Case definition of mold illness Shoemaker

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APPENDICES

1. Case definition of mold illness

The case definition of an adult mold illness patient contains two

tiers as follows: any

diagnosis of environmentally acquired biotoxin illness, including

that from mold, must

include:

(1) the potential for exposure;

(2) the presence of a distinctive grouping of symptoms; and

(3) the absence of confounding diagnoses and exposures.

This first tier of the case definition is adopted from the initial

CDC case definition of

Pfiesteria cases from 1998. The second tier of objective factors

includes three of six of

the following:

(1) HLA DR by PCR showing susceptibility;

(2) reduced levels of melanocyte stimulating hormone (MSH) in a

properly

performed specimen;

(3) elevated levels in matrix metalloproteinase-9 (MMP9) in a

properly

prepared serum specimen;

(4) deficits in visual contrast sensitivity (VCS);

(5) dysregulation of ACTH/cortisol in simultaneously obtained

specimens;

(6) dysregulation of ADH/osmolality in simultaneously obtained

specimens.

This second tier is adapted from similar use of different parameters

in illnesses such as

systemic lupus erythematosis and rheumatic fever, among others. The

case definition is

derived from looking at what thousands of mold illness patients

demonstrated that none

of the control patients demonstrated.

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For children, the case definition necessarily must exclude pituitary

hormone

abnormalities. What we have documented is an unusual increase in

autoimmune factors

in children with mold illness compared to control children. Our case

definition for

children must also account for the delay in maturation of the

neurons involved with

development of contrast. We include in our case definition in

children five elements on

the second tier, two of which must be present, including HLA DR,

antigliadins,

anticardiolipins, MMP9 elevation and MSH deficiency. This case

definition was

presented 12/16/05 at the ASTMH meetings in Washington, DC.

2. Visual Contrast Sensitivity

Testing

All subjects who normally wore corrective lenses for near-point

viewing were asked to

wear them during vision testing. The visual acuity and VCS tests

were administered

monocularly to each eye; an eye occluder was held over one eye while

the other eye was

tested. All vision tests were administered under illumination from

a " daylight "

illuminator (fluorescent source with a correlated color temperature

of approximately =

6500E K; color rendering index > 90; intensity = 1150 lux; luminance

approximately 70

foot-lamberts) in a clinical unit with normal background lighting. A

light meter was used

to insure that luminance remained constant throughout the test

sessions. A test card

holder, consisting of a face rest placed just under the cheek bones

or chin as comfort

provided, and connected by a calibrated rod to a card holder on the

distal end, was used

to position the acuity and VCS test cards at a constant distance,

previously standardized,

from the eyes (acuity - 36 cm (14 inches); contrast sensitivity - 46

cm (18 inches)).

Near Visual Acuity

The acuity test card (MIS Pocket Vision Guide, © 1997 MIS, Inc.)

contained 10 rows of

numbers in which the size of the numbers progressed from a larger

size in the top row to

a smaller size in the bottom row. Participants were asked to first

read the numbers in a

middle row. Testing proceeded to the next lower row if all numbers

were correctly

identified or to the next higher row if an error occurred. The

Snellen visual acuity of the

row (20/20 or 20/30, for example) with the smallest numbers each

identified correctly

was recorded as the visual acuity score. Two-tailed Student t-tests

0.05 were performed,

using the mean score of each participant's two eyes, to determine if

scores differed

significantly between cohorts.

Contrast Sensitivity (VCS)

The contrast sensitivity test card (Functional Acuity contrast Test,

(FACT), Stereo

Optical Co., Chicago, IL, a Gerber-Coburn Co.) contained a matrix (5

x 9) of circles

filled with sinusoidal gratings (dark and light bars). Spatial

frequency (1.5, 3, 6, 12 and

18 cycles/degree of visual arc) increased from top to bottom, and

contrast decreased from

left to right in steps of approximately 0.15 log units. The grating

bars were oriented

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either vertically, or tilted 15 degrees to the left or right. As the

investigator called out

each circle from left to right, row by row, subjects responded by

saying either: vertical,

left, right or blank. Participants were encouraged to name an

orientation if she had any

indication that the bars could be seen. Participants were given the

option to point in the

direction to which the top of the grating was tilted if she felt any

difficulty in verbalizing

the orientation; none needed this assistance. The contrast

sensitivity score for each row

(spatial frequency) was recorded as the contrast of the last test

patch correctly identified

on that row following verification by repeated testing of that patch

and the subsequent

patch. The procedure was repeated for each row in descending order.

The a priori

criterion for the inclusion of data in analyses was that the eye has

a visual acuity (Snellen

Distance Equivalent Score) of 20:50 or better, in order to avoid

confounding of the VCS

results by excessive optical-refraction error. All eyes included in

the data analyses met

the visual acuity criterion.

Data Analysis

The units of analysis for the VCS test were the mean scores of the

participant's two eyes

at each spatial frequency. Standard error of the mean was calculated

for each group of

measurements. The VCS data were analyzed using multivariate analyses

of variance

(MANOVA, with the Wilks' lambda statistic) procedures suitable for

repeated measures

with + = 0.05. The factors in the model were group and spatial

frequency. A factor for

gender was not included since there aren't any gender differences in

susceptibility to

biotoxin-induced effects shown as yet, and no gender differences in

VCS have been

reported. Results that showed a significant group-by-spatial

frequency interaction were

further analyzed in the step-down, two-tailed Student t-tests (+ =

0.05), the equivalent of

a univariate ANOVA to determine which spatial frequencies accounted

for the overall

effect.

3. Laboratory studies

LabCorp, Inc. and Quest Diagnostics, each CLIA approved, high

complexity, national

laboratory facilities. Factors analyzed included:

MSH: Alpha melanocyte stimulating hormone (MSH) is a 13 amino acid

compound

formed in the ventromedial nucleus (VMN) of the hypothalamus,

solitary nucleus and

arcuate nucleus by cleavage of proopiomelanocortin (POMC) to yield

beta-endorphin and

MSH. MSH exerts inductive regulatory effects on production of

hypothalamic

endorphins and melatonin. MSH has multiple anti-inflammatory and

neurohormonal

regulatory functions, exerting regulatory control on peripheral

cytokine release as well as

on both anterior and posterior pituitary function. Deficiency of

MSH, commonly seen in

biotoxin-associated illnesses, is associated with impairment of

multiple regulatory

functions and dysregulation of pituitary hormone release. Symptoms

associated with

MSH deficiency include chronic fatigue and chronic, unusual pain

syndromes. Normal

values of MSH established in research labs and in commercial labs

are 35-81 pg/ml. I

note that the recent shift in normal range for MSH from LabCorp to 0-

40 pg/ml was made

following the receipt of so many low values of MSH. I have

questioned both Dr. Andre

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Valcour and Dr. Marsella of LabCorp about this change; they

have received

case/control data sets from me and assure me that they will review

the adjustment of

normal ranges that were made only after lumping values for cases and

control together.

No lab, including LabCorp, can logically equate a case value of a

test with a control value

for a test.

Leptin: Leptin is a 146 amino acid adipocytokine produced by fat

cells in response to

rising levels of fatty acids. Leptin has peripheral metabolic

effects, promoting storage of

fatty acids, as well as central effects in the hypothalamus.

Following binding by leptin to

a long isoform of the leptin receptor in the VMN, a primordial gp-

130 cytokine receptor,

a JAK signal causes transcription of the gene for POMC, which is in

turned cleaved to

make MSH. Peripheral cytokine responses can cause phosphorylation of

a serine moiety

(instead of threonine) on the Leptin receptor, creating leptin

resistance and relative

deficiency of MSH production. Normal values in commercial labs show

differences

between males (5-8 ng/ml) and females (8-18 ng/ml), with levels of

leptin correlated with

BMI. In the presence of MSH deficiency, the relationship between

body weight and

leptin changes, as leptin elevation becomes disproportionate to

weight.

ADH/osmolality: abnormalities in ADH/osmolality are recorded as

absolute if ADH is <

1.3 or > 8 pg/ml; or if osmolality is >295 or <275 mOsm/kg.

Abnormalities are recorded

as relative if simultaneous osmolality is 292-295 and ADH < 2.3; or

if osmo is 275-278

and ADH> 4.0. Symptoms associated with dysregulation of ADH include

dehydration,

frequent urination, with urine showing low specific gravity;

excessive thirst and

sensitivity to static electrical shocks; as well as edema and rapid

weight gain due to fluid

retention during initial correction of ADH deficits.

ACTH/cortisol: abnormalities in ACTH/cortisol are absolute if AM

cortisol > 19 ug/ml

or < 8 ug/ml; or if AM ACTH is >60 pg/ml or < 10 pg/ml.

Abnormalities are recorded as

dysregulation if simultaneous cortisol is > 15 and ACTH is > 15, or

if cortisol is < 8 and

ACTH <40. Early in the illness, as MSH begins to fall, high ACTH is

associated with

few symptoms; a marked increase in symptoms is associated with a

fall in ACTH.

Finding simultaneous high cortisol and high ACTH may prompt

consideration of ACTH

secreting tumors, but the reality is that the dysregulation usually

corrects with therapy.

Androgens: total testosterone, androstenedione and DHEA-S provide

measurements

regarding the effectiveness of gonadotrophin secretion as influenced

adversely by MSH

deficiency. Normal ranges of these hormones in males are 75-205

ng/ml for

androstenedione, 350-1030 ng/ml for testosterone and 70-218 ug/ml

for DHEA-S.

Normal values for pre-menopausal women are 60-245, 10-55 and 48-247,

respectively.

Post-menopausal normal ranges are 30-120, 7-40 and 48-247,

respectively.

HLA DR by PCR: LabCorp offers a standard HLA DR typing assay of 10

alleles using a

PCR sequence specific chain reaction technique. As opposed to

serologic assays for the

HLA DR genotypes, the PCR gives far greater specificity in

distinguishing individual

allele polymorphisms. Linkage disequilibrium is strong in these

genotypes, with multiple

associations made to inflammatory and autoimmune disease. These

genes are part of the

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human major histocompatibility complex (MHC), also called the HLA

complex, and

located on the short arm of chromosome 6. Relative risk was

calculated, susceptible

genotypes identified, compared within each group to location and

exposure. The HLA

doesn't by itself say that mold makes a patient sick: the increased

relative risk in cases

versus controls shows the increased individual susceptibility

expressed as a statistical

ratio,

MMP9: matrix metalloproteinase 9 (gelatinase is an extracellular

zinc-dependent

enzyme produced by cytokine-stimulated neutrophils and macrophages.

MMP9 is

involved in degradation of extracellular matrix; it has been

implicated in the pathogenesis

COPD by destruction of lung elastin, in rheumatoid arthritis,

atherosclerosis,

cardiomyopathy, and abdominal aortic aneurysm. Cytokines that

stimulate MMP9

production include IL-1, IL-2, TNF, IL-1B, interferons alpha and

gamma. MMP9 is felt

to play a role in central nervous system disease including

demyelination, by generation of

myelin peptides, as it can break down myelin basic protein.

MMP9 " delivers "

inflammatory elements out of blood into subintimal spaces, where

further delivery into

solid organs (brain, lung, muscle, peripheral nerve and joint) is

initiated. Normal ranges

of MMP9 have a mean of 150, with range of 85-322 ng/ml.

C3a and C4a: Split products of complement activation, often called

anaphylatoxins.

Each activates inflammatory responses, with spillover of effect from

innate immune

response to acquired immune responses and hematologic parameters.

These short-lived

products are re-manufactured rapidly, such that an initial rise of

plasma levels is seen

within 12 hours of exposure and sustained elevation is seen until

definitive therapy is

initiated. The components increase vascular permeability, release

inflammatory elements

from macrophages, neutrophils and monocytes, stimulate smooth muscle

spasm in small

blood vessels and disrupt normal apoptosis. They also recruit

additional inflammatory

generators, such as chemokines, into action.

Anticardiolipins IgA, IgM and IgG: autoantibodies often identified

in collagen

vascular diseases such as lupus and scleroderma; often called anti-

phospholipids. These

antibodies in high titers are associated with increased

intravascular coagulation requiring

treatment with heparin and coumadin. Lower level titers are

associated with

hypercoagulability. An increased risk of spontaneous fetal loss in

the first trimester of

pregnancy is not uncommonly seen in women with presence of

cardiolipin antibodies.

This problem does not have the same " dose-response " relationship

seen with levels of

autoantibodies and illness, as does the antiphospholipid syndrome.

Anticardiolipins are

found in over 33% of children with biotoxin-associated illnesses.

Antigliadin IgA and IgG: Antibodies thought at one time to be

specific for celiac

disease. With the advent of testing for IgA antibodies to tissue

transglutaminase (TTGIgA),

gliadin antibodies are most often seen in patients with low levels

of MSH.

Ingestion of gliadin, the 22-amino acid protein found in gluten

(found in wheat, oats,

barley and rye; often added to processed foods) will initiate a

release of pro-inflammatory

cytokines in the tissues lining the intestinal tract. This cytokine

effect will often cause

symptoms within 30 minutes of ingestion that mimic attention deficit

disorder, often

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leading to an incorrect diagnosis. Antigliadin antibodies are found

in over 58% of

children with biotoxin-associated illnesses.

Vasoactive intestinal polypeptide (VIP): neuroregulatory hormone

with receptors in

suprachiasmatic nucleus of hypothalamus. This hormone/cytokine

regulates peripheral

cytokine responses, pulmonary artery pressures and inflammatory

responses throughout

the body. VIP raises intracellular levels of cAMP, a vital secondary

messenger of cell

signaling. Deficiency is commonly seen in mold illness patients,

particularly those with

dyspnea on exertion.

4. ABB`AB protocol

The foundation of these conclusions includes the use of a repetitive

exposure protocol,

called ABBÌ AB. This protocol is widely used in assessment of

causation in research,

medicine and industry; it is one of the oldest and most used in all

science. We accept the

possibility that patients with exposure to toxigenic fungi and with

multi-symptom,

multisystem illnesses could potentially be due to many sources. The

5-step protocol

answers this possibility and gives academic certainty to the cause

of illness (Figure 9, p.

10). Our baseline (Base) evaluation, including symptoms, VCS, labs

and exposures is

compared to the same parameters measured after treatment with CSM

(AC-1). After AC-

1, the improvement of the patient is documented, with reduction of

symptoms and VCS

deficits; amelioration of the multiple biomarker abnormalities is

also documented. Next,

the patient is kept away from the affected environment for at least

three days and

medication is discontinued (HOC). Here, we look at the superficial

argument often used

by defense apologists that purports to say, " Why, that dastardly

mold is everywhere; any

exposure could cause the illness. " Actually, amplified mold growth,

the true source of

illness, is rarely found in multiple environments for a given

affected individual. Then,

after documenting that nothing changed for the patient during HOC,

he is re-exposed to

the " suspect " environment (BOC) without use of CSM. The patient is

re-evaluated in

three days, ideally with symptom recording and lab evaluation done

each of the three

days.

Next, causation of the illness from exposure to indoor air in the

contaminated residence is

shown by recrudescence of symptoms and relapse in all the biomarkers

(three days is all

that is necessary: there is no dose-response relationship here, the

genetic basis for

susceptibility shows re-acquisition of the illness, complete with

abnormalities in

biomarkers with a short duration exposure isn't statistically

different from that seen with

long-term exposure). Representative VCS slides documenting

graphically the results of

the protocols we use are presented in Appendices Five 1-8.

The data from sequential monitoring during diagnostic re-exposure

demonstrate a

sequential activation of complement (C3a and C4a), cytokines

(primarily IL-1B) on Day

1; followed by a rise in leptin on Day 2; and a rise of MMP9 and

change in VEGF on

Day 3. Symptoms usually begin to recur within hours of unprotected

re-exposure, with a

fall in VCS seen usually on Day 2 to Day 3. These data were

presented at a February

2006 meeting of the American Society for Microbiology Conference on

Biowarfare and

Biodefense. Once the causative re-exposure occurs, clearly

documented by temporal

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association with the contaminated building, the patient is re-

treated, with documentation

of improvement (AC-2). For those who stay in the contaminated

environment, and many

must because of financial need, or for those who are primed for

illness following

exposure to other environments with amplified growth of mold,

prophylaxis with CSM is

offered.

The 5-step protocol has the scientific power to demonstrate

causation, as shown in

multiple studies in animals and humans. There is no known study

design that can show

greater power of causation: prospective acquisition in a time- and

location-specific

manner in a known affected, but healed individual. This approach is

not novel; it has

been routinely used outside litigation in animal and human research.

It has its basis in the

classic work by Dr. Koch, as he developed Koch's Postulates

for causation of

illness by infectious agents.