Molds grow from microscopic spores of many fungi floating in the air and when land on surfaces that are moist; none of the molds grow in the absence of moisture [1,2]. Molds are ubiquitous and found both outdoors and indoors. Outdoors, molds play a vital role in the breakdown of dead organic matters like fallen leaves and dead trees. Molds grow in humid environments outdoors; indoors, they are usually found in damp/steamy and dark areas with poor ventilation (e.g., basements, bathrooms, recently flooded areas, cluttered storages, and kitchens) [1,2]. In outdoor air, levels of molds and their components (i.e., hyphal fragments, spores, and mycotoxins), referred herein as biotoxins, remain mostly below the level of health concern, even in high humidity, due to the presence of natural light and continuous movement of air. Similarly, biotoxins are always present in indoor air entering through open windows, with foot traffic and through ventilation. The levels of biotoxins in indoor air is generally equal to or lower than found in the outdoor air [3]; however, can easily reach unsafe levels under high humidity, inadequate ventilation, and poor lighting that may lead to adverse health effects. Molds remain viable long after the source of moisture is removed resulting in biotoxins to remain in indoor air over a prolonged period of time [4].

Mycotoxins are secondary metabolites produced by many molds and capable of producing various adverse health effects on many organ systems, especially in sensitive individuals (e.g., immunocompromised and/or genetically predisposed to slow elimination, like HLA gene alleles); exposure to mycotoxins have even been linked to infants’ death [1,5-9]. In general, exposure to biotoxins to individuals living or working in mold-infested buildings can cause a variety of allergies (e.g., hay fever-type symptoms [sneezing, runny nose, red eyes], inflammation, and skin rash) [2,10-20]. Exposure to mycotoxins can cause immunotoxicity, neurotoxicity (e.g., increased anxiety, depression, and cognitive deficits), pulmotoxicity, nephrotoxicity, hepatotoxicity, and even cancer when exposed to elevated levels for prolonged periods of time [1,7-9,21].

Secondary to the exposure to mycotoxins are changes in gut microbiota (dysbiosis) with possible disruption in gut barrier function and translocation of bacterial toxins. Gut dysbiosis increases bacterial lipopolysaccharide (LPS) transport into blood which also increases bacteriotoxin and mycotoxin absorption and transport from intestine to systemic circulation and can modulate their toxicity [21-24]. LPS can also cause inflammation and increases blood brain barrier permeability; increased blood LPS levels have been observed in neurodegenerative diseases, such as Alzheimer’s and Parkinsons’s diseases [22,24]. Consequently, increased indoor bacterial growth may be of major health concern for higher bacteriotoxin and mycotoxin exposure. Therefore, closer examination of the relationship among gut dysbiosis, LPS, and higher mycotoxins absorption/exposure contributing to mycotoxin-related toxicities is warranted.

Mold-affected buildings are still a persistent and common global problem even with improved regulations and building codes [9,16,25-27]. Globally, up to 47% of the homes are reported to have dampness and molds, both in affluent and non-affluent countries [28]. Moisture and molds are found indoors in up to 27% European, 47% American and New Zealand, and 12% Chinese homes [29-40]. Problems of moisture and molds are higher in structures built of timber, drywalls and filled with insulation made of cellulose, foam, mineral wool, plastic and natural fibers, fiberglass, etc. Additionally, constructing weathertight (e.g., energy efficient) buildings to protect from elements like cold, heat, and wind exacerbate moisture indoors when compared with the structures built of concrete. Inside energy efficient buildings, humidity and moisture can build quickly favoring mold growth. This happens commonly, especially in residential buildings, due to one or combination of the reasons like poor maintenance, less than optimal air exchange from malfunctioning HVAC (heating, ventilation, and air conditioning) system or dirty air filters, blocking air exchange vents from cluttering; all leading to high moisture indoors favoring mold growth.

Growth of molds indoors may result from either a single incident like weather events and flooding, or combined effects of interrelated factors, like overcrowding, poor conditions, socioeconomic situations, globalization, urbanization, and climate change [9,16,41-45]. There is growing evidence of the relationship between climate change and increased fungal growth, sporulation, and allergies [46,47]. This relationship is likely from water and moisture intrusion into buildings from extreme weather events such as cyclones, hailstorms, storm surge, flooding, and temperature extremes, especially in climate-sensitive countries, flood-sensitive locations, and coastal communities [48-50]. A rise in fungal infections in humans [51] is observed with the rise in global temperature, humidity, and population growth (Figure 1).

Consequently, the number of buildings with biotoxin infestations are on the rise along with many of the associated adverse health effects among the inhabitants. The majority of the cases are from socioeconomically deprived neighborhoods like public housing. People living in such neighborhoods are already associated with health-related quality of life (HRQoL) affecting physical health [56] and vulnerable to any perturbation in their living environment which will likely result in much severe response to exposure to biotoxins. Reasons of high mold growth in socioeconomically deprived neighborhoods include negligence in building maintenance, each unit is usually multi-habited and poorly maintained as owners often conduct reactive maintenance waiting until the components are completely deteriorated before repairing or replacing.

Even though maintenance of housing is strongly correlated with the income of the households, a significant number of biotoxins infested buildings are also from affluent neighborhoods, mostly rental properties. Mold infestation in rental buildings from affluent neighborhoods is mostly due to negligence of landlords and/or management as they are responsible for keeping structures fit and habitable by timely and proper repairs. When leaking pipes, windows, and roofs are not fixed in timely fashion or properly, moisture may buildup in areas not directly visible, such as, inside drywalls or insulations resulting in mold growth.

To determine whether the level of molds inside a building is higher than the background (i.e., outside) due to the infestation of mold from water ingress, condensation, or dampness, one needs to identify source(s) and extent of moisture and determine presence, location, and extent of mold growth. This usually begins with a suspicion of mold due to complaints from the inhabitant(s) of an odor, moisture damage, excess humidity, damaged waterpipes/water leakage, flood, presence of molds, and/or health effects described above. At this stage, the complaint is investigated by gathering information of the incident followed by visual inspection of the premises to assess the severity of the problem. After this, additional investigations are planned, if required, to determine the presence or absence of elevated mold levels indoors by additional mold sampling, and if necessary, investigating suspected hidden areas where molds may be growing. This additional investigation is likely performed by qualified environmental specialist/consultant. In addition to determining presence of biotoxins, sometimes, samples are also taken from the area(s) of concern to determine presence and the number of colony-forming units (CFUs) of bacteria and levels of endotoxins produced.

Currently, indoor mold sampling methods are neither guided by any regulation nor well validated or standardized. There is no standardized collection protocol, and a wide variety of collection methods are used to collect molds from air, surfaces, and settled dust. In most cases, methods used by the commercial entities are proprietary and often guarded as “trade secret”; not outlined in the test results/reports, published on laboratories’ websites, peer-reviewed scientific publications, or commercially available. However, some of the common characteristics of sampling strategies depend on the size and nature of the building, available resources, availability of methods, and information needed. All mold sampling strategies are systematic in nature and generally follow recommended decision tree outlined in (Figure 2). It is important to note that such sampling only captures a small window of time, and a large temporospatial variability exists in the levels and number of species of molds inside buildings.

Urine, and sometimes blood, of inhabitant(s) are collected and analyzed to determine mycotoxins. Fecal samples are also often collected from inhabitant(s) and analyzed for commensal and dysbiotic bacteria, normal and dysbiotic molds, parasites, and several nutritional biomarkers. Collected samples are usually sent to mold testing laboratories for analysis, they utilize a variety of methods due to the lack of guidelines, regulations, and/or validated/standardized analytical methods.

Like sampling, there are neither regulatory guidelines nor well validated/standardized protocols/methods to determine and analyze molds, biotoxins, bacteria, parasites, and nutritional biomarkers in the samples collected from mold-infested buildings and their inhabitant(s). Therefore, a wide variety of methods are used by mold testing laboratories. Methods used by the commercial entities are usually proprietary in nature and often guarded as “trade secret” and publicly not available.

Samples collected from inside the buildings (air, surface and settled dust, and/or swab) are sent to mold testing laboratories for the analysis of biotoxins. These samples are analyzed for molds by either culture-based assay, non-culture-based analyses (i.e., microscopy and spore counting), or by molecular analysis (e.g., quantitative polymerase chain reaction [qPCR]); combination of methods are also used when samples are collected using specific procedures [57]. The presence and abundance of spores and hyphae in collected samples is mostly determined by counting them microscopically. The presence of bacteria in samples collected from damp, wet, and/or areas with water, is determined by a culture-based assay to determine the number of CFUs. Before deciding which analytical method to use, specific strengths and weaknesses of each method are evaluated.

Biological samples (i.e., urine, blood and/or feces) are also sent to mold testing laboratories for the analysis of mycotoxins, bacteria, parasites, and nutritional biomarkers. Blood when collected, and urine are analyzed for a list of mycotoxins using liquid chromatography/mass spectrometry (LC-MS/MS), enzyme linked immunosorbent assays (ELISA), or some other undisclosed analytical methods. There are, however, detailed analytical procedures available in the literature that can be standardized for use by the laboratories [58-62]. Fecal samples are analyzed for the presence of normal, commensal, and dysbiotic bacteria and yeast by culture-based assays; abundance of yeast is determined by microscopic counting. The presence and abundance of parasites and nutritional biomarkers in feces are also determined by commonly accepted methods.

Exposure of inhabitant(s) to molds from mold-infested buildings and their potential toxic secondary metabolite(s), i.e., mycotoxins is ascertained by determining molds and mycotoxins in biological samples. Currently there are no U.S. EPA or other federal regulations or standards for airborne molds and biotoxins; therefore, results of the sampling cannot be used to determine compliance of buildings with any regulatory standards. Similarly, there is no health-based standard for exposure to molds and mycotoxins (e.g., reference dose (RfD) or tolerable daily intake (TDI)) indoors; therefore, levels detected in biological samples cannot be used for risk assessment in a straightforward manner without further evaluation utilizing publicly available (mostly peer-reviewed scientific papers) animal and human data. Even though health impacts of exposure to mycotoxins inside residential buildings are not well studied, adverse health effects in occupational settings and in animal studies have been reported [57]. Health impacts from mycotoxin exposure vary depending on the type of mycotoxin, nature of exposure (i.e., acute versus chronic, duration, frequency), its kinetics and dose-response relationship. An interim approach for the risk assessment of mycotoxins from indoor environments can use the risk values set for ingestion (e.g., maximum levels for aflatoxins in various nuts, grains, dried figs, and milk is 0.5-15 μg/kg and for patulin in apple juice is 50 μg/kg [9]) following considerations for the differences between oral and inhalation routes of exposure, absorption, distribution, metabolism, excretion (ADME), and kinetics.

One of the issues that scientists working on indoor molds and exposure to inhabitant(s) continuously encounter is the lack of standardization of methods of sampling (e.g., air, dust, water, breathing zone air, urine, blood), storage, transport, extraction, and analysis. Each laboratory has its own “trade secret” methods which cannot be found either in peer-reviewed scientific papers or on their websites. This is very frustrating as the lack of standardization/harmonization makes scientists difficult to follow data from different laboratories when different and especially “trade secret” methods of analysis are used, raising doubts of the findings.

As mentioned earlier, variety of allergies (e.g., skin rashes, flue and hay fever-type symptoms [sneezing, runny nose, red eyes], inflammation, and respiratory and eye irritation) are known to be caused by biotoxins (i.e., hyphal fragments, spores, and mycotoxins) in people living or working in mold-infested buildings [2,10-20]. Additionally, exposure to mycotoxins can cause fatigue, nausea, immunotoxicity, neurotoxicity (e.g., dizziness, increased anxiety, depression, and cognitive deficits), pulmotoxicity, nephrotoxicity, hepatotoxicity, birth defects, and cancer usually following high levels of exposure for prolong periods of time [1,7-9,21]. Exposure to mycotoxins can also make individuals vulnerable to microbial diseases [21]. These adverse health effects may even be more pronounced in sensitive (e.g., immunocompromised, and/or genetically predisposed to slow elimination, like HLA gene alleles) individuals [1,5,7-9,55].

Even with the understanding of possible adverse health effects from exposure to molds and biotoxins, with the current understanding, numerical standards for acceptable quantities of molds in indoor environments cannot be scientifically justified. Consequently, most professional organizations like IICRC, AIHA, and ACGIH do not support numerical limits for molds in indoor environments.

There have been attempts by researchers to set up acceptable limits of molds in indoor environments based on the genus or species and adverse health effects of their biotoxins, especially mycotoxins. However, variability in mold levels from the use of different methods of measurement, made this approach challenging as results may not be comparable among mold testing laboratories. As an alternative, comparing indoor and outdoor airborne mold spores has been suggested as a more meaningful [63,64]; as in principle, amounts of molds indoors are less and of similar types than those found outdoors [3].

Assessing exposure to biotoxins, and bacteria/endotoxins is even more challenging as approaches currently used (e.g., airborne spores in breathing zone or internalized molds in feces; mycotoxins in urine and/or blood) provide only a snapshot of mold exposure without considering the long-term exposure or exposure from different environments. While results of these tests can confirm exposure, they do not provide information about when or where the exposure has occurred. To establish the source of exposure, indoor mold exposure pathways are necessary showing exposure to molds that produce biotoxins found in the exposed individual(s). Source(s) of exposure is further established by determining the amount of time inhabitant(s) spend at the building in question and at other building(s) (e.g., at work) and whether other building(s) is/are mold infested. To establish timeline and the extent of exposure to mycotoxins, kinetic information is used along with any preexisting condition(s) that may influence ADME of mycotoxins in question. Additionally, other factors are also incorporated when calculating timeline of exposure since certain genetic condition(s) like genetic predisposition at the HLA/DR gene is known to slow elimination of mycotoxins. For example, elimination of ochratoxin A and mycophenolic acid has been reported to be ~10- and ~213-fold slower, respectively, in individuals with the HLA/DR gene than those without it [55].

One of the biggest challenges in mold sampling and exposure assessment is interpretation of the results due to a lack of standardization and therefore, results can be highly variable, ambiguous, and difficult to interpret that may lead to inconsistent conclusions. These issues can easily be addressed by standardizing and validating methods, instrumentations, sampling techniques, collecting replicates (e.g., n ≥ 3), analytical procedures, and reporting of the results. These requirements should be mandated to all mold testing laboratories through regulations or by binding recommendations from a taskforce as many points of controversy arise from a lack of consistency in collection and analysis of molds, biotoxins, bacteria, and endotoxins.

We now have evidence that climate change is raising both earth temperature and humidity resulting in increased mold growth, sporulation, and fungal diseases (Figure 1). Consequently, rise in mold-infested buildings is expected, which in turn will increase the number of patients with severe mold- and biotoxin-related diseases needing better health management. Henceforth, mandating mold testing laboratories to standardize and validate methods for sampling, analysis, and reporting is required. This will help in consistency of results across the testing laboratories avoiding current controversies around results from different laboratories. Additionally, more animal studies are needed to develop inhalation exposure limits (e.g., RfC, TDI, slope factor) for molds and biotoxins, especially mycotoxins, responsible for, at least those causing severe adverse health effects. As data are being generated, an interim approach of relative abundance of molds indoors versus outdoors, NOAEL of certain mold spores and mycotoxins in animals [65], allowable levels of certain mycotoxins in food and/or drinks for ingestion [9], and other available information can be used after correction for the differences among the routes of exposure, ADME/kinetics.