Detection of Stachybotrys chartarum: The Effectiveness of Culturable-Air Sampling and Other Methods.

Abstract

In-depth microbial investigations were performed in four commercial buildings from the Mid-Atlantic region of the United States. The samples from each building were analyzed for the presence of Stachybotrys chartarum Samples were collected with bioaerosol, swab, and bulk-sampling methods. In each building, S. chartarum was present, with more than 2.8 square meters of visible mold contamination; elevated levels (more than 30 colonies) were detected in the swab and/or bulk samples. The culturable-air (bioaerosol) samples, however, rarely showed detectable levels of S. chartarum. Thus, the use of culturable-air sampling for the detection of S. chartarum often will result in a false negative finding. Investigators should use bioaerosol methods in conjunction with visual inspections, spore trap, swab, and bulk sampling.

Introduction

Fungal growth in an indoor environment is influenced by moisture, nutrients in the substrate, pH, temperature, and light. The key to fungal growth in buildings is a moisture supply. if the mold spores find suitable conditions, which could be moist building materials (e.g., wallboards, ceiling tiles, pipe insulation, etc.) with a high water activity, they will grow and damage the infected material [1]. Fungi appear on materials in a particular succession according to their minimum moisture demands: primary colonizers, Pencillium sp. and Aspergillus sp., arise first, then secondary colonizers such as Cladosoprium sp., and finally tertiary colonizers, such as species of Stachybotrys [2].

Exposure to fungi has been linked with disease, allergic reactions, and toxicosis [3-6]. The viable mold spores of many toxigenic molds, as well as their viable and nonviable fungal structures, are known to contain mixtures of toxins that cause disease and allergic reactions in sensitive individuals. Stachybotrys chartarum is evidence of a serious moisture problem (e.g., roof leaks, pipe leaks, flooding) and has serious health implications for occupants of affected buildings [2].

S. chartarum is known to grow only on wet surfaces and is able to damage various building materials. It is a strong cellulolytic mold that grows at temperatures from 2[degrees]C to 400[degrees]c (optimally at 23-27[degrees]C). Extremely wet gypsum wallboards and ceiling tiles have been observed to be damaged by S. chartarum. The presence of this mold is of particular concern because several toxic secondary metabolites (e.g., macrocyclic trichothecenes and saratoxins) have been isolated from Stachybotrys chartarum. These metabolites are responsible for dermotoxic and cytotoxic symptoms in exposed individuals [1].

Stachybotrys chartarum, a greenish-black fungus, grows on materials with a high cellulose and low nitrogen content that become water-damaged as a result of excessive humidity, leaks, condensation, or flooding [7]. Its presence is one of the clearest demonstrations that mold growth in a building is directly affecting the quality of the indoor environment, since S. chartarum is not common in the outdoors. In general, large numbers of hydrophilic fungi are indicators of extremely wet amplification sites indoors (visible or hidden), and therefore of poor indoor environmental quality Stachybotrys chartarum is a hydrophilic fungus [8].

In this study, samples from several in-depth microbial investigations of commercial buildings in the Mid-Atlantic region of the United States were analyzed for the presence of S. chartarum. The samples were collected with bioaerosol, swab, and bulk sampling methods; they were cultured either on malt extract agar (MEA) media plates or on both MEA and corn meal agar (CMA) media plates. The resulting data show that using only culturable-air (bioaerosol) sampling to detect S. chartarum in a building with extensive mold growth often results in false negative findings.

Methods

A detailed evaluation of fungal sampling results was conducted for samples collected from an office building in New York State, a detention center in Virginia, and two library buildings in New York City.

According to Guidelines on Assessment and Remediation of Stachybotrys chartarum in Indoor Environments, a publication of the New York City Department of Health, the criteria for conducting an initial site inspection include

1. the presence of visible mold,

2. evidence of water damage, and

3. symptoms in occupants consistent with an allergic or toxic response to S. chartarum.

If bulk sampling indicates the presence of S. chartarum, or if visual inspection indicates growth suggestive of S. chartarum, sampling techniques should use media with high water activity that will enhance its growth [9].

Many types of sampling media are available for the collection and identification of molds. In the first investigation, performed in an office building, the investigators used MEA sampling media to identify the fungal species present. In the surveys of the detention center and the two libraries, the investigators used both MEA and CMA media plates. MEA media plates can isolate a larger number of fungal species in a sample. CMA plates have a higher water activity than MEA plates and are considered better at isolating spores of S. chartarum.

Airborne fungi samples were collected with an Andersen N-6 single-stage viable impaction sampler (Andersen Instruments, Inc., Atlanta, Georgia), and one petri dish with sampling media was used per sample. The N-6 plate has 400 jets (openings). The pump was calibrated at a flow rate of 28.3 liters per minute (L/min). Samples were collected indoors and outdoors, with a sampling period of two minutes for each sample. All samples were collected 1.25 meters above the ground or floor surfaces. Between samples, the N-6 sampler was cleaned with 70 percent isopropyl alcohol pads. Either MEA media plates or both MEA and CMA media plates were used to retrieve culturable airborne fungus spores. At the end of each sampling day, the exposed media plates were shipped to the laboratory by overnight courier. At the laboratory, they were refrigerated and incubated at 25[degrees]C for five to seven days. The laboratory's limit of detection for bioaerosol sampling is 18 colony-forming units per cubic meter (CFU/[m.sup.3]) of air. This is calculated by the laboratory using the volume of air sampled, which is 28.3 L/min, or 0.0566 [m.sup.3] of air. The value for the limit of detection used by the laboratory is found by taking the reciprocal of the latter air volume to determine its detection limit, which is equivalent to less than one colony count.

Swab surface samples were collected from a surface 2.5 centimeters square ([cm.sup.2]) with sterlie culturettes (Becton Dickinson Microbiology Systems, Cockeysville, Maryland). Microbial bulk sampling also was performed in each building. Samples of various building materials were collected (e.g., pipe insulation, spray-on cellulose fireproofing, wailboards), and each sample was placed in an individual Whirlpak sampling bag to prevent cross-contamination. Microbial dust vacuum (micro-vacuum) samples were taken with a high-flow air pump (calibrated at 20 L/min) to pull air for one minute through open-faced mixed-cellulose-ester (MCE) filter cassettes. The pore size was 0.8 microns. Each micro-vacuum sample covered an area 100 [cm.sup.2]. At the end of each sampling day, samples were shipped to the laboratory by overnight courier. The laboratory suspended the samples in sterile distilled water, diluted serially, and inoculated the samples onto culture media plates (either MEA or both MEA and CMA). All plates were incubated at 25[degrees]C and examined for up to seven days for total CFUs and fungal species counts.

Results

Each of the four buildings investigated had over 2.8 [m.sup.2] of visible mold contamination that subsequently was found to be S. chartarum.

The office building was a four-story, 350,000-square-foot operations building with no operable windows and two large air-handling units (AHUs). It consisted mostly of open office space. A total of 47 bioaerosol samples, 33 swab surface samples, and three bulk samples were collected. Sampling was performed on two different days in late summer. On the first day of sampling, 25 bioaerosol and 15 swab samples were collected with MEA media plates. No Stachybotrys chartarum was detected in the air or swab samples. On the second day of sampling, 22 bioaerosol samples, 18 swab samples, and three bulk samples were collected (also with MEA plates). Two of the 18 swab samples yielded elevated levels of Stachybotrys chartarum (54 and 158 colonies); one bulk sample yielded 178 colonies. S. chartarum was found only in the samples taken from fiberglass pipe insulation inside the AHUs downstream of the filters. No S. chartarum was detected in the bioaerosol samples taken on the second day of sampling.

The detention center was a single-story, 55,000-square-foot brick structure with six AHUs. It consisted of classroom, office, dining, recreation, and living spaces. Sampling was performed over a two-day period during the winter. A total of 192 bioaerosol samples, 66 swab surface samples, 68 micro-vacuum samples, and 98 bulk samples were collected with MEA and CMA media plates. Stachybotrys chartarum was detected at low levels in the bioaerosol, swab and micro-vacuum samples; in the bulk samples it was found in high levels. Six of the 192 bioaerosol samples yielded S. chartarum at levels ranging from one to nine colonies. Eight of the 66 swab surface samples showed S. Chartarum at levels ranging from one to 11 colonies. Eighteen of the 68 micro-vacuum samples also yielded one to eight colonies. Thirty-seven of the 98 bulk samples yielded low to high levels of S. chartarum. The levels detected in the bulk samples ranged from one to 250 colonies. Twenty-six of the 37 bulk samples had S. chartarum levels of grea ter than 30 colonies.

The two libraries were one-story, 6,000-square-foot ([ft.sup.2]) buildings with occupied basements. Each had one AHU and consisted of common reading areas, librarians' offices, and meeting rooms. Samples were collected on two days in mid-autumn, From the first library, a total of 30 bioaerosol samples, 68 swab samples, and 24 bulk samples were collected (with MEA and CMA media plates). One of the 30 bioaerosol samples yielded one colony of S. chartarum. Twenty of the 68 swab samples yielded two to 352 colonies. S. chartarum was not detected in any of the bulk samples. Seventeen of the 20 positive swab samples had S. Chartarum levels of greater than 30 colonies,

From the second library, a total of 30 bionerosol samples, 68 swab surface samples, and 26 bulk samples were collected (with MEA and CMA plates). Two of the 30 bioaerosol samples yielded low levels of S. chartarum (one and five colonies). Seventeen of the 68 swab surface samples yielded one to 216 colonies. S. chartarum was not detected in any of the bulk samples. Eight of the 17 positive swab samples had S. Chartarum levels of greater than 30 colonies.

Discussion and Conclusions

Fungal spores decline in viability from the moment they are produced and disseminated, and the spores of some fungal species are easily damaged by light and desiccation. Aspergillus and Penicillium spores have been known to survive during exposure to the environment better than S. chartarum spores, whose viability declines very quickly [2,8]. Therefore, contamination of indoor air by Stachybotrys chartarum is considered rare. If, however, an area is identified as being contaminated with S. chartarum--or other toxigenic microbial contaminants--the situation should be remediated as soon as possible to ensure a healthy environment. The specific methods for assessing and remediating the problem should be based on the extent of the visible contamination and the underlying water damage [9].

In each of the buildings inspected for microbial contamination, the investigators identified visible mold growth of greater than 2.8 [m.sup.2], as well as water-damaged building materials. In addition, three of the four buildings were inspected in response to occupants' complaints. The fourth building was in the final stages of construction and was unoccupied at the time of the inspection.

In each building, the levels of S. chartarum detected in the bioaerosol samples were much lower than levels detected in bulk or swab samples. In addition, the number of swab and bulk samples yielding S. chartarum were greater than the number of bioaerosol samples.

None of the bioaerosol samples collected in the office building (with MEA plates only) yielded S. chartarum; however, two of the swab samples and one of the bulk samples yielded elevated levels of S. chartarum. MEA and CMA media plates were used in the detention center and each of the libraries. In the detention center, six of the bioaerosol samples yielded low levels of S. chartarum; eight swab samples, 18 micro-vacuum samples, and 37 bulk samples yielded low to high levels of S. chartarum. One of the air samples collected at the first library and two air samples from the second library yielded low levels of S. chartarum. None of the bulk samples in either library yielded S. chartarum; however, 20 swab samples from the first library and nine swab samples from the second library yielded low to high levels of S. chartarum.

In each building, bioaerosol sampling would have produced a false negative conclusion, with a finding of no S. chartarum contamination.

A microbial investigation of any building should conform to the following protocol:

* visual inspection,

* air sampling (both culturable and spore traps),

* bulk sampling, and

* Swab (or surface) sampling.

The determination of a microbial contamination problem should not be based solely on air-sampling results since air-sampling methods are prone to giving false negative results and cannot rule out contamination [9]. This caveat holds especially for S. chartarum, since the species does not readily become airborne and the spores do not survive well once they are disseminated, If S. chartarum is present in significant amounts in an indoor environment, the culturable-air (bioaerosol) samples may not show a fungal contamination problem. If, however, swab, bulk, and spore trap samples are collected in conjunction with bioaerosol samples, the presence of S. chartarum on different materials is more likely to be identified, and the spread of the fungal contamination can be better tracked. This is especially true if the mechanical (HVAC) systems are contaminated.

Acknowledgements: The authors wish to thank Dr Chin Yang of P&K Microbiology Services, Inc., Cherry Hill, New Jersey, for analytical and technical assistance.

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