1. health and fitness
2. disease

Pergamon
.I Aerosol
Su Vol. 28, No. 3, pp. 3X1&392, 1997
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( 1997 Elsewer Saence Ltd
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PII: SOO21-8502(96)00441-7
AIR SAMPLING
FOR FUNGI
IN INDOOR ENVIRONMENTS
Brian Flannigan
Department
of Biological
Sciences,
Heriot-Watt
University,
Edinburgh
EH14 4AS, U.K.
Abstract-Mould
growth in buildings is a major health issue, but most investigations
of the indoor
air spora still employ culture-based
methods. These are inadequate
for assessing exposure, since
culturable organisms comprise a small fraction of the total of potentially allergenic/toxigenic
units in
air. For epidemiological
studies, measurement
of airborne fungal biomass over extended periods
may be more relevant than total counts. Whilst (1+3)-/6D-gkKaU
has been used to assess airborne
biomass, ergosterol may be the best indicator of exposure. For case studies, patients’ serum has been
used to detect specific spores on sampler slides, and both highly specific and less specific antisera
could be used either via fluorescent antibody technique or enzyme-linked
immunosorbent
assays. In
the future, solid-phase polymerase chain reaction (PCR) may be used to detect pathogens or other
well-characterized
potentially
harmful species, and in at least some groups mycotoxin/secondary
metabolite/volatile
profiles may be used in identification.
c 1997 Elsevier Science Ltd. Ail rights
reserved
INTRODUCTION
In temperate climates, the development
of buildings with minimum energy usage has led to
“tight” air-conditioned
buildings
in which exchange of air with the outside is greatly
reduced. Water vapour, which would otherwise have been vented, condenses
on cool
surfaces in the same way as in poorly insulated buildings without air-conditioning
and
creates conditions
for microbial
growth and an associated
build-up of bioaerosols.
Although spores of fungal pathogens such as Aspevgillusfumigatus
are very seldom numerous
in indoor air and pose little hazard to healthy individuals,
there are special risks for
immunosuppressed
patients and other severely compromised
individuals.
However, it is
well known that spores of species of Aspergillus, Cladosporium and Penicillium generated in
damp buildings can cause bouts of asthma and/or rhinitis among atopic occupants.
In
addition, exposure to large indoor concentrations
of spores of a range of individual fungi,
from Cladosporium to the dry-rot fungus Serpula lacrymans, have been the cause of rare
instances of extrinsic allergic alveolitis (Flannigan
et al., 1991). As well as having a role in
such individual
cases of allergic disease, fungi are now seen as having a wider role in
respiratory
health. Several large-scale epidemiological
investigations
in North America
have noted a strong association between reported dampness/mould
in homes and reported
respiratory
symptoms (Brunekreef et al., 1989; Dales et al., 1991a, b; Spengler et al., 1991)
and, in Finland, Jaakkola et al. (1993) observed a twofold increase in respiratory symptoms
among pre-school children living in homes with reported dampness or mould. Brunekreef
et al. (1989) reported that the effect on children was of similar magnitude
to parental
smoking. Mould growth in homes is therefore a major health issue, and there is an urgent
need to obtain objective microbiological
data in order to confirm the role of moulds
indicated by these epidemiological
investigations.
THE
INDOOR
AIR SPORA
At least in North America, the general perception
of what
non-industrial
workplaces should be is of a mixture of species
air. Certainly if the buildings are air-conditioned,
or if windows
summer, the indoor counts could be expected to be somewhat
fungi such as Alternaria and Epicoccum conform to this picture,
species of Aspergillus and, especially, Penicillium outdoors to
381
the air spora in homes and
resembling that in outdoor
and doors are kept closed in
lower. Field or phylloplane
but the ratio for the sum of
that indoors may either be
B. Flannigan
382
Table 1. Mean abundance
of hyphal fragments and mam types of fungal spore in samples of
indoor and outdoor air taken on 132 days between December 1991 and September
1993 in
Ontario (Li and Kendrick, 1995)
Outdoor
Indoor
Category*
Spores (m “)
(%B
)
Spores (mm’)
(“A,)
Hyphal
146
44
457
895
41
6.3
1.9
19.8
38.8
1.8
0.3
2.6
7.9
2.8
6.5
8.9
112
74
131
1479
78
20
3.2
2. I
3.8
42.5
2.3
0.6
3.2
15.1
4.0
8.9
8.7
fragments
AItrrnu~icc
A.speryillusiPenicilli~rm
CIudo.sporium
Coprimu
Epicoccum
Gtrnotlrrmn
Lrptosphawiu
Unidentified ascosporea
Unidentified basidiospores
Other unidentified spores
* 28 other
59
1x2
65
152
206
taxa. spores of which were recorded
occasionally,
Ill
541
138
310
301
are not included
much lower or reversed (Flannigan
et al., 1991). The relative abundance
of different species
of Penicillium may also differ, with some species being more conspicuous
in indoor air and
others in outdoor air (Fradkin, 1987). A recent Canadian investigation
in which total counts
were made from exposed slides with a Samplair particle sampler (Li and Kendrick, 1995)
illustrates the relative abundance
of different categories of fungal spores, with Clndnsporium
predominating
in both outdoor
and indoor air and outdoor
concentrations
of most
categories being higher than those indoors. However, the Aspergillus/Penicillium
grouping,
generally considered largely to have an indoor origin, formed < 4% of the outdoor air
spora but nearly 20% indoors (Table 1). The study also illustrates what is not revealed by
conventional
viable sampling, viz., the relative abundance
of the spores of Ascomycetes and
Basidiomycetes,
which in this case comprised around one-third of the outdoor and one-fifth
of the total indoor air spora (Table 1). The study also emphasises the perceived role of
spores infiltrating
from outdoors in determining
much of the indoor air spora, with peak
and Alternaria, although lower, coinciding with those outdoors at
counts of Cladosporium
the height of the growing season. In contrast, three peaks of Aspergillus~Penicillium
spores
occurred indoors in January, April and September, but not outdoors.
The importance
of mould growth within buildings in contributing
to the indoor air spora
has been highlighted by, among others, Hunter et al. (1988) and Flannigan
et al. (1993). It is
not only species of Aspergillus and Penicillium that are to be found growing in buildings,
boosting the airborne spore burden. Cludosporium
spp. commonly
grow on damp indoor
surfaces, as do more hydrophilic
Phoma and Ulocllldium spp., and in very damp conditions
Stachyhotrys
atrn (syn. S. churtarum) may be prominent
(Grant et al., 1989). In an investigation of 41 homes in the American Midwest, DeKoster and Thorne (1995) noted that high
indoor viable counts were associated with high basement humidity. The mean ratio of
indoor: outdoor airborne viable fungi for basements of homes where occupants complained
of sick building syndrome (SBS) symptoms was 2.16, and in non-complaint
homes was 0.56.
The corresponding
ratios for the main floor were 0.84 versus 0.37.
ASSESSING
THE
AIR
SPORA
As has been pointed out by Strachan
et ul. (1990) failure to establish
an objective
connection between the respiratory health status of occupants of mould-affected
houses and
airborne microorganisms
in these houses may be the result of inadequate
quantification
of
the air spora to which the occupants
are exposed. Since most investigations
have only
assessed numbers of culturable organisms, they have ignored numbers of non-viable or nonculturable spores that may be as allergenic or toxigenic as their culturable counterparts
and
have as significant an effect on health. There can be both qualitative and large quantitative
Sampling
indoor
environments
383
differences between the total numbers of fungal particles (viable + non-viable) in indoor air
and those that can be collected, cultured, counted and identified on agar plates. Kozak et al.
(1979) graphically
illustrated
that the number of viable spores of individual
fungi can be
below the limit of detection by established methods although the total is sufficient to cause
a respiratory problem. Thus, they did not detect Stachybotrys atra when using an Andersen
sampler in the home of an asthmatic child, but did detect the mould by means of a rotorod
sampler. It was estimated that only l-2% of S. atra spores, which caused asthma attacks in
the child, were viable. As Kozak et al. (1979) stated, in health related studies no one
sampling technique is adequate for assessment of indoor fungi; a culture-based
system is
needed for identifying
species which may be significant for health, and a total count or
a measure of biomass is required to assess exposure. The best approach (Flannigan,
1992) is
to use the one sampler, e.g. filter sampler, liquid impinger or cyclone sampler, for collection
of all microorganisms
and divide the sample into portions for culture, total counting and/or
assessment of biomass (or any desired metabolite).
Collecting airborne microorganisms
by drawing air, at a low flow rate, through a polycarbonate
membrane
in an aerosol monitor cassette for several hours (the CAMNEA
method; Palmgren et al., 1986a, b), Strom et al. (1990) found that only a small fraction of
fungal spores in the total (washed from the membrane,
stained with acridine orange and
counted by a direct epifluorescence
technique) were culturable.
Some differences between
culturable fraction and total count in a restaurant environment
are illustrated in Table 2. As
might be expected, the counts of culturable organisms obtained by the CAMNEA method
over a period of 4 h can differ greatly from short-duration
“grab” samples taken with
a six-stage Andersen cascade impactor.
The effect of human activity on counts of viable microorganisms
in indoor air is clearly
shown by the converted Andersen sampler counts for the dining area of the restaurant, with
the largest counts occurring during the busy lunchtime period. In a more recent lo-month
investigation
of a group of Scottish houses (Flannigan
et al., 1996), the median count of
viable airborne fungi indoors, as determined
by the CAMNEA
method, was 260 colony
forming units (CFU) me3 air and that of bacteria 339 rne3. The median values for total
fungal and bacterial counts were, respectively,
13,940 spores and 88,260 cells rn- 3. On
average, the viable counts for fungi and bacteria were approximately
0.57 and 0.28% of the
corresponding
total counts. At > 175 : 1 (fungi) and nearly 360 : 1 (bacteria), the ratios of
total : viable counts are large, but not as large as in some “healthy” Swedish houses (Strom
et al., 1990), where the corresponding
ratios determined
by the two CAMNEA techniques
were 500: 1 and 2000: 1.
It is generally held that bacteria in indoor air are predominantly
Gram-positive
species
shed from the human body and that numbers of Gram-negative
bacteria are relatively
Table 2. Concentration
of airborne
microorganisms
obtained
(after Flannigan,
Concentration
Andersen
using different
1992)
sampling
methods
(CFU m 3 air)
sampling*
CAMNEA
method
Plate count
Area
Before
open
in a restaurant
DEFT
method
Mid-day
Early
evening
a.m.
212
224
141
152
129
208
3160
138
920
625
10,200
34,910
1260
5100
35
106
565
244
24
88
521
417
348
1180
2010
43,250
12,360
13,580
p.m.
a.m
p.m.
Pantry
Moulds
Bacteria
Dininy
Moulds
Bacteria
* Raw counts converted by the “positive hole” method
sites on the agar collection plates (Andersen, 1958).
to correct
for multiple
impactions
of propagules
at the same
384
B. Flannigan
Table
counts
3. Spearman
correlation
between Burkard
personal
sampler total
and Andersen two-stage sampler viable counts for indoor air in 41
homes (DeKoster and Thorne, 1995)
Category
R values
Altrrnurii~
Aspe~yillusiPeni~illilrrn
Chlosporiurn
Unclassified
Total
0.43
0.45
0.65
0.22
0.80
p values
0.02
0.01
< 0.0001
0.21
< 0.000 I
small, unless there is some amplification
site within the building, e.g. a heavily contaminated
humidifier (Flannigan,
1992). However, Gram-negative
bacteria are generally more susceptible than Gram-positive
bacteria to desiccation, so that loss of viability (culturability)
due
to continuing
exposure to a stream of sampled air after deposition on a surface (particularly
if the surface is a dry membrane rather than moist agar) is likely to account for at least part
of the difference between (a) viable counts of Gram-positive
and Gram-negative
bacteria
and (b) total and viable counts.
Flannigan
et nl. (1996) reported that the ratio between comparable
viable counts and
total counts in the indoor air of a group of Scottish houses was inconsistent,
probably
because the composition
of the air spora differs with location and different microorganisms
have different survival rates, both in the environment
and on the membrane
during the
and Penicillium may survive for long
sampling period. For example, spores of Aspergilhs
periods, even years, whilst the viability (or culturability)
of others, e.g. S. mu. may decline
very rapidly. This can make the interpretation
of results of air sampling difficult (Flannigan
and Miller, 1994). The isolation of S. utra from air samples collected on culture media
should be interpreted
differently from isolation
of Penidium
spp. on the same plates.
However, without reporting the total concentrations
of fungal spores collected, DeKoster
and Thorne (1995) stated that total concentrations,
estimated using a Burkard personal
sampler, agreed well with corresponding
concentrations
of viable spores, obtained using an
Andersen two-stage sampler (Table 3). However, although Spearman correlation
between
the counts for Cladosporium
was high, that for other categories was only moderate.
PROBLEMS
OF METHODOLOGY
Most microbiological
investigations
of indoor air still employ culture-based
methods, but
sufficient attention is seldom given to four important issues: sampler performance. temporal
variability, culture media and accurate identification
(Flannigan
and Miller. 1994). In this
last respect, too many studies identify only to the genus level and disregard the diversity of
species, their ecology and potential significance for health, especially in important
genera
such as Asperyillus and Penicillium.
Although analysis has shown that some air sampling devices can perform better than
others, the design of no currently
available
sampler can be described
as optimal on
theoretical grounds (Nevalainen
et al., 1992). All have biases and deficiencies. In a number
of laboratory
investigations,
particular samplers have been found to be better than others
for the intended purpose, e.g. the Andersen six-stage sampler for viable counts and the
Burkard 24 h sampler for total counts (Buttner and Stetzenbach,
1993). Efforts have been
made to determine the “reliability”
of various air samplers suitable for fungi by means of
side-by-side comparisons
in buildings. For example, Verhoeff et al. (1990a, b) reported that,
among air-samplers
used in side-by-side tests in houses, the Andersen N6 sampler gave the
largest colony counts and the greatest diversity of fungi. Miller (1993) attributed this to the
longer time required to sample the same volume of air as the other samplers. Noting the
temporal variability in counts for six rooms in an office building, Stanevich and Petersen
(1990) had earlier reported that the variability of raw counts from 1 min N6 samples was six
times that of 5 min samples.
Sampling
indoor
environments
385
Temporal variability is a major problem in assessing human exposure to the indoor air
spora. This is amply illustrated by Verhoeff et al. (1990b), who found that not only did the
numbers of propagules
in 2-min samples taken by an Andersen N6 (one-plate) sampler
show great variability
over relatively short periods but also that within-home
variance
approached
four times that between homes. An important
factor introducing
variability
into the nature and magnitude
of the indoor air spora is the release of fungi from carpets
and walls or other surfaces. This depends on the type and degree of activity of occupants in
the room. All activity in buildings disturbs settled spores, but cleaning, constructional
work
and any other major dust-raising
activities have a particular impact (Hunter et al., 1988).
were
Studies in an experimental
room, into which spores of Penicillium chrysogenum
introduced
and allowed to settle on a nylon carpet (Buttner and Stetzenbach,
1993)
unequivocally
confirmed that foot traffic on carpets resulted in elevated counts of airborne
spores. Buttner and Stetzenbach (1993) drew attention to the possible error introduced into
the results through the activity of investigators
at the sampling site and the re-entrainment
of settled spores by sampler exhaust air near the floor.
Differences in the size and sedimentation
rate of spores also affect what is detected in air
samples. For example, it has been demonstrated
that large Ulocladium spores released from
mould patches on walls in damp houses sediment relatively rapidly (Hunter et al., 1988) so
that, even where growth is profuse, the mould is likely to be detected in the air in quantity
only shortly after disturbance
of the growth or re-entrainment
of settled spores as a result of
activity. An investigation
over one day in a nursery school classroom (Mouilleseaux
and
Squinazi, 1991) illustrates the wide fluctuations
in numbers of airborne viable spores which
can occur as a result of variation of activity levels in a room (Fig. 1).
It has often been suggested that to circumvent this temporal variability housedust should
be sampled instead, as it provides a “memory”
of previously
airborne microorganisms
which are re-entrained
in the indoor air as a result of the activity of building occupants.
However, although the housedust mycobiota reflects that of the air, there are differences in
the relative abundance
of some types, and a Basidiomycete
common in the air within homes
in Scotland, Sistotrema brinkmannii, is only found infrequently
and in small numbers in
housedust (Flannigan
et al., 1993, 1996). This therefore argues against sampling of dust as
a substitute for air sampling. In addition, viable counts for settled dust are very much higher
than corresponding
air sampler counts for aerosolized dust (Flannigan
et al., 1994). This
suggests that many microorganisms
in dust either form aggregates or are carried on dust
particles which settle very rapidly and are too large to be respirable.
Fig.
1. Variability
in counts of viable airborne fungi during the course of a day in a nursery
classroom (after Mouilleseaux
and Squinazi, 1991).
school
B. Flannigan
386
In most investigations
of fungi in indoor air, the isolation media used favour the growth
of hydrophilic
species (Flannigan,
1992; Flannigan
and Miller, 1994) and. by being nutritionally rich, e.g. Blakeslee’s malt extract agar or modified Sabouraud
agar, may introduce
bias in favour of rapidly growing species. An international
workshop, considering the effect
of fungi in buildings on health, recommended
that such rich media should not be used
(Samson et al., 1994). Further error occurs because some fungi do not compete well with
others on isolation
plates, even if the medium is suitable for their growth, and are
consequently
not recorded as frequently as they merit, e.g. Alternariu spp. Because some
fungi have spores distinctive enough to be recognised microscopically,
two different types of
sampler operating on different principles, one for a total count of such species and one for
a viable count of those without distinctive spores, have sometimes been used in the same
health investigation
(Su et ul.. 1990; Su and Spengler. 1991).
Xerophilic species may be isolated on the media mentioned
above, but are only usually
seen when the propagules
of faster growing species are absent or few. Since xerophilic
and Wallemiu sehi are
moulds such as Eurotium spp., Aspergillus restrictus, A. penicillioides
known to be present in housedust and indoor air and are allergenic (Verhoeff et ul., 1990a, b;
Flannigan
and Miller, 1994), it has been recommended
that a low water activity medium,
e.g. dichloran-18%
glycerol agar, should be included among the isolation media used in
indoor air studies (Samson et al.. 1994).
ALTERNATIVE
APPROACHES
TO ASSESSMENT
Given the problems of assessment mentioned above, there is a strong argument for using
non-cultural
rapid methods to quantify the airborne microbial burden although, generally.
these do not allow identification
of the microorganisms
present. Such methods can be based
on chemical components,
or bio-markers.
common to organisms in particular groups, e.g.
chemical markers for peptidoglycan
or lipopolysaccharide
(LPS) in the bacterial
cell
envelope (Fox et ul., 1993; Fox and Rosario, 1994), or chitin or ergosterol in. respectively,
the walls and membranes
of hyphae and spores of filamentous
fungi and yeast cells.
Measurements
of chitin have been used as an index of fungal biomass in environments
ranging from grain (Donald and Mirocha.
1977) to wood (Swift, 1973). However, this
technique cannot be applied where insects or other arthropods are likely to be encountered.
The amount of chitin in a fragment of insect exoskeleton collected as part of an air sample is
likely to be much greater than the contribution
of all fungi in the sample. There is the
additional problem of the amount of chitin differing with species, growth conditions and the
nature of the structure (hyphal fragment or spore).
As most investigations
of fungal aerosols in indoor air are health-driven,
it can
be considered
that assessment
will best be made by measuring
some component
or
components
of the aerosol with clear biomedical effects, e.g. particular allergens. Substances
that are found in virtually
all fungi of consequence
in indoor air and which have
potent biomedical
effects are the so-called (l-3)-/j-D-glucans
in walls of hyphae and
spores. In fact, although
the predominant
linkage in such polyglucoses
is (1+3), they
are branched
mixed-linkage
polymers
with (1 +6) cross-linkages.
In mammals,
there
are receptors for fi-glucan on alveolar macrophages.
neutrophils,
basophils
and other
cells (Czop and Kay, 1991). Exposure to the glucan causes inflammation
reactions in
lymphocytes,
affects interleukin-1
secretion
via T-lymphocytes,
stimulates
bacterial
and tumour defence mechanisms, causes a decrease in numbers of pulmonary
macrophages
and inhibits phagocytosis.
A decrease in lymphocyte
numbers in the lung wall is the
opposite of the effect of exposure to endotoxin (Fogelmark
rt al.. 1994). Large concentrations of airborne
I-glucan
have been associated
with increased
reporting
of mucous
membrane irritation and fatigue by occupants of buildings in which there were greater than
normal numbers of complaints
of building-related
health effects (Rylander et al., 1992).
Disregulation
of pulmonary
macrophages
and the associated
release of inflammatory
mediators
could be responsible
for headache, fatigue and other neurological
symptoms
(Rylander, 1995).
Sampling
indoor
environments
387
Limulus amoebocyte
lysate (LAL) preparations
used for quantifying
bacterial endotoxin
(LPS) are known to be coagulated
by (l-+3)-/&glucans,
although they are lOOO-fold more
sensitive to LPS than glucans (Roslansky and Novitsky, 1991). LAL can be fractionated
to
produce a preparation
specifically sensitive to /3-glucan (Kitagawa et al., 1991). Rylander
and his colleagues (e.g. Rylander et al., 1992) have used such a fraction for quantifying
P-glucan in indoor air. The method involves collection of an air sample on a microporous
et al.,
filter similarly to the CAMNEA
method for microbiological
analysis (Palmgren
1986a, b). The glucan in the sample on the membrane
filter is extracted by autoclaving
in
a saponin solution and assayed using the glucan-specific
fraction. Bacterial endotoxin in the
same sample can be extracted in saponin at room temperature
before this, and assayed
using an endotoxin-specific
fraction (Rylander et al., 1992). Although the technique may
appear to have more relevance to health-related
studies of indoor air than traditional
methods, more research into its use is required. Because availability
of the glucan-specific
LAL is restricted, studies of indoor air in which it has been applied are limited in number
and design, and doseeresponse
experiments
with individual
species and mixtures
are
lacking, fuller evaluation
is clearly needed.
Like P-glucan in walls, ergosterol, the principal sterol in membranes
of hyphae and
spores, provides a means of assaying for fungal biomass but again gives no information
on
species present. Ergosterol measurements
have previously been used as an index of fungal
biomass in housedust (Miller et al., 1988) and what appears to be a very promising new
method for determination
of airborne
ergosterol
has now been developed (Miller and
Young, 1996) and used in investigations
of homes. Since ergosterol is stable under air-dry
conditions,
spores can be collected on a microporous
filter as in the CAMNEA
method
(Palmgren et al., 1986a, b). The sterol is extracted from the collected spores in basic aqueous
methanol, assisted by microwave heating, and then analysed by high performance
liquid
chromatography
(HPLC), gas chromatography
(GC) or GC-mass spectroscopy
(Young,
1995). Most common species in indoor air have roughly the same distribution
of spore sizes,
and the ergosterol
content
of spores of some ten common
moulds was similar after
adjustment
for size, at about 3.2 fg ergosterol mg-’ spores (Miller and Young, 1996).
Although dependent on species and analytical method, the minimum detectable number of
spores on a filter appears to be 2&100 (Young, 1955).
IDENTIFICATION
Identification
of microorganisms
presents a major problem for investigations
of indoor
air and, in many studies, the isolated fungi are assigned only to broad categories such as
“pink yeasts” or “wood-rotting
Basidiomycetes”
or to genera such as Penicillium and
Aspergillus. However, in recent cases in North America where ill-health had been attributed
by workers to their workplace, labour laws have dictated that detailed information
on the
properties of fungal species contaminating
indoor air be provided for the workers. With the
diversity of species in genera such as Penicillium and Aspergillus., and the differences in the
ecological, allergenic and toxigenic characteristics
between species in these genera, this
clearly calls for reliable identification.
Extrapolating
from a survey of toxigenic fusaria, it
can however be suggested that in the literature perhaps 50% of mould identifications
are
incorrect (Flannigan
and Miller, 1994). Accurate identification
requires skilled and experienced mycologists,
preferably with experience of fungi from food or soil. Even then, few
laboratories
have the high level of expertise required to identify, with certainty, the species
in Penicillium and other difficult genera. The problem
of dependable
identification
is
compounded
by the world-wide decline in the teaching of taxonomy
and systematics in
universities. It is becoming increasingly
evident that the numbers of individuals
with skills
in traditional
identification
methods is going to continue to decrease in the foreseeable
future. Therefore, it will be necessary to develop new methods which do not demand the
degree of training and expertise currently required. There are developments
in fields such as
medical and food microbiology
which indicate a range of approaches
that could, at least,
partly alleviate the problem.
388
B. Flannigan
Since immunological
methods are widely used in medical microbiology
and most investigations of indoor air are health-related,
it is not unnatural to consider such methods. Here,
an interesting approach involving use of serum from patients with extrinsic allergic alveolitis (EAA) symptoms to explore their home environment
was adopted by Zwick er al. (1991).
Air was sampled (10 min) with a Burkard personal air sampler and spores impacted on the
glass slide were counted; total counts ranged from < lo4 spores m-3 (“low”) to > 10’
rnp3 (“high”). Half of each spore sample area was then coated with dilutions of individual
patient’s serum, shown previously by immunodiffusion
tests to contain IgG/IgM precipitating antibodies to one or more of Aureobusidium
pullulans, Aspergillus jumigatus, “Cephalosporium”, “Penicillium
species” or the thermophilic
actinomycetes,
Faenia rectivirgula
and
Thermoactinomyces
oulgaris. The half of the spore sample area not flooded with patient’s
serum acted as a negative control. After reaction and rinsing, the whole slide was flooded
with fluorescent-labelled
anti-human
IgG/IgM antiserum and examined, after final rinsing,
for fluorescent
spores. This technique
could therefore be of value in EAA cases for
confirming
both exposure and sensitization
to airborne spores of particular
microorganisms, and also in pinpointing
areas requiring air quality control. It could also be adapted by
employing antisera raised against other species or groups of microorganisms.
For example,
monoclonal
antibodies have been produced in other fields of study, such as plant disease
and food spoilage (Dewey et al., 1993) for detecting and quantifying
particular genera, e.g.
Aspergillus,
Fusarium and Penicillium,
or particuar species, e.g. P. islandicum and Humicola
ianuginosa
(Thermomyces
lanuginosus).
Those which recognise epitopes on spore walls
could be used in a similar manner to that reported by Zwick et al. (1991) for aerometric
studies, via either fluorescent antibody technique or ELISA.
Just as in other areas of environmental
investigation,
polymerase chain reaction (PCR)
and other molecular
biology techniques
are likely to find use in the detection of wellcharacterised
toxigenic or pathogenic
microorganisms
in air. Alvarez et al. (1994) have
shown the potential of solid-phase PCR (SP-PCR) for detecting specific airborne microorganisms which might be unculturable
because of stress caused by aerosolization,
environmental exposure
or sampling.
In laboratory
experiments,
they employed
a strain of
Escherichiu
coli which contained
a plasmid with a 437-base pair insert from the silkworm
BonzhJlx mori, which was a unique marker for identification
of this strain. E. coli DHl.
Aqueous suspensions
of the bacterium
were aerosolized
and the aerosols sampled for
5 s-10 min (l-120 1) using AGI-30 all-glass liquid impingers. For SP-PCR, collection buffer
was filtered through Nytran filters, and the residue lysed before binding of DNA (with
nonspecific
DNA binding being blocked). The bound DNA was then amplified in 30
denaturation/annealing/primer
extension cycles. SP-PCR showed greater sensitivity than
was achieved by filtering corresponding
aliquots of collection buffer through a membrane
filter and incubating
that on agar medium for colony counts. Although an earlier report
that PCR would detect only viable Legionella pneumophila, Josephson rt al. (1993) showed it
could be used for detecting nonviable
E. coli, Salmonella
typhi and Shigella sonnei in
environmental
samples. As Josephson et ul. (1993) have pointed out, care must be taken in
interpreting
PCR results; positive amplification
products do not mean that target organisms are viable, only that target nucleic acid sequences are present.
Clearly, it cannot be expected that immunological
or PCR methods will be developed for
all taxa in indoor air which have some bearing on health. However, there is a real possibility
that the identity of airborne organisms belonging to some difficult genera, such as Penicilby reference to mycotoxin,/secondary
metabolite
and volatile
lium, could be confirmed
profiles. Larsen and Frisvad (1994) in whose laboratory secondary metabolite profiles have
been used successfully in chemotaxonomy
of penicillia, have pointed the way here. In
a preliminary
study of seven common indoor penicillia and Aspergillus
cersicolor,
these
authors prepared extracts from heavily sporulating
cultures on agar plates, and after
clean-up subjected the extracts to analysis by HPLC. When the HPLC traces for two
penicillia isolated from the air spora in houses and grown on Sigma yeast extract-sucrose
(SYES) and wallpaper paste (WP) agar are examined, clear differences are evident (Figs
2 and 3). The isolate of P. polonicum
produced
the mycotoxins,
penicillic acid and
Sampling
LC
j_
1000y
A
indoor
environments
389
of
of
225.5
600:
WALL
PAPER
PASTE
JTJ1AlSA.D
JTJlAlGA.
D
RGAR
600:
400:
-200:
2
-400:
.
-600:
UV
ii
26.710
<
t
j
-800:
-1000
.
i
5
:
b
Fig. 2. HPLC
LC
C
1400
SIGMA
-
.
.
.
10
.
.
.
YES
.
.
20
TImr
RGRR
.
.
_ -
<min.)
I
-
30
.
.
I
40
.
traces of cultures of Penicihm
polonicum (largely conidial) on wallpaper
and yeast extract sucrose (YES) agar (Larsen and Frisvad, 1994).
A
R
225.5
225.5
of
of
x
z
n
1200
wRu_
JTJ2AlSR.
JT.TZmlGA.
PRPER
.
-
paste agar
II
D
PASTE
AGAR
1000
800
600
400
200
0
-200
-400
SIGMR
YES
RGAR
-600
10
Fig. 3. HPLC
-20
Tfme
tmln.
_ 30
J
40
traces of cultures of Penicillium expansum (largely conidial) on wallpaper
and yeast extract sucrose (YES) agar (Larsen and Frisvad, 1994).
I
paste agar
verrucosidin
(Fig. 2) whilst P. expansum produced patulin and chaetoglobosin
X (Fig. 3). As
seen from the peak areas, the medium on which they were cultured markedly affected the
quantities of individual compounds
present. However, the extracts of both SYES and WP
cultures of P. polonicum contained the metabolites
cyclopeptin, dehydrocyclopeptin,
cyclopenal, cyclopenin, viridicatol, 3-methoxyviridicatin,
normethylverrucosidin
and puberulins,
as well as penicillic acid and verrucosidin.
In addition to patulin and chaetoglobosin
X, P.
expansum produced citrinin and traces of chaetoglobosin
C. While qualitative
differences
between the mycotoxin/secondary
metabolite profiles of the three strains of each individual
species examined were minor in some cases, large differences were sometimes noted in
others, e.g. P. commune and A. versicolor.
B. Flannigan
390
Table 4. Major
identrfied
volatile
during
compounds
produced by strains of Penrcilhn
chr~soyrnum
growth on SYES agar (Larsen and Frisvad, 1994)
P. ch,.l.soyenutn
Compound
2-methyl-1-propanol
1-heptene
3-methyl-3-buten3-methyl-1-butanol
1-pentanol
1$nonadiene
1-octen-3-01
monoterpene
I
3-octanone
3-octanol
monoterpene
2
2-methyl-isoborneol
Note: +
volatiles; -
l-01
P.
IBT
6041
IBT
4645
IBT
6183
++
+
+
++
+
+
+
+
+
+
+
+
++
++
_
++
+
+++
_
+
+
_
++
+7
++
++
++
+++
+++
++
+ + , + + + : relatrve amounts
: absent from sample.
IBT
6328
and P. commute
coll*mul,<
IBT
3468
+++
+++
_
+
++
(FID peak area) of identified
IBT
10727
+t
i
++
++
+
+
+
compounds
in sample
++
+++
+++
+
+
of collected
Larsen and Frisvad (1994) also analysed volatile compounds
emanating from agar plate
cultures of eight different species. Diffusing volatiles were collected passively on carbon
black in a tube supported
on a stainless-steel
net under the lid of each petri dish. The
volatiles were desorbed from the carbon black by elution with diethyl ether and the
resulting solutions
analysed using a gas chromatograph
with flame ionisation
detector
GC-FID, and further characterized
by mass spectrometry
(GC-MS) or Fourier transform
infrared detector (GC-FTIRD).
By way of example, the main volatile compounds
identified
from isolates of two Penidlium
spp. commonly
associated with indoor air problems in
Danish buildings
during growth on SYES are shown in Table 4. With both penicillia,
growth on SYES gave good qualitative
agreement
between strains of the same species.
Although not shown, the qualitative agreement between compounds
from cultures on SYES
and WP was less good and the quantities of volatiles produced on SYES were generally
larger than on WP (Larsen and Frisvad,
1994). illustrating
the well-known
effect of
nutritional
and environmental
factors on production
of volatile compounds
by microorganisms. Nevertheless,
there were marked differences in the major volatiles not only between
these two species, but between all seven penicillia. Since that preliminary
study, Larsen and
Frisvad (1995) have confirmed the potential value of volatile profiles from 132 isolates of 25
different terverticillate
Penidium
taxa, during growth on SYES, for identification.
Taxometric analysis showed perfect agreement
between results for volatiles and previous
classification
based on chemotaxonomy
using biosynthetic
families of non-volatile
secondary metabolites.
CONCLUSION
Sampling methods which involve culture will continue to provide valuable information
on the types of organism in indoor air but, for health related investigations,
the deficiencies
militate against their use to give quantitative
estimates of exposure. At least for epidemiological
studies, a better measure of exposure than total counts made over extended
time periods may well be fungal biomass.
AcknowledyementPI
results.
thank
Dr J. David Miller, Agriculture
Canada,
Ottawa.
for permission
to quote unpublished
Sampling
indoor
environments
391
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