Transcript Bioaerosols and Human Health

Indoor Molds and Mycotoxins
Estelle Levetin, PhD
Faculty of Biological Science
University of Tulsa
Fungi
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Abundant in the natural environment and
able to grow on many environmental and
synthetic substrates
Capable of producing secondary metabolites,
mycotoxins and VOCs
Small percent are plant or animal pathogens
In terms of human exposure, fungi can be
responsible for allergic, infectious, or toxic
diseases
Major Groups of Fungi
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Myxomycetes - slime molds
Oomycetes - water molds
Zygomycetes - bread molds, sugar fungi
Ascomycetes - sac fungi, morels, yeast
Basidiomycetes - mushrooms, puffballs
Asexual fungi (Deuteromycetes)- molds,
microfungi, anamorphic fungi, mitosporic
fungi – asexual stages of ascomycetes
Fungal Spores
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Fungi reproduce by spores
Majority of spore types adapted for
airborne dispersal
Spores unicellular to multicellular from 1
to 100 m – always microscopic
Outdoor Fungal Spores
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Amazing diversity of spores in atmosphere
Concentrations up to 200,000 spores/m3
Daytime dominated by “Dry Air Spora”
Cladosporium, Alternaria, Drechslera,
Curvularia, Pithomyces, and smut spores
Late night and early morning dominated
by basidiospores and ascospores
Rainy periods dominated by ascospores
Fungal Spores in Outdoor Air
Mold Spores in the Indoor
Environment
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Spores occur in all indoor environments
Considered contaminants indoors
Fungi can colonize countless substrates
indoors
HVAC system can also become
contaminated and even help disperse
spores
Environmental factors that influence
indoor fungal contamination
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Outdoor concentration and type
Type and rate of ventilation
Activity levels
Modern building materials
Indoor moisture levels
Typical Yearly Spore Levels
60,000
50,000
40,000
30,000
20,000
10,000
0
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Environmental factors that influence
indoor fungal contamination
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Outdoor concentration and type
Type and rate of ventilation
Activity levels
Indoor moisture levels
Modern building materials
Environmental factors that influence
indoor fungal contamination
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Outdoor concentration and type
Type and rate of ventilation
Activity levels
Indoor moisture levels
Modern building materials
Environmental factors that influence
indoor fungal contamination
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Outdoor concentration and type
Type and rate of ventilation
Activity levels
Indoor moisture levels
Modern building materials
Indoor Moisture Levels
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Availability of moisture most critical
factor in determining if fungi will grow
Leaks and moisture seeping through
walls, ceilings, basements obvious
source of problems
Courtesy of Terry Brennan
Moisture Problems
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Become worse in past 20 - 30 yrs
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increased use of washing machines,
dishwashers
vaporizers and humidifiers actively spray
droplets into the air (often contaminated)
tighter buildings for energy conservation
trap moisture
Anytime moisture available fungi will
grow
Humidity
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Indoor relative humidity
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Humid air condenses on cool surfaces
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Below 30% R.H. no mold growth and
Above 70% optimal for mold
Usually above 50% mold growth can occur
Cold windows in winter - molding and sills become wet and
suitable for fungal growth
Cold floors in winter
Cooling coils in AC units in summer
Humid air allows hygroscopic materials to absorb water
High humidity in home where
subslab ducts failed
Environmental factors that influence indoor
fungal contamination
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Outdoor concentration and type
Type and rate of ventilation
Activity levels
Indoor moisture levels
Modern building materials
Modern Building Materials
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Ceiling tiles, sheetrock, and other
building materials seem to be especially
prone to fungal contamination
High cellulose content
Many fungi are cellulose degraders in
the natural environment - perfect
substrate to culturing fungi
Objective of Study
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What happens when new ceiling tiles
are soaked with water?
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How long does it take before
contamination is visible?
What fungi are present?
Methods
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New ceiling tiles aseptically cut into 6 x 6 cm
squares and placed in sterile petri dishes
Ceiling Tile Squares (CTS) saturated with 33
ml water:
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sterile distilled water
tap water
rain water
CTS in triplicate, experiment repeated, and
extra sterile distilled water saturated CTS
Methods
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Dishes sealed with parafilm and allowed
to incubate at room temperature for a
minimum of 10 weeks
CTS were regularly evaluated by direct
microscopy of surface growth
At the end of the experiment randomly
selected CTS were ground up and
cultured
Direct Microscopy
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CTS within sealed petri dishes were
evaluated with a dissecting microscope
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Fungi were identified
After 10 weeks estimates made of percent
surface area covered by fungal growth
Results of Direct Microscopy
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All CTS showed fungal contamination
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CTS saturated with tap-water had the greatest
contamination had a mean surface coverage 65%
(range 50-90%)
CTS saturated with sterile distilled water had a
mean coverage of 60%(range 5-100%)
CTS saturated with rainwater showed the lowest
contamination with a mean coverage of 10%
(range 1-25%).
10 genera of fungi identified plus yeast
Fungal Growth on CTS
after 10 Weeks Incubation
Rainwater
Sterile Distilled Water
Tap Water
Fungi Identified by Microscopy
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Alternaria
Aspergillus
Chaetomium
Cladosporium
Drechslera
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Epicoccum
Mucor
Penicillium
Stachybotrys
Ulocladium
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Yeast
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Additional Observations
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Visible colonies of Alternaria and
Epicoccum appeared within 4 days after
water was added
Other fungi appeared much later
After 10 weeks incubation, dominant
fungal types of CTS varied greatly
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Alternaria dominant on some
Stachybotrys dominant on some
Chaetomium dominant on some
Culture Analysis
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One CTS was randomly selected from
each “water” type from each experiment
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2 sterile distilled water saturated CTS
2 tap saturated CTS
2 rainwater saturated CTS
Three additional CTS from sterile distilled
water group also selected at random
Culture Analysis Methods
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Each CTS was placed in a sterile blender
cup with 50 ml of sterile distilled water
CTS blended on high for two 15 sec
intervals
Resulting slurry was filter through 4 layers
of sterile cheese cloth
Resulting suspension was dilution plated
on MEA + strep and Cellulose Agar
Incubated at room temp for 7 to 10 days
Results of Culture Analysis
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Overall Penicillium most abundant genus on
culture plates - found on all “water” types
Several fungi that were dominant on tile
surfaces were low or absent in culture
Quantification difficult when plates overgrown
with Penicillium
Results of all media and all dilutions
combined
Mean Concentrations of Fungi Cultured from CTS
Taxa
Acremonium
Alternaria
Aspergillus
Chaetomium
Cladosporium
Drechslera
Epicoccum
Fusarium
Penicillium
Phialophora
Stachybotrys
Trichoderma
Yeast
Non-sporulating
Sterile
Distilled Water
Tap Water
Rainwater
Colony Forming Units/Tile x 106
0.25
1.00
present
0.25
0.13
present
present
present
1.40
0.50
0.32
0.25
present
0.03
0.20
present
2.23
528.55
1.50
24.00
65.75
14.20
0.25
25.00
34.50
Summary of Fungi Identified on CTS
Microscopy
Culture
Alternaria
Aspergillus
Chaetomium
Cladosporium
Drechslera
Epicoccum
Mucor
Penicillium
Stachybotrys
Yeast
Acremonium
Alternaria
Aspergillus
Chaetomium
Cladosporium
Drechslera
Epicoccum
Fusarium
Penicillium
Phialophora
Stachybotrys
Trichoderma
Yeast
Non-sporulating
Indoor Fungi
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Indoor spores generally reflects outdoors
unless there is a source of contamination
Many different types of fungi occur - 160 spp
Most common genus is Cladosporium - just
like it is outdoors – some species difference
indoors
BUT Penicillium and Aspergillus often exist at
higher concentrations indoors
Stachybotrys has received most media
attention over the past 4 years
Sampling
Andersen (N-6) sampler
Various portable spore trap impactors
Cladosporium
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Common fungal genus
occurring both indoors
and outdoors
Most abundant outdoor
spore type with a
worldwide distribution
Normally exists as a
saprobe or weak plant
pathogen
Spores are known to be
allergenic
Cladosporium spp.
Cladosporium on diffuser
Penicillium
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One of the most
common soil fungi in
natural environment
Over 250 species
Well known allergen
Some species produce
mycotoxins
Some species produce
antibiotics
Produce VOCs
Aspergillus
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Also common soil fungi
Cause rot of stored
grain
Over 150 species
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Well known allergens
Several species form
mycotoxins
Some species can grow
at high temperatures
Several species cause
infections in lung,
sinuses, and
hypersensitivity
pneumonitis
Penicillium and Aspergillus
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Small spores passively
aerosolized when spore
clusters disturbed
Spores extremely
buoyant, remain airborne
for extended time
Penicillium and
Aspergillus spores look
alike – distinguished in
culture
Pen/Asp Concentrations
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Previous and on-going studies in my lab
have focused on trying to determine baseline levels of Penicillium/Aspergillus levels
Collected multiple air samples from 12
indoor locations during Sept, Nov, Feb
along with outdoor controls
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Andersen samples for culturable fungi
Spore trap samples for total spores
Pen/Asp Concentrations
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Penicillium and Aspergillus identified in all
locations
Culture analysis identified
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23 species of Penicillium (mean 39.1 CFU/m3)
12 species of Aspergillus (mean 14.1 CFU/m3)
Spore trap samples found
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332 spores/m3 as the mean level of
Penicillium/Aspergillus type spores indoors
Stachybotrys chartarum
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Soil fungus in nature
Commonly found indoors
on wet materials
containing cellulose, such
as wallboard, jute, wicker,
straw baskets, and paper
materials
Spores in slimy mass
Thought to be allergenic
although little is known
May produce potent
mycotoxins
Stachybotrys
Indoor Air Sample (Spore Trap)
Fusarium
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Common saprobe and
important plant
pathogen
Normally found in the
soil
Indoors it is often found
in the bathroom or
other areas with high
moisture
Allergenic
Some species produce
mycotoxins
Building Related Diseases
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Allergic diseases
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Infectious diseases
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Allergic rhinitis (Hay fever)
Asthma
Allergic sinusitis
Hypersensitivity pneumonitis
Human pathogens
Opportunistic pathogens
Toxic disease
Secondary Metabolites
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Fungi produce remarkable diversity of
secondary metabolites
Often confined to one species or one
strain
Antibiotics, volatile organic compounds
(VOCs), toxins, glucans
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Fungal toxins harmful to humans or other
animals
Volatile Organic Compounds (VOCs)
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Produced by many fungi
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Earthy odor of some mushrooms
Musty, moldy smell of basements and attics
Health effects of VOCs not well studied
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Some suggest VOCs responsible for
headaches, dizziness, and eye and mucous
membrane irritation
Possibly many Sick Building symptoms caused
by VOCs
Fungal glucans
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Fungal cell wall carbohydrates
Studies suggest they have inflammatory
and/or immunomodulatory properties
Fungal Toxins
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Harmful to humans or other animals
May provide the fungus some advantage in
natural environment
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Currently we are doing some competition
assays with Stachybotrys and other fungi
Toxin types:
- Mushroom toxins formed in the fleshy fruiting
bodies of higher fungi
- Mycotoxins formed by common molds growing
under a variety of conditions
Mycotoxins
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Produced by many fungi in contaminated
foods and other substrates
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Can develop in grains or nuts in the field
Generally, mycotoxins develop in storage and
remain within the food after processing and
cooking
Many common indoor fungi are toxigenic
Some studies revealed significant levels of
mycotoxins in airborne spores
Indoor Fungi Capable of Forming
Mycotoxins
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Some Aspergillus species
 Aspergillus versicolor most widely isolated in
buildings with moisture damage
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Some Penicillium species
Some isolates of Stachybotrys chartarum
Some Fusarium species
Many other fungi that occur occasionally
Health Effects of Mycotoxins
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Acute and chronic effects on both humans and
livestock
Many are potent carcinogens
Majority of research focused on health effects
following consumption of contaminated food
Effects range from immediate toxic responses and
immunosupression to potential long-term
carcinogenic effects
Possible health effects due to airborne exposure
(exposure to airborne spores with mycotoxins)
Health effects from airborne
exposure to toxins?
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Clinical studies not completed yet
Association of Stachybotrys with health
effects in contaminated buildings but no
experimental studies with human exposure
Animal studies suggest effects of respiratory
exposure very important
Possible effects: immune suppression, rash,
headache, fatigue, sore throat, pulmonary
hemorrhage (in infants), memory loss???
We need more research studies and data!
Toxic Black Mold in the Media
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Refers to Stachybotrys chartarum
Media frenzy started with Cleveland baby
deaths and the initial CDC report in 1997
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CDC retraction in 2000 is seldom mentioned
Media frenzy stirred up again following
the 1999 lawsuit by Melinda Ballard in
Dripping Springs, Texas
Media frenzy has not stopped!
USA Weekend Cover Stories
Dorr Dearbon, MD - Cleveland
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Dorr Dearborn was one of the physicians
involved in the Cleveland baby case and
has continued studies of Stachybotrys
"There is a negative health impact of
living in a moldy environment…But the
details as to what the health effects are
and how much mold it takes – that is
what we don't know."
Malina Bill
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Introduced in Congress Oct 2002
The Malina bill will:
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Require the EPA to define what dangerous
toxic mold levels are
Create an emergency federal insurance
program, like FEMA, to pay the
astronomical costs of black mold clean up
Create a national database of homes
infected with black mold