A Brief Introduction of Mycotoxin ppt

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					A brief introduction to Mycotoxins
Dr.Kedar Karki

Secondary Metabolites
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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

Secondary Metabolites
Many have widespread commercial importance  Others have well known health effects  Included - antibiotics, toxins, alkaloids, volatile organic compounds


toxic to microorganisms  Fungal toxins harmful to humans or other animals

 Antibiotics

Fungal Toxins
 Mycotoxins

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

 Mushroom

Mycotoxins
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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

Mycotoxins
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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

Mycotoxins identified in spores
Alternaria alternata Alternaria alternata Aspergillus fumigatus Aspergillus fumigatus Aspergillus fumigatus Aspergillus fumigatus Aspergillus flavus Aspergillus parasiticus Fusarium graminearum Fusarium sporotrichioides Stachybotrys chartarum
Alternariol Alternariol monomethylether Tryptacidin Fumitremorgen Verruculogen
Fumiclavine C Aflatoxin Aflatoxin Deoxynivalenol, T-2

Deoxynivalenol, T-2 Satratoxin G, H

Water activity (aw)
A measure of the moistness of the substrate  Expressed as a decimal and directly related to substrate RH
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 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
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Other fungi survive as spores, sclerotia

fungi  control internal aw by storing glycerol

 Xerophilic

Water activities for fungi
aw
1.0 0.994 0.98 0.95 0.90 0.85 0.75 0.65 0.55 water blood sea water 1.5molal NaCl 2.8molal NaCl 6.0molal sucro saturated NaCl 22 molal glycerol DNA denatured

Solution

Material

Oomycetes meat,veggies Basidiomycetes wood and Ascomycetes bread yeasts (basid) ham yeasts (asco) salami yeasts, Penicillium salt fish Wallemia, etc

Fungi

Eurotium(Aspergillus) +

Health Effects Of Mycotoxins
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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)

History of Mycotoxins
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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

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
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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

Produced by 3 species of Aspergillus: A.

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

Effects of Aflatoxins
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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

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 (1520 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
Years 1977 1978 1980 1981 1983 1984 1990 1994 Concentration of Aflatoxin (ng/g) ppb 622 57 204 37 128 37 217 6

Economic Impact
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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

Aflatoxin B1 is Mutagenic
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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->T in the third position of codon resulting in serine instead of arginine - lab studies confirm

Claviceps purpurea

Ergot of rye

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. Anthony’s 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
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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
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Convulsive ergotism - Other toxins affect the CNS
 Hallucinations

and convulsions - loss of mental function  Feelings of burning in calves or intense cold

Alkaloids in ergot
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With modern milling techniques, ergotism rare
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Outbreak in France in 1951 - 4 deaths and 150 hospitalized 1977 in Ethiopia on contaminated barley

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Salem Witches – may have been ergotism Many alkaloids
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ergotamine and ergometrine – cause vasoconstriction Lysergic acid amides – affect CNS

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Alkaloids used in medicine – to treat migraine headaches and in childbirth

Ergot alkaloids in other fungi
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Recent studies have identified ergot alkaloids in other fungi including

Aspergillus fumigatus
 Festuclavine  Derivatives

of festuclavine

A  Fumigaclavine B  Fumigaclavine C

 Fumigaclavine

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These 4 mycotoxins found in A. fumigatus conidia

Other Mycotoxins
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.
 Today

Mycotoxin Production
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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

Aspergillus

Penicillium

Common Toxigenic Fungi
Stachybotrys

Fusarium

Ochratoxins
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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.

Patulin
Produced by a number of species of Penicillium, Aspergillus and Byssochlamys.  Most important producer is Penicillium
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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
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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
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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

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
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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.

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
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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

Zearalenone
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Produced by species of Fusarium found in moldy corn Chronic exposure is estrogenic
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Female pigs especially sensitive causing vulvovaginitis. Swelling of the vulva, enlargement of the mammary glands, enlargement of the uterus, and vaginal prolapse.

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In lower levels causes infertility, stillbirths, and small litters

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

Other fungi producing mycotoxins
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implicated in ATA, maybe?  Alternaria – tenuazonic acid – detected in commercial tomato paste  Pithomyces – sporidesmin – disease in sheep

Cladosporium – epicladosporic acid –

Summary of 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
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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Aspergillus flavus

Possible associations between inhalation of specific mycotoxins and disease
Lung cancer

Aspergillus flavus Aspergillus flavus Stachybotrys chartarum Aspergillus flavus
Aspergillus ochraceus

Colon carcinoma Lung cancer Malaise Lung cancer
Acute renal failure

Aspergillus fumigatus
Stachybotrys chartarum

Tremorgenic encephalopathy
(dementia and tremors)

Infant hemosiderosis

Health effects from airborne exposure?
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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

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Media frenzy stirred up again following the 1999 lawsuit by Melinda Ballard in Dripping Springs, Texas Media frenzy periodically re-emerges

Stachybotrys chartarum (syn S. atra and S. alternans)
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Asexual fungus Teleomorph has not been identified – recent paper using molecular data places teleomorph in ascomycete order Hyocreales (form perithecia) – possibly genus

Melanopsamma
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Stachybotrys  Memnoniella similar to Stachybotrys, but with
spores in chains
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About 10 to 12 species in the genus

Produces similar toxins Molecular data indicates it same genus Therefore many consider Memnoniella echinata as S. echinata

Natural habitat
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
 Typically

Stachybotrys chartarum

Indoor locations
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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

Spore production
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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

Stachybotrys conidiophore and
conidia

Stachybotrys trichothecene toxins
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Macrocylic trichothecenes
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Trichoverroid trichothecenes
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Roridin E and L-2 Satratoxins F, G, and H Isosatratoxins F, G, and H Verrucarins B and J
Trichoverrols A and B Trichoverrins A and B

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Satratoxins are generally produced in greater amounts than the other trichothecenes, but all compounds are produced in low quantities
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They apparently occur in all parts of the fungus

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
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Nine phenylspirodrimanes (spirolactones and spirolactams) and cyclosporin, which are potent immunosuppressive agents
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Atranones A-G Dolabellane diterpenes Stachylysin, a proteinaceous hemolysin (causes pore formation and lyses red blood cells)
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It has been suggested that the combination of trichothecenes and these immunosuppressive agents may be responsible for the observed high toxicity of this fungus

Also a hydroxymate siderophore It has been suggested these could be pathogenicity factors involved in pulmonary hemorrhage in infants exposed to S. chartarum.

Non-trichothecene toxins
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These components have a number of immune system properties
of TNFa liberation from human macrophages  Inhibition of the complement system
of the most common symptoms in moldy buildings is recurrent airways infections  It’s 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
 One

 Inhibition

Stachybotryotoxicosis history
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1930s in the Ukraine and other parts of Eastern Europe, outbreaks of a new disease in horses and other animals
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In 1938 Russian scientists determined the disease was associated with S. chartarum growing on the straw and grain fed to the animals

Irritation of mouth, throat, nose Shock Dermal necrosis Decrease in leukocytes Hemorrhage Nervous disorders Death

More history
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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
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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

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

years

and stachylysins identified in past 5

Indoor history
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Croft et al. 1986. Airborne outbreak of trichothecene toxicosis. Atmos Ennvir. 20: 549552
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Several other reports following this paper

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

Cleveland Baby Deaths
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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

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

Controversy
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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

A lot we don’t know!
There may be multiple modes of action for Stachybotrys to affect human health  Toxicosis is clearly important but immunesuppressive 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 environment…But 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
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Karki Kedar Karki Kedar Senior Vet.Officer NONE
About Dr.Kedar Karki M.V.St.Preventive Veterinary Medicine (CLSU)Philippines. Specialization in pathogenecity and immunological properties of Fusarium garaminearum Senior Vet.Officer Central Veterinary Laboratory Kathmandu Nepal