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Pergamon .I Aerosol Su Vol. 28, No. 3, pp. 3X1&392, 1997 CopyrIght ( 1997 Elsewer Saence Ltd Pnnted in Grrat Bntain All rughts reserved 0021-8502:97 $17.00 + 0.00 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. Al- though 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 the air spora in homes and non-industrial workplaces should be is of a mixture of species resembling that in outdoor air. Certainly if the buildings are air-conditioned, or if windows and doors are kept closed in summer, the indoor counts could be expected to be somewhat lower. Field or phylloplane fungi such as Alternaria and Epicoccum conform to this picture, but the ratio for the sum of species of Aspergillus and, especially, Penicillium outdoors to that indoors may either be 381
Transcript
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Pergamon .I Aerosol Su Vol. 28, No. 3, pp. 3X1&392, 1997 CopyrIght ( 1997 Elsewer Saence Ltd

Pnnted in Grrat Bntain All rughts reserved 0021-8502:97 $17.00 + 0.00 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. Al- though 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 the air spora in homes and non-industrial workplaces should be is of a mixture of species resembling that in outdoor air. Certainly if the buildings are air-conditioned, or if windows and doors are kept closed in summer, the indoor counts could be expected to be somewhat lower. Field or phylloplane fungi such as Alternaria and Epicoccum conform to this picture, but the ratio for the sum of species of Aspergillus and, especially, Penicillium outdoors to that indoors may either be

381

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382 B. Flannigan

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)

Category*

Indoor Outdoor

Spores (m “) ( %B ) Spores (mm’) ( “A, )

Hyphal fragments AItrrnu~icc A.speryillusiPenicilli~rm CIudo.sporium Coprimu Epicoccum Gtrnotlrrmn Lrptosphawiu Unidentified ascosporea Unidentified basidiospores Other unidentified spores

146 44

457 895

41

59 1x2

65 152 206

6.3 112 3.2 1.9 74 2. I

19.8 131 3.8 38.8 1479 42.5

1.8 78 2.3 0.3 20 0.6 2.6 Ill 3.2 7.9 541 15.1 2.8 138 4.0 6.5 310 8.9 8.9 301 8.7

* 28 other taxa. spores of which were recorded occasionally, 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 counts of Cladosporium and Alternaria, although lower, coinciding with those outdoors at 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 investiga- tion 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 non- culturable 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

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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 poly- carbonate 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 using different sampling methods in a restaurant (after Flannigan, 1992)

Concentration (CFU m 3 air)

Area Before open

Andersen sampling*

Early Mid-day evening

CAMNEA method

Plate count DEFT method

a.m. p.m. a.m p.m.

Pantry Moulds Bacteria

Dininy Moulds Bacteria

212 141 129 3160 920 10,200 1260 224 152 208 138 625 34,910 5100

35 565 24 521 348 2010 12,360 106 244 88 417 1180 43,250 13,580

* Raw counts converted by the “positive hole” method to correct for multiple impactions of propagules at the same sites on the agar collection plates (Andersen, 1958).

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384 B. Flannigan

Table 3. Spearman correlation between Burkard personal sampler total counts and Andersen two-stage sampler viable counts for indoor air in 41

homes (DeKoster and Thorne, 1995)

Category

Altrrnurii~ Aspe~yillusiPeni~illilrrn Chlosporiurn Unclassified Total

R values p values

0.43 0.02 0.45 0.01 0.65 < 0.0001 0.22 0.21 0.80 < 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 suscep- tible 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 sampling period. For example, spores of Aspergilhs and Penicillium may survive for long 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.

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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). Studies in an experimental room, into which spores of Penicillium chrysogenum were 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 particles which settle very rapidly and are too large to be respirable.

are carried on dust

Fig. 1. Variability in counts of viable airborne fungi during the course of a day in a nursery school classroom (after Mouilleseaux and Squinazi, 1991).

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386 B. Flannigan

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 nutri- tionally 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 moulds such as Eurotium spp., Aspergillus restrictus, A. penicillioides and Wallemiu sehi are 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 concentra- tions 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).

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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 filter similarly to the CAMNEA method for microbiological analysis (Palmgren et al., 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 experi- enced 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.

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388 B. Flannigan

Since immunological methods are widely used in medical microbiology and most invest- igations 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 alveoli- tis (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 precipita- ting antibodies to one or more of Aureobusidium pullulans, Aspergillus jumigatus, “Cephalos- porium”, “ 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 microorgan- isms, 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 well- characterised toxigenic or pathogenic microorganisms in air. Alvarez et al. (1994) have shown the potential of solid-phase PCR (SP-PCR) for detecting specific airborne microor- ganisms which might be unculturable because of stress caused by aerosolization, environ- mental 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 organ- isms 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 Penicil- lium, could be confirmed by reference to mycotoxin,/secondary metabolite and volatile 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

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Sampling indoor environments 389

LC A 225.5 of JTJ1AlSA.D 1000y j_ of JTJlAlGA. D

600: WALL PAPER PASTE RGAR

600:

400:

-200: 2

-400: ii UV 26.710 < . t

-600: j

. i -800: 5 : b SIGMA YES RGRR

-1000 - . . . . . . . . . . _ - I - . . I . . - 10 20 30 40

TImr <min.)

Fig. 2. HPLC traces of cultures of Penicihm polonicum (largely conidial) on wallpaper paste agar and yeast extract sucrose (YES) agar (Larsen and Frisvad, 1994).

LC A 225.5 x of JTJ2AlSR. II 1400 C R 225.5 of JT.TZmlGA. D

z n

1200 wRu_ PRPER PASTE AGAR

1000

800

600 400 200

0 -200 -400 SIGMR YES RGAR -600

10 -20 30 40 _ Tfme tmln. J I

Fig. 3. HPLC traces of cultures of Penicillium expansum (largely conidial) on wallpaper paste agar and yeast extract sucrose (YES) agar (Larsen and Frisvad, 1994).

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, cyclo- penal, 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.

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390 B. Flannigan

Table 4. Major identrfied volatile compounds produced by strains of Penrcilhn chr~soyrnum and P. commute during growth on SYES agar (Larsen and Frisvad, 1994)

Compound IBT 6041

P. ch,.l.soyenutn P. coll*mul,<

IBT IBT IBT IBT IBT 4645 6183 6328 3468 10727

2-methyl-1-propanol 1 -heptene 3-methyl-3-buten- l-01 3-methyl-1-butanol 1 -pentanol 1 $nonadiene 1-octen-3-01 monoterpene I 3-octanone 3-octanol monoterpene 2 2-methyl-isoborneol

++ + + +++ + + + + + + _ ++ + + +

++ ++ + + ++ +++ + _ _ _ ++ ++ +7 ++ ++

++ +++ +++ ++

+++ +t

i

++ ++ ++ +++ + +++ + + + +

Note: + + + , + + + : relatrve amounts (FID peak area) of identified compounds in sample of collected volatiles; - : absent from sample.

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 microorgan- isms. 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. Tax- ometric analysis showed perfect agreement between results for volatiles and previous classification based on chemotaxonomy using biosynthetic families of non-volatile second- ary 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 epi- demiological studies, a better measure of exposure than total counts made over extended time periods may well be fungal biomass.

AcknowledyementPI thank Dr J. David Miller, Agriculture Canada, Ottawa. for permission to quote unpublished results.

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Sampling indoor environments 391

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