Biology Advances in Applied Microbiology Fungi and The Indoor Environment (2004)

Section I.
Fungi and Sick Building Syndrome

Fungi and the Indoor Environment: Their Impact
on Human Health
J. D. COOLEY,* W. C. WONG,* C. A. JUMPER,yAND D. C. STRAUS*
*Department of Microbiology and Immunology, Texas Tech University
Health Sciences Center, Lubbock, Texas 79430
y
Department of Internal Medicine, Texas Tech University
Health Sciences Center, Lubbock, Texas, 79430
I. Introduction 3
II. Inhalation of Fungal Spores Causes Respiratory Disease in Humans 5
III. Correlation Between the Presence of Certain Fungi and SBS 9
IV. Development of an Animal Model for Allergic Penicilliosis
Induced by the Intranasal Instillation of Viable Penicillium
chrysogenum Conidia 13
V. Cellular and Humoral Responses in an Animal Model
Inhaling Penicillium chrysogenum 16
VI. Continually Measured Fungal Profiles in SBS 19
VII. Evaluation of Fungal Growth on Cellulose-Containing and
Inorganic Ceiling Tile 22
VIII. The Presence of Fungi Associated with SBS in North American
Zoological Institutions 23
IX. The Role (?) of Mycotoxins in SBS 24
References 27
I. Introduction
‘‘Yeast, molds, mushrooms, mildews, and the other fungi pervade
our world. They work great good and terrible evil. Upon them, indeed,
hangs the balance of life; for without their presence in the cycle of
decay and regeneration, neither man nor any other living thing could
survive’’ (Kavaler, 1965).
One of the most common questions asked concerning the seemingly
recent phenomenon of sick building syndrome (SBS) and fungal in-
volvement is, ‘‘Is this a new thing and why haven’t I heard of it
before?’’ Man’s realization that mold growth in his buildings is a
bad thing began over 3300 years ago in the time of Moses. Leviticus
14:33–45 (the Old Testament) describes this quite well. ‘‘If the mildew
reappears in the house after the stones have been torn out and the
house scraped and plastered, the priest is to go and examine it and, if
the mildew has spread in the house, it is a destructive mildew;
the house is unclean. It must be torn down—its stones, timbers and
all the plaster—and taken out of the town to an unclean place.’’ What is
3
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4 J. D. COOLEY et al.
truly amazing is that this course of action is not too different from what
we do today, over 33 centuries later. The building material used at that
time was ‘‘straw’’ bricks, that is, cellulose-based stones and plaster.
What of course is different today is that we now know what micro-
organisms cause these problems and we know, or at least strongly
suspect, what products these fungi produce that can cause health
problems in human beings.
One of the early scientific papers published regarding the effects of
mold spores on humans appeared in 1873 (Blackley). Charles Blackley,
who was one of the early describers of pollen skin tests, wrote about his
asthmatic responses to the inhalation of Penicillium species conidia
(spores) (Licorish et al., 1985). It now appears that the literature is quite
clear on the importance of the inhalation of fungal spores on respira-
tory disease in man. The Centers for Disease Control (CDC) recently
published a statement for the record for the United States House of
Representatives (Redd, 2002). In it they state, ‘‘While there remain
many unresolved scientific questions, we do know that exposure to
high levels of mold causes some illnesses in susceptible people. Be-
cause molds can be harmful, it is important to maintain buildings,
prevent water damage and mold growth, and clean up moldy materi-
als.’’ We have spent the last decade trying to understand the above
concepts. It is our humble hope that some of the work we have done
can help elucidate the role of fungi in the phenomenon known as sick
building syndrome.
Reports in the literature about building structures with poor
indoor air quality (IAQ) increasingly appeared soon after the mid-
1970s (Hodgson, 1992; Spangler and Sexton, 1983). SBS, a term
that is sometimes used for symptoms commonly associated with poor
IAQ, was first described in 1982. The first study examining more than
one building with SBS was published in 1984 (Finnigan et al.).
Although SBS has been difficult to define, evidence is now coming
to the fore that seems to indicate the importance of indoor fungal
growth in this phenomenon (Straus, 2001). SBS literally means
that there is something inside of said building that is actually
making people sick. These symptoms most commonly are fatigue,
runny nose, itchy eyes, sore throat, and headaches (Cooley et al.,
1998). Although no single cause for the above symptoms is likely
to be found, the presence of certain molds is becoming increasingly
associated with this phenomenon (Burrell, 1991; Cooley et al., 1998;
Dales et al., 1991; Jaakkola et al., 2002; Lehrer et al., 1983; Miller,
1992).
FUNGI AND THE INDOOR ENVIRONMENT 5
II. Inhalation of Fungal Spores Causes Respiratory Disease in Humans
Our first study examining the role of fungi in SBS was published in
1998 (Cooley et al.). In that paper we showed that there was a correlation
between certain fungi (Penicillium species [Figs. 1 and 2, see color in-
sert] in the air and Stachybotrys species [Figs. 3 and 4, see color insert]
on building surfaces) and the symptoms seen in SBS. The finding that
the elevation of culturable (viable) Penicillium species conidia in the
indoor air over those levels in the outside air caused health problems in
human beings should not have been a surprise. As mentioned previous-
ly, the inhalation of Penicillium species conidia was associated with the
initiation of an asthmatic attack as early as 1873 (Blackley). Alternaria
species spores and Penicillium species conidia were shown to provoke
immediate and delayed-type asthma in individuals already sensitized
to these organisms (Licorish et al., 1985). A 1998 study (Garret et al.)
demonstrated that childhood asthma could be correlated with expo-
sure to Penicillium species conidia levels in the air but not to visible
mold. In many of our investigations, we observed that Penicillium
FIG. 1. Penicillium chrysogenum colonies on PDA for 7 days.

FIG. 2. Penicillium chrysogenum at 400 magnification.
species tended to colonize the heating, ventilation, and air condition-

ing (HVAC) systems of buildings (Cooley and Wong, unpublished data
from ARAL database, 2003). In 2002, Gent et al., showed that infants
with high risk for development of asthma who were exposed to high
levels of Penicillium species conidia were at significant risk for persis-
tent cough and wheeze. This study was particularly interesting, be-
cause this correlation between respiratory distress and mold exposure
was valid for Penicillium species conidia but not for Cladosporium
species spores. Obviously there is something different about the genus
Penicillium that sets it apart from other fungal genera in this regard. In
1984, Fergussen et al. described for the first time Penicillium species
allergic alveolitis caused by the faulty installation of a central heating
system unit that introduced a great deal of water into a residence. In
this case the two fungal species found growing in the dwelling were
FIG. 3. Stachybotrys chartarum colonies on PDA for 14 days.
P. chrysogenum and P. cyclopium. Finally, there is one other important

respiratory disease caused by the inhalation of Penicillium species
conidia. This disease is called cheese worker’s lung or cheese washer’s
disease (Straus, 2002). This is an occupational disease that can occur in
individuals who work in the cheese industry. Occupational lung dis-
eases become more common as the world becomes more industrialized.
Some of the other more common occupational lung diseases include
maltster’s lung, farmer’s lung, bagassosis, suberosis, and wood pulp
worker’s lung. The organisms that cause the above diseases are Asper-
gillus clavatus, thermophilic actinomycetes, Penicillium frequentans,
and Alternaria species, respectively (Straus, 2002). These diseases are
all phenomena related to hypersensitivity pneumonitis (HP). HP is an
allergic reaction to a wide variety of different inhaled antigens. In the
case of cheese washer’s disease, the inhaled antigen is a fungus (either
Penicillium casei or Penicillium roqueforti, which are used to flavor the
cheese). The first report of cheese washer’s disease was in Germany in
1969 (DeWeck et al.). In this study, two individuals reported difficulty
FIG. 4. Stachybotrys chartarum at 400 magnification.
in breathing, fever, fatigue, and productive cough. These individuals

were cheese washers, and their symptoms appeared to be job related.
Because Penicillium species are grown on the surface of cheese as it is
being produced, it is necessary to have employees remove the fungi.
These individuals are, of course, called cheese washers. Cheese wash-
ing is performed by rubbing a course salt on the formed product and
then scrubbing it with a damp cloth (Marcer et al., 1996). The cheese
product itself supplies all the food and water that the fungi need to
multiply. Naturally, during this scrubbing process large numbers of
fungal conidia are emitted into the air surrounding the cheese product.
The organism most commonly found growing on the cheese in those
situations is P. casei (Schleuter, 1993). As expected, antibodies to
P. casei were detected in the sera of the two individuals described in
the above 1969 study (DeWeck et al.). Fortunately, the disease appears to
be reversible when the individual is no longer inhaling P. casei conidia
(DeWeck et al., 1969). However, if one is continually inhaling the same
type of fungal spores, ‘‘progressive pulmonary fibrosis’’ can occur with
resultant granulomatous tissue formation and subsequent shortness of
breath (Sell, 1996). Cheese washer’s disease was originally described in
Europe by DeWeck et al. (1969), but it has also been reported in the
United States. Campbell et al. (1983) examined a worker who developed
extrinsic allergic alveolitis (another term for hypersensitivity pneumo-
nitis) caused by her inhalation of a fungus that was used in the produc-
tion of the cheese and not one that grew on the cheese wheel as was
described by DeWeck et al. (1969). In this case, the cheese worker was
involved in the processing of blue cheese, which employs P. roqueforti.
Her job involved the breaking up of the blue cheese so it could be more
easily put in salad dressing bottles. This activity, of course, dispersed
high concentrations of P. roqueforti conidia into the air in her immedi-
ate vicinity. Antibodies to P. roqueforti were found in her serum
and lung washes (Campbell et al., 1983). There have been other reports
in the literature describing similar cases of cheese washer’s disease
(Guglielminetti et al., 2001; Marcer et al., 1996).
III. Correlation Between the Presence of Certain Fungi and SBS

In the 1998 study (Cooley et al.), we showed that there is a correla-
tion between the presence of certain fungi in a building and the symp-
toms associated with SBS. The symptoms associated with SBS and
described in this study can be seen in Table I. In Table I, allergic-like
symptoms were the main complaint at all of the schools, and with the
moderate to high counts of culturable Penicillium species conidia
found in the complaint areas, this is not surprising. However, with
the exception of nausea, numerous symptoms other than allergic-like
were reported at each school. This implies that there may be other
mechanisms that may be inducing adverse health effects. In fact, we
always observed a variety of different visible fungal growth on surfaces
in the schools. This 22-month study of 48 schools in the southern
United States examined buildings in which there were concerns about
poor IAQ and health. Surface samples and indoor air and outdoor
culturable air samples were taken at all 48 buildings to look for visible
fungal growth as well as culturable airborne fungal spores. Five fungal
genera were usually found in the outdoor air. They were Cladosporium
(81.5%), Penicillium (5.2%), Chrysosporium (4.9%), Alternaria (2.8%),
and Aspergillus (1.1%). Cladosporium species are commonly the
dominant fungal species in the outdoor air (Shelton et al., 2002).
TABLE I
INCIDENCES PER 100 EMPLOYEES (95% Cl) OF REPORTED COMPLAINTS AND SYMPTOMS REGARDING INDOOR AIR QUALITY (IAQ) AT 48
UNITED STATES SCHOOLS BETWEEN 1994 AND 1996
Type of symptom Incidence 95% Cl Phenomenon Incidence 95% Cl Phenomenon Incidence 95% Cl
Nasal drainage and 19.8 1.3 Discomfort When are symptoms

congestion complaints the worst?
Itchy or watering eyes 14.3 1.1 Odors 5.2 0.4 High humidity 12.0 0.9
Contact problems 5.6 1.2 Temperature (hot/cold) 7.2 0.1 Low humidity 0.0 0.0
Headaches 12.5 0.6 Noise 0.8 0.3 Spring 3.9 0.8
Sinus 10.3 0.5 Ventilation 6.1 0.3 Summer 0.0 0.0
Severe sinus 3.4 0.4 Fall 2.7 0.6
Increased airway 14.3 1.0 Onset of symptoms Winter 4.5 0.5
infections
Cough 6.5 0.6 Entering the building 3.4 0.9 Start of School 5.7 2.0
Shortness of breath 5.9 0.4 Working in the 11.0 1.7 Morning 3.4 0.4
building
Sneezing 6.8 1.0 Start of school 11.3 1.9 Afternoon 1.1 0.3
Dizziness 2.2 0.5 Monday 0.8 0.3
Fatigue 1.1 0.3 When do symptoms Late in week 0.8 0.3
go away?
Flu-like symptoms 1.8 0.6 Never 3.5 0.8 No pattern 1.1 0.3
Nausea 1.8 3.4 Leave work 2.1 0.6 Always 2.3 0.6
Allergies 17.0 1.0 Weekends 4.3 0.9 Before remedy
Asthma 1.4 0.3 Vacations 14.7 2.5 IAQ complaints 31.3 6.8
or symptoms
Other health conditions 1.2 0.5 Medications 4.4 0.2 After remedy
IAQ complaints 2.5 1.1
or symptoms
Visible surface fungal growth was observed at all 48 schools. At 20 of
the 48 schools studied, there were significantly (P < 0.0001) more
colony forming units per cubic meter of air (CFU/m3) of culturable
Penicillium species conidia in the air samples from complaint areas
as compared with the outside air samples and the indoor air samples
from noncomplaint areas (Figs. 5 and 6). At 5 of the 48 schools, there
were more (P ¼ 0.10) Penicillium species conidia in the air samples
from complaint areas when compared with the outdoor air samples and
the indoor air samples from noncomplaint areas were similar to those
in the outdoor air. However, in 11 of these schools, Stachybotrys atra
(aka chartarum) was isolated from building surfaces. Although visible
surface fungal growth was observed in the remaining 11 schools, the
culturable fungal air profiles were not significantly different between
the complaint and noncomplaint areas.
When the various schools took remedial action that resulted in an
indoor fungal profile that was similar to that observed outdoors and no
visible fungal growth was observed, the complaint profile dropped
FIG. 5. Bar graph of all air samples taken at the 48 schools.

FIG. 6. Bar graph of all air samples taken at the 20 schools where Penicillium species
were the dominant fungi.
from 31.3% to 2.5%, which was a significant (P < 0.001) decrease

(Table I). However, from our experience over the last decade, if the
building is not properly maintained, with moisture events remediated
within 72 hours, these buildings can rapidly become microbially
contaminated.
Three basic strategies should be followed to maintain building per-
formance and prevent microbial contamination: (a) routine surveil-
lance inspections and prompt response to problems, (b) adequate
preventive maintenance of the building structure as well as HVAC
and plumbing systems, and (c) adequate housekeeping including an
emphasis on proper and routine cleaning (Shaughnessy and Morey,
1999).
IV. Development of an Animal Model for Allergic Penicilliosis Induced by
the Intranasal Instillation of Viable Penicillium chrysogenum Conidia
In an effort to try to determine why the inhalation of Penicillium
species conidia could produce the symptoms associated with SBS,
we developed an animal model for allergic penicilliosis induced
by the intranasal instillation of viable (culturable) P. chrysogenum
conidia (Cooley et al., 2000). Once we were able to singly disperse
the P. chrysogenum’s conidia, we determined that there were only
approximately 25% of the conidia that were actually viable—that is,
capable of reproducing. Since we could not readily determine the
difference between a viable conidium and a non-viable conidium, we
rendered one group of conidia totally non-viable. Therefore we could
assume that any difference between the two groups could be contrib-
uted to the viability of the conidia. In this study, C57 black/6 mice were
inoculated intranasally (IN) with 104 viable (V) and non-viable (NV)
P. chrysogenum for 6 weeks. This study showed that mice inoculated
IN for 6 weeks with 104 V PC (average viability 25%) produced
significantly more IgE (total serum), peripheral eosinophils, and airway
eosinophils (Figs. 7 and 8). Except for airway neutrophilia, mice
FIG. 7. Serum levels of IgG2a (solid bars) and total IgE (shaded bars) in mice inoculated
intranasally with viable and non-viable Penicillium chrysogenum conidia once a week for 6
weeks. *P < 0.05 compared with controls. Error bars represent standard error of means (SEM).
FIG. 8. BAL fluid levels of eosinophils (solid bars) and IL-5 (shaded bars) in mice
inoculated intranasally with viable and non-viable Penicillium chrysogenum conidia
once a week for 6 weeks. *P < 0.05 compared with controls. Error bars represent standard
error of means (SEM).
receiving 104 NV P. chrysogenum IN did not demonstrate significant

increases in IgE (total serum), peripheral, or airway eosinophils (Fig. 7
and 8). However, the NV P. chrysogenum IN 104 group showed a
significant increase in total serum IgG2a and bronchoalveolar lavage
(BAL) fluid levels of interferon (IFN)-. Additionally, BAL from mice
inoculated IN with 104 V P. chrysogenum conidia demonstrated signif-
icant increases in the levels of interleukin (IL)-4 and IL-5 (Fig. 9). The
IgG2a/IgE ratio and the IFN-/IL-4 ratio were found to be lower in the
mice inoculated IN with 104 V P. chrysogenum conidia than in those
inoculated with 104 NV P. chrysogenum conidia and the control mice.
This suggests that the inflammatory response observed in the V P.
chrysogenum group was type 2 T helper cell (Th2) mediated. This
concept was supported by the demonstration that proteins extracted
from P. chrysogenum conidia incubated with serum from the V P.
chrysogenum inoculated conidia mice were IgE-specific, while the
serum from the NV P. chrysogenum group was not (Fig. 10). In this
paper we also clearly demonstrated that P. chrysogenum conidia are
easily phagocytized by mouse alveolar macrophages and degraded
FIG. 9. BAL fluid levels of IL-4 (solid bars) and IFN- (shaded bars) in mice inoculated
intranasally with viable and non-viable Penicillium chrysogenum conidia once a week
for 6 weeks. *P < 0.05 compared with controls. Error bars represent standard error of
means (SEM).
(Fig. 11). This study showed that the long-term (6-week) inhalation of V
P. chrysogenum conidia induced type 2 helper cell mediated inflam-
matory responses such as increases in total and conidia-specific serum
IgG1 and IgE. This was observed together with BAL fluid level increases
in IL-4 and IL-5, as well as airway and peripheral eosinophilia, both of
which are allergic reaction mediators.
Since the viable P. chrysogenum conidia are capable of reproducing,
these findings suggest that there is a difference between the viable
P. chrysogenum conidia and non-viable P. chrysogenum conidia. Many
researchers and clinicians in the past have been under the impression
that spores (conidia) are unlikely to release any antigens unless they
germinate, a process that requires several hours. This is because the
mucocilliary tract and the alveolar macrophages will remove most of
the fungal propagules prior to germination or attempted germination
(Platt-Mills et al., 1998).
FIG. 10. Levels of conidia-specific IgG1 (shaded bars), IgE (white bars), and IgG2a (solid
bars) in pooled serum samples from mice inoculated intranasally with viable and non-
viable Penicillium chrysogenum conidia once a week for 6 weeks. *P < 0.05 compared
with controls. Error bars represent standard error of means (SEM).
V. Cellular and Humoral Responses in an Animal Model

Inhaling Penicillium chrysogenum
We also examined the cellular and humoral responses in a mouse
model inhaling P. chrysogenum conidia (Cooley et al., 1999). The
retention of a particle is determined by the deposition and clearance
of the particle. The output of particles previously deposited in the
lungs is called clearance and refers to the process that physically
expels the particles from the lungs. This mechanism includes absorp-
tion, sneeze, cough, mucocilliary transport, and alveolar macrophage
clearance (Brain and Valberg, 1979). It should be kept in mind
that clearance is often of greater significance than deposition. There-
fore, clearance efficiency may be the determining factor for total
integrated exposure, and, consequently, the probability of a pathologic
or physiological response, especially when particles are viable conidia.
FIG. 11. Ultrastructure of alveolar macrophages taken from the BAL fluid of mice
(A) 3 hours, (B) 6 hours, and (C) 24 hours after instillation of viable conidia
(magnification 10,000). Phagocytosed conidia (arrows) at various stages of digestion
were commonly observed within phagosomes of the macrophage at all the times studied.
Temporal correlation of conidia destruction was not apparent as many macrophages
contained conidia in various stages of breakdown, even after 3 hours. Residual bodies
were present in cells at all times, typical of alveolar macrophages. (D) Ultrastructure of
Penicillium chrysogenum conidia (magnification 43,750) before instillation (spore coat
(sc) between the arrow heads and spore vacuoles (v)). This morphology is comparable to
the minimally damaged installed conidia captured in (C). The conidia in (A) and (B) are
apparently in later stages of destruction.
Continuous exposure to moderate to high levels of P. chrysogenum

conidia in a structure over a period of time will have an impact on
the clearance efficiency versus a one-time exposure. Fungi produce a
variety of secondary metabolites, including mycotoxins and fungal
volatile organic compounds (VOCs). Mycotoxins are harmful to ani-
mals and humans. In addition to mycotoxins, some VOCs produced by
FIG. 12. Female C57Bl/6 mice were inoculated IN with 1 106 Penicillium
chrysogenum spores (viability 26%), resulting in a dose of 2.6 105 CFU. At various
time periods after the acute inoculation, the mice were euthanized, the lungs and
tracheas aseptically removed, homogenized, and serial dilutions plated on SDA plates
to determine percent spore viability. Each time period had a minimum of 6 mice. The -0-
indicates no viable spores were recovered. The error bars represent the standard error of
means (SEM).
actively growing fungi are known irritants or hazardous chemicals and

may pose a health risk to building occupants and have an impact on the
clearance efficiency of the lungs (Yang and Johanning, 1997).
In this study, viable P. chrysogenum conidia were recovered
from mouse lungs, as early as 15 minutes and 3 hours through 36
hours after IN inoculation of 106 conidia (26% viability) (Fig. 12). We
demonstrated that approximately 18% of the viable conidia were
actually deposited in the mouse lungs. However, by 12 hours post-
inoculation, only 104 viable conidia were detected. These data suggest
that the mucocilliary tract had cleared most of the inoculated
conidia, but 4% of the viable conidia were housed in the airways,
probably in the alveolar spaces, where they remained viable for up to
36 hours.
One 106 doses of viable P. chrysogenum conidia induced signifi-
cant (P < 0.001) increases in tumor necrosis factor- (TNF-), while NV
P. chrysogenum conidia did not. When 1 104 doses (repeated for 3
weeks) of viable P. chrysogenum conidia were inoculated IN into mice
(C57 Black/6), significant (P < 0.05) increases in total serum IgE and
BAL IL-4 were observed, whereas this did not occur in mice receiving
1 104 NV P. chrysogenum conidia. These data also suggest that viable
P. chrysogenum conidia are capable of inducing an allergic response.
Moisture will stimulate viable P. chrysogenum conidia to attempt to
germinate. If there is a sufficient nutrient source, the conidia (spores)
will form germ tubes and hyphae and colonization will initiate. Al-
though there is enough moisture in the lungs to stimulate germination
of P. chrysogenum conidia, the temperature (37 C) and the lack
of nutrients would inhibit the formation of a germ tube (Yang and
Johanning, 1997). However, observing viable P. chrysogenum conidia
in the lungs 36 hours after instillation certainly allows enough time for
attempted germination and the production of potential antigens. In
fact, we have been able to demonstrate that the viable P. chrysogenum
conidia are capable of producing such antigens (Schwab et al., 2003).
VI. Continually Measured Fungal Profiles in SBS

In a 1999 study (McGrath et al.), we sought to answer two questions.
The first was, ‘‘when taking a culturable indoor air sample, is that
sample an accurate reflection of the air in that building or is it just a
snapshot that changes immediately after the picture is taken?’’ The
second question was ‘‘do ‘sick’ or contaminated buildings stay or
remain contaminated over an extended period of time or do they get
better and then become contaminated again?’’ To answer these ques-
tions, we compared culturable fungal air profiles measured continually
over 6 hours in a documented ‘‘sick’’ building. We measured culturable
indoor air (IDA) samples in a room experiencing IAQ problems (heavi-
ly colonized with P. chrysogenum) with fungal profiles measured
concurrently in the culturable outdoor air (ODA) samples.
Investigators often use ODA samples as a baseline measurement in
which to compare what is found in the IDA samples. Indoor/outdoor
comparisons are commonly used to document the presence or infer the
absence of indoor, biologically derived contamination (Burge et al.,
1999). However, one should use caution and compare genera and
species and not genera only.
The dominant species collected in the IDA and ODA were Alternaria
species, Cladosporium species, and Penicillium species (Fig. 13). In the
FIG. 13. Fungal concentrations measured in indoor and outdoor air. Values are mean
SEM. Each bar represents the average of all samples.
IDA, Penicillium species were consistently the dominant organisms,

ranging from 150 to 567 CFU/m3 (89.8–100% of the total viable fungi)
(Fig. 14). In the ODA, Cladosporium species were dominant in four of
the samples (40.0–70.6%), while Penicillium species were dominant
(52.7–79.6%) in two samples (Fig. 15). This study showed that as
expected, ODA fungal profiles are continually changing. Outdoor con-
centrations of some biological agents vary with time of day, wind
directions, relative humidity, and other factors. Depending on the
source of a biological agent, indoor air concentrations may be affected
by outdoor air ventilation rates, number of occupants or occupant
activity, and vibration and air movements within the structure
(Macher, 1999). The IDA fungal profiles in this particular ‘‘sick’’ build-
ing tended not to change, at least for the 6 hours we measured, which
was probably due to the very heavy colonization of Penicillium species
growing in the room. However, it has been our experience in sampling
hundreds of buildings that the IDA may only be a snapshot. If the IDA
is positive for increased fungal presence, as compared to the ODA, then
that suggests a strong indication of interior fungal growth. It should be
noted that failure to find a biological agent or related environmental
FIG. 14. Fungal profiles measured in indoor air. Values are mean SEM. Each bar
represents the mean of 3 samples.
FIG. 15. Fungal profiles measured in outdoor air. Values are mean SEM. Each bar
represents the mean of 3 samples.
condition is not absolute assurance of their absence nor the absence of

exposure or risk.
Many investigators take only total spore trap sampling and us-
ually do not take any or few culturable (viable) air sampling. The
Penicillium species and Aspergillus species conidia are single cell,
spherical in shape, 1 to 5 m in diameter, and are reported as Asper-
gillus/Penicillium-like spores. However, this can be very misleading in
that there are numerous genera of fungi that produce similar-looking
spores. With Penicillium species as one of the most common indoor
fungal colonizers, especially in the HVAC systems, the only way to
determine its presence is by culturable air sampling. Investigators can
never definitively conclude or prove that an environment is ‘‘safe’’ and
presents no risk of exposure to biological agents. In part, this means
that if the investigators have not looked for biological agents, they
cannot say that it is not there (Burge et al., 1999).
VII. Evaluation of Fungal Growth on Cellulose-Containing and

Inorganic Ceiling Tile
As we stated earlier in our introduction, mold has plagued mankind
since Moses. The construction materials used during biblical times
were ‘‘straw’’ bricks, cellulose-based stones, and plaster. As the stan-
dard of living has increased in the industrialized nations, the invention
of air conditioning, which led to increased productivity and labor-
saving products, has also allowed the industrialized nations freedom
of architectural design, ignoring thousands of years of building experi-
ence. As the post–World War II building boom started, the industria-
lized nations changed the way buildings were constructed. Inorganic
plaster-covered walls were labor intensive and expensive. To reduce
the cost of construction and meet the burgeoning demand, the use of
gypsum board (1/200 to 3/400 gypsum covered with processed-paper
[cellulose-based] to maintain its rigidity) was begun. It is inexpensive
to manufacture and relatively labor free and rapid to install. A variety
of cellulose-based compounds could be used to texture and paint it or it
could be covered with wallpaper or vinyl paper. However, in the
process, we reverted back to the ‘‘straw’’ bricks, using processed-
cellulose substances that were ideal food sources for mold. The only
thing that was lacking to induce fungal growth was moisture.
The standard of living continued to increase and there was and still
is a great demand for gypsum board. When the energy crisis occurred
in 1972, we sealed up our buildings, closed our windows, and
recirculated our air so that we could reduce the soaring energy costs.
Because we knew that mold growth in buildings was due to water
intrusion, we thought that it was essentially impossible to keep water
out of buildings. This is due to the fact that many uncontrollable events
occur (e.g., roof leaks, water pipe breaks, and floods). When these
things happen, moisture is trapped in buildings, and combined with
poor preventive maintenance and ignorance, all of the ingredients are
in place to have mold problems associated with SBS. We thus decided
to see if it was possible to develop building materials that would not
support fungal growth, even after a significant water event. The objec-
tive of this study was to examine building materials that would not
support the growth of certain fungi after a wetting event, regardless of
whether an external food source was supplied (Karunasena et al.,
2000). The growth of three fungal genera (Stachybotrys, Penicillium,
and Cladosporium) was examined on inorganic ceiling tile (ICT) and
cellulose-containing ceiling tile (CCT) (Figs. 16 and 17). Both types of
ceiling tiles were wetted and inoculated with fungal spores of the
above three genera. The study showed that the ICT did not allow for
fungal growth while the CCT did (Fig. 18). These data demonstrate that
it is possible to develop building materials that will not support fungal
growth, even after a significant water event.
VIII. The Presence of Fungi Associated with SBS in North American

Zoological Institutions
The last study we would like to discuss addressed the presence of
fungi associated with SBS in American zoos (Wilson and Straus, 2002).
One of the ideas that led to this study was a perception that it is often
difficult to get animals to breed in captivity, especially in zoos. We
wondered if chronic exposure to SBS-associated fungi could affect
breeding success or animal morbidity and/or mortality. We examined
a total of 110 sites from five zoos in the United States to determine
whether SBS-associated fungi could be isolated. We also investigated
whether the presence of said fungi could be correlated with adverse
breeding success and/or morbidity and mortality. Culturable air sam-
ples and surface samples were taken at the above zoos. High levels of
P. chrysogenum conidia were found in the air of 16 sites at all 5 zoos.
Five viable growth sites of Stachybotrys chartarum (atra) were found at
2 zoos. A number of other fungal species were recovered from all zoos.
A Fisher exact test analysis demonstrated a nonrandom, significant
(P < 0.001) relationship between sites with a record of poor animal
health and high levels of airborne P. chrysogenum conidia. This study
suggests that significant numbers of airborne fungi associated with SBS
FIG. 16. Colony-forming units of three different fungal genera on cellulose-containing

ceiling tile (CCT) and inorganic ceiling tile (ICT) exposed to fungal conidia and
incubated for 7 days. Horizontal bars represent original conidia concentrations. Error
bars represent standard deviation. The single asterisk (*) indicates a significant increase
in the number of viable spores harvested from the tiles compared to the original inocula,
and the double asterisk (**) indicates a significant decrease in the number of viable
spores harvested from the tiles compared to the original inocula. A Mann-Whitney rank
sum test was utilized (P < 0.05) to compare the inocula to the conidia harvested from the
tiles. Clado signifies Cladosporium cladosporioides, Pen signifies Penicillium chryso-
genum, and Stach signifies Stachybotrys chartarum.
are in North American zoological institutions, and therefore zookeepers

need to be aware of them and the potential problems they can cause.
IX. The Role (?) of Mycotoxins in SBS

Finally, we would like to briefly discuss the role of mycotoxin pro-
duction by fungi in the role of SBS. There is little doubt that mycotox-
ins are produced by fungi inside buildings in which the organisms are
growing. This has been shown by a number of investigators (Croft et al.,
1986; Engelhart et al., 2002; Kielsen et al., 1999; Nieminen et al., 2002;
Nikulin et al., 1994; Tuomi et al., 2000). We know that there are
FIG. 17. Colony-forming units of three different fungal genera on CCT and ICT with
400 l of TSB exposed to fungal conidia and incubated for 7 days. Horizontal bars
represent original conidia concentrations. Error bars represent standard deviation. The
single asterisk (*) indicates a significant increase in the number of viable spores
harvested from the tiles compared to the original inocula, and the double asterisk (**)
indicates a significant decrease in the number of viable spores harvested from the tiles
compared to the original inocula. A Mann-Whitney rank sum test was utilized (P < 0.05)
to compare the inocula to the viable conidia harvested from the ceiling tiles. Clado
signifies Cladosporium cladosporioides, Pen signifies Penicillium chrysogenum, and
Stach signifies Stachybotrys chartarum.
trichothecene mycotoxins on the spores of Stachybotrys atra (Sorenson

et al., 1987) and that these spores can be taken into the human
lung (Elidemir et al., 1999). In many of our examinations of tape surface
samples, we observed Penicillium species growing with Stachybotrys
atra colonies (Cooley and Wong, unpublished data from ARAL data-
base, 2003). Therefore the obvious conclusion one can draw from
this is that trichothecene mycotoxins can enter the lungs of human
beings in Stachybotrys-infested houses. There is some evidence that
implicates trichothecene mycotoxins in illnesses seen in Stachybotrys-
infested buildings and/or houses (Croft et al., 1986; Elidemir et al.,
1999; Flappan et al., 1999; Hodgson et al., 1998; Smoragiewicz et al.,
1993). Indeed, Croft et al. (2002) recently demonstrated the presence of
FIG. 18. Fungal growth on cellulose-containing ceiling tile (CCT) and inorganic ceiling
tile (ICT). CCT (B, C) and ICT (E, F) were inoculated with 3.0 104 CFU of S. chartarum
and incubated for 7 days at 25 C and 80% RH. A and D represent uninoculated CCT and
ICT, respectively. B and E represent CCT and ICT inoculated with 3.0 104 CFU of S.
chartarum plus 0 l of TSB, respectively. C and F represent CCT and ICT inoculated with
3.0 104 CFU of S. chartarum plus 400 l of TSB, respectively. These results are
representative of all of the fungi used in this study.
trichothecene mycotoxins in the urine of patients who had been ex-

posed to these compounds in mold-contaminated buildings. We know
what kinds of symptoms trichothecene mycotoxins can produce in
human beings. Phase I clinical evaluation of anguidine (a simple
trichothecene produced by Fusarium equiseti) was administered by
rapid intravenous infusion daily to a number of patients to determine
its effectiveness as an anti-cancer drug (Goodwin et al., 1978; Murphy
et al., 1978). While this compound had little or no anti-tumor activity,
it was quite toxic at some of the dosages employed. Symptoms of
toxicity included nausea; vomiting; low blood pressure; central ner-
vous system symptoms such as drowsiness, ataxia, and confusion;
diarrhea; fever and chills; burning erythema, inflammation of the mu-
cous membranes of the mouth; difficulty in breathing; and moderate
myelosuppression. Higher doses showed an association with life-
threatening liver function impairment. While we can not yet say that
mycotoxin production in our buildings is definitely responsible for
some of the illnesses seen in those living or working in Stachybotrys-
infested buildings, it is clear that exposure to these structures by
human beings is something to be avoided.
ACKNOWLEDGMENTS
The authors would like to thank the following for financial support: Assured IAQÕ,
Dallas; Texas Tech University Health Sciences Center; and the State of Texas Higher
Education Coordinating Board. We would also like to thank Enusha Karunasena, Trevor
Brasel, and Nancy Markham, and also Drs. Chris Schwab, Jim Hutson, Jim Williams,
Steve Wilson, and Jim McGrath, who helped generate the data reported here.
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Fungal Contamination as a Major Contributor to
Sick Building Syndrome
DE-WEI LI AND CHIN S. YANG
P & K Microbiology Services, Inc., 1936 Olney Ave
Cherry Hill, New Jersey 08003
I. Introduction 31
II. Effects of Indoor Fungi on Human Health 32
A. Fungal Allergies and Allergenic Respiratory Diseases 32
B. Infectious Diseases 42
C. Mycotoxins and Their Significance to Human Health 50
D. Volatile Organic Compounds (VOCs) 59
E. Glucans 62
III. Indoor Fungi 63
A. Fungal Identification 63
B. Airborne Fungi 64
C. Fungi Growing on Indoor/Building Materials 72
D. Fungal Biodiversity Indoors 74
E. Stachybotrys chartarum and Other Stachybotrys spp. 74
F. PCR and Molecular Techniques 77
IV. Ecological Factors of Fungi Indoors 78
A. Physical Factors 78
B. Building Characteristics 86
C. Succession and Changes in Indoor Fungi 91
V. Recent Studies on Limits/Exposures of Indoor Fungi 94
VI. Conclusions 96
References 97
I. Introduction
Fungi are heterotrophic eukaryotes producing exoenzymes and ab-
sorbing their nutrients by a network of hyphae and reproducing
through development of spores. They belong to Kingdom Eumycota
(Kingdom of Fungi) or Kingdom Chromista (Kendrick, 2000). However,
there is one group of organisms, which are traditionally studied by
mycologists, called pseudofungi (such as slime molds in myxomy-
cetes), that belong to Kingdom Protozoa (Kirk et al., 2001). Fungi are
a very large, diverse, and heterogeneous group of organisms found in
nearly every ecological niche (Alexopoulos et al., 1996). They play a
very important role in our ecosystem and our daily life. Fungi always
play dual roles on the earth: (a) a positive one as food, medicine, key
components in food processing, decomposers breaking down organic
matters to recycle the nutrients in the ecosystem and to form symbiotic
31
0065-2164/04 $35.00
32 LI AND YANG
relationship with other organisms; (b) a negative one as pathogens to

humans, plants, and animals; as allergens, producing secondary meta-
bolites, mycotoxins, fungal volatile organic compounds (VOCs); and as
glucans, which are detrimental to human health and building occu-
pants (Batterman, 1995; Ezeonu et al., 1994; Miller, 1992, 1993). A large
number of fungi are saprophytes or decomposers, which mainly occur
in natural environments (outdoors) such as soil and plant debris. Some
of these fungi can be found in indoor environments. One key factor that
we should keep in mind is that most indoor fungi originate from the
outdoor environment. Certain indoor fungal contaminants pose a po-
tential health risk to building occupants and may lead to sick building
syndrome (Gravesen et al., 1994; Miller, 1992, 1993; Samson et al.,
1994). Indoor fungi have attracted unprecedented attention because
of their potential health effects on humans in the last decade. Public
awareness of indoor fungi in return generates more research to eluci-
date their roles in indoor environments and human health. Indoor
fungus is not only a scientific issue but is also becoming a social issue.
Public awareness does not automatically mean a good understanding of
the indoor molds. There are still many key questions that need to be
answered to have a better understanding of the indoor mold issue.
This chapter reviews available literature on fungal contamination as
a major contributor to sick building syndrome.
II. Effects of Indoor Fungi on Human Health

A. FUNGAL ALLERGIES AND ALLERGENIC RESPIRATORY DISEASES
Allergy (Gk allos, other; ergon, work) is a disease or reaction caused
by an immunoglobin E (IgE)-mediated immune response to one or more
environmental agents, resulting in tissue inflammation and organ dys-
function, and an exaggerated and pathological variant of a normal
immune mechanism (Klein, 1990; Middleton, Jr. et al., 1988; Paul,
1989; Raven and Johnson, 1986). Fungal spores are a well known cause
of allergic diseases (Chapman, 1999; Gravesen, 1979; Horwitz and
Bush, 1997) and were identified as one of the major indoor allergens
(Burr, 1999; Pope et al., 1993; Ruotsalainen et al., 1995). Allergy is
common throughout the world. The prevalence of sensitivity to specif-
ic allergens is determined by both genetic predilection and geographic
and cultural factors responsible for exposure to the allergen (Stites and
Terr, 1991).
All fungi may be allergenic, depending on the individual, the
exposure situation, and the dose (Ruotsalainen et al., 1995). The genera
FUNGAL CONTAMINATION AS A MAJOR CONTRIBUTOR 33
of fungi, which have been reported to be allergenic, are compiled in
Table I. Since the late 1870s, when Blakeley developed symptoms of
bronchial asthma and ‘‘chest tightness’’ after inhaling spores from
Penicillium cultures, it has been believed that mold sensitization is
an important cause of respiratory allergy (Barth, 1981; Karlsson-Borgå,
1989; Salvaggio, 1986).
Allergy is perhaps the most common human reaction to airborne
fungal spores (including conidia). About 20% of the population are
allergic individuals with a genetic predisposition to produce IgE anti-
body to allergens that are either inhaled or ingested (Kaplan et al.,
1991; Tizard, 1988). The percentages of populations allergic to molds
vary from 2% to 18%, and around 80% of asthmatic patients are
allergic to molds (Flannigan et al., 1991). About 20% of the population
are atopic and easily sensitized by concentrations usually found in the
outdoor air spora (up to 106 spores/m3). These people react immediate-
ly on exposure in the upper airways with hay-fever-like symptoms or
asthma and may become sensitive to several of the allergens to which
they are exposed. The remainder of the population requires more
intensive exposure (106–109 spores/m3) for sensitization (Lacey, 1981).
The incidence and prevalence of allergic diseases is increasing
(Ruotsalainen et al., 1995). Allergies affect as many as 50 million
people in the United States, costing them up to $5 billion annually
(Jaroff, 1992), and the number is obviously much higher at present.
Asthma, rhinitis, hypersensitivity pneumonitis, and humidifier lung
are allergenic respiratory diseases that, to a certain degree, may be
related to exposure to airborne fungi.
Asthma is the most common chronic respiratory disease in all
countries. Both the severity and prevalence of persistent asthma ap-
pear to be increasing, leading to urgency in the search for its causes
(Woolcock, 1991). Four thousand people a year reportedly died from
allergic asthma attack in the United States (Jaroff, 1992). In Australia,
asthma mortality rates doubled from 1978 to 1988 (Young et al., 1991).
Immediate-type asthma symptoms were produced with both whole
spores and spore extracts of Alternaria and Penicillium (Licorish et al.,
1985; Salvaggio, 1986). Airborne fungal spores are ubiquitous (Howard,
1984) and are known in many cases to be allergenic, so it is not
surprising that mold spores are an important cause of asthma. At
present the relationship between mold spores and asthma is still poorly
understood. In Madison, Wisconsin, in a series of 100 consecutive
patients with allergic asthma, skin tests were uniformly positive to
Alternaria (Reed, 1985). Most of these patients had asthma symptoms
not only before and after the ragweed season (about August 10 to
34 LI AND YANG
TABLE I
FUNGAL GENERA REPORTED TO BE ASSOCIATED WITH ALLERGY
Fungus Order Division
Absidia Mucorales Zygomycota

Acremonium Hyphomycetes
Acrogenospora Hyphomycetes
Acrothecium Hyphomycetes
Agaricus Agaricales Basidiomycotina
Agrocybe Agaricales Basidiomycotina
Alternaria Hyphomycetes
Amanita Agaricales Basidiomycotina
Armillaria Agaricales Basidiomycotina
Arthrinium Hyphomycetes
Aspergillus Hyphomycetes
Aureobasidium Hyphomycetes
Bispora Hyphomycetes
Boletinellus Boletales Basidiomycotina
Boletus Boletales Basidiomycotina
Botrytis Hyphomycetes
Calvatia Lycoperdales Basidiomycotina
Candida Yeast
Cantharellus Aphyllophorales Basidiomycotina
Chaetomium Sordariales Ascomycotina
Chlorophyllum Agaricales Basidiomycotina
Cladosporium Hyphomycetes
Claviceps Hypocreales Ascomycotina
Coniosporium Hyphomycetes
Coprinus Agaricales Basidiomycotina
Coriolus Aphyllophorales Basidiomycotina
Cryptococcus Hyphomycetes
Cryptostroma Hyphomycetes
Cunninghamella Mucorales Zygomycota)
Curvularia Hyphomycetes
Dacrymyces Dacrymycetales Basidiomycotina
Daldinia Xylariales Ascomycotina
Debaryomyces Saccharomycetales (Yeast) Ascomycotina
Dicoccum (Trichocladium) Hyphomycetes
(continued)
TABLE I (Continued )

Didymella Dothideales Ascomycotina
Drechslera Hyphomycetes
Epicoccum Hyphomycetes
Epidermophyton Hyphomycetes
Erysiphe Erysiphales Ascomycotina
Eurotium Eurotiales Ascomycotina
Fomes Aphyllophorales Basidiomycotina
Fuligo Myxomycetes
Fusarium Hyphomycetes
Ganoderma Aphyllophorales Basidiomycotina
Geastrum Lycoperdales Basidiomycotina
Geotrichum Hyphomycetes
Gibberella Hypocreales Ascomycotina
Gliocladium Hyphomycetes
Gnomonia Diaporthales Ascomycotina
Graphium Hyphomycetes
Helminthosporium Hyphomycetes
Hypholoma Agaricales Basidiomycotina
Inonotus Aphyllophorales Basidiomycotina
Leptosphaeria Dothideales Ascomycotina
Leptosphaerulina Dothideales Ascomycotina
Lycoperdon Lycoperdales Basidiomycotina
Malassezia Hyphomycetes
Merulius (=Phlebia) Basidiomycotina
Microsphaera Erysiphales Ascomycotina
Microsporum Hyphomycetes
Monilia Hyphomycetes
Mucor Mucorales Zygomycota
Mycogone Hyphomycetes
Naematoloma Agaricales Basidiomycotina
Neurospora Sordariales Ascomycotina
Nigrospora Hyphomycetes
Oidium Hyphomycetes
Paecilomyces Hyphomycetes
Papularia Hyphomycetes
(continued)
36 LI AND YANG

Penicillium Hyphomycetes
Phoma Coelomycetes
Phycomyces Mucorales Zygomycota
Phytophthora Peronosporales Oomycota
Piptoporus Aphyllophorales Basidiomycotina
Pisolithus Sclerodermatales Basidiomycotina
Pleospora Dothideales Ascomycotina
Pleurotus Aphyllophorales Basidiomycotina
Podaxis Podaxales Basidiomycotina
Polyporus Aphyllophorales Basidiomycotina
Poria Aphyllophorales Basidiomycotina
Psilocybe Agaricales Basidiomycotina
Puccinia Uredinales Basidiomycotina
Rhizopus Mucorales Zygomycota
Rhodotorula Yeast Basidiomycotina
Saccharomyces Endomycetales (Yeast) Ascomycotina
Scleroderma Sclerodermatales Basidiomycotina
Scopulariopsis Hyphomycetes
Serpula Aphyllophorales Basidiomycotina
Sphaerotheca Erysiphales Ascomycotina
Spondylocladium Hyphomycetes
Sporobolomyces Yeast Basidiomycotina
Sporotrichum Hyphomycetes
Stachybotrys Hyphomycetes
Stemonitis Myxomycetes
Stemphylium Hyphomycetes
Stereum Aphyllophorales Basidiomycotina
Syncephalastrum Mucorales Zygomycota
Tetracoccosporium Hyphomycetes
Thermomyces Hyphomycetes
Tilletiopsis Hyphomycetes
Tilletia Basidiomycotina
Torula Hyphomycetes
Trichoderma Hyphomycetes
Trichophyton Hyphomycetes
(continued)

Trichothecium Hyphomycetes
Typhula Aphyllophorales Basidiomycotina
Urocystis Basidiomycotina
Ustilago Ustilaginales Basidiomycotina
Verticillium Hyphomycetes
Wallemia Hyphomycetes
Xylaria Xylariales Ascomycotina
Xylobolus Aphyllophorales Basidiomycotina
Chapman (1986); Ibanez et al. (1988); Latgé and Paris (1991); Santilli et al. (1990); Shen et al.
(1990); Smith (1990); Van Bronswijk et al. (1986).
September 20) but also during the time of year Alternaria spore counts
are high (July through October) (Reed, 1985). Cladosporium herbarum
has been shown to be a potential cause of allergic asthma and rhinitis
(Malling, 1990).
In a recent study, the prevalence of most building-related symptoms
was between 32% and 62%. Positive basophile histamine release
(HRT), showing serum IgE specific to one or more of the molds, was
observed in 37% of the individuals (Lander et al., 2001). The highest
frequency of positive HRT was found to Penicillium chrysogenum and
then to Aspergillus species, Cladosporium sphaerospermum, and Sta-
chybotrys chartarum (Lander et al., 2001). Savilahti et al. (2000)
showed that moisture damage and exposure to molds increased the
indoor air problems of schools and affected the respiratory health of
children.
Cladosporium, Alternaria, Penicillium, Aspergillus, and Mucor were
reported to be the commonest allergenic fungi (Furuuchi and Baba,
1986; Malling et al., 1985). Cladosporium is believed to be the most
common one causing mold allergy (Malling et al., 1985). However, the
most prevalent airborne fungi are not necessarily the most potent
allergens, at least as determined by prick testing (Terracina and Rogers,
1982). Spores of Alternaria alternata and those of the closely related
genera Stemphylium and Ulocladium are considered to be the most
important mold allergens in the United States (Hoffman, 1984;
O’Hollaren et al., 1991; Reed, 1985). Penicillium exposure was a risk
factor for asthma, while Aspergillus exposure was a risk factor for atopy
(a genetic trait of increased allergen sensitivity) (Garrett et al., 1998).
38 LI AND YANG
Chow et al. (2000) characterized Pen n 13 as a major allergen of Peni-

cillium notatum (a synonym of P. chrysogenum).
Aspergillus restrictus was demonstrated to be a potentially important
causative agent in atopic diseases when using skin prick tests and
radioallergosorbent test (RAST) on 24 patients (Sakamoto et al., 1990).
Aspergillus species and in particular Aspergillus fumigatus appeared to
be the etiological agents in various lung diseases and allergens. Inhala-
tion of low doses of Aspergillus spores may induce sensitization and
asthma in sensitive patients, while inhalation of high doses may trigger
alveolitis and farmer’s lung (Wallenbeck et al., 1991). Martinez Ordaz
et al. (2002) of Mexico found that the association of skin reactivity and
indoor exposure was significant only for Aspergillus.
Curvularia lunata was found to be a cause of allergic bronchopul-
monary disease (Halwig et al., 1985). Epicoccum nigrum was reported
to be able to colonize nasal sinuses and cause allergic fungal sinusitis
(Noble et al., 1997). Sooty molds caused allergies ranging from rhinitis
to asthma in the eastern United States (Santilli et al., 1985).
Ascospores are important airborne allergens and present unique
antigens (Eversmeyer and Kramer, 1987). Fifteen of 18 patients report-
edly reacted to Leptosphaeria ascospores (Burge, 1986). About 40% of
atopic patients reacted to at least 1 ascomycete preparation. Chaeto-
mium species, particularly C. globosum, are important ascomycetes
commonly found growing indoors on water-damaged paper and wood
products.
Basidiospores of Agaricus campestris, Coprinus micaceus, Lycoper-
don perlatum, Scleroderma lycoperdoides, and Ustilago maydis
caused allergies ranging from rhinitis to asthma in the eastern United
States (Santilli et al., 1985). Basidiospores are antigenic and can elicit
immediate skin reactivity in sensitive patients. Mushrooms and basi-
diospores are considered most likely to be of outdoor origin, although
mycelia and conidia of wood decay fungi and, occasionally, mush-
rooms of the genus Coprinus and wood decay fungi have been identi-
fied in indoor environments with a chronic water-damage history.
In a military hospital building in Finland with severe, repeated, and
enduring water and mold damage, the most abundant species was
Sporobolomyces salmonicolor. Four new cases of asthma, confirmed
by S. salmonicolor inhalation provocation tests, were found among the
hospital personnel, one of whom was also found to have alveolitis
(Seuri et al., 2000). Seven other workers with newly diagnosed rhinitis
reacted positively in nasal S. salmonicolor provocation tests. Skin
prick tests of Sporobolomyces were negative among all 14 workers
(Seuri et al., 2000).
Several epidemiologic studies concerning water damage, fungal
growth, and exposure to mold spores have been conducted in a number
of countries. The occurrence of Cladosporium, Aspergillus versicolor,
and Stachybotrys showed some value as an indicator of moisture dam-
age. Presence of moisture damage in school buildings was a significant
risk factor for respiratory symptoms in school children (Meklin et al.,
2002). The association between moisture damage and respiratory
symptoms of children was significant for buildings of concrete/brick
construction but not for wooden school buildings. The highest symp-
tom prevalence was found during spring seasons, after a long exposure
period in damaged schools (Meklin et al., 2002).
Questionnaire surveys conducted in the United Kingdom,
Canada, United States, and the Netherlands showed positive correla-
tions between self-reported allergenic respiratory symptoms and
self-reported water damage and indoor fungi problems (Andrae et al.,
1988; Brunekreef, 1989; Dales et al., 1991a; Dekker et al., 1991; Melia
et al., 1982; Strachan, 1988; Strachan and Sanders, 1989; Strachan
et al., 1990; Waegemaekers et al., 1989). Most studies identified an
association between airborne fungal spore concentrations and self-
reported allergic symptoms in the United Kingdom, Sweden, and the
Netherlands (Holmberg, 1987; Platt et al., 1989; Strachan et al., 1990;
Waegemaekers et al., 1989), but there is not always a correlation be-
tween indoor spore counts and symptoms found in research (Tobin
et al., 1987).
Yang et al. (1997) showed that the prevalence of respiratory symp-
toms was consistently higher in homes with dampness than in non-
damp homes. Dampness in the home can be used as a strong predictor
of and a risk factor for respiratory symptoms and is a considerable
public health problem in Taiwan (Yang et al., 1997). A significant
relationship was found between dampness and work-related sick
building syndrome in day-care-center workers in Taiwan (Li et al.,
1997). A significant association was found between most building-
related symptoms (BRS) and positive basophil histamine release
(Lander et al., 2001). Jacob et al. (2002) 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. Fungal allergies were more common
among children exposed to Cladosporium or Penicillium in winter or to
musty odor (Garrett et al., 1998).
In atopic children, total IgE showed a significant linear relation with
age. Prevalence of specific IgE for Cladosporium ranked first, followed
40 LI AND YANG
closely by Aspergillus and Alternaria (Nolles et al., 2001). Sensitization

to fungi is prevalent in childhood, with an age-dependent distribution
reaching maximum values at 7.7–7.8 years, followed by a decline for all
fungal sensitization with increasing age (Nolles et al., 2001).
Jaakkola et al. showed that the risk of asthma was related to the
presence of visible mold and/or mold odor in the workplace but not
to water damage or damp stains alone. The fraction of asthma attribut-
able to workplace mold exposure was estimated to be 35.1% among the
exposed (Jaakkola et al., 2002). Large airborne fungal spore concentra-
tions were recorded in association with musty odor, water intrusion,
high indoor humidity, limited ventilation through open windows, few
extractor fans, and failure to remove indoor mold growth in the homes
in the Latrobe Valley, Victoria, Australia (Garrett et al., 1998).
Aspergillus was associated strongly with work-related sick building
syndrome in day-care-center workers (Li et al., 1997). The diagnosis of
sick building syndrome related diseases, such as asthma, rhinitis, and
allergic alveolitis, can be very difficult. In the study of Thorn et al.
(1996) the symptoms of a school teacher, who was working in a school
that had indoor air quality problems on and off for several years, were
first interpreted as pulmonary embolism and later as atypical sarcoido-
sis. However, 6 years later the diagnosis of the illness was revised to
chronic allergic alveolitis.
It is important to understand that even correlations do not necessari-
ly mean causal relations. Most studies on indoor airborne fungi were
conducted without taking allergic symptoms into account. Several
recent epidemiological studies have shown that long-duration indoor
exposure to certain fungi can result in hypersensitivity reaction and
chronic diseases. Mold spore levels comparable to outside background
levels are usually well tolerated by most people. Normal or ‘‘typical’’
indoor molds may vary depending on diurnal and seasonal patterns of
outdoor fungi, weather conditions, climate variations, and geographical
regions (Li and Kendrick, 1995a).
There are other diseases caused by airborne fungal allergens, such as
rhinitis, hypersensitivity pneumonitis, and humidifier lung (Burge,
1990b; Salvaggio, 1986). A number of occupational hypersensitivity
diseases of the lung can be implicated by fungi (Table II). Hypersensi-
tivity pneumonitis, also called extrinsic allergic alveolitis, is a well-
recognized occupational disease. Hypersensitivity pneumonitis caused
by inhalation of spores from the edible mushroom Pholiota nameko
was documented by Nakazawa and Tochigi (1989). A diagnosis of
hypersensitivity pneumonitis caused by an Aspergillus species was
made by Jacobs et al. (1989). Pleurotus ostreatus was defined to be an
TABLE II
FUNGI-IMPLICATED OCCUPATIONAL HYPERSENSITIVITY DISEASES OF THE LUNG
Fungal agent Disease Source
Alternaria sp. Pulpmill worker’s lung Moldy pulpwood

Aspergillus clavatus Malt worker’s lung Moldy malt
Aspergillus fumigatus Wood trimmer’s disease Moldy timber
Aspergillus sp. Sawmill worker’s lung Moldy
Aspergillus sp. Woodchip handler’s disease Moldy woodchip
Aureobasidium pullulans Sauna taker’s lung Sauna steam
Aureobasidium pullulans Sequoiosis Moldy sawdust
Botrytis cinerea Vinegrower’s lung Moldy fruit
Farnai rectivirgula Potato riddler’s lung Straw
Cryptostrama corticale Maple bark disease Moldy maple bark
Graphium sp. Maple bark disease Moldy maple bark
Micropolyspora faeni Farmer’s lung Moldy hay
Micropolyspora faeni Mushroom worker’s lung Mushroom compost
Micropolyspora faeni Woodchip handler’s disease Moldy woodchip
Mucor sp. Woodchip handler’s disease Moldy woodchip
Penicillium casei Cheese worker’s lung Cheese
Penicillium spp. Suberosis, woodman’s disease Cork
Rhizopus sp. Wood trimmer’s disease Moldy timber
Rhizopus (Mucor) stolonifer Paprika worker’s lung Moldy paprika
Serpula (Merulius) lacrymans Dry rot lung Moldy building
allergen by Horner et al. (1988). Extrinsic allergic alveolitis caused by

spores of Pleurotus ostreatus was reported by Cox et al. (1988).
In general, the adverse effects of fungal exposure by inhalation are
related to duration and intensity. Many studies have shown that ‘‘atyp-
ical’’ mold spore levels in the indoor environment increase because of
recurrent water leaks, home dampness, and high humidity, resulting in
increases of allergies and respiratory problems (Burge, 1990a,b; Dales
et al., 1991; Flannigan et al., 1991; Johanning et al., 1993; Rylander,
1994; Solomon et al., 1978; Strachan et al., 1990; Streifel and Rhame,
1993; Tripi et al., 2000). Path analysis showed that indoor total fungal
spores, indoor Aspergillus/Penicillium, and the age of the residences
had significant direct effects on allergic symptoms (Li, 1994).
There are still significant methodological problems in the prepara-
tion and production of reliable allergen extracts from fungi as
42 LI AND YANG
compared with those from cats, dust mites, and other better-character-
ized allergens. Extracts that are available correspond poorly with the
fungi often found in indoor surveys (Horner and Lehrer, 1999). One of
the technical difficulties is to produce enough spores for allergen
extraction. Common practice in fungal allergen extraction is to use a
mixture of spores and mycelia, which was believed to be a contributing
factor to inconsistency in the low sensitivity of fungal allergenic tests.
Because of the low sensitivity of some of the commercially available
mold allergen extracts, false-negative results are not uncommon. Pa-
tients with an atopy are frequently allergic to multiple fungal species
and manifest type I reactions (asthma, rhinitis, eczema, and hay fever).
One of the reasons for the poor correlations is reportedly that fungal
allergens are extracted from mostly vegetative hyphae grown in liquid
cultures, not from spores. The differences in allergencity between
hyphae and spores should be studied.
B. INFECTIOUS DISEASES
Fungi are mostly known to cause not only allergies but also infec-
tious diseases to the skin and other body organs (Table III). Infections
caused by fungi are called mycoses. Mycoses are categorized into
endemic and opportunistic. Endemic mycosis is caused by the inhala-
tion of airborne fungal spores found in certain geographic regions
where there is a higher frequency of such fungi because of unique soil
and flora (Lacey, 1991; Pitt, 1979). Opportunistic fungal pathogens
have a great public health importance, especially in immune system
compromised individuals such as those with human immunodeficien-
cy virus (HIV) and organ transplants (Keller et al., 1999). These infec-
tions are not contagious, and the fungi are not obligatory pathogens.
Immunocompromised patients may be at an increased risk for oppor-
tunistic infections if opportunistic fungal pathogens become airborne
and their concentrations are significantly elevated in indoor air. The
major fungi causing mycosis and their medical significance are listed
in Table III.
Aspergillus fumigatus, A. flavus, and A. niger are among the fungi of
significant concern. Aspergillus fumigatus is the most important air-
borne pathogenic fungus (Brakhage and Langfelder, 2003) because of
its small respirable-size spores and its thermophilic nature (Klich and
Pitt, 1988). This is the very reason why A. fumigatus could cause a
significant problem in organ transplant wards in hospitals. Water dam-
aged materials, houseplants, soil, bird and bat droppings, organic
waste, or other organic substrates in buildings may be a source of these
TABLE III
PATHOGENIC FUNGI
Fungus Classification Disease Affected area
Absidia sp. Opportunistic Zygomycosis Face, sinuses,

Systemic (Mucormycosis, gastrointestinal
mycosis phycomycosis) tract, lungs
Cunninghamelia
sp.
Mortierella sp.
Mucor sp.
Rhizopus sp.
Syncephalastrum sp.
Basidobolus ranarum
Rhizomucor sp.
Conidiobolus
coronatus
Acremonium sp. Cutaneous Keratomycosis Eye
mycosis
Subcutaneous Maduromycetoma
mycosis
Opportunistic Systemic Lungs, deep tissue,
Systemic opportunistic body organs, blood
mycosis fungal disease
Alternaria sp. Opportunistic Systemic Lungs, deep tissue,
Systemic opportunistic body organs, blood
Arthrographis sp. Subcutaneous Dermatomycosis Skin
mycosis
Aspergillus Opportunistic Aspergillosis Lung, skin,
fumigatus Systemic mucocutaneous
mycosis tissue, any of the
body organs
Asp. flavus
Asp. niger
Asp. terreus
Asp. ustus
Aspergillus spp.
Aspergillus sp. Cutaneous Outer cutaneous Skin
mycosis mycoses
Onychomycosis Nails
Otomycosis Ear
Keratomycosis Eye
(continued)
44 LI AND YANG
TABLE III (Continued )
Aureobasidium Opportunistic Systemic Lungs, deep tissue,

pullulans Systemic opportunistic body organs, blood
Basidiobolus Rare Entomophthora Smooth skin
sp. subcutaneous basidiobolae
mycosis
Beauveria Opportunistic Systemic Lungs, deep tissue,
bassiana Systemic opportunistic body organs, blood
Blastomyces Systemic Blastomycosis Primary infection in
dermatitidis mycosis lung, may spread to
all organs, skin
lesions are common
Candida Cutaneous Intertriginous Moist skin areas: groin,
albicans mycosis candidosis glans penis, scrotum,
folds of buttocks,
under the breast,
axilla, interdigital
spaces
Candida diaper Diaper area
rash
Candidal Hands, feet, face, and
granuloma scalp
Candida Nails and skin around
paronychia and nail
onychomycosis
Mucocutaneoius Mucocutaneous areas
candidosis
Thrush Mouth and tongue
Perleche Corners of mouth
Vaginal Vagina
candidosis
Candida balinitis Glans penis
Esophageal Esophagus
candidosis
Perianal Anal ara
candidosis
Chronic
mucocutaneous
candidosis
Candida albicans, Cutaneous Onychomycosis Nails
Candida spp. mycosis
(continued)
Opportunistic Systemic Blood, heart tissue, kidney,

Systemic candidosis bladder, mucocutaneous
mycosis tissue (lungs are
colonized, but rarely
invaded)
Cutaneous Otomycosis Ear
mycosis
Candida sp. Cutaneous Keratomycosis Eye
mycosis
Cercospora Opportunistic Systemic Lungs, deep tissue,
apii Systemic Opportunistic body organs, blood
Chaetoconidium Opportunistic Systemic Lungs, deep tissue,
sp. Systemic Opportunistic body organs, blood
Chrysosporium Opportunistic Systemic Lungs, deep tissue,
parvum Systemic Opportunistic body organs, blood
Cladosporium Subcutaneous Chromomycosis Skin surface, mostly
carrionii mycosis lower extremities
Cladosporium Opportunistic Cerebral Brain or central
trichoides Systemic chromomycosis nervous system
mycosis
Coccidioides Systemic Coccidioido- Primary infection in the
immitis mycosis mycosis lung may spread to
other organs of the body;
skin lesion may be
produced
Coprinus sp. Miscellaneous Basidiomycosis
and rare
mycosis
Cryptococcus Systemic Cryptococcosis Lungs, central nervous
neoformans mycosis system, skin, any
organ of body
Curvularia Opportunistic Systemic Lungs, deep tissue,
geniculata Systemic Opportunistic body organs, blood
Drechslera Opportunistic
hawaiiensis Systemic
mycosis
Entomophthora Rare Entomophthoro- Nasal tissue and face
(conidiobolus) subcutaneous mycosis
coronata mycosis conididobolae
(continued)
46 LI AND YANG
Epidermophyton Cutaneous Tinea cruris Groin

floccosum mycosis
Tinea pedis Feet, interdigital
spaces, and soles
Tinea manuum Palms and fingers
Tinea unguium Nails
Epidermophyton Cutaneous Dermatomycoses Keratinized layers of
spp. mycosis body: skin, hair,
nails
Exophiala Subcutaneous Phaeomycotic Smooth skin
(Phialophora) mycosis Cyst
jeanselmei
Exophiala Subcutaneous Phaeomycotic Smooth skin
(Phialophora) mycosis Cyst
spinifera
Exophiala Subcutaneous Maduromycetoma
jeanselmei mycosis
Fonsecaea Subcutaneous Chromomycosis Skin surface, mostly
compactum mycosis lower extremities
Fonsecaea Opportunistic Cerebral Brain or central
pedrosoi Systemic chromomycosis nervous system
mycosis
Fonsecaea Subcutaneous Chromomycosis Skin surface, mostly
pedrosoi mycosis lower extremities
Fusarium sp. Opportunistic
Systemic
mycosis
Fusarium sp. Cutaneous Keratomycosis Eye
mycosis
Geotrichum Opportunistic
candidum Systemic
mycosis
Helmintho- Opportunistic
sporium sp. Systemic
mycosis
Hendersonula Subcutaneous Dermatomycosis Skin
sp. mycosis
Histoplasma Systemic Histoplasmosis Primary infection
capsulatum mycosis in lung
(continued)
(H. duboisii in Recticulorendothelial

Africa) system is invaded; bone
and kidney and other
organs, including the
skin, may be involved
Hortaea Superficial Tinea nigra Thick stratum corneum,
(Phaeoan- mycosis palms, and feet
nellomyces or
Exophiala)
werneckii
Loboa loboi Rare Lobomycosis Smooth skin
subcutaneous
mycosis
Malassezia Superficial Pityriasis
furfur infections versicolor
Microsporum Cutaneous Tinea capitis Scalp
audouinii mycosis
M. canis
Microsporum spp.
Microsporum Cutaneous Tinea corporis Smooth body skin
canis mycosis
M. gypseum
Microsporum spp.
Microsporum Cutaneous Tinea barbae Beard and coarse body
spp. mycosis hair
Tinea favosa Scalp, skin, and nails
Microsporum Cutaneous Dermatomycoses Keratinized layers of
spp. mycosis body: skin, hair, nails
Paecilomyces sp. Opportunistic
Systemic
mycosis
Paracoccidioides Systemic Paracoccidioido- Subclinical infection
brasiliensis mycosis mycosis in lung, mucous
membranes, and
skin are involved
Penicillium sp. Opportunistic
Systemic
mycosis
Pseudallescheria Subcutaneous Maduromycetoma
(Allescheria or mycosis
Petriellidium),
boydii
(continued)
48 LI AND YANG
Phialophora Opportunistic
parasitica Systemic
mycosis
Subcutaneous Phaeomycotic Smooth skin
mycosis Cyst
Phialophora Subcutaneous Phaeomycotic Smooth skin
repens mycosis Cyst
Phialophora Subcutaneous Phaeomycotic Smooth skin
richardsiae mycosis Cyst
Phialophora Subcutaneous Chromomycosis Skin surface, mostly
verrucosa mycosis lower extremities
Phoma Opportunistic
hibernica Systemic
mycosis
Phoma sp. Subcutaneous Phaeomycotic Smooth skin
mycosis Cyst
Piadraia hortae Superficial Black piedra Scalp and beard
mycosis
Pityrosporum Superficial Tinea Smooth body skin
orbiculare mycosis versicolor
Pseudallescheria Opportunistic
(Allescheria or Systemic
Petriellidium), mycosis
boydii
Pythium Miscellaneous Pythiosis
and rare
mycosis
Rhinosporidium Rare Rhinosporidiosis Nasal mucosa
seeberi subcutaneous
mycosis
Schizophyllum Miscellaneous Basidiomycosis
commune and rare
mycosis
Scopulariopsis Opportunistic
brevicaulis Systemic
mycosis
Scopulariopsis sp. Cutaneous Onychomycosis Nails
mycosis
Scytalidium sp. Subcutaneous Dermatomycosis Skin
mycosis
(continued)
Sporothrix Subcutaneous Sporotichosis Skin, primarily hands,

schenckii mycosis arms, and legs
Torulopsis Opportunistic
glabrata Systemic
mycosis
Trichophyton Cutaneous Tinea imbricate Smooth body skin
concentriucum mycosis
Trichophyton Cutaneous Tinea manuum Palms and fingers
rubrum mycosis
T. mentagrophyte
Trichophyton spp.
Tinea pedis Feet, interdigital
spaces, and soles
Tinea unguium Nails
Tinea cruris Groin
Tinea corporis Smooth body skin
Trichophyton Cutaneous Tinea favosa Scalp, skin, and nails
schoenleinii mycosis
Trichophyton spp.
Trichophyton Cutaneous Dermatomycoses Keratinized layers of
spp. mycosis body: skin, hair, nails
Trichophyton Cutaneous Tinea capitis Scalp
tonsurans mycosis
Trichophyton spp.
Trichophyton Cutaneous Tinea barbae Beard and coarse
verrucosum mycosis body hair
T. mentagrophytes
Trichophyton spp.
Trichosporon Superficial White piedra Beard, scalp,
beigelii mycosis pubic hair
Wangiella Opportunistic Cerebral Brain or central nervous
(Phialophora) Systemic chromomycosis system
dermatitidis mycosis
Subcutaneous Chromomycosis Skin surface, mostly
mycosis lower extremities
Maduromycetoma
Wangiella Superficial Tinea nigera Thick stratum corneum,
mansonii mycosis palms, and feet
Compiled from Campbell and Stewart (1980); Henry (1984); Howard (2003); Rippon (1988).
50 LI AND YANG
fungi (Benenson, 1990; Burge 1990a; Larsen and Frisvad, 1995).

These fungi can cause aspergillosis. In a hospital where an epidemic
of aspergillosis occurred, the source of Aspergillus spores was attrib-
uted to a defective disposal conduit door and the dispersal of a
contaminated aerosol from the ward vacuum cleaner, which had the
highest measured concentrations of Aspergillus fumigatus in or around
the building (65 colony forming units/m3 as compared with 0–6 cfu/m3
elsewhere). No further cases were identified in the hospital in
the 2 years after relevant hygiene arrangements were incorporated
(Anderson et al., 1996).
Other clinically important fungal infections include candidiasis
with local mucocutaneous or disseminated systemic organ manifesta-
tions and skin mycoses such as dermatophytoses, keratomycosis, tinea
nigra, piedra, and malassezia-caused dermatitis. Invasive fungal dis-
eases of the paranasal sinuses may also be associated with allergic
sinusitis in atopic patients (Fatterpekar, 1999). Aspergillus species
are frequently involved. Noninvasive forms may colonize body cavities
and may be asymptomatic as long as some degree of immunological
resistance can be maintained. Cryptococcus neoformans var. neofor-
mans was isolated from 20 (13%) dwellings out of 154 dwellings in the
metropolitan area of Rio de Janeiro, Brazil, comprising 5 (15.6%) of 32
dwellings of patients with AIDS-associated cryptococcosis (Passoni
et al., 1998).
Histoplasmosis is an intracellular mycotic infection of the reticuloen-
dothelial system caused by the inhalation of conidia from the fungus
Histoplasma capsulatum (Howard, 2003). Histoplasma capsulatum has
a worldwide distribution, but the Mississippi–Ohio River Valley in the
United States is a major endemic region, and the spore is occasionally
found in certain indoor environments there (Collier et al., 1998).
Coccidioides immitis causes coccidioiomycosis, a highly infectious
upper respiratory disease, and infection is caused by inhalation of its
airborne arthrospores (Howard, 2003). The disease is endemic in cer-
tain regions, mainly in desert soils and also in the air of endemic areas
in North America (Cox and Wathes, 1995). Exposure to dustborne
spores outdoors is the major risk factor of infection (Al-Doory and
Ramsey, 1987).
C. MYCOTOXINS AND THEIR SIGNIFICANCE TO HUMAN HEALTH

Another public health concern is mycotoxins produced by some
indoor fungi (Table IV). Fungi are capable of producing a number of
secondary metabolites (Nielsen, 2002). Most of these secondary
TABLE IV
COMMON MYCOTOXIGENIC INDOOR FUNGI
Fungus Mycotoxins*
Alternaria alternata Tenuazonic acid, alternatiol, alternatiol mononethyl ether,

altertoxins
Aspergillus flavus Aflatoxin B1
Aspergillus fumigatus Gliotoxin, verrucologen, fumitremorgceusins, fumitoxins,
tryptoquivalins
Aspergillus niger Naphthopyrone, malformins, nigragillin, orlandin
Aspergillus Ochratoxin A (a carcinogenic kidney toxin)
ochrrachceus
Aspergillus parasiticus Aflatoxin B1
Aspergillus versicolar Sterigmatocystin and methoxysterigmatocystin
Aspergillus ustus Austaminde, austdiol, austins, austocystins, kotanins X and Y
Chaetomium globosum Chaetoglobosins, chetomin
Cladosporium Cladosporin, emodin
cladosporioides
Emericella (Aspergillus) Sterigmatocystin, nudulotoxin
nidulans
Fusarium culmorum T-2 toxin (immunosuppressive)
Fusarium graminearum Zealralenone
Fusarium verticillioides Fumonisins
(¼ F. moniliforme)
Memnoniella Trichodermol, trichodermin, dechlorogrisseofulvins,
echinata memnobotrins A and B, memenoconol, memnoconone
Paecilomyces variotii Patulin, viriditoxin
Penicillium Auranthine, penicillic acid, verrucosidin,
aurantiogriseum nephrotoxic glycopeptides
Mycophenolic acid
Penicillium
brevcompactum
Penicillium Roquefortine C, meleagrin, chrysogin, penicillin
chrysogenum
Penicillium expansum Citrinin, patulin (nephrotoxic), cytotoxic metabolite
of unknown origin
Penicillium polonicum 3-methoxyviridicatin, verrucosidin, verrucofortine
Penicillium verrucosum Ochratoxin A (a carcinogenic kidney toxin)
Stachybotrys chartarum Macrocyclic trichothecenes: satratoxins, verrucarins,
(syn ¼ S. atra). roridins, atranones, dolabellanes, stachybotrylactones,
and lactams
Trichoderma harzianum Alamethicins, emodin, suzukacillin, trichodermin
Wallemia sebi Walleminols A and B, walleminone
*Toxins in boldface are of high potency. Compiled in part from Al-Doory and Domson, 1984; Frank
et al., 1999; Macher et al., 1999; Samson, 2000; St-Germain and Summerbell, 1996.
52 LI AND YANG
metabolites are mainly to enhance the fitness of the fungi in nature.

However, when some of these chemical compounds cause detrimental
or toxic response in higher vertebrates at low concentrations, they are
referred to as mycotoxins (Nielsen, 2002). Mycotoxicosis is defined as
the disease resulting from exposure to a mycotoxin (CAST, 2003).
Mycotoxicosis may be acute or chronic. More occult disease may
occur when the mycotoxin interferes with the immune system and
leads to a compromised immune system so as to make patients more
susceptible to infectious diseases. Major mycotoxicoses include afla-
toxicosis, ochratoxicosis, trichothecene toxicoses, citreviridin toxico-
sis, zearalenone toxicosis, fumonisin toxicosis, gliotoxin toxicosis, and
immunomodulation.
Mycotoxins’ detrimental effects on human health are at work
when they are ingested (CAST, 2003; Matossian, 1989), inhaled
(CAST, 2003; Croft et al., 1986; Johanning et al., 1993; Miller, 1993;
Smoragiewicz et al., 1993), or absorbed through skin contact (CAST,
2003; Dill et al., 1997; Singh, 1994). Historically, human exposure to
mycotoxins is mainly through ingestion of foodstuff containing or
contaminated with mycotoxins (CAST, 2003). However, because of
increases in public awareness of the health effects of indoor fungi,
inhalation of mycotoxin-containing spores of indoor fungi has become
a major public health concern in the indoor environment, and inges-
tion and dermal contact play a secondary role in indoor exposure.
There are reportedly more than 200 mycotoxins produced by various
common fungi, per the World Health Organization (WHO) Environ-
mental Health Criterion 105 on mycotoxins (Yang et al., 2002). Samson
(1992) and Smoragiewicz et al. (1993) suggested that there are more
than 400 toxic metabolites at present. The actual number of mycotoxins
is not known, but the number of fungal toxic metabolites could be
potentially in the thousands (CAST, 2003). With molecular masses
between 200 and 800 kDa (Smoragiewicz et al. 1993), mycotoxins are
not volatile at ambient temperatures (Tuomi et al. 2000). Schiefer
(1990) considered that mycotoxins generally have low volatility, and
therefore inhalation of volatile mycotoxins is not very likely. A task
group of WHO concluded that an association between trichothecene
exposure and human disease episodes is possible; however, only
limited data are available (Yang et al., 2002).
Major genera of toxigenic fungi include Aspergillus, Penicillium,
Fusarium, Stachybotrys, Memnoniella, and Claviceps (CAST, 2003).
There are other genera of mycotoxin-producing fungi. Species of 46
fungal genera have been reported to produce mycotoxins (Kendrick,
2000). Major classes of mycotoxins include aflatoxins, trichothecenes,
fumonisins, zearalenone, ochratoxin A, and ergot alkaloids
(CAST, 2003). Major toxigenic fungi and the mycotoxins produced,
as well as their health effects, are compiled in Tables V and VI. It
should be pointed out that some of the fungi in Table V are often found
indoors.
Mycotoxins are an integral part of the fungal spores or in association
with dust particles when released into the substrates. Water-damaged
building materials are often contaminated with fungi that produce
detectable levels of mycotoxins (Nikulin, 1999), which may be aero-
solized and contribute to pollution in indoor air. Sorenson et al. (1987)
showed that aerosolized conidia of S. chartarum (syn. S. atra)
TABLE V
FUNGI IMPLICATING SOME HUMAN DISEASES BECAUSE OF INVOLVEMENT OF THEIR MYCOTOXINS
Etiologic agent Disease Natural substrate
Fusarium spp. Akakabio-byo Wheat, barley, oats, rice

Fusarium spp. Alimentary toxic aleukia Cereal grains (toxic bread)
(ATA or septic angina)
Penicillium Balkan nephropathy Cereal grains
Aspergillus spp., Cardiac beriberi Rice
Penicillium spp.
Sclerotinia Celery harvester’s Celery (pink rot)
disease
Dendrodochium Dendrodochiotoxicosis Fodder (skin contact, inhaled
toxicum fodder particles)
Claviceps Ergotism Rye, cereal grains
purpurea
Fusarium Esophageal tumors Corn
moniliforme
Apergillus flavus, Hepatocarcinoma Cereal grains, peanuts
A. parasiticus (acute aflatoxicosis)
Fusarium Kashin Beck disease, Cereal grains
‘‘Urov disease’’
Aspergillus flavus, Kwashiorkor Cereal grains
A. parasiticus
Phoma sorghina Onyalai Millet
Aspergillus Reye’s syndrome Cereal grains
Satchybotrys Stachybotryotoxicosis Hay, cereal grains, fodder (skin
chartarum contact, inhaled haydust)
54 LI AND YANG
TABLE VI
MYCOTOXINS AND THEIR PATHOLOGICAL EFFECTS ON HUMANS AND ANIMALS
Mycotoxin Substrates Affected species Pathological effects
Aflatoxins Peanuts, corn, Birds Hepatotoxicity

(B1, B2, G1, wheat, rice, Duckling, (liver damage)
G2, M1, M2) cottonseed, turkey, poultry, Bile duct hyperplasia
copra, nuts, pheasant chick, Hemorrhage
various foods, mature chicken, Intestinal tract
milk, eggs, quail Kidneys
cheese, figs Mammals Carcinogenesis
Young pigs, (liver tumors)
pregnant sows,
dog, calf,
mature cattle,
sheep, cat,
monkey, human
Fish
Laboratory
animals
Citrinin Cereal grains Swine, dog, Nephrotoxicity (tubular
(wheat, barley, laboratory necrosis of kidney)
corn, rice) animals porcine nephropathy
Cyclopiazonic Corn, peanuts, Chicken, turkey, Muscle necrosis
acid cheese, swine, rat, Intestinal hemorrhage
kodo millet guinea pig, and edema
human Oral lesions
Ochratoxin A Cereal grains, Swine, dog, Nephrotoxicity (tubular
(wheat, barley, duckling, necrosis of kidney)
oats, corn), dry chicken, rat, Porcine nephropathy
beans, moldy human Mild liver damage
peanuts, cheese, Enteritis
grapes, dried Teratogenesis
fruits, wine Carcinogenesis
(kidney tumors)
Urinary tract tumors
Patulin Moldy feed, Birds Edema
rotted apples, Chicken, Brain
apple juice, chicken embryo, Lungs
wheat straw quail Hemorrhage
residue Mammals Lungs
Cat, cattle, Capillary damage
mouse, rabbit, Liver
rat, human
(continued)
TABLE VI (Continued )
Mycotoxin Substrates Affected species Pathological effects
Others Spleen
Brine shrimp, Kidney
guppie, zebra Paralysis of
Fish larvae motor nerves
Convulsions
Carcinogenesis
Antibiotic
Penicillic Stored corn, Mouse, rat, Liver damage (fatty liver,
acid cereal grains, chicken embryo, cell necrosis); kidney
dried beans, quail, brine damage; digitalis-like
moldy tobacco shrimp action on heart dilates
blood vessels;
antidiuretic edema in
rabbit skin;
carcinogenesis;
antibiotic
Penitrem Moldy cream Dog, mouse, Tremors, death,
cheese, English human icoordination, bloody
walnuts, diarrhea
hamburger
bun, beer
Sterigmatocystin Green coffee, Mouse, rat Carcinogenesis
moldy wheat, Hepatotoxin
grains,
hard cheeses,
peas, cottonseed
Trichothecenes Corn, wheat, Swine, cattle, Digestive disorders
(T-2 toxin, commercial chicken, turkey, (emesis, diarrhea,
diacetoxyscirpenol, cattle feed, horse, rat, dog, refusal to eat),
neosolaniol, mixed feed, mouse, cat, hemorrhage (stomach,
nivalenol, barley, oats human heart, intestines, lungs,
diacetylnivalenol, bladder, kidney), edema,
deoxynicalenol, oral lesions, dermatitis,
HT-2 toxin, blood disorders
fusarenon X) (leucopenia)
Zearalenone Corn, moldy hay, Swine, dairy Estrogenic effects
pelleted com- cattle, chicken, (edema of vulva,
mercial feed turkey, lamb, prolapse of vagina,
rat, mouse, enlargement of uterus)
guinea pig Atrophy of testicles
Atrophy of ovaries,
enlargement of
mammary glands
Abortion
Compiled from CAST (2003).

56 LI AND YANG
contained trichothecene mycotoxins in the laboratory. The most com-

mon toxin was satratoxin H. Lesser amounts of satratoxin G and tricho-
verrols A and B were also detected, but less frequently. They also found
that most of the airborne particles were within respirable range. Similar
experiments, conducted by Pasanen et al. (1993), demonstrated that
trichothecene mycotoxins were in airborne fungal propagules of S.
chartarum (S. atra) and could be collected on membrane filters. Con-
idia of A. flavus and A. parasiticus were reported to contain aflatoxins
(Wicklow and Shotwell, 1983). Miller (1993) also reported detection of
two mycotoxins, deoxynivalenol and T-2 toxin, in conidia of Fusarium
graminearum and F. sporotrichioides, respectively. These references
suggest that inhalation exposure to conidia may also increase the
chance of exposure to mycotoxins.
Studies indicated that some secondary metabolites of indoor air-
borne fungi could be responsible for health problems of occupants
(Croft et al. 1986; Pieckova, 2002). Croft et al. (1986) identified several
cases of mycotoxicoses caused by airborne exposure to the toxigenic
fungus S. chartarum (syn. S. atra) in a residential building. Spengler
et al. (1993) reported that a higher rate of upper respiratory tract and
lung cancer occurred among workers with a high risk of inhalation of
fungi in the grain and food handling industry.
Important indoor toxigenic fungi include Stachybotrys chartarum
(syn. S. atra), Memnoniella echinata, Aspergillus species, Penicillium
species, Fusarium species, Trichoderma species, and Paecilomyces
species. These fungi are well documented to have associations with
detrimental health effects in humans and animals by ingestion. How-
ever, many toxigenic fungi—such as Stachybotrys chartarum and spe-
cies of Aspergillus, Penicillium, and Fusarium—have been found to
infest buildings with known indoor air problems and sick building
syndrome (Croft et al., 1986; Flannigan et al., 1991; Johanning et al.,
1993).
It should be pointed out that most mycotoxin studies were con-
ducted on post-harvest stored or processed food. For better indoor
environmental quality evaluation, it is important to know whether
indoor fungi are able to produce mycotoxin in building materials or
not and under what conditions. Tuomi et al. (2000) analyzed 17 myco-
toxins from 79 bulk building materials collected from water-damaged
buildings. Their results showed sterigmatocystin was present in 24%
of the samples, trichothecenes in 19% of the samples, and citrinine in 3
samples. Aspergillus versicolor was found on most sterigmatocysin-
containing samples, and Stachybotrys spp. were found on the samples
in which satratoxins were present (Tuomi et al., 2000).
Nielsen (2002) showed that Stachybotrys chartarum produced a
number of mycotoxins on building materials at levels significantly
higher than these products by other fungi. More importantly, he dis-
covered that only 35% of the isolates of S. chartarum produced the
extremely cytotoxic satratoxins. He opined that satratoxins might not
be responsible for idiopathic pulmonary hemosiderosis (IDPH) in in-
fants and that this disease may be caused by other mycotoxins pro-
duced by S. chartarum (Nielsen, 2002). Similar results showed that
39% of S. chartarum produced macrocyclic trichothecenes (Andersen
et al., 2002). The toxicity of the isolates producing macrocyclic tri-
chothecenes is 1000 times that of other isolates, which produce atro-
nones (Jarvis 2003, per. com.). However, a recent study in Belgium
showed that in 6 IDPH cases, the isolates of S. chartarum recovered
from patients’ homes were all atronones producers (Nielsen, per. com.).
This association further raised the question whether other mycotoxins
and secondary metabolites are responsible for IDPH. To answer this
question there is no doubt that more research is necessary.
Aflatoxins are toxins discovered in 1961 from Aspergillus flavus and
A. parasiticus and considered human and animal carcinogens (CAST,
2003; International Agency for Research on Cancer, 1993). Aflatoxins
are potent liver toxins. A sublethal dose from exposure may result in
cancer (CAST, 2003). Aflatoxin-induced disease has been well docu-
mented and reviewed (Henry and Cole, 1993; International Agency for
Research on Cancer, 1993; Kurup, 1999). Aspergillus versicolor pro-
duced the mycotoxins sterigmatocystin and 5-methoxysterigmatocys-
tin, which are precursors of aflatoxins, in water-damaged materials
under field conditions and experimental conditions (Gravesen et al.,
1999; Nielsen, 2002). Trichothecene toxins inhibit protein and DNA
synthesis (CAST, 2003). The data of health effects on animals are
‘‘inadequate evidence’’ for humans (International Agency for Research
on Cancer, 1993). Macrocyclic trichothecenes, such as satratoxin H,
have not been classified. These toxins can cause alveolar macrophage
defects and may affect phagocytosis. They have been investigated for
use in cancer treatment (Goodwin et al., 1978) but also in chemical-
biological warfare.
In animal studies, all frequently isolated strains (Penicillium sp.,
Aspergillus versicolor, A. flavus, Cladosporium sphaerospermum,
and C. cladosporioides) in Slovakia produced secondary metabolites
with the strongest ciliostatic activity—their exo- and endometabolites
stopped tracheal ciliary movement in chicks for 24 h (Pieckova, 2002).
On building materials, Penicillium chrysogenum produced only few
detectable metabolites or frequently none (Nielsen, 2002).
58 LI AND YANG
Toxic metabolites from isolates of Trichoderma harzianum isolated

from the indoor environment of a building where the occupant was
suffering serious building-related ill-health symptoms damaged the
cell membrane barrier function of sperm cells (Peltola et al., 2001).
However, in Nielsen’s studies, Trichoderma spp. did not produce
detectable quantities of trichothecenes on building materials (Nielsen,
2002). Nielsen (2002) further showed that Chaetomium globosum pro-
duced high quantities of chaetoglobosins on building materials. Walle-
mia sebi, a common indoor xerophyllic fungus, was found to produce
the mycotoxins walleminol and walleminone (Frank et al., 1999).
However, reports on the biological effects of most secondary metabo-
lites are scarce, and very few of the studies evaluated the effects of
inhaled secondary metabolites (Nielsen, 2003).
Samson (1992) divided the adverse effects into four categories: acute,
chronic, mutagenic, and teratogenic. Symptoms related to mycotoxins
or toxin-containing spores (particularly those of S. chartarum) include
dermatitis; recurring cold and flu-like symptoms; burning sore throat;
headaches and excessive fatigue; diarrhea; and impaired or altered
immune function; as well as cough; irritation of eyes, skin, and respi-
ratory tract; or joint ache (Singh, 1994; Tuomi et al., 2000). Compro-
mised ability of the body to resist infectious diseases may lead to
opportunistic infections and possibly cancers. Certain mycotoxins,
such as zearalenone, have been found to cause infertility and stillbirths
in pigs (Matossian, 1989). Low-level, complex exposures from a mix-
ture of mycotoxins may have synergistic effects and may result in
central neuroendocrine-immune changes and consequently in com-
plex health reactions of the endocrine and nervous systems (Ammann,
1999). Residents or occupants who were exposed to toxigenic fungi in
water-damaged buildings might suffer from nonspecific symptoms
(Tuomi et al., 2000).
Although relationships were established to link inhalation exposure
to mycotoxin-containing fungal spores and symptoms of mycotoxicoses
in fungi-infested indoor environments (Croft et al., 1986; Johanning
et al., 1993), other possible exposure routes such as ingestion and
dermal contact are likely. Because fungal spores are ubiquitous in a
contaminated environment, the chance of ingesting toxin-containing
spores is likely to increase through eating, drinking, and smoking.
Concerns were raised that many of the data on exposures to toxigenic
molds were derived from animal toxicity studies, and these are based
primarily on ingestion (Assoulin-Daya et al., 2002). Whether these
results can be extrapolated to human health is questionable. In a review
article, Robbins et al. (2000) argued that although evidence was found
for a relationship between high levels of inhalation exposure or direct
contact to mycotoxin-containing molds or mycotoxins, and demonstra-
ble effects in animals and health effects in humans, the current litera-
ture does not provide compelling evidence that exposure at levels
expected in most mold-contaminated indoor environments is likely to
result in measurable health effects.
Novotny and Dixit (2000) reported that two fungi, Penicillium (pre-
sumptively Penicillium purpurogenum) and Trichoderma sp., were
cultured from surface samples collected in the residence where a 40-
day-old male suffered pulmonary hemorrhage following exposure to
indoor fungi and tobacco smoke. The authors thought the fungi to be
mycotoxin producers and tried to link the fungi to the pulmonary
hemorrhage. However, without properly identifying the fungi to spe-
cies, it is premature to assume the fungi were mycotoxin producers and
is difficult to establish the causal association between pulmonary hem-
orrhage, the fungi, and mycotoxins. The health effects of indoor molds
can be inconsistent; the presence of fungi was reported to be a signifi-
cant risk factor in Red Deer but not in Medicine Hat, Alberta, Canada
(Hessel et al., 2001).
Since mycotoxin production is species specific, it is crucial to inven-
tory all fungi growing in a contaminated indoor environment and
identify the fungi to species. Without knowing the fungal species that
are present, determination cannot be made whether the species are
toxigenic or not. In addition, the investigators will not be able to
ascertain whether species are able to produce mycotoxins under the
investigated conditions. Mycotoxin production can be influenced by
substrates (medium composition), temperature, water activity, and
other factors, and indoor fungi likely produce different mycotoxins
on building materials (Nielsen, 2003). Therefore it is very difficult to
evaluate the severity of indoor fungal and mycotoxin contamination.
D. VOLATILE ORGANIC COMPOUNDS (VOCS)

Actively growing fungi produce a variety of volatile organic com-
pounds (VOCs), which may produce a distinctive musty, moldy odor.
Fungal VOCs may include 3-methylbutan-1-ol, 3-methylbutan-2-ol,
fenchone, heptan-2-one, hexan-2-one, octan-3-one, octan-3-ol,
pentan-2-ol, alpha-terpineol, and thujopsene. They emit these com-
pounds into the indoor environment (Elke et al. 1999). The most preva-
lent compounds included xylene, toluene, 2-propanol, limonene, and
heptane. Formaldehyde concentrations ranging from 1.7 to 13.3 microg/
m3 and mean acetaldehyde levels ranging from <3.0 to 7.5 microg/m3
60 LI AND YANG
were reported (Reynolds et al. 2001). Larsen and Frivad (1995) studied
the in vitro production of fungal volatiles from 47 Penicillium taxa and
detected alcohols, ketones, esters, small alkenes, monterpenes, sesqui-
terpenes, and aromates. However, aldehydes were not among the VOCs
detected.
Fiedler et al. (2001) studied VOC production by Aspergillus fumiga-
tus, A. versicolor, A. niger, A. ochraceus, Trichoderma harzianum,
T. pseudokoningii, Penicillium brevicompactum, P. chrysogenum,
P. claviforme, P. expansum, Fusarium solani, and Mucor sp. More than
150 volatile substances derived from the fungal cultures have been
analyzed by head-space solid-phase microextraction (HS-SPME)
(Fiedler et al., 2001). Each species had a defined VOC profile, which
may be subject to considerable modification in response to external
factors such as cultivation on different substrata. Cultivation on differ-
ent substrata changes the number and concentration of VOCs (Fiedler
et al., 2001). Wilkins et al. (2000) studied the production of VOCs by
mold species isolated from damp buildings. They were grown on sterile
building materials and some synthetic media. Patterns of the volatile
organic compounds were very media dependent, but media, which
favor terpene biosynthesis, may give patterns unique enough for identi-
fication of dominant indoor molds (Wilkins et al., 2000). It was proposed
that species-specific volatiles may serve as marker compounds for the
selective detection of fungal species in indoor environments (Fiedler
et al., 2001). Examination of VOCs from indoor air samples may become
an important method in indoor air hygiene for the detection of type and
intensity of masked contamination by molds (Fiedler et al., 2001). Ad-
ditional fungal VOCs are compiled and listed by Ammann (2) and
Batterman (8). Almost all of the published information regarding fungal
VOCs concerns species of Penicillium and Aspergillus.
Some of the fungal VOCs have an unpleasant odor (Gravesen et al.,
1994). The musty, moldy, and earthy odors are likely to come from 2-
octen-1-ol and geosmin (1, 10-dimethyl-9 decalol) (Flannigan et al.,
1991). Ezeonu et al. (1994) identified ethanol, 2-ethyl hexanol, cyclo-
hexane, and benzene from fiberglass air duct liners colonized by As-
pergillus versicolor, Acremonium obclavatum, and Cladosporium
herbarum. Acetone and 2-butanone were only detected on agar plate
samples of A. versicolor and A. obclavatum. The 2-ethyl hexanol and
cyclohexane are eye and skin irritants, and benzene is a generally
recognized hazardous chemical. Other fungal VOCs associated
with two common indoor fungi, Penicillium and Aspergillus, have
been identified. They are 2-methyl-isoborneol, 2-methyl-1-propanol,
3-methyl-1-butanol, and 3-octanone (Gravesen et al., 1994).
Ahearn et al. (1997) found that lower concentrations of volatile
organics were released from air filter medium colonized by fungi than
noncolonized filter medium in a water-damaged office building. How-
ever, the volatiles from the colonized filter medium included fungal
metabolites such as acetone and a carbonyl sulfide-like compound that
were not present and released from noncolonized filter medium. They
suggested that the growth of fungi in air distribution systems may affect
the content of volatile organics in indoor air (Ahearn et al., 1997).
Fungal VOC levels indoors are normally low. Indoor concentrations
of total volatile organic compounds (TVOCs) were reported ranging
from 73 to 235 microg/m3 (Reynolds et al., 2001). However, microbial
volatile organic compounds (MVOCs) and metabolites of fungi de-
tected in indoor molds are considered to be a potential health hazard
(Kreja and Seidel, 2002). Their toxicological relevance and health ef-
fect, however, is largely unknown, and data are rare (Kreja and Seidel,
2002).
Although VOCs produced by Aspergillus, Penicillium, and other
fungi have been investigated extensively, little information exists on
what VOCs can be produced by S. chartarum (Gao and Martin, 2002).
Four unique VOCs—1-butanol, 3-methyl-1-butanol, 3-methyl-2-buta-
nol, and thujopsene—were detected on rice cultures of S. chartarum,
and only one of them (1-butanol) was detected on gypsum board
cultures (Gao and Martin, 2002). For a given strain, VOCs were consid-
erably different with different cultivation media (Gao and Martin,
2002). Concentration profiles of the volatile compounds varied among
compounds; however, each compound exhibited corresponding con-
centration trends between the strains (Gao and Martin, 2002). In com-
parison with their previous studies of five Aspergillus species on
gypsum board under the same experimental conditions, fewer unique
VOCs were produced by S. chartarum, and they were significantly
different. Gao and Martin (2002) indicated that VOCs produced by S.
chartarum may represent a relatively small portion of the total volatiles
present in problematic buildings where Aspergillus spp., Penicillium
spp., and other fungi frequently coexist (Gao and Martin, 2002).
Elke et al. (1999) described a new, analytically valid procedure to
assess the exposure of humans to the so-called microbial volatile or-
ganic compounds (MVOCs). The method can be used routinely for
large sample numbers and is especially valuable as a basis for further
research on the correlation between single MVOCs and indoor mold
growth (Elke et al., 1999). With the exception of 3-methylbutan-2-ol,
fenchone, nonan-2-one, octan-2-one, and thujopsene, indoor air con-
centrations of all MVOCs under investigation were significantly higher
62 LI AND YANG
inside damp and moldy dwellings. It was found that 3-methylbutan-1-

ol, hexan-2-one, heptan-2-one, and octan-3-ol were found to be most
reliable indicators for mold growth (Elke et al., 1999).
A correlation was also found between selected VOCs and the occur-
rence of mold species in mattress dust (Elke et al., 1999). Aspergillus
sp. correlated with heptan-2-one, hexan-2-one, octan-3-ol, octan-3-one,
and alpha-terpineol, while the occurrence of Eurotium sp. was
correlated with higher indoor air concentrations of 3-methylbutan-1-
ol, 3-methylbutan-2-ol, heptan-2-one, hexan-2-one, octan-3-ol, and
thujopsene (Elke et al., 1999). However, the correlation raised the
question whether mold species in mattress dust were spores or active
growth. Spores are metabolically slow or inactive and are less likely to
produce VOCs.
Indoor fungal VOCs have been suggested as possible contributors to
adverse health effects. Symptoms related to exposure to fungal VOCs
include nasal irritation and feelings of stuffiness (Flannigan and Miller,
1994). Possibly related mucous membrane and olfactory irritations
may trigger an ‘‘unpleasant odor reaction’’ and annoyance (Yang and
Johanning, 2002). Although exposure to molds can produce significant
mucosal irritation, there are very few data to suggest long-term ill
effects. More importantly, there is no evidence in humans that mold
exposure leads to nonmucosal pathology (Assoulin-Daya et al., 2002).
In a recent study, MVOC-induced DNA damage was observed under
conditions in which cytotoxic effects were observed but clastogenic
and mutagenic effects could not be detected (Kreja and Seidel, 2002).
E. GLUCANS
The polyglucose (1 ! 3)--D-glucan is a component of cell walls of
fungi, some bacteria, and plants (Rylander and Lin, 2000). (1 ! 3)--D-
Glucan has been recognized as a potential proinflammatory agent re-
sponsible for bioaerosol-induced respiratory symptoms observed in
both indoor and occupational environments (Gehring et al., 2001;
Milton et al., 2001; Rylander and Lin, 2000). Relationships between
the amount of (1 ! 3)--D-glucan and the extent of symptoms, lung
function changes, and inflammatory markers have been described. In
addition, (1 ! 3)--D-glucan can be used as a surrogate for measuring
mold biomass in field studies (Rylander and Lin, 2000).
Milton et al. (2001) developed a specific enzyme immunoassay to
quantify (1 ! 6) branched, (1 ! 3)--D-glucans in environmental
samples. The assay was highly specific for (1 ! 6) branched, (1 ! 3)-
-D-glucans and did not show any response at 200 ng/ml to curdlan,
laminarin, pustulan, dextran, mannan, carboxymethyl cellulose, and
endotoxins (Milton et al., 2001). The detection level was 0.8 ng/ml for
baker’s yeast glucan and Betafectin. A coefficient of variation of 7.8%
was obtained for (1 ! 3)--D-glucans in house dust samples. It will be
useful for the investigation of health effects from exposure to this class
of biologically active molecules (Milton et al., 2001).
Concentrations ranged from below the limit of detection to
19,013 microg/m2 (22,588 /g dust) from living room floors of 395
homes of two German cities, Erfurt and Hamburg (Gehring et al.,
2001). Associations between (1 ! 3)--D-glucan, housing characteris-
tics, and occupant behavior were found for concentrations per square
meter but not for concentrations per gram of dust (Gehring et al., 2001).
The following characteristics were associated with a significant in-
crease in beta (1 ! 3)--D-glucan levels: carpets in the living room,
keeping a dog inside, use of the home by four or more persons, use of
the living room for >180 hr/week, lower frequency of vacuum cleaning
and dust cleaning, and presence of mold spots during the past 12
months (Gehring et al., 2001).
III. Indoor Fungi

A. FUNGAL IDENTIFICATION
The modern concept of the Kingdom Fungi consists of four phyla,
Chytridiomycota, Zygomycota, Ascomycota, and Basidiomycota. In
addition, many fungi are conveniently placed in the form group deu-
teromycetes. Deuteromycetes include fungi that grow vegetatively and
may reproduce by producing asexual spores. Species of Zygomycotina,
Ascomycotina, Basidiomycotina, deuteromycetes, and myxomycetes
have all been reported from the indoor environment or identified from
indoor samples by the authors. Many important fungal traits, such as
ecological preferences, the production of mycotoxins, secondary meta-
bolites, VOCs, and the associated health effects, are species-specific. It
is therefore extremely important that fungi be accurately identified to
species. Miller (1991) stressed the importance of reliable fungal identi-
fication no matter what the identifications are used for.
Fungal classification and identification are based on morphological,
biological, molecular, genetic, and physiological characteristics.
Morphological characteristics, both macroscopic and microscopic, are
conventionally the most important in fungal classification and identi-
fication. However, there are situations in which morphological char-
acteristics cannot differentiate similar or closely related species in
large fungal genera such as Penicillium or Aspergillus. Pitt (1979) and
64 LI AND YANG
Klich and Pitt (1988) incorporated physiological and other characters

to facilitate speciation of Penicillium and Aspergillus. With the ad-
vances in molecular and genetic studies, different approaches have
been tried. However, their practical uses have been limited and the
current fungal identification is still morphology-based.
B. AIRBORNE FUNGI
Airborne fungal spores can originate from outdoor as well as indoor
sources. Many studies have been conducted for indoor and outdoor
airborne fungal spores in various parts of the world. In a well-built and
properly maintained building without a history of water damage, its
airborne fungal spores are likely from outdoor sources and should
reflect outdoor spora qualitatively and quantitatively. Only if a build-
ing experiences a significant water damage problem and subsequent
fungal growth do its airborne spores become a significant factor. The
main health concern of indoor fungal growth is the exposure of occu-
pants to airborne fungal spores and other byproducts of fungal growth
from indoors.
A number of air samplers are available for airborne fungi analysis.
However, two types of them are widely used: (a) spore trap type for
total spore count; (b) sieve to agar type (cultural method) for examina-
tion of colonies. The first involves collecting fungal spores and frag-
ments on sticky surfaces applied to a glass slide for direct fungal spore
identification under microscopes. Results are expressed as fungal
spores/m3. This yields information of total airborne fungal structures
including both viable and inviable. Such information is important for
any allergen-related health concerns, since all fungal spores and frag-
ments can be allergenic no matter whether they are viable or inviable
(including some non-sporulated ones on cultural media, such as most
basidiomycetes, obligate phytoplane fungi). However, most fungal
spores can be identified to only genus level. The second method in-
volves collecting fungal spores on growing media. After a 7-day incu-
bation, fungi are identified from colonies developed on the media and
enumerated as colony forming units (CFU)/m3. The latter one, the
culturable method, produces information very useful for assessment
of pathogenic or mycotoxingenic fungi and their health effects. This
method may not be able to quantify and characterize nonviable and
non-sporulated fungi accurately, but the fungi developed on media can
be identified to species level. The information of fungal species is
essential for assessing the health effects of fungal infestation indoors,
since pathogenicity and mycotoxingenicity are species-specific.
Molds growing indoors have been understood for some time as a
source of aeroallergens (Pope et al., 1993; Reed, 1985). Some fungi
may cause infection or allergy, depending primarily on the susceptibil-
ity of the hosts (Morey and Feeley, 1990). Outdoor sources of spores are
generally considered to be the major contributor to the indoor air spora
(Flannigan et al., 1991; Li and Kendrick, 1995a). Most of the fungi that
contribute significantly to indoor airborne spores reproduce primarily
by asexual spores (Burge, 1990a), although Chaetomium globosum,
which produces ascospores, is a common contaminant of water-
damaged paper and wood products. The indoor air environment may
be considered a walled-in portion of the outdoor. It differs in patterns
of air movement, humidity, temperature, and possibly also in gas
composition. Air movement results from ventilation, convection, heat-
ing system, cleaning activities, and movement by occupants, but air
does not move as continually—or as rapidly—over a surface as it does
outdoors. Outside air movement influences movement indoors by forc-
ing air through cracks on the windward side and sucking it out on the
leeward, with the direction of airflow changing as the wind direction
changes (Lacey, 1981). Artificial ventilation can rapidly circulate
spores through a building, but convection can also be very effective,
carrying spores from first- to fourth-floor halls within 5 min and into
rooms within 20 min (Christensen, 1950).
During summer in North America, counts of airborne spores out-
doors and indoors may roughly correspond when windows are open
(Snelly and Roby, 1979). It has also been well known for many years
that fungi readily invade and propagate in the interior of homes, and
that perennial allergic symptoms can be attributed to high concentra-
tions of such spores (Salvaggio, 1986).
Species of Cladosporium, Alternaria, Mucor, Aspergillus, and Peni-
cillium, among others, are common and are capable of reproducing
indoors when appropriate substrates and moisture are available
(Morring et al., 1983). Ten Cladosporium species, some of which are
potentially allergenic, have been isolated inside houses in Córdoba,
Spain. Only small differences were recorded among the spora in the
different rooms (Infante-Garcia-Pantaleton and Dominguez-Vilches,
1988). The genus Rhizopus was isolated mostly indoors in Barcelona,
Spain (Calvo et al., 1980). Cooley et al. (1998) reported their studies in
48 schools in the United States that the fungal genera comprised over
95% of the outdoor fungi: Cladosporium (81.5%), Penicillium (5.2%),
Chrysosporium (4.9%), Alternaria (2.8%), and Aspergillus (1.1%). At
20 schools, significantly higher concentrations of Penicillium species
were found in the air samples from complaint areas than from
66 LI AND YANG
noncomplaint areas in the schools. Ren et al. (1999) found that in the
United States, Cladosporium spp. was dominant in both indoor and
outdoor air in summer, while Penicillium and Aspergillus were domi-
nant in indoor air in winter. The dominant airborne fungal spores
indoors in Southern Ontario, Canada, were Cladosporium (38.8%),
Aspergillus/Penicillium (19.8%), Leptosphaeria (7.9%), unidentified
basidiospores (6.5%), unidentified ascospores (2.8%), Ganoderma
(2.6%), Alternaria (1.9%), Coprinus (1.8%), and Epicoccum (3.3%)
(Li and Kendrick, 1995a).
Concentrations and compositions of airborne fungal spores are de-
termined by many factors and variations. There are regional, yearly,
seasonal, and diurnal variations of airborne fungal spore populations
(Li and Kendrick, 1994). Chao et al. (2002b) showed that fungal spore
concentrations in large office buildings varied significantly with sea-
son, with a summer peak, and that concentrations of airborne fungi
were positively correlated with RH and negatively with CO2 concen-
trations. The seasonal patterns of indoor fungi are closely correlated
with outdoor fungi in residential buildings (Li and Kendrick, 1995a).
Vegetation, landscape, land usage, meteorological factors, and other
environmental factors, as well as biological characters of fungi, deter-
mine the concentration and composition of airborne fungal spores (Li
and Kendrick, 1994). These factors ultimately influence the airborne
fungi indoors. Better understanding of these variations or patterns and
other important factors is crucial for proper sampling strategies and
sample collection.
At present there are insufficient research data of dose/exposure level
and response relationship to establish practical thresholds for making
public health decisions. Although such thresholds are very important
for indoor environmental quality evaluation and investigation, it is
highly unlikely that a dose-response relationship can easily be estab-
lished because of the complexity of fungal compositions and related
allergens and secondary metabolites.
In 7 homes of patients with asthma bronchiale, the concentrations of
mesophilic fungal spores of the indoor air ranged from 100 to 1000
CFU/m3, and this was much higher than the mold counts simulta-
neously collected outdoors (Senkpiel, 1996). The major fungal species
found indoors by the investigator were Penicillium sp. > Aspergillus
sp. > Cladosporium sp., Mucor sp., Chrysonilia sp., Verticillium sp. >
Geotrichum sp., Trichoderma sp. (Senkpiel, 1996). The main cause of
fungal contamination was moist building materials on/in room walls,
insufficient air ventilation, poor maintenance of the circulating air
machines, and insufficient room hygiene (e.g., biological garbage in
the kitchen) (Senkpiel, 1996). However, the reliability of the fungal
identifications is questionable. All fungi were identified to genera. In
addition, some unusual indoor fungi, such as Chrysonilia sp. and
Geotrichum sp., were reported. Although Chrysonilia sp. has been
reportedly isolated from indoors (Samson et al., 2000), it is highly
uncommon. Geotrichum sp. typically produces slimy colonies. In fact,
it is so unusual in the air that Haugland and Vesper (2001) used G.
candidum to spike samples for quality control purposes for their Quan-
titative Polymerase Chain Reaction (Q-PCR) studies. Their detection
and identification of such fungi in the air are highly unusual unless the
fungi were misidentified. The primary characters for the identification
of Geotrichum species are their production of arthrospores. Many air-
borne fungi produce arthrospores in culture and are likely mis-identi-
fied as Geotrichum species.
The fungal spore concentrations and compositions in indoor and
outdoor air in Yokohama, Japan, were sampled and analyzed with a
Reuter centrifugal sampler (RCS) and dichloran 18% glycerol agar
(DG18) and compared with the levels assessed with potato dextrose
agar (PDA) (Takahashi, 1997). In indoor air, the fungal concentrations
were <13 to 3750 CFU/m3. Cladosporium spp. predominated, followed
by the xerophilic fungi such as the Aspergillus restrictus group,
Wallemia sebi, the A. glaucus group, and Penicillium spp. The fungal
concentrations in indoor air peaked in October. The concentrations of
fungi were significantly correlated with the indoor temperature, indoor
relative humidity, and the outdoor climatic factors, except for the
average velocity of wind (Takahashi, 1997).
Studies of airborne fungi employed several different media and sam-
plers. However, there are insufficient comparative studies to determine
their efficiencies and differences. A number of studies were conducted
within a short period of time (less than 1 year). Since airborne fungal
spores have distinct seasonality and year-to-year variations, studies for
less than 1 year may not be able to yield meaningful information for the
studied area.
Khan et al. (1999) collected air samples on rose-bengal medium for
20 minutes by using a six-stage Andersen sampler. Aspergillus spp.
were the predominant component (27.7%) of the outdoor aerospora of
Kuwait, and A. fumigatus alone accounted for 21.3% of the total
aspergilli (Khan et al., 1999). Cladosporium spp. were the major com-
ponent of airborne fungal spores indoors (22.8%), followed by Asper-
gillus (20.9%), Penicillium spp. (14.6%), and Bipolaris spp. (10.6%)
(Khan et al., 1999). Ismail et al. (1999) used the settle plate method
with Czapek-Dox agar in Uganda from March through June 1998. The
68 LI AND YANG
most prevalent fungi outdoors and indoors were Mycosphaerella,

yeasts, Penicillium, Fusarium, Aspergillus, Cochliobolus, and Alternar-
ia (Ismail et al., 1999). In Poland the concentrations of airborne fungi in
dwellings without mold problems were between 0 and 1997 CFU/m3,
while in moldy homes they were 49 and 16,968 CFU/m3, respectively.
Dominant fungi were Penicillium spp., Aspergillus spp., and yeasts.
There were as many as 167 microbial species isolated from the air of
examined dwellings by Gorny and Dutkiewicz (2002). A study of air-
borne fungi in bedrooms in 485 houses was performed over 1 year in
Melbourne, Australia. Fifty-five percent of the houses had viable fungal
propagules exceeding 500 CFU/m3, and Cladosporium and Penicillium
were identified as the most prevalent and abundant fungal genera in
indoor air (Dharmage et al., 1999). Klanova (2000) reported the total
concentrations of airborne fungi were much higher in moldy rooms than
in the reference rooms, but health complaints did not correlate with the
total concentrations of airborne fungi. All occupants of rooms where the
average concentration was 2476 CFU/m3 reported health complaints.
Sometimes samplings of airborne fungal spores may not reflect the
fungal problems indoors. Hyvarinen et al. (2001) found that some
fungal genera detected in moist building materials such as Ulocladium
and Chaetophoma were not found in indoor air. This result showed
that bulk samples of building materials provide additional mycota
information in the building (Hyvarinen et al., 2001).
Vujanovic et al. (2001) proposed that airborne fungi could be classi-
fied on the basis of the relationship between the two environmental
factors and their combinations (i.e., temperature and water require-
ments [water activity, aw]). Three different groups are proposed: (i),
represented by Emericella (Aspergillus) nidulans, A. niger, and A.
ochraceus, and characterized by sporulation that was more dependent
on temperature than on water activity; (ii), represented by A. flavus and
A. versicolor, in which sporulation was approximately equal and de-
pended on both the temperature changes and aw alterations; and (iii),
represented by Cladosporium sp., Penicillium cyclopium, and P. citri-
num, in which sporulation depended more on alteration of the aw
conditions than on temperature changes. Temperature and aw for each
of the three phases of fungal growth (i.e., germination, growth, and
sporulation) could be important for the determination of the funda-
mental niche of each fungus and its ability to form or accumulate
mycotoxins (Vujanovic et al., 2001).
Recently Gorny et al. (2002b) reported their findings of the study of
airborne fungal fragments. The study found that small fungal fragments
of Aspergillus versicolor, Penicillium melinii, and Cladosporium
cladosporioides were released into the air simultaneously with conidia
from agar and ceiling tile surfaces (Gorny et al., 2002b). However, the
results clearly showed that the release mechanisms for fungal frag-
ments and conidia are different. The release of fungal propagules
depended on the fungal species, the air velocity above the contami-
nated surface, and the texture and vibration of the substrates (Gorny
et al., 2002b). Gorny et al. (2002b) showed that fragments and conidia
of Aspergillus versicolor and Penicillium melinii shared common anti-
gens by using enzyme-linked immunosorbent assays with monoclonal
antibodies. This study clearly showed the potential biological rele-
vance of airborne fungal fragments. The presence of airborne fungal
fragments at the clearance stage of any remediation in mold-infested
buildings should not be overlooked or underestimated.
Shelton et al. (2002) evaluated 12,026 fungal air samples (9619 in-
door samples and 2407 outdoor samples) collected from 1717 buildings
located across the United States by using Andersen N6 single stage
samplers. The culturable airborne fungal concentrations in indoor air
were lower than those in outdoor air. However, Stachybotrys chartar-
um was identified in the air in 6% of the buildings studied and in 1%
of the outdoor samples. The fungal levels were highest in the fall and
summer and lowest in the winter and spring. Geographically, the high-
est fungal levels were found in the Southwest, Far West, and Southeast
(Shelton et al., 2002). However, the reliability of fungal identification
and sampling quality control of such a large project must be scrutinized
before the results and conclusions are accepted.
Viability and culturability of airborne fungi are influenced by many
factors. Environmental factors are the predominant ones. However
sampling methods, devices, and time may have significant effects on
the viability of airborne fungi. The duration of air sampling when using
Andersen, SAS, and RCS samplers varies from 1 to 10 min. Since most
airborne fungi have well defined diurnal patterns (Li and Kendrick,
1995b) and such a short sampling time or ‘‘snap shot’’ for airborne
fungi, an investigation may miss the peak periods of airborne fungi.
This kind of air sampling does not fully reflect the exposure of occu-
pants or workers to airborne fungi. This is the reason why filtration at
composting facilities is a preferred method for a long duration of
sampling for air fungal spores. In a recent study Durand et al. (2002)
showed that increased sampling time (up to 6 h) did not affect the
viability of airborne fungi collected on polycarbonate filters at 2 l/min.
At present there are a number of samplers available on the market for
collecting culturable spores (Andersen, Burkard, SAS, RCS, AGI-30,
Biosampler, etc.) and for trapping spores and hyphal fragments
70 LI AND YANG
(Burkard, Andersen, Air-O-Cell, Micro-5, Cyclex-D, Allergenco, Biosys-

tem, AGI-30, BioSampler impingers, etc.) as well as ones used for both
culturable and total spore collection purposes (bi-cassette, Button
Sampler, polycarbonate filter, etc.). Several of these are new products.
Further evaluation and validation of the new products are necessary.
Comparative interpretation of the results collected with different sam-
plers is impossible, since there is no standard sampling and analytical
protocol, and different samplers have different collection characteris-
tics. This is one more reason why determination of the exposure limits
or thresholds of airborne fungi is difficult to impossible.
Gorny et al. (2002a) found that vibration of 1Hz/14W releases the
highest number of fungal progagules of Aspergillus versicolor, Penicilli-
um melinii, and Cladosporium cladosporioides into the air. Kildesø et al.
(2002) found that release of conidia of P. chrysogenum and Trichoderma
harzianum from wet gypsum board was not significantly influenced by
repeated air disturbance on fungal growth. Penicillium chrysogenum
reached maximal sporulation at 18 to 23 days on wet gypsum board,
while T. harzianum, around 20 days (Kildesø et al., 2002).
Several published studies have dealt with the adverse effects of
airborne fungal spores indoors as related to residential characteristics
such as presence of basement, stove, carpets, humidifier, and heating
systems (Agrawak et al., 1988; Su et al., 1992). Specific indoor envir-
onments may have unique conditions that allow fungal growth to
occur. Fungal species seem to develop preferentially in kitchens, fol-
lowed by bathrooms. They occur less frequently in bedrooms, probably
as a result of the lower humidity prevailing in these rooms (Infante-
Garcia-Pantaleton and Dominguez-Vilches, 1988). Recorded spore con-
centrations in the air of some mold-affected houses during winter were
equal to or greater than those expected outside in summer (Flannigan
et al., 1991). The predominance of these fungi in the indoor atmo-
sphere has been attributed to their ability to grow on many household
items such as foods, damp leather goods, paper and cotton fabrics, and
almost any chronically damp surfaces (Vittal and Glory, 1985). Among
indoor microhabitats known to favor mold growth are garbage contain-
ers, food storage areas, upholstery, wallpaper, house plants, books,
papers, and areas of moisture such as damp basements, walls, ceilings,
shower curtains, window moldings, air conditioners, humidifiers, and
vaporizers (Morring et al., 1983; Salvaggio and Aukrust, 1981).
Observations in one climate do not necessarily apply to other cli-
mates. Indoor molds may be more important in humid climates. Occa-
sionally, in bathrooms or basements, a persistent damp area may
support enough mold growth in otherwise dry houses to liberate
sufficient spores to cause disease (Reed, 1985). Li and Kendrick (1995a,c,
1996) used CANOCO and path analysis to show that most indoor fungi
originated from outdoor sources, and both diurnal and seasonal patterns
clearly showed the close correlations of airborne fungi indoors and
outdoors. Different fungi possess different diurnal and seasonal pat-
terns, and the diurnal patterns of ascomycetes and basidiomycetes are
very different from hyphomycetes (Li and Kendrick, 1995a,b).
The significance of the diurnal and seasonal patterns in the evalua-
tion of indoor airborne fungal spores has long been overlooked. Better
understanding of the seasonal and diurnal patterns can help indivi-
duals who are doing mold exposure investigation to better understand
population dynamics of airborne fungi at a specific time, location, and
season and can assist individuals to determine the specific sampling
strategy accordingly.
Compared with outdoor air, much less research has been done on
indoor airborne fungal spores. It is partially due to the difficulty of
obtaining access to suitable residences and patients to carry out a long-
term study. In addition, there are many variables involved, such as the
structure of the houses, the furniture, upholstery, ventilation and heat-
ing systems, and the cultural background and activities of residents.
All these variables make it very difficult to design an experiment that
accounts for all important factors and the interactions among them.
Furthermore, the indoor niche is suitable for certain fungi to grow year
around, which could blur the exposure/symptom relationship.
Four recent impactor air samplers were selected for study: Samplair
(AES, Combourg, France); Air Test Omega (LCB, France); Air Samplair
Mas-100 (Merck, France); and BioImpactor 100–08 (AES) (Nesa et al.,
2001). They were compared with one another at three different hospital
sites with varying levels of air contamination. No significant difference
in the efficiency of spore recovery was found between Air Test Omega,
Mas-100, and BioImpactor, whereas Samplair was significantly less
efficient (Nesa et al., 2001). BioImpactor was then selected to represent
the three superior impactors and was compared with the single-stage
Andersen disposable sampler, the Collectron MD8 air sampler (Sarto-
rius, France), and the High Flow Air Sample (BioTest, France), which
are based on filtration and centrifugation methods, respectively. No
significant difference was observed in terms of spore recovery (Nesa
et al., 2001). On the basis of their performance, unit sampling cost,
autonomy, and simplicity of use, the authors concluded that Air Test
Omega, Air Samplair Mas-100, and BioImpactor 100–08 are suitable for
routine indoor evaluation of fungal contamination of air in hospitals
(Nesa et al., 2001).
72 LI AND YANG
Most research so far has been conducted with viable and culturable
methods (which enumerate and identify fungal colonies) and with a
variety of samplers because no standard method and instrument have
been established. It is therefore very difficult to compare the results
obtained with different protocols in a meaningful way. Research with
sampling for culturable propagules tends to seriously underestimate
actual spore levels. Airborne fungal spores may be viable, dormant,
moribound, or dead. Burge (1986) believed that viable spores are highly
selective in their cultural requirements. However, most fungi and fun-
gal spores identified in various studies mentioned in the text are ready
to grow on common fungal media unless they are dead or dormant.
Some fungi, such as certain ascomycetes and basidiomycetes, are diffi-
cult to culture on the media normally employed. In any case, spores do
not need to be viable to cause allergies. Some very important allergenic
species would, therefore, have been ignored by most studies based on
culturable methods. In the last several years several new spore-
trapping samplers were developed in addition to Burkard, Allergenco,
and Rotorod. The Burkard sampler, Allergenco, Air-O-Cell, Laro, and
Cyclex-D samplers, etc., overcome the shortcomings of the culturable
methods by permitting visual counting and identification of spores
trapped on adhesive slides or tapes. However, there is a deficiency
with these spore-trapping samplers—namely, that some fungal spores
can only be identified to the generic or group level unless they are
cultured from the spores. This problem points out the difficulty of
identifying many similar-looking spores to the generic or species level
even by highly experienced mycologists and the need to develop broad
spectrum culture techniques or media. To a large extent, both problems
remain unsolved.
C. FUNGI GROWING ON INDOOR/BUILDING MATERIALS

The detection of airborne fungi does not necessarily indicate growth
or amplification of fungi indoors. However, it is generally believed that
actively growing fungi indoors are the principal cause of the adverse
health effects because of constant exposure to indoor sources of fungal
allergens, mycotoxins, glucan, and fungal VOCs. Needless to say, it is
important to characterize indoor fungi and at the same time to identify
growth sites of fungi indoors.
Many common indoor fungi are strong deteriorating agents and have
been reported from various building materials and systems. Many
species of the genus Penicillium, commonly detected in indoor air
sampling, have frequently been referred to as food spoilage and
bio-deteriorating agents (Gravesen et al., 1994; Pitt, 1979). Pasanen et al.
(1992) demonstrated that Penicillium was the most common mesophi-
lic fungal genus in all the building materials studied (wallpaper, wood,
plywood, gypsum board, and acoustical fiber board), comprising 70%
of identified fungi. Raper and Fennell (1977) reported Aspergillus spp.
from building materials such as textiles, jute, insulation materials,
wallpaper, and other paper products. Gravensen et al. (1994) included
a list of 13 fungal species as important molds in damp buildings.
Samson et al. (1992) described 23 common fungal species in indoor
environments.
In water-damaged building materials in Denmark, the fungal genera
most frequently encountered indoors were Penicillium (68%), Asper-
gillus (56%), Chaetomium (22%), Ulocladium (21%), Stachybotrys
(19%), and Cladosporium (15%) (Gravesen et al., 1999). Penicillium
chrysogenum, Aspergillus versicolor, and Stachybotrys chartarum
were the most common species in water-damaged materials (Gravesen
et al., 1999). Stachybotrys atra was isolated with swab samples of
visible growth under wet carpets, on wet walls, or behind vinyl wall
coverings in 11 schools in the United States (Cooley et al., 1998).
Morgan-Jones and Jacobsen (1988) studied moldy carpets, plaster-
board, and wallpaper from three hotels in Florida and Georgia. The
genera of fungi identified were species of the ascomycete genus
Chaetomium; of the dematiaceous hyphomycete genera Alternaria,
Cladosporium, Stachybotrys, and Ulocladium; of the moniliaceous
hyphomycete genera Acremonium, Aspergillus, and Penicillium; and
of the pycnidial genus Phoma. In the study, 14 species, including 2 new
species of Cladosporium, in 11 genera were isolated and identified. In a
study of toxicity of moldy building materials, Johanning et al. (1998)
identified several groups of fungi from gypsum wallboard and other
building materials. The fungi identified included those described by
Morgan-Jones and Jacobsen (1988) and additional species of Aspergil-
lus, Paecilomyces, and Trichoderma. Käpylä (1985) found that pre-
dominant fungus growing on wooden frames of insulated windows
in Finland was Aureobasidium pullulans. In subartic areas, Pessi
et al. (2002) found that indoor fungi occurred infrequently in the
insulation inside insulated precast external concrete walls and that
fungal infestation in the insulation was not found to influence indoor
air in the region.
In Brazil in unusual situations, growth of Cryptococcus neoformans
var. neoformans indoors was due to the presence of avians in the
domestic environment or nearby the home. Higher incidences of cryp-
tococcosis was reported among AIDS patients residing in the dwellings
74 LI AND YANG
from which C. neoformans var. neoformans was isolated than among

AIDS patients from whose domestic environment the fungus was not
detected (Passoni et al., 1998).
In the United States, Histoplasma capsulatum and cases of histoplas-
mosis have been reported from indoor environments such as old hous-
es, church attics, chicken houses, and barns (Collier et al. 1998;
Lenhart, 1994). A primary source of H. capsulatum is soil, especially
in regions of bird or bat habitats. While wind is probably the most
important means of disseminating H. capsulatum, the fungus can sur-
vive and be transmitted from one location to another on the feet of both
birds and bats (Rippon, 1988).
D. FUNGAL BIODIVERSITY INDOORS

One hundred sixty-seven microbial species were discovered from the
air of dwellings in Central and Eastern Europe (Gorny and Dutkiecicz,
2002). Yang et al. (1993) reported that Cladosporium, Penicillium,
Aspergillus, basidiomycetes, and Alternaria were identified, by fre-
quency, as the top five fungal taxa both indoors and outdoors in the
United States. An additional 50 fungal taxa were also identified. How-
ever, Penicillium, Cladosporium, Aspergillus, basidiomycetes, and Al-
ternaria were the top five indoor fungal taxa by concentrations in a
descending order, while Cladosporium, Penicillium, basidiomycetes,
and Aspergillus were the top five outdoor taxa. Womble et al. (1999) of
the US Environmental Protection Agency (USEPA) reported that non-
sporulating fungi, Cladosporium, Penicillium, yeasts, and Aspergillus
were the five most commonly found fungal taxa indoors and outdoors,
based on the frequency of detection. The most common culturable
airborne fungi indoors and outdoors in all seasons and geographic
areas in the United States were Cladosporium, Penicillium, nonspor-
ulating fungi, and Aspergillus in a descending order (Shelton et al.,
2002). In Taiwan the predominant genera of airborne fungi are Clados-
porium, Aspergillus, Penicillium, Alternaria, and yeast (Su et al.,
2001b). Stachybotrys chartarum was identified in the indoor air in
6% of the buildings studied and in 1% of the outdoor air around the
buildings studied in the United States (Shelton et al., 2002).
E. STACHYBOTRYS CHARTARUM AND OTHER STACHYBOTRYS SPP.
Stachybotrys chartarum is one of the major species occurring on

cellulose-based building materials in indoor environments. This spe-
cies has attracted the most attention because of its ability to produce
some of the most potent mycotoxins known and its association with
infant pulmonary hemorrhage and hemosiderosis and adult nasal and
tracheal bleeding (Dearborn, 1997; Vesper and Vesper, 2002; Vesper
et al., 2001).
Stachybotrys chartarum was isolated for the first time from the lung
of a child diagnosed with pulmonary hemosiderosis in Texas (Elidemir
et al., 1999). Flappan et al. (1999) reported another case of infant
pulmonary hemorrhage associated with the presence of Stachybotrys
atra (¼S. chartarum), and mycotoxin analysis demonstrated that the
isolate was highly toxigenic. Vesper et al. (2001) characterized a hemo-
lysin, later called stachylysin, from S. chartarum and analyzed its
hemolytic activity and siderophore production. It was hypothesized
that stachylysin could be a contributing factor to infant pulmonary
hemorrhage and hemosiderosis (Vesper and Vesper, 2002).
Under field conditions, several trichothecenes were detected in each
of three commonly used building materials heavily contaminated with
S. chartarum (Gravesen et al., 1999). Under experimental conditions,
four out of five isolates of S. chartarum produced satratoxin H and G
when growing on new and old, very damp gypsum boards (Gravesen
et al., 1999). In a preliminary study conducted in a Dynamic Microbial
Test Chamber, Foarde and Memetrez (2002) showed that conidia of
Stachybotrys chartarum released from gypsum boards at low air flow
rate were positively related to air flow rate, but negatively related to
relative humidity.
Enzyme immunoassay indicated 65 of 132 (49.2%) sera tested con-
tained IgG against S. chartarum and 13 of 139 (9.4%) sera tested
contained IgE against S. chartarum (Barnes et al., 2002). Sensitivity to
S. chartarum is potentially much more widespread than previously
appreciated (Barnes et al., 2002). This fungus may affect the asthmatic
and the allergic population through both immunologic and toxic me-
chanisms (Barnes et al., 2002).
Rao et al. (2000), using an animal model, showed that methanol
extraction dramatically reduced the toxicity of S. chartarum spores,
and a single, intense exposure to toxin-containing S. chartarum spores
resulted in pulmonary inflammation and injury in a dose-dependent
manner.
Initial spore concentrations were between 0.1 and 9.3 spores/m3 of
air, and the toxicity of air particulates was correspondingly low. How-
ever, the dust in the house contained between 0.4 and 2.1 103 spores/
mg (as determined by hemocytometer counts) (Vesper et al., 2000). Air
samples taken postremediation showed no detectable levels of S. char-
tarum or related toxicity. Nine isolates of S. chartarum obtained from a
76 LI AND YANG
home were analyzed for spore toxicity, hemolytic activity, and random
amplified polymorphic DNA banding patterns (Vesper et al., 2000).
None of the isolates produced highly toxic spores (>90 g T2 toxin
equivalents per gram wet spores) after growth for 10 and 30 days on wet
wallboard, but three isolates were consistently hemolytic (Vesper et al.,
2000).
In addition to S. chartarum, several species of Stachybotrys have
been isolated and identified from the indoor environment. S. yunanen-
sis, S. nephrospora, S. microspora, S. elegans, and S. chlorohalonata
were identified and present in samples from indoor environments (Li
and Yang, 2003; Nielsen et al., 2003). Cruse et al. (2002) examined 23
isolates collected around the United States by using markers for three
polymorphic protein-coding loci and found that 2 cryptic species are
present within the species of S. chartarum. The 2 cryptic species were
indistinguishable morphologically. In another recent study, Andersen
et al. (2002) stated that there were two chemotypes: one producing
atranones, the other macrocyclic trichothecene, plus one undescribed
taxon identified based on the analysis of morphology, growth, and
more importantly metabolite production. The chemotypes and the un-
described taxon were all previously identified as Stachybotrys chartar-
um (Andersen et al., 2002). Later the undescribed taxon was described
as a new species, S. chlorohalonata (Andersen et al., 2003). Taylor et al.
(2003) developed a QPCR assay for the detection of Stachybotrys
elegans.
Characteristic symptoms and immunological tests for antibodies (IgE
and IgG) specific to S. atra and some other fungi strongly suggest
exposure to the indoor fungi. Hemorrhagic lung disease in infants
was highly associated with indoor S. chartarum exposure in a case
cluster investigation in Cleveland (Dearborn, 1997; Montana et al.,
1995) and in a case-home investigation in the midwestern United
States (Flappan et al., 1999). An epidemiological study reported a high
prevalence of pulmonary diseases among office workers of Florida
court buildings following prolonged indoor exposure to S. chartarum
and A. versicolor (Hodgson et al., 1998). Stachybotrys atra was isolated
from bronchoalveolar lavage fluid of a child with pulmonary hemor-
rhage (Elidemir et al., 1999), and S. atra exposure was found in an
infant that developed laryngeal spasm and hemorrhage during general
anesthesia (Tripi et al., 2000). In mouse studies with toxic Stachybotrys
fungi, similar effects (inflammation and hemorrhage) were observed
(Nikulin et al., 1996). Some detailed information on the latest research
of S. chartarum, mycotoxins produced, and its health effects can be
found in the review article by Kohn and Ghannoum (2003).
F. PCR AND MOLECULAR TECHNIQUES
A new methodology, Quantitative Polymerase Chain Reaction (QPCR
or sometimes called real time PCR), offers a new venue to rapidly
detect and quantify fungi with an unprecedented accuracy. Vesper
and Haugland of the US Environmental Protection Agency (US EPA)
have developed and patented primers for over 100 indoor fungal spe-
cies (personal communication) in the last several years. Detection and
quantification of selected indoor fungi is now possible by using the
QPCR. A fungus-specific PCR assay using only one primer set has been
developed for detecting indoor fungi (Alternaria chamydospora, As-
pergillus flavus, Candida famata, Cladosporium fermentans, Penicilli-
um chrysogenum, and Stachybotrys chartarum) (Zhou et al., 2002).
Their results indicated that FF2/FR1 among the 4 sets showed no
cross-amplification with non-fungal DNA. On the other hand, the
primer set cannot differentiate different species of fungi. Haugland
et al. (2002) evaluated three comparatively rapid methods for the ex-
traction of DNA from fungi for use in QPCR and developed a simple
bead milling method that provides sensitive, accurate, and precise
quantification of target fungi in air and water samples. However, dust
samples required further purification of the extracted DNA by a stream-
lined silica adsorption procedure (Haugland et al., 2002).
However, further validation of the primers of each fungal species is
necessary. Occasional cross-reactions against closely related species or
fungi within the same genus have been observed in preliminary tests by
the authors. In a recent study, Wu et al. (2002) evaluated 53 sets of
primers developed by various researchers for commonly occurring fungi
and bacteria and verified 28 sets of them. They also evaluated 7 sets of
primers for specific detection of Aspergillus fumigatus and found only 4
sets to be specific to A. fumigatus. The primers and Taqman probes
developed by Cruz-Perez et al. (2001) for detection of S. chartarum in
a QPCR analysis were found not to be specific to S. chartarum, and they
found that positive amplifications of various fragment sizes were ob-
tained for several fungal species. This study is significant for the appli-
cation of QPCR. Caution has to be taken when a set of primers is going to
be employed for the detection of a particular fungal species. Verification
of the specificity of the primers is a necessity for any application.
In a recent article, Brakhage and Langfelder (2003) reviewed signifi-
cant developments made in the last several years in understanding
the genetics of Aspergillus fumigatus and molecular techniques for
the identification of virulence determinants and manipulation of the
fungus.
78 LI AND YANG
IV. Ecological Factors of Fungi Indoors

Fungal ecology indoors is very important for the understanding
of occurrences and population dynamics of indoor molds. Some
physical/environmental factors are closely associated with fungal
growth and spore populations (Li and Kendrick, 1994, 1995b, 1995c
and 1996).
A. PHYSICAL FACTORS
1. Water and Moisture
Lawton et al. (1999) demonstrated that moisture sources were a
significant factor for mold infestation in the houses. In indoor environ-
ments, most factors necessary for dispersal, development, and coloni-
zation of fungi—such as nutrients, temperature, light, air movement—
are readily available. The only limiting factor for fungal growth indoors
is water or moisture. Modern energy-efficient buildings are sealed,
prone to moisture accumulation, and slow to dry out if water dam-
age/intrusion or dampness occurs. Since other factors for fungal growth
are present in buildings, fungi will thrive once moisture or water is
available. Rudblad et al. (2002) observed that teachers suffering from
nasal mucosal hyperreactivity in a water-damaged school persisted
over years and only slowly recovered after the water-damaged school
was successfully remediated.
Damp building materials, particularly cellulose-containing sub-
strates, are prone to fungal amplification. Fungi commonly found
are species of Penicillium, Aspergillus, Chaetomium, Ulocladium,
Stachybotrys, and Cladosporium (Graveson et al., 1999). In the United
Kingdom alone it has been estimated that 2.5 million houses are seri-
ously affected by dampness caused by condensation in 60% of cases. A
further 2 million houses suffer to a lesser extent from condensation
(Sanders and Cornish, 1982). Among tenants of houses in the public
sector in the United Kingdom there is a concern that mold growth on
damp walls may be a hazard to health (Flannigan et al., 1990). Homes
with a dampness problem showed higher average spore counts and
higher prevalence of respiratory symptoms (Waegemaekers et al.,
1989).
Water activity (aw) expresses the available water in a substrate as a
decimal fraction of the amount present when the substrate is in equi-
librium with a saturated atmosphere (an equilibrium relative humidity
of 70% around the substrate means that the substrate has a water
activity of 0.70) (Kendrick, 2000).
Grant et al. (1989) found that the minimal aw for spore germination
and growth of indoor fungi on building materials was much higher
than on fungal growth medium (MEA). On building materials, fungal
growth starts at a water activity near 0.8 (Table VII), but production of
significant quantities of mycotoxins required an aw of at least 0.95
(Nielsen, 2003). Xerophilic fungi, such as Penicillium spp. and Asper-
gillus spp., will begin to grow at aw between 0.78 and 0.90, depending
on the compositions of substrates of construction materials (Nielsen,
2003). The minimal aw required was different for spore germination
and growth of indoor fungi, and aw for fungal growth is higher than for
spore germination (Grant et al., 1989) (Table VIII).
2. T and RH
Relative humidity (RH) and temperature (T) are the most important
environmental parameters regulating spore production (Mallaiah and
Rao, 1980; Smith and Crosby, 1973). Sufficient moisture is probably the
most important factor in spore production (Lyon et al., 1984). Spore
TABLE VII
FUNGI GROWING ON BUILDING MATERIALS AND THEIR aw
Colonizer group aw range Classification Fungal example
Primary <0.80 Xerophilic/ Penicillium chrysogenum, and

colonizers Xerotolerant Aspergillus versicolor: the most
(storage fungi) common ones; A. fumigatus,
A. niger, A. sydowii, A. ustus,
Eurotium spp.,
P. brevicompactum, P. commune,
P. corylophilum, P. palitans,
Paecilomyces variotii, and
Wallemia sebi
Secondary 0.80–0.9 Mesophilic Alternaria spp., Cladosporium spp.,
colonizers Epicoccum nigrum, Phoma spp.,
and Ulocladium spp.
Tertiary >0.9 Hydrophilic Chaetomium globosum, Fusarium,
colonizers Memnoniella echinata,
(water-damage Rhizopus stolonifer,
fungi) Stachybotrys chartarum,
Trichoderma spp. (T. atroviride,
T. citrinoviride, T. harzianum,
and T. longibrachiatum)
Grant et al. (1989); Lubeck et al. (2000); Macher et al. (1999); Nielsen (2003).
80 LI AND YANG
TABLE VIII
MINIMAL aw FOR SPORE GERMINATION AND GROWTH OF INDOOR FUNGI AT 20 TO 25 C ON
GROWTH MEDIA
Minimal aw
Fungus For spore germination For growth
Alternaria alternata 0.85 0.88–0.89

Eurotium (Aspergillus) repens 0.70–0.72 0.71–0.76
Aspergillus versicolor 0.75–0.81 0.78–0.80
Aureobasidium pullulans 0.89
Cladosporium cladosorioides 0.86 0.88
Cladosporium herbarum 0.85–0.88 0.90
Cladosporium sphaerospormum 0.85
Fusarium moniliforme 0.87
Mucor plumbeus 0.93
Penicillium brevicompactum 0.78–0.84 0.81–0.82
Penicillium chrysogenum 0.78–0.85 0.79
Penicillium nigricans 0.79
Penicillium spinulosum 0.80 0.80
Phoma herbarum 0.92
Sistotrema brinkmannii 0.97
Stachybotrys chartarum 0.85–0.95 0.94
Ulocladium chartarum 0.89
Ulocladium consortiale 0.89
Compiled from Grant et al. (1989).
release of hyphomycetes was correlated with increasing T and decreas-

ing RH. The moisture content of the indoor air significantly affected all
measurable airborne concentrations (Pessi et al., 2002). Spore release of
Botrytis squamosa was promoted largely by declining RH, increasing T,
and rain but occasionally by increased RH (Sutton et al., 1978). Leach
(1975) found that spore release of Drechslera turcica and other fungi is
affected by decreasing RH but not by temperature changes. Conidia of
Cercospora asparagi were caught beginning at 07:00–08:00am when T
increased and RH fell below 90%, and the number of spores increased
drastically (Cooperman et al., 1986). Positive correlations of total air
spora, Cladosporium, and Botrytis with higher temperatures have been
documented (Beaumont et al., 1985).
Variations/fluctuations in indoor humidity and temperature have
significant effects on fungal growth (Adan, 1994; Vitanen and
Bjurman, 1995). In a bathroom situation in which transient high hu-
midities are common, dominant mycota include Alternaria, Aureoba-
sidium, Cladosporium, Phoma, and Ulocladium (Moriyama et al.,
1992; Samson et al., 2000).
Basidiospores of Paxillus panuoides were released at temperatures
above 0 C, and daily peaks were usually correlated with increased T
and decreased RH. Spore release increased from 2 C to a maximum at
37 C, then ceased at 45 C. Light and RH treatments did not significant-
ly affect spore release. Temperature was determined to be the stimulus
for the natural spore release pattern (McCracken, 1987). The humidity
factor was correlated with basidiospores (Beaumont et al., 1985). The
higher counts of basidiospores may have resulted from the higher
relative humidity and lower sunshine in 1978 in Galway, Ireland
(McDonald and O’Driscoll, 1980). Since temperature is correlated with
relative humidity, the effects of both factors could not be defined
separately in most studies.
3. Light
For a number of the perithecial ascomycetes, light is necessary to
initiate ascospore discharge (Lyon et al., 1984; Moore-Landecker,
1982). Sutton et al. (1978) suggested that light may affect the release
of conidia of Botrytis squamosa. It is found that light triggers spore
release in several fungi (Leach, 1975).
Concentrations of airborne spores are related to preceding con-
ditions affecting spore production and release (Sutton et al., 1978). In
the conidial fungi, once the spores are produced, release is
often influenced by wind velocity/air movement. In the Ascomycetes,
radiation, minimum humidity, changes in humidity, and minimum
wind velocity were all directly correlated with levels of airborne ascos-
pores (Lyon et al., 1984). These are some of the reasons why diurnal
periodicity exists. On the other hand, spores of other fungi such as
Cladosporium, Alternaria, and Helminthosporium are blown free by
wind/air movement, and this type of ‘‘dry spore’’ increases in concen-
tration with decreasing RH and increasing air movement. Thus
these species are often abundant during midday periods with maximal
sunlight.
The maturation and release of some spores such as basidiospores are
also markedly affected by the presence of free water (Lyon et al., 1984;
Salvaggio, 1986; Salvaggio and Aukrust, 1981). Under some circum-
stances, circadian rhythms in humidity and temperature interact to
82 LI AND YANG
form diurnal patterns or nocturnal increases in spore concentrations,

such as certain basidiospores (Salvaggio and Aukrust, 1981).
4. Air Movement
Air movement is the most unpredictable agent in the transport of
fungal spores. Many fungal spores are adapted for aerial dispersal. It
would appear that the horizontal distance over which individual mi-
croorganisms may be transported is largely determined by their ability
to survive in the atmosphere (Tilak, 1984).
In hyphomycetes, dry spore release is often influenced by wind
speed (Lyon et al., 1984). Botrytis squamosa spores were apparently
released at very low wind speeds (Sutton et al., 1978). Maximum
wind speed was negatively correlated with spore concentrations of
Cladosporium, Alternaria, unidentified ascospores, and unidentified
basidiospores; it was the only factor among 10 meteorological factors
significantly correlated with all four groups. Minimum wind was
directly correlated to spore counts, while maximum wind was inverse-
ly correlated (Lyon et al., 1984). Wind/air movement plays an impor-
tant role in basidiospore dispersal. After basidiospores are ejected
from basidia and drop from between the gills of the basidioma, they
are primarily dispersed by air movement (Moore-Landecker, 1982).
Wind direction had a profound effect on the airborne fungi in Galway,
Ireland (McDonald and O’Driscoll, 1980).
Although wind and air movement are known to assist in the release
and dispersal of spores in nature, their importance in spore release
and dispersal in the indoor environment has not been studied and is
poorly understood. The primary air mover in a mechanically ventilated
environment is the heating, ventilating, and air-conditioning system.
5. Substrates
Dust is ubiquitous in our daily environments and is a heterogeneous
substrate. It can be found where surfaces (hard or porous) are present:
furniture, walls, floor, ceilings, carpet, etc. It serves as a reservoir for
fungal spores and fragments. It also provides certain nutrients for fungi
to survive or grow, such as food crumbs, skin flakes, fibers, and other
organic matter. Resuspension of fungal spores from dust into air could
influence airborne fungal spore concentration and composition signifi-
cantly and subsequently affect the exposure of occupants to the aero-
solization. Individual differences in indoor conditions do not have
much influence on the diversity of the fungal flora in dust. They do,
however, influence its quantity (Rijckaert, 1981).
The most frequent fungal genera in dust were Penicillium, Eurotium,
Aspergillus, Alternaria, Epicoccum, and Cladosporium (Oppermann
et al., 2001). A total of 41 different genera/species were identified
(Oppermann et al., 2001). In the Greater New Haven, Connecticut, area
fungal composition and concentrations in house dust samples were not
correlated with those present in the indoor air (Ren et al., 1999). In dust
samples, more Mucor, Wallemia, and Alternaria species were found in
all seasons but fewer Aspergillus, Cladosporium, and Penicillium
species were found (Ren et al., 1999). Scott (2001) conducted a com-
prehensive study on indoor fungi from dust collected from 369
houses in Wallaceburg, Ontario, and found roughly 250 fungal taxa,
with the 10 most common taxa being Alternaria alternata, Aureobasi-
dium pullulans, Eurotium herbariorum, Aspergillus versicolor, Penicil-
lium chrysogenum, Cladosporium cladosporioides, P. spinulosum, C.
sphaerospermum, A. niger, and Trichoderma viride.
Engelhart et al. (2002) showed that 18% of total culturable fungi from
carpet dust samples were A. versicolor, of which 49 of 50 isolates (98%)
were found to be sterigmatocystin producers in vitro. Sterigmatocystin
could be detected at low concentrations (2 to 4 ng/g of dust) in 2 of 11
native carpet dust samples by using high-performance liquid chroma-
tography-electrospray ionization tandem mass spectrometry (Engelhart
et al., 2002).
Chao et al. (2002a) found that concentrations of total dust-borne
fungi from floors were positively related to carbon dioxide and tem-
peratures between 20 and 22.5 C. A gradual increase in total fungal
concentrations in floors was observed over a year (Chao et al., 2002a).
Total fungi isolated from chairs varied significantly by season, being
highest in September and lowest in March, and were positively corre-
lated with dust loads in floors (Chao et al., 2002a). The results suggest
the presence of seasonality of dust-borne fungi.
Higher numbers of mold isolates were associated with areas of high
shade and high levels of organic debris near the home, along with poor
landscape maintenance. Lower concentrations of mold isolates were
associated with good dust control measures. No statistically significant
correlations could be made between indoor mold isolates and any of
the following factors: number and age of the occupants, age and size of
home, month of survey, or the presence of plants (Banerjee et al., 1987).
A wide range of fungi in floor and mattress dusts was reported in
southwest Germany in winter and spring (Jovanovic et al., 2001). The
median value of CFU/g dust, collected from the floors, was 15,000
(range 0–700,000) and 28,000 (range 1500–1,350,000) for samples col-
lected from mattresses. On the other hand, Oppermann et al. (2001)
84 LI AND YANG
reported total concentrations of fungi varied from 1.4 103 to 300

103 CFU/g of dust with a geometric mean of 26.5 103 CFU/g of dust.
A total of 41 different genera/species were identified. Fungal spores
were discovered more frequently in mattresses in humid flats.
Samples of settled dust were taken by vacuuming from the carpet in
the living room of 405 homes in Erfurt (east) and Hamburg (west) and
plated on DG18 agar. No significant difference could be shown for the
total or for single genera (Alternaria, Aspergillus, Cladosporium, and
Penicillium) in concentration of spores of viable fungi in settled house
dust between Erfurt and Hamburg (Koch et al., 2000). Seasonal varia-
tion of the fungi, with a peak in August, could be identified both
indoors and outdoors. Koch et al. (2000) concluded that outdoor con-
centration is the main influence on indoor concentration of mold
spores from June to October.
Hodgson and Scott (1999) studied 243 carpet dust samples collected
from problem and control buildings in the United States. They found
that fungal concentrations, analyzed with the extraction and serial
dilution method, in dust samples from problem buildings were higher
than control samples (1.8 million CFU/g v. 30,000 CFU/g). The most
common dominant fungal group detected in problem buildings was
Pencillium, followed by Cladosporium and Aspergillus. In the control
buildings, Cladosporium was the most frequently detected dominant
taxon, followed by Penicillium, Phoma, yeasts, and Epicoccum nigrum
at equal percentages. Aspergillus was never the dominant taxon in the
control buildings. Among individual Aspergillus species, A. versicolor
was the most common species in samples from problem buildings.
Other Aspergillus species of significance included A. niger and A.
sydowii. They concluded that concentrations greater than 105 CFU/g,
and certainly those greater than 106 CFU/g, were most likely associated
with fungal contamination in buildings. However, major (20–50% of
the concentration) or dominant taxa can also serve as an indicator of
fungal contamination. Penicillium spp. and Aspergillus spp., especial-
ly A. versicolor, are generally the dominant taxa associated with
problem building samples.
Scott (2001) studied genotypic variation of two common dust-borne
Penicillium species, P. brevicompactum and P. chrysogenum. It was
found that there were two genetically divergent groups of P. brevicom-
pactum according to sequence analysis of the beta-tubulin (benA) and
rDNA loci. Authentic strains of P. brevicompactum and P. stoloniferum
clustered together in the predominant clade, accounting for 86% of
isolates (Scott, 2001). At the same time, a clonal pattern of inheritance
in P. chrysogenum was observed, and phylogenetic analyses of allele
sequences was found segregating the population into three divergent
lineages, accounting for 90%, 7%, and 3% of the house dust isolates,
respectively (Scott, 2001). Type isolates of P. chrysogenum and its
synonym P. notatum clustering within the secondary lineage con-
firmed this synonymy. No available names for the predominant and
minor lineages suggested that P. chrysogenum delineated previously is
a species complex including three taxa. Further studies are necessary
to determine the nomenclature status of the two unknown lineages of
P. chrysogenum.
Although many fungal studies of household dust are available,
Macher (2001) argued that no standard procedures for culturing fungi
from dust have been adopted widely enough to ensure the validity of
comparisons among studies. It is necessary to establish reference meth-
ods in environmental microbiology for use in the assessment of indoor
environmental quality (Macher, 2001).
Separate collection of organic and nonorganic household waste is
becoming a common form of waste management in Europe and North
America. Household organic waste indoors is a good substrate, which
might increase microbial growth in the home environment. A study in
99 homes in The Netherlands showed that increased microbial con-
taminant levels in homes are associated with indoor storage of sepa-
rated organic waste when using mold beta (1 ! 3)-glucans and fungal
extracellular polysaccharides of Aspergillus and Penicillium species in
house dust extracts as markers of microbial levels (Wouters et al.,
2000).
6. Activities of Occupants
It is generally believed that human activity in a building is a major
contributor to the fungal population indoors. However, very few stud-
ies have addressed this issue. Sessa et al. (2002) conducted a study in a
university auditorium in Rome, in an office of public buildings, and in
an apartment in the presence and absence of the buildings’ occupants,
building materials, and furnishings by using a Surface Air System
(SAS). In the presence of people and furnishings, the average concen-
trations of airborne fungi were much higher (University auditorium:
1256–1769 CFU/m3; office: 858 CFU/m3; apartment: 147–297 CFU/m3)
than in their absence (respectively: 301–431 CFU/m3; 224 CFU/m3;
102–132 CFU/m3). In a recent study, Butterner et al. (2002) showed
that after cut pile carpet was walked on for 1 minute, the airborne
conidia concentrations of P. chrysogenum were significantly higher
than the ones with vinyl tile and commercial loop pile carpet, and
the differences in concentration were often 2 orders of magnitude.
86 LI AND YANG
B. BUILDING CHARACTERISTICS
Building characteristics and designs have significant influence on
indoor aeromycota. The presence or installation of forced-air heating
systems, humidifiers, air filters, and air conditioners can lead to re-
duced concentrations of airborne fungal spores (Li and Kendrick,
1995d). Concentrations of fungi were lower in day-care centers
equipped with air conditioners/air cleaners than in centers that lacked
such equipment (Li et al., 1997). Lawton et al. (1999) found that low air
leakage and natural ventilation were not associated with higher levels
of mold growth. In the same study it was found that the presence of
wood-burning stoves and fireplaces was significantly positively
correlated to fungal infestation in residences (Lawton et al., 1999).
Hirsch et al. (2000) showed that the installation of insulated windows
and central heating systems in 98 apartments correlated with changes in
the indoor environment over 7 months. The air-exchange rate decreased
from 0.73 to 0.52 per hour, temperature increased from 13.4 to 17.5 C,
and absolute humidity increased from 4.6 g to 6.2 g H2O/kg air in. In
addition the abundance of Aspergillus fumigatus was found to increase
from 20 to 60 CFU/g carpet dust (Hirsch et al. 2000). The presence of
carpet was found to increase the concentration of airborne fungal
spores in residential buildings (Li and Kendrick, 1995d).
The concentrations of airborne fungal spores vary significantly from
room to room in some residential buildings. In the study of Li and
Kendrick (1995d), results showed that numbers of airborne fungal
spores were highest in living rooms, followed by family rooms, kitch-
ens, bathrooms, and bedrooms. However, Ren et al. (1999) found no
significant difference in concentration and composition of fungi be-
tween living room and bedroom or by season. Both concentration and
type of fungi were significantly higher in basements than other indoor
areas and outdoor air in winter. Significant seasonal variation in fungal
type was observed in living rooms, bedrooms, and outdoor air but not
in basements (Ren et al., 1999).
Wan and Li (1999) showed that the levels of bacteria and fungi
indoors were highest in day-care centers, followed by those in homes
and office buildings. However, the prevalence of airway inflammation
and systemic symptoms was higher for females in office buildings than
for those in day-care centers. That means that there are other factors to
be considered. A strong association was found between beta-1,3-glucan
and symptoms of lethargy/fatigue (Wan and Li, 1999).
Floor dust and air samples from the bedrooms of 485 houses were
collected over 1 year in Melbourne, Australia (Dharmage et al., 1999).
The dust was analyzed for ergosterol, a marker of cumulative fungal
biomass exposure. The median ergosterol level in bedroom floors was
3.8 g/g of dust. Multivariate analysis showed that several factors were
associated with lower total fungal propagules in bedroom air: a ceiling
fan, absence of visible mold, frequent vacuuming, a solid fuel fire, and
absence of pets. Total fungal propagules were lower when windows
were closed during sampling. The presence of more than one cat had
the greatest effect on total fungal propagules. Ergosterol levels were
significantly lower in homes without old fitted carpets, visible mold, or
pets and those with frequent airing and regular use of an extractor fan
in the kitchen. Old wall-to-wall carpets had the greatest effect on
ergosterol (Dharmage et al., 1999).
Poor insulation of buildings may lead to a greater fluctuation in air
temperature, condensation, and dampness in localized areas of build-
ings. A drop in the air temperature below the dew point often relates to
fungal growth inside buildings, particularly in places such as badly
insulated outer walls or room corners (Kaufhold et al., 1997). This
temporary fall below the dew point caused by temperature fluctuation
probably resulted in the building materials and wallpapers becoming
damp and subsequent fungal growth (Kaufhold et al., 1997).
Installation and operation of germicidal UV lights in central heating,
ventilation, and air conditioning (HVAC) systems of office buildings
was found feasible, cannot be detected by workers, and does not
seem to result in any adverse effects (Menzie et al., 1999). However,
the effectiveness of UV lights on fungal growth and contamination in
the HVAC system was not convincing.
The number of CFU/m3 air collected on MEA was significantly high-
er than on DG-18 (1033.5 and 846.0 CFU/m3, respectively) from 1000
homes in the northeastern United States (Ren et al., 2001). Tempera-
ture, relative humidity, seasons, and presence of cats indoors had a
statistically significant impact on the presence of fungal propagules in
indoor air. Ren et al. (2001) concluded that it is difficult to predict the
presence of fungal propagules in indoor air reliably by home character-
istics alone.
Pei-Chih et al. (2000) found that the fungal concentration for indoors
was 8946 (4372–18,306) CFU/m3 in winter and 4381 (1605–11,956)
CFU/m3 in summer. For outdoors, it was 11,464 (5767–22,788) CFU/
m3 in winter and 4689 (1895–11,603) in summer. In summer, the total
fungal concentrations, both indoors and outdoors of suburban homes,
were significantly higher than those of urban homes. The dominant
fungi contributing to this difference were Cladosporium spp. indoors
and Penicillium spp. outdoors (Pei-Chih et al., 2000).
88 LI AND YANG
1. Building Materials
Building materials such as drywall, wallpaper, insulation materials,
wood framing materials, carpet, wood flooring, and subfloor materials
provide growth substrates for fungi to colonize and develop. They also
serve as porous materials to hold water or moisture, which is another
necessary factor for the growth of fungi.
From a score system assessing the bioavailability of building materi-
als, the products most vulnerable to mold attacks were water-damaged,
aged organic materials containing cellulose, such as wooden materials,
jute, wallpaper, and cardboard (Gravesen et al., 1999).
The growth of three fungal genera (Cladosporium, Penicillium, and
Stachybotrys) was evaluated on cellulose-containing ceiling tile and
inorganic ceiling tile (Karunasena et al., 2001). The results show that
inorganic ceiling tile did not support the growth of these three fungal
genera while cellulose-containing ceiling tile did. The results demon-
strated that inorganic ceiling tile could serve as an ideal replacement
for cellulose-containing ceiling tile (Karunasena, 2001). In a controlled
laboratory study, Chang et al. (1995) found that new ceiling tiles sup-
ported the growth of Penicillium chrysogenum and P. glabrum at aw
0.85 and a corresponding moisture content >2.2%, and of Aspergillus
niger at aw 0.94 and a corresponding moisture content >4.3% on used
ceiling tiles. The same study showed that used ceiling tiles were more
susceptible to fungal growth than new ones (Chang et al., 1995).
In a study on gypsum boards just off the production line, Doll and
Burge (2001) showed that 11 fungal genera were present on new gyp-
sum board without artificial inoculation. Penicillium spp. and Asper-
gillus spp. were found at 95% RH only on the paper sides and the
number of fungi found on the new gypsum board increased with
increasing moisture content. On one occasion Stachybotrys sp. was
present on the gypsum boards (Doll and Burge, 2001). This study
showed that new gypsum boards were not free from naturally occurring
fungal spores.
Morgan-Jones and Jacobsen (1988) studied moldy carpets, plaster-
board, and wallpaper from three hotels in Florida and Georgia. They
found many fungi caused biodeterioration of paper, textiles, and plas-
ter. The genera of fungi most often identified were the ascomycete
genus Chaetomium; the dematiaceous hyphomycete genera Alternaria,
Cladosporium, Stachybotrys, and Ulocladium; the moniliaceous hy-
phomycete genera Acremonium, Aspergillus, and Penicillium; and
the pycnidial genus Phoma. In the study, 14 species in 11 genera were
isolated and identified, including two new species of Cladosporium. In
a study of toxicity of moldy building materials, Johanning et al. (1998)
identified several groups of fungi, detected satratoxin H and spirolac-
tone/lactams, and demonstrated their cytotoxicity of the materials
to cell cultures. The fungi were isolated from gypsum wallboard and
other building materials. The fungi identified included those
described by Morgan-Jones and Jacobsen (1988) plus additional species
of Aspergillus, Paecilomyces, and Trichoderma. Käpylä (1985)
found that the predominant fungus growing on wooden frames of
insulated windows in Finland was Aureobasidium pullulans. In
recent studies it was found that C. sphaerospermum out-competed
with P. chrysogenum under fluctuating aw on various plaster materials,
paints, and plasterboards. However, under constant aw, P. chrysogen-
um out-competed C. sphaerospermum (Nielsen, 2002, 2003).
Carpet is a good reservoir for fungal spores to survive and for their
redispersal into air. The conidia of P. chrysogenum were much easier
to be aerosolized from residential carpet (cut pile) by walking than
from commercial type carpet (loop pile) and vinyl tile (Buttner et al.,
2002). High indoor fungal exposures were associated with infrequent
ventilation or vacuuming, presence of pets, visible mold, and old
carpets (Dharmage et al., 1999).
All fungi found to colonize building materials are either saprophytes,
biodeteriorating agents, or both. Some fungi—such as species of Asper-
gillus, Chaetomium, Chrysosporium, Stachybotrys, and Trichoderma—
are known to be capable of degrading cellulose fibers. Although a
few fungal species have been reported in the literature, it is very likely
that any saprophytic, biodeteriogenic, or cellulolytic fungi can grow
indoors if opportunity arises and conditions are met (Burge, 1999).
Although species of the deuteromycetes are commonly detected in
moldy building materials, species of Ascomycota (such as Peziza spp.
and Pyronema domesticum) and Basidiomycota (such as species
of Cryptococcus, Rhodotorula, and Sporobolomyces) are occasionally
found on damp materials in buildings. In addition, basidiomata (fruit-
ing bodies of basidiomycota) of Coprinus spp., Pleurotus, and
Poria from various building materials, from ceiling tiles to wood pro-
ducts, have been identified (Yang, unpublished data). Mycelia and
hyphae with clamped connections, indicating basidiomycetes, are
frequently detected colonizing water-damaged wood structures and
paper products. Wood-inhabiting basidiomycetes are often wood decay
fungi. Most recently, wood decay fungi, Merulioporia incrassata and
Serpula lacrymans, were identified and detected from decayed wood
samples by using the quantitative PCR method (Yang, Li, and Lin,
unpublished).
90 LI AND YANG
2. Heating, Ventilating, and Air-Conditioning Systems (HVAC)

Heating, ventilating, and air-conditioning systems (HVAC) play an
important role (both positive and negative) in fungal infestation and
dissemination, as follows: (1) as a dispersal pathway for airborne fun-
gal spores; (2) as growth locations for fungi in the system (such as drip
pans, chill coils, and insulated or soiled duct works), if the system is
not properly maintained; and (3) as a filtration mechanism in a proper-
ly maintained system that can remove some airborne fungal spores and
reduce the airborne fungal population indoors.
Fungi have been known to grow in the HVAC system. Window air-
conditioners (AC) appear to substantially reduce indoor allergen levels
by maintaining the isolation of enclosed spaces from particle-bearing
outdoor air (Solomon et al., 1980). Central air conditioning may reduce
numbers of fungal spores in houses by 50% or more, and central
electrostatic filtration also gives significant reductions (Flannigan
et al., 1991). However, fungal growth is known to occur in poorly
maintained HVAC systems. Contaminated humidifiers and heating
equipment are also sources of allergenic fungi (Flannigan et al.,
1991). Respiratory diseases caused by contaminated air-conditioning
systems have been reported from the United Kingdom and the United
States. Acremonium, Paecilomyces, Aureobasidium, Phialophora, and
Fusarium are regularly found in humidifiers (Samson, 1986).
Heinemann et al. (1994) studied contamination of HVAC by
fungi, bacteria, and thermophilic actinomycetes. They sampled sur-
faces of filters and fans with RODAC contact plates and used the serial
dilution method for humidifier water. A wide variety of fungi were
identified. However, some of the fungi identified were likely spore
contaminants instead of fungal growth. Kemp et al. (1995) studied
fungal growth on filters of the HVAC system and reported isolation of
Aspergillus niger, A. fumigatus, Alternaria, Cladosporium, Mucor sp.,
Aspergillus sp., and Penicillium sp. However, when filters were
directly examined under the microscope, they could only confirm
growth of Aspergillus sp., Cladosporium sp., and Penicillium spp.
Buttner et al. (1999) found that fungal growth may occur on a variety
of duct materials, including bare metal, provided soiling and moisture
are present. Yang (1996) examined microscopically as well as cultured
1200 Fiberglas insulation liners from HVAC systems in the United
States and found fungal colonization and growth in approximately
50% of the samples studied. Fungal types were differentiated based
on water and humidity conditions. Species of Cladosporium and
Penicillium were primarily from areas with high relatively humidity.
Cladosporium cladosporioides, C. herbarum, C. sphaerospermum, and
P. corylophilum were the primary species identified. Species of
Acremonium, Aureobasidium, Exophiala, Fusarium, Paecilomyces,
Phoma, Rhodotorula, Sporobolomyces, and yeasts are expected in
areas subjected to frequent wetting, such as cooling coils and drain
pans.
Graudenz et al. (2002) reported a higher prevalence and a strong
association of building-related worsening of respiratory symptoms
and symptoms of rhinoconjunctivitis in the group with ventilation
machinery and ducts with >20 years of use in Brazil. The authors
found that total viable fungal spores were higher outdoors than in the
indoor samples. However, they did report that fungal growth was
detected in the ventilation systems.
Ahearn et al. (1997) studied secondary air filters in the air-handling
units on four floors of a multi-story office building with a history of
fungal colonization of insulation within the air distribution system.
Fungal mycelium and conidia of Cladosporium and Penicillium spp.
were observed on insulation from all floors and on both sides of the air
filters from one floor.
C. SUCCESSION AND CHANGES IN INDOOR FUNGI

In a chronically water-damaged environment, fungal populations are
likely to change over time. Fungi are no different from any living
organisms. They grow in an environment and compete with other fungi
and organisms. The fungal population and composition as a whole
evolve and change depending on environmental factors, interaction
with other fungi and organisms, and the biology of the fungi.
The evolution of organisms in an environment is called succession.
Fungal succession in the indoor environment is largely unknown at
this time. However, some postulations of indoor fungal succession
have been discussed (Grant et al., 1989; Singh, 1994). A knowledgeable
mycologist can use various information and means to evaluate fungal
succession in a given indoor environment. Defining fungal succession
in an indoor environment follows a forensic approach. Information and
evidence gathering is very important and should be as comprehensive
as possible. This section discusses various observations, environmen-
tal parameters, and the biology of various molds that are useful in
conducting an assessment of fungal succession in a water-damaged
environment.
Fungal growth is always the consequence of a water damage or
humidity control problem. Information regarding any water damage
92 LI AND YANG
history can provide an important clue of fungal growth. Observation of

water-damaged building materials can also shed some light regarding a
water damage history. Water is a good and common solvent and can
dissolve many substances. A building material subjected to repeated
water damage can show signs of deterioration. A ceiling tile may
become soft and deformed due to repeated water damage. Nails and
carpet strips may become rusty, stained, and weakened. If growth of
cellulolytic fungi develops, cellulose-containing building materials
can further be weakened because of degradation of cellulose fibers
and paper.
Properly identified fungi can tell a story. Fungi are no different from
plants. Water-loving plants, such as water lily or cattails, grow only in
water or swamps. On the other hand, cacti grow in dry arid environ-
ments. Identifications of moisture-loving fungi suggest the environ-
ment was wet or water damaged. Moisture-loving fungi include
species of Acremonium, Chaetomium, Fusarium, Trichoderma, Ulocla-
dium, Stachybotrys chartarum, and Memnoniella echinata. Wet
fungi—such as species of Aureobasidium, Rhodotorula, Phialophora,
Exophiala, and Sporobolomyces—generally require that substrates be
wet or water logged. On the other hand, detection of xerophilic fungal
growth suggests sufficient but low water activity in the substrates. The
condition to achieve low water substrates for growth of xerophilic
fungi is usually persistent high humidity conditions. Xerophilic fungi
include all species of the genus Eurotium, Aspergillus restrictus, A.
penicillioides, and Wallemia sebi. The two most commonly encoun-
tered indoor fungal genera, Aspergillus and Penicillium, consist of both
moisture-loving and xerophilic fungi. Aspergillus versicolor and A.
ustus are commonly detected in water-damaged building materials.
Although A. versicolor can grow at a water activity as low as 0.80, its
optimal water activity is 0.97 (Smith and Hill, 1982).
Although all indoor fungi are essentially saprobes, many of them
have unique environmental niches. Some fungi, such as species of
Aspergillus and Penicillium, are called sugar fungi (Singh, 1994)
because they exploit the sugar content of the substrates. Fungi that
can break down complex carbohydrates (such as cellulose and lignin)
or complex organics (such as wood) are usually late colonizers of the
substrates. In addition, fungi can be classified into primary colonizers,
secondary colonizers, or tertiary colonizers depending on the water
activity for their growth (Grant et al., 1989). The primary colonizers
are defined as those that grow at water activity below 0.80; secondary
colonizers grow at between 0.80 and 0.90; tertiary colonizers grow
at greater than 0.90. Therefore, moisture-loving fungi are tertiary
colonizers. Common fungal colonizers of the indoor environment,
as defined by Grant et al. (1989), are listed in Table VII. Although the
categorization of various fungi based on their water activity is useful,
further refinement is necessary. Many of the strongly xerophilic
fungi have an ecological niche that does not usually overlap with the
hydrophilic and mesophilic fungi. The authors have observed that
strong xerophilic fungi thrive in conditions subjected to long-term
high humidity conditions but not to water damage (Yang, personal
communication).
In a water-damaged environment, more than one species of fungi are
likely encountered. The types of fungi are likely to increase if the wet
conditions persist for a long time. Fungal diversity is likely to increase
when the water-damage conditions persist.
Fungi as well as mites, insects, and other arthropods are attracted to
water sources. It is commonly observed in the laboratory that some
insects and mites feed on fungal matter (Flannigan et al., 2001). These
insects and mites follow water trail and fungal growth. They also assist
in transporting and dispersing fungal spores. The presence of insects,
mites, and their fecal pellets and molt casts is a good indication of
active insect and mite colonies and long-term fungal growth.
Fungi develop different mechanisms to release and disperse their
spores (Kendrick, 2000). Some fungi—such as species of Aspergillus,
Cladosporium, and Penicillium—produce dry spores, which are re-
leased by air movement and disturbance. Some fungi can actively
discharge their spores into the air. Another group of fungi—such as
species of Acremonium, Stachybotrys, and Trichoderma—produces
wet, slimy spores. Water, insects, mites, or other arthropods and small
animals often assist their release and dispersal. Dry-spored fungi are
much more effective in the dispersal and dissemination of their spores.
Most fungi produce spores within a 2–10 m size range. However, a
few are slightly smaller than 2 m, and many are larger than 10 m.
Fungi (such as species of Aspergillus and Penicillium) that produce
smaller spores are much more prolific in their spore production. They
produce a much larger number of spores. Fungi (such as Stachybotrys
chartarum, Alternaria alternata, etc.) that produce comparatively larg-
er spores produce fewer of them. Different species of fungi grow at
different rates. Fast-growing fungi tend to produce more spores. Fungi
that produce a larger number of spores have the advantage of dissemi-
nating to a wider territory, thus increasing their chance of finding the
niche to colonize and grow.
Some fungi can cause wood decay, from brown rot, to white rot,
to soft rot. The wood decay is a slow process caused by tertiary
94 LI AND YANG
colonizers. Both brown and white rots are caused by wood decaying
basidiomycetes. Soft rot is caused by microfungi and ascomycetes
(Wang and Zabel, 1990; Zabel and Morrell, 1992). If wood structures
of a building show decay, it is an indication of a long-term problem
caused by chronic or repeated wet conditions.
V. Recent Studies on Limits/Exposures of Indoor Fungi

In southwestern Germany between November 1997 and May 1998,
the number of colony forming units was 135 CFU/m3 (range 5–17,000)
in indoor air and 145 CFU/m3 (range 15–2900) in outdoor air
(Jovanovic et al., 2001). The data suggest that an indoor-outdoor differ-
ence exceeding 500 CFU/m3 indicates an elevated mold concentration
in indoor air compared with background (Jovanovic et al., 2001). The
authors suggested that this value could be used as a temporary refer-
ence value for southwest Germany in the winter season ( Jovanovic
et al., 2001).
The CFU/g dust, collected from floors, was 15,000 (range 0–700,000),
and that collected from mattresses was 28,000 (range 1500–1,350,000)
(Jovanovic et al., 2001). Spores of mold fungi were discovered more
frequently in mattresses from humid flats (Oppermann et al., 2001). Total
concentrations of mold fungi varied from 1.4 103 to 300 103 CFU/g of
dust with a mean of 26.5 103 CFU/g of dust (Oppermann et al., 2001).
In extracts of Aspergillus fumigatus culture filtrate, two antigens
were found to be produced under all studied growth conditions (com-
mon antigens), and in the extracts of the water-soluble portion of the
mycelium, one common antigen was found (Wijnands et al., 2000). The
three common antigens may serve as marker antigens for exposure to
Aspergillus fumigatus and its products (Wijnands et al., 2000). In view
of the simultaneous presence of two of these common antigens with
Aspergillus fumigatus allergens, these two marker antigens may be
useful for estimating exposure to allergens of Aspergillus fumigatus.
Gas chromatography–mass spectrometry was used to determine the
microbial contents of building materials subjected to water damage in a
laboratory experiment and of materials collected from houses affected
by water during a flood in Klodzko in southwestern Poland (Szponar
and Larsson, 2000). Ergosterol was examined as a marker of fungal
biomass in southwestern Poland (Szponar and Larsson, 2000).
The amount of ergosterol was higher in materials that had been
exposed to water than in unexposed ones (Szponar and Larsson,
2000). The marker was stable in the building materials for at least 6
weeks at room temperature and could thus be used to reveal microbial
contamination (Szponar and Larsson, 2000). Direct measurement of 3-
hydroxy fatty acids and ergosterol in human environments could be a
useful monitoring method for potentially harmful microorganisms and
microbial constituents (Szponar and Larsson, 2000).
A fungal index or fungal detector, a simple device using spore ger-
mination of 1 to 3 fungal species as sensors for moisture, was used to
assess indoor climate and potential for fungal growth (Abe et al., 1996)
or to monitor drying of water-damaged buildings (Morey et al., 2002).
The following residential limit values (RLV) for dwellings and com-
munal premises are proposed in Poland for the concentration of air-
borne bacteria, fungi, and bacterial endotoxin: 5 103 CFU/m3, 5
103 CFU/m3, and 5 ng/m3 (50 EU), respectively (Gorny and Dutkiewicz,
2002).
Klanova (2000) proposed that any concentration of fungi in indoor
air above 2000 CFU/m3 could be a serious risk factor for health of
occupants. The proposed values for occupational exposure limit
(OEL) of bacteria, fungi, and bacterial endotoxin for industrial settings
contaminated by organic dust are 100 (3) CFU/m3, 50 (3) CFU/m3,
and 200 ng/m3 (2000 EU), respectively (Gorny and Dutkiewicz, 2002).
Allergy thresholds to common molds have been reported (Gravesen,
1979), but variations in sampling strategies and methodological limita-
tions make these very unreliable in practical settings (Dillon et al.,
1999; Miller, 1993). Therefore, the consensus is that acceptable
safe threshold limits for exposures to indoor fungi cannot be estab-
lished (Ammann, 1999; Macher et al., 1991). It is generally recom-
mended to avoid or minimize unnecessary exposures to indoor fungi
(Anonymous, 1994).
Airborne fungi could be classified on the basis of the relationship
between the two environmental factors and their combinations (i.e.,
temperature and water requirements) (water activity aw) (Vujanovic
et al., 2001). One type involves three different groups of molds, select-
ed on the basis of the quantitative and qualitative information about the
ability of fungi to sporulate under different environmental conditions:
group (i), represented by Emericella (Aspergillus) nidulans, A. niger,
and A. ochraceus, and characterized by sporulation that was more
dependent on temperature than on water activity; (ii), represented
by A. flavus and A. versicolor, in which sporulation was approximately
equal and depended on both the temperature changes and aw altera-
tions; and (iii), represented by Cladosporium sp., Penicillium
cyclopium, and P. citrinum, in which sporulation depended more
on alteration of the aw conditions than on temperature changes
(Vujanovic et al., 2001).
96 LI AND YANG
Another type is characterized by four sporulation rates with two risk

levels of mycotoxin accumulation in the spores (conidia) of each mold
species: large (Ia) and moderate (Ib) sporulation rates with a risk of
mycotoxin accumulation (aw > or ¼ 86; t > or ¼ 12 C); rare sporulation
(IIa) and absence of sporulation (IIb), without risk of mycotoxin accu-
mulation (aw < or ¼ 86; t < or ¼ 12 C) (Vujanovic et al., 2001). In
conclusion, providing a useful guide for two dimensions, temperature
and water activity, for each of the three phases of fungal growth (i.e.,
germination, growth, and sporulation) facilitates the determination
of the fundamental niche of each fungus and its ability to form or
accumulate mycotoxin (Vujanovic et al., 2001).
VI. Conclusions
Fungi are ubiquitous in nature. Fungal spores are very common both
indoors and outdoors. Fungal spores are considered to be allergens.
Some fungi are opportunistic pathogens and occasionally cause infec-
tious diseases in susceptible or immunocompromised people. Fungi
can readily grow on building materials, furniture, and other substrates
in buildings experiencing water damage/intrusion or dampness pro-
blems without immediate repairs. Subsequent proliferation of fungi
poses adverse effects on human health in the buildings.
Causal factors and agents of sick building syndrome are very com-
plex. Indoor fungi are one of the agents associated with SBS. Clinical
diagnoses of mold allergies and fungal infections are generally easier
and less complicated than emerging health concerns stemming from
such fungal metabolites as (1 ! 3)--D-glucan, mycotoxins, and fungal
VOCs. Diagnosis of SBS can be difficult and challenging. The correct
diagnosis may rely on cooperation of medical professionals with
multiple other professions so as to determine the causes of SBS.
Synergistic inhalation effects of fungal byproducts—such as myco-
toxins, -glucans, or perhaps fungal VOCs—are potentially irritating,
toxic, teratogenic, carcinogenic, and immune-suppressive. Risk assess-
ment for human exposure to fungi and their byproducts is complicated,
because it involves multiple agents, hypersensitivity reactions, and
different disease consequences. Exposure to fungi and their byproducts
at various concentrations is omnipresent in indoor environments. The
sensitivity of humans to fungi and related byproducts varies from
individual to individual. The health effects of inhalation exposure
to many fungal metabolites are not well understood. There is a great
need to better understand the adverse health effects of short-term and
long-term exposures to the fungal metabolites and to determine wheth-
er the health effects are reversible or not. It is, however, prudent to
avoid unnecessary exposures to excessive fungi and their byproducts.
At the same time, long-term research involving extensive cooperation
among several related professions is needed. Controlling indoor fungal
growth conditions is the most economical and practical approach to
maintaining a healthy environment. Indoor fungal ecology is at present
poorly studied. Future studies on indoor fungal ecology are necessary
to better understand fungal development, colonization, and succession
in the indoor environment. The information yielded from such studies
will be useful to design better buildings that are less prone to fungal
infestation.
Since the energy crisis, buildings were designed and built to be
energy efficient. At the same time it is a common practice to use new
and inexpensive materials. As a consequence, the modern buildings
are conducive to fungal growth once they experience water damage or
humidity control issues.
ACKNOWLEDGMENTS
The authors are very grateful to Mr. Brian Kerin and Mrs. Jenalynn Greer for their
assistance in preparation of the manuscript.
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Indoor Moulds and Their Associations
with Air Distribution Systems
DONALD G. AHEARN,* DANIEL L. PRICE,y ROBERT SIMMONS,*
JUDITH NOBLE-WANG,* AND SIDNEY A. CROW, JR.*
*Center for Applied and Environmental Microbiology
Georgia State University, Atlanta, GA 30303
y
Interface Research Corporation, 1603 Executive Dr.
Lagrange, GA 30241
I. Introduction 113
A. Mycotoxins 115
II. Problem Areas with Air Distribution Systems 115
A. Air Samples 116
III. Colonization of Filters 117
A. Hospitals 117
IV. Colonization of In-Duct Insulation 120
A. Metal Surfaces 124
B. Volatile Organic Carbons 125
V. Carpets and Wallboard 126
A. Stachybotrys chartarum 130
VI. Colonization of Automobile Air-Conditioning Systems 131
VII. Conclusions and Recommendations 133
References 135
I. Introduction
A wide diversity of moulds may be isolated from indoor sites and,
typically, somewhat fewer fungi can be shown to colonize indoor
surfaces. We identify fungal colonization by the presence of vegetative
growth represented by ramifying hyphae with particular attention for
conidiogenesis or dividing yeast-like cells. The colonization may be
temporal and microscopic or more extensive, obvious by sight and
smell in buildings with moisture problems. Fungi within buildings
without moisture problems exist mostly as dormant or transient forms.
Many of these transients represent airborne or food-associated moulds
from the outside environment, but indoor construction materials and
furniture may also transport and harbor dormant endogenous moulds.
Outside air typically bears a variety of mould propagules with the
potential for colonizing damp indoor surfaces (Levetin et al., 2002;
Mishra et al., 1992; Samson et al., 1994).
We have found that cellulosic ceiling tiles and paper coatings of
gypsum wallboard in the southeastern United States are especially
113
0065-2164/04 $35.00
114 DONALD G. AHEARN et al.
vulnerable to colonization by species of Acremonium, Alternaria,

Aspergillus, Ascotricha, Chaetomium, Cladosporium, Penicillium,
and Stachybotrys. These genera and Trichoderma and Syncephalas-
trum may also grow on chronically dampened wood. Minor, and some-
times extensive, mould colonizations of indoor environments may be
cryptic (e.g., air distribution systems, wall cavities) or even be ob-
servable but go unrecognized because the aesthetics of the indoor
environment for the occupant had not been unsuitably altered.
Moulds, such as Alternaria, have long been recognized as allergens,
but only about 5% of the population is estimated to show recognizable
clinical illness, and within this group, the more severe hypersensitivity
pneumonitis is rare (ACOEM, 2002). Fungal infections of humans,
perhaps with the exception of aspergillosis, are associated rarely with
moulds endogenous to indoor human habitats. In general, the classical
fungal pathogens (e.g., Coccidioides immitis) that can cause grave dis-
eases among ‘‘normal’’ or healthy individuals have an outdoor epide-
miologic association with any indoor correlations (Histoplasma and
Cryptococcus) related to deposits of bat or bird excrement. Aspergillo-
sis occurs (or is recognized) predominantly among immunocompro-
mised individuals within institutional healthcare settings. At least in
the southeastern United States, our unpublished data and reports of
others (e.g., Shelton et al., 2002) indicate that species most commonly
implicated in invasive aspergillosis (Aspergillus fumigatus, over 80%)
occur indoors at an inverse incidence to the most common species,
A. versicolor (over 90%), found indoors in hospitals and commercial
buildings (Simmons et al., 1997). We found A. fumigatus at high
incidence and densities in woodchip ground cover near entrances
and along walkways and play areas. We also found a relatively high
incidence and high densities of A. flavus associated with filters in
heating, ventilation, and air-conditioning (HVAC) systems in the
southeastern United States. These latter two species are the most com-
mon etiological agents of invasive aspergillosis, followed at a distant
third by A. niger (more often associated with otomycosis); A. terreus
has been noted only recently at an increasing incidence (Sigler and
Verweij, 2003). Infections of humans by the A. versicolor-A. sydowii
complex are rare, and Stachybotrys spp. have not been proven to cause
infections. Recovery of the various common moulds from indoor habi-
tats may not necessarily correlate with a specific incidence of disease,
including allergic fungal sinusitis, or even the diffuse complaints of
the sick building syndrome (SBS) (CDC, 2000; Hospenthal et al., 1998;
Mishra et al., 1992; Noble et al., 1997; Shelton et al., 2002; Singh and
Singh, 1999).
INDOOR MOULDS AND AIR DISTRIBUTION SYSTEMS 115
The voluminous reports on serious adverse effects of ‘‘toxic’’ and
‘‘killer moulds’’ in the lay media have caused a dramatic increase
in litigation and substantially increased costs of insurance and
mould-related remediation (see Anonymous, 2003; Belkin, 2001).
A. MYCOTOXINS
Some mould species common in indoor habitats may produce sec-
ondary metabolites during their colonization of indoor substrates. These
secondary metabolites may be toxigenic for other microorganisms.
The mycotoxins from certain moulds (e.g., Aspergillus flavus, Stachy-
botrys chartarum) are known after ingestion to be toxigenic for animals,
possibly including humans. Investigations have demonstrated that both
toxigenic and non-toxigenic genotypes exist within these morphology-
based species complexes and that toxin expression with growth on
water-damaged building materials is affected also by environmental
conditions (Andersson et al., 1997; Andersen et al., 2002; Cruse et al.,
2002; Nieminen et al., 2002; Nikulin et al., 1994; Peterson et al., 2002;
Ren et al., 1998, 1999). Reports associating toxigenic moulds, particu-
larly S. chartarum, with pulmonary hemorrhage in infants (Dearborn
et al., 1999) and neurological effects with adults (Johanning et al., 1996)
have raised recognition and controversy about potential health effects
of indoor mould. Whether the toxins are produced during colonization
of the indoor habitat in sufficient concentrations to produce hemologic
or neurologic illnesses recognizable in humans via an inhalation route
is unproven (ACOEM, 2002; CDC, 2000; Kuhn and Ghannoun, 2003;
Page and Trout, 2001). The obfuscation of the health effects of indoor
mould growth, in part, is related to the concomitant presence of the
requisite damp conditions, dust mites, other allergens such as cock-
roaches, or overall low quality of the air (e.g., high CO2) at complaint
sites. In general, these overall conditions can be associated with an
increased incidence of allergic responses and the severity of asthma
attacks (NAS, 2000). A synopsis of the mycologic status of indoor
mould investigations has been provided by Levetin et al. (2002).
II. Problem Areas with Air Distribution Systems

Our studies of indoor moulds have focused on colonization of
surfaces, particularly in association with HVAC systems. Morey and
Shattuck (1989) reviewed the major HVAC parameters that may result
in adverse air quality for building inhabitants as follows:
Systems-Design Related: Outdoor air intakes are incorrectly placed so

that they uptake exhausts or microbial-laden aerosols from plumbing
vents, cooling towers, loading areas, etc.
Construction Related: Faults in the building envelope or plumbing
leaks permit moisture to dampen susceptible substrates. Finishing
materials such as wallboard are exposed to excessive moisture from
joint compounds or fresh concrete in areas with restricted airflow. In
these instances, the air distribution system may be engineered properly
but its operation may facilitate the dissemination of fungi to susceptible
substrata throughout the building.
Building-Operation Related: The operation of the air-conditioning
system is cycled improperly so as to save energy.
Building-Maintenance Related: Air filters are not inspected or re-
placed as needed, and routine cleaning of cooling coils and condensa-
tion pans (particularly their drainage systems) is insufficient. Microbial
populations in cooling towers are not controlled. Routine cleaning
and rapid and proper response to moisture intrusions (catastrophic or
chronic) are lacking.
Building-Renovation Related: A negative indoor pressure is present
because local exhaust systems have been added without balancing
intake supply. The negative pressure results in excessive intake of
non-filtered and non-cooled air via portals aside from the designed
air-conditioning system. Renovation of interior walls has obstructed
internal air distribution.
Occupant Related: Human occupancy, or excessive electrical com-
ponents such as computers, increase ambient temperatures beyond the
capacity of the HVAC system.
All of the categories described by Morey and Shattuck (1989) indi-
rectly or directly affect dew points within the building structure and
thus microbial proliferation. Envelope leaks, chronic plumbing leaks,
catastrophic floods, and particularly operation of intake fans at speeds
excessive for the cooling capacity of the system were major factors
leading to indoor mould colonization in our studies in the southeastern
United States.
A. AIR SAMPLES
Periodic extensive sampling over several days that compares indoor
and outdoor densities of airborne fungi may provide valuable informa-
tion as to a potential source of fungi in the indoor environment. Differ-
ences between the species isolated may be as significant as differences
in densities (Samson et al., 1994). Air-sampling data alone, particularly
on a limited basis, however, can provide misleading information on the
possible relationship of indoor fungi with the health status of the
building’s inhabitants. There is general agreement that numbers of
fungi in indoor air, particularly for buildings that are not subject to
complaints of poor air quality, are usually lower than the outdoor air. A
large recent survey reported that the median indoor sample contained
about 80 CFU/m3, and outdoor samples showed median densities near
500 CFU/m3 (Shelton et al., 2002). These authors noted that no statisti-
cally significant association was observed between any common fungal
type and reported health complaints (Shelton et al., 2002). Robertson
(1997) reported that 87% of 934 indoor air samples yielded <300 cfu/
m3, whereas only 33% of 182 outdoor samples were below this number,
while 519 samples exceeded 500 cfu/m3. Because no single enrichment
medium is suitable for the growth of all fungi and because of differential
growth rates and antagonisms between various common indoor fungi—
and because systems for rapid fungal enumerations, identifications, and
differentiations between colonizing fungi and transient dormant types
from indoor reservoirs are not available—routine limited air sampling
is impractical and provides minimal useful data. For these reasons we
have made extensive use in our studies of clear adhesive-tape sampling
of surfaces and ‘‘bulk’’ sampling (actual samples may be unobtrusive
in size and be as small as several centimeters in each dimension) with
incubation in moisture chambers or purge and trap vessels (Fig. 1A–C).
The single-stage Andersen air sampler used in most of our studies is
shown in Fig. 1D.
III. Colonization of Filters

A. HOSPITALS
Simmons and Crow (1995) and Simmons et al. (1997, 1997a) found
that periodic colonization by moulds occurred on cotton-based filter
media exposed to humid air. The colonization was not restricted to
cotton filter media, however, or to fiber media with a heavy bioburden
of organic debris; colonization of polyester fibers and even the coloniza-
tion of HEPA microglass filters was observed (Fig. 2). Some unused
clean-appearing filters were colonized microscopically and sometimes
visually within 15 days. The cardboard support frames were the first to
provide visual evidence of fungal growth, followed shortly thereafter by
cellulosic filter media and usually, last, by polyester media. In several
instances, however, with two-layered polypropylene-cellulosic bag fil-
ters, the polypropylene outer layer was colonized first (Cladosporium
FIG. 1. Moisture chambers and air sampling apparatus. (A) Glove box with heater, air-
circulating fan, and soil and water reservoirs; (B) vessels with and without soil reservoirs
and various salt solutions for control of RH. Sections of various new and used materials
were suspended over the reservoirs and examined periodically for development of
microbial colonization. Chambers for certain experiments were initially sterile; (C) Purge
and trap vessel for collection of VOCs; (D) Single-stage Andersen air sampler (Grasby
Andersen, Atlanta, GA).
spp. in our studies), and fungal penetration into the inner preserved
cellulosic layer was negligible. This may have been a result of prefer-
ential adsorption of volatile organics (hydrocarbon substrates) to the
polypropylene. Our studies have indicated also that the binder for
the filter media (ethylene vinyl chloride, which is more resistant to
colonization than polyvinyl acetate) may be an additional influence on
the rate and species of fungal colonization. In general, the spectra of
species colonizing the filter frames were more diverse than those found
colonizing the filter media. Additionally, species we more often asso-
ciated with outdoor air (e.g., Alternaria, Epicoccum) colonized the
frames but not the filter media. Often the dominant species that colonized
filters in our studies were not the same as those that were dominant
colonizers of downstream in-duct insulation (Price et al., 1994; Simmons
and Crow, 1995; Simmons et al., 1997).
FIG. 2. Fungal colonization of a HEPA filter. (A) SEM of upstream side of the filter with
micro colony of Penicillium sp.; (B) Downstream side of the filter with hyphae appressed
to the microglass fibers and atypical (reduced) penicillus with conidia (examined
immediately after removal from the air duct).
Our studies (unpublished) have included examination of filters from

over 60 buildings (mainly healthcare facilities) and have included a
comparison of filters treated with a phosphated-amine preservative
(InterseptÕ, registered with US EPA for use in air-conditioning sys-
tems) versus non-treated filters. The filters were transferred to the labo-
ratory and examined microscopically. Sections were placed in moisture
chambers. On used air filters, colonization was observed mostly on the

filter media (usually cotton or polyester fibers). Entire filters with
frames were not available from all sites. For filter samples that included
the entire filter with beverage board (clay-coated, non-corrugated, re-
inforced cellulose) frame and filter media, the beverage board was often
preferentially colonized (Table I). Approximately 58% of the untreated
filters yielded fungi, versus 5% for the Intersept-treated. Among untreat-
ed filters examined, 20% were colonized on receipt, and 10.5% of these
were from hospitals. Among the phosphated-amine treated filters,
13.4% were colonized on receipt, all of which were from hospitals. After
moisture chamber exposure, 73% of the used untreated filters became
colonized within 2 weeks, versus none of the used phosphated-amine
treated filters.
In a separate set of experiments, 12 Intersept-treated filters were
placed in a hospital HVAC system and monitored for fungal coloniza-
tion over a 9-month period. Hospital engineers had reported moisture
control problems in this system (Fig. 3A, B). Filters were constructed of
solid pleated polyester cotton (80/20) media supported with beverage
board frames and were positioned as secondary bank prefilters down-
stream of the heat exchange coils, drain pan, and system fan. During
the cooling cycle, a cool mist saturated the filter frames and media.
Untreated sections of the beverage board filter frame were colonized
with Alternaria, Aspergillus, Chaetomium, Cladosporium, and Penicil-
lium following 6 weeks in a moisture-laden hospital HVAC system
with excessively low operating temperatures of 17–20 C. Exterior sec-
tions of filter frames coated with clear acrylic coating fortified with
Intersept were free of fungal colonization; however, interior sections
where the air filter media was attached to the frame were colonized
with the above-cited fungi (Fig. 3C, D). Probable areas of antagonism
between adjacent fungal colonies are suggested in Fig. 3E.
IV. Colonization of In-Duct Insulation

In several instances, extensive air sampling throughout the buildings
did not reveal evidence of the presence of fungi growing within the
HVAC systems (Ahearn et al., 1992, 1996). Activation of the units
after short periods of shutdown did not result in recoveries of fungi
from the air at the outlets that were beyond the upper range of colony-
forming units (about 25 cfu/m3) in the ambient air of the building. Only
when the unit was inactivated for weekend periods with sampling
during the first hour after activation did the numbers of recoverable
fungi significantly exceed (>300 cfu/m3) ambient air numbers. Further
TABLE I
INCIDENCE OF FUNGAL COLONIZATION ON NON-PRESERVED AND PRESERVATIVE-TREATED FILTERS*
Untreated, Intersept-treated
Untreated, colonized Intersept-treated colonized
Year filter colonized post-moisture Untreated, colonized post-moisture Intersept-treated,
examined upon receipt chamber no colonization upon receipt chamber no colonization
1995 8 29 4 1 0 11
1996 1 9 1 2 0 3
1997 5 13 1 2 0 11
1998 1 3 0 0 0 6
1999 1 6 0 1 0 3
2000 1 2 0 1 0 11
Total from 9 35 2 7 0 30
hospitals
Total
n ¼ 137 17y 62y 6 7 0y 45y
*Used, non-preserved and preservative-treated (media only) air filters were collected from HVAC systems following varying periods of use. The filters
were placed in clean plastic bags and brought to the laboratory for immediate microscopic examination. Sections of filters, 6 cm2, were placed into initially
sterile moisture chambers (IL wide-mouth polycarbonate jars with a plastic cup pedestal [bottom removed] and containing 200 ml of distilled water [>90%
RH]) (see Price and Ahearn, 1999). The samples were incubated for 72–96 h and examined with light microscopy for evidence of fungal growth.
y
Values are statistically significant with an unpaired t test (P < 0.05).
FIG. 3. (A) Improperly engineered drain pan filled with condensate; small squares
in water are bars of sanitizing agent; (B) Hyphae colonizing surface of sanitizing bars;
(C) Filters, non-preserved (left) covered with mould and preservative-treated (right) after
three weeks in filter bank; (D) Well-developed conidiophores of Aspergillus flavus on
non-preserved filter; (E) Various moulds on non-preserved filters with clear zones
suggesting antagonisms.
examinations showed that the acrylic coating of in-duct fiberglass

insulation was colonized by species of Penicillium and Cladosporium,
with apparent successional periods of conidiogenesis, which varied
with relative humidity. The time frames for colonization of various
insulation sections placed in the ducts varied with the brand and even
the placement of the fiberglass. Species of Cladosporium, Penicillium,
and A. versicolor adhered tightly to the acrylic surfaces and were not
always obvious from visual observations. Relative humidities of the
ambient air were <85%. In general, conidiogenesis by Penicillium spp.,
Aspergillus spp., and Cladosporium spp. could be detected on ‘‘tape
mount’’-microscopic examinations and specific staining of various sec-
tions of the fiberglass insulation (Fig. 4A, B). Detection of the in-duct
colonizations was difficult at several sites, because access ports were
FIG. 4. Fungal colonization of fiberglass insulation. (A) Glass filaments; (B) Same field
stained with calcofluor showing thin hyphal elements tightly appressed to glass filaments;
(C) Surface of acrylic coating of in-duct insulation, the coating was supported by a fiber
mesh with each section of the mesh supporting growth of Chaetomium sp.; (D) Enlargement
of one of the mesh areas showing mature perithecium of the Chaetomium sp.
not always available when initial air sampling was performed (Ahearn
et al., 1996, 1997). We frequently isolated A. versicolor from used and
new (less often) in-duct fiberglass insulation. In the presence of relative
humidities from about 70% to 85% (dependent on the brand of insula-
tion), colonization by the species permeated the insulation matrix (see
Ezeonu et al., 1994, 1995; Price et al., 1994). Development of micro-
scopic colonization of several types of unused in-duct insulation at low
humidities occurred by the above fungi over a 30- to 90-day period at
temperatures of 15 to 20 C both in situ and in moisture chambers. Bulk
samples of one type of fiberglass insulation from three geographically
separate buildings (Georgia, Louisiana, Texas) on transfer to moisture
chambers also demonstrated profuse production of perithecia and
ascospores by Chaetomium spp. (Fig. 4C, D).
A. METAL SURFACES
The metal ductwork itself and the supply vents can also become
colonized, mostly on painted surfaces, by species of Cladosporium
(Ahearn et al., 1991). Since this study, unpainted metal ducts also have
been found to be colonized by Cladosporium, Rhinocladiella, and
occasionally Acremonium. Volatile organics apparently adsorb not
only to fiberglass insulaton but also to the metal. Over time the aw at
FIG. 5. Fungal colonization of bare metal surface of air duct; (A) Cladosporium
sp.; (B) Acremonium sp. Growth and conidiogenesis apparently supported by adsorbed
organics from air stream.
the organic-air interphase often supports growth of C. cladosporioides
on the metal surfaces. When the metal surface is a site of chronic
condensation, Acremonium can occur (Fig. 5).
B. VOLATILE ORGANIC CARBONS

There is a general consensus in the literature that moulds, particularly
A. versicolor, one of the more common indoor fungi, have the capacity
to contribute to the indoor levels of irritant organic volatiles (Bayer and
Crow, 1993; Bjurman and Kristensson, 1992; Ezeonu et al., 1994;
Larsen and Frisvad, 1994; Pasanen et al., 1997). Often olfactory senses
are the only indication of microbial colonizations within buildings. The
spectrum of volatiles produced varies with environmental conditions
such as temperature, relative humidity, substratum, and the presence
of other organisms (Wilkins et al., 2000). We found that ethanol, ben-
zene, and the odiferous ethyl hexanol were among volatiles released
from fiberglass insulation colonized by A. versicolor and Acremonium
obclavatum (Bayer and Crow, 1993; Ezeonu et al., 1994). Pasanen et al.
(1997) found also that ethyl hexanol (2-ethyl-1-hexanol) 1-octen-len-
3-ol, 2-pentanone, 2-hexanone, 2-heptanone, 3-octanone, and 2-methyl
furan were produced following inoculum of A. versicolor.
Most of the common indoor fungi—particularly strains of Aspergil-
lus, Penicillium, and Chaetomium—perhaps with the exception of
Stachybotrys chartarum and Cladosporium cladosporioides, readily
produce olfactory volatiles with growth on a variety of substrates under
damp conditions. In some of our test systems, the numbers and con-
centrations of volatiles that were released from various substrata
decreased with development of the mixed microbial biofilms (Rose
et al., 2002).
Our preliminary studies suggest that volatiles may play a role in
growth and conidiogenesis of certain fungi within buildings. We found
that vapors of ethyl acetate, ethyl hexanol, and propanol retarded
germination of several common moulds on Sabouraud’s dextrose agar,
whereas pentane and hexane had no effect or stimulated germination
(Table II). The time for germination in the presence of acetone varied
with the species. The stimulative effect for pentane and hexane was
suggested by the trend for more rapid development of the germ tube
versus the water control. When plates of Sabouraud’s agar were inocu-
lated with selected species and incubated at 22 C to 24 C in the presence
of selected volatiles, microscopically (100) and visually detectable
growth, generally equivalent to the control, was evident by 24 h in the
presence of most of the compounds inhibitory for germination in water
TABLE II
EFFECTS OF VOLATILE ORGANIC COMPOUNDS (VOC) ON THE GERMINATION OF FUNGI
Species of Aspergillus and Penicillium*
VOC AV AN PV PCO PS PC PG PGR

y
Acetone 0 88 17 93 100 0 0 98
Ethanol 0 0 0 54 0 24 ND 0
Ethyl acetate 0 0 0 0 0 0 0 0
Ethyl hexanol 0 0 0 0 0 0 0 0
Hexane 83 100 91 97 100 100 100 100
Octane ND ND ND 92 100 ND ND ND
Pentane 100 100 100 95 100 100 67 100
Propanol 0 0 0 0 0 0 0 0
DH2O 93 100 91 100 100 100 95 100
*AV ¼ A. versicolor, AN ¼ A. niger, PV ¼ P. viridicatum, PCO ¼ P. corylophilum, PS ¼ P.

sclerotiorum, PC ¼ P. chrysogenum, PG ¼ P. glabrum, PGR ¼ P. griseofulvum, ND ¼ Not determined.
y
Average % germination: (n ¼ 10). Conidia were harvested from 5–7 days growth (25 C) on
Sabouraud’s dextrose agar, washed and suspended in 0.9% saline (106 conidia/ml); organic
compounds (100 l/10 mm diameter cellulose disks); 0.1 ml of suspension of conidia was spread
evenly on surface of Sabouraud’s agar (15 ml in 100 mm Petri dish) and 1.0 cm2 sections of agar were
transferred to prescored microscope slides that were placed in a glass Petri dish (<1.0% germination
at 0 time) containing the VOC disk; the dish was sealed and after 18 h at 25 C the number of
germinated and total conidia was determined in each of five alternate sections and the average
percent germination determined; each test system for each VOC was maintained in duplicate-
separate-sealed chambers. Data are from a representative experiment of three similar tests with varied
but overlapping spectra of species.
agar. Conidiogenesis, however, appeared to be stimulated for isolates of

A. versicolor and A. fumigatus in the presence of hexane.
V. Carpets and Wallboard

Carpets in the hallways of two of the buildings described above were
wet-cleaned over weekends and permitted to air dry with the air-
conditioning system turned off. Water from the carpets was absorbed
into the gypsum wallboard under the plastic base cove. This resulted in
the cryptic growth of Aspergillus and Penicillium under the cove on the
wallboard with conidia release at the cove borders. There was detect-
able odor in the hallways, and Penicillium spp. were recoverable in
cultures made from the carpets. The carpets (synthetic fibers) were
infrequently found to be colonized, but often they served as reservoirs
for the conidia that were seeded periodically from the colonizations
within the air ducts (Price and Ahearn, unpublished data). A relatively
new set of carpets was replaced in one building, but the new carpets
rapidly became culture positive (Ahearn et al., 1996, 1997). The pre-
sumption on the basis of positive Rodac plates (obtained by an outside
consultant) was that the carpets were supporting active growth. The
fungal colonization in the air ducts and behind the base coves was
cryptic. Upon removal of the in-duct insulation for about 9 m beyond
the air handling units, and constant running of the units over the
weekends, particularly when the carpets were cleaned, complaints of
musty odors ceased (Ahearn et al., 1996, 1997). New carpets in other
buildings were found on occasion to have had high densities of moulds
prior to their installation (unpublished data).
Incorporation of antimicrobial preservatives in the backing of com-
mercial carpet tile has been found to inhibit microbial growth, staining,
and odors (Fig. 6A) (Price et al., 1991). For example, 8-year-old carpet
tiles with the phosphated-amine preservative incorporated at the
backing were removed from a neonatal intensive care unit (NICU) in
a southwestern US teaching hospital and examined for fungal coloni-
zation and retention of inhibitory activity. Initial examination of the
soiled carpet via direct microscopy showed that the carpet was not
colonized but harbored viable conidia and other particulates. Enrich-
ment culture of small discs (4 cm diameter) of the soiled carpet tile
yielded mainly bacteria (Bacillus spp.) with colony development distal
to the actual carpet sample and no colony development on the carpet
disc sample. These results suggested that the inhibitory effect of the
preservative was still present. The soiled carpet was sprayed with a
stabilized hydrogen peroxide (3%) based carpet sanitizer, allowed to
stand 15 minutes, and vacuum extracted (Fig. 6B). The NICU carpet
tiles, cleaned with stabilized hydrogen peroxide (3%) based carpet
sanitizer, were then challenged with Aspergillus niger and Serratia
marcescens (105 cfu/ml). The 8-year-old carpet once cleaned, resisted
overgrowth by both Aspergillus niger and Serratia marcescens in the
agar overlay challenge (Fig. 6C).
In contrast to the synthetic carpets discussed above, natural fiber
carpets, particularly with jute backing and including wool and cotton,
are more readily subject to colonization by many of the common
indoor fungi. We have found species of Ascotricha and Trichoderma
colonizing wool fibers of carpets and have found Aspergillus spe-
cies colonizing the backing of non-preserved synthetic carpets. In most
instances, sources of periodic or chronic moisture intrusion were
readily associated with these colonizations (Fig. 7). Carpet pads, espe-
cially those laid directly on unsealed concrete, are particularly subject
FIG. 6. (A) Enrichment culture of non-preserved (left) and preserved (right) sections of
synthetic carpet tile challenged with Aspergillus niger. (B) Recovery of dormant or
transient microorganisms from 8-year-old preserved carpet tile (left); minimal recovery
from same carpet after cleaning with stabilized hydrogen peroxide (right). (C) Inhibition
of A. niger in enrichment agar by preservative in cleaned 8-year-old used carpet tile.
Backing of carpet (lower sections with fibers shaved off) resists fungal overgrowth (see
Price et al., 1991, for details of methods).
FIG. 7. (A) Anamorph (Dicyma ampullifera) conidiophores with conidia from adhesive
tape of wool carpet; (B) Perithecium of Ascotricha sp. (anamorph D. ampullifera) with
sympodial branching filaments; (C) Conidiophores and conidia of Aspergillus terreus
developing from bitumen layer of synthetic non-preserved carpet (three weeks in
moisture chamber).
to contamination by both bacteria and fungi. In a number of cases,

adhesive to hold the padding on the concrete or sub flooring was
subject to colonization.
A. STACHYBOTRYS CHARTARUM
Stachybotrys chartarum is not an uncommon mould in water-dam-
aged buildings and is particularly associated with gypsum wallboard
and cellulosic ceiling tiles (Fig. 8). On damp wallboard, under plastic
base coves and vinyl wall coverings, black areas with dense masses of
conidia were found on microscopic examination of tape mounts. These
colonized areas were often interspersed with sections colonized by
FIG. 8. (A) Stained ceiling tile supporting growth of Stachybotrys chartarum; (B) SEM
of conidiophores and conidia of S. chartarum on cellulose ceiling tile.
asexual stages of Ascotricha and Chaetomium, with perithecia of the
latter not uncommon. Nevertheless, swab cultures of these areas on
malt extract agar were prone to be overgrown by Aspergillus and Peni-
cillium spp. and air samples from the immediate areas, with minor
exception, failed to provide for isolation of S. chartarum, even when a
variety of media were employed. Andersen and Nissen (2000) found
that (of 22 media examined) malt extract agar supported the growth of
isolates of S. chartarum, but 2 out of 6 isolates did not sporulate. All
tested isolates of S. chartarum and C. globosum also grew on potato-
sucrose agar and V8 juice agar, but two did not sporulate. None of the 22
media were suitable for the detection of all the targeted fungi. We have
observed similar variances in germination, growth, and conidiogenesis
by these fungi on the cellulosic surfaces of wallboard (Price and Ahearn,
1999). Fine vegetative hyphae (not detectable with the unaided eye) may
permeate the cellulose coating of wallboard with about 25% water con-
tent. After three to four weeks incubation at 95% þ relative humidity
(about 30% water content), sectors may blossom with pigmented con-
idiophores bearing conidia. This occurs more rapidly in the Ascotricha
> Chaetomium > Stachybotrys. How generally this observation applies
is problematic, because we have not differentiated the metabolic types
within the S. chartarum complex (Andersen et al., 2002; Cruse et al.,
2002).
Of 17 buildings in the Georgia-Florida area in which we studied
colonizations of S. chartarum, only on three occasions could we asso-
ciate the species with the air distribution system. In one building, the
paper-glue layer under the foil coating of fiberglass insulation that
jacketed the HVAC ductwork was extensively colonized. Similarly, we
found S. chartarum under foil coating of rigid fiberglass ductwork.
These insulations, the fiberglass matrices of which yielded A. versicolor,
had been reportedly exposed to moisture during construction (Price
et al., 1995). On another occasion, we isolated S. chartarum from a
cotton rag that had been stuffed into a wall unit air conditioner to
prevent vibration. About half the buildings colonized with S. chartarum
were complaint sites (SBS) and for all but three, once the moisture and
aesthetic problems had been alleviated, complaints ceased.
VI. Colonization of Automobile Air-Conditioning Systems

Mixed fungal and bacterial biofilms occur not uncommonly within
automobile air-conditioning systems with chronic or periodic release
of noxious odors (Kumar et al., 1990; Rose et al., 2000; Simmons et al.,
1997). Air-conditioned vehicles with entrapped condensate anywhere
FIG. 9. (A) Acanthamoeba sp. cyst in a mixed bacterial biofilm on an evaporator-heat

exchanger fin; (B) Hyphae traversing open celled foam insulation surrounding the
evaporator core; (C) Mixed fungal (Rhinocladiella sp.)—bacterial biofilm on surface of
a drain tray.
in the system are subject to microbial colonization. Multiple compo-
nents—air filters, insulation foams, rubber gaskets, drain tubes, and
even the aluminum evaporator fins—may support microbial biofilms
(Fig. 9). These biofilms may develop after only short-term use of the
vehicle. The actual production of odors and fungal conidia and their
release into the passenger compartment of the automobile may be de-
pendent on the chance initiators of the diverse biofilms that can be
present. In certain instances, bacteria may predominate; in others, fungi.
The occurrence and distribution of fungal species in automobiles may
be affected by the presence of predatory Acanthamoeba spp. With
some model automobiles, the problem may be alleviated when the
automobile is parked level so that the condensation pan fully drains.
Running the AC-system blower while the automobile is parked for a
few minutes before turning off the ignition also may aid in the evapora-
tion of condensate. With certain ACs, the adoption of components non-
compliant for microbial attachment and redesign of the condensate
drainage system has been necessary. When the above approaches are
not available or economically practical, treatments to retard microbial
development in the systems may suffice (Drago et al., 2002).
VII. Conclusions and Recommendations

Fungal colonization of indoor materials is not uncommon but is
often cryptic, particularly when it occurs within the air distribution
system and within walls. Extensive use of adhesive tape-sampling,
combined with direct microscopic examination of surfaces and incu-
bation of bulk samples at specific humidity ranges, has proven helpful
in distinguishing colonizing from transient and dormant fungi.
Moisture control provides for indoor mould control; however, all
modern habitats with air-conditioning systems (or available water) will
have microenvironments that support mould growth. At some time in
the life of a structure, catastrophic or chronic water intrusions will occur
that could, if not remediated, support mould growth. Moulds, endoge-
nous to the structure or entering on a daily basis, will be present to
initiate the colonization. Halting the excess water intrusion and rapid-
ly drying the site will most often be sufficient for preventing mould
growth that could adversely impact the building’s inhabitants. The
relatively inexpensive operation of a portable dehumidifier in base-
ment areas will frequently suffice for controlling musty odors in resi-
dences following discreet incidences of water intrusion. If visible
mould colonization develops on gypsum wallboard or some other
surfaces, but structural integrity has not been compromised, cleaning
with a sanitizing agent such as a 10% bleach solution could suffice

(CDC, 2001). Such sanitation steps (which should include subsequent
drying of the material) may also be followed by application of antifun-
gal preservative coatings. If massive fungal growth is present and
structural surfaces need to be replaced, a sanitation step for dry sur-
faces should be considered, with removal of materials while surfaces
are still damp. Efficient respiratory protection that will exclude dust
particles and standard protective clothing, gloves, safety glasses, etc.,
should be sufficient for normal (excluding hypersensitive or immunos-
tressed) workers performing the remediation. If the moulds achieve
ambient particle counts of species (such as aspergilli) within a restrict-
ed airflow and cfus common to counts found for composting areas (e.g.,
10,000 cfu/m3), more stringent recommendations might be advised
(see EPA, 2001). In all instances, the air-conditioning vents to the
affected sites are sealed (including return air) immediately prior to any
remediation activities. It seems unreasonable to assume that sur-
faces, particularly nonporous types or even fabrics that have been colo-
nized by moulds, cannot be safely sanitized without major expense. In
fact, some of the more notorious moulds (i.e., S. chartarum) appear
relatively susceptible to sanitizers (Price and Ahearn, 1999).
Certain fungi are allergens, and fungal exposures may elicit low
incidences of hypersensitivity pneumonitis or allergic fungal sinusitis,
particularly in the immunocompromised individual. Although these
maladies have been more frequently recognized in the past several
decades, they have not as yet shown high incidences of definitive
associations with SBS or fungal colonization with a specific indoor
habitat (Noble et al., 1997).
Our experiences indicate that in many instances, indoor materials
can be sanitized to an acceptable level and that the use of preserved
materials combined with good maintenance can significantly reduce
the extent of indoor mould colonization. In most of our published
studies, we have examined a phosphated-amine preservative of low
water solubility and low toxicity. Foarde and Menetrez (2002) also
examined this preservative and found it effective in sealants for retard-
ing fungal development in fiberglass duct liner and on galvanized steel.
There is no question that musty indoor odors produced by mould
growth are unpleasant and can significantly reduce the quality of in-
door air for all inhabitants. For certain allergic individuals, mould
growth may make particular materials such as clothing, furnishings,
or even the habitat unacceptable. The number of individuals whose
health are significantly affected by indoor moulds is unknown, but
undoubtedly fewer people are affected than intimated in some investi-
gations and in the recent popular press.
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Microbial Cell Wall Agents and
RAGNAR RYLANDER
Department of Environmental Medicine
University of Gothenburg
Gothenburg, Sweden
I. Historical Perspective 139

II. The Nature of SBS 140
III. Indoor Microbial Contamination 142
IV. MCWA and Inflammation 143
V. Endotoxin 144
VI. (1 ! 3)--D-glucan 146
VII. Endotoxin and (1 ! 3)--D-glucan in Relation to SBS 147
VIII. Focus on Children 149
IX. Treatment and Prevention 150
References 151
I. Historical Perspective
Ill health related to indoor conditions has probably been observed
ever since humans moved into huts and other primitive dwellings.
Measures for judgment and prevention of indoor conditions were well
developed hundreds of years BC, as evidenced by the regulations laid
out in the book of Leviticus (for details see chapter ‘‘Fungi and Indoor
Environment’’). During the early days of Roman civilization there were
accounts of the effects of bad air indoors, and in the 1500s, Erasmus
Rotterdamus in his treatise ‘‘The Inn’’ vividly described the interior air
in wayside inns ‘‘But nothing is more dangerous than when many
people breathe the same air . . . and apart from all burps, the smell of
garlic and bad breath, many suffer from hidden diseases and every such
disease is contagious. . . .’’
The first scientific account of health and indoor conditions stems
from the Dutch physician van Leeuwen, who described the relation
between asthma and damp buildings in the Netherlands (van Leeuwen,
1924). Changes in ventilation characteristics to save energy, innovative
but unproven building designs, and questionable building practices
led to an almost epidemic-like reporting of symptoms and disease
indoors during the 1970s and 1980s. Initially the health problems were
poorly understood or misdiagnosed, and as always when uncertainty is
involved, they were attributed to hearsay, hysteria, or imagination.
139
0065-2164/04 $35.00
140 RAGNAR RYLANDER
It is now quite clear that the symptoms reported under the umbrella
of ‘‘sick building syndrome’’ (SBS) are real, that there is an underlying
pathophysiology, and that the causative exposure may be multifactori-
al. The presence of symptoms may be related to the exposure to chemi-
cal agents such as ozone, to physical agents such as low frequency
noise, and to microbes. The latter contain a number of specific agents
in the cell wall, microbial cell wall agents (MCWA) that have important
toxicological and immunological properties, some of which occur in a
synergistic fashion. The evidence for the importance of MCWA is now
well established, and it is gradually becoming possible to make risk
assessments based on measured values. This presentation focuses on
the particular role of MCWA in sick building syndrome. Allergic reac-
tions to specific allergens or effects caused by mycotoxins will not be
discussed.
II. The Nature of SBS

Humidity indoors is a major factor related to symptoms occurring
indoors and which have been referred to as ‘‘sick building syndrome’’
(Wieslander and Rylander, 2003). The term is a misnomer, as the
phenomenon is a conglomerate of symptoms among sick persons rather
than a particular characteristic of a building construction. What, then,
is the nature of the symptoms?
The majority of studies on indoor ill health have collected the
information on symptoms by using questionnaires. Although this in-
strument is subject to methodological errors and subjective influence,
the similarity in symptom profiles from the different investigations is
rather remarkable. The symptom profile is found in Table I.
TABLE I
SYMPTOM PROFILE FOR SICK BUILDING SYNDROME
Mucosal irritation
Skin, eyes, airways
Skin problems
Rash, itching
Systemic symptoms
Fatigue, headache, lethargy, joint pains
Neurological symptoms
Loss of sensitivity, pain
MICROBIAL CELL WALL AGENTS AND SICK BUILDING SYNDROME 141
The symptom profile comprises a series of non-specific symptoms
that are always present in the population. In studies, it is thus neces-
sary to have a non-exposed control group and compare the prevalence
of the different symptoms in the investigated building with those in
normal buildings. Another characteristic of the symptom profile is that
the symptoms may be widespread, and prevalence figures of 50–60%
have been reported.
While it was initially thought that the symptoms reflected an allergic
disease and allergic asthma in particular, it is now understood that the
underlying pathology is an inflammatory response as depicted in Fig. 1.
It is seen that the inflammatory response in the airways may have
two underlying pathologies—an allergic, IgE-driven reaction and a
non-specific inflammation caused by a toxic reaction. An inflammatory
response in the lungs may also cause systemic effects because of the
spread of inflammatory cytokines in the blood from the lung to other
organs in the body. Tiredness and headache as examples of such
systemic effects occurring as the results of effects in the brain and an
FIG. 1. Relationship between environmental exposures and different forms of

inflammation.
142 RAGNAR RYLANDER
increase in the blood level of C-reactive protein reflects the effect on the
liver. There are also data that suggest an effect on the joints, although
no clinical markers have yet been identified.
Apart from the symptoms reviewed in Table I, there is evidence that
the exposure indoors in humid buildings causes an increased risk for
infections (Husman, 1996). Because SBS is predominantly found in
buildings with humidity problems, we will in the following examine
the presence of microbes in the indoor environment.
III. Indoor Microbial Contamination

There are always a certain number of bacteria and molds in any
environment, including the indoor environment. Usually the levels
are low and do not cause any harm. This is particularly true for molds,
which are present in all environments, and fungal spores can be found
in the lungs of normal, healthy persons. Exposure to environmental
microbes takes place during normal daily activities such as when
cleaning dusty floors, emptying dustbins, or removing objects from
dusty bookshelves. Outdoor levels of bacteria and fungi depend on
climatic conditions and humid climates. Indeed, particularly when it
is windy, a large number of airborne fungal spores are present. These
normal exposures are usually not related to medical effects, except for
persons sensitized to fungi, who may develop an allergic reaction after
high level exposure or when meteorological conditions cause the dose
levels to increase very much above normal (Newson et al., 1997).
The problems start when there is an imbalance in the indoor micro-
environment. The reason is usually a dramatically increased humidity,
either generally or in specific areas of the building. An increase in
general humidity may be caused by inadequate ventilation, inhabitant
habits such as showering, or storage of humid clothes or furniture.
Specific area problems arise when there is water damage such as leak-
ing roofs or windows, burst water pipes, or basement flooding.
Under conditions of increased humidity, there is a very rapid growth
of molds. They may grow on surfaces at a material humidity above 12%
or when the air humidity exceeds about 80%. Once growth is estab-
lished, the spores remain even if desiccation procedures are under-
taken. Bacteria will grow in stagnant water such as water reservoirs in
humidifiers, open cesspits, or in bathrooms. The exposure in humid
buildings comprises a mixture of different microbial species depending
on local conditions. In some indoor environments, mold may be domi-
nant; in others Gram-negative bacteria from water reservoirs or pets are
dominant (Heinrich et al., 2001).
The most well-known effects of microbes are their capacity to settle
in the host, multiply, and cause an infection. There are, however,
specific agents in the cell wall of microbes that have the capacity to
induce an inflammatory response without the need for growth of mi-
crobes within the host. A common characteristic for these MCWA is
their extreme toxicity; they exert biological effects in concentration of
nano- and even picograms.
TABLE II
EXAMPLES OF MICROBIAL CELL WALL AGENTS (MCWA)
Source Agent
Gram-negative bacteria Endotoxin

Fungi, plants (1 ! 3)--D-glucan
Gram-positive bacteria Peptidoglycans
Gram-positive bacteria Lipoteichoic acid
A number of MCWA have been identified, as illustrated in Table II.

In relation to exposure, it is important to realize that MCWA retain
their biological activity even when the organism is dead. An estimation
of the number of viable microbes may thus be very misleading in terms
of dose estimate—the number of dead fungal spores may be millions of
times higher than the number of viable spores. In the following, we will
review the role of MCWA for the development of inflammation.
IV. MCWA and Inflammation

In an evolutionary sense, organisms have always had the need to
defend themselves against microbial products, which imply a risk for
infection or inflammation. This concept is reflected in the number of
ways that man defends himself against exposure to MCWA. This de-
fense involves attachment of MCWA to cells through particular recep-
tors, the triggering of a defense reaction through inflammatory cell
activation, and finally the breakdown of the MCWA. These defense
reactions may secondarily cause symptoms and under chronic condi-
tions, there may be clinical disease caused by the physiological and
cellular changes that take place during the defense response. A
swelling of the epithelium caused by neutrophils invading to combat
microbes may be experienced as a swelling that hampers normal
breathing, and a severe inflammatory reaction in the airways may even
lead to suffocation such as in an acute asthmatic attack.
144 RAGNAR RYLANDER
The pathogenic effects caused by microbes are not only infection

but also toxic reactions, caused by different molecular structures in
the cells. The basic defense against microbes comprises an innate
immunity recognition of microbial-line encoded molecules. This re-
presents a strategy of defense not directed against individual, highly
specialized antigens, but with a focus on a few, highly conserved
structures that are present in larger groups of microorganisms. Such
structures from microorganisms are referred to as pathogen-associated
molecular patterns (PAMP). The receptors for PAMP are referred to as
pattern-recognizing receptors (PRRs).
An important group of PRRs is the toll-like receptor (TLR) family
(Janssens and Beyaert, 2003). This class of receptors is present even in
very primitive organisms and has remained unaltered over the evolu-
tion, signifying their relevance for survival. This is in contrast to the
adaptive immunity, which developed much later during evolution.
TLRs have more than 10 different forms, specific for MCWA (Table III).
TABLE III
TOLL-LIKE RECEPTOR (TLR) SUBGROUPS AND MICROBIAL CELL WALL AGENTS (MWCA)
MCWA TLR reactive unit
Endotoxin TLR-4
(1 ! 3)--D-glucan TLR-1 þ TLR-2?
Double-stranded (ds) DNA TLR-3
Flagellin TLR-5
CpG DNA TLR-9
Peptidoglycan TLR-2 þ TLR-6
Activation of cells through the TLRs induces a defense response

characterized by activation of cellular transcription factors and expres-
sion of inflammatory cytokines (Chow et al., 1999; Lien et al., 2000). In
the following, we shall examine in detail two important MCWA—
endotoxin and (1 ! 3)--D-glucan—and describe how they induce an
inflammation.
V. Endotoxin
Endotoxin stems from the outer cell wall of Gram-negative bacteria.
These are ubiquitous in nature in terms of Enterobacter and Pseudo-
monas species on vegetation and in soil. Animals and man are impor-
tant sources in terms of the intestinal load of Escherichia coli, and
indoor contamination is due primarily to fecal contamination, parti-
cularly from pets. Gram-negative bacteria grow rapidly in stagnant
water such as humidifier reservoirs and also in organic garbage that is
kept indoors several days. Endotoxin is a lipopolysaccharide (LPS)
compound, where the polysaccharide part is responsible for the anti-
genic characteristics and lipid A for the toxic effects. Although the
chemical composition is LPS, this product is an artificial compound.
The LPS molecules in the environment are mostly connected to cells or
cell fragments, and it has been suggested to apply the term endotoxin
for this form and reserve the LPS denotation to the chemically pure
agent.
The cellular mechanisms behind the inflammagenic effects in-
duced by endotoxin are relatively well known (Martin, 2000; Schletter
et al., 1995). An important part of this event is the binding of endo-
toxin to CD14 (Dentener et al., 1993). At the molecular level, CD14
acts by transferring endotoxin and other bacterial ligands from the
circulating LPS-binding protein to the TLR-4 receptor, which together
with the MD-2 molecule activates innate host defense mechanisms,
such as release of inflammatory cytokines, and in upregulation of
co-stimulatory molecules (Miyake, 2003).
The inflammagenic properties of endotoxin contribute to lung dis-
ease in the occupational as well as the home environment. In occupa-
tional environments with high exposure levels to organic dusts,
endotoxin has been related to a variety of symptoms and clinical find-
ings such as chest tightness, decrease in lung function, and production
of inflammatory cytokines (Rylander, 2002). In the home environment,
the amount of endotoxin has been related to the severity of asthma and
chest symptoms from children (Michel et al., 1991, 1996).
Several studies have reported the effects of an acute inhalation of
pure endotoxin in humans. There are local effects in terms of an increase
in the number of neutrophils and levels of the inflammatory marker
fibronectin in the airways, a dose-related decrease in pulmonary fun-
ction, a decreased alveolar-capillary diffusion, and an increased airway
responsiveness (Herbert et al., 1992; Rylander et al., 1989; Sandström
et al., 1992). There are also systemic effects in terms of an increased
number of neutrophils in the blood, an increased amount of tumor
necrosis factor- (TNF-), myeoloperoxidase, and C-reactive protein
indicative of a general inflammatory response (Michel et al., 1996,
2001; Thorn and Rylander, 1998a). At high doses, there is also an
increase in body temperature. Symptoms are irritation in the throat,
chest tightness, tiredness, and headache. This clinical entity is referred
to as toxic pneumonitis, caused by the toxic effects of endotoxin
146 RAGNAR RYLANDER
and distinct from the infectious pneumonitis, which is caused by

microorganisms multiplying in the lung.
VI. (1 ! 3)--D-glucan

The cell wall of fungi, certain bacteria, some plants, and pollen
contains a specific polyglucose agent—(1 ! 3)--D-glucan (Fogelmark
and Rylander, 1997; Stone and Clarke, 1992). This has unique
biological properties because of the steric nature of the binding
between the polyglucose chains.
The role of (1 ! 3)--D-glucan in inflammation is still unclear.
Results from in vitro experiments and intravenous administration dem-
onstrate that it up-regulates inflammagenic cells, particularly macro-
phages (Sakurai et al., 1997). In inhalation experiments in animals,
(1 ! 3)--D-glucan does not cause the typical neutrophil response
induced by endotoxin, but it dampens the neutrophil invasion caused
by endotoxin in a dose-response fashion (Fogelmark et al., 1997). In
experiments on mice, exposure to (1 ! 3)--D-glucan caused an
increased expression of messenger RNA (mRNA) encoding interleukin
(IL)-10, a T-helper cell (Th)2 driving cytokine, whereas expression
of IL-12, promoting interferon (IFN) production, was depressed
(Wan et al., 1999). Inhalation of (1 ! 3)--D-glucan also suppressed
the formation of antibodies against inhaled ovalbumin (Rylander
and Holt, 1998).
In humans, inhalation of pure (1 ! 3)--D-glucan caused no effects
on respiratory function or airway responsiveness (Rylander, 1996).
There was a very slight increase in throat irritation and cough. In a
following study, inhalation of (1 ! 3)--D-glucan caused a decrease in
the number of blood eosinophils (Thorn et al., 2001). The TNF-
production from blood monocytes stimulated in vitro with endotoxin
was decreased as compared to the increase caused by inhalation of
saline only. These findings were confirmed in another study suggesting
that (1 ! 3)--D-glucan interferes with the ability of blood monocytes
to respond to an inflammagenic stimulus (Beijer et al., 2003).
In view of the dampening effect of (1 ! 3)--D-glucan on the inflam-
matory responses, it may be hypothesized that this depressing effect is
also responsible for a decrease in the defense against infectious agents.
This could explain the higher incidence of infections among children
in humid buildings (Husman, 1996). No experimental data to support
this hypothesis are available.
In the following, we will examine the relationships found between
SBS and the presence of endotoxin and (1 ! 3)--D-glucan indoors.
VII. Endotoxin and (1 ! 3)--D-glucan in Relation to SBS
As depicted in Fig. 1, exposure in a real-life situation comprises a
mixture of agents in different proportions depending on local condi-
tions. It is important to realize that when field studies have measured
the amount of endotoxin and/or (1 ! 3)--D-glucan, these can only be
seen as indicators of the number of (viable or nonviable) Gram-negative
bacteria or fungal cells. Conceptually, they may be the causative agent
or a surrogate for this. A conclusion concerning causality for different
agents can be drawn only if the effect measured is specific for the agent,
such as the neutrophil invasion after endotoxin exposure, which is not
present after exposure to (1 ! 3)--D-glucan, or the Th2-type response
induced by (1 ! 3)--D-glucan that is not caused by endotoxin. All data
on the relation between endotoxin and (1 ! 3)--D-glucan and the
presence of symptoms in the following must therefore be evaluated in
light of the above.
In the home environment, a relation has been found between the
amount of endotoxin in the dust and the severity of asthma in terms of
medication and clinical scoring (Michel et al., 1996). Among children,
a significant relationship has been reported between clinical asthma
scores and levels of endotoxin in their homes (Rizzo et al., 1997). In
another study, the amount of endotoxin in the house dust was inverse-
ly related to the presence of symptoms of shortness of breath, skin rash,
and cough (Park et al., 2001).
Some data suggest that the inflammation induced by endotoxin in
the home environment may be protective against atopic sensitization.
In a study among 61 infants, it was found that the risk for atopic
sensitization was inversely related to the amount of endotoxin in house
dust (Gereda et al., 2002; Gehring et al., 2002). Among children living
on farms where the prevalence of atopic sensitization is known to be
low, indoor endotoxin levels were higher than in the control group
(von Mutius et al., 2000). A relation has been reported between the
levels of endotoxin in children’s mattresses and a reduced risk for hay
fever, allergic asthma, and atopic sensitization but not for non-atopic
wheeze (Braun-Farländer et al., 2002). It was also found that blood cell
production of Th1 type inflammatory cytokines was down-regulated.
The present scenario suggests that the level of endotoxin indoors
describes the risk for airway inflammation with corresponding symp-
toms of cough, irritation, and wheeze. On the other hand, endotoxin
protects against atopic sensitization and atopic dermatitis. There is
thus a dual nature in the endotoxin-induced effects and in real life
there will be a balance between what is beneficial against atopy and
148 RAGNAR RYLANDER
what causes symptoms of airway inflammation. There is no informa-

tion on the levels at which these radically different outcomes occur.
Regarding (1 ! 3)--D-glucan, an early study on SBS showed a dose
response between the amount of (1 ! 3)--D-glucan in air samples of
agitated floor dust and the extent of eye and throat irritation, dry cough,
and itching skin (Rylander et al., 1992). A following study in a day-care
center examined the personnel before and after a building renovation
(Rylander, 1997). The values of (1 ! 3)--D-glucan in airborne agitated
floor dust were 11.4 ng/m3 before and 1.2 ng/m3 after the renovation.
Among the personnel, the number of persons with increased airway
responsiveness, indicative of airway inflammation, decreased after the
renovation. In a study on two schools, one with mold problems, the
average levels of (1 ! 3)--D-glucan in airborne agitated floor dust was
15.3 vs 2.9 ng/m3 (Rylander et al., 1998). Among children, the extent of
symptoms of dry cough, cough with phlegm, and hoarseness was higher
in the school with mold problems. The symptoms were more frequent
among atopic pupils. There was also a seasonal variation (Fig. 2).
One study examined 35 persons in homes with suspect or known
mold problems (Beijer et al., 2003). When they were divided into
those with high and low levels of (1 ! 3)--D-glucan in their homes
(6.0 vs 0.9 ng/m3 airborne agitated floor dust), the secretion of TNF
from blood mononuclear cells was higher among those with high levels
of (1 ! 3)--D-glucan in their homes. Among non-atopics, the ratio
FIG. 2. Extent of nasal irritation in schools with mold problems (squares) and control
schools (circles) during different months (after Rylander et al., 1998).
interferon /interleukin-4 (IFN /IFN-4) was also higher in homes with
high levels of (1 ! 3)--D-glucan. These data suggest that the home
exposure precipitated a Th1-like cytokine reaction pattern. In a study
on children, a relation was found between the levels of endotoxin and
(1 ! 3)--D-glucan and peak flow variability (Douwes et al., 2000).
One investigation explored the relation between different methods to
sample dust particles from a house, analyzed for the content of endo-
toxin and (1 ! 3)--D-glucan, and related the levels to the presence of
clinical markers of inflammation (Beijer et al., 2004). An association
was found between endotoxin in the air and the ratio IFN- /IL-4,
suggesting an inflammatory response of the Th1 type. In contrast, a
study on children in moldy houses reported a lower number of IFN
producing T-cells during the first year of life, suggesting that inflam-
magenic outcomes may be age dependent (Lehman et al., 2002).
Regarding (1 ! 3)--D-glucan, a relationship was found for the total
amount of IgE in serum (Beijer et al., 2004). This supports data from a
previous investigation, where a relationship was found between the
number of airborne viable molds and the amount of total IgE (Su et al.,
2004). Whether this increase signifies an increased risk for atopy and
allergy is not certain, although data from other studies suggest that the
proportion of atopic persons is higher in environments with high
exposure to molds (Thorn et al., 1998b).
The exposure to (1 ! 3)--D-glucan in the above field studies should
be interpreted as an exposure to mold cells, and it cannot be concluded
that (1 ! 3)--D-glucan is the causative agent. In that sense, the data
confirm the findings from many other studies on a relationship be-
tween mold exposure and the presence of symptoms that have been
reviewed elsewhere in this book (see the Chapter ‘‘Fungi and the
Indoor Environment: Their Impact on Human Health’’).
VIII. Focus on Children

As has been described above, the majority of symptoms in SBS are
rather unspecific and relate to inflammation in general. It is thus no
surprise that even extended symptoms among children in terms of
fatigue, headache, and irritation in the airways are easily explained
as ‘‘normal,’’ reflecting the child’s age, the school environment, the
child’s behavior in general, etc. This is particularly apparent when the
family physician has ruled out the diagnosis of allergic asthma and has
no other explanation to offer the worried parents. The following illus-
trates the severity of suffering that children may experience in humid
and moldy houses.
150 RAGNAR RYLANDER
A family with two boys (2 and 4 years old) was living in a villa where
moisture problems developed soon after the construction was finished
(Rylander et al., 1994). The boys had an irritation in the airways that
was initially looked on as ‘‘frequent colds’’ but was only later asso-
ciated with staying in the house. The symptoms disappeared during
summer vacations and when they stayed with their grandparents.
When symptoms grew worse, a skin prick test was performed; one
boy was negative to all allergens tested and the other was positive only
to house dust mite. None of them has a reaction to mold allergens. One
boy then developed several episodes of severe dyspnea and broncho-
constriction that had to be treated in the emergency room at the local
hospital. Finally, both children moved in with their grandparents and
the symptoms disappeared. After an extensive renovation of the house,
the boys returned without suffering from any symptoms.
IX. Treatment and Prevention

The presence of symptoms that by the person is related to a particu-
lar building must always be taken seriously. An inspection of the
building should aim to evaluate the presence of agents that may cause
inflammatory responses. Sources of chemical agents should not be
forgotten; examples of the many possible such agents are solvents in
connection with painting, cleaning or hobby work, ozone and particu-
lates from copying machines, and toxic substances remaining from
construction materials. Regarding MCWA, focus should be on humidi-
ty problems. The question ‘‘is there condensation on the inside of
windows?’’ has been used as a simple means to detect general humidi-
ty problems. Histories of past or present water penetration from the
outside or leaking water pipes are important in the search work. As
MCWA from bacteria and molds retain their biological activity in dead
microbial cells, water damage problems in the past may be important.
Water damage is not always visible; there may be humid spots inside
the building construction from which mold spores penetrate into the
room with normal air movements inside the building.
It is not adequate to treat the symptoms among affected persons. If
the relation between symptoms and a particular building can be estab-
lished (‘‘I am much better when I stay with my sister,’’ ‘‘Every time I
come back from the holidays I feel these symptoms start again,’’ etc.),
there is no choice but to remedy the building. If the symptoms are
present among children, they should be moved to avoid the risk for
developing atopy and possibly severe allergic disease later on in life
(Lehman et al., 2002; Savilahti et al., 2001).
There are at the present no generally accepted methods to measure the
indoor contamination with MCWA. Analysis of endotoxin or (1 ! 3)--
D-glucan can be performed on dust from the floor, airborne agitated floor
dust, airborne dust, or dust collected on surfaces suspended in the
houses. The analysis for endotoxin and (1 ! 3)--D-glucan is generally
made by using the Limulus lysate method (Thorn and Rylander, 1998),
although (1 ! 3)--D-glucan can also be determined by using an ELISA
assay against purified (1 ! 3)--D-glucan from three species of molds
(Douwes et al., 1996). Regarding the Limulus assay (Tamura et al.,
1994), there is a rather large variation between laboratories, although
the reproducibility within a single laboratory is usually quite good
(Chun et al., 2002). This variation in the methods for sampling and
analysis makes comparisons between studies difficult and constitutes
a major obstacle toward the setting of standards for preventive and
control purposes. In one study, a threshold value of 25 ng/mg floor dust
was suggested for an increase of total IgE in the serum (Beijer et al.,
2004). This number needs to be verified in future studies but could in the
meantime be used as a hypothesis for further work and risk estimations.
Experience tells us that many of the reasons for humidity problems
inside buildings are related to building practices. It might be desirable
in the future to appoint a particular person as responsible for humidity
control both in terms of building materials and construction details
to avoid problems with the finished product. The discovery of exten-
sive medical problems among inhabitants of a building, particularly
the children, is a sign that prevention and control has failed and is
not acceptable from a public health point of view.
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1230–1234.
The Role of Stachybotrys in the Phenomenon
Known as Sick Building Syndrome
EEVA-LIISA HINTIKKA
Finnish Institute of Occupational Health
Uusimaa Regional Institute of Occupational Health
Arinatie 3A, FIN-00370
Helsinki, Finland
I. Introduction 155
II. History of Stachybotrys Problems 156
III. Stachybotrys in Veterinary Medical Literature 157
IV. Biological Methods to Evaluate Stachybotrys-Contaminated
Material and Toxins 160
V. Methods to Evaluate Stachybotrys Growth 161
A. Toxic, Less-Toxic, and Nontoxic Stachybotrys Strains 161
B. Stachybotrys in Building Materials and Indoor Samples 162
VI. Airborne Stachybotrys Spores in Buildings and Stachybotrys
Toxins in Building Materials 164
VII. Mycotoxins Found in Dust and Air Samples 165
VIII. Human Stachybotrys Toxicosis Case Reports 166
IX. Conclusions 169
References 170
I. Introduction
If Stachybotrys is found in a building, special attention is directed to
this finding. In many countries today Stachybotrys is considered the
most dangerous and alarming fungus found in indoor air or in building
materials (Andersson et al., 1997).
In veterinary medicine, Stachybotrys has been described as a toxin-
producing fungus that causes diseases in domestic animals. Naturally
the evidence of a new disease-causing fungus in an outbreak where a
few animals are sick cannot withstand all scientific criteria today.
Often the clinical picture is exactly described and even laboratory
examinations are included in reports. Gradually, more information
has been gathered, and the picture of Stachybotrys mycotoxicosis in
different animals has been described in more detail.
Often mycotoxins are first detected and reported as disease-causing
agents in animals and only later has their importance to human health
been verified. Aflatoxin killed turkeys and poultry in the United King-
dom in the early 1960s, and some years later animal experiments
revealed it to be carcinogenic and therefore potentially dangerous in
155
0065-2164/04 $35.00
156 EEVA-LIISA HINTIKKA
human food. Diseases caused by toxin-producing Fusarium species

were described in domestic animals in Eastern Europe, and subse-
quently epidemics of alimentary toxic aleukia (ATA) were reported in
humans as well. Farm animals eat food that may be more moldy than
the food of people. Usually animals also live under circumstances
with higher fungal exposure than do people and thereby represent
‘‘experiments’’ never allowed in human medicine.
In this chapter the role of Stachybotrys will be described partly
through examples taken from selected papers. Less-cited papers pub-
lished in other languages than English are included in the list of
references. The picture given of so-called ‘‘sick buildings’’ and the
indoor air problems in these buildings is the sum of many physical,
chemical, and microbial factors. The roles of the various important
factors must be examined in this context.
II. History of Stachybotrys Problems

The early history of Stachybotrys goes back to the Ukraine and can be
linked to the highest political levels in the Soviet Union. Nikita
Khrushchev was ordered by Stalin to take the highest post in the
Ukraine as First Secretary of the Ukrainian Central Committee. In
his memoirs Khrushchev (1971) gives the following description of a
dangerous problem that he faced at the beginning of his new political
career in the Ukraine in 1939. Horses were dying on farms all over the
western parts of the Ukraine. No one had any idea what was making
the horses sick. It was suspected that the horses were being poi-
soned by people trying to sabotage the economy and military capacity.
Stalin was distressed by the problem. War was imminent. Jews, veter-
inarians, and professors were put in jail for this alleged poisoning of
horses.
Horses were very important in these times. They were not just live-
stock, they were what tanks, airplanes, and jeeps are today. Soviet army
transport depended almost entirely on horses. Khrushchev decided to
set up a commission to look into the mysterious deaths of the horses.
However, he was faced with a problem: there had already been several
such commissions, and when the horses kept on dying, the commis-
sions had been dissolved and their members arrested. With some
justification, it was widely thought that appointment to serve on one
of these commissions sealed a man’s fate. Khrushchev wrote, ‘‘I can’t
believe that science is absolutely helpless here. Surely if we make a
concerted effort, we can isolate and identify the cause of the deaths. I
think we should set up another commission to investigate.’’
ROLE OF STACHYBOTRYS IN SICK BUILDING SYNDROME 157
A new commission, with the president of the Academy of Sciences
as chairman, was appointed. Khrushchev promised to attend all
the plenary sessions of the commission himself and to listen to the
reports of the scientists to help avoid the commission being accused of
anything. Another precaution was taken by setting up a second com-
mission to exclude the possibility of saboteurs. Just to be absolutely
safe, a third commission, made up of Russian scientists from Moscow,
headed by Professor Vertinsky, was set up. All three groups went out to
investigate the stricken farms.
Later, one of the Ukrainian commissioners, Professor Dobrotko, came
to the conclusion that the horses were affected by a fungus that grew in
the damp hay. Dobrotko said, ‘‘When I realized this must be the cause, I
even contaminated myself with the fungus to see what would happen,
and I came down with an illness very similar to the one which was
killing the horses.’’ Professor Vertinsky, a Moscovite, was not willing to
accept Dobrotko’s view, however. To avoid a clash between the two,
Khrushchev suggested that the investigation be continued to make
certain that the answer was found.
After a long time, Vertinsky admitted that he agreed with Dobrotko’s
findings. The field investigation came to an end and a report was
written. The recommended method for stamping out the disease was
simple: keep the hay dry.
Both Professors, Dobrotko and Vertinsky, were awarded honorary
Orders. Khrushchev wrote, ‘‘We had won more than just a victory for
our agriculture. It was a moral and political victory as well. But how
many collective farm chairmen, cattle raisers, agronomists, animal
husbandry specialists, and scientists had lost their heads as saboteurs
before I stepped in and took charge of the situation.’’
III. Stachybotrys in Veterinary Medical Literature

Forgacs (1972) has written an excellent review on Stachybotrys and
its pathogenic role, covering the time up to the early 1960s.
S. alternans and Stachybotrys toxins have a prominent place in
the Russian mycotoxin literature. The textbooks on mycotoxins by
Bilaj and Pidoplishko (1970), Sarkisov et al. (1971), and Spesivtseva
(1964) contain extensive reviews of the Stachybotrys toxin research
and the clinical and pathogenic descriptions of Stachybotrys tox-
icosis. In the English mycotoxin literature, the survey by Palyusik
(1970) supplements the reviews by Forgacs summarizing the Stachy-
botrys toxin research with reference to the Russian and other Slavic
authors.
The studies on Stachybotrys toxicosis in different animals are re-

viewed by Hintikka (1977). An encyclopedic mycotoxin handbook,
Mycotoxic Fungi, Mycotoxins, Mycotoxicoses, contains references by
Hintikka (1977–1978) on descriptions of Stachybotrys toxicosis in
different domestic, wild, and laboratory animals, a short review of
human Stachybotrys toxicosis, and mycological and toxicological data
on Stachybotrys. Papers on the hemolytic, antifungal, antibacterial,
and phytotoxic effects of Stachybotrys have been published previously.
Schneider et al. (1979) described suspected Stachybotrys toxicosis in
sheep in South Africa. In this outbreak, 109 out of 568 merino sheep
died. Unseasonably heavy rainfall had lasted for over 6 months during
the entire period of harvesting, baling, and stacking of grain and straw.
The straw became wet and visibly moldy. The source of the Stachybo-
trys contamination was wheat straw containing up to 8.3 million
Stachybotrys spores per gram of ground straw. There was a clear rela-
tionship between feeding the animals with Stachybotrys chartarum–
infested cubes and the mortality of the sheep. The main clinical find-
ings were hemorrhagic septicemia and sometimes very acute or anemic
cases with progressively severe anemia.
Pathological findings of the early cases revealed a marked pulmonary
edema and congestion. There was variation in the degree and occur-
rence of hemorrhage in the respiratory organs, gastrointestinal tract,
and subcutaneous, subpleural, and subperitoneal hemorrhage. In a
feeding experiment, cubes of ground straw and wheat straw were fed
to 1-day-old ducklings and weanling rats; death occured within 6 and 9
days, respectively. Intradermally injected crude extracts of Stachybotrys-
contaminated straw cubes and straw caused severe necrotic skin
reactions in rats.
Tantaoui-Elaraki et al. (1994) and annonym Bull (1991) describe a
disease outbreak in the equine population in Morocco. Out of 242
exposed animals, 216 died rapidly, including 113 mules, 85 donkeys,
and 18 horses. The authors studied the toxicity of the eight Stachybo-
trys strains isolated from straw from the farms where Stachybotrys
toxicosis was suspected. The toxicity of these strains varied in the
different tests. In a feeding test in mice, one strain killed the animals
in a few hours, whereas four strains caused no mortality, even though
some signs of intoxication were noted. Only one of the eight strains
showed dermotoxic reactions on rat skin. The toxicity of the eight
strains to brine shrimp (Artemia salina larva) varied as well. One
isolate caused mortality to start within the first 30 minutes of contact,
and one isolate did not cause a notable increase in mortality even in 24
hours.
In Hungary, disease cases in sheep have been described by Hajtos
et al. (1983) and Harrach et al. (1983) and in horses by Harrach et al.
(1987). Stachybotrys toxicosis in calves has been described by Dzhurov
et al. (1984) in Bulgaria. In France, Stachybotrys toxicosis cases have been
described by Le Bars and Le Bars, (1991) and by Lefebrevre et al. (1993).
The nonspecific symptoms described in the first case reports of
veterinary medicine are those described by people with complaints in
buildings in which Stachybotrys has been detected in the fungal flora.
Nose bleeding, sore throat, and eye irritation were among the symp-
toms described by Croft et al. (1986) and Johanning et al. (1996).
Without doubt there are many factors in buildings, both in work
places and homes, which can underlie the symptoms that people report
in buildings with various fungal flora. If Stachybotrys contamination is
found in the close vicinity of a person complaining of more or less
defined symptoms resembling those known to be caused by Stachybo-
trys, one should keep the mind open to the possibility of Stachybotrys as
being one factor among the multifacted health problems.
Stachybotrys-contaminated material has been known to irritate the
skin of animals. The irritation is most severe at sites of contact with
Stachybotrys-contaminated material, according to Szabo et al. (1970).
When animals eat contaminated feed, such as hay or straw, their lips
and mouth are in close contact with the material. Bedding straw has
often been reported as the source for Stachybotrys contamination.
Animals with a thin coat of hair like pigs are often more affected than
animals with thick hair (Hintikka, 1977).
The early veterinary papers on Stachybotrys contamination often
mention that the lungs of animals that have been eating contaminated
feed or laying on Stachybotrys-contaminated bedding materials have
been affected. During the feeding of domestic animals there is often
considerable fungal spore exposure around the animals. Hay and other
rough forage may become dusty and heavily contaminated with various
fungi. The numbers of fungal spores increase greatly, and some of the
original species increase in number while others decrease. Hay bales
and round bales can show signs of fungal growth as miscoloured
material or visible fungal growth. Stachybotrys-contaminated hay and
straw often has the appearance of black soot, which helps to find the
suspected material. The nodes of the straw are the most heavily con-
taminated. Silage can also become contaminated with fungi if the
processing method fails.
It is known that farm workers and animal attendants may have some
health problems when handling moldy material. The animals stay in
the moldy environment longer than their attendants, and their eating
habits promote fungal exposure as they shake and sniff their feed. It is
therefore inevitable that domestic animals are inhaling various fungal
spores and that the exposure is high when the feed is moldy.
IV. Biological Methods to Evaluate Stachybotrys-Contaminated

Material and Toxins
Already some of the early Stachybotrys papers report variation in
toxin production by the different strains. Different biological methods
are used to measure different toxins or toxin combinations produced by
Stachybotrys.
The methods to evaluate the toxic or non-toxic strains vary in the
various papers. Different biological tests have been used to find out
whether the strains produce toxic metabolites in laboratory conditions
and thus cause death or some injury in the biological system. The
feeding experiment is a simple way to find out whether material con-
taminated with different Stachybotrys strains is hazardous to animals.
Korneev (1948) fed white mice with oats infested with Stachybotrys
alternans strains; the mice died after 2 to 3 days and showed catarrhal
hemorrhagic gastritis. Experiments have been conducted with mice,
guinea pigs, and other animals, even dogs, and the results have been
reviewed by Forgacs (1972). In these experiments, sterilized material
was infected with a Stachybotrys strain, and when the mold growth
was sufficient the material was fed to animals.
The irritative or dermotoxic effect of Stachybotrys toxin has been
studied especially by Soviet Union scientists. The official Soviet stan-
dard method for a skin irritation bioassay to study trichothecenes was
to rub the suspected toxic substance on the intact skin of rabbit and
repeat this after 24 hours. The reaction developed in 1 to 2 days, and the
maximum reaction usually appeared on the fourth or fifth day. The
depth and character of the inflammation process were important in
the interpretation of the results of the skin test (Hintikka 1977–78).
There are numerous modifications of the skin test.
Cell culture tests have been in wide use in mycotoxin studies, start-
ing from the 1970s by Bodon and Palyusik (1970) and by Korpinen et al.
(1974). There are a great variety of cell-culture-based methods
used for mycotoxin studies. The basic methods are described by
Gareis et al. (1999) and Hanelt et al. (1994). Protozoa (Paramecium
caudatum), brine shrimp (Artemia salina), and rice moth larva
(Corcyra cephalonica) have been used as indicators for Stachybotrys
toxins in the 1970s. For a review, see Hintikka (1977–78).
Andersson et al. (1997) introduced a method to test some mycotox-
ins: they used boar spermatozoa and compared it with the rabbit
skin test and the cell toxicity test. The inhibition of ciliostatic activity
by mycotoxins in tracheal epithelium has been used by Wilkins and
Pieckova (2002) for detecting fungal growth in building materials.
Yeast cells have also been developed as an indicator for trichothecenes
(Engler et al. 1999). Zaichenko et al. (2002) reported the results of
Stachybotrys toxicity studies in which cyanobacteria was used as
test culture.
V. Methods to Evaluate Stachybotrys Growth

A. TOXIC, LESS-TOXIC, AND NONTOXIC STACHYBOTRYS STRAINS
Biological tests give rough information about the variation of toxicity
of isolated Stachybotrys strains. When strains isolated from sources
suspected to have caused problems to humans or animals and strains
isolated during normal mycological surveys of plant or soil material are
tested in a laboratory at the same time with the same biological method,
both toxic and non-toxic strains from the same materials have been
found. Forgacs et al. (1958) studied the toxicity of 40 strains, and Yskiv
(1969) reported the toxicity results of 20 strains; they found that about
two thirds of Stachybotrys alternans strains were toxin producers
under laboratory conditions.
Similar results were found in Finland in the early 1970s by Korpinen
and Uoti (1974), when 72 Stachybotrys strains were tested in a mouse
fetal primary fibroblast culture. Forty-eight of these 72 strains were able
to produce toxic metabolites when cultivated on grain mixture. A part
of the material for this study consisted of 725 samples sent for routine
mycological examination to the Department of Plant Pathology of the
Agricultural Research Center. This part of the material was not selected
because of its moldy appearance but was collected by random sampling
of field or storage material. The other part of the material consisted of
129 samples of feed or fodder suspected to be responsible for 65 alleged
outbreaks or cases of mycotoxicoses in domestic animals. Cereal grain
for human consumption, field pea, oatmeal, grass seed, mixed feed,
and fodder were represented in the material. Both toxic and nontoxic
strains were isolated from both groups. In this study the frequency of
Stachybotrys in the randomly selected grain and field pea samples was
relatively high, 7%, as compared with the suspected samples contain-
ing 17%, but this may have been due to the isolation method. A detail
worth mentioning is that out of 68 field pea samples, toxin-producing
Stachybotrys was isolated from 13 samples (20%). For isolation, the

material was cultivated on wet cotton wool and sterile filter paper in
large Petri dishes. Fungal growth was first observed with a stereomi-
croscope. Pure cultures of Stachybotrys strains were started from the
hyphal tips and grown on oatmeal agar.
Tantaoui-Elaraki and coworkers (1994) from Morocco and Adriienko
and Zaichenko (1997) from the Ukraine have also reported variations in
the toxin-producing capacity of Stachybotrys strains.
Stachybotrys strains isolated from indoor air, surfaces, and building
material samples in houses with mold problems differ in toxin produc-
tion, like the strains isolated from plant or grain materials. Nine
Stachybotrys atra and one Stachybotrys sphaerospora strains isolated
from buildings with mold problems were grown on moist autoclaved
rice. A feline fetal lung cell line and 3-(4,5-dimethylthiazol-2-yl)-2.5-
diphenyltetrazolium bromide (MTT) tests were used to determine the
toxicity of the isolates. Crude extracts from all strains, except one
S. atra, proved to be cytotoxic. Satratoxins G and H were found
by high-performance liquid chromatography (HPLC) analysis in the
extracts of two highly toxic strains. The unusually high verrucarol
content of the hydrolyzed samples was suspected to be due to the
presence of other macrocyclic trichothecenes (Nikulin et al., 1996).
Jarvis et al. (1998) studied the toxin production of the Cleveland
isolates of Stachybotrys chartarum and Memnoniella echinata. They
cultivated the strains on rice and performed chemical and cytotoxicity
analyses of the extracts. Toxin-producing strains were found both from
the case homes and the control homes. There was a significant varia-
tion in the toxin production of a given isolate cultivated at different
temperatures.
During cultivation and storage, the strains may lose their toxin-
producing capacity (Hintikka, 1977–78). The light conditions and the
nutrients have an effect on the growth and sporulation of Stachybotrys.
Under favorable conditions, S. chartarum can sporulate under various
light exposures even in the dark. The initial growth rate and sporula-
tion of Stachybotrys are favored by light (Heinsohn et al., 1999;
Hintikka, 1977–78).
B. STACHYBOTRYS IN BUILDING MATERIALS AND INDOOR SAMPLES

1. Cultivation
Cultivation methods have been used commonly to detect and evalu-
ate Stachybotrys exposure. Suspected surfaces are sampled with sterile
cotton swabs. Various culture media with their modifications have
been widely reported to be suitable for Stachybotrys isolation. Howev-
er, some rapidly growing dominating fungi like Aspergillus can by their
overgrowth prevent Stachybotrys detection. This can present a prob-
lem. However, on nutritionally poor media, such as on wet sterilized
filter paper or on water agar (agar 15–20 g for 1 liter distilled water;
Gams et al., 1998), isolation of Stachybotrys is sometimes possible from
contaminated material.
2. Microscopy
Direct microscopy of the suspected material is recommended due
to potentially non-cultivable Stachybotrys growth. Stereomicroscopy,
light microscopy, and electron microscopy are also useful techniques.
3. Collection
Impactors that collect spores directly onto agar plates are mostly
used for sampling airborne Stachybotrys. The Andersen sampler is
often referred as the instrument for Stachybotrys and other indoor
fungi (Shelton et al., 2002). A common type of sampler collects
28.3 L/min and is run for 5–10 minutes. The resulting indoor sample
of 140–280 liters represents a very short period when compared to
the 40 m3 of air inhaled by an adult person during 24 hours. The
variation of fungal spores in indoor air is greatly affected by several
physical factors. In many papers (e.g., Wilson and Straus, 2002),
Stachybotrys have been detected successfully by surface samples, and
not by Andersen samplers, from the same source.
The CAMNEA (collection of airborne microorganisms on nucleopore
filters, estimation and analysis) technique was developed by Palmgren
et al. (1986) to determine airborne viable and non-viable fungi in highly
contaminated environments to estimate total fungal exposure.
Stachybotrys spores are slimy and therefore not easily liberated into
the air. The high water content of the substrate can prevent the libera-
tion of the spores and the outcome of experiments can thus be unex-
pected. Pasanen et al. (1993) developed a membrane filter method for
sampling of airborne Stachybotrys toxins. Toxigenic strains of Stachy-
botrys grew well and produced toxic metabolites on wallpaper, grain,
hay, and straw. Stachybotrys spores were easily liberated from wallpa-
per and grain material and could be collected on polycarbonate filters.
The method did not work on straw and hay because of their high water
content, even when Stachybotrys produced large amounts of toxins on
the original material. Stachybotrys spores very likely become airborne
when they dry.
Wilkins et al. (1998) studied respiratory effects in mice exposed to

airborne emissions from Stachybotrys chartarum. The idea of the in-
vestigation was to expose mice first to vapors and afterwards to a
combination of vapors, spores, mycelia fragments, and particles con-
taining solid metabolites. The experimental design, including an expo-
sure chamber and factors to be evaluated, was elegant. The sporulation
of Stachybotrys on malt extract agar was nevertheless limited, and the
filters designed for the determination of airborne mold spores
contained no conidia. The authors suggested that the water content of
their medium was too high.
Andersson et al. (1997) sampled building materials from water-
damaged sites of a children’s daycare center with different methods.
Microscopic analyses were carried out by light microscopy, scanning
electron microscopy (SEM), and transmission electron microscopy
(TEM). The amounts of fungal propagules were estimated by the tape
sampling method. For the isolation of different fungal species from
gypsum liner, six types of agar commonly examined in building mate-
rial studies were used. Satratoxins were analyzed from methanol ex-
tracts of building materials. Microscopic inspection revealed that
water-damaged sites contained Stachybotrys sp. as the dominant or-
ganism. In spite of the fact that Stachybotrys was detected in the
microscopic examination, it was not cultivable from the water-
damaged gypsum liner on malt extract agar (MEA) but it could be
enumerated on corn meal agar (CMA) for isolation of slowly growing
fungi. In this case, highly toxic Stachybotrys chartarum propagules of
the gypsum liner escaped culturing by routine methods, although they
were microscopically visible and dominant on the surfaces of liners,
and they were also shown to possess chemically assayable satratoxin.
Dominance of the slowly growing S. chartarum in a mold population
present on interior building materials may indicate the presence of an
old and stable microbial community.
VI. Airborne Stachybotrys Spores in Buildings and Stachybotrys

Toxins in Building Materials
Hodgson et al. (1998) described building-associated pulmonary dis-
ease in persons exposed to Stachybotrys chartarum and Aspergillus
versicolor in a courthouse and in two associated office buildings.
In bulk samples Stachybotrys chartarum was found at concentrations
of 104 to 107 CFU/cm2, and it was dominant in the molds on damaged
ceiling tiles. Stachybotrys spores were found also in settled dust.
Air sampling for culturable fungi was done in the courthouse library,
and Stachybotrys spores were found in all air samples collected
when books were being handled. Satratoxins G and H were identified
at concentrations of 2 to 5 ppm in the moldy ceiling tiles of the
courthouse.
Robertson’s (1999) case study suggests that the airborne concentra-
tions of Trichoderma and Stachybotrys spores are linked to mycotox-
icosis. The studied work environment showed airborne concentrations
of Trichoderma viride and Stachybotrys chartarum at 494 CFU/m3 and
212 CFU/m3, respectively. Active Trichoderma viride and Stachybo-
trys chartarum growth sites were documented at levels of 3.3
104 CFU/g and 2.0 107 CFU/g, respectively, and were interpreted as
typical for indoor environments.
A case report by Tripi et al. (2000) described an unexpected acute
pulmonary hemorrhage in an infant during anesthetic management.
Health inspectors found Stachybotrys chartarum in mean concentrations
of 6.29 104 CFU/m3 in the child’s bedroom.
Shelton et al. (2002) examined 12,026 fungal air samples from 1717
buildings throughout the United States. The samples were collected
during investigations of indoor air quality; 9619 were indoor samples
and 2407 were outdoor samples. Both indoor and outdoor samples
were collected with an Andersen sampler. In this extensive material,
Stachybotrys chartarum was identified in the indoor air in 6% of the
buildings studied and in the outdoor air of 1% of the buildings. The
authors suggested that the presence of Stachybotrys was not very un-
usual. Dill et al. (1997) have found Stachybotrys spores in their air
samples (see VIII). Laboratory experiments have proved that Stachybo-
trys fungi grow well on building materials and are able to produce
toxins. Pasanen et al. (1993) and Nielsen et al. (1999) have described
toxin production on different building materials artificially inoculated
with Stachybotrys. Tuomi et al. (2000) investigated Finnish buildings
with moisture problems and analyzed 79 bulk samples of moldy
interior finishing materials for 17 mycotoxins. Satratoxins G and H
were found in five samples, and Stachybotrys spp. were present in
the same samples.
VII. Mycotoxins Found in Dust and Air Samples

Mycotoxins have been reported in air samples mainly from agricul-
tural or industrial environments where people have to handle
dusty materials and the fungal concentrations are high. Ghosh et al.
(1997) reported airborne aflatoxin in grain processing in India, and
Selim et al. (1998) reported the same in Iowa in the United States.
Skaug et al. (2000) from Norway detected ochratoxin A in air samples

from cowsheds.
Lappalainen et al. (1996) found low levels of the Fusarium toxin,
deoxynivalenol (DON) in two air samples collected during grain mill-
ing on a farm in Finland. The dust samples for mycotoxin analyses
were collected with a high-volume sampler at an air-flow rate of 833 L/
min, the volume of the sample being 16–50 m3. Richard et al. (1999)
found ochratoxin A in dust samples collected from the heating ducts of
a problem household at concentrations of 306–1500 ppb. Smoragiewicz
et al. (1993) reported several trichothecenes—T-2 toxin, diacetoxyscir-
penol, roridine A, and T-2 tetraol—in the dust of ventilation systems in
office buildings of the Montreal urban area.
VIII. Human Stachybotrys Toxicosis Case Reports

Farm workers handling Stachybotrys-contaminated straw or hay
belong to the risk group for human Stachybotrys toxicosis. There is
evidence that straw or hay contaminated by Stachybotrys may be
dangerous to people handling these materials.
In the old reviews by Sarkisov et al. (1971) and Forgacs (1972),
references are made to the risks of persons handling Stachybotrys-
contaminated material. The main symptoms described were cough,
rhinitis, a burning sensation in the mouth and nasal passages, and
cutaneous irritation at the contact site of the toxic material.
According to Gajdusek (1953), cases of human stachybotryotoxicosis
have been found mainly in regions where the equine disease has also
been reported. The people affected by stachybotryotoxicosis had han-
dled Stachybotrys-contaminated hay or straw during feeding of the
animals. Gajdusek also states that human stachybotryotoxicosis has
been reported in regions in which hay or straw has been used as the
bedding material of people or as fuel to heat homes. In humans, the
symptoms described were first a rash at body sites with heavy perspi-
ration, such as the armpits. Moist dermatitis was the next state, and
this was usually succeeded by a phase characterized by dried, crusted
layers of a serous exudate. Catarrhal angina with painful, severe phar-
yngitis and a burning sensation in the nose usually followed; the nasal
exudate possibly being bloody. Fever may occur in rare instances, and
the cough varies from moderate to severe. Leukopenia has been ob-
served in some patients. O’zegovic et al. (1971) in their paper on
stachybotryotoxicosis in young cattle, state that farm workers who
were handling Stachybotrys-contaminated straw developed a disease
within 2 to 3 days.
The often-quoted paper by Croft et al. (1986) can be regarded as the
first case report on suspected Stachybotrys toxicosis in humans in the
United States. At that time, disease cases caused by Stachybotrys were
nearly unknown in the United States. A family with 5 members had
suffered from unspecific symptoms for several years. From representa-
tive samples of indoor air, the authors were able to identify spores of
Stachybotrys. Extracts of Stachybotrys-contaminated building materi-
als and dust from contaminated air ducts were toxic to mice and
contained satratoxin H, verrucarins, and trichoverrins.
A case connected with medical problems in humans and animals
was described briefly in the doctoral study of Kirsti Liukkonen (1986).
She described an outbreak in a German establishment cultivating
aquarium fish. Unexpected fish deaths were noted by the workers
cleaning tanks and transferring fish from tanks to other tanks. Some
walls in the building and the walls of the aquarium tanks were covered
with a black layer of fungal growth that was diagnosed as heavy con-
tamination by Stachybotrys spores and hyphae. The hands and arms of
the workers were red and hyperemic.
The first hospital report in the United States describing cases in
which Stachybotrys fungus had more likely been the main disease
cause was published by Etzel et al. (1998). The authors described a
rare cluster of acute pulmonary hemorrhage in infants in Cleveland,
Ohio, between January 1993 and December 1994. Ten infants with
acute pulmonary hemorrhage and hemosiderosis were seen in hospital
during the 2-year period. Stachybotrys atra spores were more frequent-
ly found in the dust and surface samples from the homes of the patients
than from the homes of controls. The mean concentration of S. atra
airborne spores in the patients’ homes was about 10 times higher than
in the control homes.
A more extensive study mainly on the same group of 37 infants includ-
ing 12 deaths in Cleveland contained clinical profiles and investigations
of pulmonary hemosiderosis (Dearborn et al., 1999). The epidemiological
investigation of pulmonary hemorrhage in infants in Cleveland found an
association with exposure to Stachybotrys chartarum and other airborne
fungi. Tobacco smoke was an additional risk factor in the presence of
S. chartarum.
Flappan et al. (1999) described a case of an infant with pulmonary
hemorrhage similar to the Cleveland cases. Fortunately it was possible
to conduct a home visit not more than 4 days after the child was
brought to the emergency ward and thereby identify the fungal contam-
ination. Because of water damage to the house, fungal growth was
visible on the ceiling and floor. In the air sample of the patient’s
bedroom, 420 Stachybotrys spores m3 were found. Stachybotrys was

also present in the dust samples from the infant’s crib and mattress. A
sample taken from the Stachybotrys-contaminated ceiling contained
large quantities of the following mycotoxins: roridin L-2, roridin E,
and satratoxin H. If the amount of Stachybotrys spores in the infant’s
bedroom was compared with a mouse experiment by Nikulin (1999),
one could extrapolate that the spore exposure of the infant had been
relatively high compared to the animal experiment. Satratoxin and
roridin were detected on building materials in this study. Nikulin
had measured the satratoxins in the spores used for the exposure
experiment. In the Flappan et al. study, an interesting detail was that
Stachybotrys was isolated also from the baby’s mattress, like in the old
Russian reports.
Elidemir et al. (1999) isolated Stachybotrys from the bronchoalveolar
lavage (BAL) fluid of a child with pulmonary hemorrhage. A 7-year-old
boy had suffered from chronic cough, recurrent pneumonia, and chron-
ic fatigue during 14 months. Stachybotrys atra was cultivated from
BAL fluid, and a moderate number of hemosiderin-laden macrophages
were found in the fluid. The home of the child was contaminated with
Stachybotrys atra; in the patient’s bedroom the wallpaper was densely
covered with mold growth. A lung biopsy to verify the growth of
Stachybotrys in the lung was not possible because of the patient’s
condition. Stachybotrys has not been reported to cause infections to
animals or to humans. In an experiment by Nikulin et al. (1997), mice
were exposed six times intranasally to Stachybotrys spores, and after 4
weeks the lungs were examined for possible Stachybotrys growth:
the results were negative. Diseases connected with Stachybotrys have
been reported to be caused by spores, toxins, or combinations of these
elements.
Dill and her coworkers (1997) described an interesting case in which
moldy plant pots heavily contaminated with Stachybotrys chartarum
caused painful inflammation on the fingertips of the 3 female workers
in a big horticulture enterprise. The pots were made of recycled paper.
The three women handling these pots developed very painful inflamed
efflorescence on their fingertips followed by scaling of the skin. When
they started to use gloves the symptoms disappeared. Masses of Sta-
chybotrys chartarum conidia on the pot walls were identified both
microscopically and by cultivation together with many other fungi.
To evaluate the inhalable spore load, air sampling was performed.
The authors did not detect Stachybotrys spores with an Andersen
sampler (28 L/min for 5 min) when using the method based on the
detection of colony-forming units. With a spore trap (particle trap)
developed by one of the authors, they succeeded in counting the
airborne particles on a sticky tape. They used the Andersen sampler
20 minutes (volume 28 L/min) to collect a sample of 566 L. Without
moving the pots, the measurements yielded levels of 30–100 Stachy-
botrys conidia per m3 of air. The concentrations of airborne conidia
increased dramatically by handling the pots: up to 7500 conidia/m3 of
air for Stachybotrys chartarum only. Dill and coworkers referred to
the earlier study by Kozak and coworkers (1980) that only 1–2% of
Stachybotrys conidia are cultivable and recommended using different
methods to evaluate the inhalable spore load.
IX. Conclusions
In indoor air—even in the indoor air of a theater building—many
actors contribute to the air quality such things as fungi, bacteria, en-
dotoxins, mycotoxins, chemicals, fibers, and dust. Also, ventilation
systems, temperature, and moisture can act as technical assistants.
Architectural structures and building materials constitute the scenery.
Stachybotrys does not present a monolog in an empty theater building.
The play on the indoor air has started, and it continues without a script
written in advance.
The following points may help in understanding the role of Stachy-
botrys in indoor air and may help prevent its growth.
1. Stachybotrys are able to produce highly toxic metabolites that can
cause fatal diseases in domestic animals.
2. In animal experiments, Stachybotrys are able, under controlled
conditions, to cause illnesses to animals, because of effect of the
toxins produced by these fungi. Inhalation as well as ingestion
and direct contact with Stachybotrys-contaminated material or
spores can lead to these diseases.
3. Some case reports and scientific studies have indicated that Sta-
chybotrys have had a role in causing diseases in humans.
4. Physicians and veterinarians are in a key position to recognize the
human and animal cases in which Stachybotrys should be taken
into account in making diagnoses.
5. Indoor air samples from houses should be representative of the air
to which the people have been exposed.
6. The biological effects of different Stachybotrys toxins and toxin
combinations as well as the possible role of Stachybotrys spores
as a disease-causing agent should be clarified.
7. Stachybotrys is unable to grow on dry clean building material.
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Moisture-Problem Buildings with Molds
Causing Work-Related Diseases
KARI REIJULA
Finnish Institute of Occupational Health
Arinatie 3A, FIN-00370 Helsinki, Finland
I. Introduction 175
II. Microbiology of Moisture-Problem Buildings 177
III. Health Outcomes in Water-Damaged Buildings 179
A. Epidemiology of Mold-Induced Symptoms 179
B. Clinical Outcomes After the Exposure to Molds 180
C. Allergies Caused by Mold Exposure 182
D. Toxic Reactions 183
E. Secondary Infections 184
IV. Diagnosis of Diseases Related to Mold Exposure 184
V. Conclusion and Future Prospects 187
References 188
I. Introduction
Moisture problems have been encountered both in workplaces and in
domestic buildings. It has been estimated that the prevalence of mois-
ture-damaged buildings is approximately 50% of all 1.2 million build-
ings in Finland (Nevalainen et al., 1998). In previous national surveys
the confirmation of moisture problems in buildings was based on an
inspection performed by an experienced building engineer. Only clear
water damage as well as those that needed an instant repair were
registered. According to these surveys, the prevalence of moisture
problems was unexpectedly high.
The high percentage of moisture problems in Finland is not excep-
tional as compared with other Western societies: water damage seems
to be common elsewhere, but there is a lack of national surveys based
on building inspections. In epidemiological studies, the presence of
moisture problems has been based on information collected from ques-
tionnaire surveys. Unfortunately, according to the present knowledge,
the prevalence of moisture damage is usually reported to be too low if
the finding is based only on reports of moldy odors or visible moisture
damage in buildings. Moisture problems can also exist in insulation
spaces, behind walls, and under the floor.
One explanation for the high proportion of moisture damage even in
modern buildings is the fact that the majority of the buildings have
175
0065-2164/04 $35.00
176 KARI REIJULA
been built after WWII in a relatively short period of time and without
sufficient knowledge of how to use concrete elements and other new
construction materials. In addition, buildings built in the 1950s and
1960s probably needed proper refurbishment during recent decades.
Unfortunately, because of the lack of knowledge of indoor air problems
associated with moisture problems and because of the recent de-
pression in national economies, construction activities have been
postponed (Haahtela and Reijula, 1997).
Second, all buildings have not been properly planned to survive
heavy rain, melting snow, and moisture, which can soak from sur-
rounding ground directly into building materials. Flat roofs, which
were built mainly in the 1970s, were more prone to suffer water leaks
as compared with slanting roofs. Water leaks and moisture in building
materials led inevitably to the growth of molds and bacteria in these
buildings.
Since the energy crisis three decades ago, buildings have been sealed
tightly. If the ventilation systems of buildings are not functioning
properly, relative humidity in the indoor environment can increase
partly because of an insufficient supply air. This in turn leads to
another form of moisture problem on the surfaces of the materials in
the building. Black spots of mold on the bathroom wall are common
examples of poorly ventilated buildings. Moisture problems caused by
increased relative humidity in addition to poor ventilation and tight
sealing have to be distinguished from moisture problems that occur
behind the surface of the walls and that are caused by water leaks and
infiltrating moisture from the surrounding ground.
If tightly sealed buildings are negatively pressurized, supply air will
infiltrate from insulation spaces through the walls, behind which there
are often water-damaged materials contaminated with microbes. On
these occasions, microbes may be transferred to indoor air. On the
other hand, exposure to microbes is less likely to occur if a sufficient
amount of supply air positively pressurizes the building, which pre-
vents contaminated air from insulation spaces to spread in room air
(Thatcher and Layton, 1995).
The aforementioned causes of moisture problems in modern build-
ings are rather common. Buildings in cold environments like in the
northern states of the United States, Canada, and Scandinavia have to
be heated during winter months, which causes accumulation of mois-
ture in poorly sealed areas of the building because of condensation. On
the other hand, in the southern states of the United States, Europe,
Australia, and Asia, cooling of indoor air associated with insufficient
MOISTURE-PROBLEM BUILDINGS WITH MOLDS 177
ventilation can lead to moisture problems appearing on surfaces of
the building that are not properly sealed against the warm temperature
outside the building. The increasing use of water at home and in
workplaces increases the relative humidity of indoor air, which is
prone to cause moisture damage if the indoor air with a high relative
humidity is able to leak into building materials (AIHA, 1996; Bornehag
et al., 2001; Verhoeff and Burge, 1997).
Unfortunately, moisture damage occurs practically in all buildings
in which improper materials have been used in areas with a constant
contact with water, where the outer surfaces of the building allow
water leaks, or where the difference is significant between the
indoor and outside temperature (Ellringer et al., 2000; Lstiburek and
Carmody, 1994; Norbäck et al., 2000). This causes water condensation
on surfaces of the building. Moreover, the risk of exposure to microbes
is remarkably increased in moisture problem buildings if the amount of
supply air is too small (Lstiburek and Carmody, 1994).
II. Microbiology of Moisture-Problem Buildings

Typical microbes encountered in water-damaged buildings in Fin-
land and elsewhere are listed in Tables I and II. These microbes are
usually not found in ‘‘healthy buildings’’ and therefore can be distin-
guished as indicator microbes in buildings with moisture problems.
Most of the fungi and bacteria in water-damaged buildings originate
from the outdoor environment. Indoor environments and building
materials with an increased humidity are fruitful habitats for certain
microbes. These microbes do not need any special requirements to
grow; water is the only critical element in the environment for fungi
and bacteria to grow on many building materials as long as the building
materials can be used as a food source.
Most of the microbes detected in water-damaged materials are fungi,
yeasts, and bacteria (e.g., actinomycetes). The identification of the
microbes detected in moisture problem buildings is crucial for the
risk assessment of the health outcomes among exposed individuals. If
there are symptomatic people who have been exposed to microbes or
microbial products, in proper clinical investigations and determination
of an occupational disease, the exact origin of the exposure should
be elucidated. The legislation of occupational diseases is usually based
on the fact that the final diagnosis of an occupational disease cannot
be verified without knowing the cause. Therefore a microbiological
investigation from the work environment is necessary for the diagnosis
178 KARI REIJULA
TABLE I
MICROBES ASSOCIATED WITH MOISTURE PROBLEMS IN BUILDINGS (SAMSON ET AL., 1994)
Require high water activity (>0.90–0.95)

Aspergillus fumigatus
Exophiala
Phialophora
Trichoderma
Ulocladium
Stachybotrys*
Fusarium*
Actinomycetes
Yeasts (e.g., Rhodotorula)
Gram-negative bacteria (e.g., Pseudomonas)
Require moderate water activity (0.90 < > 0.85)
Aspergillus versicolor*
Require low water activity (<0.85)
Aspergillus versicolor*
Eurotium
Penicillium (e.g., P. chrysogenum, P. aurantiogriseum*)
Wallemia
*Microbes able to produce mycotoxins.

Note that there are differences between species in the same genus.
of occupational disease associated with the exposure to molds in

water-damaged buildings.
The genus and species of the microbes can be confirmed if an expe-
rienced microbiologist performs the examinations. Often this is done
by a mycologist. The methods have to be standardized: how to collect
material, dust, and indoor air samples; the location where they should
be taken; how the samples should be prepared; what culture medium
has to be selected; what the length of incubation is; and what tempera-
ture is used. The risk assessment of exposed persons can be valid only
if the origin of the exposure has been fully characterized by an experi-
enced microbiologist. This emphasizes the need for multiprofessional
collaboration in investigation of buildings with moisture problems.
The investigation of the problem building has to be carried out at the
time at which the clinical examination of the symptomatic individuals
is performed.
TABLE II
LIST OF THE MOST COMMON MICROBES DETECTED IN MATERIAL SAMPLES IN
MOISTURE-PROBLEM BUILDINGS IN 1996–2002 AT THE FINNISH INSTITUTE
OF OCCUPATIONAL HEALTH, HELSINKI, FINLAND
Microbes in water-damaged material samples

Penicillium spp.
Phoma spp.
Aspergillus versicolor
Acremonium spp.
Aspergillus sydowii
Cladosporium spp.
Fusarium spp.
Trichoderma viride
Aureobasidium spp.
Stachybotrys chartarum
Chaetomium globosum
Phialophora spp.
Yeasts
Actinomycetes
III. Health Outcomes in Water-Damaged Buildings

People have always been exposed to fungi and bacteria in the outdoor
environment and indoors, even to microbes, which can be found in
water-damaged buildings. Because there is an increased incidence of
illnesses caused by moisture problem buildings infested with molds,
something must have happened in the indoor environment to bring
about this change. Either the quality or quantity of the microbes in
indoor air has changed, or the exposed subjects have become more prone
to develop symptoms. At least the number of water-damaged buildings
with microbial contamination seems to be larger than before (Bornehag
et al., 2001; Verhoeff and Burge, 1997). On the other hand, there has
recently been an increase in the prevalence of atopic persons who may
be more prone to develop symptoms in water-damaged buildings.
A. EPIDEMIOLOGY OF MOLD-INDUCED SYMPTOMS

Most of the investigations on the epidemiology of symptoms
associated with moisture problems have been based on questionnaire
surveys, which usually do not have detailed information of the
180 KARI REIJULA
building or the microbiological studies. However, only using the infor-

mation collected from the questionnaire surveys cannot elucidate an
exact etiology of the symptoms reported by the respondents. Confirma-
tion of an indoor-air related disease would require microbiological
analyses of indoor air, damaged materials, or dust from surfaces in
the problem buildings. After collection of necessary environmental
data, the final decision of a work-related disease has to come from an
experienced physician.
Bronchial wheezing has been more prevalent (OR 1.9–3.7) among
children living in damp houses in England and Scotland than among
children from houses without moisture damage (Platt et al., 1989;
Strachan, 1988, 1993). In Sweden, Andrae et al. (1988) also reported
higher prevalence of chronic cough, exercise-induced cough, and asth-
ma in children who had been exposed in moldy houses. Bronchial
wheezing (OR 1.7), cough (OR 2.1), bronchitis (OR 1.5), and asthma
(OR 1.3) were more common among children living in damp houses in
the United States (Brunekreef et al., 1989). In two different studies from
Canada, the prevalence of asthma, bronchial wheezing, bronchitis, and
cough was higher in children who had reported moisture problems in
their homes (Dales et al., 1991b; Dekker et al., 1991). In a recent
population-based incident case-control study from Finland, cases of
newly onset asthma (n ¼ 521) were associated with the presence of
visible mold and odors (OR 1.54, 95% CI 1.01–2.32) ( Jaakkola et al.,
2002).
On the other hand, Strachan and Sanders (1989) from Scotland did
not find any correlation between bedroom conditions (ambient temper-
ature and relative humidity) and pulmonary functions in 1000 children
aged 7 years. In another study on adults, Dales and coworkers (1991a)
did not find any difference in the frequency of physician-diagnosed
asthma in individuals living in damp and normal houses. However,
microbiological investigations of indoor air samples or building mate-
rials were not performed in the previous studies. The most-severe
exposure to molds and bacteria usually occurs in buildings where
building materials and indoor surfaces are contaminated.
B. CLINICAL OUTCOMES AFTER THE EXPOSURE TO MOLDS

In Finland there are more than 300,000 workplaces and over 2.3
million workers, and thousands are exposed to molds in water-
damaged workplaces. According to a recent national survey, 15% of
Finnish workers report the moldy odors associated with moisture pro-
blems in their work environment (FIOH, 2000). Over 100 new cases of
TABLE III
THE NUMBER OF NEW CASES WITH MOLD-INDUCED OCCUPATIONAL DISEASES IN FINLAND IN
1996–2001 (THE NATIONAL REGISTER OF OCCUPATIONAL DISEASES, FINNISH INSTITUTE
OF OCCUPATIONAL HEALTH, HELSINKI, FINLAND, KARJALAINEN ET AL., 2002)
Disease 1996 1997 1998 1999 2000 2001
Rhinitis 99 100 56 47 24 9
Asthma 84 56 38 34 48 66
HP
- farmers 62 57 46 116 34 57
- others* 19 11 8 7 8 16
ODTS 10 26 20 14 11 5
Total 274 250 168 218 105 153
HP ¼ hypersensitivity pneumonitis.
ODTS ¼ organic dust toxic syndrome.
*Others ¼ HP cases which have been detected in moisture-problem buildings.
mold-induced work-related diseases have been reported annually to the

Register of Occupational Diseases in Finland (Table III) (Karjalainen
et al., 2002).
Only a small portion of the individuals exposed to molds in moisture
problem buildings develop allergic diseases (i.e., a specific allergic
reaction caused by an allergen originated from either fungi or bacteria)
(Table IV) (Reijula et al., 2003). On the other hand, specific IgE-
mediated allergy caused by the two most common fungi in the Finnish
outdoor environment, namely Alternaria alternata and Cladosporium
herbarum, were fairly rare in Finnish patients who had been referred to
an allergy hospital. The prevalence of IgE-mediated allergy was 2.8%
with A. alternata and 2.7% with C. herbarum (Reijula et al., 2003).
Less than 5–10% of exposed and symptomatic workers in problem
buildings develop an IgE-mediated allergy (allergic rhinitis, asthma,
conjunctivitis, or urticaria), and only a few develop HP or ODTS.
However, over 40% of exposed individuals in water-damaged buildings
complain of respiratory or conjunctival symptoms associated with poor
indoor air quality (Reijula, 1998). It is obvious that the insufficiency in
ventilation partly explains this occurrence of symptoms. More likely the
microbes themselves, fragments of fungi, volatile organic compounds
(VOCs) emitted from the microbes, or microbial toxins cause the symp-
toms through a direct contact or irritation demonstrated among
the exposed and symptomatic subjects (Verhoeff and Burge, 1997).
182 KARI REIJULA
TABLE IV
CLINICAL SYMPTOMS AND SIGNS IN PERSONS EXPOSED TO MOLDS IN
MOISTURE PROBLEM BUILDINGS
Irritation (in over 40% after the exposure to molds)

Conjunctival (itching, burning, red/swollen eye and eyelid)
Respiratory epithelium/mucosa (watery rhinorrhea, nasal congestion or bleeding,
soreness of throat, cough)
Skin (itching, redness)
General symptoms
Fatigue, headache, nausea
Fever
Pain in joints/muscles
Allergic reactions (in less than 5–10% of symptomatic persons after the exposure)
Conjunctivitis
Rhinitis
Asthma
Hypersensitivity pneumonitis (HP)
Toxic reactions
Organic dust toxic syndrome (ODTS)
Secondary infections (caused by viruses and bacteria)
Paranasal sinusitis
Acute bronchitis
Middle ear infections
C. ALLERGIES CAUSED BY MOLD EXPOSURE

Some of the microbes found in water-damaged buildings are able to
cause allergic diseases either by an IgE-mediated (type I, ‘‘immediate’’)
or cell-mediated (type IV, ‘‘delayed’’) allergy. For instance, an exposure
to Aspergillus fumigatus in a moisture-problem building can induce
the sensitization and production of IgE-antibodies to A. fumigatus
antigens (allergens) (Kurup et al., 1992). After the primary sensitiza-
tion, the following exposure leads immediately (in 10–30 minutes) to
respiratory, dermal, or conjunctival symptoms, which appear as aller-
gic rhinitis, asthma, or conjunctivitis. Immediate allergy caused by
such fungi can be elucidated by measuring specific IgE-antibodies
to the microbe in the patient’s serum or by using the microbial antigen
in skin prick tests (Reijula et al., 2003).
HP (extrinsic allergic alveolitis) is a cell-mediated, delayed allergic
lung inflammation (Fink, 1984). In water-damaged buildings, exposure
to microbial allergens may lead to allergic reaction with fever, cough,
dyspnea, and fatigue 4–6 hours after the exposure. Typical symptoms
such as abnormalities in chest x-ray findings and impaired lung func-
tion tests reveal the diagnosis of HP caused by microbes (Terho, 1986).
The diagnosis of HP is only rarely confirmed in moldy buildings.
D. TOXIC REACTIONS
Organic dust toxic syndrome (ODTS) may occur after a high level of
exposure to toxic substances of microbes (Rylander and Haglind, 1984).
It appears with fever and may produce pathological findings in lung
function studies but only seldom causes abnormalities in chest x-ray
examinations. Therefore the diagnosis of ODTS is often based on the
history of environmental findings and the patient’s symptoms.
Stachybotrys chartarum (atra) is one of the microbes found in indoor
environments and especially in buildings with very damp conditions.
S. chartarum is capable of producing highly potent toxins (Hintikka,
1978; Johanning et al., 1993, 1998; Hintikka in this journal). Persons
occupying damp houses contaminated with S. chartarum are at risk to
develop symptoms in skin, eyes, and respiratory tract. Only a few
descriptions exist where S. chartarum has been detected in damp
houses with symptomatic individuals (Johanning et al., 1993, 1998).
In these buildings, subjects complained of headache, sore throat, flu
symptoms, fatigue, dermatitis, and general malaise. Further investiga-
tions are needed to ascertain the relationship between S. chartarum
and diseases in exposed patients. Contact with fragments of fungi
(i.e., spores and hyphae) and metabolites deliberated from the microbe
(e.g., toxins) inevitably play a significant role in the pathogenesis
of diseases associated with microbial exposure in damp houses
(Bornehag et al., 2001; Nikulin et al., 1997; Verhoeff and Burge, 1997).
Even more important is the fact that S. chartarum is able to produce
metabolites that are immunosuppressive such as cyclosporins, stachy-
botrylactones, and stachybotrylactams (Sakamoto et al., 1993). An
increased incidence of respiratory infections (e.g., sinusitis and acute
bronchitis) has recently been documented in damp houses (Husman
et al., 1993; Jaakkola et al., 1993). S. chartarum itself does not cause
infections, but it is probably able to decrease the defense mechanisms
of the protective barrier in exposed persons so that viruses and bacteria
are able to cause respiratory disorders (Sakamoto et al., 1993).
184 KARI REIJULA
E. SECONDARY INFECTIONS
In damp houses an increase in the incidence of upper respiratory
tract infections has been demonstrated, especially among children
(Husman et al., 1993; Jaakkola et al., 1993). Most likely the irritation
of the mucus membranes of the respiratory tract is due to contact with
fungal elements (spores and hyphae) and/or volatile organic com-
pounds and mycotoxins that originate from fungi. This irritation is
then able to lead to secondary infections, sinusitis, and bronchitis
caused by bacteria and viruses.
IV. Diagnosis of Diseases Related to Mold Exposure

The diagnosis of diseases associated with an exposure to molds and
bacteria in moisture problem buildings can be confirmed by using the
following strategy suggested for the personnel working in occupational
safety and health (Fig. 1). The investigation of buildings with moisture
problems infested with mold growth consists of the detailed analysis of
the building itself and the microbiology of the water-damaged moldy
materials. The clinical investigation of symptomatic persons should be
carried out at the same time. Only if the symptomatic individuals avoid
exposure, can the symptoms be prevented. Collaboration between dif-
ferent professionalists, officers, personnel of occupational safety and
health, and the representatives of the workplace is crucial (Reijula,
1998).
If a water event has occurred, it has to be repaired without a delay. If
the signs of water damage are visible, the primary cause of the moisture
problem has to be elucidated. Only by eliminating water leaks can
the problem of mold growth be avoided. People often paint spots of
molds to hide the problem. Unfortunately, this maneuver makes the
situation even worse and the problem reappears with a vengence.
Odors of mold are one of the first signs to warn the occupants about
the risk. Microbiological examinations of indoor air samples and speci-
mens collected from surfaces and from building materials can reveal
the species of fungi existing in damp houses.
If the symptoms associated with poor indoor air quality have been
reported in a water-damaged building, health care personnel should be
involved. If a moisture problem occurs in a workplace, occupational
health care professionals should evaluate the extent of the problem
(e.g., by using questionnaire surveys in problem buildings). Clinical
examinations have to be performed as a screening method to find symp-
tomatic employees (Table V). Further investigations accompanied by
FIG. 1. Strategy for personnel working in occupational safety and health to employ
when working in buildings with moisture problems.
lung function studies and provocation tests with proper test reagents
and chest x-rays should be performed in special clinics (e.g., in the
departments of occupational medicine) after the clinical screening.
IgG-antibody levels to the most common microbes detected in moldy
buildings are often measured from serum samples of persons who have
been exposed to molds. The presence of IgG-antibodies, however, only
suggests that previous exposure may have occurred to these microbes.
Unfortunately, the role of IgG-antibodies to microbial antigens in
the diagnosis of a mold-induced disease still remains uncertain.
186 KARI REIJULA
TABLE V
DIAGNOSIS OF DISEASES ASSOCIATED WITH MOLD EXPOSURE IN WATER-DAMAGED BUILDINGS
A. Determination of mold exposure

Microbiological specimens/cultures
Building materials
Dust samples
Air samples
Electronmicroscopy, immunological assays
IgG-antibodies to fungal/ bacterial antigens
B. Clinical examinations
Irritation
Differential diagnosis
Examination of eyes, nose, lungs, skin
Allergies
Asthma
‘‘Asthma routines’’ (FEV1, FVC, histamine provocation, etc.)
IgE-antibodies to molds (e.g., RAST, ELISA)
Skin prick tests with fungal allergens
Bronchial provocation with fungal allergens
Allergic rhinitis
‘‘Nasal routines’’ (smears, cells, x-rays, etc.)
IgE-antibodies to molds (e.g., RAST)
Skin prick tests with fungal allergens
Nasal provocation with fungal allergens
Hypersensitivity pneumonitis and ODTS
Clinical diagnostic criteria (e.g., Terho, 1986; Rylander and Haglind, 1984)
Lung function tests: FEV1 ¼ Forced expiratory volume in one second, FVC ¼ Forced vital capacity.
Antibody measurement from serum samples: RAST ¼ radio allergosorbent-test, ELISA ¼ enzyme-
linked immunosorbent assay.
IgE-antibodies against microbial allergens can be detected in patients

who develop IgE-mediated allergy to molds. The prevalence of IgE-
mediated allergy, however, is rather low and only seldom explains
the clinical symptoms in these patients. IgE-mediated allergy can be
investigated by using relevant microbial test reagents in skin prick tests
or by antibody measurement in serum samples.
The fact that most of the symptomatic individuals exposed in mold
houses present with nonspecific conjunctival and mucosal irritation
emphasizes the need for a careful differential diagnosis. Persons
exposed to fungal material in damp houses and complaining of symp-
toms in the lower respiratory tract have to be examined in chest hospi-
tals or at the FIOH, because several workers with allergic alveolitis and
asthma have already been found at the FIOH among persons working in
damp houses. Therefore, mold-induced symptoms can also appear as
occupational diseases.
In private buildings, evaluation of damp house problems has to be
performed by municipal inspectors or private consultants who use
valid methods to estimate the severity of the moisture problem. Micro-
biological investigations of the samples collected from indoor air, sur-
faces, and building materials should be performed only by professional
laboratories that have the necessary expertise. Primary clinical exam-
inations should be performed by municipal health care centers in
which at least one physician in each should be familiar with the health
problems associated with damp houses. After the screening, the pa-
tients should be sent to special clinics if necessary. Further scientific
studies are needed to develop proper methods for examinations of
exposed patients.
V. Conclusion and Future Prospects

Construction materials in water-damaged buildings are often con-
taminated with fungi and bacteria that may cause health problems in
exposed people. Exposure occurs if the spores, other parts of the
microbes, or their metabolic products such as volatile organic com-
pounds (VOCs) or toxins are transferred to indoor air, after which the
contact with eyes, skin, or respiratory tract occurs.
Exposure to molds may become a significant health risk and should
be prevented. Irritation of eyes and respiratory tract occurs after the
exposure to microbes or their metabolic products. Microbes from wa-
ter-damaged materials are able to cause occupational rhinitis, asthma,
HP, and ODTS. Mold-induced occupational diseases represent only the
most severe and clear cases of all work-related disorders, which have
been clinically confirmed by special clinics or occupational health care
centers. Most of the work-related disorders associated with an expo-
sure to moldy materials appear as eye and respiratory symptoms
caused by an irritation. Seldom are they seen with more-severe symp-
toms such as fever, headache, and pain in muscles and joints. Second-
ary infections are more common in persons occupying water-damaged
buildings. After correction of the water intrusion problem, the refur-
bishment of moisture-damaged buildings is the best treatment for this
increasingly common situtation.
188 KARI REIJULA
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Possible Role of Fungal Hemolysins in
STEPHEN J. VESPER* AND MARY JO VESPERy
*U.S. Environmental Protection Agency
Office of Research and Development, National Exposure Research Laboratory
26 W. M. L. King Drive
Cincinnati, Ohio 45268
y
U.S. Environmental Protection Agency, Senior Environmental Employee
26 W. M. L. King Drive
Cincinnati, Ohio 45268
I. Introduction 191
II. Biochemical Characterization of Hemolysins 192
A. Hemolysins Produced by Macro-Fungi 192
B. Hemolysins Produced by Micro-Fungi 193
C. Bacterial Hemolysins 198
D. Comparison Between Fungal and Bacterial Hemolysins 198
III. Physiological Actions of Hemolysins as Cytolysins 199
A. Cytolytic Activity of Macro-Fungal Hemolysins 199
B. Cytolytic Activity of Micro-Fungal Hemolysins 200
C. Cytolytic Activity of Bacterial Hemolysins 201
IV. Potential Roles of Micro-Fungal Hemolysins/Cytolysins in SBS 201
A. Symptoms of SBS and Possible Connection with Fungal
Hemolysin Exposure 201
B. Mode of Exposure 205
V. Why do Fungi Make Hemolysins? 205
VI. Application of Fungal Hemolysins in the Analysis of SBS 206
VII. Conclusions 207
References 208
I. Introduction
The World Health Organization (WHO) definition of sick building
syndrome (SBS) includes such symptoms in the building occupants as
headache, distraction, dizziness, fatigue, watery eyes, runny or blocked
or bleeding nose, dry or sore throat, and skin irritation (WHO, 1995).
The suspected causes of SBS include various chemicals, toxins, and
microorganisms. In this review we will focus on exposures to fungi
(molds) as causes of SBS.
Identifying and quantifying the great diversity of fungi found indoors
had been one of the main limitations to addressing the cause-effect
relationship between fungal exposures and SBS. The recent development
of quantitative PCR (QPCR) analysis of molds (US. Patent 6,387,652)
191
0065-2164/04 $35.00
192 VESPER AND VESPER
has dramatically improved fungal speciation and quantification,

resulting in a highly standardized process for describing the indoor
fungal population. Does exposure to these fungi cause SBS? In this
chapter, we will consider the hypothesis that fungal hemolysins may
play a role in some symptoms observed in SBS.
Hemolysins are molecules that are designated as such because they
have the ability to lyse red blood cells (RBCs). In this review, only
proteinaceous hemolysins will be discussed, although the authors
recognize that non-proteinaceous fungal molecules can have hemolytic
effects (e.g., T-2 toxin causes hemolysis of red blood cells) (Segal et al.,
1983). For this review of fungal hemolysins, the artificial separation of
macro-fungi (e.g., mushrooms) and micro-fungi (e.g., filamentous spe-
cies and yeasts) will be used. Throughout this review, we will compare
fungal hemolysins with bacterial hemolysins because bacterial hemo-
lysins have been more extensively studied and may provide useful
insights into the nature and activities of fungal hemolysins.
II. Biochemical Characterization of Hemolysins

There are primarily two types of hemolysins, designated alpha ()
and beta (). Alpha hemolysins cause a partial lysis of the RBCs,
resulting in a darkening of the media around a colony on sheep’s blood
agar (SBA). The beta hemolysins produce a complete lysis of the RBCs,
resulting in a clearing around the colony growing on SBA. First we will
review what is known about the biochemistry of hemolysins.
A. HEMOLYSINS PRODUCED BY MACRO-FUNGI

The first proteinaceous, macro-fungal hemolysin was named phallin
(Kobert, 1891). It is now called phallolysin and is produced by Amanita
phalloides (Wieland, 1986). The mushroom A. rubescens produces the
hemolysin rubescenslysin (Wieland, 1986). These proteins lyse RBCs
and are unstable in acids and temperatures over 65 C. The mushrooms
Pleurotus ostreatus and Agrocybe aergerita produce the hemolysins
ostreolysin and aegerolysin, respectively (Berne et al., 2002). Ostreoly-
sin has a molecular weight (MW) around 17 kDa under reducing con-
ditions, but under non-reducing conditions, 17 and 16 kDa bands were
identified. Aegerolysin has a single band of 17 kDa under reducing and
a 16 kDa band under non-reducing conditions. Wang et al. (2002)
tested 14 different mushrooms for hemolysins and found that most
produced these kinds of proteins. However, none of these compounds
were well characterized beyond suggesting that their MWs were greater
than 10 kDa (based on dialysis).
FUNGAL HEMOLYSINS 193
B. HEMOLYSINS PRODUCED BY MICRO-FUNGI
1. Yeasts
Salvin (1951) reported that hemolysins were produced by the yeast-like
phases of Histoplasma capsulatum, Blastomyces dermatitidis, Candida
albicans, and Cryptococcus neoformans. More recently, C. albicans was
shown to produce a -hemolytic agent, but it was not characterized
(Manns et al., 1994). In a more comprehensive study, Luo et al. (2001)
tested the hemolytic activity of 14 Candida species. C. albicans, C. du-
bliensis, C. kefyr, C. krusei, C. zeylanoides, C. glabrata, C. tropicalis, and
C. lusitaniae all produced alpha hemolysis at 24 h, which led to beta
hemolysis at 48 h. Candida famata, C. guilliermondii, C. rugosa, and
C. utilis produced only alpha hemolysis, and C. parapsilosis and
C. pelliculosa demonstrated no hemolytic activity.
2. Filamentous Fungi
Henrici (1939) partially purified a hemolytic agent from ground my-
celia of the filamentous fungus Aspergillus fumigatus. Asp-hemolysin
was first fully purified in 1977 (Yokota et al., 1977) and was described
as having a MW of 30 kDa with an acidic isoelectric point (pI 4.0).
Much later, cloning and sequencing allowed the actual MW to be
determined to be 14.3 kDa, containing 131 amino acids of which three
are cysteine (Ebina et al., 1994). Asp-hemolysin is a secretory glyco-
protein that binds to a specific receptor, the low-density lipoprotein
receptor (L-DLR) (Fukuchi et al., 1998) (e.g., in arterial walls) (Ebina
et al., 1983). However, there was little interest in examining whether
other filamentous fungi produced hemolysins.
`
Not until 2001 was StachylysinE recognized as a proteinaceous
hemolytic agent produced by the filamentous fungus Stachybotrys
chartarum (Vesper et al., 1999, 2001). The monomeric form of stachy-
lysin has a MW of 11.9 kDa, as determined by matrix-assisted laser
desorption ionization time-of-flight mass spectrometry (MALDI-ToF-
MS), with 114 amino acids, two of which are cysteine (Vesper et al.,
2001). More recently, chrysolysin was purified from Penicillium chry-
sogenum (Donohue et al., 2004). Monmeric chrysolysin has a MW of
2 kDa, with one cysteine, and it has a pI of 4.9. These results led us to
survey other filamentous fungi for hemolysin producers.
We found that hemolysins are produced by many of the common
indoor fungi (Van Emon et al., 2003). In this study, 90 common indoor
fungi (Table I) were grown on SBA. All species tested germinated
and grew on SBA at 23 C. However, only seven species secreted a
hemolysin at 23 C. But if those same SBA plates, with growing fungi,
TABLE I
MICRO-FUNGAL HEMOLYSIN SECRETION AT DIFFERENT TIMES AND TEMPERATURES
Incubation temperature
23 C 23 C to 37 C 37 C
Growth Hemol. Hemol. Hemol. Hemol. Growth Hemol.

Fungal speciesa day 5 day 5 day 1 day 2 day 3 day 5 day 5
Absidia corymbifera þþþb þþþ

Acremonium strictum þ *c * * no
Alternaria alternata þþ þ
Aspergillus auricomus þþ * * * no
Aspergillus caespitosus þþþ * * ** þ
Aspergillus candidus þ * * * no
194
Aspergillus carbonarius þþ * * * þ
Aspergillus cervinus þ no
Aspergillus clavatus þþ * * ** þ
Aspergillus flavipes þþ * * ** þ **
Aspergillus flavus þþ * * * þþþ ***
Aspergillus fumigatus þþ þþþ ***
Aspergillus niveus þþ * * * þþ **
Aspergillus niger þþþ ** ** ** *** þþþ ***
Aspergillus ochraceus þþ * * þ *
Aspergillus paradoxus þþ * * * no
Aspergillus parasiticus þþ þþþ **
Aspergillus puniceus þþ * * * no
Aspergillus restrictus þ no
Aspergillus sclerotiorum þþ þ **
Aspergillus sydowii þþ * * * * þþ *
Aspergillus tamarii þþþ * * ** þþþ
Aspergillus terreus þþ þþþ **
Aspergillus unguis þþ * * * þþ **
Aspergillus ustus þþ * ** ** þ
Aspergillus versicolor þ * * ** þ **
Aspergillus wentii þþ * * ** *** no
Aureobasidium pullulans þ * * no
Chaetomium globosum þþþ þþ
Cladosporium cladosporioides I þ * * * no
Cladosporium cladosporioides II þ * * * * no
Cladosporium herbarum þ * * * no
Cladosporium sphaerospermum þ * * ** no
195
Emericella nidulans þþ * * * * þþþ **

Emericella variecolor þþ * * * þ *
Epicoccum nigrum þþ ** ** ** no
Eurotium chevalieri þ * * * þ
Memnoniella echinata þþ * * * þ **
Myrothecium verrucaria þþ * * * þþþ **
Mucor racemosus þþþ no
Paecilomyces lilacinus þþ * * * þ *
Paecilomyces varioti þ * * * þþ **
Penicillium aethiopicum þþ * * þ **
Penicillium atramentosum þþ ** ** ** no
Penicillium aurantiogriseum þþ * * * no
Penicillium brevicompactum þþ * * * * no
(continued )
Incubation temperature
23 C 23 C to 37 C 37 C
Growth Hemol. Hemol. Hemol. Hemol. Growth Hemol.

Fungal speciesa day 5 day 5 day 1 day 2 day 3 day 5 day 5
Penicillium canescens þ * ** ** þ *
Penicillium chrysogeum þþ * * ** þ *
Penicillium citreonigrum þ * * * no
Penicillium citrinum þþ * * * þ *
Penicillium coprophilum þþ no
Penicillium corylophilum þ * * * no
Penicillium crustosum þþ ** ** ** no
196
Penicillium decumbens þ * * * þ *
Penicillium digitatum þ no
Penicillium expansum þþ * * ** no
Penicillium fellutanum þ * * * þ *
Penicillium glandicola þþ * * * no
Penicillium griseofulvum þ * * * þ *
Penicillium implicatum þ no
Penicillium islandicum þ ** ** ** þ **
Penicillium italicum þ no
Penicillium janthinellum þþ * * * þþ *
Penicillium lividum þ * * * no
Penicillium melinii þ * * * no
Penicillium miczynskii þþ * * * no
Penicillium olsonii þþ * * ** þ
Penicillium oxalicum þþ * * * þþþ *
Penicillium purpurogenum þ þ
Penicillium raistrickii þþ * * ** no
Penicillium restrictum þ * * * þ **
Penicillium roquefortii þþ * * * no
Penicillium sclerotiorum þþ * * * no
Penicillium simplicissimum þþ þ *
Penicillium spinulosum þþ * * * no
Penicillium variabile þ * * * no
Penicillium verrucosum þþ no
Penicillium waksmanii þþ no
Rhizopus stolonifer þþþ no
Scopulariopsis brevicaulis þþ * * * þþ *
197
Scopulariopsis brumptii þ * * * þ
Scopulariopsis chartarum þ * * * no
Stachybotrys chartarum þþ * *** *** *** þþ ***
Trichoderma asperellum þþ þþ
Trichoderma harzianum þþþ þ
Trichoderma longibrachiatum þþþ *** *** *** þþþ ***
Trichoderma viride þþþ no
Ulocladium atrum þþ þ *
Ulocladium botrytis þþ þ *
Ulocladium chartarum þþ þ
Wallemia sebi þ no
a
The Culture Collection source of each of these species can be found in Van Emon et al., 2003.
b
One plus sign indicates a small amount of growth after 5 days, two indicate moderate growth, and three indicate abundant growth.
c
One star indicates a small amount of hemolytic activity, two indicate moderate activity, and three indicate abundant hemolytic activity.
were transferred to 37 C, then 65 species secreted hemolysins. On the

other hand, only 50 of the 90 fungi were able to germinate and grow on
SBA at 37 C (Table I). Of the 50 that germinated, only 36 showed
hemolytic activity (Table I).
We are in the process of biochemically characterizing these other
micro-fungal hemolysins. So far, all of these hemolysins demonstrate a
characteristic response on SBA—that is, there is initial darkening of
the SBA medium beyond the colony. This darkened area turns bluish
green and then clears over 24 to 48 h (Van Emon et al., 2003; Vesper
et al., 2001). (This is similar to the slow reaction described by Luo et al.
[2001] for the Candida hemolysins.)
C. BACTERIAL HEMOLYSINS
Bacterial hemolysins are a fairly heterogenous group of proteins, but
they have been classified in many cases into three ‘‘families’’ of toxins.
The beta-sheet-structured (BSS) family is so named because of the abun-
dance of a -sheet structure in the monomer. An example is alpha-toxin,
produced by Staphylococcus aureus. Alpha-toxin has a molecular
weight of 33.4 kDa made up of 297 amino acids but no cysteine. It
produces oligomers that are heptamers and forms pores of 6–10 Å.
The members of the cholesterol-binding toxin family (C-BT) are
thiol-activated and bind to cholesterol in membranes. An example is
streptolysin-O, which has a molecular weight of 61.5 kDa and is made
up of 538 amino acids with one cysteine. It forms oligomers of 25 to 80
monomers and produces large pores, up to 30 nm in diameter.
The repeats-in-toxin (RTX) family is so named because the C-
terminal half of the molecule contains multiple repeats of a peptide
sequence. These hemolysins are usually produced by Gram-negative
bacteria and range in size from 102 to 178 kDa (Stanley et al., 1998).
For example, Escherichia coli -hemolysin has a MW of 110 kDa with
1024 amino acids but no cysteine. Members of this family may or may
not form oligomers. The pores produced are 10–15 Å in diameter.
A fairly comprehensive review of the bacterial hemolysins is available
(Lahiri, 2000).
D. COMPARISON BETWEEN FUNGAL AND BACTERIAL HEMOLYSINS

What little is known about the biochemistry of fungal hemolysins
is summarized in Table II. We don’t know enough yet to place the
fungal hemolysins in any of the bacterial hemolysin families. In fact,
at this time, it appears that fungal hemolysins are generally com-
posed of smaller molecular weight monomers than most bacterial
TABLE II
SOME FUNGAL HEMOLYSINS, THEIR SOURCE, MOLECULAR WEIGHT (MW),
ISOLECTRIC POINT (pI), AND REFERENCE
MW (kDa)
Hemolysin Fungus monomer pI Reference
Aegerolysin Agrocybe aegerita 16 4.85 Berne et al., 2002

Asp-hemolysin Aspergillus fumigatus 14.3 4.0 Ebina et al., 1994
Canditoxin Candida albicans ? Acidic Salvin, 1951
Chrysolysin Penicillium 2 4.9 Donohue et al., 2004
chrysogenum
Flammutoxin Flammulina 32 5.4 Bernheimer and
velutipes Oppenheim, 1987
Ostreolysin Pleurotus ostreatus 16 5.0 Berne et al., 2002
Phalloyysin Amanita phalloides 34 7.0, 7.6, 8.1 Faulstich et al., 1983
Stachylysin Stachybotrys chartarum 11.9 3.5 Vesper et al., 2001
hemolysins. They usually have more acidic isoelectric points and

contain more cysteine amino acids than bacterial hemolysins. Fungal
hemolysins are remarkably slow acting as compared with bacterial hemo-
lysins. However, like most bacterial hemolysins, fungal hemolysins are
aggregating proteins that create pores in membranes.
III. Physiological Actions of Hemolysins as Cytolysins

Hemolysins might be described more accurately as cytolysins, be-
cause they lyse many cells besides RBCs. The physiological response to
hemolysins depends on the type(s) of cell(s) affected.
A. CYTOLYTIC ACTIVITY OF MACRO-FUNGAL HEMOLYSINS

1. Rubescenslysin
The mushroom hemolysin, rubescenslysin, caused the lysis of RBCs,
as expected, but leukocytes were even more susceptible to lysis than
RBCs (Odenthal et al., 1982). This hemolysin’s broader cytolytic activity
had other effects. Skeletal muscle exposed to rubescenslysin showed a
loss of direct and indirect excitability, and rubescenslysin also caused
membrane damage in the renal parenchyma (Odenthal et al., 1982).
Dosing of experimental animals resulted in vascular permeability lead-
ing to cardiotoxicity, seizures, and ultimately hemorrhagic, pulmonary
edema and death (Odenthal et al., 1982; Seeger et al., 1981).
2. Phallolysin
Phallolysin destroys leukocytes as well as RBCs (Faulstich et al.,
1974) and is the most potent toxin produced by the mushroom
Amanita phalloides. If injected intraperitoneally (IP), it had an LD50
of 40 g/Kg in rabbits and 50 g/Kg in rats (Faulstich et al., 1983).
However, if it is consumed, phallolysin is destroyed by the stomach
acids. Therefore it does not contribute to human ingestion poisonings
(Faulstich et al., 1974, 1983).
3. Flammutoxin
The hemolysin flammutoxin was isolated from the edible mushroom
Flammulina velutipes. When injected IP into mice, it caused an immedi-
ate writhing reaction (Lin et al., 1975). It is a strong cardiotoxic protein
and results in respiratory failure and bleeding in the lungs (Lin et al.,
1974). These observations indicate that this hemolysin caused significant
membrane damage, similar to the other mushroom hemolysins.
B. CYTOLYTIC ACTIVITY OF MICRO-FUNGAL HEMOLYSINS

1. Asp-hemolysin
The broad cytolytic activity of asp-hemolysin has been the best
documented of the micro-fungal hemolysins. A dose-dependent in-
crease in cytotoxicty occurred in cultured mouse peritoneal macro-
phages exposed to asp-hemolysin (Kumagai et al., 1999). Also, when
human umbilical vein endothelial cells (HUVECs) were exposed to
asp-hemolysin at 100 g/ml, the viability of the HUVECs was reduced
by 50% after only 1 hr of exposure. These culture-based results may
explain some of the health effects observed when experimental animals
were exposed to asp-hemolysin.
Inoculation of viable spores of A. fumigatus, together with asp-
hemolysin, promoted aspergillosis infections in mice (Ebina et al.,
1982). These mice developed granulomatous lesions most commonly
in the lung and liver, with fewer in the spleen, kidney, and intestine
(Iwata et al., 1962). Injections (IP) of the purified asp-hemolysin pro-
duced hemorrhagic lesions in experimental animals (Sakaguchi and
Yokota, 1972) and an LD50 in mice of 750 g/Kg (Sakaguchi et al., 1975).
2. Stachylysin
Stachylysin injected into the earthworm Lumbricus terrestis resulted
in the leakage of hemoglobin from the animal’s vascular system (Vesper
and Vesper, 2002). We showed that stachylysin caused pores in sheep
RBC membranes (Vesper et al., 2001), but L. terrestis has no oxygen-
carrying cells and a closed vascular system. The release of erythrocruorin
hemoglobin from L. terrestis and the development of aneurism-like
structures suggested that stachylysin caused cytolytic vascular leakage,
resulting in an LD50 of 1100 g/Kg in L. terrestis (Vesper and Vesper,
2002).
C. CYTOLYTIC ACTIVITY OF BACTERIAL HEMOLYSINS

Hemolysins have been isolated and purified from many bacterial
pathogens, and they are generally important virulence factors with
broad cytolytic activities (Baker and Edwards, 1995; Bhakdi and
Tranum-Jensen, 1991; Bhakdi et al., 1990, 1996; Cavalieri et al., 1984;
Feldman et al., 1990; Grimminger et al., 1991; Johnson et al., 1985;
O’Reilly et al., 1986; Ou et al., 1988; Van Der Vijer et al., 1975). The
range of cytolytic and other effects associated with different bacterial
hemolysins is summarized in Table III, but this is by no means an
exhaustive list of either bacterial hemolysins or their effects. Additional
information can be obtained in various reviews (Bhakdi et al., 1996;
Lahiri, 2000).
IV. Potential Roles of Micro-Fungal Hemolysins/Cytolysins in SBS

It is our hypothesis that some SBS symptoms are described as flu-
and/or cold-like because some fungal hemolysins activate the same
human inflammatory system (histamine and cytokine producing cells)
as the rhino- and influenza viruses. In addition, other symptoms such
as headache, dizziness, and bleeding may be the manifestations of
some fungal hemolysin’s affect on vascular tissues. Finally, we propose
that the major mode of exposure is colonization by fungi of the human
airway, which leads to the release of hemolysins (and other toxins) into
the host.
A. SYMPTOMS OF SBS AND POSSIBLE CONNECTION WITH FUNGAL

HEMOLYSIN EXPOSURE
The World Health Organization (WHO, 1995) defined SBS as the
manifestation of at least one symptom from the following groups:
. General symptoms, such as headache, dizziness, distraction,
fatigue
. Eye irritation, such as redness, watering, itching, dryness, swollen
eyelids
TABLE III
EXAMPLES OF BACTERIAL HEMOLYSINS AND THE EFFECTS THEY HAVE ON CELLS,
ANIMALS, OR HUMANS
Hemolysin/source Effects Reference
1. Adenylate Cyclase
Toxin
Bordetella Suppression of platelet aggregation Iwaki et al., 1999
pertussis and prolongs bleeding.
2. Aerolysin
Aeromonas Histamine release from mast cells. Scheffer et al., 1988
hydrophila
3. -Toxin
Staphylococcus Apoptosis of T-lymphocytes; Jonas et al., 1994
aureus Elevation of pulmonary arterial Walmrath et al., 1993
pressure;
IL-6 induced in mice; Onogawa et al., 2002
IL-8 induced in alveolar Rose et al., 2002
epithelial cells.
4. -Hemolysin
Group B IL-8 induction; Doran et al., 2002
Streptococcus Cytolytic injury to human alveolar Gibson et al., 1999
cells and pulmonary capillary
endothelial cells leading to
hemorrhaging;
Brain invasion by breaking down Nizet et al., 1997b
blood brain barrier;
IL-6 increase; joint injury and arthritis. Puliti et al., 2000
5. -Hemolysin
Escherichia coli Microcirculatory abnormalities; Mayer et al., 1999
Release of IL-1B and IL-6; Bhakdi et al., 1996
IL-6 and IL-8 increase; Jantausch et al., 2000
Hemorrhaging; Stanley et al., 1998
Vasoconstrictor and thromboxane Walmrath et al., 1994
generation.
6. Pneumolysin
Streptococcus Release of histamine from Howell and
pneumoniae mast cells; Gomperts, 1987
IL-6 induced in mouse lung; Rijneveld et al., 2002
Increase in IL-8 but not TNF-; Cockeran et al., 2002
Ciliary slowing and Feldman et al., 1990
epithelial disruption.
7. Streptolysin-O
Streptococcus Release of IL-6 and IL-8 from Mitsui et al., 2002
pyogenes keratinocytes and endothelial cells.
. Irritated, runny or blocked nose (sometimes described as conges-
tion, bleeding nose, itchy, dry, or blocked nose)
. Dry or sore throat, sometimes described as upper airway irritation
or difficulty with swallowing
. Dryness, itching, or irritation of skin, occasionally with rash.
1. Symptoms Associated with Inflammatory Response

a. Histamine-Related Responses. The symptoms of SBS are often de-
scribed as flu- and/or cold-like, including watery eyes, runny nose, and
irritations (Harrison et al., 1992). These are common manifestations of
conditions in which mast cells in the nose, eyes, throat, and lungs are
stimulated to release histamines. These histamines attach to nearby
blood vessels, causing them to swell and secrete more fluid than usual,
resulting in watery eyes and runny nose. Histamines can also irritate
nearby nerve endings, causing itching. Do hemolysins cause histamine
release?
Bacterial hemolysins, produced by strains of E. coli, caused the
induction of histamine release from rat peritoneal mast cells and hu-
man basophilic granulocytes (Gross-Weege et al., 1988). Similarly,
Aeromonas hydrophila, Serratia marcescens, and Listeria monocyto-
genes hemolysins were shown to induce histamine release from rat
mast cells in a dose-dependent manner (Scheffer et al., 1988). Staphy-
lococcal hemolysin and Pseudomonas aeruginosa hemolysin caused
release of histamine from rat mast cells (Bergmann et al., 1989; Prevost
et al., 1998). So bacterial hemolysins cause histamine release but do
fungal hemolysins?
The induction of histamine release by fungal hemolysins has not
been as extensively studied. Phallolysin and rubescenslysin caused
disruption of rat mast cells and rapid degranulation (Seeger and
Bunsen, 1980). Asp-hemolysin caused anaphylactic shock in mice
(Iwata et al., 1962), and flammutoxin caused the release of histamines,
resulting in symptoms that were treatable with antihistamines (Lin
et al., 1975). Thus, bacterial and fungal hemolysins can stimulate
histamine release.
b. Cytokine-Related Responses. Experimental exposures of humans

to either the cold or flu virus result in the induction of cytokines
leading to the typical systemic symptoms of these diseases (Hayden
et al., 1998; Zhu et al., 1996). The cytokines, especially interleukin
(IL)-6 and also interferon (INF)- followed by tumor necrosis factor
(TNF)- and IL-8, are responsible for the virus-induced symptoms
associated with colds and flu (Hayden et al., 1998; Zhu et al., 1996). Do
hemolytic proteins also induce these same cytokines?
The cytokines induced by both bacterial and fungal hemolysins have
been measured in the tissues and cells of many different animals,
including humans. For example, Staphylococcal -toxin induced
over-secretion of IL-1 and IL-6 in cultured macrophages (Onogawa,
2002) and IL-8 in human alveolar epithelial cells (Rose et al., 2002).
Group B streptococcal -hemolysin induced IL-8 (Doran et al., 2002).
Pneumolysin introduced into lungs of mice caused a dose-dependent
increase in IL-6 (Rijneveld et al., 2002), and in human neutrophils,
an increase in IL-8 (Cockeren et al., 2002).
Cytokines were induced by the fungal hemolysin asp-hemolysin,
which caused an increase in TNF-, IL-1, IL-6, and IL-8 in human
umbilical vein endothelial cells (Kumagai et al., 2001). Mouse perito-
neal macrophages secreted TNF- and IL-1 after exposure to asp-
hemolysin (Kumagai et al., 1999). Thus the same kinds of cytokines,
especially IL-6, are induced by many bacterial and fungal hemolysins
as are induced by the cold or flu viruses. This suggests that some of the
SBS symptoms might be caused by fungal hemolysin exposures leading
to cytokine inductions.
2. Symptoms Associated with Vascular Tissue Damage
The SBS symptoms of ‘‘headache, dizziness, and nose bleeds’’ may
be associated with impairment of vascular tissues. There are many
examples of bacterial hemolysins that damage vascular tissues (Table
III). For example, alpha toxin, produced by S. aureus, caused increases
in pulmonary arterial pressure (Walmrath et al., 1993) and -hemoly-
sin, produced by E. coli, caused vascular leakage, micro-circulatory
abnormalities, and vasoconstriction (Ermert et al., 1992; Mayer et al.,
1999). Group B streptococcal -hemolysin caused a breakdown in the
pulmonary capillary endothelial cells that resulted in pulmonary he-
morrhaging, which can lead to death in infants (Nizet et al., 1996,
1997a). These kinds of vascular changes affect blood pressure and
vascular integrity that can lead to headaches, dizziness, and bleeding.
Nosebleeds, and by extension, idiopathic pulmonary hemosiderosis,
reported in Cleveland (Dearborn, 1997; Dearborn et al., 1999; Etzel
et al., 1998) and other cities (Elidemir et al., 1999; Flappan et al.,
1999), may also be manifestations of hemolysin damage to vascular
tissue. The macro-fungal hemolysins rubescenslysin (Seeger et al.,
1981) and flammutoxin (Lin et al., 1974) caused pulmonary bleeding
in experimental animals. Pure asp-hemolysin is cytotoxic for vascular
endothelial cells (Kumagai et al., 2001), leading to hemorrhagic lesions
in animals (Sakaguchi and Yokota 1972). Exposure to S. chartarum has
been shown to cause hemorrhaging in animals (Forgacs, 1972; Sarkisov
and Orshanskaiya, 1944) and agricultural workers (Hintikka, 1978;
Rylander,1994; Sorenson and Lewis, 1996). The hemolysin stachylysin
caused hemorrhaging in an animal model (Vesper and Vesper, 2002).
Thus there are a number of cases where fungal hemolysins cause
damage to or destruction of vascular tissues.
B. MODE OF EXPOSURE
There are several possible modes of exposure of humans to fungi. At
this time, direct inhalation of spores is considered the most likely.
Once inhaled, larger fungal conidia are primarily retained in the nasal
and sinus structures. Smaller conidia enter the tracheae and lungs.
Because few spores are detected in the air, many believe that any toxins
or other products in these few spores would not be adequate to cause a
health problem. Is there another possibility?
Ponikau et al. (1999) have demonstrated that more than 60 different
species of fungi were cultured from nasal secretions. Many of these
same fungi have been demonstrated to produce hemolysins (Table II).
It seems likely that these colonizing fungi would release hemolysins
(and other toxins) into their host. We found that actively growing
S. chartarum mycelia produce about 10 to 100 times more stachylysin
per mg wet weight than the conidia of the same strain (Van Emon et al.,
2003). Does the release of hemolysins occur?
Asp-hemolysin was detected in mouse kidney and cerebrum 2 days
after introduction of the A. fumigatus conidia (Ebina et al., 1982).
Stachylysin was detected by immunochemical localization around
S. chartarum conidia in mouse lung sections, 24 h (even more at
72 h) after the lungs had been instilled with S. chartarum conidia
(Gregory et al., 2003). Stachylysin was also measured in sera of rats
and humans exposed to S. chartarum (Van Emon et al., 2003). Thus,
fungal hemolysins were making their way into the bloodstream of ani-
mals and humans by some mode of exposure. We believe that colonizing
fungal mycelia may be a more significant source than inhaled spores.
V. Why Do Fungi Make Hemolysins?

One of the main advantages for microorganisms producing hemoly-
sins is the acquisition of iron that is required for their growth. There is
actually very little free iron in mammals (Bullen, 1981). Bacterial hemo-
lysins are able to lyse RBCs and thus release the iron they contain. In
fact, most infectious bacteria use this strategy to obtain iron. In many
cases, the ability of pathogenic microorganisms to acquire iron from a
mammalian host by producing a hemolysin has been shown to be of
critical importance in establishing infections (Payne and Finkelstein,
1978). In addition to obtaining iron, many bacterial hemolysins target
host immune function cells (e.g., phagocytic leukocytes, which increases
a microorganism’s chance of survival in a mammalian host). These stra-
tegies may also work for invasive fungal infections such as C. albicans and
A. fumigatus. But why do non-infectious fungi produce hemolysins?
The simple explanation is that hemolysins must provide some ad-
vantage for survival in their environment. For example, some mush-
room hemolysins have been shown to be produced only at the
initiation of fruiting body development (Berne et al., 2002). Wang
et al. (2002) demonstrated that numerous mushrooms produce pro-
teins, including hemolysins, that have insecticidal activity. The prod-
uction of hemolysins by Pleurotus and Agrocybe at the time of fruiting
might provide protection from insects. Perhaps hemolysins secreted at
ambient temperatures by microfungi are able to destroy grazing amoe-
bae or other predators. But actually very few common indoor air fungi
secrete hemolysins at ambient temperature (Van Emon et al., 2003). So
it is difficult to explain why so many micro-fungi secrete hemolysins
only when the temperature is raised near human body temperature.
We suggest that the primary role of micro-fungal hemolysins may
be assisting the colonization of the human (or mammalian) airway
(Yike et al., 2003). Most people now spend more than 90% of their life
indoors. As we continue to co-habitate with fungi, we may well be
selecting for fungi that can not only live with us but also in us. For
example, Penicillium chrysogenum is the most common Penicillium
species found indoors (Summerbell et al., 1992). We now know that
what has been called Penicillium chrysogenum is made up of a number
of genetically distinct species (Scott, 2001). However, regardless of the
genetic species of P. chrysogenum, those that grow at 37 C on SBA
produce the hemolysin chrysolysin, but those that don’t grow, don’t
produce chrysolysin (Donohue et al., 2004). Further studies of the
population diversity in fungal hemolysin secretion may lead to a better
understanding of SBS.
VI. Application of Fungal Hemolysins in the Analysis of SBS

To better understand the role of exposures to indoor fungi, such as
S. chartarum, in human health, quantifiable biomarkers of exposure
must be developed. A biomarker should be specific to a given fungus
and yet occur in all strains of the fungus. It should also be easily
measured in bodily fluids as well as environmental samples.
The low molecular mycotoxins have been considered as possible
biomarkers. For example, the trichothecenes produced by S. chartarum
have been implicated as a cause of some ill health effects in animals
(Eppley and Bailey, 1973; Forgacs, 1965), but these toxins are difficult
to measure because of their low concentrations in environmental
samples and because of their rapid metabolism and elimination from
the body (Ueno, 1983). Furthermore, not all strains of S. chartarum
produce these mycotoxins ( Jarvis et al., 1998). An ELISA test for
roridin-trichothecene IgG failed to distinguish between individuals
exposed and not exposed to S. chartarum (Trout et al., 2002).
As an alternative, we propose measuring the proteinaceous fungal
hemolysins to quantify fungal exposures. For example, stachylysin
is produced by all strains of S. chartarum (tested so far) and is essen-
tially specific to S. chartarum (Van Emon et al., 2003). Since stachyly-
sin is highly immunogenic, it was easily measured by ELISA in rats
and human serum (but was not detected in the serum of controls) and
in environmental samples like dust (Van Emon et al., 2003). We are
in the process of developing similar assays for the other hemolytic
producing fungi.
VII. Conclusions
Although this chapter has discussed exclusively fungal hemolysins
in SBS, other microorganisms and/or other products might be just as
important or even more important in causing the symptoms of SBS. In
fact, it seems likely that there are interactions of many agents and
toxins in the symptomatology of SBS. However, some points about
fungal hemolysins that are relevant to SBS include:
. Fungal and bacterial hemolysins have much in common;
. Fungal hemolysins are commonly produced;
. Fungal hemolysins could cause some symptoms of SBS;
. Fungal hemolysins might be useful as biomarkers of exposure to
indoor fungi, since they can be measured in bodily fluids and
environmental samples.
ACKNOWLEDGMENTS
This work was supported by funding from the US EPA’s National Center
for Environmental Assessment’s ‘‘Children at Risk Program,’’ which is gratefully
acknowledged.
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The Roles of Penicillium and Aspergillus in
CHRISTOPHER J. SCHWAB AND DAVID C. STRAUS
Department of Microbiology and Immunology
Texas Tech University Health Sciences Center
Lubbock, TX 79430
I. Introduction 215
II. Characteristics and Importance of Penicillium spp. in SBS 216
III. Characteristics and Importance of Aspergillus spp. in SBS 226
IV. Conclusion 230
References 231
I. Introduction
Mold or fungal growth in buildings has been a problem since Biblical
times; it was described in detail in the Bible over 3300 years ago.
However, the problem was overlooked until about the mid-1980s,
when the term sick building syndrome (SBS) was coined after buildings
were examined where people complained of various symptoms
(Finnegan et al., 1984; Hodgson, 1992, 2000; Meyer et al., 1998;
Seidner, 1999). SBS symptoms typically occur in at least 20% of ex-
posed individuals and usually abate when the person has left the
affected building for an extended period of time. However, there is
mounting evidence that some SBS symptoms do not abate quickly if
at all, leading researchers to examine the problem more closely and to
consider a broader definition. Recent evidence by numerous investiga-
tors has shown the association of various species of fungi including
Penicillium sp. and Aspergillus sp. and their spores with indoor air
quality problems and SBS (Ahearn et al., 1997; Fung and Hughson,
2003; Hodgson, 2000; McGrath et al., 1999; Mishra et al., 1992; Spengler
and Sexton, 1983; Su et al., 2001). Fungi grow in most environments
and over wide temperature ranges. Fungi also require high humidity of
50–70%, which is similar to what humans enjoy indoors. In addition,
high water activity is also required for optimal fungal spore germina-
tion and growth of the organism (Horner et al., 1995). Fungal spores
have been shown to cause allergic diseases dating back to the late 1970s
and early 1980s (Gravesen, 1979; Licorish et al., 1985). Numerous other
studies have shown the association of exposure to mold spores indoors
215
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216 SCHWAB AND STRAUS
with increased IgE in patients from sick buildings (Lander et al., 2001;
Larsen et al., 1997; Meyer et al., 1998), exacerbation of asthma symp-
toms (Delfino et al., 1997; Dharmage et al., 2002), increased emergency
room visits for asthma (Black et al., 2000; Dales et al., 2000; Ross et al.,
2000), adenoid hypertrophy (Huang and Giannoni, 2001), allergic fun-
gal sinusitis (Khan et al., 2000a), and development of atopy or asthma
(Garrett et al., 1998). Presence of moisture associated with molds
in homes and schools has also been reported to be associated with
development of allergy or asthma symptoms (Brunekreef et al., 1989;
Garrett et al., 1998; Purokivi et al., 2001; Strachan and Sanders,
1989; Taskinen et al., 1999; Verhoeff et al., 1995; Walinder et al.,
2001). This review will focus on the importance of Penicillium spp.
and Aspergillus spp. in inducing symptoms consistent with SBS and
provide experimental evidence for some of the causes of symptoms by
these organisms.
II. Characteristics and Importance of Penicillium spp. in SBS

Penicillium spp. belong to the class Deuteromycetes, which is the
same class as Aspergillus spp. (Kurup et al., 2000). Most other molds
isolated from sick buildings also fall into this class (Kurup et al., 2000).
Penicillium spp. produce multiple spores or conidia on long stalks
called conidiophores (Fig. 1A). Most species of Penicillium appear
greenish blue on various agars (Samson et al., 1977) (Fig. 1B). Penicilli-
um spp. are some of the most commonly isolated molds from contami-
nated buildings (Ahearn et al., 1997; Burge, 1990; Cooley et al., 1998,
1999; Garrett et al., 1998; Li et al., 1995; McGrath et al., 1999). Recent
evidence by our laboratory and others has correlated the presence of or
sensitization to Penicillium sp., especially Penicillium chrysogenum,
with symptoms consistent with SBS (Cooley et al., 1998; Hiipakka and
Buffington, 2000; McGrath et al., 1999), allergic asthma (Licorish et al.,
1985; Salvaggio and Aukrust, 1981), allergic alveolitis (Fergusson et al.,
1984; Guglielminetti et al., 2000), atopy (Ezeamuzie et al., 2000; Lander
et al., 2001; Nolles et al., 2001), increased lower respiratory infections
in children during the first year of life (Stark et al., 2003), and wheezing
(Gent et al., 2002) and in zoological institutions with animal health
problems (Wilson and Straus, 2002). Various Penicillium sp. have been
characterized to be prevalent in both outdoor and indoor environments
worldwide (Burge, 1992; Burge and Rogers, 2000; Dharmage et al.,
1999; Hiipakka and Buffington, 2000; Koivikko et al., 1991; Lander
et al., 2001; Larsen et al., 1997; Li et al., 1995; Mishra et al., 1992;
THE ROLES OF PENICILLIUM AND ASPERGILLUS IN SBS 217
FIG. 1. Picture of Penicillium slide showing spores and conidiophore (A) and picture
of P. chrysogenum plate (B).
Ren et al., 2001; Salvaggio et al., 1993; Shen et al., 1999b; Su et al.,
2001). Multiple people developed sensitivities to Penicillium sp. in
contaminated schools in Connecticut, where normal levels were de-
fined as less than 1000 spores/m3 of air (Santilli and Rockwell, 2003).
In another study, Penicillium sp. were the most commonly isolated
molds in indoor dust samples (Chew et al., 2003). Indoor exposure to
Penicillium sp. and Aspergillus sp. spores have been shown to be
associated with increased risk of allergic sensitization in children
(Jacob et al., 2002).
A large number of public buildings were studied for indoor air
quality by taking indoor air samples and growing cultures of organisms
from these samples. Numerous fungi were isolated from the sampled
buildings, and P. chrysogenum was one of the predominant organisms
isolated from multiple buildings (Cooley et al., 1998). Penicillium sp.
have adapted to grow at room temperature and 50% relative humidity
similar to the ideal environment most people prefer. The spores of
P. chrysogenum range in diameter from 1–5 m with an average size
of 3.5 m. This relatively small size and round shape allow spores of
this organism to penetrate the lower airways when inhaled (Brain and
Valberg, 1979). The small size of Penicillium sp. spores also allows for
them to penetrate all but the smallest pore size filters and to be airborne
for extended periods of time, allowing increased chances of inhalation
indoors. There is evidence that Penicillium spp., including P. chryso-
genum, produce mycotoxins that may be important in symptoms
associated with SBS (Nielsen et al., 1999). However, multiple allergens
have been characterized from P. chrysogenum (formerly P. notatum),
P. citrinum, P. brevicompactum, and P. oxalicum that could play a role
in inducing some of the symptoms associated with exposure to the
organism in sick buildings, many of which are proteases and are sum-
marized in the following reviews (Bush and Portnoy, 2001; Kurup,
2003; Kurup et al., 2000; Shen et al., 1999b). Monoclonal antibodies
have been produced against several of these protease allergens (Lin
et al., 2000). Increased prevalence of IgE specific for the P. chryso-
genum major allergen Pen ch 13 was found in asthmatic patients, with
higher incidence in increasing age (Chou et al., 2003), and several of
the other characterized protease allergens from Penicillium spp. also
cross-react with allergy patient sera (Chow et al., 1999; Lai et al., 2002;
Shen et al., 1991, 1996, 1999a, 2000, 2001, 2003). All of these allergens
and proteases have been isolated from mycelial cultures and not con-
idia alone. Although many of these allergens cross-react with patient
IgE, indicating sensitization, the majority of exposure to sensitizing
doses of allergens would logically occur via inhalation of conidia. In
addition, few animal studies have been conducted outside of our labo-
ratory concerning the in vivo effects of exposure to conidia or allergens
from Penicillium spp. One study characterized the lung deposition
in rabbits of Penicillium sp. spores (Thurston et al., 1979), and
another characterized experimental allergic alveolitis in guinea pigs
(Fogelmark et al., 1991). Therefore we have focused our animal studies
on the roles of allergens released by viable P. chrysogenum conidia
as opposed to conidia mixed with culture filtrates.
Recent studies by our laboratory showed an increase in serum IgE
and IgG1 in mice inoculated with 1 104 viable P. chrysogenum spores
for 4–6 weeks. Pooled IgE and IgG1 from these mice were specific for
spore-associated proteins. The mice also developed increased airway
eosinophilia and neutrophilia (Cooley et al., 1999, 2000). These studies
also showed that nonviable P. chrysogenum spores produced by expo-
sure to methanol did not induce similar allergic reactions in mice
(Cooley et al., 2000). We also inoculated C57BL/6 mice with 1 105
viable P. chrysogenum spores to determine how the mice would re-
spond to an extremely high level of spores. Although the mice did not
develop significant levels of IgE (Fig. 2) after 8 weeks of spore inocula-
tions, they did develop significant levels of IgG2a (Fig. 2) that were
specific for viable P. chrysogenum spores (Fig. 3). These mice also
developed significant lung eosinophilia (Figs. 4 and 5) and neutrophi-
lia as well as increased lung mucous production, indicating an allergic
response developed in the absence of significant IgE (Fig. 5). The
presence of IgG2a indicates that the allergic response might be shift-
ing away from a Th2-mediated response or being down-regulated.
Since the effect of ambient levels of P. chrysogenum spores had not
been studied previously in animals, we inoculated C57BL/6 mice with
1 102 viable spores that correlated with relative levels of P. chryso-
genum spores that would be encountered in normal indoor environ-
ments without contamination. The spores were cultured for a week to
lower the viability. Mice inoculated with low levels of low viability P.
chrysogenum spores did not develop any detectable immune or allergic
responses (Schwab et al., 2003). This study indicated that most people
exposed to ambient levels of P. chrysogenum spores would not be
expected to develop allergic symptoms; however, actual clinical stud-
ies examining human exposure to low levels of spores would need to
be conducted to corroborate these findings.
Previous studies indicated that several proteins with proteolytic
activity could be isolated from supernatants of viable P. chrysogenum
spores incubated in liquid media for several days and these antigens
cross-reacted with sera from mice sensitized to viable P. chrysogenum
FIG. 2. Serum IgE and IgG2a levels after 8 weeks of IN inoculations with 1 105 viable
P. chrysogenum spores. Female C57BL/6 mice were inoculated IN with 1 105
viable (VIA) P. chrysogenum (Pc) spores for 8 weeks. Blood was collected biweekly retro-
orbitally until 24 h after final IN inoculation, when the mice were euthanized. After
euthanasia, blood was collected by cardiac puncture and sera were collected. Sera were
analyzed by sandwich ELISA specific for murine IgE (A) or IgG2a (B). Bars represent
mean serum Ig concentrations (N ¼ 6). Error bars represent SEM.
spores (Cooley et al., 2000). These proteolytic enzymes or protease

extracts, termed Pen ch, were isolated for biochemical characterization
and to be studied by using a murine model. Individual proteases were
not able to be isolated, and the extracts rapidly degraded when not kept
stored at 80 C, so we used only fresh samples of protease extracts for
FIG. 3. Spore-specific IgG2a from mice sensitized to 1 105 viable P. chrysogenum

spores for 8 weeks. Female C57BL/6 mice were inoculated via intranasal (IN) instillation
with 1 105 viable Pc spores for 8 weeks. Twenty-four hours after final inoculation, mice
were euthanized, blood was drawn by cardiac puncture, and sera were collected.
Immunoplates were coated with 1 106 viable Pc spores overnight, and then sera
collected from treated mice (VIA Pc spore IgG2a) were incubated in wells with spores.
BSA was added to wells, and control serum (Control IgG2a) from mice treated only with
PBS was incubated in wells with spores to serve as negative controls. Detecting antibody
labeled with HRP was incubated in wells, and TMB was added as substrate to react with
HRP. Absorbance was detected with a plate spectrophotometer using a 450 nm filter.
Bars represent mean of duplicate wells. A single asterisk (*) indicates statistical
significance compared to the negative control (P < 0.001).
our studies (Schwab et al., 2003). Only three other allergens have
been characterized from P. chrysogenum: Pen ch 13, a 34 kDa alka-
line serine protease (Chou et al., 2002, 2003), Pen ch 18, a 32 kDa
vacuolar serine protease (Shen et al., 1999b, 2003), and Pen ch 20, a
68 kDa N-acetylglucosaminidase (Shen et al., 1999b, 1995). Inhibition
studies indicated the protease extracts to have serine protease activity,
the most prevalent of fungal proteases, caused by decreased proteolytic
activity to the inhibitor leupeptin (Fig. 6). SDS-PAGE analysis also
indicated a 52 kDa protein that reacted with sera from spore-sensitized
animals in immunoblots (Fig. 7). We developed an animal model to
study the in vivo effects of the spore-associated protease extract Pen ch.
In one study, mice were sensitized with IP injections of 20 g
Pen ch plus alum followed by two weekly inoculations with either Pen
ch or 1 104 viable or non-viable spores. Mice sensitized to Pen ch
and challenged with either viable spores or protease extracts devel-
oped increased IgE, extract-specific IgG1, and lung eosinophilia and
neutrophilia. These mice also produced increased mucus (Schwab
et al., 2003). In another recent study, mice were inoculated by different
FIG. 4. Airway (BAL) eosinophils and neutrophils after 8 weeks of IN inoculations with
1 105 viable (VIA) P. chrysogenum spores. Female C57BL/6 mice were inoculated IN
with 1 105 viable Pc spores for 8 weeks. Twenty-four hours after the final IN
inoculation, the mice were euthanized, the lungs were lavaged, and BAL cells were
mounted on slides. 1000 BAL cells were counted and differentiated. Bars represent mean
counts of cells from each group (N ¼ 6). A single asterisk (*) represents statistical
significance as compared to the control animals (PBS) P < 0.01. Error bars represent SEM.
protocols utilizing either mostly IN or IP inoculations with various

doses of Pen ch (Schwab et al., 2004). Mice sensitized by IP inocula-
tions for 5 weeks followed by IN challenge with 10 g Pen ch devel-
oped the highest levels of serum IgE and IgG1. These groups of mice
also developed significant airway eosinophilia and neutrophilia as
well as airway perivascular inflammation and mucus cell hyperplasia.
Finally, Western blot and antigen-specific ELISA analysis determined
that mice sensitized to 1 104 viable P. chrysogenum spores recog-
nized epitopes within the protease extract Pen ch when immobilized
on PVDF membranes or in immunoplates (Schwab et al., 2004). This
was an important finding, as it demonstrated that viable P. chrysogen-
um spores release or contain proteases within the spore coat to allow
presentation to the immune system by dendritic cells. Although there
is a lot of cross-reactivity among fungal allergens, the recognition of the
isolated protease extract Pen ch by spore-sensitized serum confirms the
in vivo presentation of the protease antigens. Protease-sensitized sera
also recognized and bound to immobilized P. chrysogenum spores and
Pen ch. These data confirmed the recognition of epitopes within viable
P. chrysogenum spores the same as the protease allergens that were
inoculated.
FIG. 5. Histopathological examination of lungs from mice inoculated for 8 weeks with
1 105 viable P. chrysogenum spores. Female C57BL/6 mice were inoculated IN with
1 105 viable Pc spores for 8 weeks according to a treatment protocol. Twenty-four hours
after final IN inoculation, the mice were euthanized, the lungs were lavaged, and the
lungs were removed and placed in 10% neutral-buffered formalin without inflation.
The lungs were embedded in paraffin, sliced into cross-sections, and mounted on slides.
The slides were stained with H&E and PAS to view inflammation and mucin production,
respectively. The slides were examined by a pathologist, and pictures of important areas
were made. (A, normal lung H&E 125) (B, 1 105 Pc spore-sensitized lung H&E 500)
(C, normal lung PAS 125) (D, 1 105 Pc spore-sensitized lung PAS 125).
Mice treated with only one or no IP inoculation with the protease

extract Pen ch did not develop any significant allergic effects. This was
probably due to the fact that free protein would have a more difficult
time traveling through the airways, similar to difficulties with aerosol-
ized OVA in various studies without adjuvant (Beck and Spiegelberg,
1989; Kung et al., 1994). Viable P. chrysogenum spores travel into
lower airways because of their small size, and they also act as both an
adjuvant and carrier for the protease allergens. Evidence from our
FIG. 6. Specific inhibition of Pen ch proteolytic activity by various protease inhibitors.

50 g of the Pen ch protease extract (Pc Protease) was incubated in PBS alone or with
various protease inhibitors for 1 h at room temperature. After incubation with inhibitors,
samples were assayed by the trypsinogen activation proteinase assay to determine
specific activity of samples. Values are plotted as percent activity using specific activity
of Pen ch as 100%. Error bars represent SEM of two experiments. A single asterisk (*)
represents statistical significance compared to specific activity of Pen ch P < 0.02.
(AEBSF ¼ 4-(2-Aminoethyl)benzenesulfonyl fluoride, E-64 ¼ L-trans-epoxysuccinyl-
leucylamide-(4-gaunido)-butane.)
laboratory showed that alveolar macrophages clear P. chrysogenum

spores when inhaled but that they remain intact within the cells for
up to 36 hours (Cooley et al., 2000). Our hypothesis is that viable fungal
spores are inhaled by people when in buildings where there are num-
bers of spores present because of water events, and once inhaled, the
spores attempt to germinate in vivo, allowing the release of proteolytic
allergens. The allergens are then processed by dendritic cells in the
lungs, and dendritic cells migrate to the regional lymph nodes, where
they present the protease allergen peptides to T cells. Several studies
have recently shown the ability of dendritic cells to bind to and phago-
cytize fungal hyphal filaments and spores, including those of
A. fumigatus (Bozza et al., 2002; Claudia et al., 2002; Persat et al.,
2003). Spores from A. fumigatus were also shown to activate and
mature human Langerhans cells, indicating the importance of dendrit-
ic cells in responding to fungal infections and inhalation of viable
spores (Persat et al., 2003).
FIG. 7. Western blot of Pen ch with serum from mice sensitized to 1 104 viable Pc
spores. Penicillium chrysogenum viable spore protease extract (Pen ch) was added to a
30 k molecular weight cut-off (MWCO) spin column (Millipore) to separate protease
peptides by molecular weight. 10 l of each protease sample were subjected to SDS-PAGE
and transferred to a PVDF membrane. The membrane was incubated with pooled sera from
C57BL/6 mice sensitized to 1 104 viable Pc spores for 6 weeks. Chemiluminescensce
detected a band on autoradiography film that corresponded with a protein band at 52 kDa
in the corresponding silver-stained gel as shown in the top panel. (Lane 1 ¼ MW marker,
2 ¼ denatured Pen ch > 30 k, 3 ¼ denatured Pen ch < 30 k, 4 ¼ Pen ch.)
Sensitization to the P. chrysogenum spore-specific allergens in the

protease extract Pen ch induces strong allergic inflammation in a mu-
rine model, suggesting that avoidance of P. chrysogenum spores should
help minimalize exacerbation of symptoms. Allergen avoidance is
important in fungal sensitivities. Several studies have shown the
importance of removing or reducing exposure to molds and airborne
spores with reduced symptoms (Bush and Portnoy, 2001; Matheson
et al., 2003). Although Penicillium spp. are ubiquitous and nearly
impossible to keep out of indoor environments, it would be beneficial
for buildings and homes that become contaminated with these organ-
isms to be remediated as quickly and thoroughly as possible. Also, the
use of building materials that inhibit the growth of fungi should be
incorporated as possible, as a recent study showed the effectiveness

of inorganic ceiling tile in inhibiting the growth of P. chrysogenum
(Karunasena et al., 2001). Contamination of buildings with P. chryso-
genum and other Penicillium spp. is a growing problem and will
certainly require more extensive approaches to prevent and treat
sensitization to allergens from the organism that can lead to allergic
inflammation or asthma.
III. Characteristics and Importance of Aspergillus spp. in SBS

Aspergillus spp. belong to the class Deuteromycetes, which is the
same class as Penicillium spp. (Kurup et al., 2000). Since the spore sizes
of Aspergillus spp. are similar to Penicillium spores (about 2–5 mm),
they also easily travel through the airways into the lower parts of the
lungs, allowing for development of respiratory symptoms. Several
species of Aspergillus have been associated with indoor air quality
problems, SBS, or respiratory conditions (Burge, 1990; Burr, 1999).
Aspergillus spp. isolated from contaminated buildings or associated
with SBS include A. fumigatus (Lander et al., 2001), A. terreus, A.
flavus, A. niger, A. sydowii, A. versicolor, A. flavipes, A. candidus,
and A. ustus (Fig. 8). Aspergillus spores were isolated from homes of
asthmatic patients with mold sensitivity (Beaumont et al., 1985). Damp
homes of childhood patients with respiratory symptoms were found to
contain viable spores of Aspergillus (Brunekreef et al., 1989), and they
were associated with higher rates of atopy in children (Garrett et al.,
1998). In addition, a study of over 200 German schoolchildren showed
an association of allergic disease with the presence of Aspergillus spores
in homes (Jacob et al., 2002). Allergic respiratory disease patients were
found to be sensitive to Aspergillus extracts (Ezeamuzie et al., 2000),
and high levels of Aspergillus spores in damp buildings where workers
complained of respiratory symptoms were associated with increased
histamine release from basophils when bound with specific IgE
(Lander et al., 2001). A. versicolor was recently implicated as the cause
of various respiratory symptoms in several workers in a courthouse
building (Hodgson, 1998). High levels of Aspergillus spores were
isolated from an office building with multiple complaints of respiratory
symptoms (Hiipakka and Buffington, 2000). High levels of A. flavus
and A. niger spores inside homes were shown to be associated with
atopy in asthmatic children (Li et al., 1995), and the predominant
allergen isolated from A. flavus showed cross-reactivity with allergic
patient IgE (Chou et al., 1999; Yu et al., 1999). Multiple people
developed sensitivities to Aspergillus sp. in contaminated schools in
FIG. 8. Picture of Aspergillus spp. spores and fruiting structure (A); picture of A.
versicolar grown on an SDA plate (B).
Connecticut where normal levels were defined as less than 1000

spores/m3 of air (Santilli and Rockwell, 2003). An association of sensi-
tivity to A. fumigatus extracts was also shown with atopic children by
cross-reactivity with IgE (Nolles et al., 2001).
Few animal studies have been conducted to elucidate the mech-
anism of sensitization to fungal spores and allergens released by
contaminating molds implicated in sick building syndrome. Recent
studies have characterized the allergic response of mice to A. fumigatus
conidia in which mice developed increased lung eosinophilia, epithe-

lial desquamation, and mucus production (Blease et al., 2000, 2001b;
Hogaboam et al., 2000, 2003), allergic sensitization to Aspergillus
spores in guinea pigs (Fogelmark et al., 1991), and lung deposition in
rabbits of A. fumigatus spores (Thurston et al., 1979). Another recent
study showed that carbohydrates expressed in extracts of A. fumigatus
were responsible for allergic Th2 responses in mice (Yamashita et al.,
2002). A. fumigatus is a well-characterized opportunistic mold that
plays a role primarily in immunocompromised patients, causing aller-
gic bronchopulmonary aspergillosis (ABPA) (Grunig and Kurup, 2003;
Khan et al., 2000b; Kurup and Kumar, 1991; Murali et al., 1994), and
several allergens isolated from A. fumigatus have been linked with this
disease (Banerjee et al., 1998, 2001). A. fumigatus spores were found to
release allergens after germination that reacted with human IgE (Green
et al., 2003). Recent studies characterized the induction of allergic
asthma in BALB/c mice by inoculations with recombinant A. fumiga-
tus allergens, some of which have been characterized as proteases and
reviewed in Crameri (1998) and Kurup et al. (2001, 2002). These re-
combinant allergens were shown to induce airway hyperreactivity,
airway inflammation by eosinophils, increased production of IgE, and
expression of Th2 cytokines (Grunig and Kurup, 2003; Kheradmand
et al., 2002; Kurup et al., 2001, 2002) in animal studies. Crude protease
extracts from A. fumigatus induced IL-6 and IL-8, which are proin-
flammatory cytokines, and also caused cell shrinking and cell desqua-
mation in human respiratory epithelial cell lines (Borger et al., 1999;
Kauffman et al., 2000). They have also been shown to bind human IgE
and be reactive to type I skin hypersensitivity (Kauffman et al., 1984;
Moser et al., 1992). Many of the allergens isolated from Aspergillus spp.
are proteases, have been cloned and sequenced, and have sequence
homology and cross-react with sera specific for protease allergens from
Penicillium spp. (Chou et al., 1999; Kunert and Kopecek, 2000; Lai
et al., 2002; Moser et al., 1992). Monoclonal antibodies have also been
developed to many of these protease allergens to aid in their character-
ization (Lin et al., 2000). Our lab isolated proteases from viable spores
of A. terreus, A. flavus, A. niger, A. sydowii, A. versicolor, A. flavipes,
A. candidus, and A. ustus, added these enzymes to immunoplates, and
incubated the bound proteases with serum from mice sensitized to the
protease extract Pen ch from viable P. chrysogenum spores. The Pen
ch-sensitized serum reacted with protease extracts from all Aspergillus
spp. examined except for A. flavus (Fig. 9). Studies by other investiga-
tors have also shown cross-reactivity among fungal proteases with
mouse and human sera, and sequence data also indicate that protease
FIG. 9. Cross-reactivity of various Aspergillus sp. spore-associated proteases with

IgG from mice sensitized to Pen ch. Protease extracts isolated from viable spores of
various Aspergillus sp., Pen ch, and BSA were coated on immunoplates overnight. Sera
from mice sensitized to 10 g Pen ch were incubated with each protein. Bound IgG1
was detected by addition of biotinylated anti-mouse IgG1 followed by addition of
HRP-streptavidin. Detection occurred by addition of TMB substrate. Absorbance
readings were determined by a spectrophotometric plate reader with a 450-nm filter.
Bars represent mean absorbance readings of duplicate wells minus absorbance readings
of BSA wells. A single asterisk (*) represents statistical significance as compared with
wells incubated with sera from mice treated with PBS (Control) P < 0.001.
allergens isolated from numerous Aspergillus spp. and Penicillium

spp. share similar N-terminal sequences.
The mechanism of sensitization to fungal spores and allergens has
been elucidated to some degree. The importance of chemokines, includ-
ing chemoattractant protein-1 receptor (CCR2) (Blease et al., 2000), and
C10 chemokine (Hogaboam et al., 1999) in regulating the response to
A. fumigatus spores has been shown recently in several elegant studies
and reviewed in Hogaboam et al. (2003). A recent study showed that
epithelial and endothelial cells can internalize A. fumigatus spores,
which would allow for them to interact with dendritic cells underlying
the endothelium and epithelium in the airways (Paris et al., 1997).
Several studies have recently shown the ability of dendritic cells to bind
to and phagocotyse fungal hyphal filaments and spores, including those
of A. fumigatus (Bozza et al., 2002; Claudia et al., 2002; Persat et al.,
2003). Spores from A. fumigatus were also shown to activate and mature
human Langerhans cells, indicating the importance of dendritic cells in
responding to fungal infections (Persat et al., 2003). However, many

questions are still unanswered concerning exactly how fungal spores
are processed and allergens presented to the immune system to induce
either tolerance or allergic disease in susceptible individuals.
There has been some interest in developing novel therapies to
mold sensitization. A recently published study utilized a fusion pro-
tein consisting of human IL-13 and a mutated form of Pseudomo-
nas exotoxin that significantly reduced allergic airway disease in
mice sensitized to viable Aspergillus fumigatus conidia, suggesting
a novel immunotoxin approach to treating fungus-induced allergic
inflammation (Blease et al., 2001a). Other studies have utilized chitin
microparticles (Strong et al., 2002a) and recombinant surfactant pro-
tein D (Strong et al., 2002b) to down-regulate symptoms of allergic
inflammation in mice in response to sensitization to allergen extracts
from A. fumigatus. However, a recent study showed that mice sensi-
tized to A. fumigatus extracts that developed Th2-mediated inflamma-
tion and challenged with chicken ovalbumin (OVA) did not develop
tolerance to the antigens, indicating that simply treating patients with
mold extracts after sensitization might not down-regulate ongoing al-
lergic disease in patients sensitized to molds in indoor environments
(Hurst et al., 2001). Standard treatments for symptoms of allergic sen-
sitization and asthma remain the primary therapy for patients sensi-
tized to Aspergillus and other molds (Schubert, 2000). In addition, sick
buildings contaminated with Aspergillus should be remediated as
completely and expeditiously as possible to decrease the spore burden
and lessen symptoms. Abatement of fungal spores from sick buildings
has shown some success (Bush and Portnoy, 2001), and continued
improvement in this area should be a main focus of research.
IV. Conclusion
The term sick building syndrome has only been around for about 20
years, but the number of cases reported in the literature increases
significantly each year. It is clear that Penicillium spp. and Aspergillus
spp. play a major role in causing many of the allergic and respiratory
symptoms seen in people exposed to high levels of spores and allergens
from these organisms in contaminated buildings. Although the spores
are ubiquitous and too small to filter from most indoor environments,
better control of humidity, air handling, and plumbing as well as
incorporation of mold-inhibiting building components should decrease
the incidence of complaints related to these organisms. Although many
allergens have been characterized from various species of Penicillium
and Aspergillus and important animal studies have been conducted,
much more evidence is needed to determine the exact mechanisms of
sensitization to fungal spores and allergens. Also, standard air monitor-
ing protocols, acceptable and unacceptable indoor levels of Penicillium
and Aspergillus spores, and standard abatement procedures should be
established to enable indoor air quality specialists better guidelines in
dealing with issues concerning sick building syndrome associated with
these organisms. In addition, better treatment options need to be exam-
ined for alleviating symptoms associated with mold sensitization and
allergic disease, and novel preventive or tolerance-inducing therapies
need to be developed to allow those working in contaminated or damp
and moldy environments greater protection from developing symptoms
or severe respiratory diseases. This will be an exciting area of research
for many years to come and should enhance the lives of numerous office
workers, children, and homeowners.
ACKNOWLEDGMENTS
The authors are grateful to Dr. Suzanne Graham for the histopathological analysis, to
Dr. Cindy Jumper for providing funding through an endowed professorship from Texas
Tech University Health Sciences Center (TTUHSC), and for funding provided through a
Center of Excellence award from TTUHSC.
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Pulmonary Effects of Stachybotrys chartarum in
Animal Studies
IWONA YIKE AND DORR G. DEARBORN
Case Western Reserve University
Mary Ann Swetland Center for Environmental Health
Department of Pediatrics
Rainbow Babies and Children Hospital, Cleveland, Ohio 44106
I. Introduction 241
II. In Vivo and In Vitro Studies Using Pure Trichothecene Toxins 244
III. Animal Studies Using Fungal Spores and Other
Fungus-Derived Components 245
A. The Effects of Trichothecene Toxicity 246
B. The Effects of Spore Viability 251
C. The Effects of Hemolysin 252
D. The Effects of Proteinases 256
E. Allergenicity and Antigenicity 257
F. The Effects of Volatile Organic Compounds 258
IV. Practical Considerations for Designing Animal Experiments of
Exposure to S. chartarum 259
A. Characterization of Fungal Spores 259
B. Animals 261
C. Exposure Route 262
D. Exposure Dose and Regimen 263
V. New Directions in the Investigation of the Effects of S. chartarum
in Animal Models 263
A. Toward Dissecting Pathophysiologic Mechanisms 264
B. The Acute and Chronic Effects of Lower Doses 265
C. Investigations of the Effects of Environmental Molds in the
Presence of Other Environmental Factors 266
VI. Conclusions 266
References 267
I. Introduction
The toxigenic fungus Stachybotrys chartarum (Hughes, S. atra Cor-
da) is one of several environmental fungi that can produce very potent
compounds toxic to humans and animals. The organic dust syndrome
from occupational exposures of farm workers to the toxigenic fungi
is well described and includes nasal and tracheal bleeding (in contrast
to alveolar bleeding), skin irritation, and alterations in white blood
cell counts (Hintikka, 1978). S. chartarum produces macrocyclic tri-
chothecenes that are the most potent members of a large family of
trichothecenes (Jarvis, 1991; Jarvis et al., 1995). Trichothecenes bind
241
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242 YIKE AND DEARBORN
to a single binding site on 60S ribosomes and directly inhibit either

initiation, elongation, or termination of protein synthesis depending
on which trichothecene is bound (Feinberg and McLaughlin, 1989).
Most of the investigation of trichothecenes has been supported by the
U.S. Dept of Defense, since they are suspected to be chemical warfare
agents. T-2 toxin was thought to be the active component of ‘‘yellow
rain,’’ which the United States accused the Soviet Union of using in
Vietnam and Afghanistan (Christopher et al., 1997), and Iraq has been
known to have a stockpile of trichothecenes (Zilinskas, 1997). Satra-
toxin G produced by S. chartarum was reported to be the most cytotox-
ic of eight trichothecenes tested on mammalian cells (Yang et al.,
2000). The LD50 in mice for satratoxins is <1 mg/kg (Jarvis, 1991).
Until recently, spirocyclic drimanes and their precursors produced
by S. chartarum received very little attention. Their activity includes
inhibition of proteolytic enzymes, disruption of the complement sys-
tem, inhibition of TNF- release, stimulation of plasminogen, fibrino-
lysis, thrombolysis, and cytotoxic and neurotoxic effects. They have
also been reported as endothelin receptor antagonists (Miller et al.,
2003; Nielsen, 2003).
S. chartarum is also capable of producing cyclosporin, an immune
suppressant targeting T-lymphocytes (Sakamoto et al., 1993). A novel
class of compounds, atranones, that are without known physiological
effects, has been described by Hinkley et al. (1999, 2000). The existence
of at least two (Hinkley et al., 2000; Jarvis, 2002) and possibly three
(Andersen et al., 2002) chemotypes of S. chartarum has been postu-
lated. One of them represents the isolates that produce macrocyclic
trichothecenes, and the other two produce atranones. The relationship
between these groups of isolates that appear to be genetically distinct
(Peltola et al., 2002) to two distinct phylogenetic species of S. chartar-
um postulated by Cruse et al. (2002) is not clear. The examination of
many S. chartarum isolates from the United States and Europe shows
that only about one third of the isolates produce the macrocyclic
trichothecenes (Andersen et al., 2002). In addition to mycotoxins,
S. chartarum produces proteinases that may contribute to tissue dam-
age similarly to other microbial proteinases (Kordula et al., 2002; Yike
et al., 2002). Another potentially harmful protein, stachylysin, with
hemolytic activity, has been described (Vesper et al., 2001). Also,
(1!3)--D-glucan, a cell wall component of fungi, has been linked to
the development of inflammatory reactions caused by environmental
molds (Beijer et al., 2002; Young et al., 2003).
The S. chartarum spores were not previously known to germinate
in the lung, nor is there a yeast form of this fungus. Inhalation exposure
STACHYBOTRYS CHARTARUM IN ANIMAL STUDIES 243
to the spores does not appear to cause infections; rather, it leads to
a mycotoxicosis (Section III.B).
The paucity of reports of hypersensitivity pneumonitis or allergy
from exposure to this organism may result from the lack of adequate
clinical testing, incomplete awareness, and absence of adequate envi-
ronmental detection methods. It has been reported that the spores of
S. chartarum can trigger the release of histamine in vitro by a non-IgE-
mediated pathway (Larsen et al., 1996). More recent evidence points
to strong IgE responses in animals exposed to this fungus (Korpi et al.,
2002; Viana et al., 2002). IgE antibodies binding S. chartarum proteins
have been detected in 9.4% of general population plasma specimens
tested (Barnes et al., 2002). While the microbial volatile organic com-
pounds (MVOCs) from other fungi have been investigated extensively,
information on those agents produced by S. chartarum has only recent-
ly been addressed (Gao and Martin, 2002).
Concern about S. chartarum in indoor environments surfaced in the
mid-1980s. Case reports in both residential and the non-industrial
workplaces suggested that chronic indoor exposures could result in a
variety of debilitating respiratory and non-respiratory symptoms (Croft
et al., 1986; Hodgson et al., 1998; Johanning, 1995) perhaps including
an effect on immune function (Johanning et al., 1996) and cognitive
impairment (Gordon et al., 1999). While still wet, the spores are sticky,
but when dry they are readily aerosolized and give an effective aerody-
namic diameter of 5.2 m (Sorenson et al., 1996) allowing inhalation
out to the distal airways. Aerodynamic modeling (Phalen and Oldham,
2001) predicts that this size particle has a six-fold greater likelihood
of lung deposition in the human infant lung than those of adults.
Inhalation exposure may also involve recently described micro-
particles that are significantly smaller than spores (1 m; Madsen
et al., 2003). Over the past 10 years in northeast Ohio, there have been
51 cases of acute pulmonary hemorrhage in young infants. Sixteen of
the infants have died. In November 1994, the Centers for Disease
Control and Prevention (CDC) began an investigation into the cause
of this outbreak (CDC, 1994, 1997, 2000). All but 13 of these cases have
occurred within a contiguous eight zip code area in the eastern part of
the metropolitan area. The case-control study found an association
with exposure to S. chartarum and other fungi (Etzel et al., 1998).
The association of Stachybotrys inhalation exposure with alveolar
hemorrhage in the human infant had not been reported prior to these
studies. Our experience has been limited to young infants, all but two
of whom have been <6 months old. The initial concept was that the
inhalation of Stachybotrys spores containing toxins, most notably the
trichothecene protein synthesis inhibitors, leads to focal areas of de-

creased protein synthesis (e.g., type IV collagen) in the young infant
lungs that are still growing exponentially, resulting in weakened endo-
thelial basement membranes (i.e., capillary fragility) (Dearborn et al.,
1999, 2002). Subsequent exposure to stresses that alter blood flow in
the lungs (e.g., unequal hypoxic vasoconstriction from environmental
tobacco smoke [ETS], sympathetic storm of asphyxia) could lead to
local areas of increased capillary pressure and subsequent stress hem-
orrhage of these fragile capillaries. Transmural pressures insufficient to
rupture normal capillaries may be pathogenic under these conditions
(West and Mathieu-Costello, 1995). In addition, the possible direct cell
injury from the macrocyclic trichothecenes may be exacerbated by
stachylysin, found in Stachybotrys spores (Gregory et al., 2003; Vesper
et al., 2001), along with further capillary weakening from the collag-
enolytic activity to Stachybotrys proteinases released from the spores
(Yike et al., 2002). The millimolar concentrations of satratoxin G in
these spores is five orders of magnitude greater than the EC50 for
protein synthesis inhibition (Sorenson et al., 1987). This, along with
the rapid release of toxins into aqueous media (see below), suggests
high toxicity may occur in the area around the inhaled spore.
Other cases of pulmonary hemorrhage linked to S. chartarum have
been reported (Elidimir et al., 1999; Flappan et al., 1999; Weiss and
Chidekel, 2002).
II. In Vivo and In Vitro Studies Using Pure Trichothecene Toxins

As potent protein synthesis inhibitors, trichothecenes cause severe
damage to actively dividing cells and have been investigated as potential
anti-neoplastic agents in humans; however, the efficacy/toxicity ratio
was too small for them to be of value (Jarvis and Acierto, 1989). As
recently reviewed by Bondy and Pestka (2000), they can be both im-
munosuppressive and immunostimulatory, depending on the dose and
exposure regimen. While they are cytotoxic to macrophages (Sorenson
et al., 1986), trichothecene exposure in vitro impairs or enhances mito-
gen-induced lymphocyte proliferation, depending on the dose. Simi-
larly, their dose response in vivo is biphasic for humoral immunity,
with a differential effect on immunoglobulin classes. It is notable that
the oral administration of deoxynivalenol (DON) to rats produces a
pronounced elevation of serum IgA and a concurrent depression of
IgM and IgG. This dysregulation results in immunopathology very
similar to human IgA nephropathy (Berger’s disease) and is the basis
of a widely studied rat model of IgA nephritis (Pestka et al., 1989).
Immunosuppression from trichothecene doses lower than those
causing acute cell death promotes rapid onset of leukocyte apoptosis
in parallel to the effect of cycloheximide. Immunostimulation at even
lower doses produces cytokine (e.g., IL-2, IL-6) superinduction that
stems from a differential sensitivity to protein synthesis inhibition of
cellular regulatory elements (e.g., decreasing IB synthesis resulting
in release of transcription factor NF-B) (Ouyang et al., 1996a,b).
Similar dysregulation has been observed to occur through alteration
of the Jun and Fos family proteins’ modulation of the activity of AP-1
transcription factor (Li et al., 2000).
Induction of cyclooxygenase-2 expression in macrophages by DON
is mediated by ERK and p38 but not JNK mitogen-activated protein
kinases (Moon and Pestka, 2002). The macrocyclic trichothecenes pro-
duced by S. chartarum have been found to be at least 100 times more
potent than DON (Lee et al., 1999); their cytotoxicity and apoptosis
induction appear to involve mitogen-activated protein kinases (MAPKs),
ERK, p38MAPK, and SAPK/JNK (Yang et al., 2000). Satratoxin-induced
apoptosis in a human leukemia cell line involves caspase-8 and caspase-9
activation of caspase-3 (Nagase et al., 2002). These studies have primar-
ily been performed in vitro or in ingestion animal models, and little
is known about the minimal levels required to alter immune function
with inhalation exposure of the satratoxins. Trichothecenes have also
been shown to cause membrane damage that results as a function
of the diphosphatidylcholine concentration of the membrane type
(Gyonggyossy-Issa et al., 1986; Khachatourians, 1990).
Studies of experimental inhalation exposures to the pure trichothe-
cene T-2 toxin have been done in mice, rats, guinea pigs, and swine. In
some of these experiments no lesions were found in the lung (Creasia
et al., 1987), while in others inflammatory symptoms consistent with
pneumonitis were observed (Pang et al., 1987). The LD50 was typi-
cally one order of magnitude less by inhalation compared to systemic
administration (Creasia and Lambert, 1989; Creasia et al., 1990).
III. Animal Studies Using Fungal Spores and Other

Fungus-Derived Components
Experiments employing fungal spores allow for the use of mycotox-
ins in their natural form. The slow mycotoxin release from inhaled
spores results in local lung injury, while the inhalation of pure myco-
toxins leads to their diffusion to the blood stream so rapidly that the
cell injury is to other organ systems, as shown by Thurman et al. (1988).
It appears that pulmonary exposures to spores and purified mycotoxins
have different pathological profiles even if the latter is delivered in a

particulate, crystalline form. The stream-oriented effective diameter of
dry S. chartarum spores is 5.2 microns (Sorenson et al., 1996); thus
these spores can, and experimentally do, reach the distal airways when
instilled in the trachea. Finally, the pathophysiological effects of other
components of fungal spores such as fungal proteins (Vesper et al.,
2001; Yike et al., 2002) and (1 ! 3)--D-glucan (Rylander, 1999) can be
investigated in these experimental models.
A. THE EFFECTS OF TRICHOTHECENE TOXICITY

Most of the initial studies employing the spores of S. chartarum
focused on trichothecene toxicity. Nikulin et al. (1996) compared the
effects of two isolates of S. chartarum by using 5-wk-old NMRI mice.
The isolates differed 13,000-fold in the cytotoxicity of spore extracts to
feline fetus lung cells. Satratoxins G and H, stachybotrylactone, and
stachybotrylactam were detected in the culture of the toxic isolate. No
satratoxin and only minor amounts of stachybotrylactone and stachy-
botrylactam were found in the culture of non-toxic isolate. The mice
were exposed intranasally to 1 106 spores. Only 50% (2 out of 4) of
the animals treated with the toxic isolate survived the 3-day observa-
tion period, showing 17% weight loss. The inflammation with hemor-
rhagic exudates in the lungs of animals receiving spores of the toxic
isolate of S. chartarum was described as much more severe than in
animals receiving spores of nontoxic isolate. However, these histo-
pathological differences were not quantified. Such quantification is
complicated with both intranasal and intratracheal instillation because
the variable distribution of fungal spores results in subjective scoring
systems being inaccurate. Similar histopathology was observed with
repeated exposure (6 times) to 1 103 and 1 105 spores of the same
two fungal isolates (Nikulin et al., 1997). Both doses of spores from
the toxic isolate resulted in severe lung inflammation. Only the higher
dose of the spores from the nontoxic isolate elicited pulmonary inflam-
mation, while the lungs of the mice exposed to the lower dose were
identical to those of control animals treated with phosphate-buffered
saline. No histopathological effects were detected in the thymus, spleen,
and intestine of mice. Hematological changes in these mice exposed to
both doses of toxic and non-toxic isolates included decreased platelet
counts and increases in the numbers of leukocytes and erythrocytes,
reflected in hemoglobin concentrations and hematocrit. These early
studies are limited by the lack of histological quantification and the
low numbers of animals used.
Rao et al. (2000a,b) examined the pulmonary effects of S. chartarum
spores in 10-wk-old rats. The animals were exposed intratracheally to
different doses of spores (3000–30,000/g) obtained from a highly toxic
isolate. The analysis of bronchoalveolar lavage (BAL) fluid obtained
after 24 h revealed dose-dependent increases in lactate dehydrogenase,
albumin, hemoglobin, myeloperoxidase, and leukocyte differential
counts. No statistically significant effects were observed when the
animals were treated with methanol-extracted spores. Higher doses of
intact spores but not alcohol-extracted spores led to weight loss of
treated animals. The authors concluded that pulmonary inflammation
and injury and weight losses were related to methanol-soluble toxins of
the spores. The time course of responses supported early release of
toxins, with the most severe effects occurring between 6 and 72 h after
exposure.
A series of studies conducted with Carworth Farm White (CFW) mice
and direct intratracheal instillations also focused primarily on tricho-
thecene toxicity. Many effects observed with fungal spores were paral-
leled by pure satratoxin F (Rand et al., 2002). Alveolar type II cells
and alveolar macrophages appear to be particularly sensitive. The
studies of Mason et al. (1998, 2001), McCrae et al. (2001), and Sumarah
et al. (1999) have shown that S. chartarum spores and isosatratoxin
F exposure induces significant changes in the phospholipid composi-
tion of pulmonary surfactant in BAL fluid. S. chartarum spores and
isosatratoxin F induce changes in regulation of both secretion and syn-
thesis of pulmonary surfactant and in the pattern of phospholipid tar-
geting to the pulmonary surfactant pools in mice (Mason et al., 1998).
These agents alter the activity of convertase, which is responsible for the
conversion of surfactant fractions from surface active to those metaboli-
cally used (Mason et al., 2001). These changes in mice may be due to
an increase in pulmonary surfactant phospholipids associated with
alveolar type II cell damage (McCrae et al., 2001). S. chartarum spores
and toxin induce other changes in lung surfactant phospholipid com-
position including depressed disaturated phosphotidylcholine (DSPC;
Sumarah et al., 1999), the major phospholipid responsible for main-
taining surface-tension properties of lung surfactant. Hastings et al.
(in press) have recently shown that depressed DSPC synthesis in mice
exposed to S. chartarum spores is probably related to modulation of a
key enzyme, CTP: cholinephosphate cytidylyltransferase (CPCT), in
the phosphotidylcholine synthesis pathway.
An infant rat model of pulmonary stachybotrytoxicosis describing
the effects of a single tracheal instillation of fungal spores on survival,
growth, histopathology of the lung, and respiration was developed by
Yike et al. (2001). The toxicity of the nonviable spores of a highly toxic
Cleveland isolate JS58-17 was documented by using the luciferase
translation inhibition assay (Yike et al., 1999) to be equal to 1pg of
satratoxin-G equivalents per spore (1 mM within the spore). Four-
day-old infant Sprague-Dawley rat pups were exposed to 1–8 105
spores/gm body weight (BW) via tracheostomy. Control animals
received either PBS (phosphate-buffered saline) or fungal spores whose
toxicity was undetectable after extraction with ethanol. The LD50 dose
determined in dose-response experiments was 2.7 105 spores/gm
BW. All dead pups (73% at 4 105 spores/gm BW) had extensively
hemorrhagic lungs. The period of acute toxicity as judged by reduction
in body weight and mortality lasted for 72 h after exposure. The
growth of surviving animals was impaired in a dose-dependent man-
ner. Animals exposed to 1.1 105 sp/gm BW (low mortality rate of 2%)
had changes of pulmonary function parameters (decreased respiratory
rate, higher tidal volume) consistent with increased pulmonary resis-
tance. Lung histology revealed fresh hemorrhage, hemosiderin-laden
macrophages, and evidence of inflammation including thickened alve-
olar septa infiltrated with lymphocytes and mononuclear cells. Signifi-
cant increases (P < 0.001) in numbers of macrophages (2-fold),
lymphocytes (5-fold), and neutrophils (7-fold) were found in BAL
fluid. Hemoglobin was elevated 2-fold (P ¼ 0.004). Cytokines measured
at 72 hours noted IL-1 increased more than 6-fold and TNF- 30-fold
(P < 0.001). No histopathological changes were detected in spleen,
thymus, intestines, kidneys, and brain of animals exposed to intact,
nonviable spores. Mild focal necrosis was occasionally seen in the
livers of animals treated with higher doses of spores. The numbers of
inflammatory cells declined significantly after 8 days. Since extracted
spores had minimal effect on all BAL fluid parameters, the conclusion
from these studies was that mycotoxins were primarily responsible for
the hemorrhagic and inflammatory response; however, subsequent
findings have extended this interpretation (see below).
Satratoxin and/or other trichothecenes would constitute much need-
ed biomarkers of acute exposure to S. chartarum if detectable in blood
and other body fluids. However, only about 0.13% of satratoxin-G was
recovered in ethanol extracts of whole blood of rat pups exposed
intratracheally to 4 105 spores/gm BW (equivalent to 4 105 pg of
satratoxin G/gm BW) when the animals were sacrificed immediately
following spore instillation (Yike, unpublished results). No toxin could
be detected at any other time point from 15 minutes up to 24 hr. About
5% of satratoxin G was recovered in the BAL fluid supernatants at
0 time. This amount dropped rapidly to 0.34% after 30 minutes and
declined steadily, reaching 0.02% after 24 h. These results—suggesting
a very rapid release, absorption, and metabolism of free toxin—confirm
previous studies by Craesia et al. (1987). The spores of S. chartarum
suspended in an aqueous medium (phosphate-buffered saline) release
trichothecenes within minutes, leading to the 50% loss of toxicity in less
than half an hour (Yike, unpublished). Similar observations by Jarvis
(personal communication) suggested that S. chartarum, similarly to
other fungi (Demain, 1995), releases a major portion of the trichothecene
toxin from the surface of the spores.
Immunochemical localization of satratoxin within the spores of
S. chartarum found it to be primarily along the outer plasmalemma
surface and in the inner wall layer in contrast to only modest staining
detected in hyphae (Gregory et al., 2004). In mouse lung tissues im-
pacted by the spores of highly toxic isolate JS58-17, the highest labeling
was detected in macrophage lysosomes but also along the inside of the
nuclear membrane in nuclear heterochromatin and the rough endo-
plasmic reticulum. Aveolar type II cells also showed modest labeling
of the nuclear heterochromatin and rough endoplasmic reticulum,
while there was little evidence of toxin accumulation in neutrophils,
fibroblasts, or other cells associated with the granuloma tissues sur-
rounding spores or mycelial fragments. These observations of a high
degree of cellular specificity suggest that the alveolar macrophage
plays an important role in sequestration of the spores and mycotoxins.
Alveolar macrophages but not neutrophils containing fungal spores
are frequently observed in the BAL fluid of exposed rat pups (Yike,
unpublished).
While most of the studies discussed above focus on trichothecene
toxicity, more recent findings indicate the involvement of other fungal
derived compounds (Leino et al., 2003; Murtoniemi et al., 2001;
Nielsen et al., 2002; Yike et al., 2002, 2003b). The observations of Leino
et al. (2003) conducted on mice demonstrate the lack of significant
differences in stimulating inflammatory responses in mice between
the satratoxin-producing and non-producing isolates. The increases
in inflammatory cells in the BAL fluid as well as the induction of Il-
1, Il-6, and TNF- in the lungs of mice following repeated exposure to
the spores of S. chartarum were similar for both isolates. Only one
chemokine CXCL5/LIX showed significantly higher mRNA levels after
exposure to the satratoxin producing isolate. Flemming et al. (2004)
noted lack of differences in the composition of BAL fluid from mice
exposed to low doses of high trichothecene producing (JS58-17) and
nontoxic (JS58-06) isolates of S. chartarum spores. These data agree
with observations presented in Fig. 1 and support the hypothesis that
FIG. 1. Inflammatory indices in BAL fluid from infant rats exposed to S. chartarum.
Seven-day-old pups (8 animals per experimental group) were exposed intratracheally to
4 104 spores/gm BW of two different isolates of S. chartarum, high trichothecene
producer JS 58-17 and low trichotecene-producing, highly hemolytic JS58-06, grown
either on drywall (DW) or potato dextrose agar (PDA). Control animals received
phosphate-buffered saline (PBS). Bronchoalveolar lavage was performed 48 h following
exposure. The values are expressed per milliliter of epithelial lining fluid (ELF). nd, not
detected. TNF-, Tumor necrosis factor alpha. Il1-, Interleukin 1 beta. *Significantly
different at stated P value.
TABLE I
CHARACTERIZATION OF THE SPORES FROM TWO ISOLATES OF S. CHARTARUM
Toxicity SG
equivalents
(pg/spore)
Growth Luciferase Proteolytic activityc Stachylysind

a
Strain medium ELISA translationb Units/106 spores ng/mg dry wt.
JS58-17 Drywall 0.72 0.67 0.41 1414

JS58-17 PDA 0.60 0.89 1.35 —
5
JS58-06 Drywall 4.65 10 0.04 0.82 8963
JS58-06 PDA 5.00 105 0.03 3.32 —
a
Satratoxin G ELISA assay; Chung et al., 2003.
b
Luciferase translation inhibition assay; Yike et al., 1999.
c
Enzcheck assay, Molecular Probes; Yike et al., 2002.
d
Stachylysin ELISA assay; Van Emon et al., 2003.
fungal components other than trichothecenes mediate the develop-

ment of inflammation. According to Nielsen et al. (2002), the cyto-
toxicity of S. chartarum isolates appears to be related to satratoxin
production, whereas the specific, inflammation-inducing component
from atranone-producing isolates remains obscure. We postulate that
fungal proteins described below (Section II.C,D,E; Table I, and Fig. 1)
are inducers of the inflammatory response and may directly contribute
to lung injury.
B. THE EFFECTS OF SPORE VIABILITY

Sarkisov and Orshanskaiya (1944) suggested that S. chartarum might
be infectious based on their investigation of horses exposed through
contaminated hay. However, the experimental testing of survival and/
or proliferation of the organism in the lung had not been described
until recently.
Observing that even low viability (<0.2%) spores of S. chartarum
can germinate in the lung of infant rats when instilled at high concen-
trations (Yike et al., 2003a), it became important to ascertain whether
an infection can ensue. Following instillation of viable spores (5 104
spores/gm BW) into the lungs of 4- and 14-day-old rat pups, germina-
tion was observed frequently in the lungs of the 4-day-old but rarely in
the 14-day-old pups. In the 4-day-old pups, pulmonary inflammation
with hemorrhagic exudates was observed along with a 15% mortality

rate. At 3 days following exposure, acute neutrophilic inflammation
and intense interstitial pneumonia with poorly formed granulomas
were associated with budding spores and fungal hyphae. Surviving
pups had slower weight ` gain for 7 days. Dilution plating and quanti-
tative PCR (TaqManE) analysis (Haugland et al., 1999) were used
to follow total fungal load in the rat pup lung homogenates. In the
4-day-old rat pups, viable fungi decreased rapidly and were less than
1% of the instilled load by day 7. Similarly, fungal DNA decreased
exponentially and was only 0.03% by 14 days after exposure. However,
14-day-old rat pups showed neither the lethal effects of exposures to
viable spores nor the slower weight gain, and the fungal load decreased
even more rapidly. We concluded that S. chartarum spores can initi-
ally germinate and form hyphae but even in immature rat pups do not
establish an effective infection, although a very limited persistence
cannot be excluded. Highly viable spores appear to be more detrimen-
tal to rat pups than nonviable spores in that the dose used in these
experiments led to 15% mortality, whereas a two-fold higher dose of
non-viable spores used in our previous study (Yike et al., 2001) led to
only 2% mortality. These results illustrate the significance of spore
viability in the experimental design of animal studies. Germination
and growth, however limited, could potentiate the effects of inhaled
spores if toxins are produced during these processes. This could espe-
cially be significant regarding stachylysin, which may only be released
with germination (Vesper et al., 1999, 2001). The viability of the spores
produced in our laboratory, both on drywall pieces and on PDA, re-
mains within 80–100% for fungal cultures kept at room temperature for
3 weeks.
No information regarding spore viability has been included in the
reports from other laboratories employing S. chartarum spores in
animal studies (Flemming et al., 2004; Mason et al., 1998, 2001;
Nikulin et al., 1996, 1997; Rand et al., 2003; Rao et al., 2000a,b;
Rosenblum et al., 2002; see Table II) except for the work of Leino et al.
(2003), who used -irriadiated spore preparations that most likely are
not viable.
C. THE EFFECTS OF HEMOLYSIN

Comparison of 20 S. chartarum isolates for hemolytic activity
and hydroxamate-type siderophore content found these parameters to
be increased in five of the isolates from homes of Cleveland infants
with pulmonary hemorrhage (Vesper et al., 1999, 2000). Randomly
TABLE II
COMPARISON OF EXPERIMENTAL CONDITIONS USED IN ANIMAL STUDIES OF PULMONARY EXPOSURE TO STACHYBOTRYS CHARTARUM
Toxin/ Toxin/ Dose (spores/

Fungal Growth toxicity toxicity Proteolytic gm BW)/ Administration
Reference strain media assay results activity Viability schedule Animals route
Nikulin et al., s.72 Rice HPLC 0.04 pg ND ND 50,000 acute NMRI mice Intranasal
1996, 1997 flour SG/spore 50–5000
agar repeated
0.10 pg
SH/spore
Cytotoxicity
s.29 non-toxic
253
Rao et al., California Potato Brime shrimp High ND ND 3000–30,000 CD rats Intratracheal
2000a,b isolate dextrose lethality acute
agar assay
Rosenblum California Potato Brime shrimp High ND ND 250–25,000, 3H/HeJ Intratracheal
et al., 2002 isolate dextrose lethality acute Balb/c
agar assay C57b1/6J
McCrae et al., Hawaiian Maltose ND ND ND ND 500,000/animal, CFW mice Intratracheal
2001; Mason isolate extract acute
et al., 1998 agar
Rand et al., JS58-17 Cellulose ND NDa ND ND 70,000/animal, CFW mice Intratracheal
2003 medium acute
Flemming JS58-17 Cellulose ND NDa ND ND 30–3000 acute CFW mice Intratracheal
et al., 2004 medium
JS58-06 30–3000 acute
(continued)
TABLE II (Continued)
Toxin/ Toxin/ Dose (spores/

Fungal Growth toxicity toxicity Proteolytic gm BW)/ Administration
Reference strain media assay results activity Viability schedule Animals route
Yike et al., JS58-17 Rice Luciferase 1.00 pg ND 0% 100,000–800,000, SD infant Intratracheal

2001, translation SG/spore acute rats
2003a,b assay
JS58-17 Dry wall SG ELISA 0.72 pg 0.41 Units/ 80–100% 50,000, acute
254
SG/spore 106 spores

Leino et al., s.72 Rice flour Mass 0.28 pg ND Presumed 1000 and Balb/c Intranasal
2003 agar spectroscopy SG/spore 0% 100,000/animal, mice
not detected repeated
s.29
ND-not detected.
a
Toxicity of these preparations was assumed to be close to those of Yike et al., 2001 and Chung et al., 2003.
amplified polymorphic DNA (RAPD) further supported a clustering of
these isolates in contrast to 5 isolates from homes of Cleveland control
infants (original matched infants) and 12 non-Cleveland isolates. The
S. chartarum isolate from a 7-yr-old Houston child with pulmonary
hemorrhage appears to cluster with the Cleveland case isolates. A
novel hemolysin, stachylysin, produced by S. chartarum isolate JS58-
06 from a case home, has been described by Vesper et al. (2001).
Purification of stachylysin from S. chartarum has been complicated
by apparent aggregation, with electrophoretic zymograms demonstrat-
ing at least 3 proteolytic bands in a partially purified preparation. This
together with the slow nature of the hemolytic process (hours to days)
suggest to us that proteolysis may play a significant role in the observed
hemolysis. Extracts containing stachylysin caused injury and hemor-
rhage in earth worms (Vesper and Vesper, 2002) and were cytotoxic to
the PK15 kidney cell line with an EC50 value of 150 ng/ml (Yike,
unpublished results).
Antiserum raised in rabbits against partially purified stachylysin has
been used to localize fungal proteins in spores and mycelia and in
animal lungs following instillation of S. chartarum spores (Gregory
et al., 2003). Immunoreactivity was primarily localized to the inner
cell wall (outside the cell membrane), suggesting that they are constitu-
tively produced. Spores instilled in mouse and rat lungs showed an
immunoreactivity radially decreasing out from the spores, indicating
that the proteins had diffused out of the spores. More stachylysin was
observed in the mouse lung tissue at 72 h than at 24 h, indicating that
release/production is a relatively slow process. The localization of sta-
chylysin in macrophage phagolysosomes suggests that these cells may
be involved with spore protein removal and presumed inactivation.
Stachylysin was also found in the sera of rats exposed to S. chartar-
um. The same polyclonal, affinity-purified antibodies directed against
stachylysin were used to develop a competitive ELISA (Van Emon
et al., 2003) with a sensitivity of 2 ppb. This assay was used to quantify
stachylysin in the conidia of 91 common indoor fungi. Only five other
species—Aspergillus carbonarius, Cladosporium sphaerospermum,
Memnoniella echinata, Myrothecium verucaria, and Penicillium atra-
mentosum—contained stachylysin, but at levels at least 2000 lower
than that of S. chartarum, suggesting high species specificity of the
assay. Stachylysin was detected in the sera of rats (15.7 4.6 ng/ml)
chronically exposed to low doses of S. chartarum isolate JS 58-06, in
contrast to lack of stachylysin reactivity in the sera of untreated and
PBS-exposed control animals. In addition, stachylysin reactivity was
observed with pooled sera from patients with reported indoor exposure
to S. chartarum in contrast to people without reported exposure. Fur-

ther studies using sera from individual subjects with well documented
exposure or lack thereof are needed.
While these results point to stachylysin as a potential biomarker of
acute exposure to S. chartarum, it is not clear which antigenic deter-
minants are recognized by the polyclonal antibodies, since the hemo-
lysin preparations used to develop the antiserum contain several
proteins. However, even though these polyclonal antibodies may also
recognize other antigens from S. chartarum, the high species specificity
and high sensitivity of the assay make their use very attractive for
detection of fungal exposure and as a valuable experimental tool in
further investigations of animal models.
D. THE EFFECTS OF PROTEINASES

In addition to hemolysin, other spore proteins (e.g., proteinases) may
contribute to the pathophysiology arising from spore inhalation. Pro-
teolytic activity of spore extracts analyzed by a fluorescence-based
microplate assay and zymogram gels demonstrated proteinases capable
of hydrolyzing fluorescein-labeled gelatin, collagen I, and collagen IV.
The total gelatinase activity of spore extracts can be inhibited 85% by
2.5 mM PMSF, indicating a high content of serine proteinases. Other
serine proteinase inhibitors such as Pefabloc, TPCK, and aprotinin also
are effective, while lack of inhibition with leupeptin, pepstatin, and
EDTA suggests the absence of cysteine, aspartic, and metaloprotei-
nases. Total proteinase activity of spore extracts differed between fun-
gal isolates and varied with growth conditions. Proteinase activity of
the JS58-06 isolate (highly hemolytic) was more than 2-fold higher than
that of JS58-17 (high satratoxin producer). For both isolates, the level of
proteolytic activity increased more than 3-fold when the fungus was
grown on PDA as compared with a drywall (Yike, 2002) (Table I). The
number and intensity of protein bands detected on zymograms also
varied for different isolates and different growth conditions. However,
the double band of 30 kD was found to be the most active in all spore
extracts as well as in culture media. It is likely that one of those bands
corresponds to newly described stachyrase A, a serine protease with a
broad substrate specificity that is capable of hydrolyzing several col-
lagens (type I, VI, and X), all four pulmonary proteinase inhibitors (2-
macroglobulin, 1-proteinase inhibitor, 1-antichymotrypsin, secretory
leukocyte protease inhibitor [SLPI]), and several neuropeptides (sub-
stance P, bradykinin, neurotensin, and angiotensin I and II) (Kordula
et al., 2002). Preincubation of spore extracts with 2 mM PMSF prior to
electrophoresis leads to the reduction in intensity of all of the
major proteolytic bands, indicating that most (if not all) proteolytic
enzymes present in the extracts belong to the serine class of protei-
nases. These findings were extended to in vivo studies by immuno-
chemical labeling of collagen type IV (polyclonal goat anti-collagen IV-
biotin Fab-fragments) in the lungs of mice exposed to S. chartarum
(Yike et al., 2002). In contrast to spores from C. cladosporioides, there
was significant reduction of collagen type IV in the vicinity of the
S. chartarum spores, indicating possible involvement of fungal protei-
nases in the degradation of extracellular matrix proteins either directly
through collagenolytic activity or indirectly through changes in the
proteinase-antiproteinase balance. The original concept of how S. char-
tarum produces capillary fragility (Dearborn et al., 1999, 2002) had
been that the trichothecene inhibition of protein synthesis in the still
exponentially growing lung would lead to decreased capillary base-
ment membrane proteins, especially collagen type IV, in the vicinity of
the spores. While this is still a valid concept, actual collagenolytic
destruction may be more important, especially when isolates with very
low trichothecene activity are considered. Fungal proteases may elicit
inflammatory responses via protease activated receptors (PARs), as
proposed by Kauffmann et al. (2000).
Because proteinase and hemolysin levels vary with growth condi-
tions and across isolates, characterization of the proteolytic and hemo-
lytic properties of spores also has important implications for designing
experiments (see Tables I and II and Fig. 1; also Section IV.A).
E. ALLERGENICITY AND ANTIGENICITY

Similar to other molds, S. chartarum contains proteins and other
molecules that may act as immunogens/allergens. At least two such
proteins with the molecular mass of 65 and 50 kDa have been identi-
fied by Raunio et al. (2001) in the extracts prepared from cultures
grown in cellulose broth. Two laboratories have investigated potential
allergenicity of S. chartarum in mice. Korpi et al. (2002) studied the
effects of exposure of Balb/c/cJBorn mice to aerosolized extracts from
cultures of non-toxic S. chartarum. Mice were either immunized with
ovoalbumin or fungal extract, treated with PBS, or not injected at all
(naive). Exposure to S. chartarum extract twice a week for 3 wk
increased significantly the level of total IgE in mice previously im-
munized with these extracts but also in nonimmunized mice. Mild
symptoms suggesting inflammation were observed in the lungs of ex-
posed mice with sensory irritation detected by plethysmography, while
no effect was seen in mice exposed to aerosolized ovalbumin and

phosphate-buffered saline.
BALB/c mice were also used by Viana et al. (2002) to assess the
ability of S. chartarum to cause allergic alterations in respiratory phys-
iological responses. The animals were exposed to 4 aspirations of crude
extracts obtained from the cultures of S. chartarum (combined five
isolates of S. chartarum with different trichothecene toxicity, grown
on two different media). Control animals received either bovine serum
albumin or Hanks’ balanced salt solution over a 4-wk period. Mice
exposed to the fungal extract displayed increased BAL fluid total pro-
tein and LDH and increased cell numbers, with differential cell counts
showing neutrophilia, marked eosinophilia, and increased numbers of
lymphocytes. Total IgE levels were elevated in serum and BAL fluid,
and IL-5 levels were greatly increased in BAL fluid. Exposure to fungal
extracts resulted in increased Penh (enhanced pause, indirect measure
of airway resistance; see Hammelman et al., 1997) over baseline after
the third and fourth exposures and increased responsiveness to a
methacholine aerosol challenge. Exposure to Hanks’ balanced salt so-
lution or bovine serum albumin did not alter Penh baseline, nor did
these cause an increase in airway responsiveness to methacholine.
While these results clearly show that exposure to the extracts from
S. chartarum causes allergic respiratory physiological responses simi-
lar to those observed in human allergic asthma, their physiological
relevance may be somewhat limited. Just as pure mycotoxins elicit
different symptoms when compared with fungal spores, extracts ob-
tained through spore and mycelia breakage may contain different pro-
files of antigenic proteins as compared with those released by intact
spores in vivo. Such differences between fungal antigens in vitro and
in vivo have been described before (Moutaouakil et al., 1993), and no
anti-S. chartarum antibodies were detected in the sera of rats repeated-
ly exposed to the intact spores through the nose (Leino et al., 2003;
Nikulin et al., 1996, 1997). The nature of fungal antigens eliciting the
allergic responses in these animal models has not been delineated.
Two Stachybotrys proteins reacting with IgE antibodies from human
subjects have been identified (Barnes et al., 2002).
F. THE EFFECTS OF VOLATILE ORGANIC COMPOUNDS

Production of microbial volatile organic compounds (MVOCs)
unique for S. chartarum has been recently reported by Gao and Martin
(2002). The profile of volatile compounds was highly dependent on the
culture media and varied between the three studied isolates.
Sensory irritation, bronchoconstriction, and pulmonary irritation
effects resulting from the inhalation of S. chartarum-derived vapors
were measured plethysmographically in a report published by Wilkins
et al. (1998). Little effect was seen from the vapors, in agreement with
the predicted effects of the low concentrations of volatile organic
compounds measured (Ammann, 1999). The authors concluded that
risk assessments cannot be based solely on estimated effects of emitted
MVOCs but need to take into account the effects of liberated particles
(e.g., sensitization potentials of the mold spores).
IV. Practical Considerations for Designing Animal Experiments of

Exposure to S. chartarum
Based on the observations presented above, it is clear that fungal
spores should not be treated solely as mycotoxin receptacles but rather
other characteristics such as their viability and proteolytic and hemo-
lytic activity should be considered when designing and interpreting
the results of animal studies. Quantitative characterization of these
parameters of spore preparations will allow for better comparison of
results from different laboratories. Other factors that need to be consid-
ered include the dose and route of administration, animal strains, and
species. Table II summarizes some of those parameters in published
studies describing the in vivo effects of S. chartarum spores.
A. CHARACTERIZATION OF FUNGAL SPORES

As discussed above, trichothecene content, viability, and activity of
fungal proteins are among the critical factors responsible for the ad-
verse effects of fungal spores. These factors may be affected by growth
conditions and vary greatly between different fungal isolates. Pre-
parations of spores used in animal studies should be better character-
ized with respect to their toxicity, viability, and protein content to
facilitate comparison of the results obtained by different laboratories
and to better understand the mechanisms involved in the physiological
responses.
It has recently been recognized that some isolates of S. chartarum do
not produce macrocyclic trichothecenes (Andersen et al., 2002) and
that across isolates, the levels of toxin production are highly variable
(Jarvis et al., 1998; Vesper et al., 1999). It is important that the tricho-
thecene toxicity of the spore preparations be accurately quantified. In
earlier studies of Yike et al. (2001), S. chartarum isolate JS58-17
provided by Dr. William Sorenson from NIOSH was used because of
its high cytotoxicity and documented high levels of the macrocyclic

trichothecenes rioridin L-2 and satratoxin-G and H and phenylspiro-
drimanes (Jarvis et al., 1998). The trichothecene toxicity of the spores
of JS58-17 as measured by the luciferase inhibition assay (Yike et al.,
1999) remained within a close range (0.67–0.89 pg satratoxin G equiva-
lents/spore) whether the fungus was grown on potato dextrose agar
(PDA) or drywall (Table I). No significant differences in trichothecene
toxicity were observed for the spores collected from rice, cornmeal, or
Stachybotrys cellulose medium (LabCor, Seattle, WA), averaging about
1 pg/spore (0.6–1.3 pg/spore). We have confirmed this same concentra-
tion of satratoxin-G per spore of JS58-17 isolate grown either on dry-
wall or potato dextrose agar with an anti-satratoxin-G antibody ELISA
(Chung et al., 2003; see Table I). Another isolate of S. chartarum used
to isolate hemolysin, JS58-06, grown on drywall was shown to have
about 100-fold lower trichothecene toxicity, as determined by the lu-
ciferase translation assay and calculated per wet weight of spore pre-
parations as compared with JS58-17 (Vesper et al., 1999). When the
toxicity was calculated per spore, it was only about 20 times lower
(0.04 pg/spore, Table I) suggesting that the water content of wet spore
preparations (likely to be variable) may affect the accuracy of toxicity
determination. Its satratoxin G content as determined by ELISA is only
4.65 105 pg/spore (Table I). The reason for the difference between
the two methods may relate to the presence of other trichothecenes
such as trichodermol in the spores from JS58-06 (Andersen et al., 2002;
Jarvis et al., 1998) that are detected by the luciferase translation assay
but do not bind to the antibody used to develop the ELISA that shows
high specificity towards the macrocyclic ring in trichothecene struc-
ture (Chung et al., 2003). In contrast to the constant spore content of
trichothecenes, the proteolytic activity appears to be dependent on
growth media (Table I). In addition, higher proteolytic activity seems
to parallel hemolysin content across isolates.
The effects of these differences were also observed in vivo when
7-day-old rat pups were exposed to the spores of two different iso-
lates of S. chartarum (4 104 spores/gm BW, 8 animals per group),
the high trichothecene producer JS58-17, and the hemolytic JS58-06 of
low trichothecene toxicity grown on two different media (drywall and
PDA, Fig. 1). Differences in numbers of BAL fluid neutrophils and
cytokine concentrations (TNF- and IL 1-) between JS58-17 and JS
58-06 were small within the spores from the same growth conditions.
However, all three parameters were increased (P < 0.05, Fig. 1) when
comparing the effects of each isolate grown on PDA versus drywall.
Stronger effects of fungal spores grown on PDA when more proteinases
are produced (see Table I) emphasize the importance of protein factors
in spore pathophysiology. Additionally, they show that the depen-
dence of spore content on the type of culture media needs to be
accounted for in experimental design. In summary, fungal isolates with
greater proteinase activity may have more pathogenic potency, and
isolates that lack macrocyclic trichothecenes may still be clinically
important based on their proteolytic and/or hemolytic activity.
As noted in Table II, investigators studying the in vivo effects of
S. chartarum have been growing different isolates on different media.
Nikulin et al. (1996, 1997), Leino et al. (2003), and Yike et al. (2001)
provided quantitative assessment of the trichothecene content of their
spore preparations, although different methods of toxin/toxicity deter-
mination have been used. The toxicity of the spores used by Rao et al.
(2000 a,b) was only reported as high, while Mason et al. (1998) did not
present any toxicity data. Proteolytic activity and viability have not been
determined in most studies. All of these variables make direct compari-
son of dose-response effects between investigators difficult. It would
appear that growing S. chartarum on drywall (Yike et al., 2003a,b)
or cellulose media (Flemming et al., 2004; Gregory et al., 2003) would
lead to the production of spores with characteristics similar to those
found in an indoor environment. Fungal spores derived from PDA
cultures may contain higher levels of proteinases and thus may be
more detrimental than those grown on drywall or cellulose media
(Table I, Fig. 1).
B. ANIMALS
1. Animal Species and Strains
The comparison of the studies of Yike et al. (2001, 2003a) with those
of Rand et al. (2003) and Flemming et al. (2004) suggests that mice may
be more susceptible to inhalation exposure to S. chartarum than rats,
based on the concentrations of spores used to elicit similar pathophys-
iological effects. While the effects of lower doses in infant rats are
still under investigation (Yike et al., unpublished), there have been
significant differences in susceptibility noted even within species.
Rosenblum et al. (2002) studied pulmonary responses to S. chartarum
in three different strains of mice—C3H/HeJ, Balb/C, and C57bl/6J. While
all three mouse strains showed significant dose-dependent responses to
the spores, the analyses of BAL fluid showed that myeloperoxidase
activity, albumin and hemoglobin levels, and neutrophil numbers were
significantly different among the three strains. Balb/C mice showed the
most apparent lung injury, and C57bl/6J mice showed the least. Not
surprisingly, the genetic background of animals appears to affect their
responses to mold exposure. Knowing that there are significant intra-
species differences, there may be even larger differences in mold sus-
ceptibility between different species. Although published studies have
been limited to mice and rats, each uses different strains, which also
additionally accounts for differences in quantitative results between
the laboratories (Table II).
2. Animal Age
While most studies have been conducted with juvenile and young
adult animals (Table II), Yike et al. (2001, 2002, 2003a,b) have used 4-,
7-, and 14-day-old infant rats in an attempt to investigate the vulnera-
bility of pups in parallel to the experience with human infants. Signifi-
cant age-related differences in susceptibility were observed when
comparing clearance and persistence of S. chartarum in the lungs of
4- and 14-day-old rat pups (Yike et al., 2001, 2003a), with the younger
pups being much more susceptible to fungal spores (see Section III.B).
Thus, age may be another significant variable in the experimental
design and should be taken into account when comparing different
studies.
C. EXPOSURE ROUTE
The route used to deliver the fungal spores into the lungs of experi-
mental animals can affect the effectiveness of deposition in the lower
airways. Intranasal and intratracheal (both tracheostomy and direct
instillations) have been employed in the studies of inhalation exposure
to S. chartarum (Table II). PCR enumeration of spores in infant rat lung
homogenates demonstrated that the peripheral lung deposition of
spores delivered via single intranasal instillation was highly variable,
ranging from 0.1% to 28% (Yike et al., unpublished). Although ex-
amination of lungs and BAL fluids shows that with repeated instilla-
tions all animals get exposed to enough spores to elicit similar
pathological changes, quantitative results are highly variable and often
lack statistical significance (Nikulin et al., 1997; Yike et al., unpub-
lished). The use of high numbers of experimental animals may be the
only way to overcome this problem. In addition, intranasal instillation
produces local pathology in the nose. A noninvasive, intratracheal
delivery (Brain et al., 1976) appears to be a more practical and effort/
cost effective route, especially for repeated exposures, although this
becomes much more challenging when working with infant animals.
Initial experience with intratracheal delivery directly through the lar-
ynx (after Martinez-Burnes et al., 2001) into the lungs of 7-day-old rat
pups (Yike, unpublished) resulted in levels of deposition comparable
to the results obtained with tracheostomy (Yike et al., 2003a).
D. EXPOSURE DOSE AND REGIMEN

The effects of inhalation exposure to S. chartarum are dose depen-
dent (Flemming et al., 2004; Leino et al., 2003; Nikulin et al., 1997; Rao
et al., 2000a,b; Yike et al., 2001). As shown in Table II, investigators
have used a wide range of spore exposures, ranging from 30 to 800,000
spores/gm body weight.
Initially, in an attempt to reproduce the human infant pulmonary
hemorrhage disorder with 30% mortality, we examined the effects of
spore exposures ranging from 1 105 to 8 105 per gram BW. The LD50
value for 4-day-old rat pups when using nonviable spores of JS58-17
grown on rice and containing high levels of trichothecenes was found
to be 2.7 105 spores/gm BW. Even though such high levels of
S. chartarum in indoor air are unlikely, the environmental studies do
not always provide accurate assessment of indoor spore concentrations
because of inadequate sampling and assay procedures. Most often the
estimates of S. chartarum spores in indoor air are based on enumera-
tion by culturing (Shelton et al., 2002), which can lead to as much as a
10-fold underestimate of total spores (Pasanen et al., 1993). Nonviable
spores contain toxins and are still allergenic. Regardless, the doses
used in most animal studies have been comparatively high, and study-
ing the effects of lower spore doses may be more physiologically rele-
vant. It has been proposed that the ‘‘no-adverse effect level’’ (NOAEL)
for proinflammatory cytokine production in mice ranges from 10–15
spores/gm BW (Flemming et al., 2004).
Most of the animal studies of S. chartarum have focused on acute
effects of exposure (Table II). Only Nikulin et al. (1997) and Leino et al.
(2003) have attempted both acute and chronic exposure studies. The
investigation of chronic exposure to lower doses would bring the
animal models closer to human indoor exposure.
V. New Directions in the Investigation of the Effects of S. chartarum in

Animal Models
Existing studies show that exposure of laboratory animals to toxic
isolates of S. chartarum leads to weight reduction, hemorrhage, and
lung inflammation. The in vivo effects of fungal spores extracted with
alcohol to remove toxins (Rao et al., 2000a; Yike et al., 2001) are
significantly milder than those of intact spores. However, such alcohol
extraction also leads to denaturation of fungal proteins and reduction
in spore viability. While the initial reports of Nikulin et al. (1996, 1997)
indicated that nontoxic isolates were much less potent, more recent
work (Fig. 1; Flemming et al., 2004; Korpi et al., 2002; Leino et al.,
2003) shows that even nontoxic isolates of S. chartarum can elicit
severe inflammatory symptoms similar to those observed with toxic
isolates.
A. TOWARD DISSECTING PATHOPHYSIOLOGIC MECHANISMS

The mechanisms of lung injury are complex and involve not only the
action of mycotoxins but also fungal enzymes and cell wall compo-
nents such as -D-glucan. The elucidation of those mechanisms would
not only improve our understanding of pathophysiology but also facil-
itate risk assessment especially regarding toxic and nontoxic isolates.
The increased mortality seen with viable spores (Yike et al., 2003a)
and the finding of proteinases (Yike et al., 2002) suggests viable spores
secreting active fungal proteins as an important focus.
To differentiate between the trichothecene toxins and active protei-
nacious spore components, the in vivo effects of intact, viable spores
were compared with those of autoclaved and ethanol-extracted spores.
The characteristics of the spores used in these experiments are sum-
marized in Table III. Both ethanol extraction and autoclaving denature
the proteins, even though the latter treatment retains full trichothecene
activity. The lack of proteolytic activity of autoclaved spores was con-
firmed by Enz-Check proteinase assay, and the toxicity of the spore
preparations was evaluated by luciferase translation inhibition test
(Yike et al., 1999, 2002).
TABLE III
CHARACTERISTICS OF DIFFERENT PREPARATIONS OF THE SPORES OF S. CHARTARUM
Spore components/characteristics Ethanol extracted Autoclaved Viable
(1,3,) -D glucan þ þ þ

Trichothecenes þ þ
Proteinase activity þ
Viability 80%
Seven-day-old rats were exposed intratracheally to 1 105 spores/gm
BW (toxic isolate JS58-17) and sacrificed at different time points (Yike
et al., 2003b). The inflammatory response was measured by morphomet-
ric analysis of the lung and determination of inflammatory cells in BAL
fluid. Alveolar space was greatly reduced in animals exposed to fungal
spores as compared with PBS-treated controls. The largest effects were
observed in rats treated with viable spores: alveolar space 36 hr after
treatment was 39.95%, as compared with 52.29% for animals treated
with autoclaved spores, 60.34% for animals treated with ethanol-
extracted spores, and 59.77% for PBS-treated controls. Changes in the
numbers of inflammatory cells were also most significant in animals
treated with viable spores. The difference between autoclaved and via-
ble spores further demonstrates the involvement of fungal proteins in
the inflammatory response to S. chartarum.
B. THE ACUTE AND CHRONIC EFFECTS OF LOWER DOSES

The high doses of spores required to produce frank hemorrhage in
the single-dose model are a significant shortcoming of this model
(Yike et al., 2001). The initial selection of 1 105 spores/gm BW was
made because 98% of the animals could survive and continue to grow
(Yike et al., 2001) while still showing severe symptoms of inflamma-
tion. However, experiments on mice conducted by Flemming et al.
(2004) as well as subsequent experiments in infant rats (Yike et al.,
unpublished) have found that many of the adverse effects can still be
observed with much lower doses.
After dose was reduced 20-fold (intact spores, JS58-17), the increases
in some of the inflammatory indices (30-fold increase in the numbers of
BAL fluid neutrophils) (Yike et al., unpublished) could still be ob-
served. The observation that lower doses of spores elicit effects similar
to those caused by much higher doses may stem from differential
effects of trichothecene toxins on the immune system. Low concen-
trations of trichothecenes have been shown to be immunostimula-
tory, whereas higher concentrations have immunosuppressive effects
(Bondy and Pestka, 2000). At lower spore doses, both the trichothe-
cenes and other fungal components may have more direct proinflam-
matory action, while at high doses, trichothecene immunosuppresive
effects may predominate. As mentioned above, chronic exposure stud-
ies should more closely reflect real-life conditions and provide better
insight into the practical mechanisms of pathophysiology.
C. INVESTIGATIONS OF THE EFFECTS OF ENVIRONMENTAL MOLDS IN THE PRESENCE

OF OTHER ENVIRONMENTAL FACTORS
The damp conditions where indoor molds are found are frequently
accompanied by other biological and non-biological contaminants. Those
microbial agents and their metabolites as well as other environmental
stressors such as hypoxia or tobacco smoke can contribute to adverse
respiratory effects, either through synergistic injury mechanisms or by
acting as acute environmental triggers. Because household exposure to
environmental tobacco smoke (ETS) was a significant covariant in the
case-control study of the Cleveland infants (OR ¼ 21, CI 1.07–7.5 106)
(Etzel et al., 1998) and return to ETS exposure resulted in rebleeding, it has
been proposed that ETS may be a confounding trigger of hemorrhage in
these infants.
This was investigated with 7-day-old rat pups based on additional
hypothesis that cigarette smoke exposure would significantly worsen
the respiratory dysfunction resulting from inhalation of S. chartarum
(Miller et al., 2002). Rat pups were exposed intratracheally to 50,000
spores/gm BW of highly toxic JS58-17 isolate of S. chartarum grown on
drywall. Control animals received PBS. After 48 hours, half of the pups in
each group were exposed to side stream smoke from cigarettes for three
15-min epochs. Controls were exposed to room air. Two hours after the
last exposure, respiratory function was measured by barometric plethys-
mography. Pups exposed to S. chartarum alone exhibited a 30% increase
in MV (minute volume) as compared with saline exposed controls
(P < 0.005). After smoke inhalation, MV decreased in S. chartarum–
exposed pups by 28% (P < 0.04). In contrast, smoke inhalation did not
significantly alter MV in saline-treated pups. Decrease in MV after smoke
inhalation in S. chartarum–treated pups was due to a 38% decrease in
respiratory rate (P < 0.0001) and was associated with a 220% increase in
respiratory resistance as indicated by Penh (P < 0.004). In pups treated
with saline alone, smoke exposure did not significantly alter Penh. His-
tologically, inflammation with some hemorrhage was observed in the
lungs of animals treated with S. chartarum and S. chartarum plus smoke
but no discernable differences were observed between these two experi-
mental groups. Thus the respiratory function in S. chartarum–treated rat
pups appears to be quite sensitive to acute smoke exposure.
VI. Conclusions
Animal models provide physicians and environmental scientists
with useful tools for assessing risks associated with respiratory effects
of air pollutants. The animal studies to date support the view that
pulmonary exposure to the spores of S. chartarum leads to hemorrhagic
inflammation and impairment of growth. This has been demonstrated
by increases in BAL fluid of inflammatory cells, proinflammatory med-
iators, hemoglobin, and proteins along with changes in pulmonary
surfactant. Although the earlier experiments were conducted with rela-
tively high doses, more recent findings indicate that lower doses, which
appear to be closer to the concentrations encountered in indoor air, can
elicit similar symptoms.
While trichothecene toxicity appears to be an underlying cause of
many of the adverse effects, additional factors such as fungal protei-
nases, hemolysin, and (1!3)--glucan likely contribute to the patho-
physiology. Observations with fungal isolates that produce low levels
of trichothecenes suggest that spore proteins may actually be major
determinants of lung injury.
The results of animal studies based on different experimental de-
signs are difficult to compare because of many variables including
spore toxicity, viability, and content of fungal proteins in addition to
species, strains, age of animals, and route of administration. Well-
characterized, standardized preparations of fungal spores with proper-
ties similar to those found in indoor air are needed to dissect the
practical physiological mechanisms of mold exposure. Development
of experimental conditions for chronic exposure to low doses and
inclusion of other environmental factors will make animal studies
more accurate models of human indoor exposure.
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Toxic Mold Syndrome
MICHAEL B. LEVY AND JORDAN N. FINK
Medical College of Wisconsin
9000 W. Wisconsin Avenue
Milwaukee, Wisconsin 53226
I. Introduction 275
II. Overview of Molds 275
III. Fungal Structure and Compounds 276
IV. Mycotoxins 277
A. Food Mycotoxin Effects on Animals 277
B. Mycotoxin Effects on Human Health 278
V. Building-Related Diseases 278
VI. Diseases Related to Stachybotrys 281
A. History of Stachybotrys-Related Toxicity 282
VII. Disease and Mycotoxins 282
A. Stachybotrys Mycotoxins and the Indoor Environment: A Review 284
VIII. Conclusions 285
References 286
I. Introduction
The term ‘‘toxic mold syndrome’’ has been associated in the lay and
medical literature as a constellation of symptoms resulting from
the exposure to fungal mycotoxins. The purpose of this review is to
critically examine the relationship between fungi and their expressed
mycotoxins with the development and or relationship to human
disease.
II. Overview of Molds

Fungi are eukaryotic, filamentous, and mostly spore-bearing
organisms. They may exist as saprophytes or as parasites of animals
and plants and play a critical ecologic role in the decomposition of
organic matter. Fungi, as a group, contains species of yeasts, molds,
mildews, puffballs, and mushrooms. They require moisture and an
organic food source for growth. Multiple factors that include climate,
vegetation, air temperature, and humidity may affect the concentra-
tion of fungi in the environment. Molds may reproduce by sexual
or asexual means. On contact with a moist food source, spores begin
to germinate and develop a branching system of hyphae. They may
275
0065-2164/04 $35.00
276 LEVY AND FINK
compete with bacteria for nutrients and oxygen by excreting

toxic substances including antibiotics, which in turn may kill or
inhibit bacterial growth. Fungal spores and plant pollens consti-
tute the predominant outdoor allergens as reviewed by Hardin et al.
(2003).
Indoor allergens include the proteins from dust mites, cockroaches,
pets, mice, or fungi. The prevalence and concentration of these
indoor allergens is dependent on humidity levels, ventilation, and
the presence or absence of pets or carpeting in the dwelling (Platts-
Mills, 1998). Mold spores can enter the indoor environment from the
outside through normal ventilation such as windows or air intake
systems or on the surface of people, pets, household plants, or seasonal
decorative trees. Most fungi are metabolically active over a broad range
of temperatures (Solomon et al., 1998). High moisture and relative
humidity are required for optimal growth, in the range of approximate-
ly 75%, with Stachybotrys around 93% at 25 C. Hence mold grows
well indoors in damp or wet places such as basements, windowsills,
bathrooms, shower stalls, and air conditioning or humidification sys-
tems or as a result of water damage where wet gypsum board may
provide an excellent medium for growth.
It is thought that fungi may affect human health through at least four
pathways: allergy, infection, irritation, and toxicity. The scope of this
chapter is to discuss the syndromes associated with molds and their
toxins.
III. Fungal Structure and Compounds

The fungal cell wall is composed of chitin (acetylglucosamine
polymers), glucans, polysaccharides and mucopolysaccharides, waxes,
and pigments (Kendrick, 1992). Glucans present in the fungal cell wall
are endotoxin-like substances that may stimulate the immune system
(Rylander, 1992). Fungal cell wall pigments such as melanin may be
present to protect against ultraviolet radiation (Kendick, 1992). Fungi
may release enzymes to digest organic material. As reviewed by
Burge (2001), these enzymes or their secondary metabolites may in
turn be antigenic, thereby stimulating a type I immediate hyper-
sensitivity or allergic reaction. Irritation of mucous membranes
may result from contact with volatile metabolites. Toxicity for some
forms of life may result from the produced mycotoxins or antibiotics.
Antibiotics isolated from mold cultures may be bacteriocidal or
bacteriostatic, properties that have been used medicinally to treat
infections.
TOXIC MOLD SYNDROME 277
IV. Mycotoxins
Many fungi appear to produce mycotoxins. At least 350 types of
fungi produce over 400 known toxins that have been grouped into 21
different classes (Cole, 1981). These low-molecular-weight substances
are considered to be non-volatile at ambient temperatures and are
secondarily produced as a result of food metabolization. As reviewed
by Hendry (1993), toxin synthesis may be affected by the nutritional
source, temperature, humidity, and moisture—similar factors that may
affect fungal growth. However, toxin production may require the prop-
er concentrations of other factors and conditions that are different
from those ideal for fungal growth. In the case of aflatoxins, for in-
stance, toxin production is reliant on the proper concentrations of
oxygen, carbon dioxide, zinc, and copper. Since the conditions for
mycotoxin production are usually different from those required for
fungal growth, it follows that the presence of a toxigenic mold species
in a particular environment does not necessarily implicate the
presence of mycotoxins (Cieglar et al., 1981).
Mycotoxins are not involved in nourishment for the fungus but are
thought to help reduce competition from other mold species and bac-
teria. Mycotoxins are capable of cytotoxicity that may result in the
disruption of cellular membranes and may affect the cellular processes
of protein, DNA, or RNA synthesis (Hardin et al., 2003). These actions
are not limited to molds or bacteria but may also react on the cells of
higher plants and animals including humans. There is evidence that
mycotoxins may vary their activity according to their specificity and
potency, thereby targeting or affecting different cellular processes. This
appears to be dependent on the strain or species of the toxin-producing
mold (Samson, 1992).
A. FOOD MYCOTOXIN EFFECTS ON ANIMALS

Syndromes relating to the effects of mycotoxins are well known in
the veterinary literature, where a spectrum of effects ranging from
mortality to growth retardation and reproductive abnormalities have
been observed. Animal mycotoxicosis-type outbreaks have affected
livestock in the form of sweet clover poisoning, moldy corn toxicosis,
cornstalk disease, and poultry hemorrhagic syndrome (Corrier, 1991).
The effects of the aflatoxins have been well described and are illus-
trative of the difficulties involved in the determination of mycotoxin
exposure levels and their effects. In the 1960s, an epidemic of ‘‘turkey
X’’ disease killed over 100,000 birds. The culprit aflatoxin was traced to
278 LEVY AND FINK
tainted peanuts as reviewed by Applebaum (1981). These aflatoxins are

produced by Aspergillus species; A. flavus makes aflatoxin B and A.
parasiticus produces aflatoxin B and G (IARC, 1987). These toxins are
highly hepatotoxic, immunosuppressive, mutogenic, teratogenic, and
carcinogenic (Dragan et al., 1994). Their toxicity, however, is depen-
dent on the animal species, age, and route of exposure. Farm animals
may ingest large amounts of these toxins in the contaminated feed.
Further, certain animal species may have differences in their respective
hepatocellular metabolization of the parent aflatoxin compounds
(Stuart et al., 1982). The actual levels of aflatoxin in foodstuffs are
difficult to determine, since the toxin may be found in varying amounts
within a particular feed lot, and thus quanities may be small, some-
times approaching the range of nanograms per gram of feed. If feed
contamination is high, these toxins may then become present in food-
stuffs such as milk, eggs, and meat products (Pohland, 1993). Table I
lists molds and their associated mycotoxins.
B. MYCOTOXIN EFFECTS ON HUMAN HEALTH

Historically, as reviewed by Hendry et al. (1993), it appears that
the effects of mycotoxins have been primarily related to the oral con-
sumption of moldy foods—most commonly grain. One of the earliest
descriptions of mycotoxicosis in humans dates to the Middle Ages. ‘‘St.
Anthony’s Fire,’’ which was due to ergotism from Claviceps species
in moldy rye, and presented with toxicity in both a gangrenous
form, related to C. purpura, as well as a convulsive form associated
with C. fusiformis. The ergot alkaloids that produced the syndrome
were identified in the 18th century (Kuhn et al., 2003). The disease is
rare today because of the use of fungicides and modern-day grain
storage techniques (Hussein et al., 2001).
V. Building-Related Diseases
There is increasing concern and awareness regarding the effects of
indoor molds on human health since some molds, and their associated
mycotoxins, are known pathogens as noted above. The association of
a ‘‘moldy’’ or ‘‘musty-smelling’’ building with this information has
exacerbated concerns. There has been media attention and litigation
implying severe illness as a result of indoor mold exposure, particularly
to Stachybotrys species (Hardin et al., 2003).
The recorded history of the detrimental effects of a damp or moldy
environment and its causality to human disease can be traced to
TABLE I
MOLD SOURCES AND MYCOTOXIN-ASSOCIATED DISEASES
Mycotoxin Associated disease Mold source
Aflatoxin Hepatotoxicity (Reijula and Tuomi, 2003), Aspergillus flavus

Nephrotoxicity, Carcinogenic
Citrinin Nephrotoxic (Kuhn and Ghammoum, 2003) Penicillium citrinum
Ergot alkaloids Ergotism-convulsive and gangrenous Claviceps purpurea, Claviceps fusiformis
(Kuhn and Ghannoum, 2003)
Fumitremorgens Tremors (Reijula and Tuomi, 2003) Aspergillus fumigatus
Fumonisins Hepatotoxicity, neurotoxicity, carcinogenic Fusarium moniliforme
(Kuhn and Ghannoum, 2003)
Gliotoxins Cytotoxicity (Reijula and Tuomi, 2003) Aspergillus fumigatus
Griseofulvin Teratogenic, carcinogenic, hepatotoxic Penicillium griseofulvum, Penicillium
(Burge, 2001) viridicatum
Ochratoxin A Hepatotoxic, nephrotoxic (Reijula and Tuomi, 2003) Aspergillus ochraceus
Patulin Carcinogenic, nephrotoxic (Arafat and Musa, 1995) Penicillium expansum
Sporidesmin Photosensitization (Burge, 2001) Pithomyces chartarum
Rubratoxin Hepatotoxic (Kuhn and Ghannoum, 2003) Penicillium rubrum
Sterigmatocystin Carcinogenic, hepatotoxic (Reijula and Tuomi, 2003; Aspergillus nidulans, Aspergillus
Barnes et al., 1994) versicolor, Chaetomium
T-2 Toxins Immunosuppressive, hemorrhagic disease Fusarium species
(Kuhn and Ghammoum, 2003)
Tenuazoic acid Hepatotoxin, nephrotoxic (Burge, 2001) Alternaria alternata
Trichothecenes Immunosuppressive, hemorrhagic, cytotoxicity Stachybotrys, Fusarium, Trichoderma,
(Hendry and Cole, 1993; Memnoniella echinata species,
Kuhn and Ghannoum, 2003) Myrothecium
Vomitoxin Anorexia, cytotoxic (Kuhn and Ghannoum, 2003) Fusarium graminearum
280 LEVY AND FINK
Ramazzini in the 1700s, with his description of what is now known

as hypersensitivity pneumonitis as a result of the inhalation of ‘‘moldy
hay’’ as discussed by Fink et al. (1998). Over the years it has been
difficult to find the definitive linkage between molds and their myco-
toxins with human disease. Various reports in the medical literature
have indicated, however, that the moist, damp environment created
by water leakage and subsequent mold growth in the building environs
can create adverse health effects. While initial reports have been de-
scriptive, subsequent literature has attempted to isolate the type of
mold and further analyze the mechanism responsible for the purported
symptoms. McGrath et al. (1999) measured the fungal profiles in a
building with air quality complaints and found that Penicillium spe-
cies tended to predominate and remained elevated and unchanged
over the measured period. In contrast, a building without complaints
tended to reflect the fungal profile in outdoor measured air. Wan et al.
(1999) found that in a survey of 19 buildings, dampness had a dose-
response effect on reported eye irritation, cough, and lethargy/fatigue.
Unrelated to patient symptom complaints, Gravesen and colleagues
(1999) reported on data obtained after analysis of the building materials
used in 23 mold-infested buildings. The products most vulnerable to
mold growth were organic materials containing cellulose. Penicillium
chrysogenum, Aspergillus versicolor, and Stachybotrys chartarum were
the most common mold species isolated. In materials heavily contami-
nated by S. chartarum, several trichothecenes were detected. Several of
these isolates subsequently produced satratoxin H and G under experi-
mental laboratory conditions with humid gypsum boards. Under these
same conditions, the isolated A. versicolor species produced carcino-
genic mycotoxins sterigmatocysin and 5-methoxysterigmatocystin
(Gravesen et al., 1999). Savilahti et al. (2000) reported on a cohort of
Finnish school children in which increased respiratory symptoms, in-
fections, physician visits, and use of antibiotics were notably increased
as compared with a control group attending a non-water-damaged
school. Followup 1 year after renovation of the moisture-damaged
building showed a markedly decreased prevalence of symptoms and
infections. Seuri et al. (2000) described an outbreak of respiratory dis-
eases among 14 workers at a Finnish military hospital. They found
building-related symptoms of cough in 9 workers, rhinitis in 7, asthma
in 4, and alveolitis in 1. The purported species was Sporobolomyces
salmonicolor. This was investigated immunologically and found not to
be related to an immediate hypersensitivity IgE mast cell–mediated type
of mechanism.
VI. Diseases Related to Stachybotrys
The potential role of mycotoxins, primarily from fungi of the genus
Stachybotrys, has generated increased interest among healthcare pro-
fessionals and the public and has been implicated as a cause of human
disease and known as the ‘‘fatal fungus’’ ( Environmental Health Issues
Forum, 1998).
Stachybotrys is in the class Deuteromycete and family Dematiaceae.
Interestingly, the conidial Deuteromycetes are the most important
group of airborne disseminated fungi that cause respiratory allergic
disease in humans, which would include the genera Alternaria, Cla-
dosporium, Aspergillus, and Penicillium. The spores produced by these
imperfect fungi vary in size, shape, color, texture, number of cells,
thickness of the cellular walls, and the method by which they attach
to each other and to their conidiophores. Identification of common
fungi is difficult, since fungal colony characteristics and microscopic
characteristics vary according to incubation temperature, the medium
on which the fungus is grown, strain variation, and the pleomorphic
nature of the spores (Terr, 2001).
The genus Stachybotrys contains the species atra, which is also
known in the literature as chartarum or alternans. Stachybotrys is a
saprophytic mold, which grows well on cellulose materials in areas
with low nitrogen and elevated humidity, similar to Aspergillus, Cla-
dosporium, and Alternaria species. Spores can be found on samples of
foam insulation materials, fiberboard, carpets, wallpaper, and wood-
containing products (Gravesen et al., 1999). However, it is slow grow-
ing and is rarely seen in outdoor air samples but may be found within
decaying plant residues if the conditions are wet. Agricultural pro-
ducts such as cereal grains, straw, and bean products have been noted
to have Stachybotrys growth along with other fungi. Cultures of hair
from livestock, dogs, and donkeys have also shown growth of Stachy-
botrys (Bagy, 1986). The organism can survive a wide range of tempera-
tures up to 60 C. The spores may survive winter conditions and remain
viable for years. The conidia may retain viability after transit through
the gastrointestinal tract but do not survive the conditions of compost-
ing. Disinfectants may kill the conidia and mycelia; however, cell walls
remain stable (Kuhn et al., 2003).
Indoor air sampling from homes in southern California has revealed
the low (2.9%) presence of Stachybotrys, with most cultures favoring
Penicillium and Aspergillus species. However, in water-damaged
buildings where there is significant mold present, Stachybotrys is
more readily recovered (Kozak et al., 1979, 1980). Cooley et al. (1998)
282 LEVY AND FINK
examined 48 schools in which there were concerns about air quality,

most often complaints of nasal drainage, congestion, itchy eyes, head-
aches, sinus symptoms, and increased airway infections. The authors
found that the levels of Penicillium species indoors were significantly
higher than outdoor levels, and Stachybotrys was isolated more often
from areas of wet walls, carpets, and wall coverings in a higher number
of the complaint buildings.
A. HISTORY OF STACHYBOTRYS-RELATED TOXICITY

The history of Stachybotrys-related toxicity dates to the Soviet Uk-
raine in the 1930s, when a disease affecting horses associated with
rhinitis, conjunctivitis, lip edema, stomatitis, and oral necrosis was
noted. The disease progressed to pancytopenia, coagulopathy, neuro-
logic deterioration, superinfections, and finally death (Kuhn et al.,
2003). Pathologically, the affected animals had evidence of diffuse
hemorrhages and necrosis of the gastrointestinal tract and pulmonary
edema. In the late 1930s the disease was associated with Stachybotrys
isolated in contaminated grain. Drobotko (1945) fed Stachybotrys-
contaminated straw to horses and reproduced the disease, which he
now called ‘‘stachybotryotoxicosis.’’ The disease has affected other
livestock species including sheep, cattle, swine, and deer (Hajtos
et al., 1983; Schneider et al., 1979).
Stachybotrys toxins have been studied since their isolation in the
1940s and have a chemical structure consistent with the trichothecene
class of compounds. There are 148 natural trichothecenes, of which 40
are identified as mycotoxins, produced primarily by Fusarium species
(Cole et al., 1981). Stachybotrys agents and their toxicities are listed in
Table II. Stachybotrys products and properties are noted in Table III.
VII. Disease and Mycotoxins

For mycotoxins to produce adverse health effects, there must
be adequate growth and biomass of the proper mold existing in
the appropriate conditions in which to produce adequate amounts of
adverse compounds. Absorption in toxic amounts by a susceptible
host would need to occur by either contact (i.e., dermatologic expo-
sure, ingestion in contaminated food stuffs) or inhalation of dusts with
spores, mycelial forms, or toxins themselves. Inhalation of spores,
which are often less than 10 in diameter, may allow them to reach
the small airways (O’Riordan et al., 1998). The duration of exposure in
any scenario must be adequate to deliver a toxic dose to the host.
TABLE II
STACHYBOTRYS AGENTS AND THEIR TOXICITYa,b
Agent Toxicity
Conidia Hemolytic activity

Triprenyl phenol Plasminogen activation
Enzymes Glycolysis,
-Glucanase Pentose phosphate pathway
1,3-Endoglucanases Glucose-6-phosphate
Satratoxins Protein synthesis inhibition
Cytotoxicity
IL-2 levels
Conidia, satratoxin F Alveolar type II cells
K-76-C00H Inhibits complement activity
Reduce NK cell activity
Reduce antibody dependent cell mediated
cytotoxicity
a
Terr (2001).
b
Kuhn (2003).
TABLE III
STACHYBOTRYS PRODUCTS AND PROPERTIESa,b,c
Trichothecene products affecting

protein synthesis Additional products/properties
Satratoxins F, G, H Spirolactams
Roriden E Spirolactones
Verrucarin J Phenylspirodrimanes - inhibit complement
activation
Trichoverrols A, B Cyclosporins
T-2 toxin Antibiotics with phytoxic, cytotoxic,
cytostatic properties
Stachylysin Hemolysin
Vomitoxin Resist ultraviolet light, x-ray, acid
a
Johanning et al. (1996).
b
Vesper et al. (2000).
c
Mahmoudi et al. (2000).
284 LEVY AND FINK
The amount of toxic material required to cause an adverse response

in humans is not known. However, there have been recorded cases of
human toxicity caused by trichothecene mycotoxins. Alimentary toxic
aleukia caused by Fusarium sporotrichioides in the former Soviet
Union occurred in the period around World War II and was responsible
for thousands of deaths. It presented with oral ulcers, gastroenteritis,
pancytopenia, hypotension, and death, often caused by opportunistic
infections. This disease was felt to be caused by trichothecene myco-
toxins produced by the Fusarium species in grain that had been left
unharvested because of wartime conditions (Drobotko, 1945; Kuhn
et al., 2003). Since the late 1940s there have been no further reports
of this disease.
In 1970, red mold toxicosis was reported in Japan and affected
horses, swine, and cattle. There were four human cases reported. The
patients presented with nausea, vomiting, and diarrhea, which was
due to the ingestion of grain contaminated with Fusarium graminanum
(Schneider et al., 1979).
A. STACHYBOTRYS MYCOTOXINS AND THE INDOOR ENVIRONMENT: A REVIEW

Several studies have examined the role of Stachybotrys produced
mycotoxins and illnesses related to the indoor environment. An analy-
sis of mycotoxins in crude building materials from water-damaged
buildings by Tuomi in 2000 found trichothecenes including satratoxin
G or H in 19% of samples. However, in many cases the presence of
molds thought to produce mycotoxins was not correlated with the
demonstrable presence of the toxic compounds (Tuomi et al., 2000).
Relating to human studies, Johanning et al. (1996) examined workers in
a water-damaged building contaminated with Stachybotrys. A signifi-
cant increase in lower respiratory, dermatologic, eye, and chronic
fatigue symptoms was found between employees (n ¼ 53) and controls.
In addition, CD3 lymphocytes were lower in those workers reporting a
history of respiratory symptoms (Johanning et al., 1996). Hodgson et al.
(1998) reported an outbreak of disease associated with exposure to
Stachybotrys and Aspergillus and Penicillium in a water-damaged
court house and office building. Of 14 individuals, complaints includ-
ed rhinitis, asthma, and symptoms consistent with interstitial lung
disease. Satratoxins G and H were identified in the moldy ceiling tiles.
The authors concluded that the outbreak represented a human re-
sponse to inhaled fungal toxins (Hodgson et al., 1998). A cluster of 10
cases of acute pulmonary hemorrhage and hemosiderosis in Cleveland,
Ohio, occurred among infants from January 1993 to December 1994.
Pulmonary hemorrhage recurred in 5 of the infants after they re-
turned to their home, and 1 subsequently died. The quantity of molds
including Stachybotrys was higher in the homes of infants with pul-
monary hemorrhage than in controls, and smoking increased the risk,
as reported by the Centers for Disease Control (1994). The American
Academy of Pediatrics Committee on Environmental Health
(Pediatrics, 1998) position statement noted that since little was known
about acute idiopathic pulmonary hemorrhage in infants, environmen-
tal controls to eliminate water problems and avoidance of moldy en-
vironments was to be recommended. An additional 100 cases of
pulmonary hemorrhage associated with Stachybotrys have been re-
ported, including the isolation of the organism from the lung (Elidemir
et al., 1999; Vesper et al., 2000). Published expert panels found that the
possible association between Stachybotrys pulmonary hemorrhage and
hemosiderosis was not proven (Center for Disease Control, 2000).
Several additional published articles continue to debate the signifi-
cance of Stachybotrys and other fungi in the indoor environment. Fung
et al. (1998) reviewed the published articles studying Stachybotrys and
human toxicity and found a possible association with exposure but
no firm causal relationship. Similarly Mahmoudi (2000) concluded
that the finding of Stachybotrys does not imply a cause and effect
but should alert professionals to perform more environmental testing.
A critical review by Robbins et al. (2000) found that the current litera-
ture does not provide compelling evidence that exposure to mycotoxin-
containing molds is likely to result in measurable health effects.
A literature review by Page et al. (2001) also found inadequate evidence
supporting a causal relationship between symptoms or illness among
building occupants and exposure to mycotoxins. Terr (2001) in his
comprehensive review on Stachybotrys-related disease reached similar
conclusions but notes that the endemic cases of pulmonary hemor-
rhage with or without hemosiderosis are suggestive of a link since the
in-vitro properties of satratoxin noted earlier could be consistent with
clinical disease.
VIII. Conclusions
There is considerable scientific evidence that molds, under the prop-
er growth conditions, produce mycotoxins. These mycotoxins have
numerous properties that may be detrimental to animal and human
health when ingested and possibly when inhaled in large amounts. The
growth of mycotoxin-producing molds does not always predict the
presence of mycotoxins. The levels of mycotoxins inducing toxicity
286 LEVY AND FINK
have not been determined in humans. The link between causality and
human disease remains to be determined by further study.
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Fungal Hypersensitivity: Pathophysiology,
Diagnosis, Therapy
VINCENT A. MARINKOVICH
Department of Pediatrics, Stanford Medical School
Stanford, California 94305
I. Introduction 289
II. Health Effects of Fungi 290
A. Innate and Adaptive Immunity 290
III. Clinically Relevant Characteristics of Fungi 292
IV. Diagnosis 293
A. Antibodies or Lack Thereof 293
B. Individual Variations in Response 294
C. Hypersensitivity 295
V. Symptoms 295
VI. Mycotoxins 296
VII. The Role of IgE and Non-IgE Mechanisms 297
A. Immune Complexes 298
VIII. Therapy 299
A. Environmental Molds 300
B. Food Molds 300
C. Colonization 301
D. Antifungals 302
IX. Conclusion 305
References 305
I. Introduction
The comments contained herein are those of a clinical immunologist
whose major professional activity is patient care. Over the last several
years, as the general awareness of fungal exposure as a cause of illness
has grown, more and more patients have been presenting themselves
for diagnosis and treatment after exposure to homes and offices heavily
contaminated by fungi. The Internet has come to play a major role
in the dissemination of good, and some not so good, information
about fungal diseases, and the number of patients has mushroomed.
Most American trained physicians have had little instruction in my-
cology and tend to dismiss or minimize the possibility of fungal illness
except for certain generally accepted special situations. These would
include immunologically deficient patients, Candida infections in
women, thrush in newborns, skin infections such as ringworm and
athlete’s foot, and lung infections in areas endemic for histoplasmosis
289
0065-2164/04 $35.00
290 VINCENT A. MARINKOVICH
or coccidioidomycosis. Even allergists who limit themselves to skin

tests for diagnosing hypersensitivity ignore patients complaining of
serum sickness–like symptoms (e.g., headaches, rash, malaise, joint
pain, etc.) following exposure to moldy environments and often refer
them for psychiatric care (Terr, 2001). The unfortunate patient has
nowhere to turn except to those few physicians who have listened
to their patients, believe what they say, and accept the challenge to
try to help. These physicians have gone to the Internet or the medical
literature and developed scientifically inspired diagnostic and treat-
ment programs that have proven to be helpful to their patients. I stand
among these physicians, and I would vigorously defend the scien-
tific basis and efficacy of my approach to the diagnosis and treatment
of patients suffering from exposure to high ambient levels of fungi in
indoor environments. My best thoughts on the subject are offered
herein.
II. Health Effects of Fungi

There is much about the health effects of fungi that is not under-
stood. Exposure to high ambient fungal spore levels in a water-
damaged home or building is, more likely than not, a mixed bag. Not
only are more than one fungal genus likely to be present, but bacteria
such as Legionella or Actinomyces may be present in sufficient quan-
tities to add complexity to the resulting symptoms (Fink, 1984).
Endotoxins are frequently found in abundance when Gram-negative
bacteria abound, as when sewage is the source of the water (Rylander,
2002). In addition, insects such as mosquito larvae or other mold-
feeding insects (mites) may be contributing to the airborne organic
particle burden. Even large rodent populations have been discovered
in older, run-down, water-damaged homes and buildings. All of these
can contribute to the disease patterns seen in patients exposed to
‘‘fungi’’ in water-damaged structures.
A. INNATE AND ADAPTIVE IMMUNITY

Fungal exposure itself can produce a confounding array of symptoms
as different elements of the body’s defense systems are triggered. Early
in the course of exposure, the innate immune system can be activated
as endotoxins or fungal elements enter the body tissues. This inflam-
mation can proceed without any involvement of the adaptive immune
system with its antibodies and activated T-cells (Kauffman et al., 2000).
FUNGAL HYPERSENSITIVITY 291
However, after a few days or weeks of antigen presentation on an
inflamed mucosa, the adaptive system is likely to become involved
as antibodies and T-cells specifically reactive to fungal antigens are
generated. This will add to the inflammation of the affected tissues.
Finally, fungal elements become directly involved if mycotoxins or
other inflammatory triggers are formed that can cause toxic injury to
specific organ systems. One need only be reminded of such fungal
compounds as alcohol, lysergic acid (LSD), antibiotics, cyclosporin,
or mushroom toxins to appreciate the ability of such organic molecules
to cause symptoms.
Physicians who treat patients with mold-related problems are often
challenged by the variations in the disease symptoms and the multi-
organ involvement that are presumably the result of exposure to en-
vironments heavily contaminated with fungi. They may accept the
likelihood that fungal exposure is the cause of their patient’s symp-
toms but not understand the underlying pathophysiology. Still, an
attempt is made to treat the patient, essentially by utilizing various
programs that remove the patient from the fungi. Over time, they
learn that the clinical patterns seen in such patients are consistent,
the diagnosis can be accurately made, and the response to therapy is
very good.
There are other physicians who deny that fungi as encountered
in homes or office-type work spaces are capable of causing illness.
These physicians generally are not primary caregivers and can dismiss
the patient’s complaints because of their apparent complexity with-
out a consequence. They are better designated as theorists who base
their negativity on arguments that the lack of sufficient evidence-based
proof of a causal relationship of fungal exposure to human disease
proves that such a relationship is not possible. They dismiss all
case reports (Marinkovich et al., 1975) (Fink et al., 1971), epide-
miological studies (Dearborne, 2002) (Etzel et al., 1998), and clinical
observations of experienced clinicians as worthless and such pa-
tients as malingerers or psychiatrically disturbed (Hardin et al.,
2003). They seem to lack the vision to accept the challenge of the
possibility that injury to multiple organ systems may result from expo-
sure to large amounts of fungus-derived materials (such as spores
and/or mycotoxins) in a home or office environment. They are wrong,
and they can do a great deal of harm. First in denying the patient’s
symptoms, and second by blocking disability requests from such pa-
tients injured by exposure to fungi in their workplaces. They are
guilty of using poor scientific logic because it is closed-minded. Such
thinking has no place in a medical setting where there are sick patients
who need help.
III. Clinically Relevant Characteristics of Fungi

Fungi are nature’s recyclers. They are extremely abundant in nature,
carrying the mandate to reduce all organic matter to its basic cons-
tituents. The organisms are armed with several features that allow them
to satisfy this mandate. They are microscopic cells that are nu-
merous in all climates where temperatures are above freezing; they
exist in two forms: an active, growing form and a dormant, hardy,
drought-resistant and easily wind-borne form (the spore, also known
by the scientific name of conidia). They are superbly versatile and
can grow on virtually any wet surface. They secrete their digestive
enzymes (Kurup, 2003), digest their environment, and absorb their
necessary foodstuffs from their immediate, digested environment.
Among the products of digestion are toxins (known as mycotoxins
because they are derived from fungi), which help them control the
potential intrusion of competing organisms into their space. Each of
these characteristics plays a role in the disease patterns seen in fungal
illness.
The job of fungal spores is to broadcast the organism widely in the
environment. They are tiny, lightweight, and easily airborne. They are
in all natural environments the most prevalent particles in the air
at all times. Even at the height of a pollen season, the pollen particles
are outnumbered 10 to 1 by fungal spores. The human body is marvel-
ously equipped to deal with such large numbers of potentially infec-
tious particles in the air. The filtering capacity of the nasal mucosa
easily removes the larger spores, greater than 10 microns in diameter,
from the inspired air. Once trapped on the mucosa, the tiny hairs on
mucosal surfaces (cilia) move the particles toward the throat where
they are swallowed and destroyed by the acid in the stomach. Some
of the smaller spores, less than 10 microns in diameter, may be inhaled
into the lungs (Geiser et al., 2000). However, even here the normal self-
cleansing functions of the lung, which includes its own cilia and
mucus production, are mobilized, and particles are moved upward
and swallowed. A small subset of the tiniest spores, less than 3 microns
in diameter, may be inhaled and trapped in the alveoli and terminal
bronchioles beyond the reach of the cilia. They are handled by the
scavenger cells in the lungs, the alveolar macrophages.
This is extremely important to understanding the pathophysiology
of fungal exposure, because once the fungal elements have reached
the alveoli they have entered the tissue space from which they can be
absorbed into the blood stream.
IV. Diagnosis
The diagnosis of fungal hypersensitivity syndrome rests on four
criteria: exposure to an identified heavily contaminated source, appro-
priate symptoms temporally related to exposure, high serum-specific
IgG levels to molds, and finally, a positive response to therapy. IgE
antibodies are usually not involved in hypersensitivity phenomena
secondary to exposure to high-dose antigen such as fungi, foods, and
occupational exposures to organic matter (Fink, 1984). Skin tests are
therefore of little if any value. The fourth criterion for diagnosis is
an essential feature of all medical therapy—namely, the clinical im-
provement resulting from a fungal avoidance regimen. When this con-
dition is not met, the diagnosis must be revisited. Either avoidance is
inadequate, therapy is insufficient, or the diagnosis is wrong.
A. ANTIBODIES OR LACK THEREOF

Everyone is exposed to fungi in daily living, and therefore antibodies
to fungi are found in nearly everyone. They have been shown to be
protective, except for patients whose immune systems are inade-
quate in response. These patients are extremely susceptible to fungal
infection. In such cases—for example, AIDS patients, cancer patients
(especially if on chemotherapy), transplant recipients on immunosup-
pressive drugs, and patients with acquired or congenital immune defi-
ciency, especially involving cellular immunity—fungal colonization
can be life threatening. In most healthy individuals, the constant expo-
sure to ambient fungal spore levels is handled easily by normal muco-
sal cleansing mechanisms and the ever-vigilant immune system. Ill
effects do not generally occur in the normal population. However, this
statement is not true for all otherwise ‘‘healthy’’ individuals. The
extreme example of this is seen in certain occupational fungal dis-
eases—for example, farmer’s lung (Emanuel et al., 1964), malt workers
pneumonitis (Riddle et al., 1968), etc—where enormous exposures
occur on a daily basis and virtually everyone can be symptomatic. In
such cases the inflammatory changes produced in the lungs can cause
severe destruction of lung tissue, extensive colonization of lungs with
fungi and bacteria, and slow progression to respiratory deficiency and
death (Pepys, 1969). Such patients must be treated aggressively with
complete cessation of further exposure, high doses of systemic and
inhaled antifungals (Nark et al., 2003; Stevens et al., 2000), and the
judicious use of systemic steroids to reduce inflammation and arrest
the progressive damage or remodeling of the lungs (Kaltreider, 1993).
Steroids actually encourage fungal growth by suppressing the inflam-
matory reaction, and their use must be carefully monitored to walk the
tightrope between too much steroid, encouraging fungal growth, and
too little, allowing progressive destruction of lung tissue.
B. INDIVIDUAL VARIATIONS IN RESPONSE

The levels of fungi in contaminated homes and office buildings
may be quite high but are generally not nearly as high as those encoun-
tered in the special occupational situations previously mentioned
above. Still, they are high enough to cause serious illness in non-
immuno-compromised individuals (Burr, 2001). A considerable varia-
tion in response to moldy homes among members of the family is
common. In some cases all members of the family are affected, with
some small variations in severity and in the organs infected (e.g., skin,
lungs, sinuses, gastrointestinal tract, headaches, etc.). In other in-
stances the variation in severity of illness can be considerable among
family members; one person at one extreme may be quite ill, even
disabled, while another at the other extreme has little to show for the
exposure. This is understandable in that not all rooms in the house may
be equally contaminated and those sleeping in the rooms with highest
levels of contamination are likely to have more severe symptoms.
Variations may be seen in the amount of time each individual spends
at home. And then there is genetic polymorphism, where each individ-
ual is endowed with his own unique immune responsiveness and two
individuals in the same family or bloodline may respond quite differ-
ently to the same exposure. In studies done with serum sickness in
which normal, healthy individuals were given different volumes of
horse serum intravenously, some individuals developed symptoms
(Von Pirquet, 1951) with relatively low volumes of serum while others
required 10 times more serum to show the same symptoms. The con-
clusion of these studies was that everyone was susceptible but that
there is a dose-dependent susceptibility among different individuals.
Sustained exposure to airborne fungal spores at levels far below the
occupational disease levels in otherwise normal healthy individuals
will produce symptoms in some percentage of patients. The exact
percentage of susceptible individuals is likely to be low, perhaps under
1% of the population. But with the widespread contamination of
homes and workplaces in this country—with perhaps 30% of the
schools, homes, and offices involved—the actual number of affected
individuals can easily reach 1 million. One percent of the population is
about 3 million individuals, and a third of these would be 1 million.
This is an epidemic. Unfortunately, many of these cases are not recog-
nized by the medical community and go undiagnosed and untreated.
Thanks to the Internet and the media publicizing the results of liti-
gation involving mold cases, the public’s awareness of the problem has
grown. Hopefully, this will induce more health caregivers to learn
about fungal illness.
C. HYPERSENSITIVITY
The human immune response, part of the body’s system of adaptive
immunity, can be amazingly sensitive. A person allergic to cats can
sense the presence of cat dander in a room months after the cat has
departed. And rarely, one reads of a sudden death from anaphylaxis
provoked by exposure to a tiny amount of antigen such as vespid
venom from a single bee sting or the steam rising from a fish stew, or
tiny particles of peanut contaminating a package of almonds processed
on machinery previously used to process peanuts (Samson, 1992). The
extreme sensitivity potential of the immune system is rarely seen but
frightening when it occurs. When the number of individuals exposed
to such spore levels is very high—as seems to be the case today in
homes, schools, and workplaces—a significant number of cases will
occur. To deny this is akin to denying the existence of significant
pollen or cat allergies because the great majority of people do not show
such symptoms on exposure. Genetic polymorphism is the basis for a
considerable number of differences within the human population, and
the immune response, based on the same mechanisms, shows the same
wide variations in response among individuals.
V. Symptoms
While the symptoms seen in patients exposed to high ambient levels
of fungal elements can vary a great deal among different individuals,
a fairly consistent pattern of illness is seen in patients presenting
with sufficient symptoms to warrant seeing a physician. Most patients
describe a progression of symptoms beginning a few months to a few
years after the onset of exposure (e.g., moving into a mold-infested
house). Initially the complaints are nasopharyngeal (sore throats,
hoarseness, stuffy nose, transient hearing loss) or pulmonary (cough,
wheezing, shortness of breath). With time, symptoms progress to
include headaches, fatigue, rashes, vertigo, muscle and joint pain,

fever, recurrent sinus or ear infections, etc. (Rylander, 1994). Many of
these symptoms are the result of an overactive immune system trying
desperately to overcome what it perceives to be an overwhelming
infection. The immune system generates antibodies to the absorbed
materials (or antigens). These antibodies react with the antigens to form
immune complexes, which is all part of the body’s normal immune
elimination function. These complexes are quickly taken up by scav-
enger cells, which remove the complexes from the circulation, thus
limiting their inflammatory effects. When complex formation con-
tinues over a long period of time, this clearing mechanism can become
overloaded. The complexes then remain in the blood stream, causing
myriad symptoms, known to clinical immunologists as serum sickness
or immune complex disease (Cochrane et al., 1973). To the patient, the
symptoms appear to be a severe, unrelenting flu syndrome. When one
looks up in the older literature the classical symptoms seen in serum
sickness, they are exactly those symptoms the patients with fungal
illness describe to their physician (Von Pirquet, 1951).
Since hypersensitivity states develop only after relatively long expo-
sure times, normal children under 10 years of age do not have signifi-
cant antibody titers to fungi. However, when children experience very
high exposure levels in the home or school, measurable antibody levels
appear rather quickly—that is, within a few months of exposure. Nor-
mal mature adults living in temperate or tropical climates commonly
show antibody activity toward fungi and experience symptoms follow-
ing unusual exposures. The onset of symptoms often follows exposures
by 1 or 2 days, the symptoms are not recognized for what they are, and
the symptoms are likely to be diagnosed as a virus infection.
VI. Mycotoxins
Mycotoxins are the most respected of fungal products for their po-
tential to cause serious illness through their direct biochemical action
on key body functions (Croft et al., 1986; Johanning et al., 1996; Leino
et al., 2003). The immune system is not involved. One of these, aflatox-
in, is known to be among the most potent of carcinogens. Another
group, trichothecenes, are toxins released by the fungus Stachybotrys
atra (also known as chartarum) as well as others. There is contro-
versy regarding the role of trichothecene mycotoxins in pulmonary
hemosideroisis (Dearborn et al., 1999). Other toxins can affect various
hormonal, neurological, and other body functions to produce seri-
ous health effects (Sorenson, 1999). They are so effective in certain
biological activities that they have been harnessed by the pharma-
ceutical and food industries for commercial use such as antibiotics,
immune suppressants to control graft rejection, medicine for cholesterol
control, and enzymes used in food processing and preservation. Myco-
toxins are produced by fungi under specific growth conditions, and
their role in human illness is not well understood. Exposure to certain
mycotoxins producing organisms such as Stachybotrys seem to cause
neurological damage seen as short-term memory loss, cognitive dys-
function, inability to concentrate, and ‘‘fuzzy thinking.’’ There are
common complaints of patients with fungal illness. The changes seem
to be reversible, at least in part, but they can take years to resolve.
Hyperactive immune systems responding to the influx of fungal anti-
gens following chronic exposures are much more likely to be a cause of
symptoms in most individuals.
VII. The Role of IgE and Non-IgE Mechanisms

Allergists have accepted the role that fungal spores can play in
eliciting allergy symptoms in susceptible individuals. This is akin
to the effects of other airborne organic particles such as pollen, animal
dander, and insect dust. The illness affects only individuals pro-
grammed genetically to make large quantities of specific IgE antibodies
on exposure to relatively small amounts of allergen. This is type I
immunopathology, as defined by Gell and Coomb (Gell et al., 1964)
and involves the release of pre-formed histamine and other biologically
active cytokines from sensitized mast cells and basophils. Symptoms
include watery nasal discharge, sneezing paroxysms, and itching of the
naso-oro-pharyngeal mucosa and tearing eyes and can be significantly
disabling. Symptoms disappear quickly on cessation of exposure, leav-
ing little or no residual effects. It has been suggested that perhaps
5 percent of the population may be affected in this way by fungi,
although those numbers will vary in different climates (e.g., more in
Florida than New Mexico). The great majority of patients presenting
with symptoms of fungal illness do not show IgE antibody to the fungus
(Fink, 1984). This may be the result of isotype switching from IgE to IgG
production as stimulation of the immune system increases. When this
happens, far more elaborate and damaging immune responses can be
generated by the body following exposure to large amounts of fungal
particles, especially when the exposure is chronic. These illnesses
were originally described in association with various occupational
exposures in unprotected workers such as farmer’s lung, bagassosis
in sugar cane workers, and many others. More recently such conditions
have been identified and studied in office workers whose workplace

is contaminated by fungi (especially in buildings with closed venti-
lation systems [Fink, 1984]), in individuals exposed to swamp coolers
(Marinkovich et al., 1975) or contaminated air conditioners in the
home (Banaszak et al., 1970), and in many other school, home, and
workplace exposures, generally as case reports involving a few pa-
tients per report (Cakmak et al., 2002; Dales et al., 1991; Hodgson
et al., 1998). These symptoms can occur in all individuals with normal
immunity because they are ultimately manifestations of a robust im-
mune response to a heavy unrelenting airborne fungal load with conse-
quential overload of clearing mechanisms or macrophages and the
activation of inflammatory processes.
Although the general immune response to a heavy fungal antigen
exposure may consist of all the immunoglobulin isotopes (IgA, IgM,
IgG, IgD, and IgE) plus sensitized lymphocytes or T-cells, specific IgG is
the most efficient single marker of generalized immune responses.
Specific IgG antibody levels to fungi are not diagnostic when taken
alone. However, antibody levels to fungi are directly proportional to
levels of exposure in any individual, and generally high exposure
levels result in high antibody titers. These antibody levels drop when
the patient’s exposure to the offending fungi ends. When elevated, they
are helpful in arriving at the presumptive diagnosis, and repeat mea-
surements at 4- to 6-month intervals help verify compliance with the
fungal avoidance program and help monitor the success of therapy.
A. IMMUNE COMPLEXES
The presence of antibody in the serum is not pathologic in itself.
All the immunoglobulins normally present in the tissues, with pos-
sible rare exceptions, are antibodies, and they contribute to the im-
mune state. It is only when antigens combine with antibodies that
immune complexes are formed and a potentially pathologic state is
initiated. Immune complexes are not stable, since the union is one of
complimentary surface configurational attraction between two or more
molecules produced by Van der Wall forces. The complexes can easily
be disrupted as the conditions in solution change. Changes in tempera-
tive, relative numbers of reacting molecules, their nature, the epi-
tope specificity of the reacting antibodies, etc., can all effect changes
in the size, shape, surface charges, and solubility of the complex. These
factors are the ones that determine the inflammatory potential of the
complexes formed and whether the complexes will tend to be deposit-
ed in kidneys, joints, blood vessel walls, skin, lungs, etc. (Cochrane
et al., 1973). Because of the inherent instability of immune complexes
in tissue and serum, they are difficult to study. Interest in under-
standing immune complexes was very high in the 1950s and 1960s
and quickly dissolved when IgE was discovered (Ishizaka et al., 1966)
and the attention of the immunological research teams was attracted to
the newly defined antibody responsible for classical allergy symptoms,
Type I of Gell and Coombs.
There are sound scientific reasons why specific IgG antibodies to
fungi are not always diagnostic. Some individuals with high antibody
levels to fungi remain symptom free during re-exposure. Such indivi-
duals may be less likely to produce the toxic immune complexes
required to induce symptoms by virtue of the fungal antigen epitopes
to which they respond. They may respond to minor epitopes that allow
measurement of the antibody but which do not engage in the formation
of toxic immune complexes. Other individuals may have a vast scav-
enger system, which can rapidly take up and extinguish all immune
complexes generated before symptoms can ensue. Other individuals
may satisfy the high exposure and the appropriate symptom require-
ments and have relatively low total specific antibody levels to fungi.
They may be poor antibody responders with an even lower capacity to
deal with immune complexes. The observation that they can still
experience flu-like symptoms following fungal exposure demonstrates
that a relatively low antibody level can still produce significant, dis-
abling symptoms. Mold toxins can be powerful immune suppressors. It
is sobering to remember that there would be no organ transplant pro-
gram without the availability of fungal toxins (e.g., cyclosporin). It is
possible and even likely that the fungal exposure of some patients will
include exposure to immune suppressive mycotoxins. Another cause
for low antibody levels in a symptomatic patient could be iatrogenic.
Many patients develop arthritic symptoms and present themselves to
rheumatologists who may choose to use an immunosuppressive drug
such as methotrexate to treat the arthritis. Such a drug will certainly
depress the immune response and relieve the severity of arthritic
symptoms while masking what could be the real trigger for the arthritis.
VIII. Therapy
The therapy of all hypersensitivity diseases must be based on avoid-
ance. In the case of fungi, it is important to recognize that there are
three sources of exposure: The airborne particles, mostly spores, which
result from water intrusion at home, school, and work; ingestion (as in
the enormous amounts and types of fungal products used by the food
industry); and colonization of skin, lung, sinuses, and other mucosal

surfaces.
A. ENVIRONMENTAL MOLDS
A moldy environment must be remediated or destroyed. All sources
of water intrusion have to be discovered and sealed. All wetted sur-
faces must be completely dried (e.g., both sides of sheet rock), and any
surfaces showing fungal proliferation must be replaced, including
walls, floors, carpets and pads, cabinets, furniture, etc. In many cases
of fungal hypersensitivity, the affected individual may not be able to
return to the remediated space because his sensitivity is too great for
the level to which remediation may reduce fungal efflux. There is a
simple canary-in-the-mine parallel. No amount of surveying the reme-
diated site can assure that the patient will be able to tolerate the
reduced fungal levels. More times than not, they cannot. Avoidance
may require a move to different quarters.
High-efficiency particulate air filters (HEPA) are useful in removing
spores from the air. They remove a great majority of the particles greater
than 0.3 microns in size, which includes all mold and bacterial spores.
However, a heavily mold-infested indoor space may overwhelm the
ability of a HEPA room air purifier to significantly reduce ambient
spore levels. In some cases, ozone generators have proven useful. They
must be used at full power inside sealed, vacant rooms for a full day to
significantly reduce fungal growth. It is important to stress that occu-
pants should not be exposed to ozone, which is toxic. The ozone is
unlikely to kill spores or deeply situated mycelial elements. Therefore,
the process generally has to be repeated at least bimonthly. The ozone
levels achieved in the room may not be safe at any times for individuals
with asthma, chronic obstructive pulmonary disease (COPD), or other
forms of respiratory distress; however, handling the ozoning process
(i.e., initiating and terminating the treatment) is only slightly disagree-
able to the normal individual. Mold cells are similar to human cells in
their makeup, which means that ozone levels likely to kill mold cells
are also likely to irritate the human respiratory mucosa.
B. FOOD MOLDS
Fungi are prolific enzyme and toxin generators, which is the basis for
much of their use in modern food technology. Bread will rise more
quickly and require less baking time and lower baking temperatures if
amylase is added to the dough. The amylase digests the cross-linkages
of the cellulose in the dough, making it less tough. This results in
considerable economic gain for the baking industry. Its downside is
that it has produced substantial illness in bakers, and it loads the bread
with mold products that add to the burden of a mold-sensitive individ-
ual. These additives are listed on the ingredient labels of breads as
dough conditioners or malted grains (for malt, read mold). Fruit juice
manufacturers discovered that if hemicellulases (of fungal origin) are
added to the crushed fruit prior to squeezing out the juice, the yield can
increase as much as 25%. The enzymes digest the cellulosic structure
of the fruit and allow more juice to be obtained from the cells. This is a
wonderful economic gain for juice makers but adds fungal elements to
the juice.
Citric acid is perhaps the greatest misnomer in the ingredients listing
of any food. It is added to most processed foods including soft drinks,
jams and jellies, frozen meals, etc., as a preservative. It conjures up
visions of lemons, oranges, and limes. It is none of these; it is a direct
product of Aspergillus fermentation in commercial quantities. It is a
highly impure ‘‘citric acid’’ contaminated by many other Aspergillus
products including toxins, antibiotics, etc. One wonders if pure citric
acid would confer the same excellent preservative properties as com-
mercial ‘‘citric acid’’ on foods? Again, this is an excellent product for
the food industry in extending the shelf life of foods, but it adds fungal
elements to the food.
Some foods are obligate products of fermentation such as aged
cheese (usually Penicillium), soy sauce (usually Aspergillus), chocolate
(mixed molds), and black tea (Aspergillus). While wonderful on the
educated palate, they must be eliminated in a mold-free diet. The
patient trying to avoid mold in his food must be instructed in how to
maintain a fresh food diet. This means shopping more frequently than
weekly. Farmer’s markets are often excellent sources of fresh fruits and
vegetables. This may be especially difficult in small towns where the
supermarket is the only source of food.
C. COLONIZATION
Colonization of the human mucosa is a common phenomenon that
seems to be poorly understood. The human body is colonized by
bacteria in the nasopharynx, mouth, gastrointestinal tract, skin, etc.
No one questions this, and the concept of a balanced ecology in the gut
is considered essential for proper gastrointestinal (GI) function and a
stable supply of nutrients such as vitamins. Fungal colonization is
also a widely accepted phenomenon—as for example toenail fungus,
athlete’s foot, vaginal candidiasis, chronic fungal sinusitis, and sebor-

rheic dermatitis. But the concept that fungi can become part of the
normal mucosal flora, and that once established, fungal colonization
can place a heavy burden on the body’s defenses is not always appre-
ciated. Some fungi produce toxins, and all fungi produce and secrete
enzymes into their environment. The body has to protect itself against
all foreign proteins, especially those that carry enzymatic activity. The
immune response is that protection. Foreign enzymes are known to
be among the most powerful stimulants of an immune response (Larson
et al., 1996).
Patients who have become ill from living or working in a moldy
space are often improved when they move, but their health does
not always return to normal. This means either that the fungal expo-
sure resulted in permanent damage, which is quite possible with
tissue-deposited immune complexes or certain mycotoxins, or there
is continued exposure to the fungus because of colonization. Coloniza-
tion is more likely to occur first where there has been previous
tissue damage. The nasopharynx is the first filter for airborne fungi
and would expect to be first to be colonized along with adjacent
communicating structures like the sinuses or middle ear. Colonization
is more likely to occur where there are residual scars from past
disease or surgery. This is where the body’s first line of defense—
mucous flow, ciliary action, and IgA secretory antibody function—is
likely to be missing. The lungs are easy targets for colonization when
there has been previous damage as in a past pneumonia. Children with
cystic fibrosis are virtually 100% colonized in the lungs (Etzel et al.,
1998), and patients with chronic asthma are said to be about 30%
colonized. The esophagus is a common site for colonization because
of its vulnerability to damage from hot foods, spicy foods, and acid
reflux.
D. ANTIFUNGALS
When possible, colonization is best treated topically. The oral cavity
and esophagus can be treated with liquid fluconazole (40 mg/ml) or
Itraconazole (10 mg/ml), 40 or 50 mg four times daily. Neither of these
is well absorbed in a non-acid medium, and they can be quite effec-
tive topically until they reach the stomach, where they may be ab-
sorbed. Nystatin is a non-absorbed antifungal with an excellent safety
record. It can be given as a powdered suspension in water at 500,000
units four times daily. It will continue to provide antifungal activity
throughout the GI tract as it courses its way from mouth to anus.
Fungal colonization of the nasopharynx, sinuses, and middle ear is
best treated with an antifungal nasal spray. A 2% ketoconazole suspen-
sion or a 0.01% amphotercin B solution applied generously four times
daily to the nose is effective in many cases. It must be delivered deep
into the nasal cavity and be felt passing into the pharynx. Neither will
be significantly absorbed in the pH-neutral mucosa of the nose. The
benefits of therapy are likely to be noted within a few weeks but a cure,
where therapy can be safely stopped without recrudescence of the
illness, is months and sometimes years in the future. This is likely
due to the resistance of the spore to killing with available antifungal
drugs, which means that therapy must be continued until all spores are
eliminated or germinate and become susceptible to the action of the
antifungal.
Colonization of the lungs and sometimes the sinuses requires a
systemic antifungal such as Itraconazole, ketoconazole, or voricona-
zole. Each are given at 200 to 400 mg per day (Gallin et al., 2003;
Schubert, 2000). In some cases of lung disease a nebulized antifungal
is helpful. Ketoconazole has been successfully used by nebulizing
50 mg per treatment, twice daily.
Many physicians show great concern when talking of systemic anti-
fungals because of the possibility of liver damage. This concern is
grossly overstated. Most antifungals used in high doses are given to
immunocompromised individuals with severe fungal infections in-
cluding blood-borne dissemination. Elevated liver enzymes in such
catastrophic illness is not rare and must be considered in the decision
to use such therapy. However, in my 10-year experience with antifun-
gals in immunologically normal individuals colonized by fungi, I have
yet to see a single episode of elevated liver enzymes, which can be
attributed to the use of the antifungal. Testing for liver function at 2- to
4-month intervals is recommended by the FDA. It has been reported
that in rare instances in which liver enzymes have risen, cessation of
therapy results in a rapid return to normal in all but a few rare in-
stances. The antifungals are metabolized in the liver and place some
burden on the detoxification enzymes of the liver, which are also used
to metabolize certain drugs. The use of antifungals may influence the
serum and tissue levels of such drugs, generally causing a rise in
concentration as the rate of metabolism of the drug is reduced. Such
changes can be handled by careful assessment of tissue levels of drugs
used simultaneously with the antifungals.
Fungal colonization of the GI tract is a relatively common phenome-
non encouraged by the overuse of antibiotics in medicine and their
use in the production of meat for human consumption. This usually
manifests as abdominal discomfort, heartburn, increased gastric emp-

tying time, bloating, crampy abdominal pain, and increased transmu-
cosal uptake of large food protein molecules (leaky gut). Treatment is
best begun with a non-absorbed antifungal such as Nystatin or poorly
absorbed antifungals such as miconazole or econazole. Nystatin is best
given as a powdered suspension, 2 to 3 million units per day in divided
doses (b.i.d. or t.i.d.). The miconazole and econazole are not generally
available in pure powder form from regular pharmacies and must be
formulated. This increases the cost somewhat but still leaves them far
cheaper than the newer antifungals already mentioned. They are given
in 250 mg capsules twice daily.
1. Jarisch-Herxheimer Reactions
The treatment of fungal colonization in patients hypersensitive to
fungi almost always produces a Jarisch-Herxheimer (JH) reaction if
given too aggressively. It is safest to begin with one quarter or less of
the therapeutic target dose and advance every 3 to 4 days in doubling
doses to reach the desired dose. The JH reaction can occur at the initial
dose or at any time the dose is increased. It manifests as a flu-like
reaction in its broadest sense (i.e., headache, rash, low-grade fever,
myalgia, arthritis, night sweats, malaise, cough, diarrhea, etc.). When
it appears, treatment should be stopped until the symptoms disappear
(usually 1 to 2 days), and then a lower amount should be introduced
and held there for 2 weeks before any attempt to increase the dose. It is
best to be guided by the patient, who quickly learns if there is a limit to
the dose he can tolerate, but he may subsequently have to be encour-
aged to try to take more medicine if past experience has been severe
enough to be alarming. The JH reaction can also occur when the patient
who is seemingly stable (on full dose) suddenly experiences a larger
fungal burden, such as in staying in a moldy hotel room on a trip or
following a day of spreading compost in a vegetable garden.
Colonization of the skin in the form of abscesses on the skin or
dry scaly rashes over the palms of the hands (dishydrotic eczema)
can be treated with topical antifungal creams, sometimes coupled
with systemic antifungals. The topical antifungal action on the skin
can be enhanced by use of occlusive dressings. Patients are directed
to apply the cream liberally at bedtime and then cover the lesion
with a watertight membrane (e.g., plastic food wrap), which remains
overnight.
All fungal therapy must be prolonged, often for a year or longer. This
is likely due to the resistance of fungal spores to any medicine and the
rapid reestablishment of colonization should therapy be ended too
soon. All the spores must have been shed or have germinated and been
killed by the action of the antifungal and the body’s natural defense
system before the colonization is truly ended.
IX. Conclusion
The best treatment for health problems arising from exposure to high
fungal levels is prevention. A key prerequisite to prevention is educa-
tion. Information about the nature of fungi, their presence in foods,
their rapid proliferation after water intrusion in homes, workplaces,
and schools, and their potential for health effects must be made easily
available to the general public. The Internet has already provided such
information to millions who use computers. Insurance companies
are excluding mold damage from the coverage provided in homeowner
policies, and this may alert the homeowner to the danger and to his/her
responsibility to move rapidly to minimize the effects of water leaks.
Reports in the media of litigation by celebrities experiencing fungal
illness also helps increase public awareness of the problem. Public
health service organizations have to date been more concerned to quell
the public’s concern about mold problems by suggesting that it is not
an important issue. This is a disservice. It would be far better to
acknowledge the potential health effects of mold exposure along with
suggestions for controlling mold levels in homes, workplaces, and
schools.
ACKNOWLEDGMENTS
The author is grateful to his daughter, Tess Marinkovich, for critical reading of this
manuscript and to Mary Borch for her help in preparing the text.
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Indoor Molds and Asthma in Adults
MARITTA S. JAAKKOLA AND JOUNI J. K. JAAKKOLA
Institute of Occupational and Environmental Medicine, University of
Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom
I. Occurrence of Indoor Mold and Dampness Problems 309

II. Reasons for Indoor Mold and Dampness Problems 310
III. Potential Mechanisms by Which Indoor Mold and Dampness
Problems Induce Asthma 311
IV. Assessment of Exposure in Asthma Studies 311
V. Studies on Indoor Mold and Dampness Problems and
Asthma or Wheezing 313
A. Etiologic Studies 313
B. Studies on Asthma Severity 328
C. Intervention Studies 329
VI. Methodological Considerations 330
A. Exposure Assessment 330
B. Study Design and Assessment of Asthma 331
C. Confounding 331
VII. Conclusions 332
VIII. Future Directions 333
References 333
I. Occurrence of Indoor Mold and Dampness Problems

This article will discuss exposure assessment and health effects
of indoor mold and dampness problems occurring in residences or
in non-traditional occupational situations. By traditional workplace
exposures, we mean here the farming environment and industrial ex-
posures, and by non-traditional workplace exposures, we mean indoor
dampness and molds in offices, schools, daycare centers, hospitals,
and corresponding indoor exposure situations.
Indoor dampness problems have been reported to be common in
countries around the world. For example, in the subtropical climate
in Taiwan, mold growth was reported in almost 60% of residences
(Wan and Li, 1999a). In the northeastern United States, over 50% of
households including children reported mold growth (Maier et al.,
1997), and in Canada, 38% of adult respondents across 30 communities
had home dampness and/or mold problems (Dales et al., 1991a). In
United Kingdom and Central European countries, residential dampness
problems are also common, with estimates ranging between 13%
(Brunekreef, 1992; Nowak et al., 1996) and 51% (Platt et al., 1989;
Waegemeakers et al., 1989; Williamson et al., 1997). More surprising
309
0065-2164/04 $35.00
310 JAAKKOLA AND JAAKKOLA
perhaps, indoor dampness problems have been reported to be relatively

common also in colder climates. For example, the estimated preva-
lences of residential dampness and/or molds in Sweden (Norbäck
et al., 1999), Norway (Nafstad et al., 1998), and Finland (Jaakkola
et al., 1993; Nevalainen et al., 1998; Partanen et al., 1995) have been
between 4% and 82%. Only a few studies have evaluated the preva-
lence of such problems in workplaces. In Taiwan, about 40% of day-
care centers and office buildings had mold growth (Wan and Li, 1999b).
In the city of Espoo in southern Finland, water damage had taken place
in 70% of the daycare centers, and mold odor was perceived by 6% of
the daycare center workers (Ruotsalainen et al., 1995). Because of their
universality, indoor dampness and mold problems may be of major
public health importance in different parts of the world.
II. Reasons for Indoor Mold and Dampness Problems

The main sources of moisture and dampness in buildings have been
categorized into four groups (Bornehag et al., 2001):
1. Outdoor sources of moisture (e.g., leakage of rain or snow into the
building construction and moisture absorbed from the ground).
2. Indoor activities (e.g., use of humidifiers, bathing, cooking, and
drying of wet clothes indoors).
3. Building sources (e.g., poor protection of building materials from
rain and snow at the building time, insufficient time for drying
for the concrete floors, and insufficient ventilation to eliminate
indoor humidity).
4. Accidents (e.g., leakage from pipes and flooding).
The importance of these causes varies according to the dampness-
problem building, and the main causes may differ between different
climates and countries. In Finland, the main reasons for increasing in-
door dampness and mold problems were related to the construction of
tight buildings to save energy since the 1970s and poor maintenance
of the buildings during the economic recession period in the early 1990s.
Fungi are eukaryotic microorganisms that colonize dead organic
materials in outdoor and indoor environments (Burge, 1989; Flannigan
et al., 1992). The species that are able to colonize indoor environments
can utilize nutritional sources available in indoor materials. Moisture
is the most important factor controlling fungal growth (Burge, 1989;
Flannigan et al., 1992). Because of changing indoor environmental
conditions, the colonizing process is dynamic. The indoor fungal aero-
sol consists of particles penetrating from outdoors, particles released
INDOOR MOLDS AND ASTHMA IN ADULTS 311
from active growth on indoor substrates, and reaerosolized particles
settled into dust reservoirs. Some reports have been published describ-
ing the fungal species that occur typically in water-damaged buildings
(Hyvärinen et al., 1992, Samson, 1999).
III. Potential Mechanisms by Which Indoor Mold and Dampness

Problems Induce Asthma
Indoor dampness may induce asthma through different agents, in-
cluding molds, bacteria, house dust mites, or increased emission of
chemicals from damp materials (e.g., volatile organic compounds
[VOC] and degradation products of polyvinyl chloride [PVC] floors)
(Norbäck et al., 1999). The relative importance of the causative agents
may vary in different dampness situations and in different countries.
For example, in Finland the occurrence of house dust mites is rare,
but when present they are likely to be of importance. Bacteria usually
require stagnant pools of water to grow (Rylander, 1997). The main
causes of asthma in indoor dampness situations are likely to be molds,
and potentially, emission of chemicals from damp materials.
The mechanisms through which indoor molds cause asthma are
not yet well understood, but several potential mechanisms have been
suggested. These include IgE-mediated hypersensitivity reactions to
fungal allergens, toxic effects of mycotoxins produced by fungi, and
nonspecific inflammatory reactions caused by microbial volatile organ-
ic compounds (MVOC) or fungal cell wall components such as 1,3--D-
glucan and ergosterol (Husman, 1996; Jaakkola et al., 2002a; Johanning
et al., 1999; Norbäck et al., 1999; Thorn and Rylander, 1998). It is not
known whether these mechanisms are specific for different species of
molds. However, some molds seem to have allergenic properties and are
able to produce mycotoxins and/or MVOCs (Flannigan et al., 1991;
Jaakkola et al., 2002b), so it is also possible that the same species can
induce asthma through different mechanisms. We do not know what
determines the mechanisms in each individual case, but environmental
conditions are likely to play an important role.
IV. Assessment of Exposure in Asthma Studies

Exposure to molds in health effect studies can be assessed by using
questionnaires, visual and/or engineering inspection of buildings, en-
vironmental sampling, and/or biomarkers. These methods have been
reviewed recently by Pasanen (2001). The assessment method of choice
depends on the study question and the resources that are available.
Questionnaires are the most commonly used assessment method in

studies of asthma. This is a relatively inexpensive method and allows
assessment of exposure in different environments (e.g., home and
workplace exposures) as well as assessment of past exposures. The
most common questions used to assess indoor mold and dampness
problems inquire about the occurrence of water damage or flooding,
damp or darkened stains on surfaces, mold odor, and visible mold
(Jaakkola et al., 2002a; Nafstad et al., 1998; Ruotsalamen et al., 1995).
Visual inspection consists of a walk-through assessment of the build-
ings or spaces in question. It may be accompanied by a more detailed
engineering inspection, including measurement of moisture on indoor
surfaces and in building materials, detailed physical examination of
the building, classification of moisture problems, and environmental
sampling. This method is sometimes used to validate questionnaire-
based assessment. However, to be able to use this method for valida-
tion, it is important to ensure that the conditions are similar to those at
the time of answering the questionnaire. Experts are likely to be able to
detect smaller signs of dampness and mold problems than subjects
themselves, so this method is often more sensitive than self-reports.
Environmental sampling includes air, dust, or bulk sampling fol-
lowed by laboratory analyses or in situ measurements (Pasanen,
2001). With respect to asthma and other respiratory effects, fungi in
the breathing zone are obviously the most relevant. However, there is
no agreement on what sampling method is recommended for asthma
studies. Air samples reflect the fungi at the relevant media, but dust
and bulk samples may represent better exposure over a longer period of
time and thus may be more relevant for chronic health effects. Labora-
tory analyses of the samples may include determination of spore con-
centrations, taxonomic identification of fungi, or analysis of fungal
components and products such as allergens, 1,3--D-glucan and ergos-
terol, mycotoxins, and microbial VOCs. The detailed description of the
laboratory methods is beyond the scope of this article.
Mold-specific IgG antibodies have been used as biomarkers of fungal
exposure in studies of allergic alveolitis (Eduard, 1995; Terho, 1986). In
Finland, microbe-specific antibodies have also been applied in clinical
studies of asthma. The interpretation of IgG antibodies as biomarkers of
exposure may be problematic, since such antibodies last in the blood for
months or even for years (Eduard, 1995), so it is not possible to identify
based on them when and where the person was exposed to the microbe
in question. Another potential problem is that it is not known to what
extent IgG antibody levels reflect the magnitude of exposure (Jaakkola
et al., 2002b). IgE antibodies take part in the causal pathway to asthma
rather than being indicators of exposure, and the same may be true for
some subcategories of IgG antibodies—for example, IgG4 antibodies
(Edge and Pepys, 1980; Igea et al., 1993; Tanizaki et al., 1992).
At the moment we do not know well the mechanisms through which
different molds induce asthma, or even which molds are the most re-
levant ones for the health effects. This makes the exposure assessment
in health effect studies difficult. In health effect studies, questionnaires
are usually the method of choice in large populations, but it is pre-
ferable to combine this with other exposure assessment methods in
subpopulations, if possible.
V. Studies on Indoor Mold and Dampness Problems and

Asthma or Wheezing
Several studies have related residential dampness problems to in-
creased risk of asthma and asthma-like symptoms in children (Belanger
et al., 2003; Bråbäck et al., 1995; Brunekreef et al., 1989; Dales et al.,
1991b; Dijkstra et al., 1990; Jaakkola et al., 1993; Nafstad et al., 1998;
Spengler et al., 1994; Strachan, 1989; Timonen et al., 1995; Verhoeff
et al., 1995; Waegemaekers et al., 1989; Wickman et al., 2003). The
studies conducted in adult populations are reviewed in more detail
here. They are divided into etiologic studies, studies on asthma severity,
and intervention studies. They are further categorized based on whether
the indoor dampness and mold exposure of interest is home exposure
or workplace exposure.
A. ETIOLOGIC STUDIES
1. Cross-Sectional Studies in Adults: Home Exposure
The first studies on indoor mold and dampness problems and asthma
or wheezing including adults focused on home exposure and were
reported from the United Kingdom and the Netherlands in the late
1980s (Hyndman, 1990; Martin et al., 1987; Platt et al., 1989;
Waegemaekers et al., 1989). These studies had small sample sizes,
and the results were sometimes inconclusive, but two studies sug-
gested an increased risk of asthma in relation to reported visible mold
or dampness (Hyndman, 1990; Waegemaekers et al., 1989). Since then
several cross-sectional studies focusing on home exposures in adults
have been reported. These are summarized in Table I. Some research
groups have published several reports from the same study. In those
cases, only the most recent report or the one with the largest sample
size is included in the table. The first large population-based studies
were reported from Canada (Dales et al., 1991a) and the Netherlands
TABLE I
CROSS-SECTIONAL STUDIES IN ADULTS ON INDOOR MOLD AND DAMPNESS PROBLEMS AT HOME AND ASTHMA
Author, Year, Study

Country Population Outcome Exposure OR (95% CI)
Martin et al., 300 adults Reported wheezing Any sign of dampness No significant differences
1987, UK or mold in inspection in adults
314
Platt et al., 597 adults Reported wheezing Dampness or mold No significant differences
1989, UK in inspection
Waegemaekers 328 adults Reported doctor-dg asthma Reported visible Men: 1.2 Women:
et al., 1989, mold or damp spots 4.2 (p < 0.005)
Netherlands
Hyndman et al., 345 subjects Reported ‘hidden Reported mold 2.5 (p < 0.05)
1990, UK asthma’ (¼ wheezing, dampness 2.2 (p < 0.05)
breathlessness, cough,
blocked nose)
Dales et al., 14,799 adults Reported doctor-dg asthma Reported damp spots, 1.56 (1.25–1.95)
1991a, visible mold, or flooding
Canada
Brunekreef 6,436 adults Reported attacks of Reported damp stains Women: 1.25 (0.94–1.66)
1992, shortness of breath or visible mold Men: 1.29 (0.92–1.81)
Netherlands
Pirhonen et al., 1,460 adults Reported doctor-dg asthma Reported visible mold, 1.02 (0.60–1.72)
1996, Finland mold odor, damp stains,
or water damage
Hu et al., 1997, 2,036 young adults Reported current Reported visible mold, 2.0 (1.2–3.2)
California, doctor-dg asthma water damage, 1.6 (0.7–3.8)
USA damp spots 1.3 (0.7–2.2)
Dharmage 485 adults (part Current asthma ¼ BHR Esgosterol concentration 14 n.s.
et al., 2001, of ECRHS) in methacholine challenge in dust
Australia þ reported wheezing Total viable fungal 3 n.s.
propagules in air
Comparison in both:
highest vs. lowest
quartile
Kilpeläinen 10,667 young adults Reported current Reported visible mold 2.2 (1.5–3.3)
et al., 2001, doctor-dg asthma visible mold, damp 1.7 (1.3–2.2)
315
Finland stains, or water

damage
Zock et al., 18,873 adults from Reported current Reported visible mold Asthma: 1.28 (1.13–1.46)
2002, Spain 38 centers in ECRHS asthma or BHR in BHR: 1.14 (1.01–1.29)
methacholine challenge water damage Asthma: 1.13 (0.95–1.35)
BHR: 1.15 (0.97–1.35)
OR ¼ odds ratio, CI ¼ confidence interval, ECRHS ¼ European Community Respiratory Health Survey, BHR ¼ bronchial hyperresponsiveness,
n.s. ¼ statistically nonsignificant.
(Brunekreef, 1992) in the early 1990s. They were originally designed

to study the effects of dampness problems in children, but they in-
cluded questionnaire answers also for the parents themselves. The
estimates of OR in relation to visible mold or damp stains from these
studies were from 1.25 to 1.56. The largest cross-sectional study in
adult populations published so far was based on 38 centers from the
European Community Respiratory Health Survey (ECRHS). It included
18,873 subjects (Zock et al., 2002). The OR of asthma was 1.28 in
relation to visible mold (95% CI 1.13–1.46) and 1.13 in relation to
water damage (95% CI 0.95–1.35). The study also assessed bronchial
hyperresponsiveness in methacholine challenge and found a slightly
increased risk in relation to visible mold (OR 1.14, 1.01–1.29) and
water damage (OR 1.15, 95% CI 0.97–1.35).
2. Prevalent Case-Control Studies in Adults: Home Exposure
The prevalent case-control studies on home dampness and mold
problems and asthma are summarized in Table II. Some research
groups have published several reports from the same study, and in
those cases only the most recent report or the one with the largest
sample size is included in the table. The first case-control study was
published from the United Kingdom in the late 1980s (Burr et al.,
1988). It included 72 prevalent cases of asthma and 72 controls from
a general practice register. Visible mold was reported by 26% of cases
and 13% of controls and damp patches by 39% of cases and 38% of
controls. These differences were not statistically significant. The only
statistically significant difference was observed in the occurrence of
positive skin prick tests to molds: 13% of cases versus 1% of controls
showed positive tests. However, it should be borne in mind that skin
test positivity, which indicates sensitization to molds, is actually an
early step in the disease process itself rather than an indicator of
exposure. Thus, in addition to past exposure, sensitization may also
indicate individual propensity to become sensitized and individual
susceptibility to develop asthma. Another prevalent case-control study
from the United Kingdom, published in 1997 (Williamson et al., 1997),
showed an OR of 1.93 for self-reported dampness at home but an OR of
3.03 for dampness detected in house inspection. This indicated that
the subjects tended to underestimate their exposure to dampness as
compared with expert evaluation. OR related to mold problems de-
tected in inspection was 1.35, and it increased up to 1.70 if the mold
problem, judged by the inspector, was significant. Thus there was
indication of a dose-response relation between the severity of mold
problem and the risk of asthma. Two groups from Sweden have re-
ported prevalent case-control studies on this topic (Norbäck et al.,
TABLE II
PREVALENT CASE-CONTROL STUDIES IN ADULTS ON INDOOR MOLD AND
DAMPNESS PROBLEMS AT HOME AND ASTHMA
Author, Year,
Country Study Population Exposure OR (95% CI)
Burr et al., 72 cases, Reported

1988, U.K. 72 controls visible mold 26% cases,
13% contr., n.s.
damp patches 39% cases,
38% contr., n.s.
Williamson et al., 102 cases, Reported dampness 1.93 (1.14–3.28)
1997, U.K. 196 controls In inspection:
dampness 3.03 (1.65–5.57)
mold 1.35 (0.79–2.28)
significant mold 1.70 (0.78–3.71)
Norbäck et al., 98 cases, Reported
1999, Sweden 357 controls visible mold 1.90 (0.9–3.8)
(part of ECRHS) dampness 1.80 (1.1–3.0)
damp floor 4.6 (2.0–10.5)
Thorn et al., 2001, 174 cases, 870 Reported
Sweden controls visible mold 2.2 (1.4–3.5)
dampness 1.3 (0.9–2.0)
OR ¼ odds ratio, CI ¼ confidence interval, ECRHS ¼ European Community Respiratory Health Survey.
1999; Thorn et al., 2001). Norbäck and coworkers (1999) found an OR

of 1.90 in relation to reported visible mold, 1.80 in relation to damp-
ness, and 4.6 in relation to damp floor. Based on the high risk of asthma
in relation to damp floor, they hypothesized that chemicals emitted
from the damp materials into the air could be one mechanism through
which indoor dampness problems induce asthma. The other Swedish
study made an attempt to ensure a meaningful temporal relation be-
tween the mold exposure and the onset of asthma by retrospectively
assessing exposures that had taken place the year asthma was diag-
nosed or before it (Thorn et al., 2001). Only asthmatics diagnosed
during the period 1980–94 were included to reduce potential recall
bias because of the long recall period. The OR of asthma related to
visible mold was 2.2 and to dampness problems was 1.3 in this study.
3. Cross-Sectional Studies in Adults: Workplace Exposure
Only a few studies have evaluated the relations between workplace
exposure to indoor mold and dampness problems and asthma or asth-
ma-like symptoms. The first ones were reported in the mid-1990s and
investigated asthma-like symptoms, including wheezing, as the out-

come of interest (Li et al., 1997; Ruotsalainen et al., 1995). These are
summarized in Table III. The Finnish study was conducted among
daycare center nurses (Ruotsalainen et al., 1995). It found an OR of
1.28 for wheezing in relation to mold odor and 1.66 in relation to water
damage (Ruotsalainen et al., 1995). A study from Taiwan investigated
also daycare center nurses (Li et al., 1997). The OR for wheezing was
from 1.32 to 1.39 in relation to water damage and mold problems, the
highest OR 2.87 being observed in relation to the occurrence of any
dampness indicator.
Two further studies investigated asthmatic symptoms among employ-
ees of problem buildings with dampness or mold damage and compared
these to the occurrence of symptoms among employees of control build-
ings free of such problems (Hodgson et al., 1998; Norbäck et al., 2000).
These are summarized in Table IV. Hogdson and coworkers (1998) stud-
ied two office buildings with mold problems and two control office
buildings in Florida, altogether 197 employees. OR in relation to working
in the problem offices was 2.8 for wheezing and as high as 10.4 for
chest tightness. However, the confidence intervals for these estimates
were wide because of the rather small sample size. Norbäck and col-
leagues (2000) studied 87 employees working in two hospitals with
dampness problems and two control hospitals in Sweden. The OR
of asthma symptoms—including wheezing, attacks of dyspnea, and
nocturnal symptoms—was 8.6 in relation to working in the problem
TABLE III
CROSS-SECTIONAL STUDIES IN ADULTS ON MOLD AND DAMPNESS PROBLEMS AT WORK AND WHEEZING
Author, Year,
Country Study Population Exposure OR (95% CI)
Ruotsalainen et al., 268 daycare Reported

1995, Finland center nurses water damage 1.66 (0.72–3.82)
mold odor 1.28 (0.44–3.73)
Li et al., 1997, 612 daycare Reported
Taiwan center employees visible mold 1.39 (0.84–2.29)
stuffy odor 1.38 (0.89–2.15)
water damage 1.32 (0.79–2.23)
any dampness 2.87 (1.19–6.94)
indicator
OR ¼ odds ratio, CI ¼ confidence interval.

hospitals. Again, the confidence interval was wide (1.3–56.4) because
of the small sample size.
4. Incident Case-Control Study in Adults: Workplace Exposure

When reviewing the previous studies on indoor molds and asthma in
the late 1990s, there was a lack of studies in adult populations apply-
ing incidence-design—meaning development of new asthma in adult-
hood—as well as lack of studies focusing on workplace exposures. We
conducted a population-based incident case-control study in the Pir-
kanmaa Hospital District in South Finland (Jaakkola et al., 2002a). This
district is a geographically defined area with a population of 440,913
inhabitants in 1997. During September 1997 through March 2000 we
recruited all new cases of asthma diagnosed among adults 21–63 years
old living in this area. A random sample of the source population
(residents of the Pirkanmaa District aged 21–63 years) formed the con-
trols. The diagnosis of asthma was based on the presence of asthmatic
symptoms (prolonged cough, wheezing, exercise-induced or attacks of
dyspnea, and/or nocturnal cough or wheezing) and the demonstration of
significant reversibility of airflow obstruction in lung function measure-
ments (Committee on National Asthma Program 1994; Viljanen et al.,
1982). The clinical investigations included spirometry (American
Thoracic Society 1995), accompanied by a bronchodilation test with
salbutamol (albuterol), and a 2-week diurnal follow-up of peak expira-
tory flow (PEF), the first week without any medication (if possible)
TABLE IV
STUDIES OF PROBLEM BUILDINGS WITH INDOOR MOLD OR DAMPNESS PROBLEMS AND
ASTHMATIC SYMPTOMS IN ADULTS
Author, Year,
Country Study Population Outcome OR (95% CI)
Hodgson et al., 2 mold problem Reported

1998, USA and 2 control wheezing 2.8 (0.6–17.4)
office buildings, chest tightness 10.4 (1.5–62.8)
197 employees
Norbäck et al., 2 dampness problem Reported asthma 8.6 (1.3–56.4)
2000, Sweden þ 2 control hospitals, symptoms: wheezing,
87 employees attacks of dyspnea,
nocturnal symptoms

and the second week with regular inhaled bronchodilating medication.

If these lung function tests were negative but there was still a strong
suspicion of asthma, the person underwent a 2-week treatment with
oral steroid accompanied by a further 2-week PEF follow-up and a
spirometry control.
Asthma cases were recruited through all health care facilities diag-
nosing asthma in the Pirkanmaa District, including the Tampere Uni-
versity Hospital, all health care centers, and all privately practicing
pulmonologists. In addition, the National Social Insurance Institution
of Finland that provides reimbursement for asthma medication nation-
ally invited those asthmatics who received the reimbursement right
during our study period and who had not yet participated in our study.
The medical records of all cases were checked, and those with previous
asthma diagnosis or long-term use of asthma medication were exclud-
ed. Random selection of controls was carried out from the 1997 Census,
and again, those with previous asthma were excluded. Cases and con-
trols answered a questionnaire on occurrence of water damage, damp
stains, visible mold, and mold odor at home and in the workplace
currently, 1–3 years ago, more than 3 years ago, or never. The question-
naire was modified from the Helsinki Office Environment Study ques-
tionnaire (Jaakkola and Jaakkola, 1999; Jaakkola and Miettinen, 1995)
to be used in a general population and also requested information on
potential confounders such as education, smoking habits, and other
occupational exposures. Since in the preliminary analyses there was
no trend in the risk of asthma according to the period of exposure, all of
the above periods were combined in the final logistic regression ana-
lyses. Mold odor and visible mold occurred often concurrently and
were also combined in the final analyses.
Table V presents the characteristics of asthma cases and controls.
Altogether, 521 new cases of asthma and 932 controls participated in
the study. There were more women among cases than controls. Cases
were somewhat younger, had more family history of parental atopy,
and had more often a lower level of education than the controls. Cases
were also more often smokers or ex-smokers than were controls. In
multivariate analyses, we adjusted for these factors. Table VI shows
the distributions of exposure to dampness and mold problems at work
and at home among cases and controls. The only clear differences in
exposure were observed in the occurrence of visible mold and mold
odor at work, both of which were more common among cases than
among controls.
Table VII presents the ORs of asthma in relation to different damp-
ness and mold exposure indicators adjusted for potential confounders.
TABLE V
CHARACTERISTICS OF THE STUDY POPULATION OF THE FINNISH INCIDENT CASE-CONTROL STUDY, THE
FINNISH ENVIRONMENT AND ASTHMA STUDY 1997–2000 (JAAKKOLA ET AL., 2002a)
Cases Controls
Characteristic N % N %
Total 521 932

Sex
Man 175 33.6 438 47.0
Woman 346 66.4 494 53.0
Age (years)
21–39 215 41.2 365 39.1
40–59 265 50.9 494 53.1
60–64 41 7.9 73 7.8
Parental allergic diseases
Any maternal or 186 35.7 204 21.9
paternal allergy
or asthma
Educationa
No voc. schooling 107 20.6 154 16.6
Vocational course 89 17.2 104 11.2
Vocational institution 149 28.7 271 29.2
College or university 174 33.6 399 43.0
Smokingb
No 239 46.1 487 52.4
Ex 133 25.7 203 21.8
Current 146 28.2 240 25.8
ETS
In the workplace 89 17.1 130 13.9
In the home 30 5.9 52 5.6
Pets at home
Sometimes 387 74.3 663 71.1
Any work exposurec 313 60.1 579 62.1
ETS ¼ environmental tobacco smoke.

a
Information on education was missing for 6 subjects.
b
Information on smoking was missing for 5 subjects.
c
Self-reported exposure to sensitizers, dusts, and/or fumes, except for exposure to molds.
TABLE VI
EXPOSURE TO INDOOR DAMPNESS AND MOLD PROBLEMS AMONG CASES AND CONTROLS, THE FINNISH
ENVIRONMENT AND ASTHMA STUDY 1997–2000 (JAAKKOLA ET AL., 2002a)
Cases Controls
Exposure N % N %
Work
Water damagea 63 12.1 121 13.0
Damp stains or paint peelinga 90 17.4 171 18.3
Visible molda 34 6.6 42 4.5
Mold odora 59 11.3 86 9.3
Home
Water damageb 54 10.3 103 11.0
Damp stains or paint peelingb 103 19.8 185 19.9
Visible moldb 26 5.1 56 6.0
Mold odor 54 10.4 86 9.3
a
Yes categories included only those who worked indoors at least 50% of their workday; others are
included in the no category.
b
‘Do not know’ was answered by 5 cases and 12 controls for water damage, 3 controls for damp
stains, and 1 case and 1 control for visible mold.
TABLE VII
ADJUSTED ODDS RATIOS OF ASTHMA IN RELATION TO EXPOSURE TO MOLD AND DAMPNESS
IN THE WORKPLACE AND AT HOME, THE FINNISH ENVIRONMENT AND ASTHMA STUDY
1997–2000 (JAAKKOLA ET AL., 2002a)
Exposure ORa 95% CI
Work
Visible mold or mold odor 1.54 1.01–2.32
Damp stains or paint peeling 0.84 0.56–1.25
Water damage 0.91 0.60–1.39
Home
Visible mold or mold odor 0.98 0.68–1.40
Damp stains or paint peeling 1.02 0.73–1.41
Water damage 0.90 0.61–1.34

a
Adjusted for sex, age, parental atopy/asthma, education, smoking, environmental
tobacco smoke exposure, pets, work indoors, self-reported occupational exposures,
and other exposure indicators shown in the table.
Occurrence of visible mold and/or mold odor in the workplace was
related to a significant increase in the risk of asthma with an OR of 1.54,
while dampness problems alone did not increase the risk of asthma.
None of the exposure indicators at home were related to increased risk
of asthma. There is no biologic reason to believe that the effects of
similar mold exposures at home and at work would be different. Rath-
er, the difference in effect estimates in this study are likely to be
explained by differences in exposure at work and at home. These
differences may occur in the mold species or in the extent of mold
problems at work as compared with home environments. We did not
quantify the extent of such problems, but it is plausible that in the
workplace people do not notice small dampness problems easily, for
example, because they may change work area often, and thus more
extensive mold growth may develop. In addition, it is often more
difficult to influence one’s work environment than one’s home envi-
ronment. At home people tend to pay attention to water damages and
repair them before more advanced mold problems develop, because
these damages reduce the value of the property.
Table VIII presents the adjusted risk of asthma related to visible mold
and/or mold odor in the workplace stratified according to factors that
are potentially related to susceptibility to the adverse effects of molds.
The risk was somewhat higher among women than men, it was higher
in the youngest age group as compared with the older ones, and the risk
was higher among current smokers as compared with never and ex-
smokers. The mechanisms of such susceptibility are not known at the
moment. It is possible that the young and women have more extensive
exposures, because they are often in lower positions in the workplaces
and therefore have less influence on their work environment. It is also
possible that other indoor exposures in the workplace, for example,
environmental tobacco smoke (ETS), act synergistically with mold
exposure. It has been shown that young adults often have the most
extensive ETS exposure at work (California EPA, 1997). Immunologic
and other inflammatory reactions may play a role in the sensitivity, for
example, in current smokers.
Since mold-specific IgG antibodies have commonly been applied
as biomarkers of exposure in clinical investigations of asthma patients
in Finland, we addressed the relation between microbe-specific IgG
antibodies and development of asthma in the population-based inci-
dent case-control study (Jaakkola et al., 2002b). A total of 490 cases
(94%) and 668 controls (72%) provided a serum sample for antibody
analyses. We analyzed IgG antibodies to a panel of eight microbes with
enzyme-linked immunosorbent assay (ELISA). These eight microbes
TABLE VIII
ADJUSTED ODDS RATIOS OF ASTHMA IN RELATION TO VISIBLE MOLD AND/OR MOLD ODOR IN THE
WORKPLACE ACCORDING TO POTENTIAL EFFECT MODIFIERS, THE FINNISH ENVIRONMENT AND ASTHMA
STUDY 1997–2000 (JAAKKOLA ET AL., 2002a)
Study Population ORa 95% CI
All 1.54a 1.01–2.32

Sex
Men 1.26b 0.62–2.54
Women 1.67b 1.00–2.79
Age (years)
20–29 2.98c 0.96–9.27
30–49 1.31c 0.75–2.30
50–64 1.46c 0.65–3.29
Parental atopy
Yes 1.57d 0.57–4.31
No 1.61d 1.02–2.55
Smoking
Current 3.05e 1.14–8.14
Ex 0.83e 0.32–2.10
Never 1.63e 0.92–2.90

a
Adjusted for gender, age, parental atopy/asthma, education, smoking, environmental tobacco
smoke exposure, pets, work indoors, self-reported occupational exposures, and other exposure
indicators.
b
Adjusted for age, parental atopy/asthma, education, smoking, environmental tobacco smoke
exposure, pets, work indoors, self-reported occupational exposures, and other exposure indicators.
c
Adjusted for sex, parental atopy/asthma, education, smoking, environmental tobacco smoke
exposure, pets, work indoors, self-reported occupational exposures, and other exposure indicators.
d
Adjusted for sex, age, education, smoking, environmental tobacco smoke exposure, pets, work
indoors, self-reported occupational exposures, and other exposure indicators.
e
Adjusted for sex, age, parental atopy/asthma, education, environmental tobacco smoke exposure,
pets, work indoors, self-reported occupational exposures, and other exposure indicators.
were selected based on the current knowledge of occurrence of

microbes in water-damaged buildings (Hyvärinen et al., 1992; Samson,
1999). They were Aspergillus fumigatus (mold), Aspergillus versicolor
(mold), Cladosporium cladosporioides (mold), Fusarium oxysporum
(mold), Sporobolomyces salmonicolor (yeast), Stachybotrys charta-
rum (mold), Streptomyces albus (actinomycete), and Trichoderma
citrinoviride (mold).
Table IX presents the adjusted ORs of asthma in relation to the
increasing levels of microbe-specific IgG antibodies as compared with
the lowest quartile for each antibody. Only IgG antibodies against
Trichoderma citrinoviride were related to an increased risk of
asthma. The adjusted OR for the second quartile of the antibody levels
was 1.62 (95% CI 1.12–2.35), for the third quartile 1.59 (1.07–2.34),
and for the fourth quartile 1.38 (0.90–2.12). These associations
related to the increased concentrations of IgG antibodies against
Trichoderma citrinoviride are consistent with, but do not necessarily
indicate, a causal relation between exposure to this mold and develop-
ment of asthma. For example, it is possible that this species is only a
marker for other fungi or microbes that are actually more relevant
for the health effects. Trichoderma citrinoviride has been commonly
detected in water-damaged buildings (Lübeck et al., 2000). However,
some characteristics of Trichoderma citrinoviride suggest that this
mold could induce inflammatory reactions in the airways. The dia-
meter of its spores is frequently smaller than 3.5 m (Bissett, 1984),
so they can reach small airways. Some Trichoderma species have
been found to be allergenic (Burge, 1985), and they produce mycotox-
ins including trichothecenes, gliotoxin, cyclopentane derivatives, and
peptaibols (Flannigan et al., 1991; Husman, 1996; Lübeck et al., 2000).
When growing, they may also produce different types of MVOCs
(Gravesen et al., 1994; Larsen et al., 1998). Specific IgG antibodies
against the seven other indoor dampness-related microbes that were
studied were not associated with the risk of asthma. We selected our
panel of eight microbes on the basis of the occurrence of microbes in
water-damaged buildings, but it is possible that these are not the most
relevant ones for the health effects. In addition, interpretation of IgG
antibodies as biomarkers of exposure is problematic, as discussed in
the section on exposure assessment.
In our preliminary analyses in a subpopulation of 252 cases and 600
controls, we also investigated the relations between mold-specific
IgE antibodies to six molds analyzed by the UniCAP System (Pharmacia
& Upjohn Diagnostics, Uppsala, Sweden) (Jaakkola et al., 2003a). These
molds were Aspergillus fumigatus, Cladosporium herbarum, Mucor
racemosus, Penicillium notatum, Phoma betae, and Stachybotrys atra.
A specific IgE level less than 0.35 kU/L was defined as negative. The
adjusted ORs of asthma in relation to the presence of these antibodies are
given in Table X. IgE antibodies to Cladosporium herbarum and Penicil-
lium notatum were associated with an increased risk of adult-onset
asthma.
TABLE IX
THE ADJUSTED OR OF ASTHMA IN RELATION TO MICROBE-SPECIFIC IGG ANTIBODY LEVELS, THE FINNISH ENVIRONMENT AND
ASTHMA STUDY 1997–2000 (JAAKKOLA ET AL., 2002b)
Cases Controls
Crude Adjusted Adjusted
N % N % OR 95% CI ORa 95% CI ORb 95% CI
Aspergillus fumigatus
First quartile 132 26.9 157 23.5 1.00 1.00 1.00
Second quartile 123 25.1 167 25.0 0.88 0.63–1.22 0.82 0.58–1.16 0.83 0.56–1.22
Third quartile 113 23.1 175 26.2 0.77 0.55–1.07 0.71 0.50–1.00 0.73 0.48–1.10
Fourth quartile 122 24.9 169 25.3 0.86 0.62–1.19 0.72 0.50–1.01 0.82 0.52–1.29
Aspergillus versicolor
326
First quartile 127 25.9 162 24.2 1.00 1.00 1.00

Second quartile 129 26.3 161 24.1 1.02 0.74–1.42 1.01 0.71–1.42 1.22 0.81–1.84
Third quartile 113 23.1 172 25.8 0.84 0.60–1.17 0.77 0.54–1.10 1.01 0.64–1.59
Fourth quartile 121 24.7 173 25.9 0.89 0.64–1.24 0.80 0.56–1.13 1.05 0.65–1.70
Cladosporium cladosporioides
First quartile 130 26.5 159 23.8 1.00 1.00 1.00
Second quartile 123 25.1 167 25.0 0.90 0.65–1.25 0.89 0.63–1.26 0.95 0.66–1.38
Third quartile 113 23.1 174 26.0 0.79 0.57–1.11 0.80 0.56–1.13 0.86 0.58–1.26
Fourth quartile 124 25.3 168 25.2 0.90 0.65–1.25 0.76 0.54–1.09 0.86 0.56–1.31
Fusarium oxysporum
First quartile 123 25.1 165 24.7 1.00 1.00 1.00
Second quartile 108 22.1 183 27.4 0.79 0.57–1.11 0.80 0.56–1.13 0.84 0.58–1.20
Third quartile 129 26.3 160 24.0 1.08 0.78–1.50 1.07 0.76–1.52 1.17 0.82–1.68
Fourth quartile 130 26.5 160 23.9 1.09 0.78–1.52 1.08 0.77–1.53 1.18 0.82–1.70
Sporobolomyces salmonicolor
First quartile 124 25.3 165 24.7 1.00 1.00 1.00
Second quartile 125 25.5 165 24.7 1.01 0.73–1.40 0.95 0.67–1.34 0.98 0.68–1.43
Third quartile 121 24.7 168 25.1 0.96 0.69–1.33 0.95 0.67–1.34 1.03 0.70–1.53
Fourth quartile 120 24.5 170 25.5 0.94 0.68–1.31 0.89 0.63–1.26 1.00 0.64–1.58
First quartile 134 27.3 155 23.2 1.00 1.00 1.00
Second quartile 116 23.7 172 25.8 0.78 0.56–1.09 0.77 0.54–1.08 0.80 0.55–1.16
Third quartile 119 24.3 172 25.8 0.80 0.58–1.11 0.76 0.54–1.08 0.76 0.51–1.12
Fourth quartile 121 24.7 169 25.2 0.83 0.60–1.15 0.77 0.54–1.08 0.81 0.52–1.27
Streptomyces albus
First quartile 139 28.4 150 22.5 1.00 1.00 1.00
327
Second quartile 119 24.3 171 25.6 0.75 0.54–1.04 0.72 0.51–1.01 0.71 0.49–1.02
Third quartile 119 24.3 170 25.4 0.76 0.54–1.05 0.71 0.50–1.00 0.69 0.48–1.01
Fourth quartile 113 23.0 177 26.5 0.69 0.50–0.96 0.63 0.44–0.89 0.64 0.43–0.95
Trichoderma citrinoviride
First quartile 113 23.0 176 26.4 1.00 1.00 1.00
Second quartile 136 27.8 154 23.0 1.38 0.99–1.91 1.39 0.98–1.96 1.62 1.12–2.35
Third quartile 126 25.7 163 24.4 1.20 0.86–1.68 1.26 0.89–1.78 1.59 1.07–2.34
Fourth quartile 115 23.5 175 26.2 1.02 0.73–1.43 1.06 0.74–1.50 1.38 0.90–2.12
a
Adjusted for sex, age, parental atopy, education, smoking, ETS, pets, occupational exposures, working indoors.
b
Adjusted for sex, age, parental atopy, education, smoking, ETS, pets, occupational exposures, working indoors, and other specific IgGs.
B. STUDIES ON ASTHMA SEVERITY

1. Home Exposure
Some studies have investigated the relation between indoor molds
and severity of existing asthma including adult asthmatics (Bjönsson
et al., 1995; Dharmage et al., 2002; Lebowitz et al., 1982; Ross et al.,
2000). All of them have focused on home exposure. Ross and
coworkers conducted a panel study of 57 asthmatic individuals from
Illinois, USA, 5 to 49 years of age (Ross et al., 2000). A total of 28% of
subjects reported current dampness in the home, 9% reported mold
growth during the previous 12 months, and 35% had positive allergy
skin tests for molds. Repeated indoor bioaerosol measurements were
carried out in the homes of asthmatics for 4–7 months, including
determinations of concentrations of bacteria and mold spores. Several
indicators of asthma severity were assessed: any emergency room visit
due to asthma, awakening at night because of asthma, asthma interfer-
ing with the ability to speak, missing days from work/school because of
asthma, and frequent wheezing. The strongest associations were ob-
served between the total bacteria and spore concentrations and emer-
gency room visits caused by asthma. The unadjusted ORs of emergency
visits caused by asthma were 5.8 related to increased concentrations of
total bacteria (95% CI 1.3–25.8), 2.8 related to increased total mold
spores (0.7–11.1), 3.1 related to Cladosporium (0.8–12.5), and 2.2
related to Alternaria (0.5–8.7). All of these exposures were also related
TABLE X
ADJUSTED ODDS RATIOS OF ASTHMA IN RELATION TO OCCURRENCE OF MOLD-SPECIFIC IGE
ANTIBODIES FROM THE PRELIMINARY ANALYSES, THE FINNISH ENVIRONMENT AND
ASTHMA STUDY 1997–2000 ( JAAKKOLA ET AL., 2003a)
Exposure ORa 95% CI
Aspergillus fumigatus 1.45 0.55–3.82

Cladosporium herbarum 3.00 0.69–13.03
Mucor racemosus 0.92 0.10–8.65
Penicillium notatum 1.99 0.73–5.38
Phoma betae 1.03 0.29–3.70
Stachybotrys atra 1.50 0.08–26.82

a
Adjusted for sex, age, parental atopy/asthma, education, smoking, environmental
tobacco smoke exposure, pets, and self-reported occupational exposures.
to the occurrence of awakening at night because of asthma. Increased
concentrations of total bacteria were related to all of the asthma sever-
ity indicators. Occurrence of dampness in the home alone or concen-
trations of house dust mite allergen were not related to asthma severity.
The dust mite allergen levels were generally low in this study.
Another study from Australia followed 35 young adults with current
asthma and sensitization to fungi (Dharmage et al., 2002). Their homes
were visited four times over 1 year with administration of a question-
naire and sample collection of indoor dust and air. Viable fungal
propagules and five fungal genera were determined from the air sam-
ples, and ergosterol levels were measured from the dust samples. The
subjects kept diaries on peak expiratory flow rates (PEF), symptoms,
and use of asthma medication. A total of 85% of the asthmatics were
also sensitized to house dust mites. Visible mold at home was asso-
ciated with a 1.5-fold increase in the daily PEF variability, while levels
of total fungi, specific genera, or ergosterol did not show any significant
relation to PEF variability or to symptoms.
C. INTERVENTION STUDIES
1. Workplace Exposure
Surprisingly few studies have evaluated the effects of repair mea-
sures of dampness or mold problems on symptoms and signs of
asthma in adult populations. A study from Sweden investigated 14
daycare center nurses before and 2 years after renovation of indoor
mold problems in their workplace (Rylander, 1997). Cultures of
microbes had demonstrated growth of Penicillium, Aspergillus, and
Actinomycetes on the surfaces and in the insulation material. Exposure
was assessed by measuring 1,3--D-glucan in airborne dust. The sub-
jects underwent methacholine challenge for assessment of asthmatic
signs. The outcome of interest was decrease in forced expiratory vol-
ume in one second (FEV1) (expressed as percentage of the prechallenge
post-saline value) at the dose 1.20 mg of methacholine, rather than
dose of methacholine provoking a 15% decline in FEV1 from the base-
line level (PD15), because a very sensitive measure of outcome was
needed because of the small number of study subjects. The renovation
measures led to a significant decrease in dust 1,3--D-glucan concen-
tration from 11.4 ng/m3 at baseline to 1.2 ng/m3 2 years after renova-
tion. This decline in exposure was accompanied by a change in average
FEV1 decrease in methacholine challenge from 8% to 4%, thus showing
a slight improvement in bronchial hyperresponsiveness.
VI. Methodological Considerations

A. EXPOSURE ASSESSMENT
Assessment of exposure to indoor molds in the context of health
effects is difficult, because development of diseases usually takes a
long time period, in the case of asthma probably at least months, while
the growth of indoor fungi depends strongly on the environmental
conditions and may change over time. Thus assessment of exposure
at any one point in time may not reflect precisely the exposure condi-
tions at the time of induction of the disease. Another difficulty is related
to the fact that, at the moment, it is not well known what mold species
are most relevant to the health effects or what components or pro-
ducts of fungi are responsible for development of asthma. With ques-
tionnaires, past exposures can be characterized, but it is not possible
to get more detailed information about species retrospectively.
Several studies have compared the reported presence of indoor
dampness and/or mold problems with findings in building inspections
or measurements of fungi in indoor air or dust (Dales et al., 1997;
Dharmage et al., 1999; Nevalainen et al., 1998; Norbäck et al., 1999;
Platt et al., 1989; Waegemaekers et al., 1989; Williamson et al., 1997).
Most of these have shown a relatively good agreement between these
methods of exposure assessment. It has been found that subjects tend
to underestimate dampness/mold problems at home, but this trend is
observed among both asthmatics and healthy controls, so there is no
indication of bias. Such unbiased underestimation of exposure is likely
to lead to underestimation of the true risk of asthma related to indoor
mold problems. In contrast to the other studies, one study from Canada
suggested that those with allergies overreport the presence of molds at
home (Dales et al., 1997). The Australian research group found that
visible mold is a significant predictor of the levels of fungi in indoor
environments (Dharmage et al., 1999). They concluded that direct
observation of mold growth is superior to any measure of environmental
biomass available today (Dharmage et al., 2002).
In our study the questions on indoor molds formed only one part of
the large study on environmental factors and asthma (the Finnish
Environment and Asthma Study), so no special attention was paid
to these questions over other exposures. Asthmatics and controls an-
swered the same self-administered questionnaire in a similar way.
Based on this and on experiences from earlier studies there is no reason
to suspect that there would be any bias in the answers between cases
and controls, but our risk estimates may be underestimates of the true
risk of asthma related to indoor molds.
B. STUDY DESIGN AND ASSESSMENT OF ASTHMA
Timing and accuracy in the assessment of the outcome is critical for
validity and causal inference from the results. After the onset of asthma
it becomes difficult to separate the effects of past and current exposure
on the causation on one hand and on the severity of the symptoms and
signs of asthma on the other hand. This is true particularly in the case
of exposures, such as mold, which are likely to influence both etiology
of asthma and symptoms after the onset of asthma. All of the studies on
adult populations published before our incident case-control study
were cross-sectional or prevalent case-control studies in design, so they
were not able to separate potential effects of dampness and mold-
related exposures on development of new asthma from aggravation of
asthmatic symptoms among those with an already established disease.
Almost all of the earlier studies based the diagnosis of asthma or
asthma-related symptoms on self-reports in questionnaires or inter-
views, so potentially biased reports of symptoms by those with already
established disease was of major concern.
Accurate assessment of asthma will decrease misclassification and
thus will both increase the power of the study and improve validity of
the effect estimates. In addition to including only new cases of asthma,
another strength of our study was that definition of asthma was based
on lung function measurements, which reduces potential bias related
to the outcome assessment. Among the previously reported studies
only the ones based on the European Community Respiratory Health
Survey protocol (Dharmage et al., 2001; Norbäck et al., 1999; Zock et al.,
2002) included lung function measurements, namely measurement of
bronchial hyperresponsiveness in methacholine challenge. Applying
objective and rather strict criteria for asthma in our study diminished
misclassification of the asthma status but was likely to lead to inclusion
of more severe cases as compared with earlier studies applying
less-specific criteria for diagnosing asthma.
C. CONFOUNDING
In studies of the relation between exposure to dampness and mold
problems and the risk of asthma, any determinant of asthma is a
potential confounder. A potential confounder will cause confounding
(i.e., becomes an actual confounder) when it is associated with expo-
sure to dampness and molds. A large number of indoor environmen-
tal factors—such as ETS (Jaakkola et al., 2003b), pets (Jaakkola et al.,
2002), chemical emissions from materials (Jaakkola et al., 1999), and
occupational exposures (Jaakkola et al., 2003c)—may increase the risk

of asthma in adulthood and at the same time be associated with damp-
ness problems. Also, individual factors such as age, sex, and heredity
influence the risk of developing asthma and may be related to mold
problems at home or work. In our case-control study we applied multi-
variate logistic regression analysis to take into account the effects of
many important potential confounders (e.g., age, sex, education, pets,
environmental tobacco smoke, and occupational exposures). Earlier
studies on indoor molds and asthma usually controlled for age, sex,
and/or smoking, but some have also adjusted for some measure of
socioeconomic status and housing or building characteristics.
VII. Conclusions
Based on review of the current literature on indoor molds and asth-
ma in adults it can be concluded that:
1. There is strong evidence that indoor molds at home increase the
risk of asthma. Epidemiological studies have consistently found a
relation between exposure to dampness and molds at home and
the risk of asthma in adults. The OR of asthma has ranged between
1.3 and 2.2, and there is some evidence of a dose-response relation
in which more severe mold problems are related to a higher OR.
2. There is increasing evidence that indoor molds at work increase
the risk of wheezing. The OR of asthmatic symptoms in relation to
work exposure has ranged between 1.3 and 2.8. An OR as high as
8.6 has been reported, but this was based on a small study, which
was reflected in a wide confidence interval.
3. A recent incident case-control study showed evidence that indoor
molds at work increase the risk of asthma, with an OR of 1.5. This
study also indicated that women, young adults, and smokers are
particularly susceptible to the adverse effects of workplace indoor
molds.
4. There is increasing evidence that indoor molds increase the
severity of an existing asthma.
5. There is some evidence that repair of indoor mold problems
relieves or eliminates symptoms and signs of asthma.
Overall, it can be concluded that there is increasing evidence sug-
gesting that prevention and prompt repair of indoor dampness and
mold problems in home and work environments prevent asthma in
adulthood.
VIII. Future Directions
To strengthen the evidence on the role of indoor molds for the
development of asthma in adulthood, more etiologic studies with inci-
dent cases are needed, either incident case-control studies or cohort
studies. The former design may be more efficient, since studies with
cohort design would require very big study populations and long
follow-up periods to produce equally large numbers of new cases as
incident case-control studies. There is also a lack of studies focusing on
workplace exposures, although in our incident case-control study,
workplace mold problems were a major determinant of new asthma
in adulthood.
Indoor molds could play a role in determining the prognosis of an
already established asthma, but only a few studies have investigated
the relation of dampness and mold problems with asthma severity. In
addition, there is a need for controlled intervention studies evaluating
the effects of repair measures on symptoms and/or lung function
among asthmatics.
To be able to design better exposure assessment in the health effect
studies, progress is needed in understanding the mechanisms through
which molds induce asthma and in the understanding of the mold
species that can cause the development of asthma in previously
healthy individuals. When planning exposure assessment for a health
effect study one should always take into account the study question of
interest. For example, development of new asthma is likely to require a
longer exposure period than aggravation of an underlying disease.
Given the current uncertainties, it is preferable to combine several
exposure assessment methods whenever it is feasible.
We did not find any study that had investigated potential interactions
between indoor molds and other indoor exposures. Since we found that
indoor molds are important determinants of asthma in the work envi-
ronment, it would be of special interest to evaluate potential synergistic
effects between indoor molds and ETS or other occupational exposures
in the workplaces.
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mold exposure, and asthma in the European Community Respiratory Health Survey.
Role of Molds and Mycotoxins in Being
Sick in Buildings: Neurobehavioral and
Pulmonary Impairment
KAYE H. KILBURN
University of Southern California
Keck School of Medicine
Environmental Sciences Laboratory
Alhambra, California 91803
I. Introduction 339
A. Perspective 340
B. Tight Buildings 340
II. Neurobehavioral Tests 342
A. Abnormalities From Indoor Air 342
B. Sources of Chemicals 344
III. Mold-Associated Illness 345
A. Neurobehavioral Impairment with Mold Exposure 345
B. Other Psychological Testing 348
C. Sampling of Homes for Molds 349
D. Predicting the Course 349
IV. Evidence for Cause 350
A. Studies That Link This Disorder to Molds 351
B. Air Analysis for Mycotoxins 351
C. Biomarkers for Mycotoxins 352
D. Mechanism of Mycotoxins 352
E. Probable Pathogenesis 353
V. Conclusion 355
References 355
I. Introduction
Sick (in the) building syndrome burst upon us 30 years ago during
the first Middle East energy crisis. The initial response of the medical
profession, public health officials, and many citizens was disbelief.
People with sick building syndrome were considered to have crowd
phenomena (Hefez, 1985; Landrigan and Miller, 1983) and even hyste-
ria (Small and Borus, 1983), thus resembling earlier psychic problems
associated with molds (Matossian, 1989). Studies of sickened occu-
pants of office buildings in the United Kingdom, Denmark, and
Sweden coupled with analyses of air samples showing VOCs (volatile
organic compunds) made the problem credible, and acceptance fol-
lowed (Burge et al., 1987; Hollowell and Miksch, 1981; Molhuve
et al., 1986; Wallace, 1991). Serious efforts to relate it to indoor air
339
0065-2164/04 $35.00
340 KAYE H. KILBURN
turnovers per hour followed and belatedly, after months or years of

symptoms, air turnovers were increased in some buildings (Menzies
et al., 1993). The changes did not buy amelioration, and people re-
mained ill with headaches; irritated eyes, noses and throats; extreme
fatigue; muscle pains; and impaired recall, memory, and concentration
(Burge et al., 1987; Kilburn, 2000; Molhane et al., 1986; Wallace, 1991).
A. PERSPECTIVE
The nuclear submarine ushered in a new era of indoor air concerns
in 1958 (Ebersole, 1960). In 1944, air fouled by accumulation of carbon
dioxide to above 2.5% and humidity of 100% made staying submerged
in submarines difficult despite spreading CO2 absorber and releasing
bottled oxygen. Nuclear vessels had abundant power for air condition-
ing, filtration of particles, and absorption of CO2 and stayed below
water for 60 days. Subsequently, problems shifted to Freon, the refrig-
eration gas that produced phosgene at ignition points such as burning
cigarette tips and out-gassing of solvents from deck wax and fresh paint
added to respirable particles from cigarette smoke.
Sickness and plagues in homes were described in the Bible, with
the advice being to pile belongings in the middle and set it aflame.
Dr. Theron Randolph (1945 and 1947) described a syndrome in
women and children living in Chicago in the 1940s through 1950s of
asthma, fatigue, muscle pains, intolerance to many chemicals, and
multiple chemical sensitivity (Ashford and Miller, 1998). He coun-
seled avoidance of petroleum products to which these patients were
1000 times more sensitive than usual, akin to atopy (Randolph and
Moss, 1980).
B. TIGHT BUILDINGS
Reports of people sharing tight office buildings and children in
school during Swedish winters (Dales et al., 1991; Savilathi et al.,
2000) focused on the lung with asthma and the brain with fatigue,
confusion, depression, and loss of memory and concentration. A noto-
rious example was the U.S. Environmental Protection Agency build-
ing, Waterside Mall, on the north bank of the Potomac River in
Washington, D.C. in 1987–88 (Radetsky, 1997). Hundreds of the several
thousand employees became intolerant of being in that building and
consequently had to be assigned elsewhere.
ROLE OF MOLDS AND MYCOTOXINS 341
1. Sealing Leaks
Managers responded to the 1970s energy crisis by sealing buildings
to conserve heat and cold. Windows were closed and locked and leaks
were caulked. When air exchanges were reduced the concentrations of
chemicals in indoor air increased (Molhave, 1986). Sometimes even
carbon dioxide and humidity exceeded safe levels (Gammage and
Kaye, 1985). People got sick. Initially they felt better away, but pro-
blems recurred on re-entry. Later symptoms did not remit even when
away and were triggered by perfume, cigarette smoke, diesel exhaust,
formaldehyde, and other chemicals, as well as previously inoffensive
buildings.
Complaints from people in new factory-built, manufactured homes
(Kilburn, 2000) and from those who insulated older homes by injecting
spaces between the studs with urea formaldehyde foam insulation
(UFFI) emphasized burning eyes, noses, and throats; shortness of
breath; headache; extreme fatigue and losses of recall memory; ability
to concentrate; dizziness and loss of balance; and reduced long-term
memory (Dally et al., 1981). People who worked at home on computers
and young families in first homes shared the irritation just described,
but associated problems included seizures, menstrual irregularities,
asthma, depression, falling behind in school, and losing newly
acquired capabilities (Kilburn, 2000).
2. Formaldehyde
Testing the indoor atmospheres showed formaldehyde at concentra-
tions from 0.2 to 5 ppm (Dally et al., 1981; Kilburn et al., 1985). Current
estimates of safe levels vary but have dropped to 80 parts per billion
from 1–2 parts per million in Japan (Osawa, 2003). Its sources were
carpets, drapes, particle board and pressed board, and a broad array of
forest products consisting of wood particles including sawdust held in
phenolformaldehyde resin (Ikeda and Park, 2003). This resin is cured
by adding excess formaldehyde and outgases for years (Dally et al.,
1981).
Because people complained of respiratory and central nervous sys-
tem dysfunction, testing was directed at pulmonary function and per-
formance of the brain. Both subconscious and conscious functions
were considered. The physiological tests encompassed balance, choice
reaction time, blink reflex, color discrimination, and measuring the
visual fields, hearing, grip strength, and vibration threshold (Kilburn,
2000). These are measurable functions of the brain that are important
for daily life that are rarely measured in traditional testing.
342 KAYE H. KILBURN
Spirometry—recording the delivery of a maximal breath against

time—measures volume (forced vital capacity) and flow (forced expira-
tory volume in 1 second) and flow averaged for 25% to 75% of vital
capacity. Comparison to population-based prediction equations ad-
justs for age, sex, height, and cigarette-smoking (in years) for precise
estimates. Additional testing is rarely needed as the indoor chemical
exposures decrease the flow of expired air. In contrast, to measure
brain performance takes 8 to 10 physiological and 13 psychological
function tests.
II. Neurobehavioral Tests

The neurobehavioral tests I have used are those that have contributed
most to the evaluation of over 600 individuals exposed to many differ-
ent chemicals and 25 groups of people and summarized in Chemical
Brain Injury (Kilburn, 1998). These people shared exposures to chlorine,
ammonia, hydrogen sulfide, arsenic, diesel exhaust, organophosphate,
insecticides, polychlorinated biphenyls, indoor air, formaldehyde, and
moldy buildings. Groups of people unexposed to chemicals were
matched for comparisons (Kilburn and Warshaw, 1995a; Kilburn et al.,
1998). The precision of comparisons was improved by developing eq-
uations to predict a value for each measurement by using regression
equations (Person’s test) that were based on age, sex, educational at-
tainment, height, and weight and thus adjusted for their influence. This
produced ‘‘before exposure values’’ unattainable otherwise. Next,
based on each test/measurement variability, the 95% confidence inter-
vals were defined as 1.5 standard deviations of the mean. Values that
exceeded or dropped below these levels were classified as abnormal.
To count their total abnormalities, balance was given weight of 2 and
visual field performance, 1 for each eye. Other functions had a weight
of 1, except 0.5 per side for bilateral ones. The sum of abnormalities for
subjects was averaged for a group mean, so one number was compared
for groups.
A. ABNORMALITIES FROM INDOOR AIR

The 20 persons exposed to indoor air contaminated with formalde-
hyde solvents and mold in mobile homes included adults of five
families (Kilburn, 2000). The mean values of 11 functions were signifi-
cantly different from unexposed controls (Table I). Their abnormalities
nearly equaled those of 22 workers exposed to formaldehyde in anato-
my and pathology laboratories and those fabricating fiberglass aircraft
TABLE I
COMPARISON OF NEUROBEHAVIORAL ABNORMALITIES IN 20 INDOOR AIR–EXPOSED
AND 22 FORMALDEHYDE-EXPOSED SUBJECTS
Indoor air Formaldehyde
Simple reaction time þ þ

Choice reaction time þ þ
Balance
Eyes open þ þ
Eyes closed þ þ
Blink reflex R/L 0/0 0/0
Grip R/L 0/0 þ/þ
Visual field performance R/L þ/þ þ/þ
Color discrimination R/L þ/þ þ/0
Hearing loss R/L þ/0 þ/0
Culture fair 0 þ
Digit symbol score þ þ
Vocabulary score 0 0
Peg placement 0 þ
Trail making A 0 þ
Trail making B 0 þ
Verbal recall
Immediate þ þ
Delayed þ þ
Information 0 0
Picture completion 0 þ
Similarities 0 0
þ ¼ abnormal, 0 ¼ not significantly different from unexposed referents.
cabin sections by using phenol formaldehyde. Choice reaction time,

balance (especially with eyes closed), visual fields, digit symbol, recall,
and finger tip number writing errors were abnormal. It was rarely
possible to characterize these people’s exposures to formaldehyde
and volatile organic chemicals by air measurement. Estimated doses
were below industrial exposures and for home makers nearly continu-
ous without time to deplete body stores. These impairments implied
intolerance. Frequencies of 24 of 35 symptoms, after patients rated each
complaint on a scale from 1 to 11 (10 point), averaged double those of
people unexposed to chemicals. Only 11 symptoms were not increased.
344 KAYE H. KILBURN
Feeling of anxiety, depression, anger, fatigue, and confusion were high

and vigor low when using the Profile of Mood States (POMS 1971/1981).
Their POMS scores were 4 times those of unexposed groups. Thus,
objective measurements were abnormal for balance, reaction time, and
vision performance joined by cognitive, recall, and memory functions
by elevated symptom frequencies and high adverse mood scores.
B. SOURCES OF CHEMICALS
The two main sources of chemical buildup in indoor air are (1) the
building’s construction and decorating materials including adhesives,
paints, and other applied materials; vinyl floor tile; carpets and drapes;
and (2) activities in the building such as cleaning with solvents, apply-
ing waxes, cigarette smoking, air fresheners, photocopying, photo-
graphic processing, food services, refrigeration (with Freon), and
personal adornment and odorants (perfume). A most serious toxic
exposure is from pesticides sprayed indoors in the erroneous belief
that insects can be eradicated. Sprayed (aerosolized) insecticides are
toxic to the human brain (Kilburn, 1997, 1999; Kilburn and Warshaw,
1995b). Monthly spraying builds up concentrations that outgas from
surfaces and fabrics along with decorating and construction chemicals.
1. Cleaning Agents
Cleaning agents contribute to indoor air burdens with chlorine (for the
smell of clean), phenols delivered as aerosols to shower stalls and toilets,
organic solvents to remove floor wax, ammonia sprayed to clean glass,
and degreasers that are chlorinated solvents like trichloroethylene.
Grill cooking releases acrolein, a potent aldehyde.
2. Gases
Glutaraldehyde is released in photographic processes and from cold
sterilization of instruments, catheters, and hoses. Also, cigarette smoke
contains formaldehyde and 2500 other chemicals that are not detox-
ified despite their passage in and out of smokers’ lungs. Rotten egg
gas (H2S) returns from sewers thru waste pipes when J traps dry out
or pressure builds from downstream obstruction to break water seals
in J traps. It is a frequent indoor exposure, especially when some
bathrooms or wash bowls are idle for long periods of time.
Until 1989, this was the inventory of problems from indoor air in sick
buildings (Kreiss, 1990). Awareness translated into adding air ex-
changes, vigilance to investigate odors, and care to avoid occupancy
and exposure during repair and renovation.
III. Mold-Associated Illness
Understanding, avoidance, and more air exchanges seemed to de-
crease and control problems until recently, when as many as 1.5 mil-
lion people in 500,000 homes had illnesses associated with molds
growing indoors (Sharpe, 2002). The biblical plague described by
Moses, found in Leviticus, returned with a vengeance around 1990!
People described flu-like illnesses with profound fatigue; headache;
eye, nose, and throat irritation; and sometimes bleeding from the nose,
or blood in the sputum, the urine, or the stool. These affected people
had memory loss, difficulty concentrating, dizziness and loss of bal-
ance, and feeling lost in familiar places. Shutting their eyes made
balance precarious, whether in the shower washing their hair or
moving about at night. Ability waned to recall faces, conversations,
vital data, and phone numbers (Baldo et al., 2002; Johanning et al.,
1996; Johanning and Landsbergis, 2001; Kilburn, 2000).
Asthma in children was an adverse effect of dampness and mold
growth in homes reported from Canada (Dales, 1991). They were sub-
sequently shown to have reduced blood lymphocyte counts (Dales,
1998). However, a hemorrhagic pneumonitis of 10 infants associated
with inhalation of spores of Stachybotrys atra that were found in the
lung in Cleveland, Ohio, in 1993–1994 underscored the emergence of a
new mold disorder (Etzel, 2002). Another manifestation was sick court
rooms with molds growing in Florida (Cary, 1993) and California
(Ginis, 2001). School children in Scandinavia were more likely to have
breathing difficulties in rooms excessively humid during winter
months, and many mold genera Aspergillus, Penicillium, and others
were identified (Sigsgaard et al., 2001).
A young man exposed to moldy silage developed a flu-like illness
followed by dementia with tremors. Silage grew Aspergillus flavus and
many other species of mold (Gordon, 1993). This report and others
(Auger et al., 1994) opened the door on the effects of molds in the
human brain.
A. NEUROBEHAVIORAL IMPAIRMENT WITH MOLD EXPOSURE

In January 2001, I investigated and tested 10 people in public hous-
ing in New York City who associated mold with their symptoms and ill
health. They showed slowed choice reaction time; excessive sway
speed (abnormal balance); and deficits of cognition, recall, and memory
(Table II). Black mold was growing in the moist bathrooms and bed-
rooms. By morphology and cultures, these were Stachybotyrus chartarum
346 KAYE H. KILBURN
TABLE II
NEUROBEHAVIORIAL ABNORMALITIES IN 10 NEW YORK AND 10 CALIFORNIA
EXPOSED PEOPLE
New York California

Balance
Eyes opened þ þ
Eyes closed þ þ
Blink reflex R/L 0/0 0/0
Grip strength R/L 0/0 0/0
Color discrimination R/L þ/þ þ/0
Visual fields R/L 0 0
Hearing R/L nd þ/0
Cognition
Culture fair Sc þ 0
Digit symbol Sc 0 þ
Vocabulary Sc 0 0
Verbal recall
Immediate 0 0
Delayed 0 0
Perceptual motor
Peg placement 0 þ
Trail making A 0 0
Trail making B þ þ
Finger writing errors R/L 0/0 0/0
Information 0 0
Picture completion 0 0
Similarities 0 0
Average total 9 10.5
þ ¼ abnormal, 0 ¼ not significantly different from unexposed referents,

nd ¼ not done.
(Croft et al., 2002; Kilburn, 2002). There was no air sampling to quantify
doses.
Returning to Los Angeles, I measured 10 people exposed in their
homes in Los Angeles, Phoenix, and Dallas. Within 18 months, I
evaluated 65 adults and 17 children. They painted a clear pattern of
TABLE III
ABNORMALITIES OF NEUROBEHAVIORAL FUNCTION IN 65 MOLD-EXPOSED AND 360
CHEMICALLY EXPOSED ADULTS FROM SOUTHWESTERN STATES
65 Mold 360 Chemical

Balance
Eyes opened þ þ
Eyes closed þ þ
Blink reflex R/L þ/þ þ/þ
Grip strength R/L þ/þ þ/þ
Color discrimination R/L þ/þ þ/þ
Visual fields R/L þ/þ þ/þ
Hearing R/L 0/0 0/0
Cognition
Culture fair Sc 0 þ
Digit symbol Sc þ þ
Vocabulary Sc þ þ
Verbal recall
Immediate þ þ
Delayed þ þ
Perceptual motor
Peg placement þ þ
Trail making A þ þ
Trail making B þ þ
Finger writing errors R/L 0/0 0/0
Information þ þ
Picture completion þ þ
Similarities 0 0
Average total 9.9 10.2
þ ¼ abnormal, 0 ¼ not significantly different from unexposed referents.
severe neurobehavioral impairment as compared with referent people

(Kilburn, 1998) (Table III). After individual data were adjusted for the
influence of sex, age, educational level, and other factors (Kilburn et al.,
1998), their tests showed slow simple and two-choice visual reaction
times, increased speed of body sway with eyes open that worsened
when eyes were closed, slowing of blink reflex latency, diminished
348 KAYE H. KILBURN
grip strength, increased color discrimination errors, and diminished

visual field performance frequently with constricted fields. Of these
tests, only losses of hearing were not statistically significant.
Cognitive functions for digit symbol substitution and vocabulary
were significantly reduced and Culture Fair—which evaluates problem
solving with 12 logical series, 14 maximal differences, 12 design com-
pletion, and 8 deriving and applying placements (Cattell, 1941; Cattell
et al., 1951)—was nearly abnormal. Verbal recall (Wechsler, 1945–81)
was diminished, as were picture completion (spotting missing items in
pictures) and well-known cultural information from the Wechsler
Adult Intelligence Recall-revised (WAIS-R) (Wechsler, 1981). Also,
scores on peg placement and trail making A (connecting 25 ascending
numbers) and B (alternating numbers and letters) were significantly
below the comparison group. Only finger tip number writing errors and
similarities were not significantly diminished. Children had similar
impairments that are not detailed here. Next, let us compare these
findings with prior observations that focused on cognitive and memory
functions but unfortunately did not measure balance, vision, and
choice reaction time.
B. OTHER PSYCHOLOGICAL TESTING

Psychological testing compared 10 people exposed to molds in
homes and workplaces and 10 people with apparently mild traumatic
brain injury with psychomotor, verbal learning, spatial learning, and
visuospatial memory tests and the Wechsler WAIS-III (a recent revision
of WAIS-R) (Baldo, 2002). When results of these relatively insensitive
tests were compared with normative values, no differences were found
for either group. This outcome is not surprising because of the wide
and large standard deviation of these data ranges. Although mold-
exposed patients had lower scores on the Stroop color word test and
Rey Complex Figure Test, none of the WAIS subtest scores were differ-
ent. Patients were depressed on standard scales. In a New York series,
20 patients from moldy homes had excessive physical and cognitive
symptoms and showed verbal memory impairment, trouble paying
attention, and difficulty shifting sets and integrating visual information
(Gordon et al., 2001).
Studies before ours were limited by comparing exposed subjects to
normative value ranges that are not tailored by calculating expected
scores for each subject on each measurement. Such predicted values
increase sensitivity and thus discriminatory power. A second limita-
tion is poorly selected comparisons as, for example, mold-exposed
patients to those with ‘‘mild’’ traumatic brain damage. Third, physio-
logical tests for balance, visual field performance, color discrimination,
simple and choice reaction time, blink reflex, grip strength, and hearing
profile measure realms of the brain beyond memory and cognition with
tests having less variance producing greater sensitivity (Kilburn and
Warshaw, 1995a,b; Kilburn et al., 1998, 2002). Future studies should be
designed for maximal discrimination by using such sensitive tests and
appropriate comparisons; adjusting measurements for sex, age, educa-
tional level, and other demographic variables; and expressing observed
values divided by the predicted values meaning percent predicted and
comparing these means to those for unexposed subjects by analysis of
variance to determine significance.
C. SAMPLING OF HOMES FOR MOLDS

Aspergillus species were the most frequent molds obtained from the
65 patients’ homes by applying transparent tape to surfaces, by air sam-
pling, and by growth in culture. Penicillium, Phoma, and Stachybotrys
were the most common genera.
As a biomarker, serum samples for these adults were measured for
mold antibodies (IgG, IgM, IgA, and IgE) by modified enzyme-linked
immunosorbent assays (ELISA) and for antibodies to trichothocenes, a
type of mycotoxin produced by Stachybotrys; satratoxin, a particular
trichothecene mycotoxin produced by Stachybotrys chartarum; and
aflatoxin, a mycotoxin produced by Aspergillus flavus. Fifteen medical
workers for Los Angeles and 46 community people from Wickenburg,
Arizona, without mold illness or moldy workplaces or homes were
simultaneously tested. Almost all mold-exposed and control subjects
had antibodies to all of the molds, reflecting exposure. The proportions
of the three groups with titers more than 2 standard deviations above
the means were not significantly different. Mycotoxin antibodies were
more frequent in controls than in those exposed to aflatoxin, 17%
versus 9%; trichothocenes 17% versus 13%; satratoxin 23% versus
41% (manuscript in preparation). Saliva IgA secretory antibodies did
not distinguish between the groups (Kilburn, 2003).
D. PREDICTING THE COURSE

Information about mycotoxin-induced diseases of the brain in other
animals (Hintikka, 1977) cannot predict the course of these patients or
the natural history of encephalopathy from mold, as few measurements
have been made. Thus, my findings in 8 of the 65 patients who were
350 KAYE H. KILBURN
seen again 10 to 21 months after initial evaluations are important

beacons in the darkness. All had moved to homes that were ‘‘presumed
free’’ of molds. None had improved and 7 patients had deteriorated;
indeed, often their abnormalities had doubled. One patient, a 29-year-
old woman with the most abnormalities in the group, can no longer live
independently. Therapy has been empirical, including antifungal che-
motherapy presuming fungal colonization and cholesterol binding
agents such as colestryamine that may complex trichothocenes in the
gut and prevent their reabsorption. However, there are no data to
suggest functional improvement after taking these agents or from other
treatments.
IV. Evidence for Cause

Let us consider the evidence for a causal connection of this syn-
drome to mycotoxins. The Cleveland episode of 1993–1994 showed
Stachybotrys atra in 8 of 9 homes of 10 infants with pulmonary hemor-
rhage versus 12 of 28 homes of controls (Etzel et al., 1998). Air and
surface samples for viable fungi and airborne conidia showed this
strong association. In New York City in 1995, 39 female and 14 male
office workers had sick building syndrome associated with mold
growth. Air sampling found scant Stachybotrys atra from the ground
floor, moderate amounts in the basement, but extensive contamination
of the sub-basement (Johanning et al., 1996). Growth was greatest on
moldy gypsum board and books. Satratoxin H, as well as other myco-
toxins (phenylspirodrames), was isolated by high-pressure liquid chro-
matography (HPLC) (Johanning et al., 2002). In the Phipps Apartments,
homes of the 10 New York City people I studied in 2000, molds
including Stachybotrys were identified in all (Croft, 2002). When mold
afflictions extended to the southwestern states, many mold species
were identified but without a predominance of Stachybotrys atra (per-
sonal observation). Others have found that Stachybotrys atra/chartar-
um is often well hidden in dwellings, which may explain why it is not
thought to be dominant (David Straus, personal observation).
The data from moist and moldy eastern sites have been reproduced
in the southwest. Mold species are difficult to distinguish unless air-
borne samples are obtained with Anderson separators. Despite some-
what inconsistent isolation of Stachybotrys atra, its causal role is
likely. Observation of mold growth on surfaces in homes, microscopic
identification, and culture demonstration of conidia in filters of air
(aerosols) show the genus and species of molds but do not quantify
exposure.
A. STUDIES THAT LINK THIS DISORDER TO MOLDS
Indoor dusts were vacuum collected on filters from three sites of
suspected sick building syndrome in Montreal in 1993. Dust samples
of 25 to 50 grams were extracted, partitioned, and prepared for thin-layer
chromatography (TLC) and HPLC. Two sites yielded T-2 toxin, diace-
toxyscirpenol, roridine A, and T-2 tetraol; the other site produced only
roridine A. Quantities were not estimated (Smoragiewiez et al., 1993).
Samples of building material from a water-damaged building in Fin-
land were examined by light microscopy, and viable cells were
counted and cultured. Water-extracted building materials were tested
for Limulus activity (endotoxin) and cell toxicity. Boar spermatozoa,
rabbit skin, and fetal feline lung cells showed 200 times the toxicity
from this extract as from non-moldy gypsum board. Satratoxin G & H
were assayed by HPLC. Fungi were identified as Stachybotrys atra
and showed -D-glucan-specific Limulus activity. Samples with bacte-
ria showed endotoxin-specific Limulus activity. The water-damaged
gypsum liner contained satratoxin measured by retention times by
HPLC from methanolic extracts compared with reference G and H
macrocyclic trichothecenes (Andersson, 1997).
Air samples from mold-contaminated houses in Cleveland yielded
spores that inhibited protein synthesis in an assay using translation of
firefly luciferase in rabbit reticulocytes. Compared with the standard
MTT 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, this
assay improved sensitivity by 400 fold (Yike, 1999).
A strain of Stachybotrys atra was isolated from the lung of a child
with pulmonary hemorrhage. This organism was shown to produce a
new serum chymotrypsin-like proteinase. It was demonstrated to cleave
major protease inhibitors, collagen, and biologically active peptides
found in the lung (Kordula, 2002).
Fungal fragments along with spores from three species—Aspergillus
versicolor, Penicillium melinii, and Cladosporusm cladosporiocides—
were released during fungal aerosolization from cultures on agar and
from ceiling tile surfaces. Fragments were 320 as numerous as spores
and shared antigens that showed their common origin, and it was sug-
gested by the authors that they could carry mycotoxins (Gorny, 2002).
B. AIR ANALYSIS FOR MYCOTOXINS

This evidence recommends a quantitative assay of air from homes
of symptomatic people for mycotoxins focused on measuring the tri-
chothecenes and satratoxins, specifically. The growing list of mycotoxins
352 KAYE H. KILBURN
associated with recent indoor air syndromes includes gliotoxins from

Aspergillus fumigatus (Nieminen et al., 2002) and others (Jarvis et al.,
1995; Nielsen et al., 1998; Ueno, 1977).
Inhalation exposure of 25 patients to indoor mycotoxins was assayed
by using high-volume air samplers for 24 hours onto micropore-paper
filters. The cytoxicity of mycotoxins was assayed with the MTT cell
culture bioassay coupled with identification of mold cultures. Then an
enzyme immune assay measured macrocyclic trichothecenes, and
high-pressure liquid chromatography diode array detection (HPLC-
DAD) and gas chromatograph mass spectrometer (GC-MS) analyzed
for different mycotoxins. Seven samples were highly cytotoxic, 14
moderately so, and only 4 lacked toxicity. Thus mycotoxins were
airborne along with fungal particles (Johanning et al., 2002).
Whether these mycotoxins are particle bound or free as aerosols may
be a moot point, because alveoli of human lungs receive submicronic
particles up to 5 or even 7 m mean aerodynamic diameter; 7 m is the
size of Aspergillus niger spores and the largest spore to deposit in
pulmonary alveoli. Spore size questions could be answered by analyz-
ing air sampled from above cultures growing in the laboratory. The
manner of dispersal in air may be important in devising methods to
ameliorate mold problems indoors and within walls.
C. BIOMARKERS FOR MYCOTOXINS

Clearly, recovery of mycotoxins in indoor air samples sets the stage
for chemists to seek them in human serum, blood cells, and tissue cells.
The job is far from trivial. At least 18 mycotoxins are made by Stachy-
botrys (Ammann, 2001; Bata et al., 1985; WHO, 1990). Pursuit of such
identification and sampling, first in air as shown by Johanning et al.
(2002) and Jarvis et al. (1995) and then in body fluids, would close the
ring of causation. This would help answer questions of pathogenesis
and impairment of lung, brain, and other organs of humans and do-
mestic animals (Schiefer, 1990) and hopefully propose antidotes to be
explored in evidence-based treatment trials.
D. MECHANISM OF MYCOTOXINS
The next concern is the mode of action of mycotoxins. They are
epoxides that would be postulated to bind to microtubules, mito-
chondria, and synaptic membranes (Muller et al., 1975). Poisoning of
the liver by aflatoxins is a major factor in Kwashiorkor, causing wasting
in infants (Hendrickse, 1984). The mechanism of action of these
mycotoxins is a large topic and deserves a separate review.
Since I have examined the general case of mold disease, I would like
to postulate a world view, to address how our planet is mold infested,
how molds spread, and some of their effects on other living systems.
E. PROBABLE PATHOGENESIS
The observation that indoor moisture encourages mold growth must
factor into an explanation of the mold toxicity syndrome. The change
in construction materials and assembly of office buildings and homes
since 1950 enhances opportunities for mold growth and will be de-
scribed. The second observation is that of the transoceanic transfer of
dust between continents that can seed new species in ecologic niches
such as moist inner walls and surfaces of buildings.
1. Moisture and Molds
The emerging problem in tight buildings is mold growth on walls
and ceilings. Walls and attics that do not ‘‘breathe’’ retain moisture.
High humidity encourages growth of molds on the paper enclosing
sheet rock (gypsum board), in insulation batts, and in insulation back-
ing paper. Until recently this problem was considered to be rare,
caused by humidifiers or recirculated water (Burge et al., 1985), and
not an indoor air problem. But recently many molds have been
found growing indoors and making toxins such as trichothecenes,
fumonisins, and aflatoxins.
2. Fungal Toxins
Mycotoxins include chemicals from mushrooms that are more deadly
(Goetz, 1985) than are solvents and formaldehyde found in building
materials and carpets. Molds grow on moist walls, ceilings, and floors,
making musty odors and discoloring surfaces. Molds growing in
homes, particularly on inner walls (Andersson et al., 1997; Etzel et al.,
1998; Kilburn, 2002), release toxic chemical products that can poison
the occupants. I suspect that the switch to gypsum sandwiched be-
tween layers of paper that is often recycled encouraged molds. They
grow on the moist cellulose, so paper is the medium. Tighter construc-
tion meant less venting and air circulation in walls and ceilings, so the
high humidity condensed water on paper, an ideal condition for grow-
ing molds. Contrast these methods with the earlier lath and plaster era,
in which lath walls were plastered with alkaline sand, cement, and
lime that inhibited mold growth.
The familiar green mold on bread and oranges is likely to be a mem-
ber of the genus Penicillium. A Penicillium mycotoxin is penicillin, the
354 KAYE H. KILBURN
first antibiotic (against bacteria). Many antibiotics are mold products

that kill bacteria and thus rescue many patients from infection. The
downside is that some mold chemicals, mycotoxins, have the potential
to harm people. Additionally, molds produce spores that can persist for
many centuries.
3. Dust: A Mold Source and Evidence of One Small World
Dust is not novel, but daily global contamination from foreign mold
spores is new. A storm over the Gobi desert quickly spreads dust over
China, Korea, and Japan and reaches western Canada and the United
States a week later. This observation came from an international effort
to characterize fine dust particles by using multiple land-, sea-, and
space-based instruments. Heavier particles dropped into the sea, but
sulfuric acid, mercury, and arsenic from Chinese factories and smelters
reached North America, and dust from Manchuria produced the high-
est arsenic levels ever seen in Nevada air (Dolan, 1986; Griffin, 2002).
These pollutants were examined for their potential in contributing to a
cluster of childhood leukemias seen in the United States.
Likewise, the trade winds that sweep across the mid Atlantic along
the route of Columbus pick up dust including mold spores from the
Sahara and eastern deserts that remain airborne until delivered days
later to the Caribbean Sea, Gulf states, and westward. These dust
clouds can be tracked from satellites. They contain 100 the amount
of particles found in polar air (Griffin et al., 2001a,b, 2002).
In 2002, Douglas Seba suggested that global atmospheric dust load-
ing was this millennium’s major pollutant (personal communication
with oceanographer Douglas Seba). It was tied to global warming, to
dust loads from Africa reaching Florida and the Gulf coast states,
particularly Texas, to establish a new record of dust deposition in 2000.
These observations may be tied to possible consequences of airborne
seeding of mold spores. For example, in May 1993 a new building, the
Courthouse of Martin County, Florida, north of Palm Beach, grew mold
in walls that was associated with headaches, skin rashes, nausea, and
vomiting, and some people were hospitalized (Clary, 1993). That same
year, infants had lung hemorrhages in Ohio (Etzel et al., 1998). The
new court house had a $3.5 million renovation. By 2001, across the
country, the Tulare Court House and nearby Corcoran prison caused
mold illnesses in California (they closed the court house but not the
prison) (Ginis, 2001). Other public buildings grew mold from Polk
County, Florida to Modesto, California. Thinking of dust and disease
in this light, consider the startling increase in newly diagnosed asthma,
the best known disease from inhaled particles, in the last decade.
4. Death of Coral Reefs, Asthma, and Brain Injury
Eugene Shinn, a marine biologist, observed that molds from African
dust clouds landing in Caribbean waters have been thought to cause
disease and death of coral reefs that began in the 1970s (Griffin et al.,
2001a,b, 2002). One mechanism is the death of sea urchins that normal-
ly control algae growth on coral. Another is the death of reef-building
coral caused by a soil fungus, Aspergillus sydowii, and overgrowth by
algae. Coral reefs are embattled habitats worldwide (Hirsch, 2002), even
the world’s largest living organism, Australia’s Great Barrier Reef (Los
Angeles Times, 2003). Human brains appear to share this susceptibility.
V. Conclusion
Heeding these warnings by addressing building methods, combined
with establishing protective standards, will augment personal efforts to
avoid exposure to chemicals. These actions, coupled with a long-term
vision of what’s needed for our collective health, will help create
solutions to these currently insoluble problems. Facing the simple
need for safe housing for all will open the door to solutions for stem-
ming epidemics of chemically induced brain erosion, asthma, and
immune disorders that are probably yet to peak.
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The Diagnosis of Cognitive Impairment Associated with
Exposure to Mold
WAYNE A. GORDON AND JOSHUA B. CANTOR
Department of Rehabilitation Medicine
Mount Sinai School of Medicine, New York, New York 10029
I. Introduction 361
II. What Is Neuropsychological Testing? 361
III. How Is Differential Diagnosis Used in Neuropsychology? 366
IV. Cognitive Impairment and Mycotoxin-Producing Fungi 367
V. Conclusion 373
References 373
I. Introduction
The purpose of this chapter is to provide an introduction to existing
knowledge about cognitive impairments related to exposure to
mycotoxin-producing fungi. The bulk of this information is based on
findings from neuropsychological evaluations. In preparing this manu-
script, it was assumed that our readers would know little about the
basic issues involved in neuropsychological assessment. Consequent-
ly, a general overview of the goals and methods of neuropsychological
testing will be provided first. Since differential diagnosis is an essential
component in neuropsychological assessment, especially as it applies
to persons exposed to mycotoxin-producing fungi, a discussion of the
process of differential diagnosis in neuropsychological evaluation will
follow. Finally, a review of published findings in this area will be
presented, followed by a description of our experiences in the neuro-
psychological evaluation of individuals who have been exposed to
mycotoxin-producing fungi in either their home or at work and a
review of our ongoing research in this area.
II. What Is Neuropsychological Testing?

Lezak (1995) defines neuropsychology as ‘‘. . . the area of psychology/
science that focuses on the behavioral expression of brain damage.’’
Clinical neuropsychology utilizes psychometric techniques to eluci-
date brain-behavior relationships in a systematic way. The American
Academy of Neurology (1996) has described neuropsychological test-
ing as ‘‘. . . one means of garnering quantitative information about be-
havioral changes in patients with known neurological disease or who
361
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362 GORDON AND CANTOR
are considered to be at risk for brain dysfunction.’’ The fact that neuro-
psychological tests are generally reliable, valid, and objective adds
to the soundness of the findings that are derived from their use (Amer-
ican Academy of Neurology, 1996; Lezak, 1995). It is accepted that
neuropsychological testing is useful when used for diagnostic pur-
poses, patient care, rehabilitation planning, and research (American
Academy of Neurology, 1996). The present chapter will discuss only
the diagnostic use of neuropsychological testing.
When used for diagnostic purposes, neuropsychological testing is
useful in describing the nature and extent of cognitive dysfunction,
differentiating neurological from psychiatric symptoms, differentiating
among neurological conditions, and describing recovery from or the
outcome of a neurological disorder or event (Lezak, 1995). In conduct-
ing a neuropsychological evaluation, the neuropsychologist examines
diverse aspects of cognitive function including processing speed, mo-
tor speed, attention/concentration, verbal abilities, visual perceptual
abilities, intellectual function, memory, executive functions, and
mood. A variety of tests are available to assess each of these areas,
and various testing modalities are used, including standard face-to-face
tasks administered by the examiner, computerized tests, self-report
measures, and interviews. Since the neuropsychologist may draw on
a variety of tests to examine different aspects of these domains of
function, the completion of a thorough neuropsychological evaluation
is a time-consuming process that can take at least 8 to 10 hours. Any or
all aspects of these domains of cognitive function can be affected by
cognitive impairment, since dysfunction can be related to the acquisi-
tion of information, the retention or learning of information, the ma-
nipulation and organization of information, or the communication of
information. The impact of brain damage on cognitive function is often
uneven and, as a result, some skills become impaired and others
remain preserved. Thus discrepancies in the test performance of an
individual are often used as a way of describing cognitive deficits.
There are certain types of neuropathology (e.g., mild traumatic brain
injury, exposure to toxins) in which neuropsychological testing may
be sensitive to dysfunction that cannot necessarily be detected by
other types of diagnostic procedures. Thus, cognitive impairments
may be identified with neuropsychological tests, despite evidence of
‘‘normal’’ intelligence, unremarkable neuroradiological studies, or a
clinical neurological examination in which there are no findings of
pathology. Indeed, conventional neurological diagnostic testing—such
as magnetic resonance imaging (MRI), computerized axial tomography
(CT) scans, electroencephalograms (EEGs), etc.—often fails to detect
COGNITIVE IMPAIRMENT AND EXPOSURE TO MOLD 363
impairments that are exhibited in a neuropsychological evaluation
of an individual following toxic exposure (Hartman, 1995). The validi-
ty and utility of neuropsychological tests in assessing the cognitive
functions of persons with traumatic brain injury is well documented
in the literature (e.g., Barth et al., 1989; Gronwall and Wrightson, 1974;
Hugenholtz et al., 1988; Levin et al., 1987), despite the fact that
many individuals with mild injuries show little or no evidence of
pathology on traditional imaging techniques or clinical neurological
exams (Mateer, 2000).
The ‘‘tools’’ of neuropsychologists are tests that have been standar-
dized on large groups of ‘‘normal’’ individuals. Conclusions of deficit
or impaired function are based on the comparison of the individual’s
performance with normative data or with another standard that can
serve as a guideline for describing current function, such as a discrep-
ancy between current functioning and pre-morbid performance. These
two types of comparison may provide different information and result
in different interpretations. For example, a person’s performance
may be ‘‘average’’ in comparison to the population on whom the
test was standardized but discrepant from the person’s pre-morbid
performance. There are statistical methods for judging the extent of
within-person variability on some measures. In addition, standards
of performance based on societal expectations of cognitive function
are often used. These benchmarks are grounded in expectations that
are based on level of education and notions of the cognitive compe-
tence needed to perform certain tasks/professions. For example, a
person who graduated from college and received an advanced graduate
degree is expected to function cognitively at a higher level than a
person who graduated from high school.
Except in rare instances in which a pre-morbid neuropsychological
evaluation is available, pre-morbid ability or function must be evalu-
ated by using documentation of prior abilities such as school records
and transcripts, performance on standardized tests such as American
university entrance examinations (e.g., Scholastic Aptitude Test [SAT],
Graduate Record Examination [GRE]), employment records, and the
verbal reports of others. In the absence of such documentation of prior
performance, there is no baseline that can be used as a basis with which
to compare current function. Without this baseline information, the
conclusions that can be drawn about a person are largely speculative.
In making a determination that current cognitive impairment is
related to a specific injury, medical event, or exposure to toxins, the
neuropsychologist must also rule out possible alternative explanations
that might account for the impairments. These can include medication
use, substance abuse, learning disabilities, malingering, history of

other brain injury or neurological disease, psychiatric disorders, and
general medical conditions known to cause cognitive impairment (e.g.,
certain endocrine disorders). Some aspects of gathering this infor-
mation are quite routine, such as discussing current medication use
and medication history. The neuropsychologist must be able to rule out
the potential side effects of these medications as factors that may
be influencing test performance.
However, gathering of information that may be more personal re-
quires care and in-depth discussion. For example, in ruling out such
factors as co-existing or pre-morbid neurological disorders, brain injury
or concussion, information about these issues must be systematically
collected. The nature of any pre-morbid learning disability must be
understood as it relates to present cognitive function. For example, if
the person currently reports cognitive difficulties that are different
from those that were associated with prior learning difficulties, then
it is likely that the present difficulties are of a different etiology from
those that existed in the past. Similarly, substance abuse history needs
to be evaluated in terms of frequency, duration, substance(s) used,
and date of most recent use. While the interaction between substance
use and cognitive function is well described in the clinical literature,
declines in performance are usually dose related and in many in-
stances, recovery of function has been described following a period
of abstinence. Declines in day-to-day function would also be expected
as a corollary of altered cognitive function. Thus corroboration of
the extent to which daily function may or may not have been affected
is needed in the absence of a baseline neuropsychological assessment.
Psychiatric history also needs to be explored and the nature and
extent of current mood disorders needs to be examined as part of the
neuropsychological evaluation.
There is little doubt that health difficulties, financial problems, and
other stressors related to exposure to mycotoxin-producing fungi in the
home or place of work can result in some individuals experiencing
significant psychological distress. Some people are quite overwhelmed
by feelings of their world being out of control following their exposure.
Thus, exposure can result in emotional reactivity. However, coexis-
tence of cognitive impairment with this emotional distress does not
indicate a causal relationship between the two (Kay, 1999). Indeed,
many neurological disorders result in emotional disturbances that ap-
pear to be organic in origin rather than responses to stress (Starratt,
1998). The literature on the relationship between neuropsychological
test scores and mood disorders is large and its review is beyond the
scope of this chapter. Some studies have documented an association
between the two (Cassens et al., 1990), while other research has
not (Niederehe, 1986). Some of the controversy in this area is related
to differences in the individuals studied (severe chronically depressed
individuals, episodically depressed individuals, etc.) and to the fact
that subjects were administered different tests in different studies.
A consensus regarding the nature and extent of the relationship
between affective disturbance and cognitive dysfunction has not
emerged from the literature, and there are no guidelines that relate a
specific affective disturbance of a given severity to a specific pattern of
neuropsychological dysfunction.
When possible cognitive impairment is assessed, patient behavior
during testing must also be carefully considered. The neuropsycholo-
gist will look for signs of lack of motivation to perform well, lack of
effort, or malingering that could undermine the validity of the testing.
These areas can also be assessed by using tests formulated for this
purpose. These tests typically present tasks that look difficult but are
actually fairly easy, even for a person with cognitive impairments. The
assumption is that malingerers, assuming that the test is very challeng-
ing, will deliberately perform quite poorly. This remains, however, a
difficult area to assess and one that, at present, is quite controversial.
Part of the difficulty is that no widely accepted criteria are used to
identify malingering or insufficient effort. This difficulty is complicat-
ed by the fact that the tests used to assess these domains are not
generally well standardized on large groups of either ‘‘normal’’ or
neurologically impaired persons. Most tests have been standardized
on small samples of college students who in some instances have been
instructed to behave as if they are malingering. In these instances
the scores of the ‘‘malingering’’ students were compared with those
of the non-malingering students. Differences between these two groups
were then used to determine the pattern of performance associated
with malingering (Hilsabeck et al., 2001).
Thus, in making a judgment concerning the presence of cognitive
impairment, the neuropsychologist uses information from a variety of
sources. A single aberrant score is not used as the basis for the deter-
mination of impairment. Rather, it is the pattern of performance that
forms the basis of the diagnosis. The determination of impaired perfor-
mance is based on statistical inferences regarding the variations and
discrepancies among the person’s scores on a variety of tests. Thus
performance in the ‘‘average’’ range on a series of memory tests for a
person with ‘‘superior’’ intellectual ability would constitute a statisti-
cally significant discrepancy between performance in two areas of
cognitive functioning that might, in turn, suggest the presence of

memory impairment. Isolated discrepancies or discrepancies of smal-
ler magnitude are likely to reflect normal variations in ability and
testing circumstances. This procedure forms an integral part of differ-
ential diagnosis, a technique that is used routinely in clinical neuro-
psychology and that integrates all the elements of neuropsychological
assessment described above.
III. How Is Differential Diagnosis Used in Neuropsychology?

Differential diagnosis is a widely accepted methodology in clinical
practice that is used to form the foundation for making a diagnosis. The
method is not unique to neuropsychology. It is used in many areas of
health care in which a professional gathers diverse information to
render an opinion of what diagnostic entity is the most parsimonious
explanation of what ‘‘ails the person’’ (Lezak, 1995). In adopting this
approach, the neuropsychologist must integrate information from di-
verse sources to establish whether a chain of logical causation exists
between the cognitive impairment observed in the person and the
event to which it is attributed (e.g., injury, illness, or exposure to a
toxic substance). Hartman (1999) has outlined five separate links in the
logical chain as it applies to the evaluation of persons exposed to
toxins. First is an evaluation of the person’s pre-morbid status. This
link in the chain is to establish the person’s level of achievement and
cognitive functioning prior to their exposure. The second link in the
chain involves making a determination that the person was indeed
exposed to toxins. This judgment involves the examination of data
substantiating the person’s claim of having been exposed. The patient’s
symptoms or statement that they were caused by their exposure is not
sufficient evidence of exposure. These statements must be supported
by documentation such as environmental testing and air quality re-
ports. The third link in the chain involves the determination of wheth-
er the person’s self report of cognitive dysfunction is valid. This is
accomplished by neuropsychological testing. The fourth link in the
chain is establishing the temporal relationship between the onset of
the person’s symptoms and their exposure. The key point here is that
the onset of the person’s symptoms must follow their exposure. It is not
necessarily the case that symptoms remit following removal from the
exposure as some symptoms may be permanent. Finally, the person’s
symptom report must be consistent with test findings. Diverse profes-
sionals need to be brought to bear to assist in establishing the validity
of various links in the causal chain.
IV. Cognitive Impairment and Mycotoxin-Producing Fungi
The first person evaluated by the senior author of this chapter to
determine the nature and extent of cognitive deficits secondary to mold
exposure was referred to him in 1993. The patient was well educated
and held an advanced degree from a prestigious university. At the
time of his exposure he was working for a museum. The museum
became flooded with sewage as a result of a break in a pipe. The patient
became exposed to mycotoxin-producing fungi such as Stachybotrys
atra during the clean-up of the museum. Following his exposure, he
reported feeling fatigued most of the time and having multiple upper
respiratory and sinus infections. He also reported a variety of cognitive
symptoms, including difficulty in paying attention and completing
tasks in a timely fashion, and short-term memory loss. He was no
longer able to work, and his fatigue and physical symptoms precluded
social participation. The examiner was intrigued by the correspon-
dence between his self-report and the findings of neuropsychological
testing and sought to learn more about the cognitive effects of mold
exposure by completing a literature search. The search turned up no
clinical or research studies in the area. A colleague in the Department
of Infectious Disease at Mount Sinai School of Medicine confirmed that
there was a dearth of information on this topic.
Since 1993, this situation has changed. A number of publications
have appeared documenting negative health effects secondary to mold
exposure (Augerson, 2000; Etzel, 2002; Sudakin, 1998) as well as
two studies on cognitive dysfunction associated with mold exposure
(Baldo et al., 2002; Gordon et al., 1999).
Given that the body of literature in this area is still relatively small,
some might suggest that the conclusion that exposure to mycotoxin-
producing fungi results in cognitive impairments is premature. In the
first place, no set of cognitive impairments has been identified that
is specific to mold exposure. Second, there is no known dose response
to mycotoxin-producing fungi. Third, there are no epidemiological
studies linking exposure to mycotoxin-producing fungi to cognitive
impairment. Each of these issues is addressed below.
Despite the findings described above, to date, no set of cognitive
impairments has been identified that is specific to mold exposure
or to a specific type of mold exposure. However, since responses to
different pathogens are often similar, identification of a specific set of
mold-related cognitive impairments is not necessary to affirm that
mycotoxin-producing fungi exposure can be related to cognitive
impairments.
As with many other pathogens, there is no known dose response to

exposure to mycotoxin-producing fungi. And as with other pathogens,
this does not indicate that there is no relationship between exposure and
pathology. For example, it is widely accepted that smoking causes lung
cancer and many other health-related problems. Yet the dose response
between smoking and illness remains unknown. Thus we do not know
whether a dose relationship (how many cigarettes did the person smoke
over a given period of time) or a threshold relationship (the person
smoked beyond some accepted threshold) exists linking smoking to
cancer or other health disorders. Similarly we do not know why some
people who smoke become ill and others do not. Thus factors that are
protective of the individual or put an individual ‘‘at risk’’ remain un-
known. While dose-response relationships are not needed to document
the effects of exposure, they can provide important information in
determining what levels of exposure might be deemed safe.
Finally, there are no epidemiological studies linking exposure to my-
cotoxin-producing fungi to cognitive impairment. However, epidemiolo-
gy cannot explain the effects on an individual of exposure to a given
neurotoxin. While it is true that population-based studies of samples of
individuals exposed to mycotoxin-producing fungi have not been under-
taken, it is beyond the scope of this chapter to explore the reasons that
may account for an absence of epidemiological research or to examine the
appropriate use or misuse of epidemiological methods in this regard.
Although at the present time the nature or extent of individual variation
in response to exposure to mycotoxin-producing fungi has not been
documented in large population-based samples, epidemiological studies
are not the only valid standard of evidence needed to make assertions
concerning mold-related cognitive impairment.
To date, more than 100 adults and 10 children who have been
exposed to mold have been seen in our offices. We found that there
was similarity in the self-report of symptoms and cognitive complaints
of many of the patients. Upper respiratory difficulties, pneumonia,
asthma, sinus infections, skin rashes, and gastrointestinal difficulties
were all complaints that were frequently reported. The most commonly
reported cognitive symptom was memory loss. The cognitive com-
plaints of these individuals have not always been confirmed. In some
instances individuals were found to be unimpaired or to have factors in
their medical or psychological histories that prevented associating
their cognitive complaints to their exposure. The rate of referral has
steadily increased since 1993. All individuals were given the same
basic battery that is administered to all individuals who are referred
to the Neuropsychology service in the Department of Rehabilitation
Medicine. This battery is sometimes modified to clarify diagnostic
questions or to suit the specific characteristics of the patient being
evaluated. Over the 10 years since 1993, old versions of tests have been
replaced by newer editions, which in turn have better normative data.
Some tests have been added to the basic battery over time.
In the course of our research on cognitive problems related to myco-
toxin-producing fungi exposure, we have sought to (1) document the
extent and nature of the cognitive impairment that we have seen,
(2) identify patterns of impairment, and (3) compare dysfunction
in this group to that in other diagnostic groups. Drawing on the
data described above, we have conducted two studies to date, examin-
ing cognitive impairment in persons exposed to mycotoxin-producing
fungi. The first study (Gordon et al., 1999) simply described patterns of
impairment in the first 20 patients tested between 1993 and 1998. The
second (Gordon et al., in press) compared symptom reports in a sample
of 31 mold-exposed patients to symptom reports in samples of persons
with traumatic brain injury (TBI) and with no disability and examined
which types of cognitive impairment were most prevalent in the group
using neuropsychological tests.
In the first study that we conducted (Gordon et al., 1999), the data
derived from the testing of the first 20 individuals seen that supported
the clinical impression of memory difficulties were examined. It was
decided to present the findings as a composite or ‘‘snapshot’’ of 20 cases
and not to analyze every test score (because this approach could be
characterized as data mining or as a ‘‘fishing expedition’’) but instead
to focus on examining test data that would correspond to common
complaints of the individuals tested. It was reasoned that there should
be some correspondence between self-report and test findings. Exami-
nation of the test data revealed that the level of performance on tests of
intellectual function of the individuals in the sample was in the average
range. Despite this, the distribution of scores on several other tests was
negatively skewed to the low end of the normal curve. This finding was
most apparent when performance on the Weschler Memory Scale–
Revised Edition (Wechsler, 1987) and the California Verbal Learning
Test (Delis et al., 1987) was examined. Thus these findings supported
the overall self-report of memory difficulties. In addition, difficulties
with attention/concentration and set shifting were observed.
The second study that we undertook (Gordon et al., in press)
involved an examination of the data derived from the next group of
patients seen. These patients were exposed for varying lengths of time
to mycotoxin-producing fungi such as Stachybotrys atra, Penicillium,
and Aspergillus. Some may have been exposed to additional types of
mycotoxin-producing fungi. Data collected from 31 of 38 patients were

examined. It was decided to exclude the data collected from 7 of the
individuals because of the presence of factors in their medical histories
that would compete with mold as being the most likely source of their
cognitive difficulties (e.g., psychiatric problems). In addition to exam-
ining neuropsychological test data, we compared the symptom reports
of these mold-exposed individuals to those of a sample of 47 ‘‘controls’’
(individuals with no disability) and a group of 204 individuals with
TBI. All participants with TBI met the criteria of the American Con-
gress of Rehabilitation Medicine for TBI (Kay et al., 1993). Thus all had
experienced a blow to the head or violent movement of the head
resulting in a period of loss of consciousness, loss of memory for events
immediately before or after the accident, alteration of mental state at
the time of accident (e.g., feeling dazed, disoriented, or confused), or
focal neurological deficit(s). Thirty-two percent had mild TBI (less than
20 min of altered mental status), 13% had moderate TBI (20 min to
24 hr of altered mental status), and 55% had severe TBI (24 hr or more
of altered mental status). Mean length of time since injury was 3.55 yr
(SD ¼ 5.84). The individuals in the comparison groups were selected
from a database of participants from other studies on quality of life after
brain injury conducted by the Research and Training Center on Com-
munity Integration of Individuals with TBI (RTC) at Mount Sinai
School of Medicine. ANOVA and chi-square tests indicated that
the groups were not significantly different in age, sex, or education.
Demographic data on the three samples can be found in Table I.
TABLE I
DEMOGRAPHIC DATA
Variables
Sex Years of education
12 12–16 16þ

Groups Mean age Male Female Years Years Years
Mold exposure 44.23 10 21 10 2 17

(n ¼ 31) (SD ¼ 10.05) (32.3%) (67.7%) (32.3%) (6.5%) (54.8%)
TBI 43.30 102 102 56 67 78
(n ¼ 204) (SD ¼ 10.79) (50%) (50%) (27.5%) (32.8%) (38.2%)
No disability 44.38 19 28 8 15 24
(n ¼ 47) (SD ¼ 14.48) (40.4%) (59.6%) (17%) (31.9%) (51.1%)
The symptom report of the mold-exposed group resembled that of the
TBI group much more than that of the individuals with no disability.
Some of the most frequently reported symptoms for both groups were
‘‘feeling impatient or irritable,’’ ‘‘misplacing things,’’ ‘‘thinking slowly,’’
‘‘feeling frustrated,’’ ‘‘being easily distracted,’’ ‘‘forgetting what you just
said,’’ ‘‘forgetting names of common objects or having trouble finding the
right word to express your meaning,’’ ‘‘difficulty concentrating,’’ ‘‘feeling
tired,’’ and ‘‘losing your train of thought.’’ While the mold-exposed group
reported a mean of 33 (SD ¼ 7) symptoms and the TBI group reported 37
(SD ¼ 24) on average, the controls reported a mean of only 9 symptoms
(SD ¼ 12). The differences among these means were statistically signifi-
cant (P < .001). In a previous paper, Gordon and colleagues (2000) found
that there were 25 symptoms, 23 of them cognitive, that were sensitive
and specific to individuals with TBI when compared with individuals
with spinal cord injury, liver transplant, HIV/AIDS, and a group with
no disability. In the present study, the mold-exposure group reported, on
average, about 10 of these ‘‘sensitive and specific’’ symptoms (SD ¼ 6),
and the individuals with TBI reported a mean of 8 symptoms (SD ¼ 6). The
mean number reported by the controls was 1 (SD ¼ 2.5). Obviously the
difference among these means was statistically significant (P < .001).
Although the numbers of subjects in each group were uneven, variances
across groups were not, suggesting that differences in group sizes did not
account for the differences. These findings led to the conclusion that
symptom reports, and thus what ‘‘ails’’ people who are mold-exposed,
are quite similar to symptom reports in individuals with known brain
injury. Thus the experience of mold-related cognitive impairment is
likely similar to that of TBI.
In examining the neuropsychological test data, we established criter-
ia for impairment. The group in question was of average intelligence,
and intelligence scores were normally distributed (mean Full Scale
IQ ¼ 108, SD ¼ 16) in the group. Therefore impairment on a measure
was defined as a score that fell more than 1 standard deviation below the
normative mean. A total of 25 measures derived from nine tests were
examined. All subjects exhibited impairment on at least 1 measure, with
a mean of around 7 (SD ¼ 5) impairments. Impairment was found
most often on tests of memory and tests of the executive functions
such as reasoning, mental set shifting, and critical thinking. In fact,
in both of these cognitive domains, significantly more patients exhib-
ited impairments than would have been expected by chance (P ¼ .049
to <.001). Ninety percent of the group exhibited impairments on one or
more memory measures, 45% on tests of executive functions, 39% on
tests of attention, and 17% on tests of processing speed.
It was also found that the impairments in the group were generally
not limited to one domain. Indeed, 65% of the sample had impairments
in more than one domain of cognitive function. The virtual ubiquity of
memory problems was further illustrated by the fact that, when multi-
domain impairment was found, memory was generally one of the areas
affected. All persons who exhibited attention and processing speed
impairments also had memory impairments.
Finally, significant relative impairments were found in the sample.
On the eight Memory Indexes of the WMS-III, 20% to 60% of subjects
performed significantly worse (P ¼ .034 to <.001) than would be
expected given their IQs. On all but two of these indexes, this propor-
tion was significantly greater than would be expected from population
norms (P ¼ <.05 to <.01). In each case, two to four times more subjects
had IQ–Memory discrepancies than would be expected. Thus the
mold-exposed group exhibited significantly more impairments than
would be expected, both in comparison with group norms and in
comparison with themselves.
Although the sample of individuals tended to be depressed, there
was no correlation between depression scores and the number of im-
paired scores on neuropsychological tests. Thus the extent of cognitive
impairment was not associated with the severity of depressed mood.
Results from a recent study by Baldo et al. (2002) are consistent with
the findings described above. They compared the neuropsychological
test performance of 10 mold-exposed individuals to 10 individuals
with mild TBI. The mold-exposed individuals were exposed to Stachy-
botrys atra, Penicillium, and Aspergillus, among other molds. Baldo
et al. defined impaired performance as being below the tenth percen-
tile. The most consistent impairment was found on tests of visual
memory, and the performance of the mold-exposed patients and those
with mild TBI tended to be comparable. Both groups also exhibited
similar patterns of performance on the Million Clinical Multiaxial
Inventory, and they found a weak relationship between cognitive
impairment and depression in a portion of their mold-exposed sample.
In a study of health problems in a group of persons exposed to Stachy-
botrys chartarum and Aspergillus in their work place, Hodgson and
colleagues (1998) reported no evidence of neuropsychological im-
pairment in subjects who were exposed relative to a comparison group
of individuals who were not exposed. Unfortunately, the demographic
characteristics of the two samples were not described, the battery of
neuropsychological tests administered was not described, and the test
findings were under-reported, with no descriptive statistics provided.
This paucity of information makes it difficult to evaluate the implications
of this finding. Perhaps most importantly, the authors did not state
whether the exposed individuals reported cognitive difficulties in addi-
tion to their other ailments. If the individuals who were exposed were
indeed asymptomatic—that is, did not report cognitive difficulties—
these findings are not surprising and further support the validity and
sensitivity of neuropsychological tests. The assertion has never been
made that cognitive impairments secondary to mold exposure occur in
all persons who are exposed to mold or even in a majority of cases. Rather,
they seem to be found in a minority of persons. Thus, to claim an absence
of neuropsychological impairment in a small sample of individuals ex-
posed to mold offers no evidence against (or indeed for) the idea that
mycotoxins can affect cognitive functioning.
V. Conclusion
In conclusion, initial evidence from neuropsychological research
suggests that some people experience significant, measurable cogni-
tive impairment after exposure to mycotoxin-producing fungi. Multi-
ple areas of cognitive functioning appear to be affected, although
memory seems to be the domain of cognitive function most frequently
affected. The symptoms reported are similar to those experienced by
persons with known brain injury. Further research in this area is
critical to examine the extent of the problem and its characteristics,
including potential relationships between specific molds and specific
impairments and dose responses if applicable.
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Mold and Mycotoxins: Effects on the Neurological and
Immune Systems in Humans
ANDREW W. CAMPBELL,* JACK D. THRASHER,{ MICHAEL R. GRAY,{ AND
ARISTO VOJDANI}
*
Medical Center for Immune and Toxic Disorders, Spring, Texas
{
Sam-1 Trust, Alto, New Mexico
{
Progressive Health Care Group, Benson, Arizona
}
Immunosciences Laboratories, Beverly Hills, California
I. Introduction 375
II. Water Damage and Associated Molds 378
A. Mycobiota 378
B. Mycotoxins Produced by Toxigenic Molds 378
C. Human Exposure 380
III. Symptoms, Upper and Lower Respiratory Tract 380
A. Symptoms 380
B. Upper Respiratory Fungal Infections 382
C. Lower Respiratory Tract 383
D. Proinflammatory Cytokines and Biomarkers 385
IV. IgA, IgG, and IgE Antibodies to Molds and Mycotoxins 385
A. Salivary IgA Antibodies to Molds 385
B. Serum IgA, IgM, IgG, and IgE Antibodies to Molds 386
C. Cross-Reactivity of Antibodies to Molds 387
D. Antibodies to Extracellular Polysaccharides (EPS) 390
V. Alterations in T and B Cells, Natural Killer (NK) Cells, and Other
Immune Parameters in Humans Exposed to Toxigenic Molds 390
A. Alterations in Percentage of T and B cells 390
B. Mitogen Activity 391
C. Autoantibodies 392
D. Immune Complexes 392
E. Concluding Remarks on Immunological Observations 393
VI. Neurological Abnormalities 393
A. Neurocognitive Deficits and Central Nervous System Dysfunction 394
B. Peripheral Motor and Sensory Neuropathy 396
C. Neuronal Antibodies 396
D. Demyelination of Peripheral Nerves 397
VII. Conclusion 397
References 398
I. Introduction
The potential harmful effects of exposure to molds in inhabited
buildings were recognized in early Biblical times. In the Old Testa-
ment (King James Version, Oxford 1888 Edition, Chapter XIV: Verses
375
0065-2164/04 $35.00
376 ANDREW W. CAMPBELL et al.
34 thru 47) Leviticus put forth a detailed protocol for the remediation
of contaminated structures, including the destruction of dwellings and
personal belongings if remediation failed. Currently it is recognized
that water intrusion into buildings leads to amplification of molds
(Andersson et al., 1997; Gravesen et al., 1999; Hodgson et al., 1998;
Jaakkola et al., 2002; Johanning et al., 1996; Nielsen, 2003; Peltola et al.,
2001), which often requires remediation.
Fungal fragments occur in indoor air as biocontaminants (Burge, 1990;
Gorney et al., 2002). Potentially toxic and immunogenic byproducts of
fungi and molds include mycotoxins (Croft et al., 1986; Johanning et al.,
2002; Nielsen et al., 1999; Nieminen et al., 2002; Tuomi et al., 1998,
2000); 1,3-alpha-D-glucans (Andersson et al., 1997), extracellular poly-
sacharrides (EPS) (Duowes et al., 1999; Notermans et al., 1988; Wouters
et al., 2000); exodigestive enzymes (EDS) (Monod et al., 2002), and
solvents (Claeson et al., 2002). In addition, trichothecenes, ochratoxin
A, sterigmatocystin, and other mycotoxins have been identified in venti-
lation duct dust and in the air in buildings where occupants have experi-
enced adverse health effects related to mold exposure (Croft et al., 1986;
Engelhart et al., 2003; Jarvis, 2002; Johanning et al., 2002; Nieminen
et al., 2002; Skaug et al., 2000; Smoragiewicz et al., 1993; Tuomi et al.,
1998). The worst-case scenario appears to be repeated episodes of
water damage that promote fungal growth and mycotoxin production,
followed by drier conditions leading to release of spores and hyphal
fragments (Nielsen, 2003).
Occupants of affected structures develop multiple organ symptoms
and have adverse effects of the upper and lower respiratory system,
central and peripheral nervous system, skin, gastrointestinal tract,
kidneys and urinary tract, connective tissue, and the musculoskeletal
system (Anyanwu et al., 2003a; Croft et al., 1986; Gunnbjornsdottir
et al., 1998; Gray et al., 2003; Hodgson et al., 1998; Jaakkola et al.,
2002; Johanning et al., 1996; Kilburn, 2002; Sailvilahti et al., 2000).
Human illness caused by fungi can result via one or all of the following:
(1) mycotic infections (mycoses) (Anaissie et al., 2002; Eucker et al.,
2001; Fraser, 1993; Grossi et al., 2000), (2) fungal rhino-sinusitis (Braun
et al., 2003; Ponikau et al., 1999; Thrasher and Kingdom, 2003), (3) IgE-
mediated sensitivity and asthma (Barnes et al., 2002; Lander et al.,
2001; Zureik et al., 2002), (4) hypersensitivity pneumonitis and related
inflammatory pulmonary diseases (Erkinjuntti-Pekkanen et al., 1999;
Ojanen, 1992; Patel et al., 2001; Sumi et al., 1994), (5) cytotoxicity
(Desai et al., 2002; Gareis, 1995; Jones et al., 2002; Nagata et al., 2001;
Poapolathep et al., 2002), (6) immune suppression/modulation (Berek
et al., 2001; Bondy and Petska, 2000; Jakab et al., 1994), (7) mitochondrial
MOLD AND MYCOTOXINS 377
toxicity (Hoehler, et al., 1997; Niranjan et al., 1982; Pace, 1983, 1988;
Sajan et al., 1997; Wei et al., 1984), (8) carcinogenicity (Dominguez-
Malagon and Gaytan-Graham, 2001; Schwartz, 2002), (9) nephrotoxi-
city (Anyanwu et al., 2003c; Pfohl-Leszkowicz et al., 2002), (10) the
formation of nuclear and mitochondrial DNA adducts (Hsieh and
Hsieh, 1993; Petkova-Bochatrova et al., 1998; Pfhlohl-Leszkowicz
et al., 1993). Finally, in the infectious state, molds secrete extracellular
digestive enzymes (EDE) that cause tissue destruction, angioinvasion,
thrombosis, infarction and other manifestations of mycosis (Ebina et al.,
1985; Kordula et al., 2002; Kudo et al., 2002; Monod et al., 2002; Ribes
et al., 2000; Vesper et al., 2000).
The pathological and inflammatory conditions associated with Sta-
chybotrys chartarum in infants with pulmonary hemosiderosis have
been characterized. S. chartarum isolated from the lungs of an affected
infant produced a hemolysin (stachylysin), a siderophore, and a prote-
ase (stachyrase) (Kordula et al., 2002; Vesper et al., 2000). Stachylysin
has also been demonstrated in the serum of adults ill from a building-
related exposure (Von Emon et al., 2003). In rodents, its presence has
been demonstrated by an immunocytochemical method following in-
stallation of S. chartarum spores into lungs. The hemolysin increases
in concentration from 24 to 72 hours following instillation of spores,
indicating that production/release is a relatively slow process (Gregory
et al., 2003). In addition, strains of S. chartarum produce different
quantities of toxic trichothecenes (Jarvis et al., 1998). In an earthworm
model, stachylysin increased the permeability of blood vessels, causing
leakage through the vessel endothelium and walls (Vesper and Vesper,
2002). Additionally, pathology may result from the interference of pul-
monary surfactant synthesis by S. chartarum spores and isosatratoxin-
F in juvenile mice. Ultrastructural changes in type II alveolar cells—
with condensed mitochondria, increased cytoplasmic rarefaction, and
distended lamellar bodies with irregularly shaped lamellae—have been
observed following exposure to S. chartarum (Mason et al., 1998, 2001;
McCrae et al., 2001; Rand et al., 2001). Thus, hemolysins, siderophores,
and proteases also appear to have an important role in the pathogenesis
of mold infections.
Recognizing the complexity of health problems associated with multi-
ple mold exposure, we have previously reported a multi-center investiga-
tion of patients with chronic health complaints from exposure to multiple
colonies of indoor fungi and molds. We utilized detailed health and
environmental history–gathering questionnaires, environmental moni-
toring data, physical examination, pulmonary function testing protocols,
routine clinical chemistries, measurements of lymphocyte phenotypic
markers (on T, B, and NK cells), antibodies to molds, mycotoxins,

neuronal antigen antibodies, leukocyte apoptosis, neurocognitive test-
ing, 16-channel quantitative EEGs (QEEG), nerve conduction studies
(NCS), brainstem auditory evoked potentials (BAER), visual evoked
responses (VER), and other neurological testing. The following is a sum-
mary of our findings on symptoms, pulmonary function, alterations in
peripheral lymphocyte phenotypes, autoantibodies, and neurological
abnormalities observed in patients by us and others. Currently we refer
to the illness of these individuals as a ‘‘mold mycotoxicosis’’ involving
the immune system, the lungs, the central and peripheral nervous sys-
tems, and generalized inflammatory and irritant responses to exposure to
spores, hyphal fragments, mycotoxins, solvents, and other byproducts
(e.g., EPS, EDS).
II. Water Damage and Associated Molds

A. MYCOBIOTA
Water intrusion into buildings can lead to an amplification of molds.
Molds growing on building materials (e.g., wall board, particle board,
plaster board, ceiling tiles, carpeting) are classifiable according to their
water activity, aw (Nielsen, 2003) as follows: (1) primary colonizers
have an aw of <0.8 with an optimal water requirement approaching 1
for growth. The group includes Penicillium chrysogenum and Aspergil-
lus versicolor, followed by other species of Aspergillus (niger, fumigatus,
sydowii, ustus), several Eurotium species, Penicillium species (brevi-
compactum, commune, corylophilum, pelicans), Paecilomyces variotti
and Wallemia sebi. (2) Secondary colonizers requiring a minimum of
between 0.8 and 0.9 aw include species of Alternaria, Cladosporium,
Phoma, and Ulocladium. (3) Tertiary colonizers (water-damage molds)
that require 0.9 aw or greater include the most toxic molds: Chaetomium
globlosum, Stachybotrys chartarum, Memnoniella echinata, and Tricho-
derma species (viride, citrinoviride, harzianum and longibrachiatum).
For a more detailed review, see Nielsen (2003).
B. MYCOTOXINS PRODUCED BY TOXIGENIC MOLDS

Fungi produce many metabolites, which are believed to play a cru-
cial role in their natural habitats. In addition, many of the metabolites
have been identified. Those that are toxic to animals and humans are
called mycotoxins. Paradoxically, antibiotics isolated from molds are
mycotoxins and are beneficial to humans. Table I lists the molds com-
monly found in water-damaged buildings and the toxic metabolites
TABLE I
TOXIGENIC MOLDS IN WATER-DAMAGED BUILDINGS
Mold Metabolites Health concern
Stachybotrys Spirocyclic drimanes; Pulmonary hemosiderosis;

chartarum satratoxins G, H and F; Induces proinflammatory
hydroxyroridin E, verrucarin J; cytokines
trichodermin; dolabellanes;
atrones B and C;
stachyotryamide;
stachyotrylactams; stachylysin
Alternaria Alternariols; tentoxin; Unknown
tenuissima tenuazonic acids; altertoxin I
Aspergillus Aflatoxin B1; kojic acid; Carcinogenesis;
flavus aspergillic acid; aspergillosis
3-nitropropionic acid;
cyclopiazonic acid
Aspergillus Fumigaclavines; fumitoxins; Tremors and CNS injury;
fumigatus fumitremorgens; gliotoxins; Immune damage by
tryptoquivalines; verruculogen gliotoxin; aspergillosis;
Aspergillus niger Ochratoxin A Nephropathy
Aspergillus Ochratoxin A, penicillic acid; Nephropathy
ochraceous xanthomegnin; viomellein,
vioxanthin
Aspergillus ustus Kotanins Unknown
Aspergillus Sterigmatocystin; Carcinogenesis;
versicolor 5-methoxy-sterigmatocystin aspergillosis
Penicillium Secalonic acid D Unknown
chrysogenum
Chaetomium Chaetomins; Cytoxicity; inhibition of
globosum chaetoglobosins A and C cell division
Memnoniella Griseofulvin; Unknown
echinata dechlorogriseofulvins;
trichodermin; trichodermol
Penicillium Mycophenolic acid; Toxic (mutagenic)
brevicompactum botryodiploidin.
Penicillium Patulin; citrinin; chaetoglobosin; Immune toxicity,
expansum Roquefortine C cytotoxic; tremorgenic
Penicillium Verrucosidins; Tremors; cytotoxicity;
polonicum penicillic acid; nephropathy
nephrotoxic glycopeptides
Trichoderma Trichothecenes; trichodermol; Toxicity associated
species trichodermin; gliotoxin; viridin with trichothecenes
This table summarizes the toxigenic molds found and/or identified in water-damaged buildings.
The mycotoxins isolated from the molds and their general toxic effects are also summarized. The
information in this table was obtained from the review Nielsen (2003).
(mycotoxins) that they produce with general statements on their toxic-

ity. Readers are referred to the literature cited in this chapter and in
Nielsen (2003) for more detailed information.
C. HUMAN EXPOSURE
Humans can be exposed to mycotoxins and metabolites of molds in
the indoor environment via (1) ingestion (contaminated foods, dirt, and
dust) (2) the skin (contaminated clothing and surfaces), and (3) inhala-
tion. Inhalation is the primary mode of exposure in the inhalation of
spores (3 to 7 m), hyphal fragments, and particulate matter down to
0.05 m. It has been shown that particles smaller than spores can be
shed from colonies of molds (Gorney et al., 2002; Kildeso et al., 2000).
Large quantities of particles 0.03 m can be released from colonies,
creating a 300-fold higher concentration of fungal fragments as com-
pared with the number of spores released (Gorney et al., 2002). There is
no apparent correlation between the number of particles and the num-
ber of spores. Factors that influence the release of spores and particu-
lates include low humidity (stimulates release), ventilation, external
wind speeds, human activity, and pressure shocks (e.g., elevators,
doors). Finally, because it is difficult to quantify the particulate matter
shed by colonies, very few meaningful correlations have been found
between spore concentrations and adverse health effects on humans
from indoor exposure to toxigenic molds (Nielsen, 2003). Thus biomar-
kers for molds and mycotoxins have been and need to be further
developed for exposure assessment.
One successful approach has been to use DNA adducts to determine
exposure to aflatoxin B1 (Makarananda et al., 1998) and ochratoxin A
(Pfhohl-Leszkowicz, 1993a,b). However, another effective approach has
been the development of immune assays to detect the presence of anti-
bodies to mold-specific antigens and mycotoxins. Also, an appreciation
of the adverse health effects can be obtained by utilizing neurophysio-
logical, neuropsychological, and immunological diagnostic procedures
(see below).
III. Symptoms, Upper and Lower Respiratory Tract

A. SYMPTOMS
Occupants of water-damaged buildings express multiple organ symp-
toms. Table II summarizes observations made on 209 adults exposed at
home and/or at the workplace. Complaints significantly different from
controls occurred as follows: (1) central nervous system (headache,
TABLE II
FREQUENCY OF SYMPTOMS
Mold Patients
Symptoma (N ¼ ) 209 Controls N ¼ 28 P value
Excessive Fatigue 5.8 1.9 4.3 2.1 0.0001

Headache 5.2 1.9 4.1 2 0.005
Nasal Symptoms 5.1 2.2 4.1 2 0.02
Memory Problems 5.1 2.1 3.3 1.6 0.0002
Spaciness 4.8 2.3 3.2 1.8 0.0007
Sinus Discomfort 4.7 2.2 3.6 1.8 0.01
Coughing 4.6 2.2 3.2 1.6 0.001
Watery Eyes 4.6 2.1 3.4 1.7 0.004
Throat Discomfort 4.5 2.1 3.4 1.7 0.008
Slurred Speech 4.5 2.3 3.1 2 0.002
Lightheadedness 4.4 2.2 3.2 1.4 0.006
Joint Discomfort 4.4 2.3 3.7 2.1 NS
Dizziness 4.3 2.1 3.1 1.4 0.005
Weakness 4.2 2.3 3 1.7 0.008
Bloating 4.2 2.2 3.2 1.6 0.02
Insomnia 4.1 2.2 3.8 2 NS
Weak Voice 4.1 2.2 2.8 1.4 0.003
Spasms 4 2.2 3.8 2.1 NS
Coordination Problems 4 2.2 2.9 1.4 0.01
Visual Changes 3.9 2.3 2.9 1.4 0.02
Rash 3.9 2.2 2.9 1.7 0.02
Numbness 3.9 2.2 3.4 1.7 NS
Cold Intolerance 3.9 2.4 3.1 1.8 NS
Heat Intolerance 3.8 2.4 3.6 2 NS
Chest Tightness 3.8 2,2 2.6 1.3 0.006
Chest Discomfort 3.7 2.2 3 1.3 NS
Urine Frequency 3.7 2.3 3.8 2.1 NS
Excessive Thirst 3.6 2.3 3.4 2 NS
Ringing Ears 3.6 2.2 4.4 2.4 NS
Wheezing 3.6 2 2.6 1.3 0.02
Swallowing Problems 3.2 2 3 1.7 NS
Flushing Skin 3.1 2.1 2.8 1.6 NS
Bladder Control 3.1 2 2.8 1.4 NS
Rapid Pulse 32 2.6 0.9 NS
(continued )
TABLE II (Continued)
Mold Patients
Symptoma (N ¼ ) 209 Controls N ¼ 28 P value
Palpitations 2.8 1.9 2.4 0.8 NS

Bruising 2.8 1.7 2.4 0.9 NS
Swelling Ankles 2.7 1.8 2.6 1.5 NS
Hearing Changes 2.7 1.8 2.6 1.5 NS
This table summarizes the frequency of symptoms of the 38 most frequently reported symptoms in
the patients vs the controls. To obtain these data, a total of 209 patients filled out questionnaires.
Critical t-test analysis was performed and p values are given for each symptom of patients vs controls
(NS ¼ Not Significant).
a
The symptoms compared were for females versus males. The females had significantly greater
frequency for 21 of the 38 reported symptoms (data not shown; see Results section).
short-term memory loss, lightheadedness, dizziness, blurred vision,

tinnitus, and cognitive function loss), (2) the upper respiratory tract (nasal
congestion and chronic sinusitis), (3) the lower respiratory tract (cough,
wheezing, chest tightness, exertional dyspnea, and irritation of the
throat), and (4) general ill feeling (excessive fatigue, weakness, joint
aches and pains, and rashes) (Campbell et al., 2003; Gray et al., 2003).
In addition, others have shown similar increases in the incidence of
neurological and respiratory symptoms in individuals ill from mold
exposure in water-damaged buildings (Hodgson et al., 1998; Johanning
et al., 1996; Kilburn, 2002; Vojdani et al., 2003). Vojdani et al. (2003)
reported that patients exposed to molds had significant increases in
recurrent flu-like illnesses, anxiety, and symptoms of severe allergies.
It has become increasingly obvious that exposure to multiple toxigenic
molds in water-damaged buildings leads to an increased incidence of
multiple organ symptoms in the affected individuals.
B. UPPER RESPIRATORY FUNGAL INFECTIONS

Symptoms of upper respiratory involvement include nasal conges-
tion, sinusitis, sinus pain, and nasal bleeding (chronic rhinosinusitis).
Individuals with this condition do not respond to ordinary antibiotic
therapy.
Several reports have appeared in the literature demonstrating that a
large proportion of individuals with chronic rhinosinusitis (CRS) have
infections with molds and yeast. CRS is characterized by the presence
of eosinophilic mucin, fungal hyphae, Charcot-Leyden crystals, and
the presence or absence of polyposis (Ponikau et al., 1999; Taylor et al.,
2002). The incidence of fungal involvement in different case studies was
82% to 100% (Braun et al., 2003; Dosa et al., 2001; Ponikau et al., 1999)
and 100% (Taylor et al., 2002). The fungal genera isolated and cultured
from nasal secretions include such indoor contaminants as Aspergillus
sp., Alternaria, Chaetomium, Cladosporium, Epicoccum, Penicillium,
Phoma, Trichoderma, and others (Dosa et al., 2002; Ponikau et al., 1999;
Taylor et al., 2002). The isolation of fungi and the presence of eosino-
phils and eosinophilic mucin rule out type I (IgE) hypersensitivity
(allergy) and strongly point to the role of invasive fungi as the cause of
CRS (Braun et al., 2003; Ponikau et al., 1999).
C. LOWER RESPIRATORY TRACT

Molds can cause lung disease by different mechanisms: allergic asthma
(Jaakkola et al., 2002; Zureik et al., 2002), infections (e.g., aspergillosis)
(Fraser, 1993; Sumi et al., 1994), and inflammation (e.g., hypersensitivity
pneumonitis, and farmer’s lung disease) (Fan, 2002; Ojanene, 1990,
1992; Patel et al., 2001). Chest x-rays can be used to detect pathological
changes associated with infections (e.g., aspergillosis and granuloma-
tous lesions). Pulmonary function testing (PFT) is used to diagnose
airway restriction caused by allergies to molds as well as inflammatory
conditions (hypersensitivity pneumonitis and farmer’s lung disease).
PFT measures flow rates in the airways of the lungs. The forced vital
capacity (FVC) is the maximum amount of air expelled during forced
expiration. The fraction of the vital capacity expired in one second is the
FEV1. The importance of these measurements arises from the fact that
during disease states, (e.g., asthma), the FVC may be normal while the
FEV1 is reduced because of increased airway resistance. However, these
two measurements do not discriminate between the airways of different
caliber and therefore are not able to distinguish between the status of the
large, medium, and small airways. Airborne particulate matter and
spores (bioaerosols) from fungi range from 0.03 to 10 microns. ‘‘Respira-
ble particles’’ range from 5 microns to 0.005 microns and are capable
of reaching the small airways and alveoli of lungs. Therefore, PFT
measurements used must also detect inflammatory or obstructive
changes within the small airways. The PFT measurements most suited
for small airway obstruction are FEF 75% and FEF 25–75%. These
measure the flow rates at 75% and 25–75% of the exhalation and are
indicative of air flow through the small airways. A reduction in these
PFT values is evidence of small airway obstruction. The results pre-
sented in Fig. 1 show the mean and standard deviation of PFT values in
Figure 1. The results of PFT testing on individuals exposed to molds in water-damage

structures.
individuals with symptoms of airway obstruction following exposure to

multiple colonies of molds in water-damaged buildings. The FEF 75% is
the most significantly affected parameter, demonstrating that the airway
symptoms are probably the result of obstruction of the small airways in
these individuals.
Small airway obstruction separates these patients from the typi-
cal occurrence in asthmatic patients, which is generally more global,
involving all levels of the bronchial tree. The observed small airway
obstruction indicates that particulates from <0.3 to 5 microns are being
delivered to the alveoli in the deepest regions of the lung. This model is
supported by the lack of a rise in mycotoxin-specific IgA (see Table VI)
and the findings of Rand et al. (2002, 2003) and, thus, represents the most
likely exposure route of relevance in patients exposed to indoor bioaer-
osols when multiple mold colonies are present. Therefore, the FEF 75%
appears to be a biomarker that can be used to identify injury to the small
airways as result of particulates containing mycotoxins, EPS, and EDEs
(Rand et al., 2002, 2003).
D. PROINFLAMMATORY CYTOKINES AND BIOMARKERS
Proinflammatory cytokines and other biomarkers have been demon-
strated to be elevated in the nasal lavage fluid of individuals with upper
respiratory symptoms in moldy buildings versus control subjects. Thirty-
two full-time employees in a school building contaminated with A.
fumigatus and A. versicolor, Eurotium, Exophiala, Phialophora, Rhodo-
torula, Stachybotrys, Trichoderma, Ulocladium, Willenia, and actinomy-
cetes had increased concentrations of alpha-tumor necrosis factor (TNF),
interleukin 6 (IL-6), and nitric oxide (Hirvonen et al., 1999). Furthermore,
Walinder et al. (2001) demonstrated increased concentrations of eosino-
philic cationic protein, myeloperoxidase, and albumin in the nasal
lavages of occupants in buildings with mold infestation of the gypsum
board, insulation, wallpaper, and wood. Multiple genera, including
Stachybotrys, were identified. Finally, Nielsen et al. (2001) have shown
that an extract of metabolites from Stachybotrys independent of macrocy-
clic trichothecenes and atranones is capable of inducing in vitro macro-
phage production of alpha-TNF and IL-4. This suggests that in addition
to mycotoxins, other metabolites (e.g., spirocyclic drimanes) have a role
in the nasal inflammatory process seen in mold exposure individuals
(Nielsen, 2003; Nielsen et al., 2001). Further support comes from Leino
et al. (2003), who have shown that exposure of mice to spores from
S. chartarum increases monocytes, neutrophils, and lymphocytes in
bronchial alveolar lavage fluid (BAL). The infiltration of inflammatory
cells was associated with the induction of proinflammatory cytokines (IL-
1, IL-6, TNF-alpha), chemokines (CCL3/MIP-1, CCL4/MIP-1, and CCL2/
MCP-1), and mRNA levels in the lungs. This effect was independent
of the mycotoxin satratoxin produced by this mold. Furthermore, the
effects were observed with no significant increase in IgE, IgG2a, and
IgG1 antibody levels after exposure to S. chartarum.
IV. IgA, IgG, and IgE Antibodies to Molds and Mycotoxins

Molds release antigenic determinants (e.g., EPS, EDS, and proteins)
that elicit an antigen-antibody response. In addition, mycotoxins can
act as haptens, binding to proteins, forming a new antigenic determi-
nant (NAD). The immune system then recognizes the NAD as foreign
and makes antibodies directed against the NAD.
A. SALIVARY IGA ANTIBODIES TO MOLDS

IgA antibodies are the first line of defense against foreign invasion by
preventing the attachment of microorganisms and toxins to epithelial
cells by complexing antigens (Challancombe, 1987). Recently Vojdani

et al. (2003) tested for the presence of saliva secretory IgA antibodies
against molds and mycotoxins in occupants with upper respiratory
symptoms of a water-damaged building. The patients had significantly
increased salivary IgA antibodies to Alternaria, Aspergillus, Chaeto-
mium, Cladosporium, Epicoccum, Penicillium, Stachybotrys, satratoxin
H, and other trichothecenes. It is probable that these IgA antibodies play
a role in late-phase type-1 and type-2 hypersensitivity as well as type-3
delayed sensitivities to molds and their byproducts. For example, in
farmer’s lung disease, serum IgA antibodies against A. fumigatus and
other molds are elevated and are correlated with the state of the disease
(Knutsen et al., 1994; Ojanen, 1992; Ojanen et al., 1990). In addition,
serum IgA antibodies to this organism are associated with exacerbations
of bronchopulmonary aspergillosis along with elevated IgE, peripheral
eosinophilia, and roentgenographic infiltrations (Apter et al., 1989).
B. SERUM IGA, IGM, IGG, AND IGE ANTIBODIES TO MOLDS

IgA, IgM, IgG, and IgE antibodies to 7 different molds (Alternaria,
Aspergillus, Stachybotrys, Chaetomium, Cladosporium, Epicoccum,
and Penicillium), satratoxin H, and other trichothecenes in 40 patients
with multiple organ symptoms were compared with 40 age- and sex-
matched controls (Vojdani et al., 2003). The exposed individuals occu-
pied a water-damaged building and were tested within days following
evacuation of the premises. Quantitative enzyme-linked immunosor-
bent assay (ELISA) produced the following results: (1) IgG antibodies to
the molds and the two mycotoxins were significantly elevated in the
patients versus the controls. (2) Levels of serum IgA antibodies for each
mold and the mycotoxins were significantly elevated in the patients,
with the exception of Epicoccum. The highest titers in descending
order were found for Stachybotrys, Penicillium, and Chaetomium.
(3) IgM titers were significantly elevated in these patients versus the
controls for Stachybotrys, Cladosporium, Alternaria, Aspergillus, sa-
tratoxin H, and other trichothecenes. No difference in IgM titers were
observed between patients and controls for Chaetomium, Epicoccum,
and Penicillium. (4) With respect to IgE antibodies, a significant in-
crease in titers in these patients was found only for Aspergillus and
satratoxin H. It appears from these observations that randomly selected
controls without symptoms and apparent mold exposure have low titers
of antibodies to a variety of mold and mycotoxins. However, mold-
exposed symptomatic individuals have titers that are significantly
elevated over the control values.
In another study, Vojdani et al. (2003), utilizing ELISA assay proce-
dures, tested for IgA, IgM, and IgG antibodies against S. chartarum,
A. niger, P. notatum, satratoxin H, and other trichothecenes in the
following three groups: healthy donors (N ¼ 500); 500 patients referred
to the laboratory for various diagnostic tests for illnesses without ap-
parent exposure to molds (N ¼ 500); and randomly selected patients
referred for illness associated with exposure to molds (N ¼ 500). The
results of this study are summarized in Tables III through VI. Briefly,
the concentration of IgA, IgM, and IgG antibody titers were lowest in
the blood donors, intermediate in the randomly selected patients, and
highest in the mold-exposed patients for each of the molds. With
respect to satratoxin H and trichothecene antibodies, the antibody
titers had a different distribution. When the mold-exposed patients
were compared with the healthy controls, IgG and IgM titers were
significantly elevated, while IgA titers were not. When the mold-
exposed patients were compared with the random patients, only the
IgG titers were significantly different. Moreover, on inspection of the
data on the random patients, it was noted that the standard deviation
(SD) was large and overlapped with the mean value and SD of the mold
patients. It appears from these observations that the randomly selected
patients may have been exposed to molds without recognition by the
attending physician that such exposure might have occurred. Barnes
et al. (2002) reached similar conclusions. They demonstrated IgE and
IgG antibodies to Stachybotrys chartarum in 9.4% and 42.2% of the sera
of 139 blood donors. They concluded that sensitivity to S. chartarum is
potentially much more widespread than previously appreciated. This
fungus may affect the asthmatic and allergic population through both
immunologic and toxic mechanisms. The significance of the fungus in
the milieu of allergenic fungi may need to be re-evaluated.
C. CROSS-REACTIVITY OF ANTIBODIES TO MOLDS

The use of antibodies to molds as a biomarker of exposure has been
criticized (Musmand, 2003). The critique is based on two publications.
One is an abstract the full results of which have never been published
(Halsey et al., 2001); therefore, it is impossible to determine anything
about the methods used in this paper. The second is a position paper
published on the Internet by the California Department of Public Services
in which not a single experiment was conducted. Recently the question of
cross-reactivity between mold antigens (S. chartarum, A. niger, and P.
notatum) was investigated by using affinity-purified rabbit sera (Vojdani
et al., 2004). The results of this study showed that non-immunized rabbits
TABLE III
ANTIBODY LEVELS TO PENICILLIUM NOTATUM
Healthy Mold Random Mold

Controls Patients Z P Patients Patients Z P
Antibody N ¼ 500 N ¼ 500 Score Values N ¼ 500 N ¼ 500 Score Value
IgG 620 535 2159 2458 13.7 <0.001 1383 1839 2159 2458 5.6 <0.001
IgM 692 551 1692 2442 8.9 <0.001 1241 1530 1692 2442 3.5 <0.001
IgA 640 572 1256 2163 6.1 <0.001 853 1070 1256 2163 3.7 <0.001
Mean S.D. IgG, IgM, and IgA antibody levels in ELISA units to Penicillium notatum in controls, randomly selected patients and mold-exposed patients
with Z test and P values.
TABLE IV
ANTIBODY LEVELS TO ASPERGILLUS NIGER

Antibody N ¼ 500 N ¼ 500 Score Values N ¼ 500 N ¼ 500 Score Values
IgG 618 426 1795 2316 11.1 <0.001 1349 1417 1795 2316 3.7 <0.001
IgM 782 420 1725 2449 8.5 <0.001 1177 1302 1725 2449 4.4 <0.001
IgA 732 595 1346 2456 5.4 <0.001 849 938 1346 2456 4.2 <0.001
Mean S.D. IgG, IgM, and IgA antibody levels in ELISA units to Aspergillus niger in controls, randomly selected patients and mold-exposed patients with
Z test and P values.
TABLE V
ANTIBODY LEVELS TO STACHYBOTRYS CHARTARUM

IgG 803 530 2304 2432 13.5 <0.001 973 1234 2304 2432 10.9 <0.001
IgM 629 602 1940 2478 11.5 <0.001 1115 1212 1940 2478 6.7 <0.001
IgA 665 665 1511 2660 6.9 <0.001 760 1086 1511 2660 5.8 <0.001
Mean S.D. IgG, IgM, and IgA antibody levels in ELISA units to Stachybotrys chartarum in controls, randomly selected patients and mold-exposed
patients with Z test and P values.
TABLE VI
ANTIBODY LEVELS TO SATRATOXIN H

IgG 767 641 1523 1352 11.3 <0.001 1054 1147 1523 1352 5.9 <0.001
IgM 611 648 1320 1590 9.2 <0.001 1160 1170 1320 1590 1.8 <0.060
IgA 715 588 705 868 2.1 <0.440 747 819 705 868 0.78 <0.430
Mean S.D. IgG, IgM, and IgA antibody levels in ELISA units to satratoxin H in controls, randomly selected patients and mold-exposed patients with Z test
and P values.
develop IgG antibody titers to these molds that increase in concentration

with age. The sera from these rabbits gave an impression of up to 52%
cross-reaction with Aspergillus, Penicillium, and Stachybotrys. When
using affinity-purified antibodies in cross-inhibition studies, the antigen-
ic cross-reaction between Stachybotrys and Aspergillus was between
8.6% and 12.3%, and between Stachybotrys and Penicillium extracts it
showed 9.3–9.6% antigenic similarities. Thus, for cross-reaction studies
between different molds, affinity-purified antibodies and a sensitive and
quantitative assay with natural antigens should be used. When using
such an assay, it was concluded that cross-reactions between Stachybo-
trys, Aspergillus, and Penicillium exist but are much less widespread
than previously believed. Based on these observations, antibodies to
molds and mycotoxins as developed by this laboratory methodology are
reliable biomarkers of mold and mycotoxin exposure.
D. ANTIBODIES TO EXTRACELLULAR POLYSACCHARIDES (EPS)

EPS can cause type I and type III inflammatory processes. They have
been shown to be present in mold-contaminated buildings and can be
used as a marker of mold contamination and exposure (Duowes et al.,
1999; Wouters et al., 2000). Exposure to 1–3 beta-D-glucan caused air-
way inflammation with symptoms of dry cough, phlegm, and hoarseness
(Rylander, 1997; Rylander et al., 1998). IgG antibodies in immunized
rabbits against EPS from several mold genera have been reported
(Notermans et al., 1987, 1988). The EPS antigens caused the production
of fairly specific antibodies, with some cross-reactivity as determined by
an ELISA. The EPS antigens produced by species of Penicillium, Asper-
gillus, and Geotrichum lost their immunological activity with heating at
100 C at pH 1.8. The EPS antigens from Mucor recemosus, Rhizopus
olgosporus, and C. cladosporoides were stable under the same conditions.
It appears from these data that an ELISA for antibodies to EPS released by
various molds could be developed as an additional biomarker for mold
exposure.
V. Alterations in T and B Cells, Natural Killer (NK) Cells, and Other

Immune Parameters in Humans Exposed to Toxigenic Molds
A. ALTERATIONS IN PERCENTAGE OF T AND B CELLS
Peripheral blood lymphocytes can be identified and quantified by

using fluorescent antibodies to cell surface antigens. Typical markers
for T cells are designated as CD2, CD3, CD4, and CD8. B cells are
identified by CD19 or CD20. In addition, other markers can be used to
identify activation of T and B cells, (e.g., CD25, CD26, HLR-DRþ,
CD8CD11bþ). Patients chronically ill from exposure to toxigenic molds
in water-damaged office buildings, schools, and homes have altered
percentages of lymphocyte markers in their peripheral blood when
compared with expected ranges (Gray et al., 2003). The patients had
increased B cells (CD20) (75.6%). T cell activation markers increased
for the following cell types: CD5CD25 (68.9%), CD3CD26 (91.2%),
CD8HLR-DRþ (62%), and CD8CD38 (56.6%). Decreases were observed
for CD8CD11bþ (15.6%) and natural killer (NK) cells (CD3CD16CD56,
38.5%). Moreover, Thrasher et al. (2004) found that individuals with
an ongoing exposure to molds in a water-damaged building had re-
lative increases over controls of the following: total lymphocyte count,
T cells (CD2, CD3, CD4, CD8, and CD3CD16), B (CD19) cells, and
NK cells (CD3CD16CD56).
B. MITOGEN ACTIVITY
T and B cells respond to specific and nonspecific antigens by under-
going cell division (mitogenesis). Mitogenesis responses to nonspecific
mitogens were as follows: phytohematogglutinin (PHA) was decreased
by 26.2% in mold-exposed subjects, while only 5.9% had decreased
response to Concanavalin A (ConA) (Gray et al., 2003). PHA stimulates
T cells, while Con A causes T and B cells to divide.
Mitotic responses to ConA, PHA, PWM (pokeweed mitogen), and
LPS (lipopolysaccharides) were examined in patients with an ongoing
exposure to toxigenic molds. In general, mitogenesis to PHA and Con
A was significantly elevated over controls, indicating increased
response of T cells to nonspecific antigens. In addition, mitogenic
response to B cell stimulators (ConA, PWM, and LPS) was also signifi-
cantly elevated. Although mitogenesis was increased, the patients
could be subdivided into three distinct responses to each mitogen as
follows: suppression, elevation, and extremely elevated (Thrasher
et al., 2004). Analysis of the NK cell (CD3CD16CD56) activity revealed
that 42.4% of these patients had decreased killing of target cells.
Furthermore, the CD4/CD8 (helper/suppressor) ratio was significantly
elevated.
These two studies (Gray et al., 2003; Thrasher et al., 2004) indicated
that alterations in the percentages of T and B cells, mitogenesis, and
NK cell activity ccurred in mold-exposed humans. The alterations
included an increase in activation markers, which may be a result
of antigenic stimulation. Furthermore, the changes in mitogenic re-
sponse to both nonspecific and specific mitogens indicate immune
suppression occurred in some individuals, while others experienced

immune stimulation. The decrease in NK cells and their activity may
indicate that there was a decrease in immune surveillance, which
may have importance with respect to cancer and/or infectious diseases.
C. AUTOANTIBODIES
Autoantibodies directed against self-antigens are known to occur in
a variety of autoimmune diseases and degenerative neurologic
disorders. Antinuclear autoantibodies (ANA) are the ones most com-
monly recognized and are usually associated with connective tissue
disease (e.g., lupus). However, other autoantibodies can be directed
against a variety of self-antigens and can also be used as biomarkers of
toxic exposure (Thrasher et al., 2002; Vojdani et al., 1992, 1993). Hu-
mans exposed to toxigenic molds have abnormally elevated autoanti-
bodies to the following: ANA, anti-smooth muscle, peripheral, and
central nervous system myelin and eight different neural antigens
including myelin basic protein, ganglioside G1, sulfatide, tubulin,
crystallin, filament, MOG, and MAG (Campbell et al., 2003; Gray
et al., 2003). Odds ratios for each autoantibody at 95% C.I. was signifi-
cant, showing an increased risk for autoimmunity. Autoantibodies and
autoimmune diseases are recognized as occurring from toxic exposures
(Cooper et al., 2002; Griem et al., 1998). For the significance regarding
the neural antigen autoantibodies, see Neurological Abnormalities,
Section VI.
D. IMMUNE COMPLEXES
Immune complexes occur when antigen and antibodies combine and
have been implicated in numerous immunopathologic conditions, in-
cluding systemic lupus erythematosus, rheumatoid arthritis, glomerulo-
nephritis, and infectious induced inflammation (Abbas et al., 1994).
Deposition of immune complexes can occur from cell or tissue specific
antibody-antigen reactions resulting in organ injury and/or immune com-
plex diseases (Bigazzi et al., 1986). Thus it would appear from these
observations on increased immune complexes that inflammation and
autoimmune reactions may exist in mold-exposed patients. Circulating
immune complexes containing IgG, IgM, and IgA antibodies can generate
a variety of substances associated with muscle damage and the acute
phase response that can activate the classic pathway of complement
(Sorensen et al., 2003). Autoantibodies are also known to activate the
complement system.
E. CONCLUDING REMARKS ON IMMUNOLOGICAL OBSERVATIONS
The increase in B cells, activation markers, and helper/suppressor
ratio all indicate immune activation has occurred as demonstrated by
Gray et al. (2003) and Thrasher et al. (2004). Increased activation marker
(CD45RO) has been reported for symptomatic children with exposure to
molds in contaminated homes (Dales et al., 1998). These observations
are consistent with production of proinflammatory cytokines as dis-
cussed above with antigenic stimulation. In addition, elevated immune
complexes are further support for immune activation and antigenic
stimulation. The presence of elevated immune complexes is compatible
with increased production of antibodies to mold antigens as well as the
presence of ANA, anti-smooth muscle, and anti-neural antigen antibo-
dies. The observations on immune alterations discussed above are also
consistent with the suggestion that mold exposure causes immune dys-
regulation (Hirvonen et al., 1999; Wichman, et al., 2002). Recently a
review by Anyanwu et al. (2003b) showed that natural killer cell activity
was adversely affected in patients with chronic exposure to indoor
molds and may be implicated in causing neurological abnormalities.
VI. Neurological Abnormalities

Neurological abnormalities caused by mycotoxins from molds
have been described in the literature. The neurotoxic mycotoxins
include trichothecenes, citreoviridin, patulin, fumonisin, penitrem,
verruculogen, rubratoxin, ergot alkaloids, and tremorgens.
Wilson et al. were the first to isolate a tremorgenic mycotoxin in 1964.
The mycotoxin penitrem has been shown to induce tremors and con-
vulsions in experimental animals (Hayes, 1980). Jorntner et al. (1986)
and Norris et al. (1980) studied the neurological effects of the mycotox-
ins penitrem A and verruculogen, which are known to cause a neuro-
toxicity characterized by sustained tremors. Their findings support a
primary site of action of both of these mycotoxins as being presynaptic.
Mycotoxins, being relatively nonpolar, pass through the blood-brain
barrier and thereby have access to synapses. The neurotoxic effects of
ergot alkaloids are known to affect the postganglionic parasympathetic
synapses (Berde et al., 1978).
Wang et al. (1998) in their study suggested that the primary site of
trichothecene action is the brain. Chapman (2003) reported how tricho-
thecene mycotoxins from Stachybotrys cause neurological disorders by
being neurotoxic. The clinical signs of trichothecene mycotoxicosis
include eye pain, dyspnea, tachycardia, vomiting, muscle tremors and
weakness, lack of coordination, and confusion. Patients affected devel-

op seizures, central nervous system dysfunction, hypotension, and
myelosuppression (Stahl et al., 1985). Studies have shown that expo-
sure to molds can cause optic demyelinating neuritis and multifocal
choroiditis (Campbell et al., 2003; Rudich et al., 2003).
The nephrotoxic and hepatotoxic effects of mycotoxins have been
well documented in several studies (Anyanwu et al., 2003c; Bhat et al.,
1989; Etzel et al., 1998). The mycotoxin rubratoxin was studied by
Moss (1971) and was shown to cause liver and kidney damage
and lesions of the central nervous system. Ciegler and Bennett (1980)
stated that trichothecene mycotoxins cause clinical conditions that
include skin irritations, vomiting, anorexia, diarrhea, hemorrhage,
and convulsions.
Walsh et al. (1985) reviewed a large number of patients with necropsy-
proven central nervous system aspergillosis and identified important
epidemiological, pathological, and clinical features. In their study, the
most common central nervous system lesions were subcortical hemor-
rhagic infarcts in the cerebral hemispheres or cerebellum, and they found
that the most common entry of Aspergillus into the central nervous system
was the lower respiratory tract. Aspergillosis of the central nervous sys-
tem, lungs, and at least one other organ was found in almost 66% of the
patients. Beal et al. (1982), in their neuropathological review, discovered
that the pathologic hallmark of neurologic aspergillosis cases was the
invasion of fungal hyphae into the blood vessel walls with subsequent
necrosis and thrombosis and spread into the surrounding tissues.
A. NEUROCOGNITIVE DEFICITS AND CENTRAL NERVOUS SYSTEM DYSFUNCTION

Pena (1970) noted subtle personality changes were observed as an
initial sign in cases of disseminated aspergillosis. Young et al. (1970)
noted in their study of 13 patients with disseminated aspergillosis that
all had some degree of lethargy or fatigue. Malkin et al. (1998) in their
study at National Institute of Occupational Safety and Health reported
multiple neurological symptoms in occupants of an office building
contaminated by several species of fungi, including Penicillium, As-
pergillus, Alternaria, Candida, Cladosporium, Epicoccum, Fusarium,
and Pullularia. Gordon et al. (1993) described a patient who after being
exposed to Aspergillus, Penicillium, and Rhizopus developed fatigue,
headache, progressive somnolence, slowness of thinking, and severe
tremors. The patient had coarse fasciculations of the muscles of the
face and tongue and was unable to stand unassisted. His EEG showed a
general dysrythmia consistent with a toxic encephalopathy.
Baldo et al. (2002) studied the neuropsychological performance of
10 patients exposed to molds (Stachybotrys atra, Penicillium, and
Aspergillus). The patients had a variety of symptoms: fatigue, respira-
tory problems, recurring bloody noses, nausea, frequent sore throats,
and headaches, among others. The mold-exposed patients were im-
paired on a number of cognitive measures, with the most consistent
deficits in visuospatial learning, visuospatial memory, verb, learning,
and psychomotor speed. In addition, the mold-exposed patients had
evidence of Axis I and Axis II pathology. There was a significant
correlation among patient’s scores on the Beck Depression Inventory,
with a number of neuropsychological tests falling within the impaired
range. The authors put forth a model by which to investigate the effects
of mold exposure on the central nervous system.
Crago et al. (2003) further demonstrated that measures of exposure were
highly predictive of neuropsychological test performance using two sub-
tests from the Delis–Kaplan Executive Function System (D–KEFS) to mea-
sure executive or higher-level cognitive functions. Significant predictive
power was observed for the D–KEFS Trail Making subtests of visual
scanning, letter sequencing, number–letter sequencing, and motor speed;
the D–KEFS Color–Word Inhibition/Switching subtest; the WAIS-III
Digit Symbol Coding and Symbol Search subtests; and the IVA-CPT
full-scale attention quotient and the visual and auditory attention quoti-
ents. Crago et al. (2003) also reported that significant predictive power
was found for estimates of degree of exposure and for the QEEG variables
of mean frequency delta, relative power theta, relative power alpha,
absolute power delta, absolute power theta, and absolute power alpha.
In addition, the QEEG findings in confirmed mold-exposed patients in-
dicated a restriction in the range of functioning (narrowed frequency
bands) of the frontal lobes, that is, increased (accelerated) mean frequency
of the slower frequencies (delta range) and decreased (slowed) higher
frequencies (beta range), indicating a collapse toward the middle of the
frequency spectrum. These findings, coupled with observed increased
levels of absolute and relative power theta and alpha waves in frontal
sites, indicated hypoactivation of the frontal cortex consistent with insuf-
ficient excitatory input from the reticular activating system anatomically
seated in the midbrain.
Finally, Kilburn (2002) reported on both objective neurological tests
and neuropsychological evaluation of 20 mold-exposed patients. Ob-
jective tests showed impaired balance, reaction time, color discrimina-
tion, and visual fields in the mold-exposed patients. Neuropsychological
tests showed impaired cognition, verbal recall, and trail making. Pulmo-
nary function testing showed small airway obstruction was observed in 4
patients. Longer durations of exposure and aging appeared to increase the

total abnormalities. He concluded ‘‘Moulds appear to cause chemical
encephalopathy and these abnormalities.’’
Neurophysiological effects of mold exposure have been reported in
children as compared with controls (Anyanwu et al., 2003a). Brainstem
auditory evoked response (BAER), electroencephalogram (EEG), visual
evoked potential (VEP), and somatosensory evoked potential (SSEP) were
used to test neurological abnormalities. Three of 10 children had an
abnormal EEG following mold exposure. The frontal-temporal theta wave
activity in the 10 patients seemed to indicate diffuse changes consistent
with metabolic encephalopathies. Also, 1 to 3 hertz delta activity was
asymmetric in the right hemisphere of 3 patients. BAER showed abnorm-
alities in 9 patients with both 150 and 3500 check sizes. A significant delay
in waveform V occurred in the majority of patients, representing dysfunc-
tional cognitive process and conductive hearing loss in both ears. VEP
showed clear abnormalities in 4 of the children with P100 amplitudes and
latencies decreased bilaterally. SSEP showed diffuse polyneuropathy in
three patients. The authors concluded that exposure to toxic molds can
affect neurological and behavioral status of children.
B. PERIPHERAL MOTOR AND SENSORY NEUROPATHY

Campbell et al. (2003) studied 119 patients with symptoms of neuro-
toxicity with documented measured exposure to molds. These patients
complained of fatigue, memory loss, cognitive function loss, headaches,
tremors, numbness and tingling, blurred vision, tinnitus, and muscle
weakness. Ninety-nine of these patients had abnormal clinical neuro-
logical examinations, abnormal findings on neurophysiological testing,
and elevated antibodies to neuronal antigens. Nerve conduction studies
(NCVs) revealed three groups of abnormal patients (ABM) and one
group of normal (NM): mixed sensory motor polyneuropathy (55
ABN); motor neuropathy (17 ABN); sensory neuropathy (27 ABN); and
symptoms without neurophysiological abnormalities (20 NM, controls).
C. NEURONAL ANTIBODIES
Elevated autoantibodies by ELISA to several neuronal antigens were
found in patients with documented measured exposure to molds. The
titers of the autoantibodies were significantly elevated over controls.
These included IgA, IgG, and IgM antibodies to myelin basic protein,
myelin associated glycoprotein, oligodendrocyte glycoprotein, ganglio-
side GM-1, chondroitin sulfate, crystalline, tubulin, and neurofilament.
D. DEMYELINATION OF PERIPHERAL NERVES
Campbell et al. (2003) concluded their observations on changes in
nerve conduction velocities and the presence of neural antigen autoanti-
bodies as follows: ‘‘The increased latency for motor and sensory nerves
observed in the 55 patients with mixed neuropathy is suggestive of a
demyelinating process (Busby et al., 2003).’’ This was accompanied by
a decrease in velocities for the median, ulnar, and peroneal nerves while
the tibial nerve had a decrease in the amplitude. All three sensory nerves
(median, ulnar, and superficial peroneal) exhibited increased latencies
and decreased amplitudes. Thus the polyneuropathy observed in these
patients appeared to be a demyelinating process with decreased number
and size of fibers (decreased amplitude) and chronic involvement of the
nerve (decreased velocities) (Busby et al., 2003; Steck et al., 1987).
The motor neuropathies (17 patients) had decreases in latencies (peroneal
and tibial nerves), decreased amplitudes (median and peroneal nerves),
and decreased velocities (median, ulnar, peroneal, and tibial nerves). This
appeared to be a diffuse neuropathy and may involve some demyelination
(Berger et al., 2003). Finally, the sensory neuropathies (27 patients) had
increased latencies for all three nerves, with that of the superficial pero-
neal being not significant. The increased latencies and the decreased
amplitude of the superficial peroneal suggested demyelination was
occurring (Reindl et al., 1999; Willison and Yuki, 2002).
VII. Conclusion
Forgacs noted in 1962 that mold mycotoxicosis was called ‘‘the
neglected disease.’’ The manifestations and disorders in humans
caused by molds and mycotoxins continues to be overlooked or unno-
ticed by many physicians. Each year studies continue to be published
throughout the world medical and scientific literature elucidating and
explaining the pathological processes and biomechanisms by which
exposure to molds and mycotoxins cause sickness in humans. We have
described in this chapter the most recent neuroimmune mechanisms of
disease process in humans of molds and mycotoxins. The exact
biological and chemical actions through which these mechanisms un-
fold is not completely understood. However, molds do produce meta-
bolites (mycotoxins, solvents) and shed antigenic materials (spores,
hyphae, extracellular polysaccharides, and enzymes), which are toxic
(mycotoxins) and or cause immunologic responses (antigens). Science
and medicine should continue its work in these areas and bring about
the much-needed change from ‘‘the neglected disease’’ to ‘‘the accepted
disease.’’
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Identification, Remediation, and Monitoring Processes
Used in a Mold-Contaminated High School
S. C. WILSON,* W. H. HOLDER,{ K. V. EASTERWOOD,{ G. D. HUBBARD,{
R. F. JOHNSON,{ J. D. COOLEY,{ AND D. C. STRAUS*
*Center for Indoor Air Research, Department of Microbiology and Immunology
Texas Tech University Health Sciences Center, Lubbock, Texas 79430
{
Assured Indoor Air Quality LP, 6616 Forest Park Road, Dallas, Texas 75235
{
Aerobiology Research and Analytical Laboratory, Corpus Christi, Texas 78418
I. Introduction 409
II. Environmental Survey 410
A. Results and Conclusion 411
III. Initial Indoor Air Quality Investigation 412
A. Walk Through 412
B. HVAC System 412
C. Moisture Profiles 413
D. Air and Surface Samples 413
E. Sample Identification 413
F. Other Measurements 414
IV. Results of Initial Investigation 414
A. Walk Through 414
B. HVAC System 415
C. Air and Surface Samples 415
V. Indoor Air Quality Investigation: Conclusion 415
VI. Abatement, Decontamination, and Clearance 416
A. General Considerations 416
B. Drying the Building 417
C. Containment 417
D. Removal of Contaminated Items 417
E. HVAC Reconditioning 418
VII. Clearance Testing: Methods and Results 418
VIII. Post Remediation 419
IX. Discussion 419
X. Conclusion 420
XI. Recommendations 420
References 421
I. Introduction
Microbiological, chemical, and particulate components of indoor air
all can have a potential effect on occupant health. The levels of these
components can be influenced by building design; heating, ventilation,
air conditioning (HVAC) system design; installation, operation, and
409
0065-2164/04 $35.00
410 S. C. WILSON et al.
maintenance; occupant activities; management policy/housekeeping

practices; and renovation and expansion projects. Poor indoor air
quality (IAQ) is associated with a range of human health symptoms
(Flannigan et al., 1994; Redlich et al., 1997) and with sick building
syndrome (SBS). The World Health Organization first coined this term
in 1982. SBS encompasses the effects of air toxics (airborne particulate
matter and volatile organic compounds originating from new building
materials, etc.) and in particular, the effects of airborne fungi and
their products (Cooley et al., 1998; Górny et al., 2002; Mahmoudi and
Gershwin, 2000).
The effects of fungi on human health can be broadly divided into
three categories: (1) allergic reactions, including asthma-like symptoms
(Cooley et al., 2000; Fogelmark et al., 1991; Licorish et al., 1985); (2) fun-
gal infections; and (3) responses to fungal products such as mycotoxins
and volatile organic compounds (Bondy and Pestka, 2000; Pitt et al.,
2000; Yang and Johanning, 2002). Studies on the effects of inhaled
mycotoxins have linked them to immunosuppression, liver damage,
pulmonary dysfunction, and carcinogenesis (Hollinger and Ekperigin,
1999; Jakab et al., 1994).
The presence of toxigenic molds in buildings has the potential to
negatively affect human health (Redd, 2002), and work has shown
that occupants of mold-affected buildings have significantly higher
incidences of SBS-related disorders (Johanning, 1996). Further, the
removal of toxigenic molds from affected buildings results in improved
occupant health status ( Jarvis and Morey, 2001; Sudakin, 1998).
The creation of acceptable IAQ in human-occupied dwellings can be
a complex issue. This chapter presents a case study whereby a series
of protocols was used to identify mold-related problems in a school
building complex, remediate the school to the point where IAQ was
considered acceptable, and then monitor the IAQ of the school after
remediation.
II. Environmental Survey

Management staff from a 3-year-old high school in southern Texas
had been dealing with comfort problems associated with excessive
humidity and temperature fluctuations since the opening of the school.
Concern regarding the IAQ of the building had also been expressed by
staff and students to the administration. At one point, students and
staff refused to enter the school buildings. An IAQ company was con-
tacted to investigate the problem. An initial step by the company was
to administer a survey to school staff. This survey was adapted and
IDENTIFICATION, REMEDIATION, AND MONITORING PROCESSES 411
developed from a previous National Institute of Occupational Safety
and Health (NIOSH) epidemiological survey (Crandall and Sieber,
1996). Survey questions first addressed occupant health (e.g., type,
frequency, and duration of symptoms; whether or not symptoms less-
ened after absence from building). Survey questions were then directed
towards the workplace environment (e.g., private versus open work
areas, number of people in area, physical observation of leaks in ceil-
ing, walls and floors, remediation status of leaks plus the time delay in
remediation after any leaks are observed, noted temperature swings,
status of heating system and status of maintenance schedules, etc.).
The analysis of the responses involved statistically comparing them to
responses from a database of schools that did not have an IAQ problem.
A. RESULTS AND CONCLUSION

Of the 215 survey forms distributed, 187 were returned (87% re-
sponse rate) and analyzed. The survey indicated that 28.4% of the oc-
cupants were experiencing symptoms related to the building and
9.6% of the occupants were experiencing symptoms potentially related
to the building. The incidences of both mucosal and neurological
symptom profiles were high compared with the means of other schools
without poor indoor air quality (Figs. 1 and 2). Water event scores were
FIG. 1. Mucosal symptom scores from a test school suspected of having poor indoor air
quality as compared with means and standard deviations of other schools with good
indoor air quality.
FIG. 2. Neurological symptom scores from a test school suspected of having poor
indoor air quality (IAQ) as compared to means and standard deviations of other schools
with good IAQ.
high as well, indicating nearly constant sources of moisture, which

could lead to microbial growth. In addition, the responses to the survey
indicated that strong musty odors were present, which is a common
trait of interior mold growth. It was concluded that a full indoor air
quality baseline investigation was required.
III. Initial Indoor Air Quality Investigation

The baseline investigation consisted of a number of steps. These
were as follows:
A. WALK THROUGH
An initial walk through was conducted. This is a process whereby
technicians examined the structure internally and externally for evi-
dence of mold growth and/or any obvious or subtle signs of water
intrusion (Summerbell, 1995).
B. HVAC SYSTEM
The HVAC system is the primary means for maintaining control of
moisture in a dry building. It is also the primary transport mechanism
for airborne pollutants (Hiipakka and Buffington, 2000; Simmons and
Crow, 1995). Once the conditions are suitable for fungal growth to be
established in a building, the HVAC system can transport pollutants to
other potential growth areas. The HVAC system was examined for
design flaws, construction defects, and histories of operational failures.
C. MOISTURE PROFILES
Relative moisture levels inside walls, etc., were ascertained with the
use of a moisture meter (Tramex Moisture Encounter, Tramex, Ltd.,
Littleton, CO) throughout the building.
D. AIR AND SURFACE SAMPLES

Airborne microbiological testing was performed in classrooms
throughout the school. Culturable fungal samples were collected
by using a two-stage Andersen biological cascade impactor (Thermo
Andersen, Smyrna, Georgia). The samplers were run for 5 min with a
calibrated vacuum pump (28.3 L/min) on to Sabourauds Dextrose
Agar (SDA) plates (Burge et al., 1977; Hoekstra et al., 2000) (Fisher
Scientific, Pittsburgh, Pennsylvania). An Allergenco air sampler (Mk 3.
Allergenco/Blewstone Press, San Antonio, Texas) was also run in con-
junction with the Andersen air sampler. This machine was run for
5 min. It collected non-culturable fungal spores, culturable fungal
spores, and other particulates in the air. To evaluate the findings, results
from the Andersen and Allergenco air samplers were compared with
outside air samples taken with the same instruments.
Surfaces that on visual inspection appeared to have fungal
growth were sampled with sterile swabs (Fisher Scientific, Pittsburgh,
Pennsylvania) that were then placed in a sterile container, sealed,
and labeled. A tape lift sample was also retrieved (St Germain and
Summerbell, 1996).
E. SAMPLE IDENTIFICATION
The agar plates from the Andersen sampler were incubated for 7
days at 24 C before being read. Fungal cultures were identified with
the use of reference texts after examination of colony morphology
and microscopic examination of spores and hyphae (St Germain and
Summerbell, 1996; Sutton et al., 1998). Cultures employing Potato
Dextrose Agar (PDA) and Malt Extract Agar (MEA) were also used
where necessary to sub-culture and identify unknown fungi. The num-
ber of different fungal species was recorded as well as the numbers of
individual colonies within each species. Results were expressed as the
total number of colony forming units (CFU) retrieved per cubic meter of
air. Microscope slides from the Allergenco air sampler were read at
400 on an Olympus BH laboratory microscope (GMI Inc., Clearwater,
Minnesota). Results included the identification and the enumeration of
fungal spores, expressed as spores per cubic meter of air.
From surface samples, the tape lift was stained with lactophenol
cotton blue (Fisher Scientific) and the microscope slides were then
read at 400 on an Olympus BH laboratory microscope. Swabs were
placed in 10 ml sterile tubes and had 5 ml of phosphate buffered saline
(PBS) added to them. After a period of 5 h, the swabs were serially
diluted in PBS, and 0.1 ml of these dilutions was then added to SDA,
PDA, and MEA plates. The media were incubated for 7 days at 24 C.
After this period, the plates were read by using the same procedures as
for the Andersen sampler agar plates.
F. OTHER MEASUREMENTS
Temperature and relative humidity measurements were taken to
determine whether the dew point had been reached inside the build-
ing. Particulates in sizes ranging from 0.3 m to 5.0 m were also taken
in conjunction with the airborne samplings to determine whether there
was a high particulate count in the air. An airborne particle counter
(APC-1000 Biotest Diagnostics, Denville, New Jersey) was used to
count particles relative to four thresholds: >0.3, 0.5, 1.0, and 5.0 m.
IV. Results of Initial Investigation

A. WALK THROUGH
Sagging and stained ceiling tiles and a rusted ceiling grid system and
light fixtures indicated an excessively wet environment. Stachybotrys
sp. growth was discovered in several locations, primarily on the insu-
lation of the chilled water piping system located in various mechanical
rooms. Rust was also observed on structural members. Other general
deficiencies observed were the presence of roof leaks and cracks in
masonry walls caused by foundation movement. A physical examina-
tion of the fiberglass insulation above the ceiling tiles showed the
presence of moisture, which may have been trapped by the insulation
material. Loose floor tiles and adhesive seeps were also noted. It ap-
peared that the overall building design and the placement of vapor
retaining insulation allowed condensation to take place in interstitial
spaces.
B. HVAC SYSTEM
The conclusion was that the design of the outside air system in
providing ventilation, coupled with the building air conditioning sys-
tem design, was insufficient to properly condition and/or treat the
outside air. This created conditions in the building that led to discom-
fort, condensation, and fungal proliferation. Specific issues were: the
dew point of the outside air for ventilation was not sufficiently de-
pressed. The chilled water system piping was undersized. The air
handling unit (AHU) control valves were leaking and not seating prop-
erly. The set point of the chilled water was too low in an attempt to
overcome undersized piping and marginal primary/secondary loop
design/installation, resulting in improper operation. The fiberglass
insulation material on piping was not appropriate for hot and humid
environmental conditions. There was poor workmanship in the instal-
lation of the insulation system and sealing of the vapor barrier.
The condensate drain systems did not have a sufficient slope or were
leaking, resulting in air conditioning pan overflows. The control sys-
tems sequences of operation of the outside air system were allowing
non-conditioned air to enter the building during the ‘‘off’’ period.
C. AIR AND SURFACE SAMPLES

Table I shows some representative findings from the air sampling.
The 31 samples taken did not suggest a contaminated environment;
total CFU counts inside were lower than outside. Cladosporium sp.
were the dominant fungi in the outside air. With the exception of one
room, Cladosporium sp. were dominant in all inside samples. Results
of the spore traps revealed that inside counts were a reflection of
outside counts.
Culturable fungal growth sites were found on a number of substrates.
These included Stachybotrys sp. and Aspergillus sp. Growth sites were
found on chilled water piping, on ceiling insulation, and on a variety of
other surfaces.
V. Indoor Air Quality Investigation: Conclusion

Although there was not an indication of a mold problem from the air
samples, the findings from the walk through and the results of the
HVAC profiles led to the conclusion that the inability of the HVAC
system to effectively dehumidify indoor air meant that all surfaces and
materials were at risk of condensation and fungal growth. A decision
TABLE I
PRE-REMEDIATION MICROBIOLOGICAL AIR SAMPLE RESULTS FROM LOCATIONS IN A HIGH SCHOOL
SAMPLED WITH AN ANDERSEN AIR SAMPLER
Fungal genera
Location Cladosporium Penicillium Aspergillus Alternaria Bipolaris
OAS 1 182 21 28
OAS 2 91 28 7
OAS 3 238 42
OAS 4 287 42
Library 91 21
Gym 28 14 28
Cafeteria 182 21 14
Band 28 7
L204 161 21
L203 126 49
L201 182 14
L105 84
L101 28 21
D204 56 14
D202 14 14
D111 35 7 7
OAS ¼ outside air sample. Results expressed in Colony Forming Units/m3 air.
was made to address the cause of the excessive moisture and to

commence with the remediation process.
VI. Abatement, Decontamination, and Clearance

A. GENERAL CONSIDERATIONS
All work was directed to creating an environment free of mold
growth sites and one where air temperatures were between 72 and
76 F and relative humidity between 45% and 55%. Importantly, fresh
air was to be conditioned to 45 F dew point and approximately 70 F
before being distributed to the occupied space (American Society for
Heating Refrigeration and Air-Conditioning Engineers, 1989). All work
was conducted after students and staff vacated the building.
B. DRYING THE BUILDING
Removal of excess absorbed moisture in building materials inside
the building and the lack of conditioned or dehumidified outdoor
air for ventilation was temporarily addressed with the use of eight
portable desiccant dryers (Munters Titanium Silica Gel Desiccant
wheel, Texas Manufacturing Center, Selma, Texas) connected to the
existing outside air intakes. The desiccant driers lowered the vapor
pressure in the building, thereby facilitating drying of structural com-
ponents (poured roof deck, fireproofing, masonry construction, etc.)
and contents.
C. CONTAINMENT
Prior to remediation, areas were selected and put into containment
as per the Environmental Protection Agency (2001) and the New York
City Department of Health guidelines (1993). Inside the contained
areas, high-efficiency particulate air (HEPA) filter negative air systems
were installed whereby air was drawn from the outside of the contain-
ment area into the containment area, circulated, then exhausted di-
rectly to the outside air. This system allowed for any fungal spores
liberated during remediation to be collected in the HEPA filters. Once
this was completed, remediation began. Personnel working inside
containment were required to wear TYVEKTM protection suits (Texas
America Safety Company, South Brownwood, Texas), rubber gloves,
and full-face respirators fitted with P100 filters (National Institute of
Occupational Safety and Health, 1995).
D. REMOVAL OF CONTAMINATED ITEMS

The removal of mold-contaminated materials can have a direct posi-
tive effect on IAQ (Ellringer et al., 2000), therefore all visible mold
growth sites were removed. This included the removal of all contami-
nated building materials, ceiling tiles, fiberglass insulation above both
drywall and lay-in ceilings, and chilled water insulation. Carpet was
replaced with vinyl composition tile in most areas. Areas around the
contaminated items were also cleaned with a 70% alcohol solution
(Steri-Fab, Yonkers, New York). The ceiling tiles were replaced with a
moisture-resistant tile (1729A HumiGuard, Armstrong World Industries
Inc., Minneapolis, Minnesota).
E. HVAC RECONDITIONING
All air handler units (AHU) and ductwork were cleaned in accor-
dance with industry specifications (National Air Duct Cleaning Asso-
ciation, 1992). This included fan coils and air handler units. The
condensate drain system for the fan coil units was redesigned and
replaced, drain receivers were relocated, and trap primers were
installed. All interior surfaces of the AHU casing and the first 120 of
supply air duct were coated with an anti-microbial agent (Aegis Envi-
ronmental [Atlantic] Ltd., Nova Scotia, Canada). However, note that
this anti-microbial treatment is still under evaluation by the Environ-
mental Protection Agency. The coils were coated with an anti-foulant
(First Strike Micro Coat, Controlled Release Technologies, Inc.,
North Clearwater, Florida). The fiberglass jacket for the chilled water
insulation system in the building was replaced with a phenolic foam
(Extol of Ohio, Norwalk, Ohio) with an all-service jacket (ASJ). The
condensation drain lines were insulated with Armaflex (Armstrong
World Industries, Lancaster, Pennsylvania). A portion of the supply
air was redirected to the space above the ceiling to keep the building
elements in that space environmentally stable. An engineered outside
air ventilation system that incorporated desiccant dryer technologies
(Munters Titanium Silica Gel Desiccant Wheel, Texas Manufacturing
Center, Selma, Texas) was permanently installed to replace the porta-
ble desiccant dryers. This conditioned the outside air for ventilation to
a 45 F dew point and approximately a 70 F dry bulb temperature
before being distributed to air handling units and fan coil units serving
the occupied spaces.
VII. Clearance Testing: Methods and Results

After each area had been remediated, a preliminary clearance test
was performed, which involved the use of spore traps, tape lifts, and
most importantly, a practical assessment of the remediation effort in
conjunction with a visual inspection of the remediated area. Spore trap
levels of indoor airborne fungi needed to be equal to or lower than the
outdoor levels, with a similar rank order and composition (or less than)
of mold types present in outside air. If a spore trap result was consid-
ered unacceptable, the area was re-examined for fungal growth; the air
in the area was also ‘‘scrubbed’’ with a negative air machine for a total
of 24 h. The area was then re-sampled. If an area was judged acceptable,
it would then be taken out of containment. This judgment was a
subjective one based on the air sample results and an evaluation of
the remediation work. At this point workers were no longer required to
use respirators and wear protective clothing such as chemical protec-
tion suits and rubber gloves. Final cleaning and general maintenance
was then conducted, at the completion of which a final clearance test
was performed. This involved the use of culturable and non-culturable
fungal sampling and an overall consideration as to the success of the
remediation effort.
VIII. Post Remediation

After completion of the remediation and cleanup process, the
school was handed back to the school administrators. An ongoing
monitoring system was then initiated. The school was examined se-
quentially at 30-day intervals during which time air samples were
collected and physical inspections conducted.
IX. Discussion
The school was typical of many schools in this geographic region in
that the buildings are well sealed to conserve energy costs. This type of
construction has been associated with poor IAQ (Addington, 2001). In
a well-sealed building, a defective HVAC system has the potential to
cause widespread problems. This situation can be exacerbated by local
water intrusions resulting from roof leaks, etc. Remediation can be an
expensive and time-consuming process, therefore it is advisable that
strict attention is paid to the HVAC operations and to any evidence of
moisture intrusion. It is axiomatic that the control of moisture leads to
the control of mold. The high numbers of defects in the HVAC system
were the principal reason for the moisture levels that allowed mold to
grow in this school.
The remediation process did not include the cleaning of contents
inside the school by the remediation company. This was conducted
by the custodial and maintenance staff of the school with conventional
cleaning techniques. General guidelines for contents cleaning have
been proposed (EPA, 2001), and work has been conducted in this
laboratory regarding appropriate cleaning methodology (Wilson et al.,
in press). A significant issue in the mold identification process was the
disparity between results of the microbiological air samples and the
observations of mold growth sites and the results of the mold survey.
This is because air samples are often not representative (Burge, 2001).
Some of the reasons for this are that the process of sporulation is not
constant with certain fungal species. Sporulation is triggered by a
number of factors such as temperature and humidity (Yang and

Johanning, 2002), and air sampling may take place during intervals
between sporulation. Also, some fungi such as Stachybotrys chartarum
produce a relatively wet and heavy spore compared with other fungi
such as Penicillium sp., which may partially account for the low rate of
capture of this organism in air samplers. The culturability of fungal
conidia also plays a role, although this issue is addressed if a spore trap
is used in conjunction with a culturable air sampler. A separate issue
is that there are no universally accepted levels for interpreting air
samples. Currently the best approach is to compare the inside samples
to the outside samples. Inside air should reflect the outside air in terms
of types, levels, and rank orders of fungal organisms. Indoor air samples
should not be interpreted in isolation. They need to be considered as a
tool but not an autonomous criterion for passing or failing the air
quality in buildings. Air sampling was employed as part of the clear-
ance process in this school. However, the data were interpreted in the
light of the limitations described above. Because of the limitations of
the air sampling process, locating hidden mold in affected buildings
still remains a challenge for indoor air investigators. While the occu-
pant survey is one tool, others include visual inspections and/or the
taking of air samples of building cavities (WallChek, SKC, Eighty Four,
Pennsylvania). In the future, techniques that identify microbial VOC
(Gao and Martin, 2002) and the analysis of fungal DNA in air samples
may lead to improved detection rates of hidden mold (Cruz-Perez et al.,
2001; Roe et al., 2001).
X. Conclusion
In this case study, a mold-related epidemiological survey in conjunc-
tion with indoor air quality technician observations was effective
in identifying mold problems in a contaminated high school. Tech-
niques such as dehumidification of the outside air for the HVAC sys-
tem, the reconditioning of the HVAC system, and the physical removal
of mold growth sites were used in the remediation process. Post reme-
diation monitoring processes indicate that these techniques have been
successful.
XI. Recommendations
HVAC systems need to be carefully evaluated with regard to their
potential for creating excessive moisture in well-sealed schools. Air
samples are prone to false negatives and should be interpreted cautiously
when assessing mold contamination of school buildings.
ACKNOWLEDGMENTS
The authors would like to thank Drs. Chunfa Wu and Larysa Andriychuk for critical
review of the manuscript, Mr. Curtis Carriker and Mr. Matt Fogle for laboratory expertise,
and the staff of Assured Indoor Air Quality Limited, L.P., for their support and valuable
input. Drs. Wilson and Straus were supported by a Center of Excellence grant from Texas
Tech University Health Sciences Center. Dr Straus was also supported by a grant from the
State of Texas Higher Education Coordinating Board.
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The Microbial Status and Remediation of Contents in
Mold-Contaminated Structures
STEPHEN C. WILSON AND ROBERT C. LAYTON
Center for Indoor Air Research
Department of Microbiology and Immunology
Texas Tech University Health Sciences Center
Lubbock, Texas 79430-6591
I. Introduction 425
II. Should We Be Concerned About Contents Contamination? 426
A. Visible Fungal Growth on Contents 426
B. Fungal Conidia 427
C. Mycotoxins 429
D. Conclusion 430
III. Properties of an Ideal Cleaning/Sterilization Technique 431
IV. Cleaning/Sterilization Agents 431
A. Sodium Hypochlorite 431
B. 70% Ethanol 432
C. Gamma-Irradiation 432
D. Ozone 432
E. Other Techniques 433
V. Post Cleaning 433
VI. Conclusion 434
References 434
I. Introduction
The World Health Organization first coined the term ‘‘sick building
syndrome’’ (SBS) in 1982. It is a broad label for a range of human health
symptoms associated with poor indoor air quality. Public awareness of
SBS has been growing for a number of years, and it is a significant issue
in many buildings, including schools, offices, and private homes (Red-
lich, 1997). Initially, investigations centered on airborne particulate
matter and volatile organic compounds that originated from new build-
ing materials, office equipment/furnishings, etc. More recently, the
focus in this area has been on the effects of airborne fungi and their
products (Chapman, 2003; Mahmoudi and Gershwin, 2000).
While it has been acknowledged that damp and moldy structures
are potentially harmful to the occupants (Redd, 2002), a related major
and largely uninvestigated subject also relevant to the occupants of
425
0065-2164/04 $35.00
426 WILSON AND LAYTON
mold-contaminated structures is the status of their contents inside these

structures. The possibility exists that the contents may be contaminated
by fungal colonies/conidia and/or mycotoxins.
There have been no previous investigations into relationships be-
tween contents from a mold-contaminated structure and human
health. There are two stumbling blocks for an investigation of this
nature.
(1) No accurate or comprehensive testing methodologies are available
for determining the levels of fungal conidia, fragments, and potentially
harmful metabolites on contents that are inside mold-contaminated
structures. For example, sample collection techniques such as tape lifts
(St. Germain and Summerbell, 1996) and a mini-vacuuming technique
with Air-O-Cell slides (Zefon International, St Petersburg, Florida) may
be used to collect fungal conidia and other debris from sample sub-
strates and examined through a light microscope, but the results of
these techniques only represent a limited area of the contents.
(2) These results are difficult to interpret; it is important to be able to
distinguish between normal environmental levels of conidia and levels
from a contaminated structure. This is a totally subjective exercise at
present.
This dearth of information can create a serious concern for the
occupant when articles of sentimental value or great expense are
in question. In this article, we will discuss the issue of contents
potentially being contaminated by mold conidia and/or mycotoxins,
present some recent research we have performed in this area, and
finally, examine some sterilizing procedures that have been found to
be effective in eliminating fungal conidia and mycotoxins on home
contents.
II. Should We Be Concerned About Contents Contamination?

A. VISIBLE FUNGAL GROWTH ON CONTENTS
In our work we have seen visible fungal growth on a wide range of
substrates, such as shoes, books, clothing, shelving, and wardrobes. This
potentially can pose a threat to human health. However, the severity of
the threat depends on the type and amount of fungi growing on the
contents. In general, visible fungal growth should be removed or treated
effectively, but one of the factors to be taken into consideration with
regard to treating visible fungal growth is the possibility of regrowth of
either the same organism or different ones a short time later (Price and
Ahearn, 1999).
MICROBIAL STATUS AND REMEDIATION OF CONTENTS 427
B. FUNGAL CONIDIA
How many conidia of which particular fungal species constitute a
health threat to which particular individual? Simply put, the answer is:
we do not know. In light of this deficiency, the cautious working
principle we use when looking at contents contamination is that very
high numbers of conidia on items should be considered as potentially
contributing to poor human health.
To gain some information on this subject we conducted two studies
that examined (1) the sporulation characteristics of Stachybotrys
chartarum, colloquially known as black mold, under conditions of
normal room temperature and humidity, and (2) conidia burdens on
the contents of contaminated and non-contaminated structures.
1. Experiment 1: Sporulation Characteristics of

In the first experiment, replicated three times, we took sterile sheet-
rock, 6 square centimeters in area, and inoculated them with 1 106
conidia of culturable S. chartarum. The sheetrock was then placed in
a large Petri dish containing 30 mL of sterile water. The Petri dishes
were then placed inside three closed chambers. Two chambers were
1 cubic meter in volume, and one chamber was 0.5 of a cubic meter
in volume. The chambers were kept at 25 C and between 40% and
60% relative humidity. We closely surrounded the sheetrock square
with two circles of Potato Dextrose Agar (PDA) plates. These were
replaced on a daily basis for 28 days, and at the end of this period
the sheetrock was covered with confluent growth of S. chartarum.
The plates taken out of the chambers were incubated at 25 C for
7 days; after this time any fungal colonies on the plates were identi-
fied and enumerated. Every time the chamber doors were opened for
plate replacement, air movement occurred. The results were surprising
to us. On no occasion was there any growth of S. chartarum on the
plates. These results indicate that under these conditions, the conidia
observed on the sheetrock colonies had not left the sheetrock. This
raises the intriguing possibility that it is not conidia but some other
component of S. chartarum that human occupants are exposed to in
S. chartarum–contaminated structures. However, it is also worth not-
ing that the sheetrock was placed face up on the base of the chamber.
Conceivably, contaminated ceiling tiles would allow for better conidia
dispersal because of the effects of gravity. This is only speculation at
this time.
2. Experiment 2: Conidia Burdens on the Contents of Contaminated

and Non-Contaminated Structures
In the second experiment, we sampled the contents of four schools
from south Texas. Two schools had a documented widespread mold
contamination problem. The other two schools previously had a docu-
mented mold problem but had been remediated 6 months beforehand
and according to ongoing mold surveys and sampling results, did not
currently have a mold-contamination problem. All schools had been
unoccupied for 1 month at the time of sampling.
A total of 200 contents were sampled from each school. The samples
were divided into 10 classrooms per school and 20 contents per class-
room The contents per classroom were: 5 chairs selected at random,
5 desks selected at random, 5 books selected at random, 1 computer
keyboard, 1 slide screen, 1 phone, 1 whiteboard, and 1 screen projector.
These last miscellaneous items were selected on the basis of our previ-
ous observations of contaminated schools, which showed that these
areas often had growth sites. Tape lifts were taken from an area of
3 square inches from the same locations on the samples. All samples
were viewed at 400 magnification on a light microscope. The micro-
scope slides were categorized on the basis of the number of Penicilli-
um/Aspergillus-like conidia, Alternaria sp., Cladosporium sp., and
Bipolaris sp. conidia, because these are the most common conidia
found on contents in our experience. Special note was made of any
conidia found belonging to the genera Stachybotrys and Chaetomium.
The categories were as follows:
1. Zero conidia
2. Very light: slides having 5 conidia or less
3. Light: slides having 5–50 conidia
4. Moderate: slides having 50–100 conidia
5. Heavy: slides having greater than 100 conidia
Mean numbers of contents per category per mean classroom were
compared to see if there was a difference between the schools (Table I).
A one-way analysis of variance (ANOVA) or a Kruskal-Wallis one-
way analysis of variance on ranks (SigmaStat Version 2.0, SPSS Inc.
Chicago, Illinois) was used for the analysis. The results showed that (1)
the majority of classrooms in both groups had levels of conidia that
were in the zero to very light categories; (2) there were significant
differences (P < 0.05) between individual schools in the zero, very
light, and light categories, but these differences did not show any
identifiable trends (and interestingly, when the pre-remediated and
TABLE I
COMPARISON OF CONIDIA BURDENS ON CONTENTS BETWEEN 4 SCHOOLS IN THE SOUTH TEXAS REGION
School A School B School C School D

Conidia level
category Mean SE Mean SE Mean SE Mean SE
Zero 16.5a 0.4 14.5b 0.60 16.7a 0.70 12.9b 1.40

a b a
Very Light 0.7 0.30 5.1 0.70 3.1 0.70 6.9b 1.40
Light 2.3a 0.60 0.4b 0.30 0.1b 0.10 0.2b 0.20
Moderate 0.4 0.13 0 0 0 0 0 0
Heavy 0 0 0 0 0.1 0.1 0 0
Means represent the number of contents per categories per mean classroom. N ¼ 200 contents per
school (20 contents per classroom, 10 classrooms per school). Schools A and B had been remediated
at least 6 months beforehand, schools C and D had a documented mold contamination problem.
Different superscripts indicate a significant difference at the P ¼ 0.05 level.
post-remediated schools were combined, there were no significant

differences (P < 0.05) between them); and (3) a low number of class-
rooms from the remediated group had conidia levels in the moderate
category, and one classroom from the remediated group had contents in
the heavy category.
Our conclusions were that (1) the lack of difference between the two
groups in the zero, very light, and light categories may have been a
genuine finding; alternatively, the random sampling tape lift technique
may have been too limited to detect any meaningful differences; and (2)
the number of classrooms that had conidia levels in the moderate and
heavy categories were too few in number to be able to state that there
was a definite effect from the remediation process.
These results again illustrate the nature of the obstacles we face
when trying to determine acceptable levels of conidia on contents.
C. MYCOTOXINS
We know it is possible for both fungal colonies/conidia and myco-
toxins to be present together (Sorenson et al., 1987), but we do not know
if mycotoxins can be present alone. Presently there are no practical or
commercial tests for the determination of mycotoxins on contents.
There are some analytical procedures available (e.g., high-performance
liquid chromatography and mass spectrometry), but these are mostly
used in an experimental setting.
Fungi produce a wide range of mycotoxins. With regard to mold-

contaminated structures, much attention has been given to a family of
mycotoxins known as the trichothecenes. These are produced by a
number of different fungi such as Stachybotrys sp. and Fusarium sp.,
among others. In one study, a type A trichothecene preparation called
anguidine was injected into humans (Wannemacher and Wiener,
1997). The subjects developed central nervous system and dermal
disorders. These symptoms are similar to those reported by occupants
of mold-contaminated buildings. However, it must be noted that many
factors can contribute to these symptoms. The primary route of expo-
sure to potential mycotoxins in a sick building is inhalation; the routes
of exposure in the experimental work on humans and animals have
been intraperitoneal and intravenous injections, ingestion or inhala-
tion. It has been stated that it is difficult to compare these different
exposure modes (Assoulin-Daya et al., 2002). However, other work has
shown that the effects of inhaled mycotoxins can be just as severe as
with other routes of exposure (Creasia et al., 1987). The data are still
being gathered, and accordingly, it is still too early to state unequivo-
cally that mycotoxins cause demonstrable health effects in occupants
of toxigenic mold-contaminated structures (Chapman, 2003).
Are mycotoxins present on contents that do not have visible mold
growth? Currently this is unknown. The theoretical possibility exists,
and one postulate is that mycotoxins may separate from the fungi and
become airborne on fungal fragments. Because these fragments can be
extremely small in size—as small as or smaller than the particulates in
cigarette smoke, for example—they can float throughout the dwelling,
possibly propelled by an air equilibration process that takes place
when doors or windows are opened. The fragments eventually coalesce
with other dust particles, which makes them heavier, allowing for them
to land and settle on home contents. This theory has not been proven
and even so, if this were possible, the amount of fungal fragments
containing mycotoxins may be extremely small. However, mycotoxins
can be detrimental to experimental animals in very low quantities
(Korpinen, 1974).
D. CONCLUSION
Visible fungal growth should be removed or treated. Regarding con-
idia burdens and mycotoxin loads on contents, it is unknown if they
present a danger to human health, and accurate measurement of both is
not achievable at this stage. A conservative approach is to assume that
the potential is there and to clean or remove contents that are in
contaminated structures. This conclusion leads to the search for an
ideal cleaning/sterilizing technique.
III. Properties of an Ideal Cleaning/Sterilization Technique

Optimally, an ideal sterilization technique should have the following
properties:
. The ability to inactivate fungal conidia and fungal fragments
. The ability to inactivate mycotoxins
. The ability to penetrate into inaccessible nooks and crannies of
items
. Be safe to use regarding user health
. Be safe to use on the item.
At first glance some of these properties appear to be mutually
exclusive, and this has been the major challenge in our search for an
ideal sterilization technique. In this article we discuss four common
cleaning agents.
IV. Cleaning/Sterilization Agents

A. SODIUM HYPOCHLORITE
Sodium hypochlorite has been shown to possess bactericidal and
fungicidal properties against certain organisms (Abdel-Mallek et al.,
1995; Rutala and Weber, 1997). One study showed that it had mixed
results against Penicillium roqueforti (Bundgaard-Nielsen and Nielsen,
1996). With regard to mycotoxins, 20 ml of a 1.3% concentration of
sodium hypochlorite is sufficient to inactivate 20 g of pure aflatoxin
(WHO 1980). However, this technique has a proviso that 5% acetone
is used as a rinse afterwards, because a carcinogenic by-product,
2,3-dichloro aflatoxin B1, is formed out of the inactivation process.
Sodium hypochlorite has also been quoted as being able to inactivate
trichothecene mycotoxins at a concentration between 3% and 5%
(Wannemacher and Wiener, 1997; WHO 1980).
It is worth noting that sodium hypochlorite can be highly corrosive
and can give off vapors that are detrimental to human health. When
used in high concentrations, protective clothing should be worn, and
the use of respirators with a suitable filter is highly recommended.
Splash-protective goggles should be used when using any liquid chem-
ical overhead. Users should read the Material Data Safety Sheet
(MSDS) carefully before using it.
A commercial preparation of sodium hypochlorite, Clorox, when

used as per the manufacturers instructions at about 5% concentration
in water, has been shown to be effective in inactivating aflatoxins in
one limited study (Yang, 1972). The concentration of sodium hypo-
chlorite in Clorox is 5%. This may be preferable to a more concentrated
sodium hypochlorite solution, because in the concentrated form it can
produce fumes that can be too strong for people to use for any length of
time.
B. 70% ETHANOL
This is commonly used as a sterilizing agent. One advantage it has is
that it does not have the strongly corrosive properties of sodium hypo-
chlorite. It has been shown that 70% ethanol at a 10-minute exposure
level was effective in inactivating vegetative cells and conidia of a wide
range of molds (Bundgaard-Nielsen and Nielsen, 1996). Ethanol has
also been noted as being able to dissolve certain types of mycotoxins
such as trichothecenes (Cole and Cox, 1981), and this may facilitate
their removal.
C. GAMMA-IRRADIATION
A number of studies have examined the effects of gamma-irradiation
on fungal conidia and mycotoxins (Aziz et al., 1997; Blank and Corrigan,
1995; Saleh et al., 1988). It is a routinely used sterilizing procedure
for a wide range of food items. The technique has a high penetration
power. Experimental results show that it has mixed results with regard
to inactivating mycotoxins. Adjusting relative humidity during irradi-
ation appears to play a role (Uralova et al., 1987), although some
studies with moisture levels in grains have not shown any significant
differences (Hooshmand and Klopfenstein, 1995). It is possible that a
large increase in relative humidity is required for the treatment to
become effective. If mycotoxin inactivation is not a concern, then this
technique appears to be quite effective, although attention must be
given to the degrading effect of gamma-irradiation on items.
D. OZONE
Ozone is a strong oxidizing agent that can denature items. There
appear to be three variables to take into account when evaluating the
efficacy of an ozone treatment. They are (1) the amount of ozone, (2) the
duration of ozone treatment, and (3) the relative humidity. Results from
past work have shown that in a gas-phase, it has not been particularly
effective against mold conidia or colonies on building substrates (Cole
and Fuarde, 1999). However, it has been successful against aflatoxins
when delivered at a high dosage and in a rapid manner (McKenzie
et al., 1997).
E. OTHER TECHNIQUES
Ultraviolet light, high-energy electron beams, hypochlorous acid,
hydrogen peroxide, and steam cleaning are other techniques that are
either used commercially as general sterilizing agents or have been
shown to be effective to a degree against mold conidia and or mycotox-
ins. There are many other agents that potentially can be very efficient
fungicides.
A recent study (Górny et al., 2002) showed that fungal colonies can
aerosolize large numbers of antigenic fungal fragments simulta-
neously with fungal conidia into the environment. These fragments
were shown to be much smaller than fungal conidia. It is possible that
these fragments also carry mycotoxins and that they contribute to
fungal contamination of contents. The cleaning techniques discussed
above may be effective against fungal fragments, but this remains to be
examined.
V. Post Cleaning
After cleaning/remediation has been performed, our practice is to
inform the occupants involved that they are the final judges as to the
success of the cleaning technique. This is for two reasons: first, because
of the limitations of current testing techniques, it is possible that
biological (e.g., human) indicators can be more sensitive as indicators
of contents contamination; and second, variation between people in
terms of sensitivity to mold contamination is addressed. Unfortunately,
this approach cannot distinguish between a genuine sensitivity to
mold contamination and an imagined sensitivity. The psychological
issues involved in mold contamination may interfere significantly with
judgments on the efficacy of mold remediation techniques.
After contents are cleaned they will consequently be exposed to
fungal conidia, hyphae, and perhaps mycotoxins from the remediated
environment. Ideally this will only be a background level and impor-
tantly, the types of conidia, etc., will differ from those that were in the
pre-remediated environment.
VI. Conclusion
The obstacles regarding the determination of levels of conidia/
mycotoxins and fungal fragments on contents are such that it is possi-
ble we will never be able to know what those levels are. The strategy we
promote in light of this major deficiency is to assume that the contents
can potentially have high numbers of conidia/mycotoxins/fragments
and to use a cleaning technique on them. No technique is perfect,
although a number will inactivate conidia and mycotoxins from a wide
range of organisms. The occupant should use the contents after clean-
ing and make a personal decision as to the efficacy of the cleaning
technique.
ACKNOWLEDGMENTS
The authors would like to thank the staff of the Center for Indoor Air Research for their
invaluable help and the staff at Assured Indoor Air Quality Ltd, Dallas, for their field data
and financial support.
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Chapman, J. A. (2003). Stachybotrys chartarum (chartarum = atra = alternans) and other
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W. P., Voss, K. A., Plattner, R. D., Kubena, L. F., and Phillips, T. D. (1997). Oxidative
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Specific Detection of Fungi Associated
With SBS When Using Quantitative Polymerase
Chain Reaction
PATRICIA CRUZ AND LINDA D. STETZENBACH
Harry Reid Center for Environmental Studies
University of Nevada, Las Vegas
Las Vegas, Nevada 89154
I. Introduction 437
II. Quantitative Polymerase Chain Reaction (QPCR) 439
III. Primer Selection 440
A. Primer Design 440
B. Optimization and Sensitivity 441
C. Specificity Testing 441
IV. Sample Collection and Processing 442
V. DNA Extraction and Purification 442
A. PCR Inhibition 443
VI. DNA Amplification 444
VII. Quantitation Standards 444
VIII. Conclusion 446
References 447
I. Introduction
Fungi are ubiquitous in indoor and outdoor environments, occupying
natural and manmade habitats (Stetzenbach, 1997). Paint, wallpaper,
cellulose products, and wood derivatives can be colonized and dam-
aged by fungi, especially under humid or wet conditions. Colonization
and dispersal of fungi in indoor environments such as the workplace,
schools, and living quarters provide an environment with an increased
possibility of occupant exposure and adverse health effects (Buttner
et al., 2001). In addition, toxins produced by certain molds can cause
health effects on direct contact with the skin or when they are inhaled
or accidentally ingested (Burge and Otten, 1999). Rapid identification
and quantitation of fungi in indoor environments are necessary
for assessment of contamination levels and to estimate the exposure of
occupants. However, monitoring is hampered by the scarcity of methods
that provide precise, accurate, and representative exposure estimates
for airborne and surface-associated fungi (ACGIH, 1999). Traditional
monitoring for fungi and other biocontaminants relies on sampling the
air and surfaces followed by analysis of samples either microscopically
or by culturing on artificial growth media. Culture-based analyses are
437
0065-2164/04 $35.00
438 CRUZ AND STETZENBACH
limited to detection of those microorganisms that proliferate under the

nutrient and temperature conditions provided in the laboratory (Buttner
et al., 2002). In addition, faster-growing fungi may out-compete slower-
growing fungi, and it may take days or weeks to culture the organisms of
interest. Culture-based methods underestimate the concentration of
biocontaminants because only culturable cells are enumerated and
identified, while those that are not culturable remain undetected (Cox,
1989). However, some fungi are capable of causing health effects wheth-
er they are in the culturable or the non-culturable state (Levetin, 1995).
Microscopy-based assays can be used to detect total fungal populations
in a given sample regardless of the physiological state of the organisms,
thereby permitting the detection of both viable and nonviable cells.
However, these analyses are imprecise, laborious, and time-consuming,
and identification is generally limited to the genus level (Buttner et al.,
2002). In addition, microscopic analyses of fungal cultures are
essentially subjective, making intra-laboratory comparisons difficult
(Stetzenbach, 2002). The inaccuracy of traditional methods and the long
analytical time required to characterize bioaerosol and surface contam-
inant concentrations by these methods underscore the need to develop
new analytical techniques that can provide fast, reliable data for fungal
exposure monitoring (Buttner et al., 2001).
Methods for enhanced detection of fungi and other microorganisms
have been developed that increase the sensitivity and specificity of
detection while reducing analysis time (Buttner, 2002; Stetzenbach,
2002). Polymerase chain reaction (PCR) is an enzymatic process capa-
ble of rapidly amplifying specific DNA sequences (Saiki et al., 1985).
This technique can be used to detect specific microorganisms by am-
plifying DNA sequences unique to the organism of interest regardless
of the physiological state of the organism. This is especially useful in
the detection of slow-growing organisms, non-culturable organisms, or
those that are difficult to grow in the laboratory (Buttner, 2002). For
example, PCR has been used to detect aerosolized bacteria when cul-
ture results were negative (Alvarez et al., 1994). PCR has been shown to
enhance detection of fungi in pure culture and/or collected from sev-
eral matrices (Bock et al., 1994; Haugland and Heckman, 1998; Kappe
et al., 1996; Makimura et al., 1994; Spreadbury et al., 1993). Conven-
tional PCR analysis requires the use of agarose gel electrophoresis with
ethidium bromide staining for visualization of results, quantitates
the target organism based on end-point detection, and estimates the
amount of template from the band intensity of the gel (Alvarez
and Toranzos, 1997). Limitations of conventional PCR include the
necessity of post-PCR manipulations and its semi-quantitative nature.
SPECIFIC DETECTION OF FUNGI USING QPCR 439
In addition, DNA size markers eletrophoresed in parallel with the
target amplicon do not constitute definite proof of target amplification.
A fluorogenic nuclease assay in conjunction with a fluorescence
detector was developed as a means to amplify and quantitate PCR
products, eliminating the need for post-PCR manipulations (Heid
et al., 1996). This technology also has a high degree of sensitivity
because it is capable of detecting as little as one cell of the target
organism and offers a linear range of five orders of magnitude. Quanti-
tation is more accurate and precise than end-point methods, and the
process is amenable to high sample throughput. Fungi such as Asper-
gillus fumigatus (Cruz-Perez et al., 2001a; Loeffler et al., 2000) and
Stachybotrys chartarum (Cruz-Perez et al., 2001b; Haugland et al.,
1999) have been detected and quantitated by using real-time quantita-
tive PCR (QPCR). The use of real-time PCR continues to gain accep-
tance in the scientific community for the detection of microorganisms
as more genome sequences become available in the genetic data-
bases and more primer and probe sequences are developed for targeted
microorganisms.
II. Quantitative Polymerase Chain Reaction (QPCR)

To use the PCR technique for the detection of a particular microor-
ganism, DNA sequence information is obtained and sequences unique
to the microorganism or group of microorganisms must be identified.
Oligonucleotide primers are selected, synthesized, and ` then tested for
specificity, selectivity, and sensitivity. The TaqManE real-time QPCR
utilizes a fluorescently labeled oligonucleotide probe that anneals be-
tween the selected primers as the amplification reaction proceeds,
allowing for the determination of starting copy number of target DNA
(Sequence Detection System; Applied Biosystems, Foster City, CA).
This probe also provides confirmation of the product because it will
only be cleaved and, therefore,
` fluoresce in the presence of the target
organism. The TaqManE assay has been previously validated for the
detection of bacterial DNA (Bassler et al., 1995; Desjardin et al., 1998;
Nogva and Lillehaug, 1999) and has been demonstrated to rapidly and
accurately detect fungi in indoor environments (Cruz-Perez et al.,
2001a,b; Haugland et al., 1999; Leenders et al., 1999; Vesper et al.,
2000). Other QPCR instruments rely on the use of the fluorescent dye
SYBR Green or two fluorescently labeled probes that serve as hybridi-
zation probes to the double-stranded DNA amplified (LightCycler;
Roche, Indianapolis, Indiana; R.A.P.I.D. System; Idaho Technology,
Inc., Salt Lake City, Utah). These instruments have been utilized in
the detection of Aspergillus fumigatus and Candida albicans (Loeffler

et al., 2000), Staphylococcus aureus (Hein et al., 2001) and Bacillus
anthracis (Higgins et al., 2003).
III. Primer Selection

A. PRIMER DESIGN
Primers may be designed for the detection of a given fungal genus
(genus-specific primers) or for the detection of a single fungal species
(species-specific primers). The nuclear small-subunit ribosomal DNA
sequences (i.e., 18S rRNA gene) may be used for the design of genus-
specific primers because they contain sequences that are highly con-
served among members of the same genus but are highly variable
between different genera. The internal transcribed spacer regions and
intergenic spacer of the nuclear rRNA gene may be used for the design
of species-specific primers because they are highly variable within a
genus or among populations (White et al., 1990). The design of species-
specific primers is a difficult task because of the similarity (homology)
in the DNA of closely related species. The first step in the design of
primers and probes is to find a DNA region unique to the organism(s) of
interest. DNA sequences may be obtained from a DNA repository such
as GenBank and compared against all other sequences available with a
sequence homology computer program such as the Basic Local Align-
ment Search Tool algorithm (BLAST) (Altschul et al., 1990). The
unique DNA region is then imported into a primer design software
package such as Primer Express (Applied Biosystems). Primer design
programs are based on the parameters selected for melting temperature,
amplicon size, base content, and primer/probe length. Primers and
probes should be designed following the recommendations of the soft-
ware package manufacturer as well as those suggested by the manufac-
turer of the QPCR thermocycler to be used for amplification. One or
more primer/probe sets are generally produced by the computer
program, with a score indicating the relative probability of success of
the set. Primer and probe sequences may also be found in the scientific
literature; however, care must be taken to identify the primer set as
being compatible with QPCR and to corroborate that the primers have
been extensively validated for their specificity and sensitivity. This
validation consists of testing numerous strains of the target organism
as well as related and non-related microorganisms to determine cross-
reactivity and the lower detection limit of the primer set. Custom-made
primers and probes can be obtained from any of many commercial
molecular biology sources in a variety of concentrations (yield scales)
and purifications.
B. OPTIMIZATION AND SENSITIVITY

The amplification reaction should be optimized for maximum ampli-
fication of DNA extracted from the target organism. It is suggested that
primer optimization consists of testing combinations from 50 nM to
900 nM concentrations for the forward and reverse primers with DNA
from the target organism. When more than one primer set is being
tested for the same target organism, the sensitivity of detection may
be a useful tool in the selection of one primer set over the other.
Dilutions of control DNA are amplified with both primer/probe sets
to determine which set has a lower detection limit (i.e., which set is
more sensitive). To corroborate the specificity of the primer set, the
DNA from several different strains or isolates of the target organism
should be amplified. It is advised that prior to initiating DNA extrac-
tion experiments the identity of the target organism be confirmed by a
mycologist. Otherwise, a sample of the organism of interest may be
obtained from a reputable source or from a culture repository such as
the American Type Culture Collection (ATCC; Manassas, VA).
C. SPECIFICITY TESTING
Although a sequence comparison may demonstrate that the designed
primer set will amplify only the target organism, it is imperative that
the primers and probe be challenged in the laboratory with DNA
extracted from other (non-target) organisms to confirm that they will
not cross-react with non-targets. Closely related as well as distant
organisms should be included in these validation tests. Fungal spores
may be collected for DNA extraction by swabbing the surface of a
colony (Cruz-Perez et al., 2001b), flooding with buffer, and agitating
the agar plate where the colony is growing (Crow et al., 1994), or
inverting onto a funnel and tapping the agar plate where the colony
is growing (Cruz-Perez et al., 2001a). Following extraction, the fungal
DNA from each challenge organism is subjected to QPCR amplification
with the designed primers and probe for the target organism. If the
primer set is specific for the target organism, it will not amplify the
DNA from the organisms selected for specificity testing. To corrobo-
rate that a negative QPCR result is due to the absence of cross-reactivity
and not to the absence of DNA, a method must be used to deter-

mine the presence of DNA on specificity testing samples. This may be
accomplished with an ethidium bromide dot quantitation method or by
determination of absorbance at 260 and 280 nm (Ausubel et al., 1995).
The appropriate controls must be included with these protocols.
IV. Sample Collection and Processing

Surface sampling methods in which the sample is resuspended in a
liquid, and air sampling that delivers the sample into a liquid or onto
a filter, are compatible with PCR amplification. Samples collected
with bioaerosol samplers such as liquid impactors, liquid impingers,
filter cassettes, and electrostatic precipitators may be analyzed by PCR
(Buttner, 2002). For the detection of fungi by QPCR, blood specimens
(Loeffler et al., 2000), water samples (Brinkman et al., 2003), wine
(Phister and Mills, 2003), and air samples collected by filtration and
liquid impingement (Vesper et al., 2000) have been used. DNA extrac-
tion methods developed for field sample processing should be capable
of processing a sample volume that is representative of the original
sample. Research has shown that concentration by filtration onto a
filter membrane of large volumes of liquid samples (i.e., 9 to 30 ml)
and subsequent elution of spores from the filter into a smaller volume
(i.e., 500 l) increases the sensitivity of detection of the target organ-
ism by PCR (Buttner et al., 2001). A disadvantage of maximizing
target detection in this fashion is that although sample concentration
will increase the amount of DNA in the PCR reaction, it may also
increase the amount of PCR inhibitors present. Therefore additional
manipulations may be required for the removal of PCR inhibitors prior
to amplification.
V. DNA Extraction and Purification

Several methods have been tested for fungal DNA extraction. Van
Burik and coworkers (1998) compared mechanical disruption by using
bead beating, grinding with a mortar and pestle, sonication, and enzyme
lysis. Other researchers have used detergents (Velegraki et al., 1999);
heat (Foster et al., 1993); a combination of detergent, enzyme lysis, and
heat (Cruz-Perez et al., 2001a,b); and freeze-thaw (Kutchma et al., 1998).
It is recommended that the DNA extraction protocol be followed by a
DNA purification procedure to eliminate or reduce any PCR inhibitors
that may be present. This applies not only to environmental or field
samples but also to pure cultures, because it has been demonstrated
that fungal isolates such as Aspergillus fumigatus and Stachybotrys
chartarum contain PCR inhibitors (Cruz-Perez et al., 2001a,b). Several
commercially available kits have been tested for concentration and
purification of fungal DNA (Cruz-Perez et al., 2001a,b). These methods
are based on a variety of principles to achieve DNA purification such
as alcohol precipitation, salt precipitation/filtration, silica mem-
brane spin columns, magnetic beads, anion exchange resins, and bind-
ing to glass particles. The effect of sample volume on the efficiency of
extraction/purification protocols in removing PCR inhibitors has been
studied, and the efficiency of removal of PCR inhibitors is dependent
on the volume of sample extracted (Cruz-Perez et al., 2001b). Proces-
sing low sample volumes may minimize PCR inhibition, but a low
percentage of the sample is amplified and the detection sensitivity is
reduced.
A. PCR INHIBITION
Several researchers have studied the agents that act as PCR inhibitors
(Lee and Cooper, 1995; Rossen et al., 1992; Wilson, 1997). Reagents
used in the laboratory, food constituents, and environmental back-
ground material are common PCR inhibitors. They generally act by
interfering with the DNA extraction process, degrading or trapping
the target DNA, or affecting the DNA polymerase that catalyzes the
amplification reaction. Samples may be purified or diluted in an at-
tempt to remove or reduce such` inhibitors. In either case, an internal
positive control (IPC) (IPC-VICE Probe, Applied Biosystems) may be
incorporated into the PCR reaction to avoid false-negative results and
determine whether the samples contain PCR inhibitors. This internal
positive control consists of control DNA and control primers and
probe. This fluorescent probe is labeled with a specific dye and should
be used only with reactions containing a target DNA probe labeled with
a different fluorescent dye to allow for the differentiation of fluorescent
signals generated during amplification. A known amount of positive
control DNA is amplified with the sample, and inhibition is observed
by a change in the quantitation of positive control DNA. The assump-
tion of the IPC is that the inhibitors affecting the target DNA will affect
the positive control DNA in a similar fashion. Sample dilution may be
used to minimize the effect of the inhibitors present, but this also
results in dilution of the target DNA and a reduction in the sensitivity
of detection.
VI. DNA Amplification

Polymerase chain reaction is the exponential, in vitro amplification
of DNA. For the reaction to proceed, the reaction mix must contain
fungal DNA template, buffer, MgCl2, deoxynucleotide triphosphates
(dATP, dCTP, dGTP, and dUTP), DNA polymerase, forward and re-
verse primers, and fluorescent probe. Reaction volumes typically range
from 10 to 50 l. Theoretically, the larger the DNA volume the greater
the possibility of detecting
` an organism with a low starting copy num-
ber. The TaqManE technology has an incorporated PCR carryover
prevention component that ensures that any amplified DNA acciden-
tally introduced in the reaction will be destroyed prior to initiation of
the amplification reaction.
It is recommended that an internal positive control complete with
DNA, primers, and probes be incorporated into the QPCR reaction to
determine whether samples contain PCR inhibitors. The IPC is normal-
ly obtained with a fluorescent probe containing a dye different from
that of the target DNA probe to allow for the differentiation of fluores-
cent signals generated during amplification. This constitutes a reaction
similar to that of multiplex PCR and prevents false-negative results.
QPCR (i.e., TaqMan) cycling conditions generally consist of 2 min at
50 C (for the activation of the carryover prevention enzyme), 10 min
at 95 C (for the activation of the polymerase), and a two-step PCR
consisting of 40 cycles of 15 sec at 95 C followed by 1 min at 60 C
(for double-stranded DNA separation, annealing of the primers and
extension of the template DNA).
VII. Quantitation Standards

DNA amplification via QPCR results in the production of a fluores-
cent signal that is monitored at each cycle of the amplification reaction
(Fig. 1, see color insert). The fluorescence generated is proportional
to the starting copy number of the target organism, and the results
are observed in real time. Using the concentrations assigned to each
standard, the software employed with the thermocycler constructs a
standard curve of Ct value versus concentration (Fig. 2, see color
insert). Ct refers to the PCR cycle at which fluorescence (i.e., amplifica-
tion product) is first detected and is inversely proportional to the initial
DNA template concentration. Concentration values for the unknown
samples are extrapolated from the standard curve by the software
and reported as the mean of two replicates. Various quantitation
strategies have been used with QPCR (e.g., competitive PCR, absolute
FIG. 1. DNA amplification plot of standards of known template concentration

monitored with a fluorescence detector. Fluorescence (y-axis) is monitored at each cycle
of the amplification reaction (x-axis).
FIG. 2. Standard curve of threshold cycle (Ct) value versus concentration. Ct refers to
the PCR cycle at which fluorescence is first detected; it is inversely proportional to the
initial DNA template concentration. Concentration values for the unknown samples (grey
circles) are extrapolated from the standard curve by the software and reported as the
mean of two replicates (black circles indicate standards).
quantitation, relative quantitation). The use of absolute standards re-

fers to amplification of a known concentration of target DNA and
allows for the most accurate quantitation of the target organism. The
preparation of absolute quantitation standards has been performed by
enumeration of spores with an electronic particle sizer, a methodology

that is faster and more accurate than microscopic enumeration methods
(Buttner et al., 2001; Cruz-Perez et al., 2001a,b). Absolute quantitation of
fungal DNA can be accomplished by obtaining a spore suspension of the
target organism, enumerating the suspension with a method capable of
measuring total (viable and non-viable) organisms, performing serial
dilutions of the spore suspension, extracting the DNA with the same
extraction protocol used for unknown samples, and amplifying the DNA
standards at the same time as the unknowns. Fungal spores are har-
vested from pure cultures of the target organism to prepare standards
of known concentration. Fungal colonies containing spores that tend to
clump may have to be harvested by flooding the plate, while powdery
colonies may only necessitate inversion of the plate onto a funnel. Care
must be taken, especially with spore harvests generated by agitation or
swabbing, to remove any spore and/or hyphal fragments present in the
spore suspension as they may interfere with the spore enumeration
process. Sucrose density centrifugation may be used for purification of
spore suspensions. Effectiveness of the cleaning method may be as-
sessed before and after cleaning by enumeration of the spore suspension
with an electronic particle counter. Fungal reference books (e.g., Pitt and
Hocking, 1997; Samson et al., 2000; Sutton et al., 1998) should be used to
determine an average spore-size range for the target organism. Once the
concentration of total spores per volume in the spore suspension is
determined, aliquots are serially diluted in buffer and the DNA is ex-
tracted. Standards (e.g., 100 to 105 templates/PCR reaction) are amplified
in duplicate or triplicate with replicate unknown samples. After ampli-
fication, the data are analyzed by using the software provided with the
thermocycler. The concentration of the standards is designated, and the
software constructs a standard curve of Ct value versus concentration.
Concentration values for the unknown samples are extrapolated from
the standard curve by the software and reported as the mean of two
replicates. To test the efficiency of the quantitation standards, additional
spore suspensions from the target organism can be enumerated with an
electronic particle counter and compared with QPCR quantitation.
VIII. Conclusion
The use of QPCR for the detection and enumeration of fungi of
interest in air and surface samples provides enhanced capabilities for
indoor monitoring. Additional work is needed to improve the removal
of PCR inhibitors and increase the applicability of QPCR for the detec-
tion of fungi in indoor environments. The use of QPCR in building
surveys will assist in the assessment of contamination in schools, office
buildings, and residences and provide data that can be used to develop
risk assessment and risk management guidance for building owners
and occupants.
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INDEX
A air samples from, 116–17, 118

colonization of filters in, 117–21,
Abatement, decontamination of molds in 298, 376
buildings, 415–18 colonization of in-duct insulation in,
AHU maintenance in, 418 120–26
drying in, 417 fungi in, 113–34
HEPA filters in, 417 hospital, 117–21
moisture resistant building materials metal surface colonization in,
in, 417 124–25
removal of building material in, 417 problem areas with, 115–17
ABPA. See Allergic bronchopulmonary VOC in, 125–26
aspergillosis Air handling unit (AHU), 415, 418
Absidia Alimentary toxic aleukia (ATA), 156
allergy associated with, 34 from Fusarium sporotrichioides, 284
infectious disease associated with, 43 Allergic alveolitis. See Hypersensitivity
Acanthamoeba pneumonitis
auto air-conditioning systems with, Allergic bronchopulmonary aspergillosis
131–133 (ABPA), 228
Acremonium Allergic penicilliosis
allergy associated with, 34 animal model developed for, 13–16
infectious disease associated with, 43 BAL fluid levels with, 14–15
moisture problem buildings with, 179 IgE serum levels with, 13–15
Acrogenospora intranasal induction of, 13–16
allergy associated with, 34 mice with, 13–16
Acrothecium viable v. non-viable Penicillium in,
allergy associated with, 34 13–16
Actinomycetes, 290 Allergies
Aflatoxins Alternaria causing, 34, 40, 41, 114, 281
from Aspergillus, 57, 155, 349 Aspergillus causing, 227–28
detection using DNA adducts, 380 fungi caused, 32–63
inactivation of, 431–33 IgE associated with, 32–33, 37, 226–27,
turkey X disease from, 277–78 311–12, 385–86
Agaricus incidence increase pf, 33
allergy associated with, 34 Th2 response, 228, 230
Agrocybe Alternaria
allergy associated with, 34 allergy associated with, 34, 40, 41, 114
Agrocybe aergerita, 192 asthma provoked by, 5, 33
AHU. See Air handling unit colonization of filters with, 118, 120
AIDS patients indoor air with, 11, 65, 68, 281
in hypersensitivity, 293 infectious disease associated with, 43
Air distribution systems. See also Heating, mycotoxins from, 51
ventilation, air conditioning occupational lung disease from, 7
451
452 INDEX
Alternaria (Cont.) cytolytic activity of, 200, 203, 204
occupational lung disease provoked by, low density lipoprotein receptor
7, 41, 181 mechanism of, 193
outdoor air with, 9, 11, 19–21, 281 molecular weight of, 193
Alveolar macrophage Aspergillosis, 50, 200, 228, 381
BAL fluid with, 17 allergic bronchopulmonary (ABPA),
clearance with, 16 228, 385
ultrastructure of, 17 Aspergillus, 227
Amanita aflatoxins from, 57, 155, 277–78
allergy associated with, 34 allergy associated with, 34, 37,
Amanita phalloides, 192 39–41, 228
phallolysin from, 192, 200 animal studies on, 227–29
Amanita rubescens asthma provoked by, 226
rubescenslysin from, 192, 199, 204 carpets with, 126–130
American Academy of Neurology, 361–62 colonization of filters with, 120
American Academy of Pediactrics ecological factors influencing, 78–80, 83,
Committee 84, 86, 90, 92–94
toxic mold syndrome, indoor indoor air with, 9, 11, 65–70, 226, 415
environment findings from, 284–85 indoor building materials with, 73
American Type Culture Collection infectious disease associated with,
(ATCC), 441 43, 50
ANA. See Antinuclear autoantibodies moisture problem buildings with,
Animal models, 266–67 178, 179
BAL fluid levels with, 14–15 molds, causing cognitive impairment
cellular responses in, 16–19 and, 369, 372, 396
development of, 13–16 mycotoxins from, 51, 52, 56, 57, 228
eosinophilia in, 15, 228 occupational lung disease provoked by,
humoral responses in, 16–19 7, 41
IgE serum levels in, 13–15 outdoor air with, 9, 11, 22
viable v. non-viable Penicillium in, proteolytic allergens from, 228–29
13–16 sick building syndrome (SBS) and,
Animal toxicosis 226–30
from Fusarium, 284 Aspergillus ustus, 378
from Stachybotrys, 157–60 Aspergillus versicolor, 280, 324, 351, 378
Animals respiratory symptoms from, 226
Stachybotrys influencing, 155–60 sterigmatocysin from, 280
Antifungals Asthma, 300, 311–32, 345. See also Molds,
in treatment of fungal hypersensitivity, indoor, causing asthma
293–94, 302–5 Alternaria causing, 5, 33
Antinuclear autoantibodies (ANA), 393 Aspergillus causing, 226
Armillaria bronchial hyperresponsiveness (BHR),
allergy associated with, 34 315, 316
Arthrinium damp buildings related to, 139,
allergy associated with, 34 309–11, 376
Arthritis Finnish Environment, Asthma Study,
in hypersensitivity, 299 319–27
Ascotricha IgE, as fungal marker for, 325
carpets with, 127, 129 ATA. See Alimentary toxic aleukia
Asp-hemolysin, from Aspergillus ATCC. See American Type
fumigatus, 193, 200 Culture Collection
INDEX 453
Atranones, from Stachybotrys chartarum Candida albicans, 193
in pathway inhibition, 242 detection of fungal species using QPCR
Aureobasidium and, 439–40
allergy associated with, 34 Cantharellus
infectious disease associated with, 44 allergy associated with, 34
moisture problem buildings with, 179 CBT. See Cholesterol binding toxin
Auto air-conditioning systems CCR2. See Chemoattractant
fungi in, 131–33 protein-1 receptor
CCT. See Cellulose-containing ceiling tiles
Ceiling tiles. See Cellulose-containing
B ceiling tiles; Inorganic ceiling tiles
Cellulose-containing ceiling tiles (CCT)
Bagassosis, 7, 297 fungi with, 22–24, 26
BAL. See Bronchoalveolar lavage Centers for Disease Control (CDC)
BHR. See Bronchial hyperresponsiveness mold exposure recognized by, 4
Bispora toxic mold syndrome, indoor
allergy associated with, 34 environment findings of, 284–85
Blood–brain barrier, 395 Chaetomium
Boletinellus allergy associated with, 34
allergy associated with, 34 colonization of filters with, 120
Boletus indoor building materials with, 73
allergy associated with, 34 Chartarum. See Stachybotrys
Botrytis Cheese washer’s disease. See Cheese
allergy associated with, 34 worker’s lungs
Bronchial hyperresponsiveness (BHR) Cheese worker’s lungs
molds, indoor causing asthma, Penicillium causing, 7–9, 41
315–16 Chemical Brain Injury, 342
Bronchoalveolar lavage (BAL), Chemoattractant protein-1 receptor
247, 384 (CCR2), 229
alveolar macrophage with, 17 Chemokines
fluid level increase of, 14–15, 19 C10, 229
Stachybotrys in, 168, 247–49, 261–62, chemoattractant protein-1 receptor
265, 267 (CCR2) and, 229
Building materials, 341, 355, 425. See also in sick building syndrome (SBS),
Cellulose-containing ceiling tiles; 229, 384
Inorganic ceiling tiles Chlorophyllum
fungi in, 72–74 allergy associated with, 34
Stachybotrys, toxins in, 164–65 Cholesterol binding toxin (CBT), from
Streptococcus, 198, 204
Chronic obstructive pulmonary disease
C (COPD), 300
Chronic rhinosinusitus (CRS), 381
Calvatia Chrysolysin, from Penicillium
allergy associated with, 34 chrysogenum, 193, 206
Cancer Chrysosporium
mycotoxins and, 26, 377 indoor air with, 9, 11
Candida infectious disease from, 45
allergy associated with, 34 outdoor air with, 9, 11
infectious disease associated with, Cladosporium
44–45 allergy associated with, 34, 37, 39
454 INDEX
Cladosporium (Cont.) Corn meal agar (CMA), 164
ceiling tile with, 23–25 CRS. See Chronic rhinosinusitus
colonization of filters with, 120 Cryptococcus
ecological factors influencing, 78–82, 84, allergy associated with, 34
88, 90, 91, 95 infectious disease from, 45, 50
indoor air with, 11, 12, 65–68, 70, Cryptostroma
281, 415 allergy associated with, 34
indoor building materials with, 73 Cunninghamella
infectious disease associated with, 45 allergy associated with, 34
moisture problem buildings with, 179 infectious disease from, 43
mycotoxins from, 51, 57 Curvularia
outdoor air with, 9, 11, 12, 19–21, 281 allergy associated with, 34, 38
respiratory distress from, 6 Cytokine release
VOC from, 60 with hemolysins, 203–4
Cladosporium cladosporioides, 257, in hypersensitivity, 297
324, 351 with interleukins, 203–4, 228, 230, 243,
proteases in, 257 260, 384–85
Cladosporium herbarum, 325 Cytolysins. See Hemolysins
Claviceps
allergy associated with, 34
mycotoxins from, 51, 52, 278 D
Claviceps fusiform, 278
Claviceps purpura, 278 Dacrymyces
Cleansing/sterilization agents allergy associated with, 34
ethanol, 432 Daldinia
gamma-irradiation, 432 allergy associated with, 34
hydrogen peroxide, 433 Debaryomyces
ozone, 432–33 allergy associated with, 34
sodium hypochlorite, 431–32 Deoxynivalenol (DON), 166, 244
ultraviolet light, 433 macrophages and, 245
Clearance Detection of fungal species using QPCR,
definition of, 16 437–47
fungi’s impact on, 18 for Aspergillus fumigatus, 439–40
CMA. See Corn meal agar assay sensitivity in, 439, 441
Cognitive impairment associated with assay specificity in, 441–42
molds. See Molds, causing benefits of, 446–47
cognitive impairment for Candida albicans, 439–40
Colony forming units (CFU), 414 DNA amplification methodology in, 444
Conidia burden studies DNA extraction/purification methods in,
molds, remediation/monitoring and, 442–43
428–29 DNA primer design in, 440
Coniosporium DNA primer design software for,
COPD. See Chronic obstructive fluorogenic nuclease assay in, 439
pulmonary disease GenBank DNA repository for, 446–47
Coprinus internal positive controls in, 443, 444
allergy associated with, 34 limitations of cultural analyses,
infectious disease associated with, 45 437–38
Coriolus limitations of traditional PCR, 438
allergy associated with, 34 PCR inhibitor contamination in, 443
INDEX 455
quantitation standards used in, 444–46 Exophiala
sample collection methods in, 442 infectious disease associated with, 46
unique DNA sequence information used moisture problem buildings with, 178
in, 439 Extracellular polysaccharides (EPS),
Dicoccum 391–92
Didymella
allergy associated with, 35 F
Differential diagnosis, 361
DNA adducts, 380 Farmer’s lung, 7, 41, 297, 383
DON. See Deoxynivalenol Finnish Environment, Asthma Study,
Drechslera 319–27
allergy associated with, 35 environmental analysis of buildings
infectious disease associated with, 45 in, 312
IgG v. IgE biomarker analysis in, 312–13
visual inspection of buildings in, 312
E Flammulina velutipes
flammutoxin from, 200, 203, 204
ECRHS. See European Community Flammutoxin
Respiratory Health Survey cytolytic activity of, 200, 203, 204
EDS. See Exodigestive enzymes from Flammulina velutipes, 200
Endotoxin Fluconazole
from Enterobacter, 144 therapy, for fungal hypersensitivity, 302
Limulus test for, 351 Fomes
from Pseudomonas, 144–45 allergy associated with, 35
SBS with, 144–49, 148 Fonsecaea
Enterobacter infectious disease from, 46
endotoxin in, 144 Formaldehyde, 341
Environmental tobacco smoke (ETS), 266, Freon, 340
321, 323 Fuligo
Eosinophilia/neutrophilia allergy associated with, 35
mice with, 15, 228 Fungal hypersensitivity. See
in SBS, 219, 221, 222, 228 Hypersensitivity, fungal
Epicoccum Fungi
allergy associated with, 35 air distribution systems with, 113–34
Epidermophyton air movement influencing, 82
allergy associated with, 35 airborne, 64–72
infectious disease associated with, 46 allergies from, 32–63, 182–83, 186
EPS. See Extracellular polysaccharides Alternaria, 5
Ergosterol, 312 asthma from, 33
Erysiphe auto air-conditioning systems with,
Escherichia coli building characteristics influencing,
repeats-in-toxin (RTX) from, 198 86–91
ETS. See Environmental tobacco smoke building materials with, 72–74
European Community Respiratory Health carpets with, 126–131
Survey (ECRHS), 315–17 changes in, 91–94
Eurotium Cladosporium, 6
allergy associated with, 35 clearance influenced by, 16, 18
Exodigestive enzymes (EDS), 376 clinical outcomes from, 180–82
456 INDEX
Fungi (Cont.) moisture problem buildings with,
contamination from, 31–97 178, 179
ecological factors of, 78–94 mycotoxins from, 51, 52, 56, 282
epidemiology of symptoms from, Fusarium oxysporum, 324
179–80 Fusarium sporotrichioides, 284
exposure studies on, 94–96
glucans in, 62–63, 267, 311, 312, 329,
376, 391 G
growth on ceiling tiles of, 22–23
hospitals with, 117–21 Ganoderma
human activity influencing, 85 allergy associated with, 35
human health impacted by, 3–27, Geastrum
32–63 allergy associated with, 35
humidity and, 142 GenBank DNA repository
HVAC with, 6, 12, 87, 90–91, 114 for detection of fungal species using
IAQ with, 4, 63–78 QPCR, 440
identification of, 63–64 Geotrichum
indoor biodiversity of, 74 allergy associated with, 35
infectious diseases from, 42–50 indoor air with, 66–67
inhalation of, 5–9 infectious disease associated with, 46
introduction to, 3–4, 31–32 Gibberella
Leviticus and, 3, 215, 345, 375–76 allergy associated with, 35
light influencing, 81–82 Gliocladium
moisture and, 78–79, 175–87, allergy associated with, 35
300, 417 -D-glucan
mycoses from, 42–49 microbial cell wall agents (MCWA),
mycotoxins produced by, 17, 50–59, 147–49, 242, 264, 267, 311–12,
115, 282 329, 391
ODTS from, 183 Glucans
PCR studies of, 77–78 fungi with, 62–63, 267, 311–12, 329,
Penicillium, 4–8 376, 391
physical factors of, 78–85 Gnomonia
pollen skin tests and, 4 allergy associated with, 35
relative humidity influencing, Graphium
SBS correlated with, 3–5, 9–12,
23–24, 31–97
Stachybotrys, 5, 7, 8, 74–76 H
Stachybotrys atra, 11
structure of, 276 Head-space solid-phase microextraction
substrates influencing, 82–85 (HS-SPME), 60
succession in, 91–94 Heating, ventilation, air conditioning
temperature influencing, 79–81 (HVAC), 409–10, 412–13, 415, 419
toxic reactions from, 183–84 colonization of filters in, 120,
ultraviolet lights influencing, 87 298, 376
VOC produced by, 17, 59–62, 312 fungi in, 6, 12, 87, 90–91, 114
wallboard with, 126–31 preventive maintenance of, 12
Fusarium Helminthosporium
allergy associated with, 35 allergy associated with, 35
infectious disease associated with, 46 infectious disease associated with, 46
INDEX 457
Hemmorhagic pneumonitis, 345 HEPA filter, 300
Hemolysins, bacterial, 198 High-efficiency particulate air filter. See
beta-sheet-structured (BSS), 198 HEPA filter
cholesterol binding toxin (CBT) form Histamine release (HRT)
of, 198 allergy with, 37
cytokine release with, 203–4 with hemolysins, 203
cytolytic activity, 201–2 Histoplasma
fungal forms, comparison with, infectious disease associated with, 46
198–99 Histoplasmosis, 46
histamine release with, 203 HIV. See Human immunodeficiency virus
repeats-in-toxin (RTX) form of, 198 HP. See Hypersensitivity pneumonitis
vascular tissue damage with, 204 HRT. See Histamine release
Hemolysins, fungal, 377 HS-SPME. See Head-space solid-phase
, forms, 192–93 microextraction
aegerolysin, from Agrocybe Human immunodeficiency virus (HIV)
aergerita, 192 fungi influencing, 42
asp-hemolysin, from Aspergillus Human umbilical endothelial cells
fumigatus, 193, 200 (HUVEC), 200
biomarker exposure assays, 206–7 HUVEC. See Human umbilical
from Candida sp. yeasts, 193, 198 endothelial cells
chrysolysin, from Penicillium HVAC. See Heating, ventilation, air
chrysogenum, 193, 206 conditioning
comparison with bacterial forms of, Hypersensitivity, fungal, 289–305,
198–99 376, 385
cytokine release with, 203–4 AIDS patients and, 293
exposure modes with, 205 antifungals in treatment of, 293–94,
flammutoxin, from Flammulina 302–5
velutipes, 200, 203–4 arthritis and, 299
histamine release with, 203 bacterial v., 290
inflammatory response activation in, 201 characteristics of fungi in,
iron release from RBCs with, 206 292–93
molecular weights of, 192 cytokine release in, 297
ostreolysin, from Pleurotus diagnosis of, 293–96
ostreatus, 192 environmental molds in, 300
phallin, 192 foodstuffs and, 277–78, 300–1
phallolysin from Amanita phalloides, IgE role in, 297–98, 311, 376
192, 200 immune complex disease and, 296,
properties of, 199 298–99
rubescenslysin from Amanita rubescens, immune response in, 290–91,
192, 199, 204 293, 376
SBS, possible involvement with, 191–92 Jarisch Herxheimer reaction in, 304
SBS, symptoms comparison with, mold colonization in, 300–1
201–4 mycotoxins in 277–79, 291, 296–97,
secretion at variable time/temperature, 299, 311
194–97 serum sickness in, 296
stachylysin, from Stachybotrys skin hypersensitivity, type I in, 228
chartarum, 193, 200–1, 205, 242, susceptibility to, 294–95
255–56, 267, 377 symptoms of, 294–96
vascular tissue damage with, therapies for, 297–305
204–5 trichothecenes in, 296–97
458 INDEX
Hypersensitivity pneumonitis (HP), continually measured fungi profiles in,
376, 383 19–22
cheese washer’s disease as, 7, 41 formaldehyde in, 333–334, 344
Hypholoma fungi influencing, 4, 63–78
allergy associated with, 35 glutaraldehyde with, 344
leakage/flooding affecting, 310–11, 320,
341, 367, 411
I mycotoxins and, 56
neurobehavioral testing, 342–44
IAQ. See Indoor air quality Penicillium in, 9, 11–12, 19–21,
ICT. See Inorganic ceiling tiles 65–70
IgA. See Immunoglobulin A physiological testing, 341–42
IgE. See Immunoglobulin E reports of, 4
IgG. See Immunoglobulin G sources of moisture affecting, 310–11
IgM. See Immunoglobulin M spirometry testing, 342
IL. See Interleukins symptomology in, 341
Immune complex disease, 394 tight buildings and, 340–42
IgG hypersensitivity in, 298–99 VOC from, 339
Immune response to molds, 297–99, Inonotus
302–5, 376. See also Hypersensitivity, allergy associated with, 35
fungal; Molds, influencing Inorganic ceiling tiles (ICT)
neurological/immune systems composition of, 22–23
T/B cell response in, 392–94 fungi with, 22–24, 26, 427
Immunoglobulin A (IgA), 244, 349, 385 Interferons, 203–4
Immunoglobulin E (IgE) Interleukins (IL), 203–4, 228, 230, 243,
allergy caused by, 32, 311–12, 385–86 260, 384–85
Aspergillus influencing, 226–27
as cross-reaction marker, 218–22, J
226–27, 229, 243
floor dust influencing, 151 Jarisch-Herxheimer reaction
as fungal marker for asthma, 325 in fungal hypersensitivity, 304
in hypersensitvity, 297, 311, 376
mice with, 13–15, 19
Stachybotrys influencing, 75, 257, 258 K
Immunoglobulin G (IgG)
IgG serum in mice and, 13–15 Ketoconazole
as marker for fungal exposure, 297–98, therapy, for fungal hypersensitivity, 303
312–13, 323–25, 385–86
Stachybotrys influencing, 75–76 L
Immunoglobulin M (IgM)
as biomarker, 349, 385–86 L-DLR. See Low-density
Indoor air quality (IAQ), 280–81, 313–28, lipoprotein receptor
351–52, 376, 412 Langerhans cell
Alternaria in, 9, 11, 19–21, 281 activation by Aspergillus fumigatus,
Aspergillus clavatus in, 9, 11, 22 224, 229–30
building materials affecting, 72–74, Leakage/flooding, affecting indoor air
130–31, 162–65, 310–11, 355 quality, 310–11, 320, 341, 367, 411
Cladosporium in, 9, 11–12, 19–21, 281 Legionella, 290
cleaning agents in, 344, 431–33 Leptosphaeria
climate influencing, 70–71, 309–10 allergy associated with, 35, 38
INDEX 459
Leptosphaerulina Moisture problem buildings, 310–11.
allergy associated with, 35 See also Finnish Environment,
Leviticus, on fungi, 3, 215, 345, Asthma Study
375–76 allergies from mold in, 182–83, 186
Lipopolysaccharide (LPS), 145 clinical outcomes of mold from, 180–82
Low-density lipoprotein receptor disease diagnosis from mold exposure
(L-DLR), 193 in, 184–87
LPS. See Lipopolysaccharide energy crisis influence on, 176
Lung diseases epidemiology of mold symptoms from,
occupational, 7–9, 41 179–80, 341
Lycoperdon Finland with, 175
allergy associated with, 35 health outcomes from, 179–84, 181, 182
high percentage of, 175–76
introduction to, 175–77
M microbiology of, 176–79
molds in, 175–87, 300, 419
Malassezia ODTS from, 183
allergy associated with, 35 Stachybotrys in, 183
infectious disease associated with, 47 toxic reactions from, 183–84
Malt extract agar (MEA), 164, 413 Molds
Maltster’s lung, 7 antifungals and, 302–5
MCWA. See Microbial cell wall agents in environment, 300
MEA. See Malt extract agar in foodstuffs, 300–1
Memnoniella immune response to, 297–98,
mycotoxins from, 51–52 302–5, 376
Merulius overview of, 275–76
allergy associated with, 35 toxicity, effect on health of,
Microbial cell wall agents (MCWA) 277–79, 299
children influenced by, 148–50 Molds, causing cognitive impairment,
endotoxin, 144–49 361–73, 396–97
examples of, 143 Aspergillus and, 369, 372
-D-glucan, 147–49, 242, 264, 267, detection of dysfunction from,
311–12, 329, 391 362–63, 367
historical perspective on, 139–40 differential diagnosis and, 361, 366
indoor microbial contamination and, dose response to exposure in, 367–68
142–43, 143 emotional disorders and, 364–65
inflammation and, 143–44 epidemiological studies/exposure in,
PAMP and, 144 367–68
SBS and, 139–51 logical chain deduction in diagnosis
TLR and, 144 of, 366
treatment for, 150–51 mycotoxins and, 362–63, 367
Microbial volatile organic compounds neuropathology and, 362
(MVOC), 243, 311, 325, 412 neuropsychological assessment and,
analysis of, 243, 258–59 361–66, 370–73
Microsphaera normal intelligence assessment tools in,
Microsporum Penicillium and, 369, 372
allergy associated with, 35 scholastic aptitude tests and, 362–63
infectious disease associated with, 47 Stachybotrys atra and, 369, 372
Mitogenesis, 391–393 Stachybotrys chartarum and, 372
460 INDEX
Molds, causing cognitive lymphocyte marker changes with,
impairment (Cont.) 391–92
statistical treatment of study data with, mitogenesis changes with, 391–93
370–71 mycotoxins in, 378–80, 379
studies on exposure to, 369–73 neurological responses/abnormalities to,
symptomology of, 368–69, 371–73 394–98
TBI patients, compared to, 369–71, 373 neurophysiological tests for, 396–97
Molds, indoor, causing asthma. ochratoxins in, 380
See also Asthma pulmonary function testing for,
bronchial hyperresponsiveness (BHR) 383–84
and, 315–16 symptoms/frequency in, 376, 380–83
damp buildings related to, 139, T/B cell response, 392–94
309–11, 376 water damage and, 375–80
exposure assessment for, 311–13, 330 Molds, mycotoxins, role in SBS, 25–27,
Finnish Environment, Asthma Study, 51–52, 56, 165–66, 339, 352–53. See
319–27 also Sick Building Syndrome (SBS)
fungi associated with, 5–6, 33 assay sensitivity and, 351
in-home cross-sectional studies with, biomarker assays in, 206–7, 349, 352
313–316 building materials and, 355
in-home prevalent case-control studies causal evidence of, 350–51
with, 316–17 chemical sensitivity and, 340
mechanisms of, 311 cleaning agents with, 344, 433–35
mycotoxins in, 325 dust and, 354, 432
occurrence worldwide, 309–10 formaldehyde with, 333, 341, 344
Penicillium causing, 5–6, 33 freon with, 340
reasons for problems with, 310–11 growth in buildings of, 350
severity study of, 328–29 human correlation studies on, 252,
study design considerations for, 330–32 255–56
workplace cross-sectional studies with, immunopathology of trichothecenes
317–19 from, 244, 265
workplace incident case-control study indoor environments with, 278–81
with, 319–21, 323–25 inflammatory indices in, 250
workplace intervention studies of, 329 inhalation exposure to, 243–44, 430
Molds, influencing neurological/immune introduction to, 155–56
systems leukocyte apoptosis from, 245, 378
aflatoxins in, 380 lymphocyte proliferation by
antibody markers for, 391–92, 397 trichothecenes, from, 244
aspergillosis from, 381 microscopy for, 163
Aspergillus and, 396 moisture problem buildings with,
autoantibody creation by, 393 178–79, 183
blood-brain barrier and, 395 MTT bioculture assay and, 350, 352
chronic rhinosinusitus from, 381 MVOC from, 243, 258–59, 410
clinical/analytical tests employed, physiological tests with, 349
377–78 potency of trichothecenes, from, 241–51,
cross-reactivity markers for, 391–92, 397 258–60
demyelination due to, 397–98 probable pathogenicity of, 353–55
health/environmental history studies prognosis for, 349–50
on, 377–80 protein synthesis inhibition by
immune system response to, trichothecenes from, 241–42,
384–91, 394 244, 257
INDEX 461
proteins/proteinases in spores from, sample culturing in, 413–14
256–57, 260–61, 267, 377 sampling air/surfaces in, 413, 427
psychological evaluation with, 348–49 sporulation studies in Stachbotrys
pulmonary effect in animals, 241–44 and, 427
sampling tests for, 349, 351 Stachybotrys and, 414
SBS symptoms correlated with, 5, 205 symptoms resulting from, 411–12
SBS with, 155–169 Monilia
SEM evaluation of, 164 allergy associated with, 35
spore characterization in, 251, MTT bioculture assay, 350, 352
259–61, 264 Mucocilliary transport, 16
spore viability in, 251–52 Mucor
stachylysin from, 193, 200–1, 205, 242, allergy associated with, 35
255–56, 267, 377 indoor air with, 65
symptoms in, 263–64, 282, 345–48 infectious disease associated with, 43
TEM evaluation of, 164 Mucor racemosus, 325
toxic products from, 283 MVOC. See Microbial volatile
toxicity differences in, 161–62, 259–61 organic compounds
toxicosis case reports for, 157–60, Mycobiota, in water damage, 378
166–69, 252, 256 Mycogone
trichothecenes from, 241–42, 244, allergy associated with, 35
258–60, 280, 296, 349, 430 Mycoses, 376
veterinary medical literature with, definition of, 42
157–60 fungi causing, 42–49
view of, 7–8 Mycotoxicosis, 376
wall board with, 130–31, 427 Mycotoxins, 57, 228, 275, 277–78, 361,
Molds, remediation/monitoring, 409–20. 376. See also Molds, mycotoxins, role
See also Cleansing/sterilization agents in SBS
abatement, decontamination of, 415–18 in asthma, 325
air handling unit (AHU) considerations cancer and, 26, 377
in, 415 in cognitive impairment, 362–63, 367
allergic reactions with, 410 common, 51
building survey limitations and, diseases from, 53, 279
420, 426 fungi produced, 17, 50–59, 115
building survey results and, 414, 416, gliotoxin, 325
426–27 health significance of, 50–59, 301–2
cleansing/sterilization agents in, hepatotoxic effects of, 395
431–33 human studies with, 430
clearance testing for, 418–19 in hypersensitivity, fungal, 277–79, 291,
conidia burden studies and, 428–29 296–97, 299, 311
contamination concerns for, 426–31 indoor air and, 56, 115, 312, 351–52
fungal infections with, 410 nephrotoxic effects of, 395
HVAC in, 409–10, 412–13, 415, 419 neurological effects of, 395
indoor air quality evaluation for, 412–14 in neurological/immune systems,
indoor air quality results for, 414–15 378–80
moisture profiles in, 413 pathological effects of, 54–55
mycotoxins from, 410, 429–30 peptaibols, 325
NIOSH environmental survey in, in remediation/monitoring, 410, 429–30
410–12, 420 from SBS, 24–27, 51–52, 56, 165–66,
post-remediation activities in, 433–34 349, 352–53, 429–30
post-remediation measures for, 419–20 toxicity of, 26, 299, 395
462 INDEX
N P
Naematoloma Paecilomyces
National Institute of Occupational Safety infectious disease associated with, 47
(NIOSH), 411 PAMP. See Pathogen-associated
Natural killer cells (NK), 378, 392 molecular patterns
Neonatal intensive care unit (NICU), 127 Papularia
Neuropsychological assessment, allergy associated with, 35
361–66 Pathogen-associated molecular patterns
Neurospora (PAMP), 144
allergy associated with, 35 PCR. See Polymerase Chain Reaction
NICU. See Neonatal intensive care unit PCR inhibitors
Nigrospora in Aspergillus fumigatus, 443
allergy associated with, 35 in detection of fungal species, 443
NIOSH. See National Institute of in Stachybotrys chartarum, 443
Occupational Safety PDA. See Potato dextrose agar
NK. See Natural killer cells Peak expiratory flow (PEF),
Nystatin 319, 329
therapy, for fungal hypersensitivity, PEF. See Peak expiratory flow
302, 304 Pen ch allergen
from Penicillium chrysogenum, 218,
220–25
O protease inhibition of, 224
Penicillium, 379
OAQ. See Outdoor air quality allergic alveolitis from, 6, 219
Ochratoxins allergy associated with, 33, 36, 37,
detection using DNA adducts, 380 39, 41
Odds ratio (OR), in health surveys, 315, asthma provoked by, 5–6, 218
317, 319, 321–22, 324, 326–28 carpets with, 126
ODTS. See Organic dust toxic syndrome cheese worker’s lungs from, 7–9, 41
Oidium colonization of filters with,
OR. See Odds ratio, in health surveys ecological factors influencing, 78–80,
Organ transplants 83–84, 88, 90, 92, 93
fungi influencing, 42 HVAC colonization by, 6, 420
Organic dust toxic syndrome (ODTS) indoor air with, 11–12, 65–70, 218
moisture problem buildings with, 183 indoor building materials with,
Outdoor air quality (OAQ), 280–81 72–73, 353
Alternaria in, 9, 11, 19, 20–21, 281 infectious disease associated with, 47
Aspergillus clavatus in, 9, 11, 22 moisture problem buildings with,
Chrysoporium in, 9, 11 178–79
Cladosporium in, 9, 11–12, 19–21, 281 moisture with, 19
continually measured fungi profiles in, molds, causing cognitive impairment
19–22 and, 369, 372, 396
Penicillium in, 9, 11–12, 19–21 mycotoxins from, 25–26, 51–52, 56,
sources of moisture affecting, 57, 59
310–311 outdoor air with, 9, 11–12, 19–21
Ozone, as fumigant, 300, 432–33 viable v. non-viable, 13–16
limitations of, 432–33 view of, 5–6
INDEX 463
VOC with, 60–61, 126 SBS, 150–51
zoos with, 23 three strategies for, 12
Penicillium casei, 7–9 Pseudomonas
Penicillium chrysogenum, 193, 206, 378 endotoxin in, 144–45
chrysolysin from, 193, 206 Psilocybe
Pen ch allergen from, 218, 220–25 allergy associated with, 36
SBS, role of, 216, 218–19, 222, 280–81 Puccinia
Penicillium melinii, 351 allergy associated with, 36
Penicillium notatum, 325 Pulmonary effects of Stachybotrys, in
Penicillium roqueforti, 7 animal studies, 241–44, 247–49
PFT. See Pulmonary function testing Pulmonary function testing (PFT),
Phallolysin 383–84
from Amanita phalloides, 192
cytolytic activity of, 200
Phialophora Q
infectious disease associated with, 48
moisture problem buildings with, 178 QPCR. See Quantitative Polymerase Chain
Pholiota nameko Reaction
allergy associated with, 40 Quantitative Polymerase Chain Reaction
Phoma (QPCR), 67, 77–78. See also Detection
allergy associated with, 36 of fungal species using QPCR
infectious disease associated with, 48 in fungal speciation/quantification,
moisture problem buildings with, 179 437–47
Phoma betae, 325
Phycomyces
allergy associated with, 36 R
Phytophthora
allergy associated with, 36 Radioallergosorbent test (RAST), 38
Piptoporus Randomly amplified polymorphic DNA
allergy associated with, 36 (RAPD), 252, 255
Pisolithus RAST. See Radioallergosorbent test
allergy associated with, 36 RBC. See Red blood cells
Pleospora RCS. See Reuter centrifugal sampler
allergy associated with, 36 Real-time PCR, 439
Pleurotus Red blood cells (RBC), 192, 199, 200, 205
allergy associated with, 36, 40 Repeats-in-toxin (RTX)
Pleurotus ostreatus, 192 hemolysins, form of, 198
Podaxis Respiratory distress
allergy associated with, 36 Penicillium associated with, 6,
Polymerase Chain Reaction (PCR) 216–18
in fungal speciation/quantification, Reuter centrifugal sampler (RCS), 67
191–92, 437–47 Rhizopus
Polyporus allergy associated with, 36
allergy associated with, 36 Rhodotorula
Poria allergy associated with, 36
allergy associated with, 36 Rotterdamus, Erasmus, 139
Potato dextrose agar (PDA), 260–61, RTX. See Repeats-in-toxin
413, 427 Rubescenslysin
Prevention from Amanita rubescens, 192
fungal growth, 23 cytolytic activity of, 199, 204
464 INDEX
S history of, 3
IgE cross-reaction, in detection of,
Saccharomyces 218–22, 226–27, 229, 243
allergy associated with, 36 indoor air quality with, 215–16, 225–26,
Satratoxin, 244, 246–49, 256, 260, 280, 339–40, 350–52, 410
284, 349, 350–51, 377, 386 indoor air samples in, 218, 351–352, 410
SBA growth medium, 192–93, 198, 206 inflammation of, 143–44
SBS. See Sick building syndrome MCWA and, 139–51
Scanning electron microscopy (SEM), 164 microbial contamination and,
Scleroderma 142–43
allergy associated with, 36 mold problems associated with, 23
Scopulariopsis MTT bioculture assay, 350, 352
allergy associated with, 36 mycotoxins in, 24–27, 299, 429–30
infectious disease associated with, 48 nature of, 140–42
SEM. See Scanning electron microscopy Penicillium chrysogenum, role in, 216,
Serpula 218–19, 222, 280–81
allergy associated with, 36 Penicillium inhalation and, 13, 206,
Serratia 215–18
carpets and, 127 prevention of, 150–51
Serum sickness Stachybotrys evaluation and, 160–64
hypersensitivity and, 296 Stachybotrys in, 155–69, 345, 430
Sick building syndrome (SBS), 339, 425. symptoms of, 3–4, 9–10, 140–41, 191,
See also Molds, mycotoxins, role in 201–4, 216, 341
SBS; Molds, remediation/monitoring toxicosis case reports for, 166–69
abatement procedures for, 230–31, treatment of, 150–51
415–18 zoo fungi associated with, 23–24
airborne Stachybotrys and, 164–65 Siderophores, 377
Apergillus inhalation and, 206, Skin hypersensitivity, type I, 228
215–16, 345 Sphaerotheca
Aspergillus, importance in, 226–30 allergy associated with, 36
assay sensitivity in, 351 Spondylocladium
bacteria correlated with, 201–2, 203–5 allergy associated with, 36
biomarker assays for hemolysins in, Spore characterization, in Stachybotrys,
206–7, 349 251, 264
Candida inhalation and, 206 Sporobolomyces
chemokines in, 229, 384 allergy associated with, 36, 38, 280
children influenced by, 148–50 Sporobolomyces salmonicolor, 324
continually measured fungi profiles in, Sporotrichum
endotoxin of, 144–49 Stachybotrys. See also Stachybotrys
eosinophilia/neutrophilia in, 219, chartarum
221–22, 228 airborne, 164–65
freon in, 340 allergenicity/antigenicity of, 257–58
fungi correlated with, 3, 9–12 allergy associated with, 36
Fusarium inhalation and, 430 animal toxicosis from, 157–60
-D-glucan related to, 147–49 BAL fluid with, 168, 247–49,
hemolysins in, 191–92, 201–6 261–62, 267
histopathological examination of lungs biological methods to evaluate,
in, 223 160–61
historical perspective on, 139–40 building surface with, 11
INDEX 465
ceiling tile with, 23–25, 130–31 ETS with, 266
dust with, 165–66 experimental considerations in study of,
ecological factors influencing, 78–80, 259–63
88–89, 92–93, 266 exposure to, 205, 243, 262–63
environmental tobacco smoke with, 266 molds, causing cognitive impairment
experimental considerations in study of, and, 372, 396
259–63 PCR inhibitors in, 443
exposure to, 205, 243, 262–63 sporulation studies in, 427
growth evaluation of, 161–64 Stachylysin, from Stachybotrys chartarum,
history of, 156–57 193, 255–56, 267, 377
horses killed by, 156–57 cytolytic activity of, 200–1, 205,
human toxicosis from, 166–69 242, 255
HVAC colonization by, 420 species specificity of, 255
indoor building materials with, 73, zymograms of, 255
130–31, 162–65 Stachyrase, 377
indoor environments with, 23–25, Staphylococcal -toxin, 198,
74–76, 278–80, 415 202, 204
inflammatory indices in, 250 Staphylococcus aureus
ingestion of spores from, 430 beta-sheet-structured (BSS) hemolysin
molds, remediation/monitoring and, 414 from, 198
potency of satratoxin from, 244, 246, staphylococcal -toxin from, 198
256, 260 Stemonitis
potency of trichothecenes from, 244, allergy associated with, 36
258–60, 280, 284–85, 296–97, 325, Stemphylium
349, 376, 430 allergy associated with, 36
protein synthesis inhibition by Stereum
trichothecenes from, 241, 242, allergy associated with, 36
244, 257 Sterigmatocysin, from Aspergillus
pulmonary effects, in animal studies, versicolor, 280
241–44 Streptococcus
spore characterization in, 251, 264 cholesterol binding toxin (CBT)
spore proteins/proteinases in, 256–57, from, 198
260–61, 267, 377 Streptomyces albus, 324
sporulation studies in, 427 Suberosis, 7
symptoms in, 263–64, 282 Syncephalastrum
toxic agents associated with, 283 allergy associated with, 36
toxic products from, 283
Stachybotrys atra, 11, 325, 345, 349, 367.
See also Stachybotrys chartarum T
building surface with, 11
molds, causing cognitive impairment T-2 toxin, 242, 351
and, 369, 372, 396 T helper cell, 14–15, 146, 392–94
Stachybotrys chartarum, 324. See also TBI. See Traumatic Brain Injury
Stachybotrys atra TEM. See Transmission
allergenicity/antigenicity of, 257–58, electron microscopy
377, 386 Tetracoccosporium
animal studies on, 245–59, 262 allergy associated with, 36
cyclosporin from, 242 Th2 response, allergic, 228, 230
cytotoxicity of isolates from, 255 Thermomyces
ecological factors influencing, 266 allergy associated with, 36
466 INDEX
Thermophilic actinomycetes Trichothecium
occupational lung disease provoked allergy associated with, 37
by, 7 Tumor necrosis factor (TNF), 203–4, 384
Tilletia TVOC. See Total volatile organic
allergy associated with, 36 compounds
Tilletiopsis Typhula
TLR. See Toll-like receptor
TNF. See Tumor necrosis factor
U
Toll-like receptor (TLR)
MCWA and, 144
UFFI. See Urea formaldehyde foam
Torula
insulation
Ultraviolet light
Total volatile organic compounds
in cleansing/sterilization, 433
(TVOC), 61
fungi influenced by, 87
Toxic mold syndrome, 275–86
Urea formaldehyde foam insulation
air sampling in, 280–82
(UFFI), 341
building-related diseases in, 278–80
Urocystis
foodstuffs and, 277–78
mycotoxins in, 275, 277–78, 282
Ustilago
overview of molds in, 275–76
Penicillium chrysogenum, role in,
280–81
toxicity, effect on health of molds in, V
277–79
Transmission electron microscopy Vascular tissue damage
(TEM), 164 with hemolysins, 204–5
Traumatic Brain Injury (TBI), Verticillium
Trichoderma indoor air and, 66
allergy associated with, 36 VOC. See Volatile organic compounds
indoor building materials with, 73 Volatile organic compounds (VOC)
moisture problem buildings with, fungi produced, 17, 59–62, 312, 339
178–79 indoor, 61
mycotoxins from, 51, 56, 58 odor of, 60
VOC from, 60 Voriconazole
Trichoderma citrinoviride, 324–25 therapy, for fungal hypersensitivity, 303
Trichophyton
infectious disease associated with, 49 W
Trichothecenes, from Stachybotrys
chartarum, 244, 258–60, 280, 284–85, WAIS-R. See Wechsler adult intelligence
296–97, 325, 349, 376, 430 recall-revised
immunopathology of, 244, 265 Wallemia
leukocyte apoptosis by, 245 allergy associated with, 37
lymphocyte proliferation by, 244 moisture problem buildings with, 178
neurotoxic nature of, 395 mycotoxins from, 51
potency of, 244–51, 247, 258–60 Water intrusion, 376
protein synthesis inhibition by, 241–42, Wechsler adult intelligence recall-revised
244, 257 (WAIS-R), 348
INDEX 467
WHO. See World Health Organization Y
Wood pulp worker’s lung, 7, 41
World Health Organization (WHO), 191, Yeasts, 192
201, 410, 425
Z
X
Zoological institutions
fungi associated with SBS in, 23–24
Xylaria
Xylobolus
CONTENTS OF PREVIOUS VOLUMES
Volume 41 Microbiological Production of Lactic Acid

John H. Litchfield
Microbial Oxidation of Unsaturated
Fatty Acids Biodegradable Polyesters
Ching T. Hou Ch. Sasikala
Improving Productivity of Heterologous The Utility of Strains of Morphological
Proteins in Recombinant Group II Bacillus
Saccharomyces cerevisiae Samuel Singer
Fermentations
Phytase
Amit Vasavada
Rudy J. Wodzinski and
Manipulations of Catabolic Genes for A. H. J. Ullah
the Degradation and Detoxification
of Xenobiotics INDEX
Rup Lal, Sukanya Lal, P. S. Dhanaraj,
and D. M. Saxena
Aqueous Two-Phase Extraction for Volume 43
Downstream Processing of Production of Acetic Acid by
Enzymes=Proteins Clostridium thermoaceticum
K. S. M. S. Raghava Rao, N. K. Rastogi, Munir Cheryan, Sarad Parekh,
M. K. Gowthaman, and Minish Shah, and Kusuma Witjitra
N. G. Karanth
Contact Lenses, Disinfectants, and
Biotechnological Potentials of
Acanthamoeba Keratitis
Anoxygenic Phototrophic Bacteria.
Donald G. Ahearn
Part I. Production of Single Cell
and Manal M. Gabriel
Protein, Vitamins, Ubiquinones,
Hormones, and Enzymes and Marine Microorganisms as a Source of
Use in Waste Treatment New Natural Products
Ch. Sasikala and Ch. V. Ramana V. S. Bernan, M. Greenstein,
and W. M. Maiese
Biotechnological Potentials of
Anoxygenic Phototrophic Bacteria. Stereoselective Biotransformations in
Part II. Biopolyesters, Biopesticide, Synthesis of Some Pharmaceutical
Biofuel, and Biofertilizer Intermediates
Ch. Sasikala and Ch. V. Ramana Ramesh N. Patel
Microbial Xylanolytic Enzyme System:
INDEX
Properties and Applications
Pratima Bajpai
Volume 42
Oleaginous Microorganisms: An
The Insecticidal Proteins of Assessment of the Potential
Bacillus thuringiensis Jacek Leman
P. Ananda Kumar, R. P. Sharma, and
V. S. Malik INDEX
469
470 CONTENTS OF PREVIOUS VOLUMES
Volume 44 Formation of Flavor Compounds
in Cheese
Biologically Active Fungal Metabolites
P. F. Fox and J. M. Wallace
Cedric Pearce
The Role of Microorganisms in Soy
Old and New Synthetic Capacities of Sauce Production
Baker’s Yeast
Desmond K. O’Toole
P. D’Arrigo, G. Pedrocchi-Fantoni,
and S. Servi Gene Transfer Among Bacteria in
Natural Environments
Investigation of the Carbon- and Xiaoming Yin and G. Stotzky
Sulfur-Oxidizing Capabilities of
Microorganisms by Breathing Manganese and Iron:
Active-Site Modeling Solid-State Respiration
Herbert L. Holland Kenneth H. Nealson and
Brenda Little
Microbial Synthesis of d-Ribose:
Metabolic Deregulation and Enzymatic Deinking
Fermentation Process Pratima Bajpai
P. de Wulf and E. J. Vandamme
Microbial Production of Docosahexaenoic
Production and Application of Acid (DHA, C22:6)
Tannin Acyl Hydrolase: State Ajay Singh and Owen P. Word
of the Art
P. K. Lekha and B. K. Lonsane INDEX
Ethanol Production from Agricultural

Biomass Substrates Volume 46
Rodney J. Bothast and Badal C. Saha
Cumulative Subject Index
Thermal Processing of Foods,
A Retrospective, Part I: Uncertainties
Volume 47
in Thermal Processing and
Statistical Analysis Seeing Red: The Story of Prodigiosin
M. N. Ramesh, S. G. Prapulla, J. W. Bennett and Ronald Bentley
M. A. Kumar, and
Microbial=Enzymatic Synthesis of Chiral
M. Mahadevaiah
Drug Intermediates
Thermal Processing of Foods, Ramesh N. Patel
A Retrospective, Part II: On-Line
Recent Developments in the
Methods for Ensuring
Molecular Genetics of the
Commercial Sterility
Erythromycin-Producing Organism
M. N. Ramesh, M. A. Kumar,
Saccharopolyspora erythraea
S. G. Prapulla, and M. Mahadevaiah
Thomas J. Vanden Boom
INDEX Bioactive Products from Streptomyces
Vladisalv Behal
Volume 45 Advances in Phytase Research
Edward J. Mullaney, Catherine B. Daly,
One Gene to Whole Pathway:
and Abdul H. J. Ullah
The Role of Norsolorinic Acid in
Aflatoxin Research Biotransformation of Unsaturated
J. W. Bennett, P.-K. Chang, and Fatty Acids of industrial Products
D. Bhatnagar Ching T. Hou
CONTENTS OF PREVIOUS VOLUMES 471
Ethanol and Thermotolerance in Volume 49
the Bioconversion of Xylose
Biodegredation of Explosives
by Yeasts
Thomas W. Jeffries and Yong-Su Jin Susan J. Rosser, Amrik Basran, Emmal
R. Travis, Christopher E. French,
Microbial Degradation of the and Neil C. Bruce
Pesticide Lindane
Biodiversity of Acidophilic Prokaryotes
(-Hexachlorocyclohexane)
Brajesh Kumar Singh, Ramesh Chander Kevin B. Hallberg and
Kuhad, Ajay Singh, K. K. Tripathi, D. Barrie Johnson
and P. K. Ghosh Laboratory Birproduction of Paralytic
Microbial Production of Oligosaccharides: Shellfish Toxins in Dinoflagellates
A Review Dennis P. H. Hsieh, Dazhi Wang, and
Garry H. Chang
S. G. Prapulla, V. Subhaprada, and
N. G. Karanth Metal Toxicity in Yeasts and the Role of
Oxidative Stress
INDEX S. V. Avery
Foodbourne Microbial Pathogens and the
Volume 48 Food Research Institute
Biodegredation of Nitro-Substituted M. Ellin Doyle and Michael W. Pariza
Explosives by White-Rot Fungi: Alexander Flemin and the Discovery
A Mechanistic Approach of Penicillin
Benoit Van Aken and J. W. Bennett and King-Thom Chung
Spiros N. Agathos
INDEX
Microbial Degredation of Pollutants in
Pulp Mill Effluents
Pratima Bajpai Volume 50
Bioremediation Technologies Paleobiology of the Archean
for Metal-Containing Sherry L. Cady
Wastewaters Using Metabolically
A Comparative Genomics Approach
Active Microorganisms
for Studying Ancestral Proteins
Thomas Pumpel and
and Evolution
Kishorel M. Paknikar
Ping Liang and Monica Riley
The Role of Microorganisms in Ecological
Chromosome Packaging by
Risk Assessment of Hydrophobic
Archaeal Histones
Organic Contaminants in Soils
Kathleen Sandman and John N. Reeve
C. J. A. MacLeod, A. W. J. Morriss,
and K. T. Semple DNA Recombination and Repair
in the Archaea
The Development of Fungi:
Erica M. Seitz, Cynthia A. Haseltine, and
A New Concept Introduced
Stephen C. Kowalczykowski
By Anton de Bary
Gerhart Drews Basal and Regulated Transcription
in Archaea
Bartolomeo Gosio, 1863–1944:
Jörg Soppa
An Appreciation
Ronald Bentley Protein Folding and Molecular
Chaperones in Archaea
INDEX Michel R. Leroux
Archaeal Proteasomes: Proteolytic Volume 52
Nanocompartments of the Cell
Soil-Based Gene Discovery: A New
Julie A. Maupin-Furlow,
Steven J. Kaczowka, Mark S. Ou, Technology to Accelerate and Broaden
and Heather L. Wilson Biocatalytic Applications
Kevin A. Gray, Toby H. Richardson, Dan
Archaeal Catabolite Repression: A Gene E. Robertson, Paul E. Swanson, and
Regulatory Paradigm Mani V. Subramanian
Elisabetta Bini and Paul Blum
The Potential of Site-Specific
INDEX Recombinases as Novel Reporters in
Whole-Cell Biosensors of Pollution
Volume 51 Paul Hinde, Jane Meadows, Jon
Saunders, and Clive Edwards
The Biochemistry and Molecular
Biology of Lipid Accumulation Microbial Phosphate Removal and
in Oleaginous Microorganisms Polyphosphate Production from
Colin Ratledge and James P. Wynn Wastewaters
John W. McGrath and John P. Quinn
Bioethanol Technology: Developments
and Perspectives Biosurfactants: Evolution and Diversity in
Owen P. Ward and Ajay Singh Bacteria
Raina M. Maier
Progress of Aspergillus oryzae Genomics
Masayuki Machida Comparative Biology of Mesophilic and
Thermophilic Nitrile Hydratases
Transmission Genetics of Microbotryum Don A. Cowan, Rory A. Cameron, and
violaceum (Ustilago violacea): Tsepo L. Tsekoa
A Case History
E. D. Garber and M. Ruddat From Enzyme Adaptation to Gene
Regulation
Molecular Biology of the Koji Molds William C. Summers
Katsuhiko Kitamoto
Acid Resistance in Escherichia coli
Noninvasive Methods for the Hope T. Richard and John W. Foster
Investigation of Organisms at
Low Oxygen Levels Iron Chelation in Chemotherapy
David Lloyd Eugene D. Weinberg
The Development of the Penicillin Angular Leaf Spot: A Disease Caused by the
Fungus Phaeoisariopsis griseola (Sacc.)
Production Process in Delft, The
Netherlands, During World War II Ferraris on Phaseolus vulgaris L.
Under Nazi Occupation Sebastian Stenglein, L. Daniel Ploper,
Oscar Vizgarra, and Pedro Balatti
Marlene Burns and
Piet W. M. van Dijck The Fungal Genetics Stock Center: From
Genomics for Applied Microbiology Molds to Molecules
Kevin McCluskey
William C. Nierman and
Karen E. Nelson Adaptation by Phase Variation in
Pathogenic Bacteria
INDEX Laurence Salaün, Lori A. S. Snyder, and
Nigel J. Saunders
CONTENTS OF PREVIOUS VOLUMES 473
What Is an Antibiotic? Revisited LuxS and Autoinducer-2: Their
Ronald Bentley and J. W. Bennett Contribution to Quorum Sensing and
Metabolism in Bacteria
An Alternative View of the Early History
Klaus Winzer, Kim R. Hardie, and
of Microbiology
Paul Williams
Milton Wainwright
Microbiological Contributions to the
The Delft School of Microbiology, from the
Search of Extraterrestrial Life
Nineteenth to
Brendlyn D. Faison
the Twenty-first Century
Lesley A. Robertson
INDEX
INDEX
Volume 54
Volume 53 Metarhizium spp.: Cosmopolitan Insect-
Pathogenic Fungi – Mycological
Biodegradation of Organic Pollutants in
Aspects
the Rhizosphere
Donald W. Roberts and
Liz J. Shaw and Richard G. Burns
Raymond J. St. Leger
Anaerobic Dehalogenation of
Molecular Biology of the Burkholderia
Organohalide Contaminants in the
cepacia Complex
Marine Environment
Jimmy S. H. Tsang
Max M. Häggblom, Young-Boem Ahn,
Donna E. Fennell, Lee J. Kerkhof, Non-Culturable Bacteria in Complex
and Sung-Keun Rhee Commensal Populations
William G. Wade
Biotechnological Application of
Metal-Reducing Microorganisms l Red-Mediated Genetic Manipulation of
Jonathan R. Lloyd, Derek R. Lovley, and Antibiotic-Producing Streptomyces
Lynne E. Macaskie Bertolt Gust, Govind Chandra, Dagmara
Jakimowicz, Tian Yuqing,
Determinants of Freeze Tolerance in
Celia J. Bruton, and
Microorganisms, Physiological
Keith F. Chater
Importance, and Biotechnological
Applications Colicins and Microcins: The Next
An Tanghe, Patrick Van Dijck, and Generation Antimicrobials
Johan M. Thevelein Osnat Gillor, Benjamin C. Kirkup, and
Margaret A. Riley
Fungal Osmotolerance
P. Hooley, D. A. Fincham, M. P. Mannose-Binding Quinone Glycoside,
Whitehead, and N. J. W. Clipson MBQ: Potential Utility and Action
Mechanism
Mycotoxin Research in South Africa
Yasuhiro Igarashi and Toshikazu Oki
M. F. Dutton
Protozoan Grazing of Freshwater Biofilms
Electrophoretic Karyotype Analysis in
Jacqueline Dawn Parry
Fungi
J. Beadle, M. Wright, L. McNeely, and Metals in Yeast Fermentation Processes
J. W. Bennett Graeme M. Walker
Tissue Infection and Site-Specific Gene Interactions between Lactobacilli and
Expression in Candida albicans Antibiotic-Associated Diarrhea
Chantal Fradin and Bernard Hube Paul Naaber and Marika Mikelsaar
Bacterial Diversity in the Human Gut The Inactivation of Microbes by Sunlight:
Sandra MacFarlane and Solar Disinfection as a Water
George T. MacFarlane Treatment Process
Robert H. Reed
Interpreting the Host-Pathogen Dialogue
Through Microarrays
INDEX
Brian K. Coombes, Philip R. Hardwidge,
and B. Brett Finlay
PREFACE
Humankind has known about health problems associated with in-

door mold growth since the time of Moses, over 3300 years ago. We
know this because it is accurately described in the Old Testament of
the Bible, in Leviticus 14:33–45. Of course, in that time, people had
no idea why indoor mold proliferation was undesirable—they just
knew empirically that it was.
Today we know what organisms cause indoor mold growth, and in
many cases we know what products these organisms produce that
can cause adverse human health effects. For instance, we now know
that the inhalation of high concentrations of fungal spores or conidia
can cause allergic rhinitis, hypersensitivity pneumonitis, and asthma
exacerbation. The role of the inhalation of mycotoxins in the adverse
health effects seen in sick building syndrome (SBS) is still in question,
but we believe that much progress will soon be made in the under-
standing of this important issue.
It is well known that fungi can cause human infection, but that is
not usually what occurs in SBS. However, people in ‘‘sick buildings’’
can suffer fungal lung infections, but this most commonly occurs
in those who are immunocompromised. This is most normally
seen in the form of an Aspergillus lung infection known as
aspergillosis.
In SBS, of course, it is not the building that is sick but the people who
live, work, or go to school there. What, then, is sick building syndrome?
The definition varies depending upon who is defining it. The term
probably came into being in 1982 in an attempt to describe a number
of symptoms that occurred in abundance in a group of individuals that
worked in the same buildings. These symptoms occurred in concert
when said individuals entered the buildings and abated when they left.
These symptoms include, but are not limited to, eye irritation, dry or
sore throat, nasal congestion, skin rash, memory loss, nose bleeds,
headache, balance difficulties, and extreme fatigue.
While it is clear that no one cause of SBS exists, scientific data are
now available that demonstrate that indoor fungal growth is an extre-
mely important component of this phenomenon. This book deals
xv
xvi PREFACE
primarily with the role of fungi and their aftermath in SBS. It is hoped
that the chapters in this book will answer some of the riddles posed
by SBS.
David C. Straus

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Section I.

Fungi and Sick Building Syndrome

FIG. 1. Penicillium chrysogenum colonies on PDA for 7 days.

FIG. 2. Penicillium chrysogenum at 400 magnification.

species tended to colonize the heating, ventilation, and air condition-

FIG. 3. Stachybotrys chartarum colonies on PDA for 14 days.

P. chrysogenum and P. cyclopium. Finally, there is one other important

FIG. 4. Stachybotrys chartarum at 400 magnification.

in breathing, fever, fatigue, and productive cough. These individuals

III. Correlation Between the Presence of Certain Fungi and SBS

Nasal drainage and 19.8 1.3 Discomfort When are symptoms

FIG. 5. Bar graph of all air samples taken at the 48 schools.

from 31.3% to 2.5%, which was a significant (P < 0.001) decrease

receiving 104 NV P. chrysogenum IN did not demonstrate significant

V. Cellular and Humoral Responses in an Animal Model

Continuous exposure to moderate to high levels of P. chrysogenum

actively growing fungi are known irritants or hazardous chemicals and

VI. Continually Measured Fungal Profiles in SBS

IDA, Penicillium species were consistently the dominant organisms,

condition is not absolute assurance of their absence nor the absence of

VII. Evaluation of Fungal Growth on Cellulose-Containing and

VIII. The Presence of Fungi Associated with SBS in North American

FIG. 16. Colony-forming units of three different fungal genera on cellulose-containing

are in North American zoological institutions, and therefore zookeepers

IX. The Role (?) of Mycotoxins in SBS

trichothecene mycotoxins on the spores of Stachybotrys atra (Sorenson

trichothecene mycotoxins in the urine of patients who had been ex-

Blackley, C. H. Experimental researches on the causes and nature of catarrhus aestivus

relationship with other organisms; (b) a negative one as pathogens to

II. Effects of Indoor Fungi on Human Health

Fungus Order Division

Absidia Mucorales Zygomycota

Fungus Order Division

Fungus Order Division

Fungus Order Division

Chow et al. (2000) characterized Pen n 13 as a major allergen of Peni-

closely by Aspergillus and Alternaria (Nolles et al., 2001). Sensitization

Fungal agent Disease Source

Alternaria sp. Pulpmill worker’s lung Moldy pulpwood

allergen by Horner et al. (1988). Extrinsic allergic alveolitis caused by

Fungus Classification Disease Affected area

Absidia sp. Opportunistic Zygomycosis Face, sinuses,

TABLE III (Continued )

Fungus Classification Disease Affected area

Aureobasidium Opportunistic Systemic Lungs, deep tissue,

Fungus Classification Disease Affected area

Opportunistic Systemic Blood, heart tissue, kidney,

TABLE III (Continued )

Fungus Classification Disease Affected area

Epidermophyton Cutaneous Tinea cruris Groin

Fungus Classification Disease Affected area

(H. duboisii in Recticulorendothelial

TABLE III (Continued )

Fungus Classification Disease Affected area

Fungus Classification Disease Affected area

Sporothrix Subcutaneous Sporotichosis Skin, primarily hands,

fungi (Benenson, 1990; Burge 1990a; Larsen and Frisvad, 1995).

C. MYCOTOXINS AND THEIR SIGNIFICANCE TO HUMAN HEALTH

Alternaria alternata Tenuazonic acid, alternatiol, alternatiol mononethyl ether,

metabolites are mainly to enhance the fitness of the fungi in nature.

Etiologic agent Disease Natural substrate

Fusarium spp. Akakabio-byo Wheat, barley, oats, rice

VI. (1 ! 3)--D-glucan