization . However, we found that allergic 

sensitization was significantly associated with 

exposure to one or more genera of indoor 

mold spores, even after adjustment for house 

dust mite exposure . The effect strengthened 

when we restricted our study population to 

children who had lived in the same home 

since birth. Furthermore, our study suggests 

that high indoor spore counts might increase 

the prevalence of allergic symptoms in all 

children whether they are sensitized or not . 

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