Molds are microscopic fungi, ubiquitous in nature that grow on any moist and/or damp surface containing organic matters. In the presence of considerable moisture (≥ 60% relative humidity) and appropriate temperature (25-30°C, some molds are capable to grow at refrigerator temperatures as well), almost any organic substance will support mold growth. Molds, therefore, can grow on materials like raw and cooked food, wood, paper, carpet, drywall, insulation, plants, soil, wallpaper, fabric, and upholstery (https://www.fsis.usda.gov/food-safety/safe-food-handling-and-preparation/food-safety-basics/molds-food-are-they-dangerous). Body of molds is called thallus, which can range in size and cellularity (single cell or multicellular). Cells within thallus are coated with a wall made of a strong polysaccharide called chitin. Long, branched filaments of molds are called hyphae that form the tangled web, called mycelium, giving mold the characteristic fussy or cottony appearance. Hyphae are made of an outer cell wall and an inner lumen. Molds produce microscopic reproductive bodies (spores) ranging 2-10 μm in size; several types of sexual and asexual spores are produced by molds for their propagation. Spores formed by hyphal fragmentation are called arthrospores, spores surrounded by a thick wall before hyphal fragmentation are called chlamydospores, and spores developed within a sac (sporangium) at the tip of hyphae are called sporangiospores (https://www.uwyo.edu/virtual_edge/lab13/fungi.htm). Spores float in both outdoor and indoor air and start growing into molds when land on moist surfaces; none of the molds grow in the absence of moisture.

Hydrolytic enzymes are secreted from the tips of the hyphae that break complex organic matters into simpler substances for absorption by hyphae, in this way, molds play a major role in decomposing organic materials and recycling nutrients in the ecosystems. Usually during late in growth, many filamentous molds produce complex secondary metabolites, notable among them are antibiotics and mycotoxins. Secondary metabolites are not directly involved in molds’ normal survival and/or growth but often linked to their development by stimulating sporulation, improving survivability of spores, and enhancing the fitness of fungal community through ecological interactions with other organisms [1,2].

Outdoors, molds breakdown dead organic matter like fallen leaves and dead trees. Indoors, under favorable conditions, molds grow and damage common materials like wood, paper, and organic fibers. Indoors, molds remain viable long after the source of moisture is removed resulting in molds and their components (i.e., hyphal fragments, spores, and mycotoxins), referred herein as biotoxins, with the potential to cause adverse health effects, remain in indoor air for an extended period of time [3]. Molds grow outdoors in humid environments and 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].

Levels of biotoxins in outdoor air do not normally rise above the health concern, even in high humidity, due to wind mediated dispersal, heat and sun light mediated degradation, and relatively brief durations of exposures of most of the individuals. Indoors, the presence of mold spores is generally not problematic [4], as they are always present, unless they land on a wet or damp surface and begin growing. Levels of biotoxins in indoor air are mostly lower or equal to that of the outdoor air [5]; however, can easily reach unsafe levels from the growth of mold spores under high humidity, inadequate ventilation, and poor lighting, with possibility of causing adverse health effects [6]. This conclusion is supported by the fact that many people, especially susceptible individuals (e.g., immunocompromised and/or genetically predisposed) living or working in mold-infested buildings commonly complain of a variety of adverse health effects. These adverse health effects range from allergies (e.g., hay fever-type symptoms [sneezing, runny nose, red eyes], inflammation, and skin rash) to adverse effects, less commonly, to many organs and systems [1,2,7-24]. Exposure to mycotoxins has been reported to 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 [1,18,21,23,25]. Furthermore, exposure to mycotoxins can make individuals vulnerable to microbial diseases [25].

Global prevalence of buildings with dampness and molds is ~47%; as high as 27, 47, 47, and 12% of homes in Europe, America, New Zealand, and China, respectively, have been reported to have moisture and mold issues [26-40]. Mold-affected buildings are persistent and common global problems with evidence of an upward trend due to factors like overcrowding from increasing world population, poor living conditions, socioeconomic conditions, globalization, urbanization, energy efficient buildings, and climate change [15,23,41-51]. Consequently, a rise in fungal infections among humans [52] is seen with climate change (i.e., rise in global temperature, humidity, and population growth). Due to these factors, a further increase in mold-infested buildings and rise in biotoxin-related health effects is anticipated.

To assess mold infestation inside a building, levels of several mold spores and hyphal fragments are determined in air and dust and compared with the background (i.e., outside). Similarly, to determine if exposure to the occupants has occurred and crossed the threshold of adverse health effects, several biological samples (e.g., urine, feces, and blood [rarely]) are collected and analyzed for molds, mycotoxins, and bacteria [24]. Additionally, to cover other microorganisms that may be responsible for adverse health effects, samples are also collected and analyzed for the presence of pathogenic bacteria and endotoxins. This paper reviews specific molds, mycotoxins, and bacteria that the collected samples are routinely analyzed for and/or detected in the collected samples during the inspection of buildings for mold infestation and exposure of occupants.

Approximately 5 g of dust samples are collected from inside the buildings that are representative of majority of the indoor areas of the building in question, for example, dust by vacuuming, from the ceiling fans, from vacuum cleaner, from heating, ventilation, and air conditioning (HVAC), air inlets/air outlets/filters, or by gathering settled dust from other objects. Samples are preserved and placed into sterile containers and shipped to a microbiology laboratory for the analysis using mold-specific genes with quantitative real-time polymerase chain reaction (qPCR). Samples are routinely analyzed for 36 mold species, 26 of these species are known to thrive in water damaged buildings, designated as “Group 1” mold species, and 10 species are found inside all buildings, with or without water damage, designated as “Group 2” mold species (Table 1). Based on the abundance, buildings are scored for environmental relative moldiness index (ERMI) using the difference between “Group 1” and Group 2” log-transformed values [53]. The list contained 11 species of Aspergillus, 7 species of Penicillium, 4 species of Cladosporium, 2 species of Scopulariopsis and Rhizopus, and 1 species of Acremonium, Alternaria, Aureobasidium, Chaetomium, Epicoccum, Eurotium, Paecilomyces, Stachybotrys, Trichoderma, and Wallemia. Abundance of two species of nonpathogenic molds (Aspergillus flavus, Penicillium purpurogenum) is also determined. In addition to determining ERMI from the generated data, these authors opine that each mold should also be assessed for its toxicity and potential health hazards by knowing number of spores and biotoxins they produce, and their ability to cause short- and long-term adverse health effects mentioned in the preceding section (see “Introduction” above).

Other samples from inside of buildings are also collected to determine mold spores, hyphal and insect fragments, pollen, and yeast. For this purpose, air and dust samples are collected using sticky slides, tape-lifts, and swabs [24]. The collected samples are analyzed for fungal spores, fungal structures, hyphal fragments, pollen, insect fragments, skin fragments, and fibrous particulate matters/fibers by optical microscopy. (Table 2) shows routinely detected mold spores inside the buildings; the list presents molds that are detected inside of even one out of over 800 buildings sampled by the Mold Case Consulting in the last four years. Molds with no or unknown pathogenicity are not described in this paper.

Acremonium
Acremonium grows moderately rapidly with compact and moist colonies containing loose cottony white, gray, or rose hyphae when fully grown. Acremonium grows well indoors under very wet conditions. Acremonium causes nausea, vomiting, and diarrhea in occupants of infested buildings (https://hsrm.umn.edu/node/901). Acremonium infection occurs mostly to immunocompromised individuals. Acremonium is associated with endocarditis, pneumonia, meningitis, cerebritis, peritonitis, arthritis, osteomyelitis, sepsis, and infection of gastrointestinal (GI) tract, eyes, skin, nails, and mouth; because of disseminated infection fungus reaches bloodstream and spread throughout the body [54-58]. It triggers type I allergies (e.g., hay fever, asthma, rhinitis, eye infections, dermatitis, and onychomycosis) and type III hypersensitivity; pneumonitis [57-59]. Type I allergic reactions are mediated by IgE antibodies whereas, type III reactions are mediated by IgM and IgG antibodies.

Several species of Acremonium can produce colonies in human lungs producing pulmonary fungal ball (aspergilloma, a mass of fungus that grows in a lung cavity) sometimes even in immunocompetent individuals; Acremonium can grow colonies in the GI tract and produce bezoars [54]. Acremonium is also known to produce trichothecene mycotoxins which are potent inhibitors of DNA, RNA, and protein syntheses and are known carcinogens [60,61]. Trichothecenes are often linked to both acute and chronic toxicoses including alimentary canal toxic aleukia (nausea, vomiting, diarrhea, leukopenia, hemorrhaging, skin inflammation, and sometimes death) and Kashin-Beck disease [62]. For details about trichothecenes, readers are directed to “Description of the Mycotoxins…” section below.

Alternaria

Alternaria grows rapidly, colonies are grayish white in color at the beginning and darken later, becoming greenish black or olive brown with a light border. Alternaria often grows on carpets, textiles, and horizontal surfaces such as window frames [63]. Alternaria can grow at temperatures ranging from 1–35°C and pH between 2.5–10, with optimum growth at 20–25°C. Spores of Alternaria are large and readily found in air samples, they are capable of depositing in the nose, mouth, and upper respiratory tract [64,65]. They usually attack immunocompromised individuals undergoing prolong steroid treatment or those with very weak immune system. Species of Alternaria can cause hypersensitive pneumonia, bronchial asthma, and allergic sinusitis and rhinitis [66]. They can cause sores in the nose, ulceration of skin, and nail infections. Acute symptoms include edema and bronchospasms, chronic cases may develop pulmonary emphysema [67]. More than 70 mycotoxins have been reported to be produced by Alternaria [68]. Mycotoxins produced by Alternaria are suspected to be mutagenic, genotoxic, teratogenic, fetotoxic, estrogenic, cytotoxic, and carcinogenic [69,70].

Arthrinium

Arthrinium colonies grow rapidly into grape-like masses that appear woolly or cottony white with brown spots. Arthrinium is widespread saprobe commonly found feeding/decomposing dead and decaying wood, leaves, litter, and other organic matter. It is often detected in air near grassy places, especially in fall when plants start to die due to cold weather. At least one species of Arthrinium is allergenic. Arthrinium produces many secondary metabolites (e.g., xanthones, peptides, diterpenes, sesquiterpenes, and quinines). Mycotoxin, 3-nitropropionic acid (3-NPA), a highly toxic compound, produced by Arthrinium, has been reported to cause noninflammatory encephalopathy, preceded by vomiting, nausea, diarrhea, that may lead to coma and death in 10% of the exposed individuals and in some individuals may result in lifelong disabilities [71-73].

Arthrospores

Arthrospores are formed by hyphal fragmentation and are capable of producing allergies in certain individual, especially those prone to getting allergies (e.g., immunocompromised individuals).

Ascospores

Ascospores are sexual spores of Ascomycetes which grow well under a variety of conditions and commonly found growing indoors on damp materials. Ascospores can be single- or multi-cellular and extremely variable in size and shape. Many Ascospores are allergenic, commonly cause coughing, wheezing, nasal congestion, and eye irritation; they are considered potential opportunistic pathogens and producers of toxins. Ascospores have not been studied extensively and therefore only a few are known to cause diseases; however, Ascospores can cause severe irritation and have high allergenic potential - can severely damage organs following long-term exposure [19].

Aspergillus

Aspergillus colonies are generally fast growing, may be woolly or cottony in texture, and in the shades of green, brown, or black. It is found on organic materials like soil, plant debris, compost, stored grain, wood, paper, house dust, ventilation systems, water-damaged building materials (e.g., clothing, carpets). Out of about 300, 16 species of Aspergillus have been reported to cause human diseases. Aspergillus is a common cause of extrinsic asthma, symptoms include edema and bronchospasms, chronic cases may result in pulmonary emphysema. Aspergillosis is the second most common fungal infection requiring hospitalization in the United States. A. fumigatus releases copious amounts of spores which become airborne and can reach alveoli following inhalation. Many Aspergillus species produce mycotoxins (e.g., aflatoxins, ochratoxin A [OTA], sterigmatocystin) capable of causing developmental toxicity, stunted growth, immune suppression, DNA damage, hepatotoxicity, nephrotoxicity; aflatoxins are known hepatocarcinogen. Aflatoxins also cause occasional outbreaks of acute aflatoxicosis that may result in death shortly after exposure [74-81; https://www.adelaide.edu.au/mycology/]. Aflatoxins are known to cause mutation in p53 gene which is also known as the guardian of genome [82]. For details about aflatoxin, OTA, and sterigmatocystin, readers are directed to “Description of the Mycotoxins…” section below.

Aureobasidium

Aureobasidium grows moderately rapidly and is covered with slimy masses of spores. Aureobasidium is yeast like, start with cream to pink in color and become dark brown and velvety with age. It is detected in soil, freshwater, marine estuary sediments, plants, and woods. Aureobasidium spores can be transferred by water droplets when wet and by wind when dry. This mold is, therefore, widespread inside buildings where moisture accumulates, particularly in bathrooms and kitchens, on shower curtains, tile grout, windowsills, textiles, and liquid waste materials. Aureobasidium causes type I allergies and Type III hypersensitivity pneumonitis; it has also been isolated, rarely though, from skin lesions, keratitis (inflammation of the cornea), spleen abscess, and blood of immunocompromised individuals [83; https://library.bustmold.com/aureobasidium/].

Basidiospores

Basidiospores are sexual spores produced by Basidiomycetes, ubiquitous in gardens, forests, and woodlands. Basidiospores are produced on club shaped cells called basidia, each containing four small outgrowths, sterigmata, each producing one spore. Basidiospores are often actively and forcefully released into the surrounding air. They cause type I allergies, type III hypersensitive pneumonitis, and are rare opportunistic pathogens causing eye, skin, and nail infections [84]. Cryptococcus belongs to Basidiomycete, some of which are known to cause severe forms of meningitis called cryptococcosis, especially in immunocompromised individuals, with fatality rates of 9% in high-income regions to over 70% in sub-Saharan Africa [85]. Basidiospores produce many mycotoxins like amanitins, monomethyl-hydrazine, muscarine, ibotenic acid, and psilocybin.

Bipolaris

Bipolaris grows rapidly, even in semi-dry environments, to initially white to grayish brown velvety to woolly turning into olive green to black colonies with raised grayish peripheries as they mature. Bipolaris is a ubiquitous dry spore producing mold; spores are disseminated by wind. It is often found on decaying plants, grasses, and in soil. Bipolaris causes infections of cornea, skin, heart, bone, lungs, and central nervous system causing brain lesions often in immunocompromised but also in young and healthy individuals (https://library.bustmold.com/bipolaris/). In some individuals with severe allergies, large spores of Bipolaris can lodge and attach to the mucus in the sinuses or upper respiratory tract and grow, producing perpetual allergy that can progressively and permanently damage sinuses. Bipolaris, along with Aspergillus, produce mycotoxin sterigmatocystin that is known to cause liver and kidney damage in animals following oral dosing [76; https://www.adelaide.edu.au/mycology/]. For details about sterigmatocystin, readers are directed to “Description of the Mycotoxins…” section below. At least two other mycotoxins (prehelminthosporal and sorokinianin) are also produced by Bipolaris, whose effects on humans are not known [86].

Botrytis

Botrytis initially appears as a white growth on plants that turns to gray quickly. Botrytis produces "dusty" smoky-gray spores that spread by wind or water. Spores of Botrytis can remain dormant on plant surfaces for a very long period of time, sometimes throughout the plants’ life. Botrytis has a low prevalence, both outdoors and indoors. Even with low prevalence, Botrytis causes sensitization with a high prevalence. Species of Botrytis (e.g., B. cinerea) causes allergic rhinitis in children and adults, asthma and hypersensitivity in children, and pneumonitis in individuals working in horticulture/cultivation and harvesting of grapes [87].

Cephaliophora

Cephaliophora has been isolated from soil and animal dung, and found to grow on teak, cotton, poplar, and other plants [88-91]. It is also called “cup fungi” due to macro-structure it forms which is like the shape of a cup [92]. Cephaliophora grows vegetative hyphae which is colorless with copious branching and produces pale to brownish spores [93]. Cephaliophora has been reported to cause mycotic keratitis in humans [94].

Cercospora

Cercospora is a plant parasite, it causes leaf spot mostly in higher plants. Distinctive spores of Cercospora spread through wind and is commonly found outdoors in agricultural areas, especially during harvest; it seldom grows indoors. Cercospora species (e.g., C. apii) has been isolated from patients with cutaneous and subcutaneous lesions involving face, ears and nasal mucosa, failure of therapeutic interventions can lead the infection for many years [95]. Cercospora does not produce any known potential toxins.

Chaetomium

Chaetomium is readily found growing on the damp or water damaged drywalls. Chaetomium produces cottony and white to grey to olive color colonies and grows rapidly. Chaetomium is the third most common indoor mold [96,97]. Chaetomium is allergenic and causes skin and nail infections [98,99]. C. globosum is known to cause pulmonary mycosis, severe brain abscesses, and fatal cerebral infections in immunocompromised individuals [100]. C. globosum is also one of the primary Chaetomium found in the homes of asthmatics individuals [101] with reported invasive Chaetomium infections in the lungs [102]. Chaetomium is known to inhibit cell division and is shown to be fatal to rodents [103]. Chaetomium produces several mycotoxins (e.g., sterigmatocystin, O-methylsterigmatocystin, chaetochromin, chaetoglobsins A and C, chaetocin, chetomin, cochliodinols, and mollicellin G), many are developmental toxicants and carcinogens [104]. For details about sterigmatocystin and chaetoglobsins A, readers are directed to “Description of the Mycotoxins…” section below. These mycotoxins are extremely toxic and potentially fatal to cattle when present as contaminants in feed; Chaetomium extract caused spleen, liver, and kidney toxicity in mice [105-107]. One study isolated 25 secondary metabolites from building materials that are produced by the strains of C. globosum alone including major (chaetoglobosin A, C and F, chaetomugilin D, and chaetoviridin A) and minor (chaetomugilin I, chaetoviridin E, azaphilones, and other chaetoglobosins) mycotoxins [108]. Strains of C. globosum and other species of Chaetomium have been reported to produce many other secondary metabolites (e.g., chetomin, chaetocin, cochliodinol, epipolythiodioxopiperazines, xanthones, anthraquinones, chromones, depsidones, terpenoids, and steroids) [97,108,109].

Chrysonilia/Neurospora

Chrysonilia/Neurospora grows fast to form pink colonies; Neurospora is holomorph of Chrysonilia (http://website.nbm-mnb.ca/mycologywebpages/Moulds/Chrysonilia.html). Both Chrysonilia and Neurospora are found on the soil surface after grassfire in forest. A single case of endophthalmitis (inflammation of the intraocular cavities) with Chrysonilia is reported [110].

Cladosporium

Cladosporium is relatively slow growing powdery or velvety olive-green to olive-brown colonies, grows well between 0-35°C. Some species of Cladosporium infect plants or other mold pathogens; some live on plants without causing infections, and some can cause mycosis of lungs, eyes, skin, and nails in mostly immunocompromised individuals [111]. Cladosporium is the most frequently detected molds found in ~70% of the houses tested [112,113]. Indoors, it is often detected in dirty refrigerators, on moist window frames, paint, paper, fabrics stored under humid conditions, and ventilation systems. The optimal temperature for Cladosporium growth is 18-28°C; however, some species can live below freezing temperatures with the ability to reproduce even on frozen meat [114]. Cladosporium spores are easily airborne and transported over long distances. Cladosporium is a well-known allergen, commonly causes hay fever and asthma. In immunocompromised individuals, Cladosporium may cause corneal infections and mycetoma involving cutaneous and subcutaneous tissue, fascia, bone abscesses, granulomata, and draining sinuses [115].

Coelomycetes

Coelomycetes is a term used for asexual forms of Ascomycota and Basidiomycota (previously known as anamorphs) that produce conidia (mitospores) within fruiting bodies called conidiomata [116]. Coelomycetes grow on plants or other fungi and are ubiquitous in soil, salt and freshwaters, and in sewage [117]. There are reports of increasing cases of cutaneous/subcutaneous and invasive disease, keratitis, and deep tissue infection, especially in immunocompromised individuals [118,119].

Corynespora

Corynespora is a plant pathogen that causes leaf-spotting and rarely causes human infections [120-122]. However, Corynespora has been reported to cause serious subcutaneous infection in humans [122-125].

Curvularia

Curvularia grows rapidly to form white to pinkish gray woolly colonies which turn to olive brown or black upon maturation. Spores of Curvularia are relatively large and often remain in the nose or sinuses of humans following inhalation. Curvularia is found in soil, plant debris, stored grains as well as often indoors – mostly on wooden structures. Curvularia is a plant pathogen [126]. Curvularia is an allergenic and opportunistic pathogen, especially among immunocompromised individuals. Curvularia occasionally causes onychomycosis, mycetoma, keratitis, sinusitis, mycetoma, pneumonia, endocarditis, peritonitis, and cerebral abscess, mostly in immunocompromised individuals but also in healthy individuals [127-130].

Epicoccum

Epicoccum grows rapidly at 37°C (with capability to grow from –3°C to 45°C) producing woolly, cottony, or felty yellow, orange, red or pink colonies that turn greenish brown to black with aging. It is occasionally present in dust collected from inside the buildings and frequently found in indoor air. Epicoccum spores survive for extended periods of time in dry weather and grow under optimal conditions. Epicoccum causes skin infection, respiratory tract allergies like rhinitis, sinusitis, and asthma; systemic diseases from Epicoccum are rare [131]. Elevated spores of Epicoccum are known to induce or worsen asthma attacks in children (https://www.inspq.qc.ca/en/moulds/fact-sheets/epicoccum-purpurascens); they cause severe allergies like hypersensitivity, pneumonitis, and allergic fungal sinusitis in 5-7% of global population [131]. Epicoccum produces a wide array of secondary metabolites [132].

Eurotium

Eurotium is the sexual state of Aspergillus containing characteristic whitish to bright yellow spherical fruiting bodies. Aspergillus, when growing for a long period of time on nutrition rich substrate that is conducive for the conversion to sexual phase, produces related Eurotium. It is one of the most frequently found mold constituents found in household dust [131]. It generally grows on substrates low in moisture like stored grains, textiles, leather, and materials coated with resins and lacquers, like furniture. Allergenicity and toxicity of Eurotium closely resemble Aspergillus; Eurotium has not been studied separately from its asexual stage, i.e., Aspergillus. Still species of Eurotium are known to cause opportunistic infections of ears, eyes, skin, mycetoma, asthma, bronchopulmonary mycosis, cerebral abscess (in one healthy individual) [133,134].

Fusarium

Fusarium grows rapidly to form white, tan, cream, yellow, salmon, cinnamon, pink, red, violet, or purple colonies. Fusarium is found in soil, plants, and grains. Its growth requires very wet conditions and normally does not appear in air samples. Fusarium produces many bioactive and volatile metabolites; some produce strong musty smell in water-damaged building materials. Fusarium is allergenic and often causes eye, skin, and nail infections; it readily infects burn victims.

Species of Fusarium produce three of the most important classes of mycotoxins: trichothecenes (nivalenol, deoxynivalenol, T-2 toxin, fusarenon-X, and related compounds), fumonisins (B1, B2, B3 and B4 with B1 and B2 are classified as possible human carcinogens), and zearalenones and four less studied but emerging mycotoxins: fusaproliferin, beauvericin, enniatins, and moniliformin [135-137]. Other potentially important secondary metabolites not exclusively produced by Fusarium include: acuminatum, butenolide, culmorin, cyclonerodiol, equisetin, fusaprolieferins, fusarochromanones, fusaric acids, fusarins, napthoquinones, sambutoxin, and wortmannin [138].

Fumonisins are associated with esophageal cancer in humans from ingestion of Fusarium contaminated corn and classified as Group 2B carcinogens (possibly carcinogenic to humans) [135]. Fumonisins are poorly absorbed with oral bioavailability of <5% and the absorbed fraction is rapidly distributed and eliminated in bile following glucuronidation; elimination half-life (t_½) of fumonisins has been reported 0.3 to 3.2 h [139-140]. Fumonisins cause liver and kidney toxicity and cancer in rodents; they are also neurotoxic. More recently, fumonisins have also been implicated in birth defects (neural tube) in humans and animals. Fumonisins produce these effects by inhibiting activity of ceramide synthase, which converts sphinganine to sphingosine resulting in increased tissue concentration of sphinganine, thus affecting cellular growth, differentiation, and cell communication resulting in toxicity and carcinogenicity [75,141]. To ensure safety, a maximum tolerable daily intake (TDI) for all fumonisins was set at 2 μg/kg [142] and for FB1 and FB2 at 1 μg/kg [143]. For details about trichothecenes and zearalenones, readers are directed to “Description of the Mycotoxins…” section below.

Hyphal fragments

Hyphal fragments or mycelia are fruiting structures of molds. They normally settle quickly and therefore are found in indoor dust; they are also found in outdoor air. Their presence in indoor air, especially in large quantities, is suggestive of active mold growth. Hyphal fragments may cause allergic reactions in some individuals.

Insect fragment

Insect fragment, presence of large quantities of carpet beetle larvae hair, fly hair, and insect scale are indicative of enough moisture to support insect and mold growth even if not visible. Insect scales and hair can be highly allergenic, causing itching, redness, and irritation; they can also be contact and/or respiratory sensitizer resulting in severe allergic reactions involving immune system, such as swelling and difficulty breathing.

Mucor

Mucor is a member of the class of fungi known as Zygomycete (see below for details about Zygomycete) which grows rapidly to dark gray or light olive gray colonies. Mucor is one of the most quickly invading and spoiling molds for many kinds of stored food products. Mucor is often found in dust of inside buildings, stored grains, hay, and horse manure. High concentrations of Mucor spores are frequently detected in indoor air samples, accumulated dust in HVAC systems, and poorly maintained carpeting. High exposure to Mucor spores (through inhalation, ingestion, surgical and accidental wounds, ears, nose, nails, and eyes) can cause extrinsic allergic alveolitis, a type III allergic response to exposure associated with elevated temperature, flu-like symptoms, general malaise, difficulty breathing, and asthma. In immunocompromised individuals, Mucor can cause severe infections characterized by vascular invasion, thrombosis, infarction, and tissue necrosis (https://library.bustmold.com/mucor/). Mucor can cause mucormycosis (infection mostly of respiratory and central nervous system but may also be of skin, GI tract). Mucormycosis pneumonia is the most dangerous Mucor-related infections usually associated with pulmonary or nose, paranasal sinuses, and brain, with prolonged neutropenia, elevated serum iron in almost always in immunocompromised individual but have also been reported in immunocompetent individuals. The mortality rate of mucormycosis pneumonia is 60% or greater, much higher than many other molds [144]. Mucor infection in healthy individuals is rare [145-147].

Nigrospora

Nigrospora grows rapidly to white woolly colonies turning to gray and eventually black upon maturation. Nigrospora is ubiquitous, especially in warm climates. It grows on decaying plant material and soil; its spores are dispersed by an active discharge mechanism without the aid of wind or rain. It normally does not grow indoors. Nigrospora is allergenic with rare cases of human infection causing allergies of the respiratory tract as well as of skin, nail, and eye, especially in immunocompromised individuals [148-150].

Oidiodendron

Oidiodendron is detected in living and decomposing plants, animals, soil, wood, decomposing human hair, and indoor air and dust samples. At least one species of Oidiodendron has been reported to cause atopic eczema in one individual [151].

Paecilomyces

Paecilomyces is regularly found in soil and dust and less often in air; several species cause food spoilage detected in edible oils, peanuts, margarine, cereals, bread, and meat products. Paecilomyces can also grow indoors on water damaged building materials and is commonly detected in indoor samples. Paecilomyces can grow in the presence of moisture at temperatures ranging from 1°C to 60°C and even can withstand 80-100°C for a brief period of time, up to 15 min [131,152]. Paecilomyces can also grow on creams, lotions, cosmetics, plastics, vinyl and diagnostic materials even when containing antifungal agents [131]. Paecilomyces is an opportunistic pathogen [153] responsible for pulmonary, cutaneous infections, endocarditis, peritonitis, and sinusitis; some species of Paecilomyces cause pneumonia. Paecilomyces causes infections in organ transplant, HIV and immunosuppressed patients and detected in respiratory secretions, tissue biopsies, blood, and isolated from abscesses [154].

Particulate matters/fibers

Depending on the size and shape, fibers/particulate matters (airborne particles) can reach and deposit in various regions of the respiratory tract [155]. Larger particles are deposited in the extra thoracic region of the respiratory tract, coarse particles (PM₁₀) in the tracheobronchial region, and the smallest particles (PM_2.5) in the pulmonary region. The smallest particles can reach the alveolar region and exchange with blood. Toxicity of the airborne particles depends on their chemical composition and their capacity to produce reactive oxygen species, they may be mutagenic, carcinogenic, or acutely inflammatory, depending on their organic, elemental, and water-solubility characteristics [155].

Papulaspora

Species of Papulaspora have been reported to cause ocular or systemic infection resulting from exposure [156].

Penicillium

Penicillium grows with a velvety, wooly, or cottony texture, colonies grow rapidly to initially white and with time turn to blue green, gray green, olive green, yellow, or pinkish in color. It is normally found in soil, food, cellulose, paint, grains, and compost. Inside buildings, Penicillium is found in wallpaper, carpet, and inside the duct insulation, it is often found inside water damaged buildings [107,157]. Genus Penicillium is comprised of over 300 species and is one of the most frequently detected molds in the world. Spores of Penicillium are easily airborne and inhaled by inhabitants. Many species of Penicillium are known to cause human diseases including skin allergy, keratitis, penicilliosis, mycosis, otomycosis, allergic alveolitis, hay fever, asthma, and hypersensitive pneumonitis in susceptible individuals [158,159]. Long-term exposure can lead to chronic sinusitis. Penicillium exposure can worsen symptoms and lead to health complications in people with immune disorders and/or genetically predisposed to mold toxicity. Penicillium species produce more than 30 different mycotoxins (e.g., citrinin, cyclopiazonic acid, OTA, patulin, penicillic acid, penitrem A, roquefortine, frequentin, palitantin, mycophenolic acid, viomellein, gliotoxin, citreoviridin, and rubratoxin B) [160-162]. For details about citrinin, OTA, mycophenolic acid, and gliotoxin, readers are directed to “Description of the Mycotoxins…” section below.

Pestalotia/Pestalotiopsis

Pestalotia/Pestalotiopsis is a plant pathogen found on plant leaves, stems, twigs, and barks as lesions or gray spots. Some species of Pestalotia/Pestalotiopsis can grow on synthetic polymer found inside many buildings. Pestalotia/Pestalotiopsis has been reported to cause keratitis in an older individual [163]. No additional information about allergenicity, toxicity or adverse health effects of Pestalotia/ Pestalotiopsis is available.

Rhizopus

Rhizopus is often found on bread, fruits, in soil and dust that looks like a dense layer of cotton. Rhizopus is a member of the class of fungi known as Zygomycete (see below for details about Zygomycete). Rhizopus is a fast-growing deep grey to black mold that can grow in a wide range of settings and in harsh conditions [164]. Rhizopus often causes spoilage of food and is pathogenic to humans. It is allergenic, especially to sensitive individuals, causes coughing, wheezing, runny nose; in immunocompromised individuals, it can cause mucormycosis. Rhizopus infection in healthy individuals is rare [145-147].

Periconia

Periconia forms pale to dark brown spores; Periconia is difficult to differentiate from Smuts, Myxomycetes and other molds that produce brown spores [165,166]. It is commonly found outdoors and less indoors; only 1-5% (~0.6%/m³) of total spores usually belong to Periconia [168,169]. Periconia is allergenic and known to cause keratitis [169]. Periconia produces several bioactive compounds (e.g., periconin A, B, C, D, coumarin, benzaldehyde, piperine, taxol) with antibacterial, antifungal, anticancer activities [170-174].

Phaeotrichoconis

Phaeotrichoconis is a plant pathogen found as endophytic fungi on healthy leaves [175]. Species of Phaeotrichoconis are found in Africa, Asia, Australia, North America, and South America [175]. Phaeotrichoconis crotalariae has been reported to cause mycotic keratitis in animals [176].

Pithomyces

Pithomyces produces cottony suede-like fast growing white to cream or olive colonies that turn tan to brown with age. It grows optimally at ~24°C and above 80% relative humidity, mainly on decaying plants, grasses, and soils [177]. Pithomyces may grow on paper, but they are not prolific indoors. Pithomyces is pathogenic, reported to cause sinusitis, peritonitis, onchomycosis, and asthma, especially in immunocompromised individuals [178-181]. One study found Phitomyces more frequently in higher concentrations in homes with asthmatic children than in homes without asthmatic children [19]. Pithomyces produces mycotoxin sporidesmin A which causes liver damage and facial eczema in animals [177].

Pollen

Pollen is plant particles, not mold spores. Pollen is not restricted outdoors, and at least some typically find their way indoors and frequently detected in indoor air samples. Presence of pollens in indoor air when they are not expected in outdoor air is likely indicative of dust and pollen reservoir inside the building such as in a dirty HVAC system. Levels of pollen vary widely, even indoors, with season, wind, weather, temperature, rainfall. Pollen is the most common cause of allergies globally. Pollen often causes runny nose, itchy and/or watery eyes, sore throat, cough, and decreased sense of taste and/or smell, they may also trigger respiratory illness and/or some forms of asthma.

Rust

Rust grows on grass, flowers, trees, and living plant materials, it doesn’t grow indoors without the presence of host plants. It produces red, rusty to orangish spores. Rust causes type I allergic reactions in humans.

Scopulariopsis

Scopulariopsis grows moderately rapidly at 25°C to white velvety to powdery textured colonies which become light brown to tan as colonies mature. In indoors, it is found on drywalls, cellulose board, wallpaper, wood, mattress dust, carpets, shoes, and wood pulp [182-184]. It is commonly found indoors. Certain species of Scopulariopsis may cause nail infection, pulmonary mycoses, infection of soft tissues, bones and rarely pneumonia, keratomycosis, otitis, and septicemia, especially in immunocompromised individuals [185,186]. Many species of Scopulariopsis can release garlicky smelling arsine gas from the growing substrate that contains arsenic [187].

Smuts

Smuts form black powdery spore masses resembling soot, and therefore, called smuts. They are indistinguishable from Myxomycetes and Periconia under microscope at 600x magnification. Smuts are plant pathogens requiring living host (e.g., corn, grass, weeds, flowering plants, and other fungi) to complete their life cycle and distributed by wind [188,189]. They are therefore usually not found growing indoors. Smuts are type I allergens in immunocompromised individuals or those who work or live near farms infested with smuts causing asthma, bronchitis, hay fever, hypersensitivity pneumonitis [190-193].

Sporidesmium

Sporidesmium infects dead plants in pasture. Sporidesmium produces sporidesmins (a potent hepatotoxin) that causes facial eczema in sheep and cattle [194].

Stachybotrys

Stachybotrys rapidly grow to produce cottony white colonies that turn to dark green and black upon maturation. Stachybotrys is not commonly found outdoors. Indoors, it flourishes on water damaged cellulose rich materials such as drywalls, ceiling tiles, cellulose-containing insulation, and wallpaper and is commonly associated with a multitude of illnesses. Stachybotrys produces many mycotoxins like trichothecenes (e.g., Satratoxin F, G, H, Isosatratoxin F, Roridin A, E, H, L-2, Verrucarin A, J). Stachybotrys exposure has been reported to cause debilitating respiratory symptoms, including, pathological changes in the lungs at even low concentrations [195-197]. Allergic sensitization, inflammation, and cytotoxicity of the respiratory tracts of animals have been reported from the exposure to biotoxins of S. chartarum [197-201]. S. chartarum has been linked with infant pulmonary hemosiderosis at six locations (Cleveland, Texas, Kansas City, Belgium, and Quebec) [9,198,202,203]. For details about trichothecenes, readers are directed to “Description of the Mycotoxins…” section below.

Stemphylium

Stemphylium grows rapidly forming velvety to cottony gray, brown, or brownish-black colonies. Stemphylium grows in soil, wood, and decaying vegetation; some species grow on leaves. Stemphylium is a plant pathogen. Stemphylium rarely grows indoors, however, it is detected in dust that is tracked indoors with foot traffic. Stemphylium is one of the most important fungal allergens in the world causing type I allergies that include rhinitis and asthma in children, and angioedema, conjunctivitis, allergic sinusitis, and bronchopulmonary mycosis in sensitive individuals [204; https://newtonlaboratory.com/mold/stemphylium/].

Syncephalastrum

Syncephalastrum belongs to the class Zygomycetes and order Mucorale. Syncephalastrum is often responsible for opportunistic fungal infections in immunocompromised individuals [205,206]. Syncephalastrum usually causes skin and nail infection [207], there have been reports of mucormycosis in immunocompromised individuals [208] with potentially fatal outcomes [206].

Tetraploa

Tetraploa colonies are brownish in color. Tetraploa grows at the base of leaves and stems just above the soil on many plants and trees. It has been reported to cause keratitis and subcutaneous infection [209-211].

Torula

Torula forms dark brown to black velvety colonies [212,213]. It grows on soils, dead wood, leaves, food, hay, textiles, and is found in the air and frequently detected, in small amounts, in indoor air and sometimes outdoors [214-218]. Indoors, Torula grows on cellulose-based materials. Torula causes type I allergies and may cause hay fever and asthma. Torula produces several bioactive secondary metabolites [219-221].

Triadelphia

Triadelphia is found on rotting wood or other plant materials, often submerged in water except for a few species known to be opportunistic human pathogens isolated from clinical samples [222-224]. Triadelphia grows slowly forming velvety colonies with white tufts, starting as uncolored and later becoming greenish grey to dark brown/grey, and finally to brown/black with whitish margins and abundant sporulation; colonies grow best around 30°C. Infections occur mostly in immunocompromised individuals including those with diabetes, or those on chemotherapy and may involve lungs and brain [223].

Trichoderma

Trichoderma grows fast at 25–30°C, some species grow well at 45°C to initially transparent to white colonies which turn to compact or loose clusters of green, yellow, or white in color. Trichoderma is frequently found in soil, decaying dead trees, pine needles, paper, and inside buildings; it often grows on other fungi. Spores of Trichoderma are spread through air. Trichoderma is an opportunistic pathogen that causes allergies, sinusitis, brain abscess, liver infection, stomatitis, hypersensitive pneumonitis, skin infections and disseminated infections, mostly in immunocompromised and organ transplant recipients [225,226]. Infections caused by Trichoderma are rare, however increasing. Trichoderma is associated with hyalohyphomycosis and nosocomial infections traced to contaminated solutions used in hospitals. Several species of Trichoderma produce mycotoxins like trilongins, trichothecenes, and gliotoxin [227,228]. Trilongins block potassium and sodium ion channels and can affect the heart, lungs, and nervous system [227]. Gliotoxin affects the immune system and inhibits phagocytosis and acts as an immunosuppressor [228]. For details about trichothecenes, gliotoxin, readers are directed to “Description of the Mycotoxins…” section below.

Trichothecium

Trichothecium is widely distributed and found on decaying vegetation, foodstuffs, and in soil. It forms powdery colonies, initially white and later turns pale pink to peach in color [229]. Several secondary metabolites including trichothecene (first isolated and named after this mold) are produced by Trichothecium [229]. For details about trichothecenes, readers are directed to “Description of the Mycotoxins…” section below.

Unidentifiable spores

Unidentifiable spores are considered allergenic.

Wallemia

Wallemia is found in soil, on fruits, dry foods, dairy products, textiles, and hay; it also grows on materials with high sugar and salt content like sugary foods and salted meats [230-232]. It grows on materials with low water activity [131]. Wallemia is commonly detected in dust collected from inside buildings [131]. Wallemia is allergenic and known to cause rare infections both in healthy and immunocompromised individuals [230,233].

Yeast

Yeast is found worldwide in varied natural habitats. It is present on the skin and in the GI tracts, where yeast may act as parasites or have symbiotic relationship with the host. Colonies of yeast grow rapidly, they may appear smooth and glabrous, pasty, moist, or dry and most are white to cream in color, but some may be tan, pinkish, or orange in color. The most common fungal infections in humans are yeast infections. Yeast infections range from localized cutaneous or mucocutaneous lesions, to fungemia or systemic mycoses. Some yeasts are also allergenic and multiple exposure may lead to hypersensitivity. Additionally, yeasts may be allergenic to susceptible individuals at sufficient levels.

Zygomycetes

Zygomycetes is a fast growing mold, commonly found in soil or on decaying plants or animal material. Zygomycetes often overgrow and/or inhibits the growth of other molds growing nearby. Spores of Zygomycetes are transmitted by wind and infection occurs through inhalation, damaged skin, and/or ingestion. Species of Zygomycetes (e.g., Rhizopus, Mucor) cause infections and disease, called zygomycosis [234], generally in immunocompromised individuals [235-237]. Zygomycosis rarely occurs in normal individuals. Risk factors for zygomycosis are diabetes mellitus, neutropenia, sustained immunosuppressive therapy, chronic prednisone use, iron chelation therapy, broad-spectrum antibiotic use, severe malnutrition, and breach in the integrity of primary cutaneous barrier such as trauma, surgical wounds, needle sticks, or burns. The most common are rhinocerebral, pulmonary, GI, cutaneous, and disseminated zygomycosis as well as allergies [235-237]. Some of the species of Zygomycetes cause angioinvasive disease that often leads to thrombosis, infarction of involved tissues, and tissue destruction; diseases are mediated by several proteases, lipases, and mycotoxins [147].

From suitable places inside buildings, suspected ideal for bacterial growth, samples are collected and placed into sterile containers and sent to microbiology laboratory for the identification and enumeration of culturable bacteria (i.e., colony forming units or CFU/mL). Level of endotoxins produced by the bacteria is also determined and reported. As the focus of this paper is mold and mycotoxins, we are only presenting a list of bacteria and endotoxins in (Table 3) for completeness. These are detected inside of even one out of over 800 buildings sampled by the Mold Case Consulting in the last four years without any further discussion. For details, see [24].

Urine (rarely blood, only when warranted) samples are collected from the inhabitant(s) of the buildings in question and analyzed for 11 mycotoxins produced by at least 5 genera of molds (Table 4). These are considered primary molds and mycotoxins of concern for human health. For details of the methods, see [24].

Mycotoxins consist of several hundred identified toxic compounds that are naturally produced by certain molds [23]; only those which are commonly detected in occupants with potential toxicity are mentioned here. According to WHO [23], mycotoxins can cause a variety of adverse health effects ranging from acute poisoning to long-term effects such as immune dysfunction and cancer, posing a serious health threat to both humans and animals [238,239]. Based on the adverse health effects, aflatoxin, fumonisins, trichothecenes, OTA, zearalenones, and patulin are recognized as the most important mycotoxins [240]. The International Agency for Research on Cancer (IARC) has classified several mycotoxins (e.g., aflatoxin B1, aflatoxin B2, aflatoxin G1, and aflatoxin G2) as Group 1 (known) human carcinogen and several (OTA, fumonisin B1, and fumonisin B2, aflatoxin M1) as Group 2B (possibly) human carcinogen [241,242]. The most frequently detected mycotoxins were OTA, CTN, and MPA followed by gliotoxin, and aflatoxin M1 in samples collected from over 2000 occupants from across the United States who lived in over 800 suspected mold-infested buildings.

Individuals hypersensitive to mycotoxin are at higher risk of developing toxicities against aforementioned agents. Symptoms of mycotoxin poisoning depend on the type of mycotoxin involved, dose, frequency, and duration of exposure as well as gender, health, and age of the exposed individual. Exposure to mycotoxins can also increase the vulnerability to microbial diseases [25,243,244]. Mycotoxins have been reported to impair barrier function of epithelial cells, including cells that make blood brain barrier, resulting in inflammatory changes and neurological effects in healthy individuals and exacerbation of many inflammatory conditions, like pneumonia, chronic fatigue [1,16,25,245-248].

Mycotoxins are easily absorbed following inhalation, dermal, and oral routes and reported to cause innate immune activation, neural, cognitive, and emotional dysfunction [21,249]. There is now mounting evidence, both from animal and human (epidemiological) studies, that exposure to molds and mycotoxins in indoor settings can cause adverse health effects [250].

Aflatoxin

Aflatoxins are among the most poisonous mycotoxins that are produced by certain species of Aspergillus, mainly A. flavus, A. parasiticus and A. nomius [23,251,252]. Out of over groups of 20 known aflatoxins, B1 (most potent), B2, G1, and G2 are the four major ones for toxicity [253]. Aflatoxin M1 is the hydroxylated metabolite of B1 formed during fermentation by A. parasiticus and produced during in vivo metabolism and excreted via urine (major elimination route) and milk (minor elimination route); they have also been detected in human breast milk, infant formula, cow’s milk, and dairy products [254-258].

Aflatoxins cause hepatotoxicity, immunotoxicity, developmental toxicity, and are mutagens, thus are carcinogens. According to the European Food Safety Authority (EFSA), even exposure of as low as 1 ng/kg/day can increase the risk of developing liver cancer [259]. Liver toxicity of aflatoxin is considered a crucial issue; aflatoxicosis, characterized by liver damage, with acute symptoms of edema, hepatitis, hemorrhagic necrosis of liver and profound lethargy, while chronic effects include immune suppression, growth retardation, and cancer [260-268]. Ingestion of as little as 2 mg/day of aflatoxin for a month can lead to acute hepatitis and death [260,263,270].

Immunotoxicity of aflatoxins in humans is characterized by downregulation of interleukin-4 (IL-4) and upregulation of tumor necrosis factor-alpha (TNFα) secretion; Aflatoxin B1 is a known suppresser of immunity by decreasing protective effects of vaccines [271-273]. Aflatoxins can also enter developing fetus in humans by crossing placenta and have been detected in cord blood [274-278]. Increased preterm birth and late-term miscarriages have been reported from maternal exposure to aflatoxins [279]. Aflatoxin B1 and M1 are classified as Group 1 and Group 2B carcinogens, respectively by the IARC [78,79,280]. Carcinogenicity of aflatoxin B1 is partly due to its ability to cause DNA damage and mutation [281-283]; they have also been reported to cross blood brain barrier causing damage to mitochondrial DNA of brain cells [284,285].

The most common route of entry of aflatoxin into the human body is ingestion followed by inhalation. Once absorbed, aflatoxins are metabolized by microsomal mixed-function oxidase, cytochrome P450 3A4 and 1A2, mostly in the liver, to form reactive epoxide intermediates which are responsible for DNA mutation and cellular dysregulation, following depletion of glutathione, through binding reactive metabolites to proteins, RNA, and DNA [79,82,286,287]. The absorbed aflatoxins are relatively rapidly excreted mainly through bile. In urine, aflatoxin B1 is excreted as aflatoxin M1. Elimination t_½ of aflatoxin M1 in humans is ~8 h [288]; elimination t_½ of aflatoxin-albumin adduct from blood in humans has been reported to be 30-60 days [267].

Chaetoglobosin A

Chaetoglobosin A is produced by molds (e.g., Chaetomium, Penicillium), primarily of the genus Chaetomium [289,290]; for details of secondary metabolites produced by Chaetomium, see section on Chaetomium above. Chaetoglobosin A belongs to cytochalasans, a highly diversified group of fungal secondary metabolites, which display a broad range of bioactive properties. Cytochalasans have been shown to act as phytoalexins, hamper cholesterol biosynthesis, act as an immunosuppressor at high doses, interfere with glucose transport by human erythrocyte membranes, inhibit secretion of thyroid hormones, inhibit postmitotic cytoplasmic cleavage of HeLa cells, inhibit cell movement, inhibit ciliary beating in chicken tracheal organ culture [96,103,291]. Cytochalasans also facilitate fungal virulence (https://metacyc.org/META/NEW-IMAGE?type=PATHWAY&object=PWY-7612). Chaetoglobosin A has been reported to be highly acutely toxic to rats when administered subcutaneously killing all of them at the lowest dose of 2 mg/kg tested; LD₅₀ in mice was ~7 mg/kg (male) and ~18 mg/kg (female) with 5 mg/kg dose causing visceral congestion, necrosis of the thymus and spleen tissues and degeneration of spermatocytes in testes [103]. Oral doses were far less toxic (>400 mg/kg), likely due to low oral absorption and/or first-pass metabolism [103]. A 2-week dosing of 30 ppm chaetoglobosin A to mice in diet (~6 mg/kg/day) caused liver injuries, bone marrow aplasia, and atrophy of lymphatic tissue [109]. Reproductive and developmental toxicities (increased resorptions, decreased fetal body weight, and fetal survivability) have been reported in mice dosed with either ~2 or ~6 mg/kg/day chaetoglobosin A from gestational days 0-18 [109]. A slight increase in mutation frequency was observed in a mouse mammary cancer cell line [292].

Citrinin

Mycotoxin citrinin (CTN) is produced by species of many molds including Penicillium, Aspergillus and Monascus [25,293]. CTN has some antibiotic properties against gram-positive bacteria, but it has high nephrotoxicity and therefore, never used as a drug. Although, the major target organ for CTN toxicity is kidney, it is also hepatotoxic, embryocidal, fetotoxic, immunotoxic; cause toxicity to bone marrow and modulate immune system [294-303]. Kidney damage by CTN is characterized by enlarged kidney, hydropic degeneration, loss of brush border, and pyknotic nuclei in the proximal tubules [304]. CTN inhibits key enzymes in cholesterol biosynthesis, reducing concentration of serum testosterone and causes hypocholesterolemia [305]. Swelling of the kidneys and acute tubular necrosis was observed in animals dosed with acutely toxic doses (LD₅₀ oral and subcutaneous = 50 and 35 mg/kg) of CTN [303,304,306-309]. The TDI of CTN is set to 0.2 μg/kg by the European Food Safety Authority and Deutsche Forschungsgemeinschaft [310-312].

Following absorption, CTN is extensively metabolized to dihydrocitrinone (DHC) and excreted in urine as the main metabolite, along with parent CTN [313-314]. DHC is less toxic than CTN and the presence of DHC in urine or blood is used as a biomarker of exposure to CTN [315,316]. In human volunteers, cumulative (CTN+DHC) urinary excretion over 24 h has been reported to between 33 and 71% with urinary elimination t_½ of ~7 h for CTN and ~9 h for DHC; plasma elimination t_½ of CTN is ~9 h [314].

Enniatin B

Enniatins (ENNs) are produced by several Fusarium species, 29 of them are known [317]; the most important ENNs is ENN B based on incidence and observance. ENNs inhibit acyl-CoA: cholesterol acyl transferase activity resulting in oxidative stress [318,319]. ENNs are cytotoxic through the disruption of normal physiological concentrations of Ca²⁺, Na⁺, K⁺ across membranes by disrupting ionic selectivity, which is debilitating for mitochondrial membranes causing uncoupling of oxidative phosphorylation [320,321]. The non-functioning of mitochondria leads to cell cycle disruption and apoptotic cell death [319,322-329].

ENN B alters cellular energy metabolism and reduces cell proliferation, increases apoptosis, and necrotic cell death; alteration in energy metabolism is by effecting mitochondrial membrane permeability transition and hence its function [330]. ENN B inhibits multidrug resistance associated protein-1 (ABCG2) and P-glycoprotein (ABCB1) efflux pumps [331-333]. Cytotoxicity is observed at low micromolar concentrations in animal cell lines (i.e., mouse macrophages, porcine kidney cells, Spodoptera frugiperda cell line, [SF-9 cells]), and reduce motility of boar spermatozoa [334]. ENN B is an endocrine disruptive chemical, produces adrenal toxicity [335]; toxicity of ENN B has been reported to enhance in the presence of other ENNs or other mycotoxins [329]. Once absorbed, ENN B is detected in all tissues and in blood with the highest concentrations in adipose tissue and liver indicating its bioaccumulation in lipophilic organs [336]. Elimination t_½ of ENN B has been reported to be ~5 h in mice [337] and ~1.6 h in pigs [338].

Gliotoxin

Gliotoxin is produced by several fungal species belonging to genus Aspergillus (e.g., A. flavus, A. fumigatus, A. niger, A. terreus), Eurotium (e.g., E. chevalieri, E. Rubrum), Neosartorya (e.g., N. pseudofischeri), Trichoderma (e.g., T. virens), and some species of Penicillium, Acremonium and Alternaria; spores of A. fumigatus is the most clinically relevant source of gliotoxin [339-344]. Gliotoxin is an epipolythiodioxopiperazine (ETP) derivative that exerts profound immunosuppressive effects by inhibiting cellular functions of B, and T lymphocytes, macrophages, and apoptosis of immune cells [345-348]. The presence of a disulfide bridge in ETP makes gliotoxin to inactivate proteins at high concentrations by covalently binding with thiol groups of proteins and generating reactive oxygen species by redox cycling [349,350]. At relatively low concentrations (IC₅₀ of 50–100 nM), gliotoxin selectively inhibits activation of nuclear factor-kappa B (NF-κB), preventing induction of intercellular adhesion molecule 1 (ICAM-1) demonstrated using ICAM-1 promoter-reporter luciferase gene and determing the reporter activity by tumor necrosis factor-alpha (TNFα), interleukin -1 (IL-1), and phorbol myristate acetate (PMA), all of them are NF-κB activators [351].

Gliotoxin suppresses phagocytosis by interfering with phosphatidylinositol 3,4,5-trisphosphate production which impairs the ability of macrophages to recognize and destroy invading pathogens [352]. Exposure to gliotoxin is hypothesized to result in colonization and virulence of A. fumigatus [348,353-355]. In mice, administration of gliotoxin caused immunosuppression resulting in the establishment of invasive aspergillosis with A. fumigatus; in humans, gliotoxin is known to slow ciliary action and damage epithelium of the respiratory tract [348,353-355]. Gliotoxin can lead to invasive aspergillosis, caused by the opportunistic mold A. fumigatus, that typically reside in the pulmonary system, the ears, the eyes, or the nails; gliotoxin is detected in serum of patients with invasive aspergillus [356]. Aspergillosis is a devastating disease for immunocompromised individuals (e.g., organ transplant recipients, those with AIDS, cancer, receiving bone marrow transplant, patients undergoing chemotherapy) caused by gliotoxin frequently resulted in renal failure with mortality rate of ~90% [349-351,357-360]. A. fumigatus is also frequently detected in sputum of patients with chronic respiratory diseases like cystic fibrosis [361,362].

Mycophenolic acid

Mycophenolic acid (MPA) is produced by many species of Penicillium, it is a potent immunosuppressant often used to prevent rejection in renal transplant patients [363,364]. MPA is a selective and non-competitive inhibitor of the inosine monophosphate dehydrogenases (IMPDH), an enzyme involved in de novo biosynthesis of guanosine nucleotide, the only pathway of purines synthesis in B and T lymphocytes [365]. Inhibition of IMPDH blocks cell proliferation by blocking DNA and RNA biosynthesis [364,366]. Inhibition of proliferation of both B and T-cells suppresses lymphocytes that identify mold toxins and increases risk of opportunistic infections [367-369]. Following oral administration, MPA is rapidly absorbed from the small intestine reaching maximum blood concentration in 60 to 90 min. with an average oral bioavailability of ~94% in healthy human volunteers [370]. MPA is rapidly metabolized to an inactive glucuronide conjugate by isoforms of the UDP-glucuronosyltransferases in the liver, kidney, and intestine [371,372]. At least three minor metabolites of MPA have also been identified in humans [373].

Plasma elimination t_½ of MPA in healthy volunteers is 17.9 h after oral administration (https://go.drugbank.com/drugs/DB01024). Between 8 and 12 h after oral administration, an average of 37% of people display a secondary peak of MPA in plasma, representing absorption of the intestinal bacterial deglucuronidated metabolites of MPA through enterohepatic circulation accounting for up to 40-60% of the total circulating dose. Metabolites of MPA is predominantly eliminated in urine (~93% of the dose), fecal elimination accounts for ~6% of the dose [374; https://go.drugbank.com/drugs/DB01024].

Human leukocyte antigen (HLA) genes are a family of genes on the human chromosome 6 responsible for making a group of cell-surface proteins, i.e., HLA complex, which are essential for removing either infected cells or producing antibodies in response to invading/foreign antigens [375]. HLA genes have many alleles which play key role in presenting endogenous and exogenous peptides to T-cells for fine-tuning of the adaptive immune response; mutations at the HLA gene alleles result in slow response to eliminate MPA from the body [376-378]. People with genetic predisposition at the HLA genes have shown to eliminate MPA extremely slowly from their body (~213-fold slower with the t_½ of ~160 days instead of ~0.75 day in individuals without genetic predisposition at the HLA genes) [24].

Ochratoxin A

Ochratoxins are produced by species of Eurotium, Aspergillus, Fusarium and Penicillium. There are three generally recognized ochratoxins, designated A, B and C. Ochratoxin A (OTA) is the most toxic, followed by OTB and OTC. Kidney is the primary target organ of OTA toxicity through lysis of tubular cells [379]. OTA is nephrotoxic to every animal species tested to date and is most likely toxic to humans; elimination of OTA is the slowest in humans than any other species examined [http://www.ictm.com/Reports/ereport_Vol3No3.pdf; 380]. Endemic Balkan nephropathy, a progressive chronic nephritis in people living in areas bordering Danube River, is speculated to be caused by ochratoxins contaminated food as evidenced by the presence of ochratoxins in serum of families with endemic Balkan nephropathy and urinary tract tumors than in unaffected families [381-384]. In addition to being a nephrotoxin, OTA is also hepatotoxic, immune suppressant, potent teratogen, mutagen, and carcinogen [385,386]. OAT has been placed under cancer category 2B, possible human carcinogen, by the IARC [386].

Mode of toxicity of OTA is not completely understood and appears to be quite complex. Inhibition of energy production (e.g., mitochondrial ATPs), inhibition of the synthesis of proteins (e.g., enzymes that synthesize phenylalanine-tRNA, phenylalanyl-tRNA synthetase), stimulation of lipid peroxidation, induction of oxidative stress, DNA adducts formation, apoptosis, cellular necrosis, and cell cycle arrest are possible reasons of OTA’s toxicity [25,74,80,369,387-396].

Following absorption, OTA is distributed at a high concentration in the kidney, the major target organ [397]; OTA has also been reported to cross placenta and found twice as high in fetal than maternal serum in swine and humans [398]. OTA remains highly bound to plasma proteins (99.9% of the circulating OTA remains protein bound with highest binding affinity to human serum albumin), oral bioavailability is between 40 and 60% in animals and ~93% in humans [396,399-401]. Based on lipophilicity and high blood partition of OTA and lungs being highly perfused organ, high absorption of OTA is also expected from the lungs following inhalation exposure. OTA is poorly metabolized and slowly excreted with a plasma t_½ of ~6 days in rats, 19-21 days in monkeys, and ~36 days in humans; elimination occurs in the form of parent OTA and following hydrolysis of the peptide bond, presumably by carboxypeptidases produced by the intestinal microflora [288,394,401-404].

Based on the lowest observed adverse effect level (LOAEL) of 8 μg/kg/day in pigs that caused renal malfunction in a dose-response study [405], regulatory agencies have set TDI of OTA between 3 and ~17 ng/kg/day, 4 ng/kg/day intake is considered with negligible cancer risk [406-413].

Roridin E

Roridin E is a trichothecene produced by various species of Fusarium, Myrothecium and Stachybotrys. Roridin E can cause respiratory and olfactory toxicity as many other trichothecenes, it may also disrupt DNA, RNA synthesis, and inhibits protein biosynthesis by preventing peptidyl transferase activity and are known carcinogen [60,61]. Elimination t_½ of roridin E is estimated using quantitative structure activity relationship (QSAR) modeling to be ~8 h in healthy humans (https://www.mycocentral.eu/mycotoxins/719). Disruption of DNA, RNA, and protein biosynthesis impact every cell in the body, cells with the highest rate of mitosis are the most susceptible targets of trichothecene. Even low levels of exposure to trichothecenes can result in immune suppression, neurological issues, endocrine disruption, cardiovascular issues, and GI distress [195,197]. For details about trichothecenes, readers are directed to “trichothecenes” section below.

Sterigmatocystin

Sterigmatocystin is produced by several species of Aspergillus, Bipolaris, Botryotrichum, Humicola and Penicillium with Aspergillus being the main producer of sterigmatocystin [414]. Acute toxicity of sterigmatocystin fall under U.S. EPA category II (i.e., oral LD₅₀ ≤500 mg/kg), LD₅₀ following intraperitoneal dosing to rats and monkeys is 60-85 mg/kg and 32 mg/kg, respectively [415]. Sterigmatocystin has been found to affect immune function [416,417]. Sterigmatocystin has been reported to covalently bind to DNA and forms DNA adducts; sterigmatocystin-DNA adducts have been detected in blood and urine of patients with liver and stomach cancer and with liver cirrhosis [414,418-421]. Sterigmatocystin is mutagenic, teratogenic, and carcinogenic (lung and liver cancer) in experimental animals at doses ranging from 5 ng/kg (subcutaneous) to 3-30 mg/kg (oral) [422-426]. Sterigmatocystin has been reported to generate liver and lung tumors following a single subcutaneous dose of 5 ng/kg to newborn mice and confirmed as a putative causal factor for lung adenocarcinoma in mice [424,425]. Sterigmatocystin has been classified as Group 2B human carcinogen by the IARC [427].

Trichothecenes

Trichothecenes are not measured directly as they are a family of more than 200 mycotoxins, several of the commonly detected mold  species indoors produce one or more of trichothecenes (for details, see “Description of Commonly Detected Molds…”, above), therefore, we consider it will be helpful for readers to provide information about the trichothecenes as a group here in addition to the mycotoxins analyzed in urine and/or blood. Trichothecenes are produced by some species of Acremonium, Cephalosporium, Cylindrocarpon, Fusarium, Mycothecium, Phomopsis, Spicellum, Stachybotrys, Trichoderma, Trichothecium, and Verticimonosporium [61,62,136,137,227,229,342, 428-430].

Each trichothecene has a common 12,13-epoxitrichothec-9-ene structure (Figure 1), the epoxy group is highly reactive site of trichothecenes allowing them to cause oxidative damage due to the generation of free radicals [431,432]. Based on the substitution pattern of R, H, OH, O, Acyl groups on 12,13-epoxitrichothec-9-ene, trichothecene are classified into Types A, B, C, and D [433,434]. For Type A trichothecenes, substitution at C-8 is either a hydroxyl group (e.g., neosolaniol), an ester (e.g., T-2 toxin), or no oxygen substitution (e.g., trichodermin, 4,15-diacetoxyscirpenol, and harzianum A). For Type B trichothecenes, substitution at C-8 is a keto (carbonyl) group (e.g., nivalenol, deoxynivalenol, and trichothecin). Type B trichothecenes produced by Fusarium typically have hydroxyl group at C-7; however, this is absent in trichothecenes produced by other genera. Trichothecenes in Type C have an epoxide between C-7/C-8 (e.g., crotocin). Trichothecenes in Type D have an additional ring connecting to C-4 and C-15 positions of the 12,13-epoxitrichothec-9-ene (e.g., roridin A, verrucarin A, satratoxin H) [435]. This classification, however, misses some other structural features like all trichothecenes (Types A and B) produced by Fusarium contain a hydroxyl or an acetyl group at C-3 position and trichothecenes (Types A, B, C and D) produced by Trichoderma, Trichothecium, Myrothecium or Stachybotrys lack oxygen at C-3 position [436-438].

Trichothecenes exert multiorgan effects, e.g., anorexia, loss of weight, growth retardation, immunodeficiency (through decreasing lymphocyte production in hematopoietic organs), cardiovascular alterations, nervous system disorders, decreased reproduction, bone marrow damage, skin toxicity, hemostatic derangements, and alimentary toxic aleukia (mycotoxin-induced conditions like nausea, vomiting, diarrhea, leukopenia, hemorrhaging, skin inflammation, and sometimes death) [60,62,195,197,439-442]. Following dermal or oral exposure, trichothecenes can cause irritation, burning and itching, rash or blisters, and bleeding from skin or intestinal mucosa. Exposure to eyes can cause burning, tearing, pain, conjunctivitis, and blurred vision [441]. Trichothecenes are immunostimulatory at low doses and immunosuppressive at high doses [60,428,441]. Individuals with chronic exposure to the mycotoxins report dermatitis, cough, rhinitis, nose bleeds, burning sensation in the mouth and nasal passage, cold and flu, headache, general malaise, fever, chronic fatigue, irregular menstrual cycle, and premature ovarian failure. Unlike many other mycotoxins, metabolic activation is not needed for trichothecenes to exert their toxicity; they directly react with cellular components through their highly reactive epoxide moiety. Trichothecenes are potent inhibitors of DNA, RNA, and protein syntheses, and mitosis; some of them are also known carcinogen [60,61,244].

Trichothecenes are cytotoxic which is due to their ability to inhibit protein synthesis by binding to ribosomes interfering with the active site of peptidyl transferase at the 3'-end of large 28S ribosomal RNA and inhibit initiation, elongation or termination of protein synthesis causing polyribosome disaggregation. Cells with the highest rate of mitosis, including GI mucosal cells (enterocytes), lymphoid tissues, and bone marrow, are the most susceptible to this effect. Binding to ribosomes also activates signaling downstream resulting in immune response and apoptosis; apoptosis can also be induced through the generation of reactive oxygen species. Additionally, trichothecenes can alter membrane structure resulting in increased lipid peroxidation and inhibition of electron transport in mitochondria [60,441,443-445].

Trichothecenes are easily absorbed following inhalation, dermal, and oral routes of exposure due to their lipophilicity. They are metabolized mainly by cytochrome P-450s and carboxylesterase in liver; other tissues (e.g., kidney, spleen, and intestine) also show some metabolic activity. Trichothecenes are biotransformed into less-toxic metabolites following hydrolysis, hydroxylation, de-epoxidation, and glucuronidation and excreted in urine and feces [440,441].

Verrucarin A

Verrucarin A is a trichothecene produced by Fusarium and Aspergillus. As other trichothecenes, verrucarin is neurotoxic, immunotoxic, cytotoxic, potent protein synthesis inhibitor. Verrucarin A is partially absorbed in dogs following oral administration and excreted with an elimination t_½ of about 2 h [446]. Verrucarin is considered among some of the most toxic trichothecenes and more toxic than simple trichothecenes [439,442]. For details about trichothecenes, readers are directed to “trichothecenes” section above.

Zearalenone

Zearalenone is produced by Fusarium molds. Zearalenone is an endocrine disruptor, it is similar in chemical structure to estrogen and therefore, binds with estrogen receptors affecting reproductive organs [447,448]. It also affects the male reproductive system [449]. Zearalenone is also an immunotoxicant and hepatotoxicant [450]. Zearalenone exposure decreases fertility, precocious puberty, change weight of thyroid, adrenal, and pituitary glands, alter progesterone and estradiol levels in serum, cause fibrosis and hyperplasia in the uterus, breast cancer, endometrial carcinoma, and liver damages which may lead to liver cancer [244]. One study found higher levels of zearalenone in urine and serum of autistic children than their healthy siblings [451]. Zearalenone has relatively low acute toxicity with LD₅₀ >2000 mg/kg (EFSA 2011b). EFSA set TDI for zearalenone to 0.25 μg/kg [452-454].

Zearalenone is extensively absorbed and metabolized by oral, dermal, and inhalation routes. Reduction of zearalenone results in formation of α-zearalenol, which is more estrogenic, and β-zearalenol, which is less estrogenic than zearalenone. Efficient glucuronidation of zearalenone in the small intestine and liver significantly reduces the amounts of unconjugated (i.e., receptor-active) parent compound that can reach the circulation. Cytochrome P-450-mediated oxidation produces catechol metabolites that are subject to redox cycling to reactive quinones [288]. Zearalenone is eliminated with an elimination t_½ of ~86 h in pigs and <24 h in humans with high oral bioavailability [454,455].

Fecal samples are collected from the occupant(s) of the buildings in question and analyzed for bacteria (expected, commensal, and dysbiotic) and yeast (normal and dysbiotic) by culturing in appropriate growth media or via optical microscopy. As the focus of this paper is mold and mycotoxins, we are only presenting a list of commensals and dysbiotic bacteria in (Table 5) for reference only. These are detected in even one out of over 2000 occupants lived in over 800 buildings sampled by the Mold Case Consulting in the last four years without any further discussion. For details of the methods, see [24]. Dysbiosis is an alteration in gut microbiome that increases intestinal permeability increasing the transfer of bacterial endotoxins via portal vein into liver [456]. Endotoxins in liver, activate liver resident macrophages (i.e., Kupffer cells) and promote release of proinflammatory cytokines (e.g., TNFα, IL-1, and IL-6 leading to a cascade of immune responses and inflammation [457-459].

Gut microbes are good sources of secondary metabolites, such as short chain fatty acids (SCFAs) and lipopolysaccharides, however, in the event of alteration in gut microbiota, gut dysbiosis occurs. In a healthy state, gut microbiota help maintain the basic functions, whereas dysbiosis alters metabolism that can lead to diseases like metabolic syndrome, cardiovascular, GI, neurodegenerative diseases, and cancer [460,461]. Additionally, gut dysbiosis increases the intestinal permeability of bacterial liposaccharides (LPS). Infections with several species of toxic bacteria or occurrence of microbiota dysbiosis, secondary to mycotoxin exposure, likely contribute to several chronic human diseases such as diabetes, colorectal cancer, and degenerative neurological diseases (e.g., Alzheimer’s and Parkinsons’s diseases) [462-464]. Pathogenesis of Alzheimer’s disease (AD) is linked to strong inflammatory responses leading to higher amyloid beta (Aβ) formation [465,466]. The brains of AD patients contain 5–10 times more bacterial LPS than healthy brains [467]. A high cholesterol diet may cause increased sequestration of mycotoxins on the surface of cholesterol leading to increased absorption of various mycotoxins [468]. Additionally, binding of mycotoxins to lipoproteins in human and animal plasma is linked with induction of hypercholesterolemia [469]. Hypercholesterolemia, non-alcoholic fatty liver disease (NAFLD) from metabolic syndrome, diabetes, and inflammation also modifies blood brain barrier (BBB), increasing permeability of mycotoxins across BBB into brain [470]. Higher penetration of mycotoxins to central nervous system due to LPS-induced inflammation and increased blood brain barrier permeability also exacerbate neurological effects of mycotoxins. Additionally, enhanced absorption of lipoprotein-sequestered mycotoxins from intestine will lead to their increased bioavailability to target organs [462,464,469-472].

Globally, inhalation as well as ingestion of mycotoxin, bacterial infections, and gut dysbiosis resulting in increased systemic LPS levels are of major health concern. In developing countries, exposure to mycotoxins and LPS is primarily from ingestion of contaminated food. In developed countries, inhalation exposure from inhabiting mold-infested energy efficient buildings is of more concern, which is predicted to be similar to exposure from diet in developing countries, than exposure through food which is normally significantly less due to strict regulations in developed countries [462,464]. Mycotoxins and LPS once absorbed, either from ingesting from intestine, with high lipids-containing diets, or from inhalation when inhabiting mold-infested building, find their way to target organs. Gut dysbiosis results in systemically increased inflammatory metabolites and cytokines which increase BBB permeability and results in increased transport of immune cells and mediators of neuroinflammation into the brain [470]. Mold exposure to individuals with gut dysbiosis may lead to increased delivery of mycotoxins to the brain from increased BBB permeability. Additionally, enhanced absorption of mycotoxins sequestered in lipids can cause other adverse health effects mentioned above. Therefore, excessive bacterial growth indoors and fat rich diet may be of major concern for mycotoxin exposure, either by ingestion or inhalalation, warranting a closer investigation of LPS and mycotoxin nexus in mycotoxin-exposed individuals.

Risk assessment is a process of integrating hazard identification with dose-response and exposure assessment (https://www.epa.gov/risk/conducting-human-health-risk-assessment). The first step in the risk assessment is the identification of hazard, i.e., whether exposure to something of concern can increase the incidence of (specific) adverse health effects and effects, if seen only in experimental animals, are likely to occur in humans. Hazard identification also considers pharmacokinetics, pharmacodynamics, mode-of-action in experimental animals, and relevance to humans. For example, ∝_2u-globulin (a male rat-specific protein) related kidney toxicity and cancer in male rats has no concordance with humans and mice are much more resistant to aflatoxin B1-induced liver cancer than rats [473,474]. Based on the information presented here, many molds and mycotoxins commonly detected in samples collected from the suspected mold-infested buildings and residents are identified to cause adverse health effects including some causing cancer to animals and humans (see above for details).

The second step of the risk assessment is the determination of the dose-response (or exposure response) relationship, i.e., degree of adverse health effects at different exposure (dose), normally in experimental animals. Terminologies frequently used in assessing dose-response relationships include: 1) no-observed adverse effect level (NOAEL), the highest exposure level (dose) at which no statistical or biological significant increase in the frequency and/or severity of adverse effects is observed in exposed than non-exposed (control) populations; 2) lowest-observed-adverse-effect level (LOAEL) when some adverse effect(s) is/are observed at the lowest dose tested; 3) benchmark dose (BMD), determined by mathematical modeling, as an alternative to NOAEL.

The LOAEL, NOAEL, or statistical lower confidence limit of BMD that is used as the point of departure for extrapolation to lower doses. The NOAEL (preferred), LOAEL (when NOAEL is not achieved), or lower confidence limit of BMD is used to calculate maximum acceptable dose of a toxic substance, also called reference dose (RfD) or reference concentration (RfC) for inhalation exposure, or acceptable/tolerable daily intake (ADI/TDI), by applying uncertainty factors (UFs). The UFs are generally applied in the order-of-magnitude to account for the variability and uncertainty from differences between test animals and humans (generally 10-fold) and variability within the human population (generally another 10-fold). When LOAEL, instead of NOAEL, is used, another 10-fold UF is used; use of lower or higher UFs depend upon the quality of the generated toxicological, toxicokinetic, and toxicodynamic data in experimental animals as well as human relevance of the effects in animals. For most of the mycotoxins determined in urine samples collected from the residents of the suspected mold-infested buildings and presented in (Table 4), dose-response data already exist and TDI developed (Table 6). For additional information readers are directed to Borchers et al. [475] publication.

Unfortunately, extremely limited dose-response relationship data are available for molds, even in experimental animals. There are only a few animal studies where animals were dosed with known number of spores and adverse health effects determined. For example, Flemming et al. [476] conducted a study in rats to determine NOAEL of S. chartarum. They dosed rats with 30, 300, and 3000 S. chartarum spores/g bodyweight via intratracheal instillations and determined inflammatory biomarkers in the lung lavage. The NOAEL of the study was < 30 S. chartarum spores/g. Using this NOAEL, one can calculate TDI by dividing NOAEL in animals with UF. A UF of 10 is used to account for the possible differences in responsiveness between animals and humans, another UF of 10 is used to account for variation in susceptibility in human population. The UF of 100 is usually appropriate for many chemicals; however, for chemicals with less complete data, e.g., use of short-term study, as the case for Flemming et al. [476] study, an additional UF of 10 is required (leading to a UF of 1000) (https://www.epa.gov/iris/reference-dose-rfd-description-and-use-health-risk-assessments). Based on this risk assessment, TDI of S. chartarum in humans is <30 spores/kg bodyweight.

The third step in risk assessment is to determine the extent of exposure which entails measuring or estimating magnitude, frequency, and duration of exposure and sometimes estimating future exposures from certain environments, e.g., molds, bacteria, and related biotoxins when living in a mold-infested building. For the assessment of exposure to molds and biotoxins in indoors settings, levels of molds, bacteria, endotoxins are determined in air, dust and other samples collected from suspected mold-infested buildings, see (Tables 1-5) for details. Currently there are no regulations or standards for molds indoors including no threshold limit values for airborne mold or mold spore concentrations (https://www.epa.gov/mold/mold-testing-or-sampling#regs; https://www.osha.gov/mold/standards). Frequency and duration of exposure are estimated based on the duration of residence and normal time spent daily inside the building. For mycotoxins in urine and blood (rarely conducted) establishing systemic exposure, pharmacokinetic information is used to further adjust the actual systemic dose. This is especially helpful when samples are collected weeks or months after moving out from the mold-infested to non-mold-infested buildings to estimate the systemic exposure at the time of residing in the building in question [24].

As part of the fourth step in the risk assessment all the information gathered in the first three steps is summarized and integrated to synthesize overall conclusion about risk. Therefore, in the absence of any regulatory guidelines, several approaches can be used to determine if exposure has crossed the threshold of the adverse health effects. One approach that many mold-testing companies advocate is setting arbitrary number of spores in the air to classify the level as normal (<500 spores/m³) or high (>500 spores/m³). However, we consider a more scientific approach using the fact that levels of molds and spores indoors are mostly lower or equal to that found outdoor [5]. This approach compares the levels of spores indoors with that of the outdoors to make conclusions about overall risk based on the severity of adverse health effects rendered by the mold(s) and related biotoxin(s) in question [4,477]. For mycotoxins, on the other hand, systemic exposures are used to calculate risk based on the hazard (i.e., toxicity) and dose-response information as shown above and in (Table 6) along with other scientific approaches when such data are not available.

Out of an estimated 1.5–5.0 million fungal species [481,482], only several hundred can cause diseases in humans, primarily in immunocompromised and critically ill individuals, with only a very few can affect healthy individuals. The number of at-risk populations is unfortunately increasing globally over time due to the rise in immunocompromising diseases like diabetes along with genetic predisposition and environmental factors resulting in increased exposure to fungi and related health complications [24,480-482]. Among the environmental factors, there is growing evidence of a positive relationship between climate change and increased mold growth, sporulation, and allergies [41,43,485-487]. Recent data corroborate with the fact that a rise in fungal infections in humans has been observed with the rise in global temperature, precipitation, flooding, population, and urbanization [52,485,488,489].

As mentioned above, biotoxins (i.e., hyphal fragments, spores, and mycotoxins) cause a variety of allergies in people living or working in mold-infested buildings [2,8,10-16,19,20,22]. In addition to allergies, exposure to mycotoxins can cause many systemic toxicities to organs, e.g., respiratory and nervous systems, liver, kidney, and developing fetus; several of them are known (Group 1) or possibly (Group 2B) carcinogens [1,18,21,23,25]. Exposure to mycotoxins can also make individuals vulnerable to microbial diseases [25]. 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 [1,18,21,23,24,195].

Fungal (including mold) infections are among the most difficult diseases to manage, and invasive fungal infections cause significant morbidity and mortality [490-492]. High complexity in managing fungal infections is mostly due to nonspecific clinical presentations leading to poor diagnosis, lack of an array of antifungal agents, toxicity of the available antifungal medicines, and need of a prolong therapy [490-492]. It is estimated that globally, fungal infections occur to at least 13 million people and cause more than 1.5 million deaths every year [491]. In 2018, in the USA alone, approximately 666,235 fungal infections were diagnosed out of 35.5 million inpatients costing $6.7 billion; additionally, 6.6 million fungal infections were diagnosed in outpatients [491]. Approximately 76% of fungal infections were from Aspergillus, Pneumocystis, and Candida [491].

Assessing exposure to biotoxins, and bacteria/endotoxins, especially in indoor settings is challenging with the currently employed approaches. These approaches (e.g., airborne spores in breathing zone or internalized molds in feces; mycotoxins in urine and/or blood) only give a snapshot of exposure to biotoxins without providing trends of 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 occurred. Source(s) of exposure is/are established by determining the proportion of time occupant(s) spend(s) at the building in question and at other building(s) (e.g., at work) and level of molds in other building(s). The timeline and extent of exposure to mycotoxins is determined by using kinetic information of mycotoxin(s) in question and any preexisting condition(s) of occupant(s) that may influence absorption, distribution, metabolism, and excretion (ADME) of mycotoxin(s), e.g., genetic predisposition. Elimination of OTA and MPA slows by ~10- and ~213-fold, respectively, in individuals with specific genotype of HLA/DR gene [24]. Some physicians claim to treat mold toxicity through the process of “detoxification” by dosing activated charcoal orally, in most cases, long after the cessation of exposure. They claim that activated charcoal mobilizes mycotoxins from different organs, where they may be sequestered, and bring them to the GI tract, where they bind with the orally dosed activated charcoal. Activated charcoal is commonly used in emergency to treat certain kinds of poisoning as it absorbs poisons present in the GI tract, mostly in stomach, and prevents their absorption into the body. There is no research that backs the claim that activated charcoal actively extract mycotoxins from the body; elimination of mycotoxins from the body is governed by ADME.

A rise in buildings with mold infestation is expected with rise in both temperature and humidity from the climate change, consequently, an increase in the number of patients with severe mold related diseases is also expected, thus warranting better health management. To address indoor mold-related illness, there is an urgent need to streamline the process of testing suspected mold-infested buildings by standardizing collection, analysis, and reporting of the results for the consistency and comparison of results across the testing laboratories. Additionally, the use of better markers of mold-related illness in humans are needed along with generation of animal and human (e.g., epidemiological studies) data. Quality animal studies are needed, at least for molds responsible for severe adverse health effects, to develop RfDs/RfCs, ADI/TDI for both mold components (e.g., spores, hyphal fragments) and mycotoxins, especially following inhalation exposure. A careful examination of factors that may influence the severity of illness, like dysbiosis of gut microbiota, endotoxin, other chemicals, and nutrition, is needed for better understanding of mold- and mycotoxin-related adverse health effects in humans. While data for the relevant route(s) of exposure are being generated, we can use risk values set for ingestion presented in this paper along with other available information like 0.5-15 μg/kg for aflatoxins in nuts, grains, dried figs, and milk and 50 μg/kg for patulin in apple juice [23] after incorporating differences in ADME between oral and inhalation routes of exposure. For this paper, we have used data of samples collected from over 800 suspected mold-infested buildings and over 2000 residents from the whole United States with the objective of compiling findings for use by the researchers and those working in this area.

Authors acknowledge David Merchwart of Bio Genesis Industries for using data generated by the Mold Test Company.