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CIRS: Part 3-The Biotoxin Pathway & Tier Two Diagnosis

CIRS: Part 3-The Biotoxin Pathway & Tier Two Diagnosis

CIRS: An Examination of Chronic Inflammatory Response Syndrome from Exposure to Recovery (Part 3-The Biotoxin Pathway & Tier Two Diagnosis )

Chronic Inflammatory Response Syndrome (CIRS), biotoxin-related illness, sick-building syndrome, and mold illness...these are all names for a condition caused by exposure to biotoxins (biological toxins). CIRS affects many systems of the body, often causing numerous, seemingly unrelated symptoms.1 To learn what biotoxins are and how you can get exposed, please read part one of this series. The second part of this series covers tier-one CIRS diagnosis. And this post will address tier two diagnosis. My goal for these articles is to educate and empower so you can reverse the root cause of your condition and reclaim your joy in living.

What is the Biotoxin Pathway and How Does It Relate to Tier Two CIRS Diagnosis?

The biotoxin pathway is the mechanism of chronic inflammatory response syndrome (CIRS) in the body. For most people, the immune system tags biotoxins as foreign invaders. They’re then broken down and cleared from the body through the liver.

For others, including those with a genetic susceptibility, the body is unable to identify and tag biotoxins for elimination, so they circulate unchecked. If exposure continues, biotoxins begin to build up in the body, binding to toll receptors on many kinds of cells, especially fat and endothelial cells.

The binding of biotoxins to toll receptors causes cytokine production. Cytokines are messenger proteins that signal an inflammatory immune response, often causing flu-like symptoms as well as other symptoms typical in CIRS. You can read about CIRS symptom clusters in part two of this series.

The continuous circulation and binding of biotoxins to toll receptors and the resulting continuous production of cytokines causes inflammation to cascade out of control, leading to dysregulation of many biological processes further along the biotoxin pathway. This dysregulation is measurable and is used in tier two diagnosis to help verify whether or not you have CIRS.

Tier two diagnosis includes a series of tests, including bloodwork, that measures specific markers of inflammatory damage. Not all tests are required for all patients. It’s important to work with a Shoemaker-certified practitioner or other skilled physician who’ll determine what tests to order and explain your results. The following tests are used to help make a tier two CIRS diagnosis:

Visual Contrast Sensitivity (VCS) Test

Biotoxins disrupt nerve cell function. That is why tier two screening commonly starts with the Visual Contrast Sensitivity Test (VCS). Contrast is your ability to see an edge such as a black picture frame on a white wall. The VCS test measures your vision at low contrasts and is an indicator of proper neurologic function.

For VCS testing to be accurate, your vision (natural or corrected) has to be 20:50 or better. If you have glasses, you’ll wear them during the test. While VCS is most accurate when done in your physician’s office, there’s an online test available that you can take right now.

92% of those with CIRS fail the VCS test. 8% of people with CIRS such as teenagers with great vision will pass the test anyway. And 1% of people that don’t have CIRS will still fail the VCS. If you fail the VCS test and your cluster analysis was positive, there’s a 98.5% chance you have CIRS.2

Human Leukocyte Antigen (HLA) Genetic Test

Human leukocyte antigens are inherited genes (one from each parent) that help control the process of clearing toxins through your adaptive immune system. These genes are responsible for antibody formation when an antigen (foreign invader like a biotoxin, bacteria, or virus) enters your body.

In some CIRS patients, there are genetic mutations that disable this process, leaving biotoxins free to move through the body. It’s estimated that 24% of the population has this genetic susceptibility.3 These mutations can explain how only one member of an entire family might be sick after exposure to a water-damaged building. Your practitioner may order this genetic test to confirm or rule out an HLA mutation.

Increased Complement Split Products C3a and C4a

Within the innate immune system, the complement system enhances (or complements) your body’s ability to clear pathogens and dead or dying cells through the regulation of inflammation. In chronic inflammatory response syndrome, the complement system is upregulated, causing elevated levels of C3a and C4a. The result is hypoperfusion (reduced blood flow) with symptoms that may include fatigue, cognitive impairment, and respiratory problems.

C4a is commonly elevated within 12 hours of biotoxin exposure and remains high until therapy is started. For those who’ve recovered from CIRS, a single 15-minute re-exposure can cause elevated C4a levels within four hours, making complement split product testing useful to assess remediation efficacy and building safety.4 This test measures the amount of C4a or C3a circulating in your bloodstream. Normal C4a range is 0-2830 ng/ml and normal C3a range is 0-940 mg/ml.

Transforming Growth Factor Beta-1 (TGF-b1) and T-Regulatory Cells

TGF Beta-1 is a cytokine that both initiates and suppresses the inflammatory immune response. It plays a key role in regulating the innate immune system by controlling growth, differentiation, activation, and death of immune cells.

In CIRS, TGF-b1 is elevated, which impairs the function of T-regulatory cells and may lead to fibrosis, skin and liver changes, other lung-related diseases, and autoimmunity.5 TGF-b1 may also contribute to cognitive difficulties resulting from a leaky blood-brain barrier that allows inflammatory agents into brain tissues. The normal range for TGF-b1 is <2380 pg/ml.

Matrix Metallopeptidase-9 (MMP-9 )

Matrix metallopeptidase-9 is an enzyme involved in breaking down and repairing tissues via the extracellular matrix and is especially important in vascular remodeling. When the innate immune system is dysregulated and elevated cytokines bind to white blood cells, MMP-9 is over-released into the bloodstream. It then breaks down proteins in cell walls, weakening them to the point of destabilization. The cell walls become permeable, and inflammatory agents are free to enter the tissue and cause damage. In CIRS, MMP-9 is elevated above the normal  range of 85-332 ng/mL.6


Leptin is produced in fat cells and is the hormone that tells the brain you’re full. In CIRS, leptin is elevated, damaging leptin receptors on the hypothalamus and causing leptin resistance. This resistance causes the body to hold and store fatty acids as fat. Rapid weight gain that diet and exercise don’t correct is often seen in CIRS patients as a result.

Also in the hypothalamus, leptin activates pro-opio-melanocortin (POMC) neurons, which release alpha melanocyte stimulating hormone (MSH). Because of leptin-receptor damage, POMC pathway communication is impaired and MSH production is reduced, leading to the loss of hormonal regulation.7 Normal leptin ranges are 0.5-13.8 ng/mL for males and 1.1-27.5 ng/mL for females.

Melanocyte Stimulating Hormone (MSH)

Melanocyte stimulating hormone is a neuro-regulatory hormone that modulates other endocrine hormones and plays a part in regulating mucus membranes. Low MSH causes CIRS symptoms to cascade out of control. It’s linked to leaky gut, inflammatory bowel disease, and malabsorption. Loss of pain control is common as is the development of Celiac-related antibodies. Low MSH can upregulate aromatase, which increases estrogen and lowers testosterone and DHEA. In mold-related CIRS, MSH will be low in over 95% of patients8. The normal range for MSH is 35-81 pg/mL.

Multiple Resistant Coagulase Negative Staph (MARCoNS)

MARCoNS are staph bacteria often found in the nasal passages of CIRS patients. Overuse of antibiotics has allowed these bacteria to develop antibiotic resistance. In addition, they produce and surround themselves in biofilm, which antibodies can rarely penetrate. Within the biofilm, they’re free to live in undetected colonies, acting like multicellular organisms, which can turn on certain genes and affect gene expression.

MARCoNS and MSH have a bidirectional relationship. MSH is meant to protect the mucus membranes in the nose from colonization, but MSH deficiency breaks down the protective barrier. Up to 80% of MSH-deficient patients will have colonized bacteria compared to normal controls.9 Once colonized, MARCoNS suppresses MSH by releasing small proteins called hemolysins that destroy red blood cells. The bacteria then consume the iron, depleting your body’s required supply. A deep nasal swab is cultured to test for the presence of MARCoNS in your nose.

Vasoactive Intestinal Peptide (VIP)

Vasoactive intestinal peptide (VIP) is a neuro-regulatory hormone produced in the hypothalamus, the  gut, and the pancreas. VIP is a vasodilator, regulating inflammation and blood flow, and plays a significant role in intracellular communication and regeneration. When VIP levels are properly regulated, it suppresses cytokines and TGF-b1 and modulates T-regulatory cells. VIP levels are reduced in CIRS, causing dysregulation of these processes. Reduced VIP may lead to chemical sensitivity, fatigue, joint pain, cognitive impairment, depression, and shortness of breath.10 VIP therapy is used in the final step of the Shoemaker Protocol to treat CIRS. Normal VIP levels are 23-63 pg/mL.

Antidiuretic Hormone (ADH) and Serum Osmolality (ADH/Osm)

ADH is also known as vasopressin. This hormone is released from the pituitary when receptors in the brain detect an increase in osmotic pressure in the blood. ADH prevents water loss by the kidney, decreasing plasma osmolality, which is the body’s electrolyte-water balance. More than 80% of CIRS patients have ADH dysregulation11 that can cause dehydration, electric shock, excessive thirst, increased urination, and migraine headache. Normal ADH levels range from 1.0-13.3 pg/mL while normal osmolality ranges from 280-300 mOsm/L.

Adrenocorticotropic Hormone (ACTH) and Cortisol

ACTH is a hormone that controls the production of cortisol. ACTH operates through the hypothalamic-pituitary-adrenal axis, a hormonal feedback system that regulates the body’s response to stress.

It works when the hypothalamus is activated and releases a hormone that tells the pituitary to cleave proopiomelanocortin (POMC) into ACTH, MSH, and beta-endorphins. ACTH then stimulates the release of cortisol from the adrenals, which are found on top of the kidneys. Cortisol then inhibits the release of ACTH through a negative feedback loop, restoring homeostasis.

Chronic stress or inflammation disrupts this feedback, leading to low ACTH and cortisol. In CIRS, ACTH and cortisol may rise initially but will drop below the normal range in nearly 85% of CIRS cases.12 Normal cortisol ranges are 4.3- 22.4 ug/dL while ACTH ranges between 8-37 pg/mL are normal.

Vascular Endothelial Growth Factor (VEGF)

Vascular endothelial growth factor is a polypeptide that encourages the growth of blood vessels in order to increase blood flow to your tissues so they get enough oxygen. VEGF acts through receptors found on endothelial cells, which line the inner surface of blood vessels.

As it relates to the biotoxin pathway, VEGF initially spikes when high TGF beta-1 and cytokine levels attract white blood cells to capillaries, restricting blood flow and lowering oxygen levels in your tissues. VEGF rises in an attempt to increase blood flow and oxygen delivery through hypoxia inducible factors (HIF).

Over time, however, this process fails and VEGF levels decrease, causing cognitive impairment, fatigue, muscle cramps, and shortness of breath13. If you have CIRS, your VEGF levels may be lower than 31. Normal levels are between 31-86 pg/mL.

Antigliadin Antibodies IgG/IgA

Gluten sensitivity is one of many autoimmune responses associated with CIRS. Antigliadin antibodies are produced in response to the presence of wheat gliadin in the body. In biotoxin illness, dysregulation of  T regulatory cells  and low MSH14 upregulate this response, often leading to leaky gut from increased cellular wall permeability. Although elevated AGA is not specific to celiac disease, if your AGA levels are high, you should be tested for celiac disease and eliminate gluten permanently if you’re positive. Your AGA levels should be under 20 units with no antibodies detected.

Plasminogen Activator Inhibitor-1 (PAI-1)

Plasminogen activator inhibitor-1 is secreted by endothelial and fat tissue and is a marker for increased blood coagulation. PAI-1 increases clotting and inhibits fibrinolysis (the process that prevents blood clotting), increasing the risk of thrombosis (blood clots in veins or arteries). MMP-9 and PAI-1 together may let inflammatory agents into the subintimal space, causing fibrosis and atherosclerosis. In CIRS, PAI-1 is elevated above the normal range of 5-40 mg/l in cases.15

Anticardiolipin Antibodies (IgA, IgG, IgM)

Anticardiolipin antibodies affect the cell membrane, causing increased risk for abnormal bleeding and increased clotting. Bloody nose, miscarriage, thrombosis, low platelet counts, stroke, and pulmonary emboli and hypertensiona are common consequences of elevated anticardiolipin antibodies16. IgA levels should be less than 11 APL (IgA phospholipid units), IgG levels less than 14 GPL (IgG phospholipid units), and IgM levels less than 12 MPL (IgM phospholipid units).  

von Willebrand Profile

The von Willebrand Profile is an antigen test that measures the amount of von Willebrand factor, a protein that helps the blood to clot. Elevated levels of C4a can cause acquired von Willebrand syndrome, leading to abnormal bleeding and clotting.

MRI with NeuroQuant

MRI with NeuroQuant is an FDA-approved computer program that compares an individual’s brain structure volume measurements to those of a healthy population. Significant brain changes resulting from blood-brain barrier permeability are associated with biotoxin-related illness17. These changes, including microscopic changes in brain volume that are imperceptible to a radiologist, are detected by NeuroQuant.

Stress Echocardiogram

An echocardiogram is used to test resting pulmonary arterial pressure and  compare it to post-exercise pressure. An increase greater than 8 in pulmonary arterial systolic pressure is a marker for CIRS.


Transcriptomics measures mRNA and iRNA to monitor what genes are actively expressing, revealing abnormalities that result from the dysregulation of the biotoxin pathway in CIRS18. CD244, CD3D, CD48, CD52, granzymes, defensins, and the Ikaros family are immune regulators that are triggered differently in CIRS. New research in transcriptomics is streamlining CIRS diagnosis and treatment analysis. Some CIRS experts, including Dr. Shoemaker, are hopeful transcriptomics could replace much of the Tier I and II diagnostic process. A CIRS-specific transcriptomics test called the GENIE (Gene Expression: Inflammation Explained) is now available.

Mold Specific Quantitative PCR (ERMI)

The Environmental Relative Mold Index (ERMI) is a test for your home or any building you’re exposed to that may have water damage. ERMI is a DNA-based test that samples the air to identify specific mold species in your home. Although each recovering CIRS patient is different when it comes to their sensitivity, the ERMI test is a good indicator as to whether or not a building is safe. For most people, the ERMI score should be below two before re-entering a building.


If these tests confirm a tier two diagnosis, the 12-step Shoemaker Protocol is followed in sequential order to correct the dysregulated inflammatory response. My next article will talk about CIRS treatment using the Shoemaker protocol, including how continued laboratory testing is used to confirm successful completion of each step in the protocol.

Through the steps of the Shoemaker treatment protocol, the inflammatory markers inspected during tier two diagnoses are systematically corrected to reverse the biotoxin pathway and clear the biotoxins from the body, restoring homeostasis and health as long as there is no re-exposure. There is hope for full recovery, so look for my next blog post, the final in this four-part series.


  1. Shoemaker RC. Surviving Mold: Life in the Era of Dangerous Buildings. Otter Bay Books: Baltimore, MD, 2010.