Mycotoxins

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Mycotoxins
2
Secondary Metabolites

• Organic compounds which have no direct role in
  major metabolic pathways
• Fungi produce remarkable diversity of these
  secondary metabolites
• May serve to discourage predators or suppress
  competition
• Formation quite specific, often confined to one
  species or just one strain
• Thousands of secondary products from fungi have
  been analyzed and characterized

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Secondary Metabolites

• Many have widespread commercial importance
• Others have well known health effects
• Included - antibiotics, toxins, alkaloids,
  volatile organic compounds
• Antibiotics toxic to microorganisms
• Fungal toxins harmful to humans or other animals

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Fungal Toxins

• Mycotoxins formed by hyphae of common molds
  growing under a variety of conditions
• Mushroom toxins formed in the fleshy fruiting
  bodies of higher fungi

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Mycotoxins

• Mycotoxins produced by many fungi growing in
  contaminated foods and other substrates.
• Small molecules low MW
• Generally, the highest levels in substrates with
  high water activity and warm temperatures.
• Can develop in grains or nuts in the field due to
  metabolism of pathogenic or saprobic fungi on the
  living plant
• Forage grass may contain mycotoxins because of a
  pathogenic fungus or a fungal endophyte

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Mycotoxins

• More commonly -- mycotoxins develop in storage
  and remain within the food after processing and
  cooking
• Many common indoor environmental contaminants are
  toxigenic - able to produce toxins
• Some studies revealed significant levels of
  airborne mycotoxins in occupational settings,
  offices, and even homes
• Mycotoxins are not volatile so exposure must be
  in airborne spores

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Mycotoxins identified in spores
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Water activity (aw)

• A measure of the moistness of the substrate
• Expressed as a decimal and directly related to
  substrate RH
• If substrate RH 95, aw 0.95
• Water activity of pure water 1.0
• Fungi can grow under low aw

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Low water activities

• Limits to growth 1.0 to 0.55
• Animals function at 0.99
• Many plants wilt at 0.98
• Most bacteria 0.95 and higher (some extreme
  exceptions among halophilic bacteria)
• Fungi down to 0.65
• Xerophilic fungi
• control internal aw by storing glycerol
• Other fungi survive as spores, sclerotia

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Water activities for fungi

• aw Solution Material Fungi
• 1.0 water Oomycetes
• 0.994 blood meat,veggies
  Basidiomycetes
• 0.98 sea water wood and
  Ascomycetes
• 0.95 1.5molal NaCl bread yeasts (basid)
• 0.90 2.8molal NaCl ham yeasts
  (asco)
• 0.85 6.0molal sucro salami yeasts,
  Penicillium
• 0.75 saturated NaCl salt fish
  Wallemia, etc
• 0.65 22 molal glycerol Eurotium(Aspergillus
  )
• 0.55 DNA denatured

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Health Effects Of Mycotoxins

• Acute and chronic effects on both humans and
  livestock
• Mycotoxins are believed to be among the most
  potent known carcinogens
• Majority of research focused on health effects
  following ingestion of contaminated food
• Effect range from immediate toxic responses and
  immunosupression to potential long-term
  teratogenic, estrogenic, and carcinogenic
  effects.
• Possible health effects due to airborne exposure
  (exposure to airborne spores with mycotoxins)

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History of Mycotoxins

• Turkey X Disease killed over 100,000 young
  turkeys in 1960 in England
• Affected turkeys stopped eating, became
  lethargic, suffered hemorrhages under the skin,
  and died
• Autopsies showed livers had undergone extensive
  necrosis, kidneys developed lesions
• Partridges, pheasants, ducklings, and other
  animals also affected
• Only factor in common with all the poisonings was
  Brazilian peanut meal as a feed supplement.
• Toxin isolated from feed associated with fungal
  contaminant Aspergillus flavus

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Aflatoxins

• Aflatoxin A (Aspergillus) - fla (flavus) - toxin
• Four toxins soon identified Aflatoxin B1, B2,
  G1, G2 - blue or green florescence under UV-light
• Today known to be 10 aflatoxins
• Aflatoxin B1 most important - highly carcinogenic
  and widespread occurrence in foods

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Sources of Aflatoxins

• Produced by 3 species of Aspergillus A. flavus,
  A. parasiticus, A. nomius
• Aspergillus flavus a common saprobe that occurs
  on grains and legumes in storage
• A. parasiticus most toxigenic species
• Aflatoxins not only toxic but also carcinogenic

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Aflatoxin Production

• Aflatoxins are produced under certain conditions
  only by some strains
• Nontoxigenic strains of A. flavus used to prepare
  fermented foods in the Orient

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Effects of Aflatoxins

• Even when levels not toxic, prolonged exposure
  caused liver cancer in every species of lab
  animal tested
• Believed responsible for high rates of liver
  cancer in population groups in Asia and Africa
  where contaminated food is often consumed
• Toxic effects shown in India in 1974 when
  hundreds were poisoned by eating corn containing
  aflatoxins - 106 people died
• Most important crops - peanuts and corn

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Aflatoxin Levels

• Levels permissible in foods subject to legal
  limits in many countries
• Today, foods most frequently contaminated,
  routinely screened before processing or sale
• Permissible limits generally quite low (15-20
  parts per billion)
• Some scientists feel that no detectable levels of
  aflatoxins should be permitted because of the
  carcinogenic effects

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Average yearly level of aflatoxin contamination
from corn grown along the coastal plain of Georgia
ppb
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Economic Impact

• Because of enforced limits the presence of
  aflatoxins can have serious economic implications
• In 1980 nearly 66 of random corn samples from
  North Carolina had concentrations exceeding 20
  ppm resulting in a 31 million loss to producers
  and handlers.
• When cows and goats are fed grains contaminated
  with aflatoxins, they produce milk with
  aflatoxins - As a result, limits exist for
  livestock feed

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Aflatoxin B1 is Mutagenic

• Hepatocellular carcinoma (HCC) is a major cause
  of mortality in certain areas of the world
• About 50 of the HCC cases in parts of the world
  where food is contaminated with AFB1show a
  mutation in codon 249 of p53 tumor suppressor
  gene
• Mutation consists of transversion of G-gtT in the
  third position of codon resulting in serine
  instead of arginine - lab studies confirm

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Claviceps purpurea Ergot of rye
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Ergotism

• Ergot contains a number of toxic alkaloids, if
  harvested with the grain and milled into the
  flower - it can cause a disease called ergotism
• During Middle Ages called Dancing Mania and
  St. Anthonys Fire
• Ergotism can also occur in grazing animals that
  forage on contaminated grain
• Many different alkaloids --- cause many
  different effects

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Alkaloids in Ergot

• Gangrenous ergotism - Some of the alkaloids
  constrict blood vessels and impair circulation
• Feelings of burning in calves or intense cold
• In extreme conditions can result in gangrene
• Limbs may drop off or require amputation
• Convulsive ergotism - Other toxins affect the CNS
• Hallucinations and convulsions - loss of mental
  function
• Feelings of burning in calves or intense cold

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Alkaloids in ergot

• With modern milling techniques, ergotism rare
• Outbreak in France in 1951 - 4 deaths and 150
  hospitalized
• 1977 in Ethiopia on contaminated barley
• Salem Witches may have been ergotism
• Many alkaloids
• ergotamine and ergometrine cause
  vasoconstriction
• Lysergic acid amides affect CNS
• Alkaloids used in medicine to treat migraine
  headaches and in childbirth

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Ergot alkaloids in other fungi

• Recent studies have identified ergot alkaloids in
  other fungi including Aspergillus fumigatus
• Festuclavine
• Derivatives of festuclavine
• Fumigaclavine A
• Fumigaclavine B
• Fumigaclavine C
• These 4 mycotoxins found in A. fumigatus conidia

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Other Mycotoxins

• Today over 400 mycotoxins have been identified
  from 150 species of fungi with new ones
  discovered each year
• Species of Aspergillus, Penicillium, Fusarium,
  Alternaria, Cladosporium and Stachybotrys form
  mycotoxins.

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Mycotoxin Production

• Can occur in one strain of a species, other
  strains of the same species not toxigenic
• Fungi from air samples in homes tested for
  mycotoxin production using tissue culture of
  human fibroblast cells
• In multiple isolates of a single species (up to
  12) some produced mycotoxins, others did not in
  my lab 1/3 isolates were toxin forming
• Warm temperatures and abundant moisture promote
  mycotoxin production

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Common Toxigenic Fungi
Aspergillus
Penicillium
Stachybotrys
Fusarium
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Ochratoxins

• Produced by species of Aspergillus such as A.
  ochraceus
• Most important is Penicillium verrucosum which
  occurs on grains
• Ochratoxin A a nephrotoxin responsible for
  nephropathy in pigs and probably humans
• It is immunosuppressive and also assumed to be
  carcinogenic.

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Patulin

• Produced by a number of species of Penicillium,
  Aspergillus and Byssochlamys.
• Most important producer is Penicillium expansum.
• Fungus causes a soft rot of apples toxin found
  in apple juice
• Patulin first attracted attention as an
  antibiotic in 1943 no current interest in
  antibiotic properties.

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Trichothecenes

• Produced by several species of Fusarium
• One of the most toxic is T-2
• Believed T-2 responsible for outbreak of
  Alimentary Toxic Aleukia (ATA) in Siberia during
  and after WWII
• In some areas 10 of the population developed the
  disease and in most cases it was fatal

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Alimentary Toxic Aleukia

• ATA characterized by nausea, vomiting,
  hemorrhages in many organs, bleeding from nose
  and throat, bloody diarrhea, low leukocyte count,
  exhaustion of bone marrow.
• About a third of deaths due to strangulation from
  internal swelling of throat
• Years later scientists made the connection
  between the disease and consumption of moldy
  grain
• Symptoms appeared when people ate 2 kg of moldy
  grain, 6 kg was lethal. Similar hemorrhagic
  syndrome in animals called moldy corn toxicosis

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Vomitoxin

• Produces vomiting in pigs at low concentrations
• Much less toxic than T-2 but is immunosuppressive
• Contaminates corn, barley, and wheat
• Permissible limits are 0.3 ppm for flour and 0.1
  ppm in bread or breakfast cereal
• During recent wet growing season, Ontario farmers
  lost 17 million on a wheat harvest contaminated
  with vomitoxin.

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Macrocyclic Trichothecenes

• More toxic than T-2
• Produced by Stachybotrys chartarum (S. atra) and
  also by species of Myrothecium.
• Specific toxins are Satratoxins, Verrucarins, and
  Roridins
• Fungi are cellulose decomposers and found growing
  on hay or straw stored under poor conditions.

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Macrocyclic Trichothecenes

• Responsible for the deaths of many horses, but it
  can also affect cattle, pigs, sheep, poultry
• Complex of unpleasant symptoms like ATA.
• Stachybotrys found indoors in many locations
  growing on ceiling tiles and wallboard
• Concern about airborne inhalation of spores

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Yellow Rain

• During the Viet Nam war, there was concern that
  the Viet Cong were using chemical weapons against
  the US as well as the population in Laos and
  Cambodia
• Victims were sprayed with a yellow rain
• Symptoms were like ATA (possibly some evidence of
  trichothecene toxins in some of the victims.
  However, the issue was not clear cut)
• Little evidence Viet Cong using chemical weapons
• On September 13, 1981, then-U.S. Secretary of
  State Alexander Haig accused the Soviet Union of
  supplying trichothecene mycotoxins to the
  Communist regimes in Vietnam and Laos for use in
  counterinsurgency warfare
• Samples of some yellow rain deposits later shown
  to be largely made up of pollen - cleansing
  flight theory 2002 yellow rain in India
• This issue over yellow rain in Viet Nam has never
  been resolved

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Zearalenone

• Produced by species of Fusarium found in moldy
  corn
• Chronic exposure is estrogenic
• Female pigs especially sensitive causing
  vulvovaginitis.
• Swelling of the vulva, enlargement of the mammary
  glands, enlargement of the uterus, and vaginal
  prolapse.
• In lower levels causes infertility, stillbirths,
  and small litters

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Fumonisins

• Produced by Fusarium species on moldy corn
• Implicated in cases of esophageal cancer in
  humans and other cancer in animals

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Other fungi producing mycotoxins

• Cladosporium epicladosporic acid implicated
  in ATA, maybe?
• Alternaria tenuazonic acid detected in
  commercial tomato paste
• Pithomyces sporidesmin disease in sheep

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Summary of 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)

41
Health effects from airborne exposure

• Some epidemiological studies linked inhalation of
  mycotoxins with human disease
• Lack of adequate data on exposure, intake,
  excretion, metabolism
• Little information available on amounts of
  mycotoxins in air
• Experiments with animals show extreme toxicity to
  alveolar macrophages caused by several different
  mycotoxins

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Possible associations between inhalation of
specific mycotoxins and disease
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Health effects from airborne exposure?

• 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 periodically re-emerges

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Stachybotrys chartarum (syn S. atra and S.
alternans)

• Asexual fungus
• Teleomorph has not been identified recent paper
  using molecular data places teleomorph in
  ascomycete order Hyocreales (form perithecia)
  possibly genus Melanopsamma
• About 10 to 12 species in the genus Stachybotrys
• Memnoniella similar to Stachybotrys, but with
  spores in chains
• Produces similar toxins
• Molecular data indicates it same genus
• Therefore many consider Memnoniella echinata as
  S. echinata

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Natural habitat

• Typically a soil fungus, common on decaying plant
  substrates, decomposing cellulose (hay, straw),
  leaf litter, and seeds
• One recent paper indicating it is a pathogen on
  soybean causing root lesions an forming toxins
  in plant

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Stachybotrys chartarum
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Indoor locations

• Commonly found indoors on wet materials
  containing cellulose, such as wallboard, jute,
  wicker, straw, and paper
• Also found on wood and wood paneling and on
  general organic debris
• Wet conditions absolutely required
• Hidden in ceiling, walls, floors with little
  visible evidence within the interior of the room
  but spores can contaminate interior through holes
  or cracks in the building materials aided by
  negative pressure
• Spores can also be transported by air handling
  system

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Spore production

• Dark grey to black (dark green, brown) conidia
  produced by cluster of cylindrical phialides
• Conidia ovoid about 10-12 mm in length
• Conidia roughened to warty to ridged when viewed
  at 1000x
• Conidia produced singly and successively into a
  slime droplet that covers the phialides.
• Suggested to be dispersed by insects in the
  natural environment
• Eventually slime dries and the conidia can become
  airborne

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Stachybotrys conidiophore and conidia
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Stachybotrys trichothecene toxins

• Macrocylic trichothecenes
• Roridin E and L-2
• Satratoxins F, G, and H
• Isosatratoxins F, G, and H
• Verrucarins B and J
• Trichoverroid trichothecenes
• Trichoverrols A and B
• Trichoverrins A and B
• Satratoxins are generally produced in greater
  amounts than the other trichothecenes, but all
  compounds are produced in low quantities
• They apparently occur in all parts of the fungus

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Macrocyclic trichothecenes

• Highly toxic compounds with a potent ability to
  inhibit protein synthesis
• Numerous studies have demonstrated the toxicity
  of toxins from S. chartarum on animals and animal
  and human cells
• Satratoxin is the most cytotoxic of
  trichothecenes tested on mammalian cells, even
  more toxic than T-2 toxin associated with ATA
• LD50 in mice for satratoxins is 1 mg/kg

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Other Stachybotrys toxins non MTR

• Nine phenylspirodrimanes (spirolactones and
  spirolactams) and cyclosporin, which are potent
  immunosuppressive agents
• It has been suggested that the combination of
  trichothecenes and these immunosuppressive agents
  may be responsible for the observed high toxicity
  of this fungus
• Atranones A-G
• Dolabellane diterpenes
• Stachylysin, a proteinaceous hemolysin (causes
  pore formation and lyses red blood cells)
• Also a hydroxymate siderophore
• It has been suggested these could be
  pathogenicity factors involved in pulmonary
  hemorrhage in infants exposed to S. chartarum.

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Non-trichothecene toxins

• These components have a number of immune system
  properties
• Inhibition of TNFa liberation from human
  macrophages
• Inhibition of the complement system
• One of the most common symptoms in moldy
  buildings is recurrent airways infections
• Its been suggested that metabolites which
  inhibit the complement system may be involved
  since the complement system is an important part
  of our defense against bacteria

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Stachybotryotoxicosis history

• 1930s in the Ukraine and other parts of Eastern
  Europe, outbreaks of a new disease in horses and
  other animals
• Irritation of mouth, throat, nose
• Shock
• Dermal necrosis
• Decrease in leukocytes
• Hemorrhage
• Nervous disorders
• Death
• In 1938 Russian scientists determined the disease
  was associated with S. chartarum growing on the
  straw and grain fed to the animals

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More history

• Intensive studies showed the toxicity of
  Stachybotrys in animals horses actually fed
  cultures - often resulting in death
• Russians called disease stachybotrytoxicosis
• Disease later reported in other farm animals
  (cattle, pigs, sheep, poultry) from other parts
  of the world but not from N. Am.
• In the late 1930s, the disease reported in humans
  working of farms in Russia. People affected
  handled infected hay or feed
• Symptoms rash, dermatitis, pain and
  inflammation of mucous membranes, conjunctivitis,
  chest tightness, cough, bloody rhinitis, burning
  eyes, throat, nasal passages, etc
• Outbreak in Hungary in 1977 and 1996 report in
  Germany

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Toxins identified

• In 1973, Eppley Bailey were the first to
  isolate trichothecenes from S. chartarum
• They found the known components, trichodermol and
  roridin E and three novel components, named
  satratoxins H, G and F.
• Structures determined 4-7 years later
• Toxins continue to be isolated from Stachybotrys
• Atranones and stachylysins identified in past 5
  years

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Indoor history

• Croft et al. 1986. Airborne outbreak of
  trichothecene toxicosis. Atmos Ennvir. 20
  549-552
• Over 5 yr period, family complained of headaches,
  sore throats, recurring colds, flu symptoms,
  fatigue, dermatitis, and general malaise
• Air sampling showed S. chartarum spores
• Growing on moist organic material in cold air
  duct and on wood fiber ceiling material
• Home had chronic moisture problems
• Extracts from duct debris had satratoxin H ,
  verrucarin B, verrucarin J, and the trichoverrins
  - also were toxic to test animals
• When mold problems corrected symptoms gone
• Several other reports following this paper

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Cleveland Baby Deaths

• 1993-1994 cluster of cases of pulmonary
  hemorrhage (hemosiderosis) in infants
• All the homes of the infants had high levels of
  total fungi and S. chartarum (based on air and
  surface sampling)
• Homes had water damage
• Stachybotrys isolates from homes produced
  trichothecenes
• Similar cases published in the late 1990s
• 1997 CDC report suggested an association

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More evidence

• Unrelated case of a 7 year old with various
  symptoms cough, fatigue, fever, recurrent
  pneumonia
• Stachybotrys identified in fluid washed from
  lungs
• His home damaged from a flood and Stachybotrys
  (and other fungi) growing on wallpaper near his
  bedroom
• Child became symptom free when moved

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USA Weekend Cover Stories
63
Controversy

• Can Stachybotrys chartarum cause pulmonary
  hemorrhage? Can it effect human health in indoor
  environments?
• Most feel there is insufficient evidence to prove
  a link
• In 2000 CDC retracted previous statement and
  issued reports critical of the study done in
  Cleveland and concluded the association was not
  proven
• 2004 IOM of NAS link to toxin-related symptoms
  not proven

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A lot we dont know!

• There may be multiple modes of action for
  Stachybotrys to affect human health
• Toxicosis is clearly important but
  immune-suppressive compounds may also be
  important, especially in infants
• Hemolytic compounds also important. Some suggest
  maybe we should consider this a pathogen!

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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
  mold 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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Summary

• Health effects on indoor exposure to Stachybotrys
  not proven
• Data since 1930s suggests that we should not be
  handling material contaminated with Stachybotrys
  without safety equipment
• Indoor environments contaminated with
  Stachybotrys are not healthy especially for
  children