How To Assess and Measure Exposure to Indoor Pollutants such as Mold

From the World Health Organization in its report WHO Guidelines for Indoor Air Quality: Dampness and Mould, published July 16, 2009

2.4 Exposure assessment

Exposure can be defined as an event during which people come into contact with a pollutant at a certain concentration during a certain length of time (WHO Regional Office for Europe, 2006a). In most circumstances, however, exposure defined in this way cannot be determined confidently, and exposure indicators are used instead. Thus, when the word exposure is used without qualification in this document, it refers to indicators. The indicators of exposure in indoor environments used most commonly are derived from answers to questionnaires. A more objective approach (but not necessarily a more valid one; see below) might be to measure the airborne or surface concentrations of indoor pollutants, such as the amount of pollutant per cubic metre of air or per gram of house dust. These are, however, generally relatively crude proxies of the true exposure and thus lead to at least some misclassification of exposure and subsequent bias.

The relative lack of knowledge about the role of specific exposures in health problems related to house dampness is due mainly to a lack of valid, quantitative methods for assessing exposure, particularly of bioaerosols. This may explain the relatively large number of studies that have failed to demonstrate a direct association between bioaerosol concentrations and health effects in damp indoor environments (see Chapter 4). This section discusses the issues of exposure assessment related to observed and perceived dampness and of bioaerosol measurements.

Measurement of humidity in the air and of the moisture content of building materials is discussed in Chapter 3.

2.4.1 Measurement of indicators of dampness

Occupants’ perceptions are the basis used for assessing house dampness in most epidemiological studies, and questionnaires are therefore often the method chosen. The questions typically elicit information on whether conditions such as leaks, flooding, wet basements, window condensation, visible fungal growth or mouldy odours are or have been present. Sometimes, the extent of water damage and damp is also assessed. Prevalence estimates may vary widely, however, depending on the way in which such questions are framed, the type of question, the level of detail requested and the judgement of the people filling in the questionnaire.

Reliance on self-reporting, which is by definition subjective, may be a source of error in cross-sectional studies, as demonstrated by Dales, Miller and McMullen (1997), who reported that under some conditions people with allergies are more likely than non-allergic people to report visible fungal growth. Other studies have shown that such bias is unlikely (Verhoeff et al., 1995; Zock et al., 2002). To overcome the problems associated with the bias of self-reporting, trained inspectors have been used in several studies to visit houses and assess indoor dampness, including its severity. This method has the advantage of being more objective and allows a more standardized approach; nevertheless, it is often a single snapshot, which lacks the longer perspective of the occupants. These differences in approaches may lead to different estimates of the prevalence of house dampness, as demonstrated in several comparisons of the two methods (see section 2.1).

Measurements of humidity in air, the moisture content of building materials, their interrelationships and their relationship with indoor climate dynamicsmore generally are discussed in Chapter 3.

2.4.2 Measurement of microorganisms and microbial agents

The assessment of indoor concentrations of microorganisms presents distinct challenges. Pathogenic microorganisms may be hazardous at extremely low levels, while other organisms may become important health hazards only at concentrations that are orders of magnitude higher. Some organisms and spores are extremely resilient, while others are inactivated during sampling. Certain fungal spores are easily identified and counted, while many bacteria are difficult to characterize.

Sensitive, specific methods are available for quantifying some microbial agents, while there are no good methods for others. Many of the newly developed methods (e.g. measurement of microbial agents such as fungal (1→3)-β-Dglucans or fungal extracellular polysaccharides; see below) have not been well validated and are often unavailable commercially. Even with some well-established methods (e.g. the Limulus amoebocyte lysate assay for measuring bacterial endotoxins; see below), significant variations in concentrations have been found (Thorne et al., 1997; Chun et al., 2000; Reynolds et al., 2002). Issues about the storage and transport of bioaerosol samples have often not been addressed, although these conditions can affect the activity of some biological agents, such as endotoxins (Thorne et al., 1994; Douwes et al., 1995; Duchaine et al., 2001).

Furthermore, not all the biological agents that might be associated with damp indoor environments and their health effects may have been identified.

Most studies of dampness and health have focused on visible fungi or water damage, and in most of these studies exposure was assessed from questionnaires. The extent to which questionnaire reports of fungal growth correlate with actual exposure to relevant fungal components is, however, not known. In studies with objective measurements of fungal concentrations, spores were generally cultured from indoor air (Garrett et al., 1998) or from settled dust (Jacob et al., 2002). The section below gives the options available for measuring concentrations of microorganisms (particularly fungi) in indoor air.

2.4.2.1. Culture-based methods

Airborne concentrations of microorganisms can be studied by counting culturable propagules in air samples or settled dust samples. Sampling of culturable microorganisms is based on impaction (in which microorganisms are collected from the airstream due to an inertial force that deposits them onto a solid or semisolid collection surface), liquid impingement (in which inertia as a principle force collects microorganisms in a liquid medium) or air filtration (separation of microorganisms from the airstream by passage through a porous medium such as a filter). After sample collection, colonies of bacteria and fungi are grown on culture media at a defined temperature for the length of time required for colony development (usually 3–7 days). Colonies are counted manually or by image analysis techniques. To date, no standard methods are available for detecting and enumerating fungi in indoor environments, which significantly limits the potential for comparing data from different studies. International standards are, however, being prepared by the International Organization for Standardization (ISO) technical committee 147/SC on indoor air for sampling by filtration and impaction and for the cultivation of fungi (ISO 16000-16, -17,-18).

Counting culturable microorganisms has some serious limitations. These include poor reproducibility; selection of certain species because of, for example, the choice of sampling method, culture media or temperature chosen; and the lack of detection of non-culturable and dead microorganisms, cell debris and microbial components, although they too may have toxic or allergenic properties.

In addition, no good methods for sampling personal air for culturable microorganisms are available, and air sampling for more than 15 minutes is often not possible, whereas air concentrations usually vary widely over time (see section 2.4.5). Nevertheless, counting culturable microorganisms is potentially a very sensitive technique, allowing the identification of many different species. Traditional culture methods have proven to be of limited use for quantitative assessment of exposure. Culture-based techniques thus usually provide qualitative rather than quantitative data. The former can, however, be important in risk assessment, as not all fungal and bacterial species pose the same hazard. Furthermore, a qualitative comparison of indoor and outdoor microbiota (in samples collected at the same time) may provide important information about potential indoor sources of contamination. More extensive reviews of techniques for sampling and culturing microorganisms are available (Eduard, Heederik, 1998;Macher, 1999).

2.4.2.2. Non-culture-based methods

In non-culture-based methods, organisms are enumerated regardless of their viability. Non-culturable microorganisms are generally sampled by air filtration or liquid impingement methods. Microorganisms can be stained with a fluorochrome, such as acridine orange, and counted under an epifluorescence microscope (Thorne et al., 1994).

Slit impaction on glass slides and staining with lactophenol blue is a common method for microscopic determination of the total concentration of fungal spores. The possibility of classifying microorganisms taxanomically is limited because little structure can be observed. Electron microscopy or scanning electron microscopy allows better determination (Eduard et al., 1988; Karlsson, Malmberg, 1989). Bacteria collected on impingers or filters can be counted by flow cytometry after staining with 4/,6-diamino-2-phenylindole or by fluorescent in situ hybridization (Lange, Thorne, Lynch, 1997).

The main advantage of microscopy and flow cytometry is that both culturable and non-culturable microorganisms can be quantified, selection effects are limited, personal air sampling is possible, the sampling time can – for many microorganisms– be varied over a wide range, and results are available quickly.

The disadvantages include the unknown validity of these techniques, lack of detection of possibly relevant toxic or allergenic components or cell debris, limited possibilities for determining microorganisms, laborious and complicated procedures, and high cost per sample of the more advanced methods. An extensive review of microscopy and flow cytometry methods for counting nonculturable micro organism has been published (Eduard, Heederik, 1998). Little or no experience has been gained in non-industrial indoor environments with more advanced non-culture-based methods, such as scanning electron and epifluorescence microscopy and flow cytometry. Therefore the usefulness of these methods for indoor risk assessment is unknown.

2.4.2.3. Methods for assessing microbial constituents

Constituents or metabolites of microorganisms can be measured to estimate microbial exposure, instead of counting culturable or non-culturable microbial propagules. Toxic (e.g mycotoxins) or pro-inflammatory components (e.g endotoxins) can be measured, and non-toxic molecules can be used as markers of large groups of microorganisms or of specific microbial genera or species. The availability of methods, such as those based on the polymerase chain reaction (PCR) and immunoassays, has opened new avenues for detection and identification of species, regardless of whether the organisms are culturable.

Markers for the assessment of fungal biomass include ergosterol, measured by gas chromatography–mass spectrometry (Miller, Young, 1997), and fungal extracellular polysaccharides, measured in specific enzyme immunoas says (Douwes et al., 1999). These allow partial identification of the mould genera present. Volatile organic compounds produced by fungi, which may be suitable markers of fungal growth (Dillon, Heinsohn, Miller, 1996; Moularat et al., 2008b), are usually measured in air samples by gas chromatography with or without mass spectrometry or high-pressure liquid chromatography. Other agents, such as (1→3)-β-Dglucans (Aketagawa et al., 1993; Douwes et al., 1996) and bacterial endotoxins, are measured because of their toxic potency. Endotoxins are measured with a Limulus amoebocyte lysate test, prepared from blood cells of the horseshoe crab, Limulus polyphemus (Bang, 1956). Analytical chemistry techniques with gas chromatography–mass spectrometry for quantifying lipopolysaccharides have also been developed (Sonesson et al., 1988, 1990); however, these methods require special extraction procedures and have not been widely used. Two methods for measuring (1→3)-β-D-glucans have been described, one of which is based on the Limulus amoebocyte lysate assay (Aketagawa et al., 1993) and the other on an enzyme immunoassay (Douwes et al., 1996).

PCR techniques are available for the identification of species of bacteria and fungi in air (Alvarez et al., 1994; Khan, Cerniglia, 1994), and several quantitative PCR methods have been validated for use in the indoor environment. For example, real-time PCR methods have been described to detect and quantify Cladosporium (Zeng et al., 2006) and Aspergillus (Goebes et al., 2007) at the genus level. Similar methods have been developed for measuring species of common indoor fungi (Vesper et al., 2005; Meklin et al., 2007). PCR methods allow the assessment of large groups of microorganisms. For instance, a quantitative PCR method is available for measuring 36 indicator species commonly associated with damp houses in the United States and has been used to define an “environmentalrelative mouldiness index” for houses in that country (Vesper et al., 2007). PCR methods for quantitative assessment of exposure to fungi and other microorganisms have significant advantages, including sensitivity and specificity.

Also, they can be used for quantitative assessment; they provide results relatively quickly; they can be used to measure a wide range of microorganisms, both genus and species; and they are independent of the culturability of the organism. Most methods for measuring microbial constituents (with the exception of that for bacterial endotoxins) are experimental and have yet to be used routinely or are unavailable commercially. Important advantages of these methods include the stability of most of the measured components, allowing longer sampling times for airborne measurements and frozen storage of samples before analysis; the use of standards in most of the methods; and the possibility of testing for reproducibility. These methods do not, however, leave fungal isolates for further investigation.

2.4.3 Measurement of indoor allergens

Antibody-based immunoassays, particularly enzyme-linked immunosorbent assays, are widely used to measure aeroallergens and allergens in settled dust in buildings. These assays involve use of antibodies specific to the target allergen and an enzymic reaction with a substrate for detection. In radioimmunoassays, radiolabelling is used for detection. The house dust mite allergens Der p I, Der f I and Der p/f II have been widely investigated and the methods well described (Luczynska et al., 1989; Price et al., 1990; Leaderer et al., 2002). Methods for assessing exposure to allergens from rodents (Swanson, Agarwal, Reed, 1985; Schou, Svendson, Lowenstein, 1991; Hollander, Heederik, Doekes, 1997), cockroaches (Pollart et al., 1994) and storage mites (Iversen et al., 1990) have been published.

Methods for measuring fungal allergens are not widely available, mainly because of difficulties in manufacturing and standardizing fungal allergen extracts(see section 2.3.1). Nonetheless, some enzyme-linked immunosorbent assays have been described in the literature, and a commercial assay is available for A. alternata allergen (Alt a I). A comparison of several monoclonal and polyclonal antibody-based assays for measuring Alt a I (including a commercially available method) showed, however, wide disparity (Barnes et al., 2006). It is therefore unclear whether these assays provide valid estimates of the true Alternaria allergen concentrations in indoor samples.

2.4.4 Strategies for monitoring exposure

In addition to questionnaires, personal or environmental monitoring is commonly used for exposure assessment. Although monitoring can potentially result in a more valid, accurate assessment, this may not always be the case. Validity is strongly dependent on the sampling strategy chosen, which in turn depends on a large number of factors, including: the type of exposure and disease or symptoms of interest; whether the health outcomes are acute or chronic (e.g. exacerbation versus development of disease); whether the approach is population- or patient-based; suspected variations in exposure over both time and space and between diseased and reference populations; the methods available to assess exposure; and the costs of sampling and analysis.

2.4.4.1. What should be measured?

With regard to health problems associated with indoor air, many exposures should be considered, as it is often unclear which microorganisms or agents are causing the symptoms or diseases. Some studies are conducted specifically to assess which exposures are contributing to the development of symptoms. In practice, both funding and the availability of methods for measuring agents are limited, as many methods are not commercially available and are used only in research, severely limiting the possibility of measuring all agents of interest.

2.4.4.2. How useful are routinely collected data?

Data collected for use in monitoring may be of limited value in epidemiological studies. For example, monitoring is often done in areas where the concentrations are likely to be highest, to ensure compliance with exposure limits. In contrast, epidemiological studies require information on average concentrations. Special surveys may therefore be necessary, with random sampling, rather than relying on data collected during monitoring.

2.4.4.3. When should sampling be done?

To the extent possible, samples should be taken so that they represent the true exposure at an appropriate time. For acute effects, exposure measured shortly before the effects occur is the most useful. The situation is more complicated for chronic effects, as, ideally, exposure should be assessed before the effects occur and preferably at the time they are biologically most relevant – that is, when the exposure is considered the most problematic or when people are most likely to be exposed. This is possible only in prospective cohort studies or in retrospective cohort studies in which information on past exposure is available; even then, it is often unclear when people are most likely to be exposed to the agent of interest. In cross-sectional studies, exposure measurement can be valuable for assessing past exposure, but only when the environment has not changed significantly.

2.4.4.4. How many samples should be taken?

Measures of exposure should be sufficiently accurate and precise that the effect on disease can be estimated with minimal bias and maximum efficiency. Precision can be gained (i.e. measurement error can be reduced) by increasing the number of samples taken, either by increasing the number of people for whom exposure is measured or by increasing the number of measurements per person. In population studies, repeated sampling is particularly effective for exposures known to vary more widely over time within people than among people. If the within-people variation is lower than that between people, repeated measurements will not reduce the measurement error significantly. If there is known within- and between-person variation (from previous surveys or pilot studies, for example), the number of samples required to reduce bias in the risk estimate by a specific amount can be computed in the manner described by Cochran (1968) (see also section 2.4.5).

2.4.4.5. Should settled dust or airborne samples be taken?

In many studies, reservoir dust from carpets or mattresses is collected, and the concentrations are usually expressed in either weight per gram of sampled dust or weight per square metre. Although both measures are generally accepted, the latter may better reflect actual exposure (Institute of Medicine, 2004). The advantage of settled dust sampling is the presumed integration over time that occurs in deposition of the pollutant on surfaces (Institute of Medicine, 2000). Micro organisms can also proliferate in carpets, provided there is sufficient access to water; however, surface samples allow only a crude measure that is probably only a poor surrogate for airborne concentrations.

Airborne sampling requires very sensitive analytical methods. In addition, for an accurate assessment, large numbers of samples must be collected, as the temporal variation in airborne concentrations is probably very high (see section 2.4.5). Airborne sampling after agitation of settled dust has been used in some studies (Rylander et al., 1992, 1998; Rylander, 1997b; Thorn, Rylander, 1998), but it is questionable whether this results in a more valid exposure assessment. Therefore, exposure assessment is generally uncertain and this may obscure exposure–response relationships in epidemiological studies.

The recently described dustfall collector, a simple passive tool for long-term collection of airborne dust, combines the two methods to some extent (Wurtz et al., 2005). Collectors are placed on shelves or cupboards at least 1.5 m above the floor and receive airborne dust by sedimentation; they can be used for up to several months. This method is not affected by short-term temporal variance in airborne concentrations and is probably a better surrogate for airborne exposures relevant to indoor health. The dustfall collector is cheap to produce and simple to use, and the microbial levels measured with this device appear to correlate with the degree of moisture in school buildings. Although the initial results look promising, more validation is required to assess the usefulness of the device for measuring indoor exposure.

2.4.4.6. Should ambient or personal airborne sampling be conducted?

In general, personal measurements best represent the risk of the relevant exposure, and personal sampling is therefore preferred to area sampling. Modern sampling equipment is now sufficiently light and small that it can be used for personal sampling, and several studies of chemical air pollution have demonstrated its feasibility both indoors and outdoors (Janssen et al., 1999, 2000). Personal sampling might not always be possible, however, for practical reasons, such as being too cumbersome for the study participants or a lack of portable equipment for making the desired measurements (of viable microorganisms, for example). Nonetheless, it is expected that greater use of new, sensitive exposure assessment methods, including quantitative PCR techniques to measure indoor microbial concentrations (see section 2.4.2), will overcome some of these constraints, in particular when used in combination with passive personal samplers.

2.4.5 Problems in measuring indoor exposure

Exposure to microorganisms in the indoor environment is most frequently assessed by counting culturable spores in settled dust or the air, but this approach has serious drawbacks (see section 2.4.2). Perhaps the most important problem, which has rarely been acknowledged in the literature, is that air sampling for more than 15 minutes is often not possible, since air concentrations usually vary a great deal over time. The few studies in which repeated measurements were made of fungi in air or in settled dust showed considerable temporal variation in concentrations, even over short periods (Hunter et al., 1988; Verhoeff et al., 1994b). The variation in the concentrations of isolated genera was even more substantial (Verhoeff et al., 1994b; Chew et al., 2001).

It has been suggested that in order to achieve a ratio of 3–4 for within- and between-house variation in concentration, which appears to be realistic for culturable indoor fungi (Verhoeff et al., 1994b), 27–36 samples should be taken per house. This is necessary for reliable estimates of the average concentration in an epidemiological study with less than 10% bias in the relationship between a health end-point and the exposure (Heederik, Attfield, 2000; Heederik et al., 2003).

Thus, unless many samples are taken per house, sampling of culturable organisms will probably result in a poor quantitative measure of exposure, leading to a nonspecific bias towards the null. This might explain why most studies that included measurements of culturable fungi found no association with symptoms (in contrast to reported mould). The issue is particularly relevant for measurements of viable microorganisms; nonetheless, similar problems may exist for airborne measurements of other bioaerosols, such as house dust mite allergens, endotoxins and fungal (1→3)-β-D-glucans, as the airborne concentrations of these agents are also likely to be characterized by high temporal variation. This problem can be overcome by increasing the sampling time (up to several days or weeks), which is feasible for most bioaerosols, except viable microorganisms. This is often considered to be impractical, and therefore most exposure measurements continue to involve surface sampling, which is generally less affected by temporal variation. Surface sampling, however, may be a poor proxy for airborne concentrations (see above).

As no health-based exposure limits for indoor biological agents have been recommended, interpretation of concentrations is difficult, particularly in case studies. Therefore, strategies to evaluate indoor concentrations (either quantitatively or qualitatively) should include comparisons of exposure data with background levels or, better, comparisons of the exposure levels of symptomatic and non-symptomatic persons or in damp and non-damp buildings. A quantitative evaluation involves comparisons of concentrations, whereas a qualitative evaluation could consist of comparisons of species or genera of microorganisms in different environments. Because of differences in climatic and meteorological conditions and in the measurement protocols used in different studies (e.g. viable or non-viable sampling or by type of sampler or analysis), reference material in the literature can seldom be used.