Indoor Molds and Mycotoxins

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Indoor Molds and Mycotoxins

• Estelle Levetin, PhD
• Faculty of Biological Science
• University of Tulsa

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Fungi

• 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

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Major Groups of Fungi

• 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

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Fungal Spores

• Fungi reproduce by spores
• Majority of spore types adapted for airborne
  dispersal
• Spores unicellular to multicellular from 1 to 100
  ?m always microscopic

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Outdoor Fungal Spores

• 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

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Fungal Spores in Outdoor Air
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Mold Spores in the Indoor Environment

• 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

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Environmental factors that influence indoor
fungal contamination

• Outdoor concentration and type
• Type and rate of ventilation
• Activity levels
• Modern building materials
• Indoor moisture levels

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Typical Yearly Spore Levels
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Environmental factors that influence indoor
fungal contamination

• Outdoor concentration and type
• Type and rate of ventilation
• Activity levels
• Indoor moisture levels
• Modern building materials

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Environmental factors that influence indoor
fungal contamination

• Outdoor concentration and type
• Type and rate of ventilation
• Activity levels
• Indoor moisture levels
• Modern building materials

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Environmental factors that influence indoor
fungal contamination

• Outdoor concentration and type
• Type and rate of ventilation
• Activity levels
• Indoor moisture levels
• Modern building materials

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Indoor Moisture Levels

• Availability of moisture most critical factor in
  determining if fungi will grow
• Leaks and moisture seeping through walls,
  ceilings, basements obvious source of problems

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Courtesy of Terry Brennan
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Moisture Problems

• Become worse in past 20 - 30 yrs
• 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

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Humidity

• Indoor relative humidity
• Below 30 R.H. no mold growth and
• Above 70 optimal for mold
• Usually above 50 mold growth can occur
• Humid air condenses on cool surfaces
• 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

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High humidity in home where subslab ducts failed
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Environmental factors that influence indoor
fungal contamination

• Outdoor concentration and type
• Type and rate of ventilation
• Activity levels
• Indoor moisture levels
• Modern building materials

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Modern Building Materials

• 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

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Objective of Study

• What happens when new ceiling tiles are soaked
  with water?
• How long does it take before contamination is
  visible?
• What fungi are present?

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Methods

• 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
• sterile distilled water
• tap water
• rain water
• CTS in triplicate, experiment repeated, and extra
  sterile distilled water saturated CTS

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Methods

• 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

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Direct Microscopy

• CTS within sealed petri dishes were evaluated
  with a dissecting microscope
• Fungi were identified
• After 10 weeks estimates made of percent surface
  area covered by fungal growth

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Results of Direct Microscopy

• All CTS showed fungal contamination
• 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

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Fungal Growth on CTS after 10 Weeks Incubation
Rainwater
Sterile Distilled Water
Tap Water
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Fungi Identified by Microscopy

• Alternaria
• Aspergillus
• Chaetomium
• Cladosporium
• Drechslera

• Epicoccum
• Mucor
• Penicillium
• Stachybotrys
• Ulocladium
• Yeast

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Additional Observations

• 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
• Alternaria dominant on some
• Stachybotrys dominant on some
• Chaetomium dominant on some

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Culture Analysis

• One CTS was randomly selected from each water
  type from each experiment
• 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

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Culture Analysis Methods

• 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

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Results of Culture Analysis

• 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

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Summary of Fungi Identified on CTS
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Indoor Fungi

• 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

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Sampling
Andersen (N-6) sampler
Various portable spore trap impactors
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Cladosporium

• 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

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Cladosporium spp.
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Cladosporium on diffuser
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Penicillium

• 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

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Aspergillus

• Also common soil fungi
• Cause rot of stored grain
• Over 150 species
• Well known allergens
• Several species form mycotoxins
• Some species can grow at high temperatures
• Several species cause infections in lung,
  sinuses, and hypersensitivity pneumonitis

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Penicillium and Aspergillus

• 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

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Pen/Asp Concentrations

• Previous and on-going studies in my lab have
  focused on trying to determine base-line levels
  of Penicillium/Aspergillus levels
• Collected multiple air samples from 12 indoor
  locations during Sept, Nov, Feb along with
  outdoor controls
• Andersen samples for culturable fungi
• Spore trap samples for total spores

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Pen/Asp Concentrations

• Penicillium and Aspergillus identified in all
  locations
• Culture analysis identified
• 23 species of Penicillium (mean 39.1 CFU/m3)
• 12 species of Aspergillus (mean 14.1 CFU/m3)
• Spore trap samples found
• 332 spores/m3 as the mean level of
  Penicillium/Aspergillus type spores indoors

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Stachybotrys chartarum

• 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

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Stachybotrys
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Indoor Air Sample (Spore Trap)
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Fusarium

• 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

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Building Related Diseases

• Allergic diseases
• Allergic rhinitis (Hay fever)
• Asthma
• Allergic sinusitis
• Hypersensitivity pneumonitis
• Infectious diseases
• Human pathogens
• Opportunistic pathogens
• Toxic disease

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Secondary Metabolites

• Fungi produce remarkable diversity of secondary
  metabolites
• Often confined to one species or one strain
• Antibiotics, volatile organic compounds (VOCs),
  toxins, glucans
• Fungal toxins harmful to humans or other animals

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Volatile Organic Compounds (VOCs)

• Produced by many fungi
• Earthy odor of some mushrooms
• Musty, moldy smell of basements and attics
• Health effects of VOCs not well studied
• Some suggest VOCs responsible for headaches,
  dizziness, and eye and mucous membrane irritation
  
• Possibly many Sick Building symptoms caused by
  VOCs

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Fungal glucans

• Fungal cell wall carbohydrates
• Studies suggest they have inflammatory and/or
  immunomodulatory properties

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Fungal Toxins

• Harmful to humans or other animals
• May provide the fungus some advantage in natural
  environment
• 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

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Mycotoxins

• Produced by many fungi in contaminated foods and
  other substrates
• 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

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Indoor Fungi Capable of Forming Mycotoxins

• Some Aspergillus species
• Aspergillus versicolor most widely isolated in
  buildings with moisture damage
• Some Penicillium species
• Some isolates of Stachybotrys chartarum
• Some Fusarium species
• Many other fungi that occur occasionally

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Health Effects of Mycotoxins

• 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)

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Health effects from airborne exposure to toxins?

• 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!

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Toxic Black Mold in the Media

• Refers to Stachybotrys chartarum
• Media frenzy started with Cleveland baby deaths
  and the initial CDC report in 1997
• 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!

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USA Weekend Cover Stories
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Dorr Dearbon, MD - Cleveland

• 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 environmentBut the details as to what the
  health effects are and how much mold it takes
  that is what we don't know."

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Malina Bill

• Introduced in Congress Oct 2002
• The Malina bill will
• 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