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Characterisation of exposure to airborne fungi: Measurement of ergosterol
Journal of Microbiological Methods 63 (2005) 185 – 192 www.elsevier.com/locate/jmicmeth
Characterisation of exposure to airborne fungi: Measurement of ergosterol Enric RobineT, Isabelle Lacaze, Ste´phane Moularat, Se´bastien Ritoux, Marjorie Boissier Laboratoire de Microbiologie des Environnements Inte´rieurs, Centre Scientifique et Technique du Baˆtiment, 84 avenue Jean Jaure`s, Champs sur Marne. 77447 Marne la Valle´e Cedex 02, France Received 16 February 2004; received in revised form 14 March 2005; accepted 14 March 2005 Available online 21 September 2005
Abstract In order to gain a clearer understanding of the level of fungal air contamination in indoor environments, we have adapted and tested a method to evaluate fungal biomass. Liquid phase chromatography (HPLC) of ergosterol, a component of the cell membrane of microscopic fungi, was employed. This method permits the detection and identification of ergosterol molecules at a concentration of 40 Ag/ml (n = 33, r = 5). By combining this assay with a rotating cup collection apparatus, it was possible to measure fungal flora levels with a limit of quantification of 0.4 ng/m3 or a theoretical value of 150 spores per cubic meter (m3). Measurements of ergosterol levels performed on different sites showed that this method reflected the different situations of exposure of occupants to airborne fungal flora. D 2005 Elsevier B.V. All rights reserved. Keywords: Mould; Bioaerosol; Ergoste´rol; Indoor air quality
1. Introduction No figures are available on the prevalence in France of fungal contamination in indoor environments, although a recent bibliographical study by the
T Corresponding author. Scientific and Technical Building Institute (CSTB), Sustainable Development Department, Health and Building, 84 avenue J. Jaure`s, Champs sur Marne F-77447 Marnela-Valle´e Cedex 02, France. Tel.: +33 1 64 68 82 66; fax: +33 1 64 68 88 23. E-mail address: [email protected], [email protected] (E. Robine). 0167-7012/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2005.03.008
Institut National de Sante´ Public du Que´bec (2002) reported the extent of the risks linked to the presence of moulds in these environments. The development of moulds inside dwellings is a phenomenon which occurs in both old buildings and recent constructions. These buildings may constitute becological nichesQ for the development of these micro-organisms, influenced by various factors which include design and construction, shapes and configurations, materials and structures, the type of use to which a building is put and its conditions of maintenance (Hyva¨rinen et al., 2002).
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E. Robine et al. / Journal of Microbiological Methods 63 (2005) 185–192
The contamination of buildings raises problems both with respect to both the deterioration of materials and structures and a not inconsiderable health risk because these bio-contaminants can, under certain circumstances of exposure, give rise to the onset of disease: allergies, infections, toxi-infections. (Kuhn and Ghannoum, 2003). Indeed, the inhalation of spores, mycelial fragments or particles of contaminated materials or dust may have a variety of effects on health. These events seem to correlate with an increase in the surface area that is contaminated in the building. Most epidemiological studies have demonstrated a link between the prevalence of respiratory symptoms and the presence of excessive moisture and moulds in these areas. However, very little data are available concerning exposure to moulds in an indoor environment, particularly because of the problems inherent in measuring these biological contaminants (Flannigan, 1997; Madelin, 1994). Thus, with respect to the dose of exposure, few reliable data are available at present to establish a threshold below which there are no effects on health, nor is there a list of reference documents that allows an assessment of the level of risk as a function of the species of moulds encountered. The definition of biological indicators and the development of appropriate measurement methods form part of an essential preliminary stage in achieving an assessment of the level of exposure of occupants. Metrological efforts must address not only the development and improvement of sampling techniques but also the development or adaptation of labelling or indirect recognition methods based in particular on metabolic products or specific cell constituents. Thus, conidia and mycelial fragments, the vectors of mould dispersion, contain an ergosterol ester which is common and specific to most microscopic fungi. Assay of this molecule has mainly been used to determine the contamination of solid substrates such as cereals or soils, . . .(Seitz et al., 1979; Grant and West, 1986; Zill et al., 1988; Gessner and Schmitt, 1996). More recently, this molecule was measured in dust, the products of construction and the indoor air in dwellings (Axelsson et al., 1995; Miller and Young, 1997; Pasanen et al., 2000). In the context of this study, we used ergosterol as a marker of the presence of fungi in indoor air. The aim
was to design and develop in the laboratory a methodology that could evaluate all microscopic fungi and to validate this technique in situ.
2. Materials and methods 2.1. Biological material 2.1.1. Choice of strains The tests were performed using a strain from the collection held by the Institut d’Hygie`ne et d’Epide´miologie de Mycologie (Mycological Hygiene and Epidemiology Institute) in Brussels, IHEM: Aspergillus niger 05077. This strain was stored in distilled water. A subculture to encourage sporulation was performed on Sabouraud agar prior to each use. The final culture was obtained after 7 days at 25 8C on oat agar media. 2.1.2. Counting of moulds The concentration of the fungal suspension was measured by turbidimetry. This method applies the principle of the light diffusion of particles in a liquid suspension. An HI 93703 (Hanna Instruments) model was employed. The measurement range of this apparatus is between 10 3 and 1 FTU (formazine turbidity unit). A correlation between the spore concentration and the turbidity of the suspension (Fig. 1) was established. The number of spores was evaluated using a Thoma cell. The conidia were counted by observation under a microscope in 5 squares representing a volume of 1/250 mm3. 2.2. Assay method for ergosterol The measurement principle was based on the UV absorbance of ergosterol at 282 nm. The ergesterol esters contained in the fungal cell membrane were released and transformed into alcohol by saponification. The isolated compound was then analysed by liquid phase chromatography. The extraction protocol consisted in placing contaminated substrates in 2 ml methanol and then subjecting them to ultrasounds (BRANSON 2510, Bransonic). The extract was collected and centrifuged at 6000 rpm for 15 min (EBA 8 S, Hettich). The supernatant was filtered (inorganic membrane filter ANOTOP, 0.2 Am What-
E. Robine et al. / Journal of Microbiological Methods 63 (2005) 185–192
187
Fig. 1. Relationship between turbidimetry and the concentration of a spore suspension.
man, VWR International) then assayed under high performance liquid chromatography (HPLC). The system was calibrated using a commercial extract of ergosterol with 99.4% purity (Fluka). The water system used was equipped with a LICHROSPHER RP18 column of 100 2 (Merck). Detection was ensured by a UV detector (Waters 996 Photodiode Array Detector). The entire apparatus was piloted by Millennium 32 software. We employed a mobile phase composed exclusively of methanol (methanol rectapur, Fisher) in an isocratic mode. The flow rate of this phase during use was equal to 1.5 l/min, with a resulting pressure of 1400–1500 PSI. When not in use, the column was stored in a methanol/water mixture (40 / 60), the water to prevent
drying and the methanol to prevent the proliferation of micro-organisms in the column. 2.3. Collection of airborne fungi 2.3.1. Use of the rotating cup system A cup coated with polyurethane foam and mounted on the axis of an electric motor was rotated at high speed within a cylindrical cavity (CIP 10, Arelco). The ambient air was aspirated via a selector. This selected the inhalable fraction of particles. The air was then exhausted via a tangential outlet on the wall of the apparatus (Fig. 2). The cup containing the particles collected was then processed so as to measure the quantity of ergosterol
Fig. 2. Diagram of the rotating cup apparatus and selector of the inhalable fraction (in compliance with NF norm X 43-262).
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sampled and consequently the concentration in the ambient air. Measurements were expressed in nanograms ergosterol per cubic metre of air. This apparatus complies with the requirements of the French norm NF X 43-262: bGravimetric determination of alveolar deposits of particulate pollutionrotating cup methodQ (Courbon et al., 1988; AFNOR, 1991). 2.3.2. Sampling using an impactor An impactor (Andersen Instruments Incorporated) was used to determine fungal contamination of the air. Level no. 4 was employed, the air being aspirated at a rate of 28.3 l/min for 2 min, (aperture diameter 0.71 mm; d 50 = 3.3 Am). The particles were collected on Sabouraud culture medium. The colonies were counted after 7 days of incubation in the dark (25 8C).
3. Results and discussion 3.1. Characterisation and validation of the technique 3.1.1. Uncertainty and limits of quantification For a standard range of ergosterol solutions at concentrations of between 0 and 10 mg/l, the relationships between peak surface areas and ergosterol concentrations were linear, with a coefficient of correlation, r 2, of 0.99. The dispersion of the analytical results was calculated from 31 operations performed in an identical fashion using a standard solution at 6 mg/l. It was accepted that a method was repeatable when the ratio between the standard deviation and the mean was low. The coefficient of variation thus calculated, lower than 1%, appeared to be highly satisfactory. Furthermore, using ergosterol solutions at concentrations Table 1 Stability of ergosterol in spores of Aspergillus niger after 19 and 27 days of storage and 7 days of collection Storage of sample
Mean (mg/l) Standard deviation No. of samples
Effect of collection
T0
T 19 days
T 27 days
T0
7 days of functioning
0.359 0.042
0.294 0.064
0.375 0.019
0.279 0.006
0.237 0.087
8
3
3
3
3
Table 2 Simultaneous measurements of outdoor airborne ergosterol Sampler
Ergosterol (ng/m3)
1 2 3 4 5 Mean Standard deviation
1.57 1.71 2.31 1.79 1.94 1.87 0.28
decreasing as from 1 mg/l, the mean value of the limit of quantification was evaluated at 0.04 mg/l (n = 33; r = 0.005). The calculated variation of coefficient for this limit was 12.5%. 3.1.2. Storage of samples and verification of the impact of rotation at collection After sampling, the cup coated with polyurethane foam was sent directly to the laboratory. It was necessarily protected and stored in an airtight packaging, protected from light. Ergosterol is a stable molecule, which mainly degrades when it is exposed to UV light. For example, a (standard) ergosterol solution can be stored at 4 8C protected from light for a period of two months without deteriorating. Nevertheless, we checked the stability of samples during the period of storage. Thus, 14 sampling cups were artificially seeded with the same quantity of spores (A. niger) and then placed in a cold room at 4 8C for 19 and 27 days. When compared with the reference, no significant changes in ergosterol concentrations were observed during these periods of storage (Table 1). Table 3 Comparison between airborne ergosterol concentrations and occasional measurements of cultivable fungal flora Sample date
07/03/01 07/31/01
CFU/m3 Min–max, n =3 38–42 292–358
Ergosterol Types of moulds 3 isolated Mean (ng/m ) from 07/30 to 08/06/01 40 320
08/02/01 1073–1257 1190 08/03/01 425–470 440
2.74 F 0.52 Majority species 95%: Cladosporium sp, Penicillium sp, and Aspergillus sp Minority species 5%: Ulocladium sp, Alternaria sp and sterile mycelium
E. Robine et al. / Journal of Microbiological Methods 63 (2005) 185–192
We also verified that the collection method did not affect the ergosterol content in conidia. An identical and known quantity of spores was distributed between six cups. Three were placed in the apparatus and rotated for 7 days protected from any fungal pollution, while the other three were stored at 25 8C for the same period. We did not note any significant differences in concentrations (Table 1).
189
Thus the collection method did not appear to degrade the ergosterol molecules. 3.1.3. Dispersion of measurements In order to evaluate the dispersion of measurements between different systems, we placed five sets of apparatus on the same outdoor site and collected ergosterol over a period of two weeks (198 m3). The
Fig. 3. (a) Measurement of airborne cultivable fungal flora over 1 day. (b) Measurement of airborne cultivable fungal flora over more than one week.
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cumulated uncertainty of sampling and analysis spread over a range of 19%. Although this test was subject to spatial fluctuations in concentrations on the site, it allowed an appreciation of the relatively good dispersion of measurements for environmental sampling (Table 2). 3.2. Comparison between ergosterol measurements and cultures The outdoor concentration of airborne ergosterol was measured by correlation with cultivable fungal flora. Ergosterol measurements were performed after 7 days of sampling and, during the same period, four occasional samples were collected using the impactor. The results are shown in Table 3. For a mean value of 498 CFU/m3 [40–1190], we measured a concentration of 2.74 ng ergosterol/m3. A previous study by Miller and Young (1997) had estimated the mean level of ergosterol per spore in three majority species at 2.45 ng/spore. For Alternaria alternata IHEM 3183, we measured (results not published) 4.62 pg/spore (n = 6, r = 1.54). This higher level was linked to the large size of Alternaria spores. This difference linked to the dimensions of conidia had already been observed in other species by the same author.
If we consider the respective percentages of the different species isolated (95% and 5%), it could be estimated that the mean quantity of ergosterol per spore was 2.56 pg, or a theoretical value of 1070 spores/m3. This theoretical concentration is double the mean value of the cultivable fraction. This difference was probably linked to the non-cultivable fraction collected. In addition, outdoor concentrations fluctuate markedly, variations of a factor of 2– 3 being observed over a day and a factor of nearly 100 over a week (Fig. 3a,b). The values for air contamination obtained by culture principally enables an instantaneous assessment of the level of contamination. Sampling rarely exceeds 2 min of collection and, depending on the apparatus employed, represents a maximum of 0.2 m3 air sampled. In contrast, the measurements of ergosterol covered a week of sampling, or nearly 100 m3 of air. 3.3. Measurements on site Air samples were collected on different sites: in a laboratory (double-flow ventilation system) with HEPA filters, in an occupied open-plan office (double-flow ventilation system), in six dwellings and in two schools.
Fig. 4. Measurements of airborne ergosterol present inside and outside different premises. Indoor samples in an area were collected at 1 metre from floor level. A further, reference measurement was always performed outside under the same conditions.
E. Robine et al. / Journal of Microbiological Methods 63 (2005) 185–192
The ergosterol concentrations measured during this study reached a maximum of 6.47 ng/m3 (Fig. 4). Several different types of air contamination were observed on five sites (dwellings 4, 5; schools A, B and the office); the outdoor and indoor concentrations did not differ significantly. The premises tested were, in principle, exempt of any endogenous sources of fungi. The source of most of the microscopic fungi present in indoor air was principally from outdoors. The level of contamination attained in these environments was therefore mainly the result of seasonal variations (Fig. 5), as the highest outdoor fungal concentrations are recorded in the summer. On four sites (1, 2, 6 and the laboratory), we observed levels which were lower indoors than outdoors. There were no endogenous fungal sources in these premises. The ventilation system (and the air conditioning when relevant) ensured a reduction of approximately 50% in the transfer of outdoor fungal pollution. The laboratory, which was equipped with a specific filtration system (very high efficiency filters) totally restricted the penetration of all fungal contaminants. In the case of dwelling 3, the indoor concentration was double the outdoor level of contamination. A source of contamination was clearly present on the
191
premises: contaminated materials, plants or contamination of the ventilation system. Thus the principal situations for indoor contamination were examined. Ergosterol measurements appeared to constitute a robust method capable of quantitatively evaluating levels of airborne fungal flora.
4. Conclusion The aim of this study was to establish a measurement system capable of estimating total levels of airborne fungal flora. A method for the assay of a specific cell component in fungi was adapted and developed accordingly. By combining this assay with a collection apparatus validated elsewhere, it was possible to measure total levels of fungal flora. When compared with traditional methods involving cultures, the assay of ergosterol levels is an integrated measurement. Culture enables an instantaneous assessment of the level of contamination, while the measurement of ergosterol can evaluate nearly a week of cumulated exposure. Regarding the detection of the cultivable fraction, the method proposed remains less sensitive,
Fig. 5. Outdoor measurement of airborne fungal biomass, year 2002–2003, on the Champs-sur-Marne site (France).
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with a limit of detection close to 150 spores/m3 (0.4 ng/m3, sampling of 100 m3). However, the fraction of non-cultivable spores can also cause immunoallergic or even toxic symptoms. Furthermore, the biological contamination of premises is a discontinuous phenomenon subject to aeraulics and the abrupt formation of bwavesQ of pollution which are difficult to measure by an instantaneous collection of the cultivable fraction. In addition, the ergosterol measurements carried out on different sites showed that they reflected the different situations of exposure of their occupants to airborne fungal flora. The use of this technique thus appears to be well suited to the quantitative evaluation of individual exposure. It should however be backed up by a qualitative approach; in this case, cultures should be employed, as only they allow identification of the most allergenic and/or toxinogenic moulds.
References AFNOR, 1991. Qualite´ de l’air, air des lieux de travail. NF X 43262 (Oct. 1990). Tome 3, 170 – 181. Axelsson, B.-O., Saraf, A., Larsson, L., 1995. Determination of ergosterol in organic dust by gas chromatography–mass spectrometry. J. Chromatogr., B, Biomed. Sci. Appl. 666 (1), 77 – 84. Courbon, P., Wrobel, R., Fabries, J.F., 1988. A new individual respirable dust sampler: the CIP 10. Ann. Occup. Hyg. 32 (1), 129 – 143.
Flannigan, B., 1997. Air sampling for fungi in indoor environments. J. Aerosol Sci. 28 (3), 381 – 392. Gessner, M.O., Schmitt, A.L., 1996. Use of solid-phase extraction to determine ergosterol concentrations in plant tissue colonized by fungi. Appl. Environ. Microbiol. 62 (2), 415 – 419. Grant, W.D., West, A.W., 1986. Measurement of ergosterol, diaminopimelic acid and glucosamine in soil: evaluation as indicators of microbial biomass. J. Microbiol. Methods 6, 47 – 53. Hyva¨rinen, A., Meklin, T., Vepsa¨la¨inen, A., Nevalainen, A., 2002. Fungi and actinobacteria in moisture-damaged building materials — concentrations and diversity. Int. Biodeterior. Biodegrad. 49, 27 – 37. Institut National de Sante´ Public du Que´bec, 2002. Les risques a` la sante´ associe´s a` la pre´sence de moisissures en milieu inte´rieur. document de´pose´ a` Sante´com, p. 163. http://www.sante´com. qc.ca. Kuhn, D.M., Ghannoum, M.A., 2003. Indoor mold, toxigenic fungi, and Stachybotrys chartarum: infectious disease perspective. Clin. Microbiol. Rev. 16 (1), 144 – 172. Madelin, T.M., 1994. Fungal aerosols: a review. J. Aerosol Sci. 25 (8), 1405 – 1412. Miller, J.D., Young, J.C., 1997. The use of ergosterol to measure exposure to fungal propagules in indoor air. Am. Ind. Hyg. Assoc. J. 58, 39 – 43. Pasanen, A.L., Kasanen, J.-P., Rautiala, S., Ika¨heimo, M., Rantama¨ki, J., Ka¨a¨ria¨inen, H., et al., 2000. Fungal growth and survival in building materials under fluctuating moisture and temperature conditions. Int. Biodeterior. Biodegrad. 46, 117 – 127. Seitz, L.M., Sauer, D.B., Burroughs, R., Mohr, H.E., Hubbard, J.D., 1979. Ergosterol as a measure of fungal growth. Phytopathology 69, 1202 – 1203. Zill, G., Engelhardt, G., Wallnffer, P.R., 1988. Determination of ergosterol as a measure of fungal growth using Si 60 HPLC. Z. Lebensm.-Unters. Forsch. 187 (3), 246 – 249.
Characterisation of exposure to airborne fungi: Measurement of ergosterol Enric RobineT, Isabelle Lacaze, Ste´phane Moularat, Se´bastien Ritoux, Marjorie Boissier Laboratoire de Microbiologie des Environnements Inte´rieurs, Centre Scientifique et Technique du Baˆtiment, 84 avenue Jean Jaure`s, Champs sur Marne. 77447 Marne la Valle´e Cedex 02, France Received 16 February 2004; received in revised form 14 March 2005; accepted 14 March 2005 Available online 21 September 2005
Abstract In order to gain a clearer understanding of the level of fungal air contamination in indoor environments, we have adapted and tested a method to evaluate fungal biomass. Liquid phase chromatography (HPLC) of ergosterol, a component of the cell membrane of microscopic fungi, was employed. This method permits the detection and identification of ergosterol molecules at a concentration of 40 Ag/ml (n = 33, r = 5). By combining this assay with a rotating cup collection apparatus, it was possible to measure fungal flora levels with a limit of quantification of 0.4 ng/m3 or a theoretical value of 150 spores per cubic meter (m3). Measurements of ergosterol levels performed on different sites showed that this method reflected the different situations of exposure of occupants to airborne fungal flora. D 2005 Elsevier B.V. All rights reserved. Keywords: Mould; Bioaerosol; Ergoste´rol; Indoor air quality
1. Introduction No figures are available on the prevalence in France of fungal contamination in indoor environments, although a recent bibliographical study by the
T Corresponding author. Scientific and Technical Building Institute (CSTB), Sustainable Development Department, Health and Building, 84 avenue J. Jaure`s, Champs sur Marne F-77447 Marnela-Valle´e Cedex 02, France. Tel.: +33 1 64 68 82 66; fax: +33 1 64 68 88 23. E-mail address: [email protected], [email protected] (E. Robine). 0167-7012/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2005.03.008
Institut National de Sante´ Public du Que´bec (2002) reported the extent of the risks linked to the presence of moulds in these environments. The development of moulds inside dwellings is a phenomenon which occurs in both old buildings and recent constructions. These buildings may constitute becological nichesQ for the development of these micro-organisms, influenced by various factors which include design and construction, shapes and configurations, materials and structures, the type of use to which a building is put and its conditions of maintenance (Hyva¨rinen et al., 2002).
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E. Robine et al. / Journal of Microbiological Methods 63 (2005) 185–192
The contamination of buildings raises problems both with respect to both the deterioration of materials and structures and a not inconsiderable health risk because these bio-contaminants can, under certain circumstances of exposure, give rise to the onset of disease: allergies, infections, toxi-infections. (Kuhn and Ghannoum, 2003). Indeed, the inhalation of spores, mycelial fragments or particles of contaminated materials or dust may have a variety of effects on health. These events seem to correlate with an increase in the surface area that is contaminated in the building. Most epidemiological studies have demonstrated a link between the prevalence of respiratory symptoms and the presence of excessive moisture and moulds in these areas. However, very little data are available concerning exposure to moulds in an indoor environment, particularly because of the problems inherent in measuring these biological contaminants (Flannigan, 1997; Madelin, 1994). Thus, with respect to the dose of exposure, few reliable data are available at present to establish a threshold below which there are no effects on health, nor is there a list of reference documents that allows an assessment of the level of risk as a function of the species of moulds encountered. The definition of biological indicators and the development of appropriate measurement methods form part of an essential preliminary stage in achieving an assessment of the level of exposure of occupants. Metrological efforts must address not only the development and improvement of sampling techniques but also the development or adaptation of labelling or indirect recognition methods based in particular on metabolic products or specific cell constituents. Thus, conidia and mycelial fragments, the vectors of mould dispersion, contain an ergosterol ester which is common and specific to most microscopic fungi. Assay of this molecule has mainly been used to determine the contamination of solid substrates such as cereals or soils, . . .(Seitz et al., 1979; Grant and West, 1986; Zill et al., 1988; Gessner and Schmitt, 1996). More recently, this molecule was measured in dust, the products of construction and the indoor air in dwellings (Axelsson et al., 1995; Miller and Young, 1997; Pasanen et al., 2000). In the context of this study, we used ergosterol as a marker of the presence of fungi in indoor air. The aim
was to design and develop in the laboratory a methodology that could evaluate all microscopic fungi and to validate this technique in situ.
2. Materials and methods 2.1. Biological material 2.1.1. Choice of strains The tests were performed using a strain from the collection held by the Institut d’Hygie`ne et d’Epide´miologie de Mycologie (Mycological Hygiene and Epidemiology Institute) in Brussels, IHEM: Aspergillus niger 05077. This strain was stored in distilled water. A subculture to encourage sporulation was performed on Sabouraud agar prior to each use. The final culture was obtained after 7 days at 25 8C on oat agar media. 2.1.2. Counting of moulds The concentration of the fungal suspension was measured by turbidimetry. This method applies the principle of the light diffusion of particles in a liquid suspension. An HI 93703 (Hanna Instruments) model was employed. The measurement range of this apparatus is between 10 3 and 1 FTU (formazine turbidity unit). A correlation between the spore concentration and the turbidity of the suspension (Fig. 1) was established. The number of spores was evaluated using a Thoma cell. The conidia were counted by observation under a microscope in 5 squares representing a volume of 1/250 mm3. 2.2. Assay method for ergosterol The measurement principle was based on the UV absorbance of ergosterol at 282 nm. The ergesterol esters contained in the fungal cell membrane were released and transformed into alcohol by saponification. The isolated compound was then analysed by liquid phase chromatography. The extraction protocol consisted in placing contaminated substrates in 2 ml methanol and then subjecting them to ultrasounds (BRANSON 2510, Bransonic). The extract was collected and centrifuged at 6000 rpm for 15 min (EBA 8 S, Hettich). The supernatant was filtered (inorganic membrane filter ANOTOP, 0.2 Am What-
E. Robine et al. / Journal of Microbiological Methods 63 (2005) 185–192
187
Fig. 1. Relationship between turbidimetry and the concentration of a spore suspension.
man, VWR International) then assayed under high performance liquid chromatography (HPLC). The system was calibrated using a commercial extract of ergosterol with 99.4% purity (Fluka). The water system used was equipped with a LICHROSPHER RP18 column of 100 2 (Merck). Detection was ensured by a UV detector (Waters 996 Photodiode Array Detector). The entire apparatus was piloted by Millennium 32 software. We employed a mobile phase composed exclusively of methanol (methanol rectapur, Fisher) in an isocratic mode. The flow rate of this phase during use was equal to 1.5 l/min, with a resulting pressure of 1400–1500 PSI. When not in use, the column was stored in a methanol/water mixture (40 / 60), the water to prevent
drying and the methanol to prevent the proliferation of micro-organisms in the column. 2.3. Collection of airborne fungi 2.3.1. Use of the rotating cup system A cup coated with polyurethane foam and mounted on the axis of an electric motor was rotated at high speed within a cylindrical cavity (CIP 10, Arelco). The ambient air was aspirated via a selector. This selected the inhalable fraction of particles. The air was then exhausted via a tangential outlet on the wall of the apparatus (Fig. 2). The cup containing the particles collected was then processed so as to measure the quantity of ergosterol
Fig. 2. Diagram of the rotating cup apparatus and selector of the inhalable fraction (in compliance with NF norm X 43-262).
188
E. Robine et al. / Journal of Microbiological Methods 63 (2005) 185–192
sampled and consequently the concentration in the ambient air. Measurements were expressed in nanograms ergosterol per cubic metre of air. This apparatus complies with the requirements of the French norm NF X 43-262: bGravimetric determination of alveolar deposits of particulate pollutionrotating cup methodQ (Courbon et al., 1988; AFNOR, 1991). 2.3.2. Sampling using an impactor An impactor (Andersen Instruments Incorporated) was used to determine fungal contamination of the air. Level no. 4 was employed, the air being aspirated at a rate of 28.3 l/min for 2 min, (aperture diameter 0.71 mm; d 50 = 3.3 Am). The particles were collected on Sabouraud culture medium. The colonies were counted after 7 days of incubation in the dark (25 8C).
3. Results and discussion 3.1. Characterisation and validation of the technique 3.1.1. Uncertainty and limits of quantification For a standard range of ergosterol solutions at concentrations of between 0 and 10 mg/l, the relationships between peak surface areas and ergosterol concentrations were linear, with a coefficient of correlation, r 2, of 0.99. The dispersion of the analytical results was calculated from 31 operations performed in an identical fashion using a standard solution at 6 mg/l. It was accepted that a method was repeatable when the ratio between the standard deviation and the mean was low. The coefficient of variation thus calculated, lower than 1%, appeared to be highly satisfactory. Furthermore, using ergosterol solutions at concentrations Table 1 Stability of ergosterol in spores of Aspergillus niger after 19 and 27 days of storage and 7 days of collection Storage of sample
Mean (mg/l) Standard deviation No. of samples
Effect of collection
T0
T 19 days
T 27 days
T0
7 days of functioning
0.359 0.042
0.294 0.064
0.375 0.019
0.279 0.006
0.237 0.087
8
3
3
3
3
Table 2 Simultaneous measurements of outdoor airborne ergosterol Sampler
Ergosterol (ng/m3)
1 2 3 4 5 Mean Standard deviation
1.57 1.71 2.31 1.79 1.94 1.87 0.28
decreasing as from 1 mg/l, the mean value of the limit of quantification was evaluated at 0.04 mg/l (n = 33; r = 0.005). The calculated variation of coefficient for this limit was 12.5%. 3.1.2. Storage of samples and verification of the impact of rotation at collection After sampling, the cup coated with polyurethane foam was sent directly to the laboratory. It was necessarily protected and stored in an airtight packaging, protected from light. Ergosterol is a stable molecule, which mainly degrades when it is exposed to UV light. For example, a (standard) ergosterol solution can be stored at 4 8C protected from light for a period of two months without deteriorating. Nevertheless, we checked the stability of samples during the period of storage. Thus, 14 sampling cups were artificially seeded with the same quantity of spores (A. niger) and then placed in a cold room at 4 8C for 19 and 27 days. When compared with the reference, no significant changes in ergosterol concentrations were observed during these periods of storage (Table 1). Table 3 Comparison between airborne ergosterol concentrations and occasional measurements of cultivable fungal flora Sample date
07/03/01 07/31/01
CFU/m3 Min–max, n =3 38–42 292–358
Ergosterol Types of moulds 3 isolated Mean (ng/m ) from 07/30 to 08/06/01 40 320
08/02/01 1073–1257 1190 08/03/01 425–470 440
2.74 F 0.52 Majority species 95%: Cladosporium sp, Penicillium sp, and Aspergillus sp Minority species 5%: Ulocladium sp, Alternaria sp and sterile mycelium
E. Robine et al. / Journal of Microbiological Methods 63 (2005) 185–192
We also verified that the collection method did not affect the ergosterol content in conidia. An identical and known quantity of spores was distributed between six cups. Three were placed in the apparatus and rotated for 7 days protected from any fungal pollution, while the other three were stored at 25 8C for the same period. We did not note any significant differences in concentrations (Table 1).
189
Thus the collection method did not appear to degrade the ergosterol molecules. 3.1.3. Dispersion of measurements In order to evaluate the dispersion of measurements between different systems, we placed five sets of apparatus on the same outdoor site and collected ergosterol over a period of two weeks (198 m3). The
Fig. 3. (a) Measurement of airborne cultivable fungal flora over 1 day. (b) Measurement of airborne cultivable fungal flora over more than one week.
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cumulated uncertainty of sampling and analysis spread over a range of 19%. Although this test was subject to spatial fluctuations in concentrations on the site, it allowed an appreciation of the relatively good dispersion of measurements for environmental sampling (Table 2). 3.2. Comparison between ergosterol measurements and cultures The outdoor concentration of airborne ergosterol was measured by correlation with cultivable fungal flora. Ergosterol measurements were performed after 7 days of sampling and, during the same period, four occasional samples were collected using the impactor. The results are shown in Table 3. For a mean value of 498 CFU/m3 [40–1190], we measured a concentration of 2.74 ng ergosterol/m3. A previous study by Miller and Young (1997) had estimated the mean level of ergosterol per spore in three majority species at 2.45 ng/spore. For Alternaria alternata IHEM 3183, we measured (results not published) 4.62 pg/spore (n = 6, r = 1.54). This higher level was linked to the large size of Alternaria spores. This difference linked to the dimensions of conidia had already been observed in other species by the same author.
If we consider the respective percentages of the different species isolated (95% and 5%), it could be estimated that the mean quantity of ergosterol per spore was 2.56 pg, or a theoretical value of 1070 spores/m3. This theoretical concentration is double the mean value of the cultivable fraction. This difference was probably linked to the non-cultivable fraction collected. In addition, outdoor concentrations fluctuate markedly, variations of a factor of 2– 3 being observed over a day and a factor of nearly 100 over a week (Fig. 3a,b). The values for air contamination obtained by culture principally enables an instantaneous assessment of the level of contamination. Sampling rarely exceeds 2 min of collection and, depending on the apparatus employed, represents a maximum of 0.2 m3 air sampled. In contrast, the measurements of ergosterol covered a week of sampling, or nearly 100 m3 of air. 3.3. Measurements on site Air samples were collected on different sites: in a laboratory (double-flow ventilation system) with HEPA filters, in an occupied open-plan office (double-flow ventilation system), in six dwellings and in two schools.
Fig. 4. Measurements of airborne ergosterol present inside and outside different premises. Indoor samples in an area were collected at 1 metre from floor level. A further, reference measurement was always performed outside under the same conditions.
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The ergosterol concentrations measured during this study reached a maximum of 6.47 ng/m3 (Fig. 4). Several different types of air contamination were observed on five sites (dwellings 4, 5; schools A, B and the office); the outdoor and indoor concentrations did not differ significantly. The premises tested were, in principle, exempt of any endogenous sources of fungi. The source of most of the microscopic fungi present in indoor air was principally from outdoors. The level of contamination attained in these environments was therefore mainly the result of seasonal variations (Fig. 5), as the highest outdoor fungal concentrations are recorded in the summer. On four sites (1, 2, 6 and the laboratory), we observed levels which were lower indoors than outdoors. There were no endogenous fungal sources in these premises. The ventilation system (and the air conditioning when relevant) ensured a reduction of approximately 50% in the transfer of outdoor fungal pollution. The laboratory, which was equipped with a specific filtration system (very high efficiency filters) totally restricted the penetration of all fungal contaminants. In the case of dwelling 3, the indoor concentration was double the outdoor level of contamination. A source of contamination was clearly present on the
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premises: contaminated materials, plants or contamination of the ventilation system. Thus the principal situations for indoor contamination were examined. Ergosterol measurements appeared to constitute a robust method capable of quantitatively evaluating levels of airborne fungal flora.
4. Conclusion The aim of this study was to establish a measurement system capable of estimating total levels of airborne fungal flora. A method for the assay of a specific cell component in fungi was adapted and developed accordingly. By combining this assay with a collection apparatus validated elsewhere, it was possible to measure total levels of fungal flora. When compared with traditional methods involving cultures, the assay of ergosterol levels is an integrated measurement. Culture enables an instantaneous assessment of the level of contamination, while the measurement of ergosterol can evaluate nearly a week of cumulated exposure. Regarding the detection of the cultivable fraction, the method proposed remains less sensitive,
Fig. 5. Outdoor measurement of airborne fungal biomass, year 2002–2003, on the Champs-sur-Marne site (France).
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with a limit of detection close to 150 spores/m3 (0.4 ng/m3, sampling of 100 m3). However, the fraction of non-cultivable spores can also cause immunoallergic or even toxic symptoms. Furthermore, the biological contamination of premises is a discontinuous phenomenon subject to aeraulics and the abrupt formation of bwavesQ of pollution which are difficult to measure by an instantaneous collection of the cultivable fraction. In addition, the ergosterol measurements carried out on different sites showed that they reflected the different situations of exposure of their occupants to airborne fungal flora. The use of this technique thus appears to be well suited to the quantitative evaluation of individual exposure. It should however be backed up by a qualitative approach; in this case, cultures should be employed, as only they allow identification of the most allergenic and/or toxinogenic moulds.
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