Molds, Mycotoxins and
Public Health
Michael R. Gray, MD, MPH, CIME
Internal and Occupational Medicine
Emergency Medicine & Toxicology
In collaboration with
Kaye Kilburn, MD
Internal Medicine and Neurotoxicology
Robert Crago, Ph.D.
Psychology, Neurophysiology and
Neurotherapy
ImmunTox, LLC
PO Box 2050 Benson, Arizona 85602
11/11/02 APHA Philadelphia
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1
Case Study: Tucson Bank, 1994
• Bank Manager
• 9–10 Other Employees
– Similar symptoms
– 49 y.o. Married Female
• Shortness of breath
– Symptoms
• Cognitive problems
• 18 month hx arthritis
• Periorbital edema
(wheelchair bound)
• Skin rashes limited to
• General malaise
exposed skin.
• Excessive fatigue
• Workplace related
• Cognitive dysfunction
• Worsened on Mondays
w/ memory loss
• Improvement on
• Difficulty concentrating
weekends & vacations
• Multiple other
symptoms
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2
Case Study: Tucson Bank, 1994
• Clinical Findings
• Environmental findings
– Urticarial & granulomatous
skin changes
– Small airway obstruction &
reactivity
– Immunologic hyper
activation & simultaneous
suppression
– Multiple inflammatory
symptoms
– Elevated EBV IgG
antibodies
– Cognitive deficits
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– Presence of multiple
filamentous molds
– Pipes leaking in common
wall to air handler &
bathroom
– Elevated Stachybotrys
spore counts detected in:
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• Air and surface
• Highest on the manager’s
desktop
• All employees passed this
desk to go to break room
3
Hidden Fungi
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4
Historical Perspective
• Biblical Warning
• Occupational Disease
– If your house be
contaminated with
plagues, molds, and
Leprosy, put the
contents in the middle
and set it aflame
--Leviticus
– Brown Lung Disease
– Farmer’s Lung
– Pigeon Breeder’s
Disease
– Yellow Rice Disease
• Robigalia- April 25th
– 7th to 8th Century BC
– To protect trees & grains
from rusts or mildew
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Toxic Effects of Mycotoxins in
Humans
• Trichothecenes
– “This family of mycotoxins causes multiorgan
effects including emesis and diarrhea, weight
loss, nervous disorders, cardiovascular
alterations, immunodepression, hemostatic
derangements, skin toxicity, decreased
reproductive capacity, and bone marrow
damage”
• Medical Aspects of Chemical and Biological
Warfare, Chapter 34: Trichothecene Mycotoxins,
1997
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Fungi Producing
Trichothecene Toxins
•
•
•
•
•
•
•
Fusarium
Trichothecium
Myrothecium
Cephalosporium
Stachybotrys
Verticimonosporium
Cylindrocarpon
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Trichothecenes
• Major category of mycotoxins
• 1st case of mycotoxicosis
– 1932
– USSR
– Alimentary toxic aleukia
• 60% mortality
• Major chemical warfare agent
– Yellow Rain
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Fungi Common in Sick Building
Syndrome
Alternaria
Trichoderma
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Acremonium
Stachybotrys
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Epicoccum
Penicillium
9
Top Twenty Symptoms (N=195)
38
Headache
45
21
Intense Fatigue
83
24
Spaciness
56
26
Memory Problem s
57
32
Nasal Sym ptom s
56
31
Throat Discomfort
34
21
Coughing
47
29
Lightheaded
33
29
Watery eyes
42
26
Sinus Discomfort
29
36
Dizziness
27
Insom ina
17
34
Coordination Probs
19
28
20
23
Slurred/Word Find
11/11/02 APHA Philadelphia
0
10
20
30
Daily to Almost Daily
31
42
12
Cold Intolerance
20
28
Rash
Muscle Spas m
Several Times a Week
45
21
Weak Voice
Once a Week
31
30
11
Joint Discomfort
Several Times a Month
51
Bloating
One a Month
36
ImmunTox,
40 Property
50 60 70of 80
90 100 110LLC
120 130 140 150
10
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Diz zines
We ak V o
ic
e
ation pro
b lems
Ras h
sm
0
t
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Fatigu e
Headac h
e
Mem ory
Problem
s
Nas al S
ympto ms
Spa cine
ss
Coughin
g
es
Sinus D
isc omfort
Wa tery E
y
Throat d
is co mfor
Lighthea
de d
J oint Dis
c omfort
Ins omnia
Bloati ng
s
Slurred /W
ord Find
ing
Coordin
Mus cle S
pa
Cold Into
leran ce
Compared Symptom Profiles
156 Exposed Patient’s Compared to 28 Referent Patient’s
Means
6
5
4
3
2
1
Non Exposed
Exposed
11
Historical Perspective
• Spring 1998
– CDC MMWR 9 cases
hemorrhagic pneumonitis in Infants
• Stachybotrys chartarum found in lungs
– Stachybotrys chartarum (atra) present
in homes
– In National Survey Ruth Etzel, MD, Ph.D.
finds 115 cases of hemosiderosis associated
with mold exposure (mortality approx. 15%)
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Stachybotrys chartarum
• Isolated from lung of a child with
pulmonary hemosiderosis
– Strain JS5106
– Stachyrase A
•
•
•
•
•
New Chymotrypsin-like Serine proteinase
Cleaves major protease inhibitors,
Several active peptides, and
Collagen
All above found in the lung
– Infection and Immunity, Jan 2002, p 419-423
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Immune Effects
• Increased B-cells
• Excessive T-cell activation
– CD3+CD26+(TA1) excess
– CD3+HLA-DR+ excess
• Suppressor cell activation
– CD8+CD38+ excess
– CD8+HLA-DR+ excess
– Increased Interleuken 2 Receptors on T-cells
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Immune Activation Effects
Exposed
Exposed Patients
Patients to
to Normal
Normal Ranges
Ranges for
for Activation
Activation Markers
Markers
50.000
50.000
45.000
45.000
40.000
40.000
35.000
35.000
Exposed
Exposed Mean
Mean
Min
Normal
Min Normal
30.000
30.000
25.000
25.000
Mean
Mean Normal
Normal
Max
Max Normal
Normal
20.000
20.000
15.000
15.000
10.000
10.000
5.000
5.000
%%
CCDD
2200
++((
BB C
Ce
el ll
%%
lss)
)
C
CDD
5
5++
CCD
D2
255
++((I
I LL
22))
%%
CCDD
33++
CCD
D2
266
++
%%C
CD
D3
3++
HHL
LAA
--DD
RR+
+
%%C
CD
D8
8++
CCD
D3
388
++
%%C
CD
D8
8++
HHL
LAA
--DD
RR+
+
0.000
0.000
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Immune Depression Effects
%CD3-CD16+CD56+ Natural
Killer Cells as % Tot Lymph
%CD8+CD11b+ Human
Leuko Compliment Receptors
45.0
20.0
20
45
18
40
16
35
12.5
14
12
25
10
8
6
25.0
30
7.7
20
15
5.0
5.0
5.3
Low
Normal
Exposed
Mean
10
4
5
2
0
0
Low
Normal
Exposed
Mean
11/11/02 APHA Philadelphia
Mean
Normal
High
Normal
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Mean
Normal
High
Normal
16
Immune Suppression
Lymph Stimulation by
Phytohemagglutinin (PHA)
Concanavillin Stimulation
400
200
300
150
200
100
100
50
0
Low Normal
0
Low Nor ma l
Expose d Me a n
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Me a n Nor ma l
High Nor ma l
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Exposed
Mean
Mean Normal
High Normal
17
Immune Effects
• Increased markers of autoimmunity
– Anti CNS myelin antibodies
– Anti PNS myelin antibodies
– Anti smooth muscle antibodies
– Anti nuclear antibodies
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Immune Effects (n=195)
Autoimmune
Autoimmune Antibodies
Antibodies
Patients
Patientsw/
w/Pos
PosAutoantibodies
Autoantibodiesof
of195
195Total
TotalPatients
Patients
120
120
100
100
Pts
Pts at
at 256
256
Pts
at
128
Pts at 128
80
80
60
60
Pts
Pts at
at 64
64
Pts
at
32
Pts at 32
40
40
20
20
00
Pts
Pts at
at 16
16
CNS
CNS
Myelin
Myelin
IgG
IgG
CNS
CNS
Myelin
Myelin
IgA
IgA
CNA
CNA
Myelin
Myelin
IgM
IgM
Periph
Periph
Nerve
Nerve
IgG
IgG
11/11/02 APHA Philadelphia
Periph
Periph
Nerve
Nerve
IgA
IgA
Periph
Periph
Nerve
Nerve
IgM
IgM
40
40
35
35
30
30
25
25
Pts
Pts atat40
40
Pts
Pts atat80
80
20
20
15
15
Pts
Pts atat160
160
Pts
Pts atat320
320
10
10
55
00
Anti-nuclear
Anti-nuclearAb
Ab
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Anti-Smooth
Anti-SmoothAb
Ab
19
Factors affecting respirability
• Particulate size
– Greater than 5
microns
• Non-respirable
• Except if aspect ratio >
1:3
– 5 microns to 0.005
micron
• Considered respirable
• Reaches alveoli
• Passive filtration is
useless
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Pulmonary Effects of Spores
Smokers and Nonsmokers
45
40
41
35
35
30
31
25
29
29
20
15
10
14
5
0
Critical
<30
V Severe
30 - 45
Severe
46 - 55
Moderate
56 - 65
Mild
66 - 80
Normal
>80
Degree of Small Airway Obstruction Percentage of Predicted FEF 75%
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Pre and Post Bronchodilator FEF 75%
Combined Smokers and Non-Smokers
80
70
60
50
40
30
20
10
0
Critical
Very severe
Severe
Pred Pre FEF75%
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Moderate
Mild
Pred post FEF75%
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22
Pre Dilator Degree of Severity
Smokers vs. Nonsmokers
25
24
20
15
10
17
21
24
23
14
10
8
5
4
4
0
Critical
<30
V Severe Severe Moderate
30 - 45
46 - 55 56 - 65
Nonsmokers
11/11/02 APHA Philadelphia
25
2
Mild Normal
66 - 80
>80
Smokers
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Mean Pre and Post FEF 75%
Nonsmokers vs. Smokers
80
60
Nonsmokers
40
Smokers
20
0
Pre FEF75%
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Post FEF75%
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Pre and Post FEF 75% by Smoke Pack
Years
70
60
50
40
30
20
10
0
> 20 Years
11 to 20
Pred Pre FEF75%
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6 to 10
1 to 5
Pred post FEF75%
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Pulmonary Mycotoxicosis
•
Pulmonary Nodules
– Associated with intense chest
pain
– Aspergillus common, but not
only cause
– Presence across fissure line
implies “kissing” lesions
reflecting exodigestion—
secretion of digestive
enzymes
– Antifungals effective, but often
induce toxicity (liver, bone
marrow, kidneys vulnerable)
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Symptom Profiles – Dr. Crago
Low in energy
Headaches
Remembering things
Trouble conc
Worrying
Blank Mind
Double-check
Feeling weak
Feeling tense
Pt Answering 2
Feeling blocked
Pt Ansering 3
Easily annoyed
Pt Answering 4
Dif making decisions
Trouble falling asleep
Feeling blue
Slowly for correctness
Nervousness
Muscle soreness
Hot/cold spells
Tingling
Loss sexual interest
0
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3
6
9
12
15
18
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21
24
27
Kilburn Neurotox Data
• The following data is Dr. K. Kilburn’s
analysis of 43 mycotoxin exposed patients
and 202 non-exposed referent subjects as
precent of predicted (PFTs), means,
Standard Deviation (SD), and P values by
analysis of variance
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Demographic Data
43 exposed Mean +/_ SD
202 referent Mean +/-SD
48.2 +/_ 13.9
46.6 +/- 20.6
0.624
15 +/- 2.6
12.9 +/- 2.3
0.0001
Age
(years)
Education (years)
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P value
29
Reaction Times (ms)
43 exposed Mean +/_ SD
202 referent Mean +/-SD
P value
Simple
104.0 +/_ 6.6
99.9 +/- 3.7
0.0001
Choice
102.6 +/- 6.6
100.0 +/- 3.7
0.0001
Reaction Tim e
104
102
100
98
96
Simple Reaction Choice Reaction
Time (ms)
Time (ms)
P=0.0001
P=0.0001
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Balance Sway Speed (cm/sec)
43 exposed Mean +/_ SD
202 referent Mean +/-SD
P value
Eyes Opened
140.5 +/_ 49.9
100.2 +/- 2.5
0.0001
Eyes Closed
168.7 +/- 102.7
103.1 +/- 26.7
0.0001
200
150
100
50
0
Eyes Opened
P=0.0001
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Eyes Closed
P=0.0001
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Blinking Reflex Latency R-1 (ms)
43 exposed Mean +/_ SD
202 referent Mean +/-SD
Right
113.9 +/_ 10.8
99.4 +/- 14.6
0.0001
Left
115.1 +/- 11.4
96.4 +/- 13.2
0.0001
120
115
110
105
100
95
90
85
4 3 EXPOSED M EAN
2 0 2 REFERENT M EAN
Right
P=0.0001
11/11/02 APHA Philadelphia
P value
Lef t
P=0.0001
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Color Perception Score
43 exposed Mean +/_ SD
202 referent Mean +/-SD
P value
Right
69.6 +/_ 40.8
102.6 +/- 51.1
0.0001
Left
75.8 +/- 48.8
102.6 +/- 51.1
0.002
120
100
80
60
4 3 EXPOSED MEAN
40
2 0 2 R EFERENT MEAN
20
0
Right
P=0.0001
11/11/02 APHA Philadelphia
Left
P=0.002
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Visual Performance
43 exposed Mean +/_ SD
202 referent Mean +/-SD
P value
Right
87.5 +/_ 23.9
100.0 +/- 22.8
0.006
Left
90.0 +/- 20.9
101.1 +/- 21.7
0.008
105
100
95
43 EXPOSED MEAN
90
202 REFERENT MEAN
85
80
Right
P=0.006
11/11/02 APHA Philadelphia
Left
P=0.008
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Grip Strength (lbs)
43 exposed Mean +/-SD
202 referent Mean +/-SD
P value
Right
91.6 +/_20.0
99.3 +/- 17.5
0.012
Left
86.8 +/_ 22.5
99.1 +/- 17.5
0.0001
100
98
96
94
92
90
88
86
84
82
43 EXP OS ED MEAN
202 REFERENT MEAN
80
Right
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P=0.012
Lef t
P=0.0001
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Verbal Recall
43 exposed Mean +/_ SD
202 referent Mean +/-SD
P value
Immediate
76.7 +/_ 23.4
99.8 +/- 31.1
0.0001
Delayed
68.7 +/- 38.2
99.9 +/- 41.3
0.0001
100
90
80
70
60
50
43 EXPOSED MEAN
40
202 REFERENT MEAN
30
20
10
0
Immediate
P=0.0001
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Delayed
P=0.0001
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Information, Pictures, Similarities
43 exposed Mean +/_ SD
202 referent Mean +/-SD
P value
Information
88.9 +/_ 34.7
101.5 +/_ 39.4
0.052
Picture Completion
78.8 +/_ 30.7
99.3 +/_ 32.2
0.0002
Similarities
95.5 +/_ 25.8
98.1 +/_ 41.2
0.691
120
100
80
60
40
20
0
Information
P=0.052
11/11/02 APHA Philadelphia
Picture
Completion
P=0.0002
Simularities
P=0.691
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Profile of Mood States
43 exposed
Mean +/_ SD
202 referent
Mean +/-SD
59.7 +/- 41.8
21.0+/- 31.6
0.0001
Tension
16.1 +/- 8.5
9.1+/- 5.8
0.0001
Depression
14.6 +/- 12.2
8.1+/- 9.3
0.0001
Anger
13.4 +/- 10.1
8.3+/- 7.9
0.0003
Fatigue
15.2 +/- 7.3
7.6+/- 6.1
0.0001
10.3
+/- 6.7
18.3
+/- 6.3
0.0001
12.9+/- 6.9
6.1+/- 4.5
0.0001
Score
Vigor
Confusion
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P value
38
Mechanism for Neurotoxic
Effects of Micotoxins
• Fumonisin B1 (FB1) enzyme inhibitor
blocking biosynthesis of sphingolipids and
accumulations of sphiganine
• Resultant brain hemorrhage
(leukoencephalomalacia) (Wilson, 1992)
• Sterigmatocystin interferes with
sphyngosine metabolism: an essential
component of neuronal membranes
• Tremulo- and Vomitoxins are mycotoxins
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Fungal Effects
• Neurological Component
– Cognitive dysfunction
• Confusion and
spaciness
• Memory loss
• Spatial disorientation
• Dyslexia
• Seizures
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• Attention Deficit Disorders
– Suspect intrusion of
paroxysmal increased
brain wave frequency
and activity
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Mycotoxin Effects
• Pulmonary
• Neurological
–
–
–
–
–
–
Behavioral changes
Cognitive changes
Ataxia
Convulsions
Multiple sclerosis
Optic neuritis
• Immune suppression
• Interferes with protein
synthesis
11/11/02 APHA Philadelphia
– Small airway
obstruction
– Hypersensitivity
pneumonitis
– Hemorrhagic
pneumonitis
• Cardiovascular
– Vascular permeability
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Mycotoxin Intoxication
• Acute and Chronic Mycotoxicosis can cause
serious and sometimes multisystem diseases
with severe and sometime fatal outcomes.
• The possibility of Mycotoxin intoxication should
be considered when an acute disease occurs in
several persons when there is no evidence of
infection with a known etiologic agent, and no
improvement in the clinical picture follows
treatment.
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Assays for Trichothecenes
• Protein Translation assay in Airborne Particulates
– Rapid, inexpensive assay using firefly luciferase has
been developed to detect and quantify the fungusderived toxicity of airborne particulates
– Entire testing procedure can be accomplished in
2 hours
– Authors demonstrated T-2 toxin, Satratoxin, and DON
inhibit luciferase mRNA in a cell free rabbit
reticulocyte system
– Results are highly reproducible
– Applied and Environmental Microbiology, Jan, 1999, p. 88-94
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Symptoms of Sick Building
Syndrome
• Head, Eyes, Nose,
Throat
–
–
–
–
–
–
–
–
–
Headache
Dizziness
Dry eyes
Watery eyes
Itchy eyes
Epistaxis
Stuffy nose
Rhinorrhea
Alopecia
11/11/02 APHA Philadelphia
• Gastrointestinal
– Nausea & Vomiting
– Diarrhea
– Constipation
• Respiratory
– Cold & flu symptoms
– Cough
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Fungal Exposure & Health
Effects
• Pathogenic infections
• Allergic illnesses
from fungi
• Hypersensitivity
pneumonitis
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Conclusion
• Acute and Chronic Mycotoxicosis can cause
serious and sometimes multisystem diseases
with severe and sometime fatal outcomes
• The possibility of Mycotoxin intoxication should
be considered when an acute disease occurs in
several persons when there is no evidence of
infection with a known etiologic agent, and no
improvement in the clinical picture follows
treatment
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Afterword:
Be Not Afraid
To Look For
Zebras
--Anon
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Molds, Mycotoxins and
Human Health
Michael R. Gray, MD, MPH, CIME
Internal and Occupational Medicine
Emergency Medicine & Toxicology
In collaboration with
Kaye Kilburn, MD
Internal Medicine and Neurotoxicology
Robert Crago, Ph.D.
Psychology, Neurophysiology and
Neurotherapy
ImmunTox, LLC
PO Box 2050 Benson, Arizona 85602
11/11/02 APHA Philadelphia
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48