Protocols For Investigation Of Indoor Fungal Amplifiers

The purpose of these protocols

The following protocols are not intended to be read as background information, but rather are designed to serve as "recipes" assisting the investigator in detecting potentially problematic indoor fungal amplifiers. Each protocol is numbered and is referred to by number in the text. This device, although necessary for brevity and procedural clarity, may make the text difficult to read discursively, and it is recommended that a printout of the Outline (above) be made and kept to one side to be used as a guide to the numbers. Each protocol has a preamble partially outlining its rationale.

The intended user of these protocols is a knowledgeable member of any of the various professions (occupational health and safety inspector, building inspector, mycologist, health professional, building engineer, industrial hygienist, etc.) who may be called in to investigate a building with a possible fungal amplification problem. Such a building may either be associated with health complaints, or may be a place where mold growth has been noted visually or detected through some sort of routine sampling procedure. It is anticipated that the professional doing the inspection will either have a competence in environmental mycology or will engage the collaboration of someone who does. No information on indoor fungal identification is provided here.

Outline

1. Why investigate?

2. Building history investigation

3. Building visual inspection

4. Air sampling

5. Examination for evidence of fungi in settled materials

6. Dust vacuum sampling

7. Direct microscopic mold or dust examination

8. Culture of swabs, scrapings, or surface dust; use of contact culture plates

9. Extraordinary physical search: examination of difficult sites within building

10. Mold (non-Stachybotrys) source location and cleanup

11. Minimal Stachybotrys, source not located, likely indoors

12. Low to high Stachybotrys, source not located, likely indoors

13. Minimal Stachybotrys, source not located, outdoors or indoors

14. Low to high Stachybotrys, source not located, outdoors or indoors

15. Ergosterol or glucan sampling

16. Mycotoxin sampling from air/dust and bulk

17. Humidifier water direct microscopy

18. Humidifier water culture

19. Cytotoxicity testing

This material is not intended to assist in the search for opportunistic fungal pathogens in hospitals. In that situation, the search may be directed towards numerically rare types of fungal propagules, whereas in general indoor fungal amplification problems, numerically abundant propagule types are most often the object of concern. Detection strategies in the two cases are necessarily quite different.

Several of the protocols are directed towards the diagnosis of infestations caused by Stachybotrys chartarum (= S. atra). It is singled out not because of its relatively high toxicity, but rather for purely technical reasons: unusual techniques are often needed to detect it accurately. Unlike other toxigenic molds associated with indoor proliferation, it frequently is represented in the environment predominantly by conidia and other materials which, although actively toxic, do not grow on fungal culture media. The majority of conidia in many amplifiers appear either to be non-viable or to be otherwise inhibited in germination. Only uncommonly are numbers of colonies obtained which give a fair approximation of the amount of S. chartarum present. In some cases, air or dust sampling may yield no S. chartarum at all, yet considerable quantities of still-toxic conidia may be evidenced on direct examination of dust, or may be found within a wall cavity of a room whose occupants have been distressed. The episodic culturability of this species necessitates several atypical search strategies, outlined in protocols 11-14. Other common toxigenic indoor molds tend to be culturable, although in a few cases growth may be contingent on the water activity of the medium used. Protocols for these molds are generally less elaborate.

How to use these protocols

Begin at #1 and follow the suggestions in that section which direct you to other protocols that may apply. For each protocol you are directed to, read the short introductory paragraph(s) and then each of the numbered subsections below it (e.g., 8, 8a, 8b, 8c, 8d). Choose only the ones that apply to your situation and follow them, ignoring the ones that do not apply. The subsections will tell you which other numbered protocols to go to. A suggested protocol marked with an asterisk (*) is usually a valuable technique which is very labor-intensive, knowledge intensive, or costly, and which would ordinarily be done only after more simple techniques had proven unable to resolve the situation. Techniques marked with an asterisk can therefore ordinarily be ignored in early investigations, and in investigations which turn out to be straightforward, lacking unusual complicating factors. Special symbols and expressions used in the protocols are summarized below.

For Symbol/expression Explanation, see table above

#1: Why Investigate?

Investigation of a potential fungal contribution to indoor air problems may follow either 1) the appearance of symptoms compatible with exposure to fungal bioaerosols (see below) or 2) the detection of potentially problematical fungal material in a building environment. Apart from the necessarily rapid diagnosis and cleanup of gross contamination, fungal investigation should not normally proceed in isolation from investigation of other parameters influencing air quality (see protocol #2).

1a. Symptoms noted: If symptoms compatible with exposure to fungal bioaerosols are reported (e.g., specifically building- or room-associated non-specific respiratory symptoms, such as rhinitis, cough, wheeze, sometimes accompanied by headache; exacerbation of asthma in persons already known to be asthmatic; in less common cases of heavy exposure influenza-like symptoms including fatigue, muscle cramps; in connection with Stachybotrys strong mucous membrane irritation usually presenting as burning sensation of eyes (may be recorded by physician as conjunctivitis) and/or throat; in infants less than 18 months old subjected to extreme quantities of Stachybotrys, pulmonary hemosiderosis associated with pulmonary haemorrhage) then check building history (#2) and do visual inspection (#3); if these do not reveal obvious source of problem, then do air sampling (#4) or thorough dust sampling (#6*). Also, where inspection and history do not reveal evident problem, but a hidden problem is reasonably suspected based on correlation of symptoms and exposure to the affected building/room(s), do examination of settled materials (#5).

1b. No symptoms noted: If no symptoms, take no action unless:

i) moderate to large quantity of mold seen (do #2,3,7,8*,4*,5*) or

ii) high counts revealed in preliminary or routine air samples (do #2,3,5*) or

iii) Stachybotrys detected in preliminary or routine air samples, dust samples, sampled visible mold or cultured swabs, surface materials or contact plates (proceed according to instructions for Stachybotrys in protocol #4 for air samples, #6 for dust samples, #7 for macroscopically or microscopically visible Stachybotrys, and #8 for swab/surface material cultures and contact plates), or

iv) extensive water damage to finely divided cellulosic materials (e.g., wallpaper, wallboard covering paper, carpet with jute backing, paper covering of fibreglass insulation bats) observed in situations where the hidden components of the material (e.g., wallboard paper inside wall cavity, backing of the waterstained carpet, inner surface of insulation-covering paper) may be moldy, especially if a musty odour is present. Thoroughly do #2, #3; if there is a musty odour indicating fungal/other microbial degradation, consider doing #4,5,9 on the presumption that some occupants probably have unelucidated symptoms or will develop them in the future. If there is no musty odour and thorough non-destructive searching reveals no evident mold, consider proactively doing #4 and surface dust analysis components of #5 to rule out significant repositories of hidden molds or, at a lesser level of vigilance regarding health and building maintenance where no further action is taken, at least instructing responsible individuals to monitor for development of symptoms in occupants. Ensure building maintenance prevents exacerbation of any leaks/flood sources which may have been responsible for the water damage seen.

#2: Building History

Building history (general): Indoor air problems may have various etiologies, and should not be assumed automatically to be of microbial origin. Important factors such as non-fungal chemical contamination (e.g., volatile organic compounds of industrial or commercial origin, tobacco smoke, automobile exhaust, ozone, nitrogen dioxide, carbon monoxide) are outside the scope of these protocols, as are the potential effects of sociological issues such as labour/management dispute on perceived environmental tolerability. Also beyond the scope of these protocols, but possibly germane in investigated cases, are the evaluation of bacterial diseases, allergies and intoxications (including endotoxin exposure), viral diseases, arthropod (e.g., insect, mite) allergies, protozoal allergies, and allergies to any aerosolized animal and plant parts or products. A thorough investigation should consider as many of these factors as may be applicable.

Building history (relevant to fungal growth): inquire about sites associated with symptoms, any previous flooding, window or roof leaks, or other water damage, seasonal humidity, fires, winter condensation, summer condensation on cold plumbing fixtures and pipes, frequency of carpet cleaning, duct cleaning, HVAC maintenance; humidifier, aerosolizer or mister type and maintenance; long-time colonization of attic or other areas by birds or bats. Interpret any water damage seen in visual inspection (#3) as a historical indicator.

2a. History suggests area of readily discernible mold growth, e.g., water damaged surfaces or stored materials, poorly maintained ducts or HVAC, etc: perform visual inspection (#3) wherever not done already.

2b. History suggests hidden locus of mold growth, e.g., previously flooded wall cavity or false ceiling, previously flooded carpet backing, unshielded cold water pipes or leaky pipes in false ceilings, previous fire extinguished by water, possibly affecting wall cavities, insulation materials present inside ducts. In these and similar cases, where occupants have or claim to have symptoms which at least in part, in the judgement of a person experienced with indoor mold infestations, are plausibly connected to mold exposure (see #1, above), or where musty odour signals microbial activity, do extraordinary physical search (#9). If this search does not reveal area of mold growth but symptoms, odours or other conditions remain strongly suggestive of the presence of mold, perform air sampling (#4) and evaluate molds growing in samples ecologically in order to determine the likely nature of the colonized substrate. Also, use samples from different areas to determine if a particular area appears to be associated with high colony counts. Where water damage or other factors suggest a possible hidden locus of mold growth, but area is not associated with symptoms and is unlikely to be a source of mold inoculum to areas associated with symptoms (e.g., is not connected to vents which may distribute mold inoculum to rooms where symptoms have been noted), and no musty odour is present, consider the suggestions in #1biv, above.

2c. History suggests long-time bird or bat colonization of attic or elsewhere: inspect visually (#3) while wearing HEPA filter respirator; or call expert.

2d. History not suggestive or information lacking or incomplete: if symptoms are plausibly adduced (as per #2b, above) or if physical conditions are suggestive of a potential mold problem, rely on #3, 4 (or 6), 5*. Procedures #4 and 6 (air or dust sampling) are relatively labor intensive and should be undertaken only where the source of this problem is not immediately evident upon walk-through (#3) coupled with microscopic (#7) visual inspection.

#3: Visual Inspection

Visual inspection (for mold-related signs and conditions): inspect for mold growth or water damage on walls, especially in basement, on windowframes and on carpets (check backing in waterstained areas if possible), on ceiling tiles, as well as on any currently or formerly damp material made of fibrous cellulose (wallpaper, books, papers), and all accessible HVAC components, especially plenums; also check for any substantial earthen spaces indoors such as unfinished basements or crawlspaces, extensive indoor plantings, contiguous greenhouses, attics with resident or seasonal birds, bats or other animals (wear HEPA filter respirator when checking if building history (#2) indicates long-term bird or bat breeding or roosting in attic); and any other likely mold sources.

3a. Visual inspection reveals mold, suspected mold or debris accumulation which might signify or contain mold: take scrapings or transparent tape mount for direct microscopic examination (#7); also, optionally, for most thorough investigation, take scrapings, swabs, or contact plates for culture (#8*). If attic or other area contains substantial amounts of bird or bat guano, consult expert (e.g., medical mycologist working for or recommended by your provincial, territorial or state public health laboratory). Bat guano may resemble heaps of sawdust. Although usually originating in an attic, it may sift down through floor cracks or wall cavities into lower floors, including the basement, and even these distal areas may be infested with virulently pathogenic fungi, especially in geographic areas endemic to Histoplasma capsulatum or Blastomyces dermatitidis (consult medical mycologist if unsure).

3b. Visual inspection reveals no mold, but reveals current or formerly damp materials or earthen spaces as indicated above in introductory paragraph for #3: if symptoms reported, rely on #2,4,5. If substantial earthen area was found, then ensure that any air sampling includes samples taken near this area to probe its significance as a source of mold propagules. Earth may also be analysed using same techniques as dust analysis (#6*). If no symptoms reported, see #1biv.

3c. Visual inspection reveals neither mold nor current/former dampness nor substantial indoor soils: if symptoms present in occupants rely on #2,4,5,6*. If no symptoms present, and no other evidence of indoor fungal amplification or microenvironments conducive to mold growth exists, take no further action.

#4: Air Sampling

Vacuum/culture devices such as RCS, Andersen, and slit-to-agar samplers are recommended for air sampling in public buildings and residences; note that current Health Canada guidelines are based on 4 min samples with the RCS sampler. Where no such device is available (e.g., in remote areas), settle plates may be used for preliminary study but should be interpreted by an expert familiar with their limitations and with common indicator species of indoor mold proliferation; even as interpreted by such an expert, settle plates may reliably indicate only extreme (very moldy, very clean) environments, while low to moderate indoor mold growth may give settle plate results which are difficult to interpret. See Sampling methodology for fungal bioaerosols and amplifiers in cases of suspected indoor mold proliferation) [link] for further background information on vacuum/culture and settle plate sampling.

In any case where the indoor environment is suspected to be the source of mold-related problems, analysis of air samples should concentrate on counting and identifying molds of indoor origin or indoor accumulation, not transitory molds of outdoor origin. This is accomplished by a species-by- species comparison with outdoor control air samples: disregard any species found indoors at a level similar to or lower than its contemporaneous outdoor level (exceptions: Stachybotrys, and in the extremely unlikely event of their growth, classic pathogenic fungi such as Histoplasma, Blastomyces, Coccidioides, or Cryptococcus neoformans) unless other evidence indicates the species is nonetheless proliferating indoors. Species need not be named in these comparisons, but isolates must be recognized as conspecific based on mycological analysis of macroscopic or microscopic characters. Identification of Aspergillus species, Penicillium subgenera and genera of other numerically significant fungi is recommended. Minor species present in proportions below 1% can be ignored provided any Stachybotrys, Aspergillus fumigatus, Aspergillus flavus or other species known to be particularly noxious is detected.

It is critical that outdoor controls should be taken well away from the building being tested or any similar buildings, and should be taken upwind of any possible air outflows. Heavily contaminated buildings will significantly adulterate "outdoor controls" and render them useless unless proper procedures are followed. Sample outdoors at least 6, preferably 10 m windward of building of concern (and any similar buildings) if possible; investigators using electrical devices should carry enough all-weather extension cord to facilitate this. Where buildings are closely crowded, air sampling on the windward side of the roof may be tried, but if high levels of molds ordinarily associated with indoor sources are obtained, further investigation should be undertaken to determine the prevalence of these species outdoors in the general area (square kilometre) of the sample.

In general, well established, medically meaningful benchmarks for acceptable indoor airborne mold propagule levels are lacking; in their absence, the best benchmark to use for the detection of indoor mold amplifiers is that no species should be at significantly greater levels indoors than outdoors (test significance with chi-squared test where applicable). A species more prevalent indoors than out is usually proliferating indoors. For example, if Cladosporium cladosporioides, despite normally being more abundant outdoors than indoors, makes up 20% of the outdoor spora and 40% of the indoor spora in a case under investigation, indoor amplification is likely and is probably occurring in an excessively moist area. Only rarely will outdoor spora sediment out and accumulate indoors to the extent that species of outdoor origin appear to be more common indoors than out. This may, however, occur near large outdoor composting facilities if the still air of buildings acts as a sedimentation sink for the propagules blown in from the composting area.

The indoor proliferation of mold species generally indicates a building maintenance problem. Since exposure-related health risk itself is currently difficult or impossible to quantify for indoor molds, a better approach is to use mold sampling to detect building maintenance problems and to correct them with the assurance that any genuinely associated, mold-related health problems will be corrected at the same time.

Benchmarks or action levels based on absolute rather than relative numbers are a frequently discussed scientific ideal, but most such benchmarks heretofore proposed are based on unwarranted assumptions or have arbitrary elements. In recent years, most authorities have declined to give uniform and absolute standards for acceptable fungal propagule levels in indoor air. Indoor/outdoor comparisons are generally superior in the detection of potentially problematic proliferations. In some cases, counts of molds of indoor origin in buildings with air quality problems have been compared to average mold levels in complaint-free buildings. Such comparisons with "normal" air spora levels are best interpreted in combination with indoor/outdoor comparison and amplifier detection as outlined above. At best, studies comparing buildings under investigation with results from known problem and non-problem buildings must be interpreted with caution. Not only must technology be highly standardized with that used to compose the baseline data, but also, the environments studied (e.g., type of building, geographic area) should be highly comparable. Some tentative benchmarks based on airborne fungal levels in essentially proliferation- free, normal Canadian office buildings were originally proposed by Health Canada (1) and appear in a slightly revised version in the most recent version of the document (Davies et al., 1995 --see Section 3.2 of that document).

Growth media for air sampling fall into two physiological categories: high water activity (general fungal growth media such as malt extract agar or colony diameter restricting media such as Littman oxgall or rose bengal agar), and low water activity (medium with high solute concentration, e.g., dichloran 18% glycerol agar, used for detecting fungi growing on very dry material such as dust components and not growing well on general fungal media). Since many common indoor molds grow on these low water activity media, their use in indoor mold sampling has increased in recent years. The usage of various media in indoor fungal sampling is further discussed in Sampling methodology for fungal bioaerosols and amplifiers in cases of suspected indoor mold proliferation) [link]. Antibacterial antibiotics are usually incorporated into the media in mycological studies.

4a. Air sampling with vacuum/culture device shows molds of indoor origin present and exceeding benchmark (e.g., significantly higher than outdoor levels for same species; in the case of Stachybotrys, any isolation is above benchmark and should trigger further investigation). Go to #10, and, if any Stachybotrys present, go to #4b - 4f and follow the most fitting choice.

4b. Minimal Stachybotrys found indoors but not found in outdoor controls. (Minimal = a vanishingly small number detected, for example, a single isolated colony found under any circumstances, or two widely dispersed colonies found in a large series of samples from different rooms. Note that "colony" means an actual fungal colony observed on the sampling medium after incubation, not a CFU per cubic metre of air in calculation). Go to #11.

4c. Low to high numbers of Stachybotrys colonies found ("low to high": at least three colonies found in a multi-room sample or two colonies from a single room; or more colonies found under any circumstances) but either not found in outdoor controls or found in outdoor controls at statistically significantly lower levels (chi-squared test). Go to #12.

4d. Minimal Stachybotrys found (see 4b for definition of minimal) and also found in outdoor controls at minimal or higher level. Go to #13.

4e. Low to high (see #4c for definition) numbers of Stachybotrys found and also found in outdoor controls at insignificantly differing (chi-squared test) or higher levels. Go to #14.

4f. Air sampling shows molds of indoor origin absent or below benchmark; no Stachybotrys present. Rely on #2,3,5 (6*) to detect any temporarily poorly disseminating amplifiers.

#5 Examination For Evidence of Fungi in Settled Materials

Air sampling using culturing techniques has traditionally been favoured over direct sampling of potential fungal substrates and sediments indoors. This type of air sampling has the strength that it reflects (to a reasonable degree of approximation) the number of live fungal propagules a person's respiratory system is exposed to. Its disadvantages include its insensitivity to non-viable propagules, its reliance on potentially fluctuant airborne propagule populations, its short sampling times making phenomena like diurnal cycles of propagule release difficult to discern, and its inability to discern the source of indoor propagules. On the other hand, the direct sampling of sediments and solid or liquid fungal substrata may facilitate the detection of non-viable fungal propagules, the confirmation of sites of fungal growth, the quantifying in situ of deleterious fungal chemicals or of chemicals indicative of fungal biomass, and the discernment of patterns of fungal propagule deposition over moderately long periods of time. Note that significant biofilms and potentially contaminated standing water ordinarily should not be present in a building and should ordinarily be remediated immediately upon discovery. Sampling in such cases is only to establish possible links between growth at these sites and airborne or sedimented inoculum sampled elsewhere, and to correlate the degree of irritation associated with the species detected and the severity of symptoms claimed by affected individuals.

5. Examination for evidence of fungi in settled materials: dust, biofilms, and standing water. Initial procedures where symptoms absent, analytic resources limited, or preliminary survey being conducted: on representative surfaces with relatively recent dust deposition (e.g., windowsill or desk corner with few weeks/months dust but not cobwebby cabinet top or refrigerator bottom with years of dust accumulation) and, where applicable, within air ducts and HVAC coils, etc., do #7, 8*; visually inspect any functioning humidifier, aerosolizer, vaporizer or mister and if water not clear then take sample for microscopic examination (#17) and culture (#18)

5a. Examination for evidence of fungi in settled materials: dust, biofilms, and standing water. Rigorous procedure where symptoms warrant investigation and resources permit: do all procedures outlined in #5, as well as #6. Certain procedures currently under applied research investigation may become increasingly used in these investigations, including fungal ergosterol or glucan sampling (#15) or, in environments with mycotoxigenic species present or expected, direct mycotoxin sampling (#16) or cytotoxicity testing (#19).

#6 Dust Vacuum Sampling

Collect floor or carpet dust using a suitable vacuum device; also compare outdoor dust control*. On subsample of bulk dust perform direct microscopic examination (#7); also do dilution series in sterile water or other appropriate diluent, beginning with a measured quantity, e.g., 1 g dust suspended in 10 ml fluid (1:10 suspension), subsampling 1 ml of suspension to 9 ml fluid to make a 1:100 suspension, and likewise 1:1000, 1:10000 and 1:100,000*. Plate 0.1 ml from each suspension onto high water activity and low water activity growth media as per procedure #4, with replication. Analyse CFU/g dust based on counts on the dilution which has more than 25 and less than 100 colonies per plate, or a number close to one of these figures. Estimate number of molds of indoor origin, as gauged by species- by-species comparison with outdoor controls. Species need not be named in these comparisons, but isolates must be recognized as conspecific based on mycological analysis of macroscopic or microscopic characters. Identification of Aspergillus species, Penicillium subgenera and genera of other numerically significant fungi is recommended. Minor species present in proportions below 1% can be ignored provided any Stachybotrys, Aspergillus or other pathogenic or toxigenic fungus known to be of concern are detected.

Note that some workers sample dust by plating it directly on growth medium (often Dichloran 18% glycerol medium, which favours the growth of dust- associated xerophiles), since making aqueous suspensions may be difficult. Others prefer to gain an approximate measure of human exposure to settled dust by disturbing dusty materials and air-sampling with a volumetric sampler. Quantitatively different results will be obtained with different techniques. In water dilutions and in air sampling of disturbed dust, clumps of hydrophobic spores or conidia may remain largely intact, yielding colonies which actually reflect agglomerations of potential colony forming units. In dilutions where a wetting agent (e.g., dimethyl sulfoxide) is used, they may break up and give a stronger appearance of heavy infestation.

6a. If molds of indoor origin are present at negligible levels or below benchmark (see below), take no further action unless indicated by other procedures. The presence of any quantity of Stachybotrys always warrants further investigation--go to 6b. Re benchmark: there are currently no proposed benchmarks for such studies, but experimental studies possibly yielding such benchmarks for different dust sampling techniques are in progress. If available, please use. In the meantime, experienced evaluators may judge whether molds are present at unusual levels suggestive of indoor amplification, or whether individual species or species associations suggestive of indoor proliferation are present. If so, go to 6b.

6b If molds of indoor origin are present at levels indicative of substantial indoor amplifiers or at levels above benchmark (see comment on benchmark under 6, above) for total fungal count, or individual species of concern, or ecological categories (such as toxigenic fungi) of concern, or if species composition of dust spora suggests indoor proliferation, then go to appropriate subprocedure of #6b:

6bi. Molds other than Stachybotrys at levels considered to be of potential concern (see comment on benchmark under 6a, above): go to #10.

6bii. Minimal Stachybotrys found indoors but not found in outdoor controls. (Minimal = a vanishingly small number detected, for example, a single isolated colony found under any circumstances, or two widely dispersed colonies found in a large series of samples from different rooms. Note that "colony" means an actual fungal colony observed on the sampling medium after incubation, not a CFU/g in calculation). Go to #11.

6biii. Low to high numbers of Stachybotrys colonies found ("low to high": at least three colonies found in a multi-room sample or two colonies from a single room; or more colonies found under any circumstances) but either not found in outdoor controls or found in outdoor controls at statistically significantly lower levels (chi-squared test). Go to #12.

6biv. Minimal Stachybotrys found (see #6bii for definition of minimal) and also found in outdoor controls at minimal or higher level. Go to #13.

6bv. Low to high (see #6biii for definition) numbers of Stachybotrys found and also found in outdoor controls at insignificantly differing (chi- squared test) or higher levels. Go to #14.

#7 Direct Microscopic Mold or Dust Examination

Mount dust sample, mold scrapings, transparent tape sample, or surface scrapings on a microscope slide with a hydrophobicity-reducing mounting medium (e.g., water + Roccal or other laboratory detergent; 25% sodium hydroxide; or ethanol followed immediately by water) and examine under 10X and 40X for characteristic structures of fungi, particularly fungi of known concern. In surface scrapings, a site of active fungal growth, i.e., an amplifier, is recognized by the presence of hyphae and (in most cases) conidiophores as well as conidia of the same species. Other fungal sporulating structures such as ascomata may also be present. Heavy deposits of conidia or spores alone may signify either heavy deposition from another source, or an old, inactive amplifier in which structures of active growth have broken down; alternatively, it may simply indicate that the surface sampled has not been abraded aggressively enough to detach hyphal structures from the substratum. Taking these and any other salient factors into account, the experienced investigator should make a judgement about whether the material examined is from an amplifier. For general remediation of mold amplifiers, see Section 5.1 of Davies et al. (1995). A specialized remediation protocol for Stachybotrys has been formulated (New York City Dept. of Health, 1993).

7a. Microscopic mount shows Stachybotrys from amplifier site: if site is known, go to Stachybotrys remediation protocol (New York City Dept. of Health, 1993); if unknown, or if more sites may exist, go to 7c.

7b. Microscopic mount shows other mold from amplifier site: clean as warranted, based on type and extent of mold (see Section 5.1 of Davies et al. (1995)).

7c. Microscopic mount shows no evidence of an amplifier but Stachybotrys conidia present, or, mount shows evidence of a Stachybotrys amplifier for which the location cannot readily be traced (e.g., conidiophores from an unknown source present in bulk dust sample): rely on #2,3,5, repeating or extending if necessary, and, failing these, #9 or expert consultation, to locate source. When found, go to Stachybotrys remediation protocol (New York City Dept. of Health, 1993).

7d. Microscopic mount shows no evidence of an amplifier, but significant levels of fungal material present, or, mount shows evidence of an amplifier for a fungus other than Stachybotrys but the exact location of this amplifier is not apparent: rely on #2,3,4*,5 (extended if necessary), #6* to assist in locating amplifier site(s). If an amplifier is located, go to remediation protocol (Section 5.1 of Davies et al. (1995)).

7e. Microscopic mount negative for fungal structures or nearly so. Take no further action unless another procedure (#3,4,5,6*) indicates significant indoor mold proliferation in other locations.

#8 Culture of Swabs, Scrapings, or Surface Dust; Use of Contact Culture Plates

Surface dust culture, swabs, scrapings, or contact plates will all suffice to detect fungi on surfaces, but will not distinguish between species growing on the surfaces and species which have merely been deposited there as inactive propagules. These techniques are substantially interchangeable, and their use should be tailored to the situation and the sampling materials available.

Sufficiently large quantities of dust or other material which can be suspended more-or-less evenly in water can be cultured by dilution series as outlined in #6. Swabs should be streaked on mesic and xeric* media (see #4) as per normal microbiologic inoculum-attenuation procedure (e.g., limiting the original swab streak to 1/3 of the plate and cross-streaking with a sterile loop through three or four partial rotations of the plate). Small pieces from scrapings, particularly from moldy-looking areas, should be suspended in sterile water; the suspension should be vigorously agitated or sonicated, and plated out directly on mesic and xeric* medium. Contact plates should be applied to surfaces where mold growth or deposition is suspected, and incubated at room temperature (or, where invasive opportunistic pathogens are specifically being sought, at 37 degrees C) for at least 7 days.

8a Culture shows Stachybotrys in scrapings, swabs, or contact plates; to find source rely on #2,3,5,7, repeating orextending if necessary, and failing these, #9 or 9a or expert consultation. For Stachybotrys in dust, proceed as per #6aii-6av as appropriate, or, where outdoor controls are lacking, consider proceeding as per #6*. If original Stachybotrys in swabs, scrapings or contact plates was minimal, seen only as 1 or 2 colonies in a large sample, AND #9/9a fails to show evidence of Stachybotrys, then do new 3,4 (or 6*),5. If these are again positive but minimal, consult outdoor controls from #4 or 6 and proceed as per #6bii or 6biv, whichever is more appropriate. If repeats are again positive but levels rise beyond minimal, then consult outdoor controls and proceed according to #6biii or 6bv, whichever is more appropriate. If repeats are negative, then disinfect site where Stachybotrys was isolated but, where no symptoms are reported, take no further action. If symptoms are reported and #9a has been done, consider doing #9. If #9 and all other procedures following the initial minimal isolation of Stachybotrys are negative, but symptoms continue to be reported, consider monitoring again in 2 - 3 months in addition to testing non-fungal potential causes of symptoms.

8b. Scraping, swab, contact plate or surface dust culture grows profuse toxigenic or allergenic mold but not Stachybotrys: rely on #7 to confirm growth at sampling site. If #7 positive, see instructions for #7. If #7 negative (e.g., if growth is derived from small, sedimented conidia obscured by debris), rely on #2,3,5 to locate site of growth and also disinfect broad area around positive sample site as per Section 5.1 of Davies et al. (1995). Select additional sample sites in similar or nearby areas and proceed according to #5,7,8. Reiterate until no more sites of heavy mold inoculum are detected.

8c. Scraping, swab, contact plate or surface dust sample grows sparse toxigenic or allergenic mold but not Stachybotrys: rely on #7; if #7 is positive for growing mold, then disinfect site as per Section 5.1 of Davies et al. (1995). If #7 is negative, then rely on #2,3,5 and results of any previous #4 or 6 to indicate whether further action is necessary. If no evidence of significant indoor mold proliferation, take no further action. If there is evidence of indoor mold proliferation or of a history suggestive of conditions conducing to mold growth, search for source with extended #3, 5; consider #9a or 9 where walls, ceilings or carpets have been flooded.

8c. Scraping, swab, contact plate, or surface dust culture heavily positive for antibiotic-resistant bacteria or yeast, with few or no molds present or with wet-spored molds predominant, e.g., Fusarium, Exophiala. Such findings usually indicate a very moist environment, likely occurring indoors in conditions of high humidity, flooding, or condensation which will also lead to the growth of other molds in less moist habitats. Rely on #3, 4 (or 6*), 5 to detect degree of mold growth and sites where it occurs. Remediate conspicuously moist areas immediately (Section 5.1 of Davies et al. (1995)). Note that dry house dust will often yield large numbers of yeasts, but will also generally yield a moderate to high proportion of dry-spored molds in the sample. Such findings do not necessarily indicate excessive moisture, but rather may indicate persistence of outdoor air yeasts in dust sediments.

8d. Scraping, swab, contact plate or surface dust culture negative for mold or nearly so. Take no further action unless another procedure (#3,4,5,6*) indicates significant indoor mold proliferation in other locations.

#9 Extraordinary Physical Search: Examination of Difficult Sites Within Building

Previous flooding may have resulted in accumulations of mold conidia within wall or false ceiling cavities, in wall or duct insulation, on the backings of carpets, or in other inconvenient locations. Walls and false ceilings are sufficiently enclosed to serve as humid chambers promoting mold growth, yet are often sufficiently porous to allow the dissemination of conidia or toxic or antigenic hyphal or conidial fragments. Actively growing mold may produce offensive volatile odor substances. Flooding history or the results of air, dust, or surface samples may suggest that easily accessible sites are not growing enough mold to explain the observed mold levels or mold- related symptoms, and that a possible cryptic mold reservoir exists. In such cases, difficult samples, requiring entry of ordinarily inaccessible spaces, must be undertaken.

9a. History (#2) or physical inspection (#3) of site suggests previous or current flooding in poorly accessible site, e.g., wall cavity, false ceiling interior, wall or duct insulation, carpet backing: expose a representative portion of the environment (e.g., wall interior or carpet backing) in area of concern, perform #7, 8* in newly exposed environment. If available, a rigid endoscope (borescope) may be used to examine cavity interiors with minimal damage; perform 7, 8* adjacent to borehole. Where material suggestive of fungal amplifiers or spore deposition is seen using endoscope, drill additional holes as necessary and perform #7, 8*.

9b. In sites where no historical information is available and no water damage is recalled or seen, but where investigation is still warranted by symptoms or other concerns, perform one or more of the following tests with stringency appropriate for the degree of health concern:

9bi) Wall cavities. A low stringency test is to check a few representative wall cavities behind switch face plates. Use an alcohol- disinfected, flamed, bent wire to scrape small amounts of material from back or front of wall cavity, being careful to avoid electric wires (fuses or breakers corresponding to these switches should be turned off), and perform #7 and 8*. This test is subject to false negative results if amplifiers are discontinuously distributed but can detect gross, confluent contamination or heavy general spore accumulation. More stringently, use a rigid endoscope (borescope) as outlined in #9a or cut an approximately 15 cm X 15 cm hole in representative wall sections and inspect visually. Perform 7, 8* on exposed interiors, and in particular on suspected amplifiers.

9bii) Ducts. Use existing access openings or a rigid endoscope (borescope) to inspect duct interiors. Perform 7, 8* adjacent to access hole or borehole. Where material suggestive of fungal amplifiers or spore deposition is seen at a distance from original entry point, use elongated scraping device or drill additional holes as necessary and perform #7, 8*. Holes can be sealed with a plug.

#10 Mold [Non-Stachybotrys] Source Location and Clean-up

Procedures such as air sampling and dust sampling do not directly disclose the location of amplifiers, that is, sites of indoor mold proliferation. If potentially problematic levels of molds of indoor origin are revealed by these techniques, the sources of these mold propagules must be discovered. Stachybotrys is treated separately because a stringent cleanup protocol has been described.

10a. Find source of (non-Stachybotrys) mold: rely on #2,3,5 to suggest location (follow any procedures branching from #5); if necessary perform extended #3 and 9a* or 9b*; when source found remediate (see Section 5.1 of Davies et al. (1995)).

#11 Minimal Stachybotrys, Source Not Located, Likely Indoors

Stachybotrys occurs on cellulose that has become moist. Although it may occur indoors in large quantities on structural materials and bulk stored materials, it may also occur sporadically on small cellulosic substrata, or may occur in low abundance in marginal habitats. For example, a library may contain a very low number of previously water-damaged books which may still shed a few Stachybotrys conidia. A few Stachybotrys isolates in an air or dust survey may derive from such origins. Likewise dust of outdoor origin may serve as a reservoir for a small number of Stachybotrys conidia indoors, and one or two colonies may turn up on indoor surveys even when contemporaneous outdoor control samples are negative.

The isolation of a small number of Stachybotrys colonies is difficult to interpret. One of the attributes rendering Stachybotrys distinctive is the low viability of its conidia encountered in indoor situations. Hundreds of conidia seen in a direct examination may show only 2 - 3% viability in culture, and culture techniques alone may profoundly underestimate the presence of this organism, and therefore its toxic effect. Dead conidia apparently remain toxic for some time, as do conidial fragments. Undoubtedly the decline in conidium viability occurs over time, and most or all conidia are likely viable when they are fresh. Yet Stachybotrys material investigated by indoor mold researchers tends to show low culture viability. It may be that this highly toxic material is less likely than other environmental contaminants to be degraded into an inoffensive form after it has lost viability.

One or two colonies of Stachybotrys in an indoor survey may be the viable representatives of large numbers of non-viable propagules, thus indicating a serious level of contamination. Or they may reflect fortuitous isolation from a minor source. In either case, repeat air or dust samples analysed by culture may be false-negative, and the best procedure to use for followup of positive cultures is a search for possible Stachybotrys sources. Only when this search gives a negative result is it reasonable to attempt repeat culture samples. This is done to exploit the possibility that, if significant amounts of Stachybotrys are present, they may reoccur at least some of the time in culture samples. When better techniques have failed, that is, this imperfect technique may be used because of the chance that it may yield valuable information.

11. Rely on #2,3,5 (and failing these, #6*) to suggest site of Stachybotrys growth. If #3 is negative, and 5 or 6 positive for visible conidia or culturable propagules, perform extended 3 and 9*; if all tests negative, repeat #4 or do 6 (repeat #6 if it was the original source of Stachybotrys isolation) in area where Stachybotrys was detected. If repeat is negative, disregard Stachybotrys; if repeat #4 or 6 is positive, but minimal (a single colony or two widely dispersed colonies in a large series of samples), rely on direct microscopy of dust as per #7 to determine if dead Stachybotrys conidia present; also perform #9* or 9a if not performed already; if no Stachybotrys conidia or sources evident, disregard Stachybotrys. If conidia evident in #7, do extended #3 and 9; if no source found in these procedures, call expert. If #6 is positive for low (three or more colonies overall or two clustered colonies; or a single colony in a small sample) to moderate or high, follow procedure #12 if outdoor controls negative or 14 if outdoor controls positive.

#12 Low to High Stachybotrys, Source Not Located, Likely Indoors

The finding of low to high Stachybotrys colonies strongly implies that an indoor amplifier is likely to exist. In such cases, it is necessary to find the amplifier.

12. Rely on #2,3,5 (and if these fail, #6, repeated if done only once before) to suggest site of Stachybotrys growth; if source found, remediate as per protocol (New York City Dept. of Health, 1993); if not found, perform extended #3 and 9 or 9a. If all tests negative, repeat #4 or do 6 (repeat #6 only if done only once before; if you already have a result of a repeat trial, rely on that); if repeat #4 or 6 negative, consider monitoring on one or more further occasions or, where symptoms are of concern, consult expert; if repeat #4 or 6 positive, further extend #2,3,5,9 exhaustively or consult expert.

#13 Minimal Stachybotrys, Source Not Located, Outdoors or Indoors

See comments under #11 on the significance of minimal numbers of Stachybotrys colonies. Note that Stachybotrys is common in certain agricultural situations, such as decaying straw, especially in horse barns. It may also occur on other natural moist cellulose substrata, and hence may appear in outdoor control samples.

13. Low-level or distant outdoor Stachybotrys source is possible, rely on #2,3,5 (and if these fail, #6, repeated if done before) to clarify indoor levels and sources. If new outdoor controls and #2,3,5 are negative and new indoor dust sample (#6) positive, but minimal (a single colony or two widely dispersed colonies in a large series of samples), consider monitoring on one or more further occasions; if outdoor controls negative but indoor levels are low (three or more colonies overall or two clustered colonies; or a single colony in a small sample) to moderate or high, the likelihood of an indoor source is heightened: follow procedure #12. If outdoor control is positive but indoors negative, disregard indoor Stachybotrys procedures (if outdoor source yields high levels, consider searching for and eliminating it). If both outdoor and indoor samples are negative, discontinue Stachybotrys procedures.

#14 Low to High Stachybotrys, Source Not Located, Outdoors or Indoors

See comments under #13 on possible occurrence of Stachybotrys in the outdoor environment.

14. Controls of previous studies have indicated that a nearby or relatively high-level outdoor source of Stachybotrys is possible. Nonetheless, an indoor source also remains possible where conducive habitat conditions exist or have existed; an outdoor amplifier may have served as an inoculum source for indoor colonization. Rely on #2,3,5,9a* to detect any indoor amplifier; if original Stachybotrys-positive controls in procedures #4 or 6 were within 6 m of the test building or downwind of possible air outflow from building, repeat #4 or do 6 (repeat #6 only if done only once before; if you already have a result of a repeat trial, rely on that) with outdoor controls in more distant, windward (#4) or normally windward (#6) sites. If new outdoor controls are negative and indoors positive for Stachybotrys at any level, go to #12. If outdoor levels in repeat still differ insignificantly from (chi- squared) or exceed indoor levels, discontinue indoor analysis or, if symptoms are of concern, consult with expert. If new outdoor controls are positive for Stachybotrys but indoor levels are significantly greater (chi- squared), then go to #12. If new indoor and outdoor tests are both negative for Stachybotrys, discontinue analysis.

#15. Ergosterol or glucan sampling

Ergosterol is a cell membrane component (analogous to human cholesterol) which is specific to fungi. Quantitative analysis of ergosterol in an environment can be used to estimate the level of fungal inoculum present. It cannot identify whether this inoculum derives from indoor amplifiers, sedimentation of outdoor air spora, or fungi in imported mud and debris (e.g., from shoes, dog or cat feet). Nonetheless, markedly high indoor levels generally indicate problematic fungal amplifiers, and this inference is easily confirmed with species-by-species analysis of a small number of air or dust samples.

For further information see Miller et al. (1988).

Beta 1,3-glucan: This is another fungal biochemical, a cell wall component, which may be measured in an attempt to assess the total quantity of viable and non-viable fungal biomass. A well established protocol for interpreting Beta 1,3 glucan results from allegedly mold-contaminated buildings does not yet exist, but is an area of active research.

#16. Mycotoxin sampling from air or dust

In certain cases, significant levels of a particular mycotoxin may be expected a priori to occur in an environment, either because the associated mold is known to be present in large quantity or because aerosols deriving from a substrate for toxigenic mold growth (e.g., peanut or corn dust in the case of Aspergillus flavus) are suspected to be present. Direct mycotoxin detection mechanisms ranging from simple medium tests to complex chromatography and spectrometry may be used to determine mycotoxin levels. Generally, the use of such techniques requires extensive preliminary studies, combined with expert analysis and adequate facilities. A large literature exists on analysis techniques for various mycotoxin classes, but review of this literature is beyond the scope of this article.

In many contaminated indoor environments, the substances responsible for causing symptoms are imperfectly known and cannot be detected directly. Most molds produce complex mixtures of mycotoxins, and may produce toxic proteins and irritating antigens in addition to the classic small mycotoxin molecules. Hence direct mycotoxin monitoring is only applicable where there is a clear environmental dominance of an individual toxin-producing species, or a special concern regarding a particular toxin or class of toxins. An example of such a concern is the possibility of exposure to aflatoxins predisposing workers to liver cancer in a peanut processing factory where occupant symptoms and adverse health effects suggest exposure to mycotoxins.

#17. Humidifier water direct microscopy

Water from humidifier and misters may contain profuse growth of microorganisms, including molds and yeasts in addition to bacteria and protozoans. Clean humidifier water should appear clear. If it does not, the cause of turbidity may be biological or chemical. Chemical turbidity usually consists of a sediment of calcium carbonate crystals; in areas where water is high in calcium, this sediment may heavily encrust evaporation- based humidifiers. Chemical turbidity arising from the suspension of fine carbonate particles may be difficult to distinguish from biological turbidity. Microscopy and culture may be used to make this distinction.

Take water in a sterile container. Make mounts and examine microscopically, looking for distinctive fungal filaments, yeast cells and conidia. Centrifugation may be used to concentrate the biological matter for examination.

#18. Humidifier water culture

Plate water directly onto any general fungal growth medium (ideally media restricting colony diameter such as Littman oxgall agar, rose bengal agar, or inhibitory mold agar), necessarily containing antibacterial antibiotics. Prior centrifugation should not be necessary as inoculum is dense in significantly contaminated humidifiers. Indeed, a dilution series (as #6, but simply dilute aqueous suspensions 1:10, 1:100 and so on) may be desirable to obtain well separated, countable colonies relatively free of overgrowth by antibiotic-resistant bacteria. Bacteriological evaluation and endotoxin evaluation (matters beyond the scope of this article) should also be considered.

#19. Cytotoxicity testing

Dust collected by means of a vacuum device may be tested directly for toxic components without targeting particular toxins. A technique for accomplishing this utilizes a sensitive cultured human cell line and exposes it to chemical extracts of the dust. Miller et al. (ref# 12 in my version-- Miller, LaFlamme, Sobol et al. 1988) adapted a technique employing human HeLa cells for dust cytotoxicity studies; the reader is referred to that publication for further information.

Cytotoxicity analysis detects overall levels of toxins in dust, not just mycotoxins, and the relative importance of mycotoxins in known cytotoxic samples must be inferred from fungal analysis (culture and/or direct microscopy) of the same samples.

References

Davies, R., R.C. Summerbell, D. Haldane, A. Dufour, K. Yu, I. Broder, R. Dales, J. Kirkbride, T. Kauri, and W. Robertson. 1995. Fungal contamination in public buildings: a guide to recognition and management. 76 pp. www.hc-sc.gc.ca/datahpb/dataehd/English/catalog/bch_pubs/fungal_contamination.htm

Health Canada. 1993. Indoor air quality in office buildings: a technical guide. A report of the Federal-Provincial Advisory Committee on Environmental and Occupational Health. 55p.

Miller, J.D., A.M. LaFlamme, Y. Sobol, P. Lafontaine, and R. Greenhalgh. 1988. Fungi and fungal products in some Canadian houses. Int. Biodeterior. 24: 103-120.

Nathanson, T. 1993. Indoor air quality in office buildings: a technical guide. Health Canada, Ottawa. 55 pp.