AIR AND SURFACE SAMPLING MEASUREMENTS OF FUNGAL CONTAMINANTS IN INDOOR ENVIRONMENTS P Cruz Perez, JL Henry, AK Klima-Comba and LD Stetzenbach Harry Reid Center for Environmental Studies, University of Nevada, Las Vegas, NV, USA ABSTRACT Research was conducted to evaluate surface sampling in conjunction with air sampling for the detection of fungi in indoor Residential units in a condominium complex with reported water intrusion problems were selected for sampling. Air sampling for culturable fungi was conducted with an Andersen impactor sampler supplied with malt extract agar amended with chloramphenicol (MEAC). Surface samples consisted of carpet dust and swabs. To improve the isolation of Stachybotrys spp., surface samples were cultured using a cellulose-based agar, and replicate MEAC plates were incubated at 45 C for the isolation of Aspergillus fumigatus. Tape impression samples were also collected and viewed with light microscopy. The results of this study demonstrated that air sampling alone did not always indicate the presence of a fungal contamination problem and that the use of surface sampling in conjunction with air sampling enhances the detection of fungal contaminants in indoor INDEX TERMS Bioaerosols, Fungi, Biocontamination, Measurement methods, Residences INTRODUCTION Numerous building materials and furnishings can be colonized and damaged by fungi, especially under humid or wet conditions. Some fungi can produce toxins that can cause health effects upon direct contact with skin, inhalation or ingestion (Burge and Otten, 1999). However, the presence of surface-associated fungi in indoor environments may go undetected if traditional air sampling methods with culture analysis are the only monitoring methods used. The detection of airborne and surface-associated fungal contaminants in indoor environments is necessary for risk assessment and to determine the extent of remediation for contaminated One fungus of concern in contaminated building materials is Stachybotrys. Stachybotrys chartarum is a toxigenic fungus that has been associated with the emergence of health effects in exposed individuals (Croft et al., 1986; Johanning, et al., 1996). It is a slow growing organism that can colonize wet materials composed of cellulose. However, this organism is often underestimated in traditional air sampling with culture analysis due to the tendency of its spores to clump in oily masses. In addition, the presence of S. chartarum may also be underestimated with the use of routine laboratory culture media due to its specific nutrient and humidity requirements and the competition with other fungi (Sorenson, et al., 1987). Another fungus of concern in indoor environments is Aspergillus fumigatus, an allergenic, thermophilic organism that grows in damp environments and has been implicated in several respiratory diseases (Kurup and Kumar, 1991). A. fumigatus is also an opportunistic pathogen affecting immunocompromised and cystic fibrosis patients (Yang and Johanning, 1997; Contact author email: stetzenl@unlv.edu 420
Kwon-Chung and Bennett, 1992). However, A. fumigatus is often underestimated in traditional culture analyses due to its elevated growth temperature requirement. In this study, air and surface sampling was conducted to evaluate detection methodologies for fungal contaminants, particularly Stachybotrys spp. and A. fumigatus, in the indoor environment. METHODS Sampling site. A total of 92 residences in a condominium complex with reported water intrusion problems in Las Vegas, Nevada (USA) were visually examined for fungal contamination. These residences were inspected for water staining indicative of water intrusion events. Sixty of these residences were selected for biological testing consisting of air and surface sampling. Culture media. Malt extract agar (Difco Laboratories, Detroit, MI) amended with chloramphenicol (100 µg/ml, final concentration) (MEAC) and cellulose agar (formulation courtesy of Drs. B. Jarvis, University of Maryland, College Park, MD and Wm. Sorenson, NIOSH, Morgantown, WV) amended with chloramphenicol (100 µg/ml, final concentration) (CAC) were used for the isolation of culturable fungi. Air sampling. Air samples for culturable fungi were collected at the approximate height of the breathing zone (1.5 m) using the Andersen single-stage impactor sampler (Graseby Andersen, Atlanta, GA) operated at a flow rate of 28.3 liters/min for 2 min (0.057 m 3 of air per sample). Samples were collected using MEAC. The sampler was decontaminated with a 95% ethanol wipe between each sample location and allowed to dry. Reference samples were collected from the outdoor air. Negative controls consisting of field and laboratory blanks were also collected. All agar plates were taped, bagged, and transported to the laboratory for incubation at 23 C for a minimum of 5 days. Fungal colonies were enumerated and identified by macroscopic and light microscopic morphology. The number of colonies per plate was corrected for coincidence with the positive hole correction method as per manufacturer s instructions, and the number of colony forming units per cubic meter of air sampled (CFU/m 3 ) was calculated. Surface sampling Carpet dust. Surface sampling for culturable fungi from carpets was performed using vacuum sampling with an individual field filter cassette (field monitors, 0.45 µm pore size, Millipore Corp., Bedford, MA) attached to a vacuum pump. The protective cap on the sampling port of the filter cassette was removed and the surface of the carpet was sampled. Surfaces were sampled until a sufficient quantity of material was collected as determined by visual examination of the filter cassette. Because the collected material was weighed and the organisms present were reported as organisms per gram of dust, the surface area sampled was not determined. The collection efficiency of this method will vary by carpet style and type, and the flow rate of the vacuum pump used. After sampling, the protective caps were replaced and the outside of the cassette was decontaminated with an ethanol wipe and allowed to dry. Each cassette was labeled, taped, placed in a plastic bag and transported to the laboratory. Sample processing consisted of aseptically removing the filter from the cassette, weighing the collected material and blending it with a measured amount of 0.01M phosphate buffer with 0.05% tween (ph 7.0; PBT) in a sterile test tube. The material was mixed by vortexing for one minute followed by serial dilution in PBT and spread plating onto MEAC and CAC plates. One series of MEAC plates was incubated at 23 C for a minimum of 5 days and another series of MEAC plates was incubated at 45 C for the isolation of Aspergillus 421
fumigatus. CAC plates were incubated at 23 C for a minimum of 10 days for the isolation of Stachybotrys spp. Swab samples. Smooth surfaces were sampled using a sterile calcium alginate swab (Fisherbrand, Fisher Scientific, Pittsburgh, PA). Surfaces sampled included areas of suspected fungal contamination determined by visual inspection such as heating ventilating air conditioning (HVAC) return air registers and window sills. Sample collection was performed in a manner so as to ensure that a representative sample of the suspected material was collected. Therefore, the organisms present were reported as organisms per sample, and the surface area sampled or sampling pattern were not recorded. The collection efficiency of this method will vary by surface material composition and the amount of sample collected. Each swab was placed in an individual sterile test tube, labeled and transported to the laboratory. Sample processing consisted of adding a measured amount of PBT and vortexing followed by serial dilution in PBT, spread plating and incubating as described above for carpet dust samples. Tape impression. Smooth surfaces with visible staining or suspected fungal contamination were sampled using a piece of transparent tape. A 3 cm piece of tape was gently pressed with the adhesive side down against the surface to be sampled. The tape was then removed and placed onto a labeled, clean glass microscope slide and transported to the laboratory in a plastic slide container. The tape sample was stained with lacto phenol cotton blue (Medical Chemical Corp., Torrance, CA) and viewed using light microscopy. Fungal structures observed were identified by microscopic morphology. RESULTS Sampling site. Of the 60 residences sampled, 33 residences were found to contain visible fungi resulting from water damage and/or elevated airborne fungal concentrations compared with outdoor controls. Sixteen residences were found to contain surface or carpet fungal contamination and/or an unusual composition of fungal populations. Air sampling. Air samples were collected at 103 locations from the 60 residences visited (Table 1). Fungal concentrations ranged from below the limit of detection (<18 CFU/m 3 ) to >10 4 CFU/m 3. Several of the samples collected were overgrown by rapidly growing genera such as Trichoderma. No Stachybotrys or Aspergillus fumigatus were isolated from air samples. Surface sampling. At 83 of the 103 locations, surface samples were collected in the vicinity of an air sample. Culturable fungal concentrations detected with carpet dust samples ranged from below the limit of detection (<1.18 10 3 CFU/m 3 )to >3.53 10 7 CFU/g. Swab samples detected culturable fungal concentrations ranging from below the limit of detection (<20 CFU/sample) to 9.00 10 5 CFU/sample. Although the air samples showed no indication of the presence of the fungi Aspergillus fumigatus or Stachybotrys, these organisms were isolated from several of the surface samples (Table 1). A. fumigatus was detected in three carpet dust samples at concentrations ranging from 4.32 10 2 to 1.03 10 3 CFU per gram of dust material. Stachybotrys spp. were isolated from 30 of the 65 carpet dust samples at concentrations ranging from 3.79 10 2 to 8.21 10 4 CFU/g. Stachybotrys spp. were isolated at a concentration of 20 CFU/swab in 1 of 17 swab samples. 422
Table 1. Detection of Stachybotrys spp. and Aspergillus fumigatus at indoor residential locations (percentage of positive samples is indicated in parentheses). Sample type Number of samples Number of samples positive for: collected Stachybotrys spp. A. fumigatus Air 103 0 (0%) 0 (0%) Surface Carpet dust 65 30 (46%) 3 (5%) Swab 17 1 (6%) 0 (0%) Tape impression 14 2 (14%) 0 (0%) DISCUSSION The results of this study demonstrated that surface sampling was useful for the detection of Stachybotrys spp. and A. fumigatus in water damaged residential Previous work in our laboratory has also shown only a limited isolation of Stachybotrys from culturable air samples using cellulose agar and malt extract agar. The use of a spore trap sampler can overcome the limitations encountered in detecting Stachybotrys by culture-based air sampling. Unfortunately, spore trap sampling prevents discrimination of some genera (e.g., Aspergillus and Penicillium) and does not facilitate speciation. The results of the present study demonstrated that often air sampling did not indicate the presence of a fungal contamination problem. Therefore, surface sampling should be used in conjunction with air sampling because air sampling alone may not be sufficient for the detection of biocontaminants in indoor CONCLUSION AND IMPLICATIONS As demonstrated in this study, traditional culture-based air sampling may fail to detect fungi colonizing building materials and furnishings. Because these organisms can become airborne resulting in exposure to building occupants, it is necessary to detect biocontaminants for the purpose of risk assessment and to determine the extent of remediation for biocontaminated materials. The use of both surface and air sampling, the use of various growth media (such as cellulose agar), and the use of various incubation temperatures (such as 45 C) should be implemented in order to enhance the detection of fungal contaminants in indoor REFERENCES Burge, HA and Otten, JA. 1999. Fungi. In Bioaerosols. Assessment and Control, Macher, J Amman, HA, Burge, HA, et al., eds. Cincinnati, OH: American Conference of Governmental Industrial Hygienists, pp. 19-1 19-13. Croft, WA, Jarvis, BB and Yatawara, CS. 1986. Airborne outbreak of trichothecene toxicosis. Atmospheric Environment. Vol. 20, pp. 549-552. Johanning, E, Biagini, R, Hull, D, et al. 1996. Health and immunology study following exposure to toxigenic fungi (Stachybotrys chartarum) in a water-damaged office environment. International Archives of Occupational and Environmental Health. Vol. 8, pp. 207-218. Kurup, VP and Kumar, A. 1991. Immunodiagnosis of aspergillosis. Clinical Microbiological Reviews. Vol. 4, pp. 439-456. Kwon-Chung, KJ and Bennett, JE. 1992. Medical mycology. Philadelphia, PA: Lea & Febiger. Sorenson, WG, Frazer, DG, Jarvis, BB, et al. 1987. Trichothecene mycotoxins in aerosolized conidia of Stachybotrys atra. Applied and Environmental Microbiology. Vol. 53, pp. 1370-1375. 423
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