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Mycotoxin Testing
What are Mycotoxins?
Mycotoxins (“Mold toxins”) are small and light weight molecules produced by molds as part of their metabolism. Some of the toxins have the secondary effect of being toxic to other molds, which enables them to better compete for food, moisture and surface area. Mycotoxins interfere with RNA synthesis and may cause DNA damage. Mycotoxins, even in minute quantities, are fat-soluble and readily absorbed through the intestinal lining, airways, and skin. (2) One of the most toxic substances known to man, Aflatoxin, occurs associated with fungi that grow on nuts, seeds and grains. Aflatoxin is suspected to be the main culprit in the rise in peanut allergies, although scientific explanation remains elusive. 
We have come a long way in understanding “toxic black mold,“ those molds that have been attributed as producing significant health effects associated with mycotoxins ("mold toxins"). While there were previous incidents of black mold exposures for workers and animals, there was little publicity or awareness until the well-publicized occurrence in Cleveland Ohio in 1994 that resulted in a rash of infant deaths. The understanding of the cause of death has since been refuted as not including the other factors involved including heavy exposures to tobacco smoke and possible exposures to bacterial endotoxins or other pathogenic substances. (1) However, that finding does not diminish the risk of mycotoxins and their significance on human health.
aflatoxins are most commonly associated with peanuts
Many mycotoxins are proven carcinogens in laboratory animals or from epidemiological studies. The cancers generally affect the digestive organs (e.g. liver or gall bladder) or kidneys. Foods commonly associated with molds (e.g. nuts and cheeses) are required by the FDA to be routinely tested for the most harmful and common toxins from food molds. Organic foods such as spices and herbs have been found to contain higher levels of mold toxins (but within FDA health standards) due to the absence of the use of any fungicide.(3) 

Mycotoxins are also suspected to have endocrine disruptive effects, particularly targeting estrogen, androgen and thyroid hormones either by mimicking the hormone or by binding to and thereby blocking the function of certain hormones.(4)  Strictly speaking, endocrine disruption is a “non-toxic” effect.

The most common mycotoxins are Aflatoxin, Trichothecenes, Ochratoxin, and Gliotoxin. Some mycotoxins may be generated from the normal and healthy conditions in the body. For example, Candida spp., which is commonly found in the body can produce Gliotoxin. (8)

A majority of mycotoxins do not appreciably bioaccumulate and are believed to have a short-half life in the body and begin to be excreted as quickly as 24 hours after exposure. Trichothecenes (linked predominantly with the mold common "black mold" Stachybotrys spp.) have a short half-life of a few hours in the body. Because of the short residency time in the body, the clay and charcoal binders and other health supplements sold for “mold detoxification” are unproven and generally considered to be one of many naturopathic scams. One exception is Ochratoxin, which has an estimate half-life of 35 days in the body due in part to its unusual ability to bind to plasma proteins.(5)

It has been reported that 25% of people have a gene (HLA-DR) that predisposes a person to the effects of mold toxins. This gene reputedly causes a misprocessing of antigens, which inhibits the immune system from reacting correctly to infections and toxins.(6)

Molds do not always produce mycotoxins (or spores). In the ideal environment, they can grow “fat and happy.” However, in a majority of cases there is sufficient competition between molds and bacteria for food and sufficient environmental disturbance to cause both mold spore and mycotoxin production. One study estimated that as Stachybotrys dries, the mycotoxin production increases up to 40,000 times.(7) Mycotoxin production can vary within a species depending on a combination of poorly understood environmental factors including the substrate, type of food source, amount of light, temperature as well as the maturity of the colony.(12) Mycotoxin production can also vary within a species or even within a strain of species, particularly as observed for Stachybotrys spp., further complicating the difficulties with studies of the risk.(9)
Mycotoxins explain the hallucinogenic properties of certain mushrooms associated with recreational drug use.

Additional substances released by degrading mold include enzymes and glucans, both of which can be irritating and evoke an immune system response. (1) At this time, these substances would appear to be secondary to or part of the exposure response to mycotoxins.

Mycotoxins should not be confused with mold VOCs (Volatile Organic Compounds), which are also produced by mold. It is generally well established that humans, pets and even building products emit some of these compounds. There is no evidence that they cause health concerns as they are present in water damaged and moldy buildings at levels well below toxic levels. (2) Human sensitivity in the detection of certain mold VOCs such as found in the so-called “musty odor” rivals or even exceeds some analytical detection methods! (12) 

How to Test for Mycotoxins 
Unfortunately, current laboratory detection methods do not allow for reliable detection of mycotoxins in an air sample. For environmental testing, the principal means of testing these trace substances is with a sterile gauze wipe of a dense accumulation of dust. A target sample of at least 3 grams of dust is needed to achieve the recommended detection limit, which can be difficult to obtain in clean homes. This method of dust testing in a home remains relatively expensive, generally starting at about $400 per sample. Fortunately, it has been reported that composite samples from multiple locations of the home are appropriate and reliable predictors of exposure. Therefore, typically only one dust sample is needed unless there are distinctly different mold problems within a large home. The identification of specific species of mold in dust can be diagnostic for fingerprinting purposes. However, like mold spore air tests, there are no health standards and little published data on normal background levels. Therefore, unless a sample results are very low or very high, the test results are rather subjective in nature and can be difficult to interpret.Testing of mycotoxins in blood serum, nasal fluid and urine can be performed by a doctor. The cost is about $400-700, depending on the specific test utilized. You can also order a urine test kit online. The methodology for testing of mold toxins in body fluids remains controversial. According to Quackwatch.org, the prevailing medical opinion is that it is of unproven diagnostic utility and that health standards for mold toxins in urine have not been developed. There is no assurance that mycotoxins are being efficiently excreted in a sick individual. Furthermore, these tests have not been approved by the Center for Disease Control (CDC) or FDA. While the tests have not been scientifically proven based on the rigorous and legally-defensible validation requirements of the medical field, a test could provide anecdotal and useful information in some circumstances, particularly if a very low or high result is obtained or if a carefully controlled methodology is utilized and applicable excretion rates are accounted for. If someone has persisted for years with undiagnosable or untreatable symptoms by conventional medicine, it is understandable if people look to unproven medicine for answers. 

One published article demonstrated an anecdotal connection between the tissue cultures of surgically removed nasal polyps with mycotoxins in urine samples. The cultured mold type Aspergillus Niger is a producer of Ochratoxin, which was found elevated in the patient’s urine sample.(10)

Mycotoxin testing of blood has proven problematic. In a study of the blood levels of trichothecene in 33 patients, the results did not show any significant difference between mold symptomatic individuals compared to the 17 non-symptomatic persons used as controls, possibly explained by the rapid removal from the blood. (18)

A study of people suffering from CRS determined that viable mold was recovered from the sinuses, including Aspergillus (flavus, niger, fumigatus, versicolor), Chaetomium, Fusarium, Penicillium and Trichoderma. In addition, other trapped mold particulate was recovered including hypha, conidia and spores. However, positive swab cultures from nasal mucous were found in 91% of patients and 91% of healthy individuals used as controls, indicating their mere presence is not diagnostic of a problem. However, a study of nasal washings showed that mycotoxins could be detected in the infected individuals but was not present in the healthy control group. This suggests the nasal washing method may be the most reliable of all the proposed medical mycotoxin tests.(17)

The testing for antibodies to mold (immunoglobulins) is less expensive than for mycotoxins. However, these tests are not helpful except in the case of very unusual circumstances that cause unusual molds to grow since nearly everyone has been exposed to and therefore carries antibodies to all the major types of mold. Blood tests for specific antigens or antibodies have been shown to be useful in the diagnosis of some fungal infections, but these are the exception not the rule. (3) One laboratory advertises for blood serum testing for identification of 12 common antibodies to mycotoxins for a cost of $380. This laboratory operates offshore and does not include any official certifications. These tests are problematic because there are a number of interferences, people develop antibodies from low-level background exposures and a lack of discrete specificity among most antibodies (e.g. many antibodies cannot be conveniently linked to specific mold types, Histoplasma spp., a disease associated with bird and bat droppings being a notable exception).(19)

Unfortunately, current laboratory detection methods do not allow for reliable detection of mycotoxins in an air sample. For environmental testing, the principal means of testing these trace substances is with a sterile gauze wipe of a dense accumulation of dust. A target sample of at least 3 grams of dust is needed to achieve the recommended detection limit, which can be difficult to obtain in clean homes. This method of dust testing in a home remains relatively expensive, generally starting at about $400 per sample. Fortunately, it has been reported that composite samples from multiple locations of the home are appropriate and reliable predictors of exposure. Therefore, typically only one dust sample is needed unless there are distinctly different mold problems within a large home. The identification of specific species of mold in dust can be diagnostic for fingerprinting purposes. However, like mold spore air tests, there are no health standards and little published data on normal background levels. Therefore, unless a sample results are very low or very high, the test results are rather subjective in nature and can be difficult to interpret.







Mycotoxins and Nanotoxins
Mycotoxins are not only linked to spores, conidia and hyphae (mold structures of growth visible under a normal microscope). Recent research interest has increased in fine mold fragment cell debris, enzymes and other metabolic residues (crudely known as mold “nanotoxins”). The black mold Stachybotrys chartarum as well as other molds and bacteria produce large quantities of ultrafine nanometer range fragments (0.03-0.3 microns in diameter) when compared to airborne spore counts. These nanotoxin particles are too small to be seen and identified in a standard air test. However, the number of nano-sized mold fragments present is estimated to be least 300 times greater than the spore counts. The respiratory deposition of these fine fungal fragments has been measured at 230 times that of spores.(20)

Due to the difficulty in measuring nanotoxin fragments, additional research is needed to understand how much health impact is associated with these nearly invisible mold particles. Bulk mycotoxin dust samples include the mycotoxin contributions from these tiny particles in addition to the larger mold spores, hyphae and conidia. In certain situations where a mold source has been eliminated, the larger cellular mold particulate may be degraded but a substantial amount of smaller mold nanotoxins may remain from the initial stage of the decomposition process.

A Case Study on Mycotoxin Testing
A recent mold remediation industry webinar featured a case study involving mycotoxins. A house was known to have persistent water-damage problems from mold for a number of years, causing chronic illness of a resident. After the problem was corrected and all mold cleaned up, the resident’s symptoms did not clear up. Mold spore testing indicated the house was fine with no evident mold problems. Follow up dust testing discovered elevated levels of mycotoxins in the home. A subsequent thorough inspection of the home found that a custom-built wood enclosure was built over a sump pump in the basement. The malfunction of the sump pump had caused saturation of the interior cabinet of the wood, causing suspect mold and bacteria growth. Unfortunately, testing of the wood was not performed. The resident was also not medically examined for fungal infection. After the cabinet was removed and the house cleaned, a re-test of the dust was performed, resulting in non-detectable levels of mycotoxins. (21) Due to the insufficient control or evaluation of the variables in this case study including testing for bacterial endotoxins, it was inconclusive whether the mycotoxins could be linked to past problems, the moldy wooden cabinet or the resident’s health problems. A valuable opportunity to better understand mycotoxins was lost!
Conclusions
Mold spore testing of air samples will remain the dominant and most reliable measure of assessing mold impact in the near future. Dust testing has some practical uses but will remain cost-prohibitive for most typical mold assessment and remediation options. There is a need for the development of new test methods involving mold degradation products including nanotoxins. Medical test methods involving mycotoxins remain costly and largely experimental. Those of us in the industry can look forward to and hope for new and more affordable test methods to be developed with an increase in sensitivity as well as a better understanding that the role that mycotoxin particulate may play in health effects.

An improved understanding of how mold affects that body demonstrates that correcting a mold problem will not necessarily result in a reduction of chronic health symptoms. People who live in a mold remediated home should be evaluated by a doctor. Clinical or subclinical respiratory infections could persist, particularly if a resilient biofilm is present. Antifungals may be prescribed to eliminate any low level infections but sometimes surgical polyp removal or other invasive procedures may be required. One study of mycotoxin exposed individuals showed that 90% had a dramatic improvement in health after medical treatment.(22)

There are many diseases and disorders that have not been adequately explained to date. We should keep an open mind to mycotoxins or other mold-related health impacts being linked to their cause.

Tri-Tech can provide environmental mycotoxin testing services for projects where other test methods have proven unsatisfactory or insufficient to assess the mold concern in question. Tri-Tech can also provide an expert second opinion for mold assessments and remediation projects and advise whether mycotoxin testing may be necessary.


References

1.Toxic Effects of Indoor Molds, Official Journal of the American Academy of Pediatrics, 1998.
2.Building Dampness and Its Effect on Indoor Exposure to Biological and Non-Biological Pollutants, World Health Organization, 2009.
3.Determination of Aflatoxin B1 Levels in Organic Spices and Herbs". The Scientific World Journal, 2013.
4.Endocrine Activity of Mycotoxins and Mycotoxin Mixtures, Food & Chemical Toxicology, 2016 
5. Ochratoxin A and Human Health Risk: A Review of the Evidence; Critical Reviews of Food Science and Nutrition, 2016.
6.Mold‐sensitivity In Children with Moderate‐Severe Asthma is Associated With HLA‐DR and HLA‐DQ , European Journal Of Allergy And Clinical Immunology, 2010.
7.Abstract of Unpublished Paper Presented at American Academy of Allergy, Asthma and Immunology Annual Meeting, 2001. 
8.Thrombosis and Haemostasis,”. Department of Anesthesiology and Intensive Care E-Publication, University of Muenster, 2010.
9.Mycotoxin Production by Indoor Molds. Fungal Genetics and Biology, 2003.
10.Chronic Illness Associated With Mold and Mycotoxins: Is Naso-Sinus Fungal Biofilm the Culprit? National Institute of Health, 2013.
11.Detection of Mycotoxins in Patients With Chronic Fatigue Syndrome, Toxins (Basel/Switzerland), 2013. 
12.Spectrum of Noninfectious Health Effects From Molds, Official Journal of The American Academy of Pediatrics, 2006.
13.Further Evidence for Allergic Pathophysiology in Allergic Fungal Sinusitis. Laryngoscope, 1998
14.Neurologic and Neuropsychiatric Syndrome Features of Mold and Mycotoxin Exposure, Toxicology and Industrial Health, 2009.
15.Exposure Assessment to Mycotoxins in Gluten-Free Diet for Celiac Patients, Food and Chemical Toxicology, 2014.
16.Fusarium Spp. and Storage Fungi in Suboptimally Stored Wheat: Mycotoxins and Influence on Wheat Gluten Proteins, Mycotoxin Research, 2003.
17.Mycotoxin Detection in Human Samples from Patients Exposed to Environmental Molds. International Journal of Molecular Sciences, 2009.
18.Immune Response Among Patients Exposed to Molds, International Journal of Molecular Sciences, 2009.
19.Clinical Use of Immunoassays in Assessing Exposure to Fungi and Potential Health Effects Related to Fungal Exposure, Annals of Allergy, Asthma & Immunology, 2004.
20.Fungal Fragments as Indoor Air Biocontaminants, Applied Environmental Microbiology, 2002.
21.Latest Research Demands New Risk Assessments: Mycotoxins in Mold Environments; Julie And Bill Nicoll, Restoration and Remediation Magazine, March 2020.
22.Neurological Disease After Mold Exposure, Immune Risks & Response to Biofilm-Focused Antifungal Therapy; Proceedings of the 52nd Annual Interscience Conference on Antimicrobial Agents and Chemotherapy Conference; 2013.

Stachybotrys
Fruiting structure of the black mold
Stachybotrys developing mold spores
Mycotoxin Symptoms
Mycotoxin poisoning is generally well understood from ingestion from moldy foods, particularly by persons who have allergies or other sensitivities. Symptoms are generally consistent with general food poisoning symptoms including vomiting, nausea and diarrhea. Fortunately, due to modern food production procedures and testing requirements, acute mycotoxin poisoning from ingestion of contaminated food is rare. 

Occupational exposures and symptoms vary widely, depending on the route of exposure (e.g. inhalation or dermal contact). Generally, inhalation of mycotoxins will cause as a minimum respiratory irritation (e.g. cough or severe congestion). Direct contact will cause physical irritation (e.g. itching, burning, blisters etc.). Low level chronic exposures can cause a number of different health symptoms and diseases, which is reviewed further here.