INTRODUCTION — Mold is ubiquitous outdoors, and it is readily introduced or physically transported into the home, work, and school indoor environments.
This topic will focus strictly on the detection of mold in indoor environments.
A more general discussion of assessment of allergens in the indoor environment is found separately. (See "Allergen sampling in the environment".)
TERMINOLOGY — The generic term "mold" encompasses many types of fungi, a diverse class of eukaryotic micro-organisms that live on organic nutrients. The kingdom Fungi is comprised of plants without leaves, flowers, or roots that reproduce from spores and includes yeasts, molds, smuts, and mushrooms (table 1).
Molds lack chlorophyll and vascular tissue. They range in form from a single cell to a body mass of branched filamentous hyphae that spread into and feed off of dead organic matter or living organisms.
PROPERTIES OF MOLDS — Molds produce specialized, uniquely structured, fruiting bodies called spores that are generally invisible to the naked eye (table 1). The unique features of these spores are used to speciate the mold by microscopic, immunologic, and molecular biology techniques.
The majority of the spores produced by molds range in size from 2 to 20 microns in diameter and 1 to 100 mm in length. These spores can become readily airborne. The spores appear as oblong and irregular biomasses, with visually characteristic shapes, sizes, and colors that can be discriminated from each other microscopically (figure 1 and figure 2 and picture 1). Morphologically, the spore surface under a microscope may appear as smooth, granular, warty, spiny, cup-shaped, pie-crust shaped, or reticulate (figure 3).
Mold spores need a relative humidity >65 percent, a temperature between 50 to 90°F (10 to 32°C), and organic matter as their nutrient base to grow. When a spore germinates, it produces hyphae or branches that digest organic matter on the growing surface.
The general appearance of mold varies widely in texture from a filamentous cottony or granular display to a leathery or smooth velvety surface. Mold can present as colorless or with a white, gray, brown, black, yellow, green, or fluorescent hue (picture 1). As mold grows, it often exudes a "musty" odor that results from volatile organic compound vapors that are released.
There are approximately 100 indoor molds that have been identified as potentially hazardous to human health, although only a small subset of these are commonly found in the air and dust in indoor environments (table 2 and table 3) [1,2]. The classification of medically important molds, some that are known to elicit immunoglobulin E (IgE) antibodies in humans, is presented in the table (table 4).
DETECTION OF INDOOR MOLD GROWTH — There is often mold growth where there has been water damage or constant high humidity. Invisible spores can be transported indoors on clothing, shoes, or pets, or they may blow in through open doors, windows, or ventilation systems. Spores can survive on wood, paper, carpet, soil, plants, fabrics, and other surfaces. As they settle on a surface with the right humidity and temperature, mold spores begin to germinate, gradually producing an increasingly expanding network of hyphae [3].
Odors — The nose may be the first sensory organ to identify a mold infestation as a result of the pungent organic compounds molds release into the air. Any musty odor raises suspicion that a major mold contamination is present, even if it cannot be readily seen.
Visual signs — An initial indoor environmental assessment involves a visual inspection of the floor, walls, windows, and air handling system. Mold can sometimes be visually detected as a discolored surface. The environment should also be inspected for sources of dampness or standing water. Visible mold can be remediated without further testing or analysis. (See 'Prevention and remediation' below.)
INDICATIONS FOR ASSESSMENT — Before pursuing a professional assessment of mold in an indoor environment, it is important to understand the limitations of these analyses. In the United States (and many other countries), there are no federal limits for acceptable levels of mold spores in any indoor environment. Thus, the demonstration of mold in an indoor environment does not provide definitive information for making decisions about remediation. However, it can provide additional information about the presence of mold in an indoor environment if simple visual inspection is equivocal or negative and the nose detects a mold problem.
Therefore, the following criteria should be met before professional evaluation of the home and work/school environment for mold is performed:
●The patient has a confirmed condition known to be related to fungus, such as a hypersensitivity disorder (eg, asthma, allergic rhinitis, hypersensitivity pneumonitis, or sinusitis) or a severe fungal infection (especially in an immunocompromised individual).
●There is evidence of exposure to a specific fungus that is known to cause the condition the patient has (table 3). (See 'Detection of indoor mold growth' above.)
Guidance for health care providers in determining when a home assessment for potential mold exposure is warranted have been published [4]. This is based on questions, shown in the table, that can help identify an association between mold exposure and symptoms (table 5) [5]. The indoor environments to which the patient is exposed should be evaluated for growth of the implicated species of fungi if these two criteria are met. Sampling and testing are generally not necessary if visual inspection reveals mold, since laboratory testing will likely not change the decision to remediate the environment. However, further assessment is indicated if no mold is seen but musty odors suggest mold contamination or if there is a known history of water damage in that environment.
ASSESSMENT FOR MOLD — The systematic process of assessing an environment for the presence of mold involves two phases:
●Sampling of the environment
●Analysis to detect evidence of specific mold(s)
In general, no single laboratory collection method or analytic test can be used to definitively type and quantify mold in an environment, since molds are continually changing with time.
We recommend that the person performing the assessment be a certified industrial hygienist, since this ensures a defined level of training and knowledge.
Sampling methods — A number of environmental sampling methods are used to assess the burden of mold in an environment [6]. The four principal collection techniques include:
●Obtaining a bulk "reservoir" dust from the floor or ventilation system
●Sampling of a piece of the physical surface that is discolored
●Performing a residual surface swipe on a suspected contaminated surface
●Obtaining an air sample using one of several multistage samplers that are often equipped with a nutrient-filled culture plate
Bulk reservoir dust collection — A bulk surface "reservoir" dust specimen is a sample of accumulated particulate released from the air over time. It can be collected using a clean vacuum device that is equipped with any of a number of new collection unit attachments (eg, nozzle sock) [7].
Sampling should be performed where individuals spend the majority of their time in the environment (eg, bedroom, family room, kitchen, study) and where the symptoms are believed to be induced. For practical reasons, the mattress in a bedroom is vacuumed first, followed by upholstered furniture, and then carpeting. The bare floor adjacent to a major item of upholstered furniture is vacuumed if carpet is not present. A global specimen may be taken from the major areas where the individual spends most of their time and where they feel mold contamination is most likely if cost is an issue. However, separate testing of rooms allows more definitive testing of specific areas.
The collected material may include associated debris (sand, human and animal hair, textile fibers, lint, human dander, coins, or insect parts). Once collected, the bulk dust is processed by passing it through a 50-mesh metal sieve. This removes irrelevant particulate and allows 300-micron diameter and smaller particles including mold spores <20 microns to pass through.
A second form of bulk sampling involves removal of a piece of actual discolored surface material from the wall or floor. Often material from different sites is evaluated. Each is stored in a sterile plastic bag prior to evaluation. These specimens are more problematic to test, as they are heterogeneous, and there is no reference method for processing them for culture or microscopic analysis.
Once in the laboratory, a portion of the sample can be mounted on a silicone grease or petroleum gel-covered slide, stained, and examined microscopically for the presence of characteristically shaped fungal spores. This method provides a measure of the total spore count. It is more difficult to perform with a piece of wallboard or carpet on which mold contamination is suspected.
A second portion of the bulk dust may be evaluated in a microbiologic assay for viable mold spores. One method involves distributing 5 mg of sieved fine dust from the bulk specimen in a physiologic buffer onto medium in a culture plate that contains antibiotics to prevent bacterial growth. Mold spores, if present and viable, grow into colonies that can be visually enumerated at 24, 48, or 72 hours. Some normative data are available on the number of viable colonies per gram of surface dust in homes across the United States [1].
Surface wipe sampling — Mold that settles out from air onto various indoor surfaces can be collected using a surface swab or clear adhesive tape transfer technique. Contents on the swab are released into a physiologic buffer (eg, phosphate-buffered saline) and delivered onto a culture plate in a manner similar to that used for the bulk dust sample. The adhesive tape contact sample can alternatively be stained and analyzed for spores microscopically. The smoother the surface being sampled, the greater the efficiency of adhesive tape collection. Swab collection techniques are sometimes problematic in that the spores may be damaged during sampling, which compromises viability. Both swab and tape sampling are inexpensive, readily mastered, and nondestructive sampling techniques. However, they are highly variable in terms of their ability to collect reproducible test specimens that produce comparable colony counts upon analysis. If the surface being evaluated is a filter, then vacuum and swab samples were superior to the cut and elute collection method in terms of recovery and diversity of mold species [8].
Sampling of air — Aerosolized spores can be sampled by gravity samplers, inertial impactors, or filtration devices [6]. The suction impactors are generally preferred for mold evaluation, since they have the highest efficiency of mold spore collection and they allow automated monitoring over a multiday period.
●The gravity sampler allows particles to fall on a collecting surface, usually an adhesive-coated glass slide or an open culture plate containing various media (eg, chloramphenicol/potato dextrose agar and malt extract agar) [9]. Gravity generally plays a small part in the collection, because air is never still. The larger the particle size, the more efficient the collection in a gravity sampler.
●Inertial impactors depend on particle motion for collection. They either pass airborne particles across a stationary surface using a fan or suction apparatus or they move the collection surface through the air at a constant speed. Collection efficiency of inertial impactors depends on the particle size, with smaller particles remaining in the air stream and having a lower probability of impacting on the collection surface that is coated with a sticky substance (eg, thin layer of silicon grease). Rotation impactors, such as the rotarod, liquid impingers, and suction impactors, are included in this group. The rotarod uses a 1.2 mm-wide plastic rod with the leading edge that is greased. It is satisfactory for collecting particles down to 10 microns in diameter, which includes some larger mold spores.
●Suction impactors include slit samplers (eg, Burkard spore trap) that draw measured quantities of air across a narrow slit onto a moving drum over a 24-hour or seven-day sampling period. Alternatively, cascade impactors (eg, Andersen sampler) draw air over a large entrance orifice and accelerate it stepwise through plates of increasingly smaller pore or sieve sizes. Increasing the speed causes smaller particles to be trapped more efficiently. These samplers discriminate between particle sizes and are better at trapping small mold spores than the inertial impactors or gravity samplers. Petri dishes filled with media nutrients can also be placed in the Andersen sampler to directly collect viable spores with different particle sizes for culturing and further analysis.
Air sampling methods are unfortunately prone to contamination by collection of molds that may enter the air space but that are not representative of the molds that have caused the environmental contamination of concern. This can occur as a result of open doors or windows that allow contaminating or irrelevant molds into the air space during the sampling period. In addition, different molds are more or less effectively trapped, depending on their size and shape. Moreover, air sampling may sometimes be inexact, because spores can be in an inactive form at the time they are collected, since biologic activity requires humid, warm, nutrient-filled conditions that may not be present at the time of sampling. These inactive spores are not contributing to mold contamination observed in an environment at the time of sampling, but they can become activated when cultured and will be included in the quantified spore count.
Analysis methods — Once the specimen has been collected, it can be analyzed by bioassay, biochemical assay, culture, direct microscopy, nucleic acid analysis, or immunologic assay to identify and quantify the specific molds in the sample. In selected environmental testing laboratories, liquid chromatography high resolution mass spectrometry is being used to more precisely analyze toxins and allergens in foods and environmental extracts. Only a few of these methods are discussed, since a combination of these, often involving purification from a culture, are most widely used to speciate and quantify the mold content of indoor environments where visual inspection has failed to detect a mold infestation.
Direct microscopy — Visual identification of mold should be performed by a certified technician who is familiar with the morphologic forms of different fungal spores. Direct microscopic examination is used to quantify the total number of fungal spores on a slide. One limitation of this technique is that it quantifies the total fungal spore burden (both viable and inactive spores), since only a percentage of fungal spores that are counted will be viable and therefore contribute to widespread mold contamination of the environment.
Culture — Culture is commonly performed, regardless of the collection method, to expand the spore number so that other laboratory techniques can more readily detect the various molds present. A nutrient medium, with antibiotics such as penicillin and gentamicin to prevent bacterial growth, allows growth of viable spores. The individual colonies are then identified macroscopically, microscopically, biochemically, immunologically, or based on their nucleic acid profile. The number of viable mold spores are then reported at 24 or 48 hours as colony counts or colony-forming units (CFUs) per gram of bulk dust or colonies per cubic meters of air sampled.
The culture procedure has technical limitations that can lead to underestimates of the actual spore levels for several reasons:
●A high number of bacteria can occasionally exceed the capacity of the antibiotics in the medium to inhibit growth. This will result in the plated medium becoming covered with bacterial colony clusters, which are morphologically distinct from mold colony-forming units. A result of this nature will be reported as "high bacterial contamination, mold could not be assessed in this specimen."
●Some spores have highly specialized growth conditions that are not mimicked by the contents of the culture plate.
●Certain spores produce inhibitors that can prevent growth.
●Overcrowding induces soluble inhibition factors, and growth of colonies may diminish as a result of contact suppression.
●The collection process often has a desiccating effect that reduces the viability of some spores.
●Most spores have limited viability periods when airborne, and these time intervals vary among the different spore types and release times during the day.
Nucleic acid analysis — A mold-specific, quantitative polymerase chain reaction (PCR) assay is a laboratory tool for assessing the content of mold in extracts and surface dust. One group used a PCR-based assay to detect the presence and relative levels in house dust of 26 mold species associated with water damage and 10 mold species not associated with water damage. An environmental relative moldiness index (ERMI) was developed using the level of these molds. These 36 molds were measured in the dust from 271 homes of children with asthma. The ability of ERMI level to predict the development of respiratory illness (wheeze and/or rhinitis) was then compared with a binary classification of homes as either moldy or non-moldy, based on onsite inspection [10,11]. The visual onsite home inspection was not predictive of respiratory illness, while the ERMI was able to predict the occurrence of illness in homes containing mold associated with water damage.
Immunologic assays — Monoclonal antibody-based immunoenzymometric assays are available to quantify the level of selected individual mold allergens in extracts of surface dust or cultures. "Indicator" allergen assays for Aspergillus fumigatus (Asp f group 1 [Ribotoxin]), Alternaria alternata (Alt a group 1 allergen [acidic glycoprotein]), and antigens of Aspergillus versicolor and Stachybotrys chartarum can be performed and are sometimes useful. However, the technique has a serious limitation in that nutrient requirements are not always optimal for efficient growth of these molds and expression of their allergenic proteins. Thus, Aspergillus fumigatus or Alternaria alternata in particular may be present in the specimen but their specific allergenic proteins are not expressed and thus undetectable by immunoassay. For this reason, quantitative immunoassay analysis is not performed as a routine clinical test but rather is considered a research tool.
Mass spectrometry assays — The use of mass spectrometry of extracted and trypsinized mold fragments can provide more precise proteomic evaluation of the mold composition in food and environmental samples. Spore and hyphae proteins are typically extracted in buffer containing 2.5 percent trifluoroacetic acid. The resulting supernatants are then dried and washed with 90 percent acetone. Protein content of these is then determined using liquid chromatography high resolution mass spectrometry. Research using these methods has detected toxins in foods and has shown that allergenic Alternaria spore and hyphae proteomes appear to be distinct from each other [12,13]. Mass spectrometry methods are best used in research investigations of toxins and allergenic mold proteins in foods and biological extracts used in the diagnosis and treatment of allergy. They are rarely used in routine evaluation of indoor environments.
PREVENTION AND REMEDIATION — Academic and federal agencies have developed guidelines for homeowners on the issues of mold, moisture, and health in the home [14,15]. Information is also available at the United States Environmental Protection Agency website.
Since mold is ever-present in environments, the absolute level of spore contamination can be used to make decisions about costly remediation of indoor environments. Mold contamination should be expected in cases of water damage. Caution, however, should be exercised when interpreting mold spore or mold-specific allergen levels, as these are rarely quantitative, and they are often generated using samples that may not be representative of the entire environmental area of concern. Direct comparison of levels against outdoor spore counts is also not generally useful. Thus, relative levels of mold spores or allergen levels need to be judged conservatively and where possible within the context of an onsite environmental survey.
Reducing humidity by increasing ventilation, covering cold surfaces such as water pipes with insulation, and increasing the air temperature to reduce surface humidity are inexpensive actions that can help discourage mold expansion (table 6). Plumbing leaks and other sources of dampness or standing water should also be fixed.
Visible mold can be remediated using several simple measures (table 6). The least expensive and most effective means of removing surface mold involves scrubbing contaminated nonporous hard surfaces with detergent and water and then drying the area completely. Disinfectants or biocides, such as a diluted chlorine bleach solution (eg, 10 percent), are not usually necessary. Any contaminated areas in which mold has embedded itself, such as a porous wall, floor, carpet, or upholstered area, need to be removed or replaced. A certified industrial hygienist is preferred if professional remediation is required.
SUMMARY AND RECOMMENDATIONS
●Every indoor environment has some mold present, and moisture control is the key to mold growth control. Mold spores need a relative humidity >65 percent, a temperature between 50 to 90°F (10 to 32°C), and organic matter as their nutrient base to grow. (See 'Properties of molds' above.)
●The initial inspection for indoor mold includes examination for musty odors and visible mold on surfaces. Visible mold can be remediated without further testing or analysis. (See 'Detection of indoor mold growth' above.)
●There are no federal limits for acceptable levels of mold spores in any indoor environment. Thus, mold spore confirmation testing does not provide definitive information for decisions about remediation. (See 'Indications for assessment' above.)
●A professional assessment for mold in the indoor environment is not indicated unless a patient has a confirmed condition known to be related to fungus and there is evidence of exposure to the specific fungus that causes that condition. The indoor environments to which the patient is exposed should be evaluated for growth of the implicated species of fungi if these two criteria are met. Sampling and testing is generally not necessary if visual inspection reveals mold. However, further assessment is indicated if no mold is seen on visual inspection but a musty odor suggests mold contamination. (See 'Indications for assessment' above and 'Assessment for mold' above.)
●Measures that can be taken to discourage mold growth in the indoor environment include reducing humidity by increasing ventilation, covering cold surfaces such as water pipes with insulation, and increasing the air temperature. Washing nonporous surfaces with detergent and water and drying the area effectively address surface mold contamination. Contaminated porous surface areas in which the mold is embedded must be removed. (See 'Prevention and remediation' above.) (table 6).
